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Metabolomics of type 2 diabetes mellitus in sprague dawley rats—in search of potential metabolic biomarkers.

1. Introduction

2.1. multivariate statistical analysis, 2.2. screening of differential metabolites, 2.3. weekly changes of the potential biomarkers, 2.4. metabolic pathway discovery and analyses of differential metabolites, 2.5. potential biomarker verification, 3. discussion, 4. methods and materials, 4.1. experimental design, 4.2. induction of type 2 diabetes mellitus, 4.3. terminal studies, 4.4. analysis of serum samples, 4.5. untargeted gcxgc-tofms approach, 4.5.1. gcxgc-tofms analysis, 4.5.2. peak identification, 4.6. data clean-up, 4.7. metabolic biomarker discovery and pathway analysis, 4.8. statistical analysis, 5. conclusions and recommendations, supplementary materials, author contributions, institutional review board statement, informed consent statement, data availability statement, acknowledgments, conflicts of interest.

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Ndlovu, I.S.; Tshilwane, S.I.; Vosloo, A.; Chaisi, M.; Mukaratirwa, S. Metabolomics of Type 2 Diabetes Mellitus in Sprague Dawley Rats—In Search of Potential Metabolic Biomarkers. Int. J. Mol. Sci. 2023 , 24 , 12467. https://doi.org/10.3390/ijms241512467

Ndlovu IS, Tshilwane SI, Vosloo A, Chaisi M, Mukaratirwa S. Metabolomics of Type 2 Diabetes Mellitus in Sprague Dawley Rats—In Search of Potential Metabolic Biomarkers. International Journal of Molecular Sciences . 2023; 24(15):12467. https://doi.org/10.3390/ijms241512467

Ndlovu, Innocent Siyanda, Selaelo Ivy Tshilwane, Andre Vosloo, Mamohale Chaisi, and Samson Mukaratirwa. 2023. "Metabolomics of Type 2 Diabetes Mellitus in Sprague Dawley Rats—In Search of Potential Metabolic Biomarkers" International Journal of Molecular Sciences 24, no. 15: 12467. https://doi.org/10.3390/ijms241512467

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• Published: 21 August 2024

Inhibition of Caspase-1-dependent pyroptosis alleviates myocardial ischemia/reperfusion injury during cardiopulmonary bypass (CPB) in type 2 diabetic rats

• Wenjing Zhou 1 , 2   na1 ,
• Yingya Yang 1   na1 ,
• Zhouheng Feng 2 ,
• Yu Zhang 2 ,
• Yiman Chen 1 ,
• Tian Yu 2 &
• Haiying Wang 1  

Scientific Reports volume  14 , Article number:  19420 ( 2024 ) Cite this article

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• Medical research
• Molecular medicine

Cardiovascular complications pose a significant burden in type 2 diabetes mellitus (T2DM), driven by the intricate interplay of chronic hyperglycemia, insulin resistance, and lipid metabolism disturbances. Myocardial ischemia/reperfusion (MI/R) injury during cardiopulmonary bypass (CPB) exacerbates cardiac vulnerability. This study aims to probe the role of Caspase-1-dependent pyroptosis in global ischemia/reperfusion injury among T2DM rats undergoing CPB, elucidating the mechanisms underlying heightened myocardial injury in T2DM. This study established a rat model of T2DM and compared Mean arterial pressure (MAP), heart rate (HR), and hematocrit (Hct) between T2DM and normal rats. Myocardial cell morphology, infarction area, mitochondrial ROS and caspase-1 levels, NLRP3, pro-caspase-1, caspase-1 p10, GSDMD expressions, plasma CK-MB, cTnI, IL-1β, and IL-18 levels were assessed after reperfusion in both T2DM and normal rats. The role of Caspase-1-dependent pyroptosis in myocardial ischemia/reperfusion injury during CPB in T2DM rats was examined using the caspase-1 inhibitor VX-765 and the ROS scavenger NAC. T2DM rats demonstrated impaired glucose tolerance but stable hemodynamics during CPB, while showing heightened vulnerability to MI/R injury. This was marked by substantial lipid deposition, disrupted myocardial fibers, and intensified cellular apoptosis. The activation of caspase-1-mediated pyroptosis and increased reactive oxygen species (ROS) production further contributed to tissue damage and the ensuing inflammatory response. Notably, myocardial injury was mitigated by inhibiting caspase-1 through VX-765, which also attenuated the inflammatory cascade. Likewise, NAC treatment reduced oxidative stress and partially suppressed ROS-mediated caspase-1 activation, resulting in diminished myocardial injury. This study proved that Caspase-1-dependent pyroptosis significantly contributes to the inflammation and injury stemming from global MI/R in T2DM rats under CPB, which correlate with the surplus ROS generated by oxidative stress during reperfusion.

Introduction

The incidence of diabetes is on the rise, particularly among younger individuals. Global reports indicate that the global count of individuals aged 20–79 diagnosed with diabetes was 425 million in 2017, projected to escalate to 629 million by 2045 1 . Type 2 diabetes (T2DM) constitutes over 90% of all diabetes cases. Among individuals aged 65 and above with T2DM, 70% succumb to cardiovascular ailments, a rate 2–4 times higher than their non-diabetic counterparts. Consequently, T2DM, serving as a significant risk factor for cardiovascular maladies, presents a substantial menace to worldwide public health.

Several studies demonstrate that T2DM patients undergoing cardiopulmonary bypass surgery (CPB) suffer more severe myocardial injury compared to non-diabetic counterparts. This heightened vulnerability is attributed to T2DM-induced metabolic dysregulation, exacerbating myocardial sensitivity to ischemia–reperfusion (I/R). Hyperglycemia, insulin resistance, and abnormal lipid metabolism typical in T2DM foster oxidative stress and inflammation, thereby worsening myocardial damage. In T2DM rat models, mitochondrial dysfunction triggers increased myocardial cell apoptosis, larger infarction areas, and more pronounced cardiac dysfunction, with necroptosis playing a pivotal role. Post-ischemia–reperfusion, T2DM patients exhibit elevated serum levels of inflammatory cytokines such as IL-1β and IL-6, which further amplify myocardial injury. Thus, strategies to mitigate myocardial I/R injury in T2DM patients are urgently needed to enhance their postoperative outcomes.

Numerous studies indicate that T2DM patients suffer more severe myocardial injury after cardiopulmonary bypass surgery (CPB) compared to non-diabetic individuals 2 , 3 . This susceptibility is linked to T2DM-related metabolic disruptions that increase myocardial sensitivity to ischemia–reperfusion (I/R). T2DM is typically associated with hyperglycemia, insulin resistance, and abnormal lipid metabolism, leading to oxidative stress and inflammation, which worsen myocardial damage 4 , 5 , 6 . In T2DM rat models, impaired myocardial mitochondrial function results in heightened myocardial cell apoptosis, larger infarction areas, and more severe cardiac dysfunction, with necroptosis playing a crucial role 7 . Following ischemia–reperfusion injury, T2DM patients exhibit significantly higher levels of inflammatory cytokines like IL-1β and IL-6 in their serum compared to healthy controls, further exacerbating myocardial injury 8 . Therefore, effective strategies are urgently needed to reduce myocardial ischemia/reperfusion injury in T2DM patients and improve their postoperative outcomes.

Cell pyroptosis is a programmed cell death mode triggered by caspase-1 or caspase-4/5/11 activation through the inflammasome pathway, leading to GSDM family-mediated inflammatory necrosis. Its hallmark feature is the formation of membrane pores, releasing inflammatory contents and initiating robust inflammatory responses 9 , 10 . Recent studies underscore its role across various diseases, including cardiovascular, neurological, metabolic, and infectious disorders 11 , 12 , 13 . Increased cell death-associated molecules in myocardial cells of T2DM rats link closely with diabetic cardiomyopathy, atherosclerosis, and coronary heart disease 14 , 15 , 16 . In H9C2 myocardial cells under high glucose conditions, cell death exacerbates hypoxia/reoxygenation injuries 17 . Diabetes-related complications also involve caspase-1-driven cell death in liver ischemia–reperfusion injury, aggravating hepatic dysfunction. Strategies targeting ROS and NLRP3 inflammasome activation show promise in mitigating liver I/R injury in diabetic patients 18 . Activation of the NLRP3/caspase-1/IL-1β pathway contributes significantly to renal tubular injury in diabetic nephropathy (DN). Inhibiting NLRP3 or caspase-1 expression deactivates inflammasomes, protecting renal tissue and suggesting a therapeutic target for DN treatment 19 . Modulating the miR-214-3p/caspase-1 axis holds potential in reducing neuronal cell death, offering a strategy to mitigate diabetic brain damage 20 .

During cell apoptosis, reactive oxygen species (ROS) serve as vital inflammatory signals, promoting NOD-like receptor protein 3 (NLRP3) activation via pathways like nuclear factor-κB (NF-κB) and thioredoxin-interacting protein (TXNIP). This process plays a pivotal role in inducing cell apoptosis and fostering inflammatory responses 21 , 22 . NLRP3 collaborates with apoptosis-associated speck-like protein (ASC) and procaspase-1, forming the NLRP3 inflammasome—a central component in cell apoptosis. This inflammasome significantly contributes to myocardial ischemia–reperfusion injury, where its activation triggers myocardial cell apoptosis and elicits inflammatory responses. Effective mitigation of myocardial ischemia–reperfusion injury and reduction of myocardial cell apoptosis can be achieved by inhibiting the caspase-1-dependent cell pyroptosis pathway, thus affording myocardial protection.

Hence, the intricate interconnection among myocardial ischemia–reperfusion injury, type 2 diabetes mellitus, and cell pyroptosis warrants thorough exploration. Unraveling their mechanisms and identifying corresponding therapeutic approaches holds paramount importance for preventing and managing associated disorders. This study delved into the involvement and potential mechanisms of caspase-1-dependent cell pyroptosis in T2DM rat models subjected to CPB-induced whole-heart ischemia–reperfusion injury.

Animal care and ethics statement

This study is reported in accordance with ARRIVE guidelines. All animal experiments were ethically approved by the Institutional Animal Care and Use Committee of Zunyi Medical University and were conducted following the guidelines outlined in the National Institutes of Health Guide for the Care and Use of Laboratory Animals. Adult male Sprague–Dawley rats (200–250 g) were procured from an authorized breeder and housed at the accredited Animal Center of Zunyi Medical University. The rats were accommodated in a controlled environment with a 12-h light/dark cycle, receiving standard rodent diet and unrestricted water access. They were housed in groups of four per cage, furnished with ample space and bedding material, and underwent a one-week acclimatization period prior to experimentation. The cages were maintained with daily cleaning routines, and the animals were continuously monitored for any signs of distress or illness throughout the experimental period.

Establishment of T2DM rat model

Adult male Sprague–Dawley rats, SPF grade, weighing approximately 300 g, were maintained on a standard diet for one week while monitoring their health status. Subsequently, they received a high-fat and high-sugar diet for four weeks. Following a 12-h fasting period, a 0.1% STZ solution was prepared and administered intraperitoneally at a dose of 35 mg/kg. Blood glucose levels were assessed after 72 h, and rats displaying blood glucose concentrations > 16.7 mmol/L were confirmed as T2DM rats. Model validation was further conducted through glucose tolerance testing.

CPB procedure

Following intraperitoneal injection of a 2% pentobarbital solution (50 mg/kg) for anesthesia, the rat was positioned supine on a board with its limbs and head secured.

Following the administration of pentobarbital, we assessed the depth of anesthesia by monitoring respiratory rate, heart rate, and reflex responses. Surgery was initiated only when the animals exhibited a complete loss of reflex responses and stable vital signs. Throughout the surgical procedure, animals were continuously monitored for signs of pain or distress, including changes in respiratory rate, heart rate, and reflex responses. Additional doses of anesthetic were administered if any signs of pain or awakening were observed. Utilizing a pediatric laryngoscope, tracheal intubation was achieved using a 16G catheter needle. Mechanical ventilation was initiated through connection to a small animal ventilator. A 26G catheter was inserted into the tail vein to facilitate continuous fluid infusion and drug administration. Cannulation of bilateral femoral arteries (22G) was performed; one side was linked to a real-time arterial blood pressure monitor, while the other served for blood reflux during CPB surgery. The right jugular vein was catheterized and positioned for optimal drainage. After catheter placement, a left sternal incision exposed the chest cavity, and the ascending aortic root was ligated following the third rib's excision. The rat's membrane oxygenator was connected to a blood reservoir and peristaltic pump. Before liver heparinization (4 mg/kg) via the tail vein, tubing was primed with a solution. Target flow rate, adjusted to attain 100 ml/kg·min-1, was continuously monitored alongside mean arterial pressure (MAP) and heart rate. Blood gas analysis was performed to ensure stable circulation and internal environment during CPB, maintaining MAP ≥ 60 mmHg and Hct ≥ 20%. Adjustments were made if MAP < 60 mmHg, including increasing perfusion flow rate. The sham group experienced continuous circulation for 150 min post 10 min of simulated surgery, while the ischemia/reperfusion group maintained the target flow rate for 10 min before ascending aortic root clamping. After 30 min of cardiac arrest, temperature was gradually raised, the aorta opened, and circulation sustained for 120 additional minutes until experiment termination.

Temperature control during CPB

For inducing myocardial ischemia, ice chips were placed around the heart, simultaneously deactivating overhead light and the animal warming blanket, thereby reducing temperature to 28–30 ℃. Ice chips were removed and overhead light reinstated upon reperfusion initiation. Lab temperature was elevated, and the warming blanket activated to restore rectal temperature to 32–34 ℃.

Animal grouping

Nor+Sham (S) (n = 18) and T2DM+S (n = 18) Groups: The experiment terminates after 150 min of surgery with a target flow rate of 100 ml/kg·min-1.

Nor+I/R (n = 18) and T2DM+I/R (n = 18) Groups: Following a 10-min CPB period, the ascending aorta is clamped to induce 30 min of global cardiac ischemia. Subsequently, upon aortic release, the experiment concludes after 120 min of surgery.

T2DM+I/R+NAC (n = 18) and T2DM+I/R+Normal saline (NS) (n = 18) Groups: Prior to ischemia induction, NAC (150 mg/kg) or NS is continuously infused via the tail vein at a rate of 1 ml/h for 30 min. After a 10-min CPB interval, the ascending aorta undergoes clamping, leading to 30 min of global cardiac ischemia. The experiment culminates after 120 min of surgery upon aortic release.

T2DM+I/R+VX-765 (n = 18) and T2DM+I/R+DMSO (n = 18) Groups: VX-765 (16 mg/kg) or DMSO (0.5 ml/kg) is injected into the abdominal cavity 30 min prior to ischemia induction. After 10 min of CPB, the ascending aorta experiences clamping, inducing 30 min of global cardiac ischemia. Subsequently, upon aortic release, the experiment concludes after 120 min of surgery.

In our experiment, there were eight groups, with each group consisting of 18 rats for the electron microscopy (n = 6), infarct size (n = 6), and protein quantification experiments (sharing the same heart sample with the immunofluorescence experiment) (n = 6). Therefore, the study required a total of 36 normal rats and 108 T2DM rats. The mortality rate for normal rats undergoing CPB-I/R modeling was 5.56%, while the mortality rate for T2DM rats was 12.04%. If a rat died during the modeling process, an additional rat was included to maintain the sample size.

Myocardial infarction size assessment

The extent of myocardial infarction was determined employing the TTC (2,3,5-triphenyltetrazolium chloride) staining method. A 1% TTC staining solution was prepared and prewarmed to 37 °C in a light-resistant water bath prior to application. Upon experiment completion, intact rat hearts were promptly excised and placed in pre-cooled 4 °C PBS buffer. Following drainage of residual blood from the heart chambers, hearts were dried with blotting paper, then cooled at − 80 °C for 12 min.

Subsequently, hearts were sectioned into five uniformly thick segments across the heart's cross-section. These sections were then incubated in a pre-warmed TTC dye solution at room temperature (37 °C) for 30 min. After incubation, heart sections were rinsed with PBS, blotted with absorbent paper, and gently shaped before being fixed in formalin for 24 h and subsequently photographed. Image J software facilitated the calculation of the myocardial infarction area, presented as a percentage of the total area to depict the extent of myocardial infarction.

Transmission electron microscopy

In this study, transmission electron microscopy (TEM) was employed to investigate the ultrastructure of left ventricular myocardial tissue in rats. The tissue was initially fixed in 2.5% glutaraldehyde, followed by post-fixation with 1% osmium tetroxide. Subsequent steps included dehydration through an ethanol gradient, embedding in epoxy resin, and sectioning into thin slices using an ultramicrotome. These sections were stained with uranyl acetate and lead citrate prior to examination with a transmission electron microscope. Through this process, various ultrastructural changes were observed and analyzed, encompassing modifications in mitochondrial morphology, myofibril structure, and the identification of cellular damage indicators like swelling or vacuolation.

Flameng score

The Flameng score stands as a widely accepted approach for quantifying mitochondrial impairment within cardiac tissue. This method holds significance as a tool to gauge the extent of myocardial injury across diverse cardiac conditions. An experienced electron microscopist, unaware of the experimental contexts and treatment groups, is tasked with determining the score. This measure is employed to ensure both objectivity and precision in assessment. The employed Flameng score variant adheres to a 0–4 grading system:

Score 0: Normal mitochondrial configuration, characterized by orderly cristae and intact outer and inner membranes.

Score 1: Minimal mitochondrial impairment, marked by slightly disorganized cristae and/or minor swelling or vacuolization.

Score 2: Mild to moderate mitochondrial damage, indicating moderately disrupted cristae and/or moderate swelling or vacuolization.

Score 3: Moderate to severe mitochondrial damage, showcasing severely disrupted cristae and/or pronounced swelling or vacuolization.

Score 4: Severe mitochondrial damage, manifesting as complete cristae disruption and/or fragmentation of the mitochondrial matrix.

Myocardial ROS assessment

Upon reperfusion completion, left ventricular myocardial tissue was collected and embedded using OTC embedding solution. Subsequently, the myocardial tissue was sectioned into 10-µm-thick segments using a frozen microtome. These sections were directly affixed to slides. To initiate the assessment, the sections underwent three 5-min washes with pre-warmed PBS at 37 °C. A 10 μmol/l solution of DHE probe, similarly warmed to 37 °C, was then applied onto the sections. The sections were incubated within a light-resistant humidified chamber at 37 °C for 30 min. Following the incubation period, three additional washes with PBS at 37 °C (5 min each) were executed under light-protected conditions. Post-washing, the sections were allowed to dry before being treated with DAPI dye and enclosed with coverslips. The sections were subsequently examined and imaged using fluorescence microscopy. Utilizing Image J software, the average fluorescence intensity was assessed and recorded.

Quantification of myocardial Caspase-1 immunofluorescence

The tissue sections were initially fixed using 1.4% paraformaldehyde for a 15-min duration. Subsequent steps involved sequential washes with PBS to enhance cell membrane permeability: incubation with 0.2% Triton X-100 for 10 min, followed by three PBS washes, each lasting 5 min. For primary antibody application, a dropwise addition of diluted primary antibody was performed, and refrigeration was maintained overnight at 4 °C. The sections were subsequently washed three times with PBS for 15 min each time. After preparing and applying the secondary antibody, sections were placed in a humid chamber for a 1-h incubation at room temperature. This was followed by three PBS washes, each lasting 15 min. Upon completing these procedures, the sections underwent observation and photography via fluorescence microscopy.

For each heart, 5–6 images were captured and analyzed to ensure comprehensive coverage and accurate representation of the tissue. Each heart was sectioned into several regions: apex, mid-ventricle, and base. From each section, images were taken from both the endocardial and epicardial regions. This systematic approach ensured that different areas of the myocardium were represented, capturing the heterogeneity within the heart tissue. The images were analyzed using ImageJ software to quantify fluorescence intensity. The mean fluorescence intensity for each image was calculated. These values were then averaged to obtain a single representative value for each heart, which was used for statistical analysis. This process was performed separately for both ROS and caspase-1 immunofluorescence measurements.

Western blot analysis

Rat left ventricular myocardial tissue samples were collected and homogenized in RIPA buffer supplemented with protease inhibitors. The protein content was quantified utilizing the Bradford assay, and equivalent protein quantities were loaded onto a 10% SDS–polyacrylamide gel for electrophoresis. Following gel electrophoresis, proteins were transferred onto a PVDF membrane, which was then subjected to a 1-h blocking step at room temperature using 5% non-fat milk. Subsequently, primary antibodies (β-Actin: Affinity, AF7018, 1:5000; NLRP3: Affinity, DF7438, 1:1000; GSDMD: Affinity, DF12275, 1:1000; por-Caspase1: Affinity, AF5418, 1:1000; Caspase p10: Affinity, AF4022, 1:500) specific to the protein of interest within rat left ventricular myocardial tissue were employed for an overnight incubation at 4 °C. This was followed by a 1-h incubation at room temperature with a secondary antibody (Goat Anti-Rabbit IgG(H+L) HRP: MULTI SCIENCES, GAR0072, 1:0000) conjugated to horseradish peroxidase. The PVDF membrane was subsequently processed using an enhanced chemiluminescence system and analyzed through chemiluminescence detection. Protein expression levels were quantified via densitometric analysis performed using ImageJ software. To ensure reliability, the experiments were conducted in triplicate using distinct samples. Due to budget constraints and in an effort to conserve antibodies, the blots were trimmed before hybridization with antibodies. Images of all blots with membrane edges visible were included in the Supplementary Information file .

Blood sample collection and ELISA assay

Heparinized tubes were employed to collect blood samples from experimental animals, subsequently centrifuged at 1500 g for 10 min at 4 °C, yielding plasma. The concentrations of cardiac enzymes, encompassing creatine kinase-MB (CK-MB) and lactate dehydrogenase (LDH), alongside inflammatory cytokines such as tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6), were determined via commercially available ELISA kits (BioLegend, San Diego, CA, USA) following the manufacturer’s guidelines. The procedure involved the coating of 96-well plates with specific capture antibodies corresponding to each target molecule, left to incubate overnight at 4 °C. After buffer washing, the plates were blocked with 1% bovine serum albumin (BSA) for 1 h at room temperature. Subsequent steps included addition of plasma samples and standard solutions into the wells, followed by a 2-h incubation at room temperature. The wells were then subjected to a sequence of washing and incubation with biotinylated detection antibodies for 1 h at room temperature. Streptavidin–horseradish peroxidase (HRP) conjugate was applied for a 30-min incubation, followed by a final washing step. The plates were developed using 3,3′,5,5′-tetramethylbenzidine (TMB) substrate, and absorbance was quantified at 450 nm using a microplate reader. Quantification of cardiac enzymes and cytokines relied on a standard curve generated from known protein concentrations. All samples were analyzed in duplicate, and mean values were derived. The intra-assay and inter-assay coefficients of variation (CVs) were both less than 10% and 15%, respectively, indicative of robust assay precision.

Statistical analysis

GraphPad Prism version 8.0.2 (GraphPad Software, La Jolla, CA, USA) was employed to analyze data from Western blot findings, immunofluorescence intensity, myocardial infarction area, mitochondrial Flameng score, heart rate, blood pressure, and temporal hemoglobin alterations. Prior to analysis, normality was assessed via the Shapiro–Wilk test, and homogeneity of variances was determined using Levene’s test. For comparative analyses, the unpaired Student's t-test was employed when comparing two groups, while one-way ANOVA followed by Tukey's multiple comparison test was utilized for comparisons involving more than two groups. Outcomes are presented as mean ± standard error of the mean (SEM). Statistical significance was denoted at a level of P < 0.05.

The hemodynamic and internal environment effects of global MI/R in a cardiopulmonary bypass model of T2DM rats

Figure  1 A, B displays the outcomes of glucose tolerance tests (IPGTT & OGTT). Following the administration of a 20% glucose solution via gavage or intraperitoneal injection, both normal and T2DM rats exhibited a substantial increase in blood glucose levels, which gradually decreased after 30 min. Remarkably, T2DM rats consistently displayed higher blood glucose levels exceeding 16.7 mmol/l (P < 0.05) compared to normal rats, indicating impaired glucose tolerance and confirming the effectiveness of the model. Mean arterial pressure (MAP) in all groups demonstrated a gradual decrease over time, remaining above 60 mmHg at all time points. Compared to the T0 group, T1–T4 groups exhibited significant decreases in MAP (P < 0.05) (Fig.  1 C). Similarly, Fig.  1 D reveal that heart rate (HR) significantly decreased in T1-T4 groups compared to the T0 group (P < 0.05). The I/R groups all achieved complete cardiac arrest at T2 and were successfully resuscitated at T3, approaching the S group at T4. Figure  1 E illustrate that the hematocrit (Hct) of all groups remained above 20% at all time points, despite a significant decrease at T1 compared to T0 (P < 0.05) due to the dilution effect of the priming solution. Overall, these findings confirm the stable hemodynamics and internal environment of T2DM rats and normal rats during CPB.

T2DM rats and normal rats exhibit same changes in hemodynamics and internal environment during CPB. ( A ) Blood glucose level in intraperitoneal glucose tolerance test (IPGTT). ( B ) Blood glucose level in oral glucose tolerance tests (OGTT). ( C ) Changes in Mean Arterial Pressure (MAP) among different groups of rats. ( D ) Changes in heart rate (HR) among different groups of rats. The heart rate (HR) and blood pressure are monitored via the femoral artery rather than electrocardiography. The observed drop in HR to zero in the control group occurs during aortic cross-clamping, when the heart temporarily ceases to eject blood. This is an artifact of the monitoring method and not an indication of actual cardiac arrest. ( E ) Changes in hematocrit (Hct) among different groups of rats. Compared with the Nor group, a represents P < 0.05. Compared to the T0 time point within the same group, b indicates P < 0.05.

The severity of myocardial ischemia/reperfusion injury was greater in T2DM rats compared to normal rats

The infarct area was found to be larger in both the Nor+I/R and T2DM+I/R groups compared to their respective S group (P < 0.05), as determined through TTC staining. Furthermore, the T2DM+I/R group exhibited a significantly greater infarct area than the Nor+I/R group during the ischemia/reperfusion (I/R) state (P < 0.05), as depicted in Fig.  2 A, B, indicating a more severe local myocardial injury. The application of ELISA facilitated the detection of cardiac enzymes CK-MB and cTnI release within rat plasma. The release of both CK-MB and cTnI was notably elevated in the T2DM+S group when contrasted with the Nor+S group (P < 0.05). Similarly, the release of CK-MB and cTnI was markedly higher in both the Nor+I/R and T2DM+I/R groups compared to their corresponding S group (P < 0.05). In particular, the T2DM+I/R group displayed a notably higher release of CK-MB and cTnI than the Nor+I/R group in the I/R state (P < 0.05), as illustrated in Fig.  2 C, D.

Myocardial injury following global ischemia/reperfusion in T2DM rats and normal rats. ( A ) Comparison of infarct area in rat hearts after TTC staining among different groups, where the white area represents the myocardial infarct region and the red area represents the normal myocardial region. ( B ) Statistical results of Myocardial infarct area in different groups of rats. ( C ) and ( D ) ELISA detection results of myocardial enzymes (ck-mb, ctni) in different groups of rats. ( E ) Transmission electron micrograph of cardiac tissue, with the enlarged view of the red-framed area on the left shown on the right side. The arrows indicate damaged mitochondria. ( F ) Statistical results of myocardial mitochondrial scoring in different groups of rats. *P < 0.05, **P < 0.01, ***P < 0.001.

The ultrastructure of cardiac myocytes was examined using transmission electron microscopy (Fig.  2 E). Subsequent to global ischemia/reperfusion induced by CPB, both the Nor+I/R and T2DM+I/R groups exhibited myocardial fiber disorder, breakage, and interstitial edema. Upon higher magnification, the Nor+S group displayed regular mitochondrial morphology with few instances of rupture. Conversely, the Nor+I/R and T2DM+I/R groups presented the disappearance of mitochondrial ridges, loss of mitochondrial membrane integrity, and mitochondrial swelling and rupture, highlighting severe local cellular damage and compromised myocardial integrity. These alterations were accompanied by early indicators of cell death, including nuclear swelling and chromatin marginalization. The evaluation of mitochondrial scoring, as shown in Fig.  2 F, unveiled a notably higher mitochondrial score in the T2DM+S group compared to the Nor+S group (P < 0.05). Furthermore, substantial mitochondrial damage was evident in both the Nor+I/R and T2DM+I/R groups, reflected by an increased mitochondrial score compared to their corresponding S groups (P < 0.05). Notably, the T2DM+I/R group exhibited more severe mitochondrial damage than the Nor+I/R group during the I/R state (P < 0.05), indicating a critical local response to ischemia–reperfusion injury.

The extent of cellular apoptosis activation in T2DM rats subjected to CPB-induced global ischemia/reperfusion surpassed that observed in normal rats

Reactive oxygen species (ROS) production was evaluated through fluorescence microscopy employing DHE and DAPI staining (Fig.  3 A, B). The outcomes revealed heightened ROS generation in T2DM+S rats in comparison to Nor+S rats (P < 0.05). Moreover, both Nor+I/R and T2DM+I/R rats exhibited elevated ROS production relative to their respective S groups (P < 0.05). Notably, during the I/R state, T2DM+I/R rats displayed a more pronounced rise in ROS production than Nor+I/R rats (P < 0.05), contributing to further myocardial damage and dysfunction. Immunofluorescence staining was utilized to assess the relative fluorescence intensity of caspase-1 (Fig.  3 C, D. Findings demonstrated escalated caspase-1 expression in T2DM+S rats compared to Nor+S rats (P < 0.05). Similarly, both Nor+I/R and T2DM+I/R rats displayed heightened caspase-1 expression in contrast to their corresponding S groups (P < 0.05). Remarkably, in the I/R state, T2DM+I/R rats exhibited a more substantial upsurge in caspase-1 expression than Nor+I/R rats (P < 0.05), indicating enhanced local inflammation and pyroptosis, exacerbating myocardial injury.

The oxidative stress response following CPB in T2DM rats and normal rats. ( A ) Rat myocardial tissue ROS immunofluorescence staining images for each group, with red fluorescence representing ROS and blue fluorescence representing cell nuclei. ( B ) Statistical results of relative ROS fluorescence intensity in rat myocardial tissue for each group. ( C ) Rat myocardial tissue caspase-1 immunofluorescence staining images for each group, with red fluorescence representing caspase-1 and blue fluorescence representing cell nuclei. ( D ) Statistical results of relative caspase-1 fluorescence intensity in rat myocardial tissue for each group. *P < 0.05, **P < 0.01, ***P < 0.001.

Western blot analysis unveiled augmented expression of NLRP3, pro-caspase-1, caspase-1 p10, and GSDMD across all groups relative to Nor+S rats (P < 0.05) (Fig.  4 A–E). ELISA results (Fig.  4 F, G) indicated amplified release of IL-1β and IL-18 in T2DM+S rats compared to Nor+S rats (P < 0.05). Furthermore, both Nor+I/R and T2DM+I/R rats showed increased release of IL-1β and IL-18 in comparison to their corresponding S groups (P < 0.05). Notably, T2DM+I/R rats displayed a more pronounced elevation in release of IL-1β and IL-18 compared to Nor+I/R rats during the I/R state (P < 0.05), reflecting extensive local myocardial damage and systemic inflammatory response.

The myocardial cell apoptosis status following CPB in rats with T2DM and normal rats. ( A ) Representative Western blot bands of apoptosis-related proteins in rat myocardial tissue from each group. ( B – E ) Statistical results of expression levels for each apoptosis-related protein. ( F ) and ( G ) Statistical results of ELISA measurements for IL-1β and IL-18 levels in rat groups. *P < 0.05, **P < 0.01, ***P < 0.001.

Inhibiting caspase-1 activation mitigates myocardial injury in T2DM rats with MI/RI

We then used the caspase-1 inhibitor VX-765 to assess the impact of caspase-1 inhibition on MI/RI-induced myocardial injury in T2DM rats. VX-765 is a prodrug that requires activation by cellular esterases to convert into its active form, VRT-043198, which effectively inhibits caspase-1 23 . Due to this requirement, VX-765 (16 mg/kg) was dissolved in DMSO (0.5 ml/kg DMSO diluted with NS to 0.9 ml) and administered intraperitoneally 30 min before ischemia to ensure its conversion to the active form prior to myocardial I/R injury. This method allows sufficient time for the prodrug to be activated within the target cells. The T2DM+I/R+VX-765 group displayed a smaller myocardial infarction area, in contrast to the control group (P < 0.05) (Fig.  5 A). Transmission electron microscopy depicted disorganized and swollen myocardial fibers across all groups, with varying mitochondrial damage; however, the T2DM+I/R+VX-765 group exhibited notably reduced mitochondrial impairment (P < 0.05) (Fig.  5 B). The caspase-1 inhibitor VX-765 significantly attenuated caspase-1 activation and downregulated its downstream targets, p10 and GSDMD (P < 0.05), while NLRP3 and pro-caspase-1 expression remained unaffected (Fig.  5 C–G). Moreover, ELISA analysis of plasma samples revealed diminished levels of IL-1β and IL-18 in the T2DM+I/R+VX-765 group, along with decreased CK-MB and cTnI levels, compared to the control group (P < 0.05) (Fig.  5 H–K). These results imply that caspase-1 inhibition might mitigate myocardial injury in T2DM rats during MI/RI, potentially by suppressing the inflammasome activation pathway.

Inhibiting caspase-1 can alleviate myocardial damage in CPB-induced cardiac injury in T2DM rats. ( A ) Comparison of myocardial infarction areas between the VX-765 treatment group and the non-treatment group in rats. ( B ) Transmission electron micrograph of cardiac tissue, with the enlarged view of the red-framed area on the left shown on the right side. The arrows indicate damaged mitochondria, and statistical results of myocardial mitochondrial scoring. ( C ) Representative Western blot bands of apoptosis-related proteins. ( D – G ) Statistical results of expression levels for each apoptosis-related protein. ( H – K ) Statistical results of ELISA measurements for IL-1β, IL-18, CK-MB and cTnI levels. *P < 0.05, **P < 0.01, ***P < 0.001.

ROS induction significantly contributes to cell apoptosis during myocardial ischemia/reperfusion injury in T2DM rats

N-acetylcysteine (NAC) was employed to mitigate reactive oxygen species (ROS) in myocardial cells. The relative ROS fluorescence intensity in T2DM+I/R+NAC myocardial tissue notably decreased compared to the control group (P < 0.05), indicating effective ROS reduction by NAC in T2DM+I/R+NAC rat myocardial cells (Fig.  6 A, B). Furthermore, caspase-1 expression in T2DM+I/R+NAC myocardial tissue significantly decreased compared to the control group (P < 0.05), as indicated by relative caspase-1 fluorescence intensity among groups (Fig.  6 C, D).

Inhibition of ROS by N -acetylcysteine. ( A ) Rat myocardial tissue ROS immunofluorescence staining images, with red fluorescence representing ROS and blue fluorescence representing cell nuclei. ( B ) Statistical results of relative ROS fluorescence intensity in rat myocardial tissue. ( C ) Rat myocardial tissue caspase-1 immunofluorescence staining images, with red fluorescence representing caspase-1 and blue fluorescence representing cell nuclei. ( D ) Statistical results of relative caspase-1 fluorescence intensity in rat myocardial tissue. *P < 0.05, ***P < 0.001.

Myocardial infarction area in T2DM+I/R+NAC rats was notably smaller than in the control group (P < 0.05), as indicated in Fig.  7 A. Transmission electron microscopy images of myocardial tissue revealed disordered myocardial fiber arrangement, interstitial edema, rupture, and varying degrees of mitochondrial damage including mitochondrial ridge disappearance, loss of mitochondrial membrane integrity, and swelling. The mitochondrial injury score illustrated significantly reduced mitochondrial damage in the T2DM+I/R+NAC group versus the control group (P < 0.05) (Fig.  7 B). The T2DM+I/R+NAC rat myocardial tissue exhibited markedly diminished expression of NLRP3, pro-caspase-1, caspase-1 p10, and GSDMD compared to the control group (P < 0.05), as presented in Fig.  7 C–G. ELISA outcomes demonstrated considerable reduction in plasma IL-1β and IL-18 levels, and significantly reduced release of CK-MB and cTnI in T2DM+I/R+NAC rats compared to the control group (P < 0.05), as detailed in Fig.  7 H–K.

Inhibit ROS-induced cell apoptosis on myocardial injury in T2DM rats following CPB ischemia–reperfusion. ( A ) Comparison of myocardial infarction areas between the NAC treatment group and the non-treatment group in rats. ( B ) Transmission electron micrograph of cardiac tissue, with the enlarged view of the red-framed area on the left shown on the right side. The arrows indicate damaged mitochondria, and statistical results of myocardial mitochondrial scoring. ( C ) Representative Western blot bands of apoptosis-related proteins. ( D – G ) Statistical results of expression levels for each apoptosis-related protein. ( H – K ) Statistical results of ELISA measurements for IL-1β, IL-18, CK-MB and cTnI levels. *P < 0.05, **P < 0.01, ***P < 0.001.

This study investigated the impact of global myocardial ischemia/reperfusion (MI/R) on hemodynamics and the internal milieu within a cardiopulmonary bypass (CPB) model using type 2 diabetes mellitus (T2DM) rats. Our findings revealed that T2DM rats exhibited impaired glucose tolerance but maintained stable hemodynamics and internal conditions during CPB. However, T2DM rats experienced heightened myocardial ischemia/reperfusion injury compared to normal rats. This was evident through substantial lipid deposition, disrupted myocardial fibers, fragmentation, and interstitial edema, which underscores the exacerbated local myocardial injury and critical local cellular damage due to T2DM-mediated metabolic disturbances. Moreover, the activation of cellular apoptosis was significantly more pronounced in T2DM rats during CPB-induced global ischemia/reperfusion, accompanied by elevated generation of reactive oxygen species and increased caspase-1 expression. Notably, the marked increase in ROS levels in the myocardial tissue of T2DM rats highlights the local oxidative stress contributing to myocardial injury. Elevated caspase-1 expression suggests enhanced local inflammation and cell death via pyroptosis, further aggravating myocardial damage. The significantly higher levels of cardiac enzymes (CK-MB and LDH) and inflammatory cytokines (TNF-α and IL-6) in T2DM rats indicate extensive local myocardial damage and a pronounced systemic inflammatory response. Thus, T2DM rats subjected to CPB-induced global cardiac ischemia/reperfusion displayed exacerbated myocardial tissue damage and inflammatory responses, possibly due to heightened cell necroptosis activation, potentially linked to excessive ROS production during reperfusion.

This investigation establishes that type 2 diabetes mellitus (T2DM), accompanied by metabolic syndrome, involving chronic hyperglycemia, insulin resistance, and lipid metabolism disorders, heightens not only the vulnerability to myocardial ischemia and cardiomyopathy but also diminishes the innate myocardial safeguards and tolerance against ischemia/reperfusion (I/R) injury. Consequently, this cascade contributes to the development of heart failure and sudden cardiac death 24 .

In this study, by utilizing transmission electron microscopy to scrutinize myocardial tissue, we identified substantial lipid deposition within myocardial cells of T2DM rats. However, these rats exhibited no pathological changes indicative of diabetic cardiomyopathy (DCM), such as interstitial fibrosis or myocardial remodeling, when compared to normal rats 25 , 26 . Conversely, T2DM+S rats displayed intensified mitochondrial damage in comparison to Nor+S rats. This heightened damage can be ascribed to various pathways, including the chronic hyperglycemia-induced advanced glycation end products (AGEs) pathway, the protein kinase C (PKC) pathway, and heightened mitochondrial metabolism. These factors contribute to the accumulation of mitochondrial reactive oxygen species (ROS), alterations in respiratory function and membrane potential, ultimately culminating in mitochondrial dysfunction 27 .

The study successfully established the T2DM model, confirmed through subsequent transmission electron microscopy analysis, which unveiled significant pathological alterations in the mitochondria of T2DM+S rats. These modifications encompassed mitochondrial swelling, cristae disappearance, and a higher score of mitochondrial injury. These findings signify that the T2DM-associated metabolic syndrome can trigger myocardial mitochondrial dysfunction even before the conventional pathological manifestations of DCM, like interstitial fibrosis and myocardial remodeling. Moreover, the T2DM+S cohort demonstrated heightened expression of cell apoptosis-related proteins and the release of inflammatory factors in comparison to the Nor+S group. This observation implies that T2DM itself possesses the potential to activate cell apoptosis.

Caspase-1-dependent pyroptosis represents a mode of cell demise triggered by caspase-1 activation in response to heightened reactive oxygen species (ROS) levels 28 , 29 . This investigation revealed a substantial elevation in the expression of pyroptosis-associated proteins—NLRP3, pro-caspase-1, caspase-1 p10, and GSDMD—alongside the release of inflammatory cytokines IL-1β and IL-18 across all rat groups. Particularly, in comparison to their respective “S” groups, both the Nor+I/R and T2DM+I/R groups exhibited heightened protein expression. Notably, the T2DM+I/R group demonstrated a more pronounced increase, indicating an escalated activation of caspase-1-dependent pyroptosis, thereby culminating in heightened inflammatory damage. In our study, we observed an increase in the inactive form of caspase-1 in the ischemia–reperfusion (I/R) groups (Fig.  4 ). This finding may be attributed to the early initiation of inflammasome activation during the ischemic phase induced by CPB. It is hypothesized that the accumulation of inactive caspase-1 forms part of the preparatory phase for subsequent inflammatory responses during reperfusion. This phenomenon has been supported by previous studies suggesting that inflammasome components, including caspase-1, undergo initial accumulation and activation under ischemic conditions 30 , 31 . Therefore, the observed increase in inactive caspase-1 forms in our study underscores the complex interplay between ischemia and subsequent inflammatory cascades in myocardial ischemia–reperfusion injury.

VX-765, an extensively selective and safe caspase-1 inhibitor, is a bioavailable small molecule that underwent testing in a phase II human clinical trial for epilepsy treatment 32 . Notably, it effectively diminishes plasma IL-1β and IL-18 levels, exhibiting anti-inflammatory effects across diverse disease processes. Despite its demonstrated efficacy, the exact mechanism of action remains enigmatic 33 , 34 . To effectively inhibit caspase-1, VX-765 must undergo enzymatic conversion to VRT-043198. In this study, 16 mg/kg of VX-765 was dissolved in DMSO (0.5 ml/kg DMSO+NS diluted to 0.9 ml) and administered intraperitoneally 30 min before ischemia to ensure its transformation into its active form prior to myocardial I/R 35 .

The results demonstrate that VX-765 effectively mitigated myocardial injury in T2DM rats subjected to global ischemia/reperfusion (I/R). VX-765 restrained caspase-1 activation, leading to reduced expression of caspase-1 p10 fragment and downstream GSDMD. This, in turn, caused a decrease in the release of IL-1β and IL-18, while leaving the expression of upstream proteins NLRP3 and pro-caspase-1 unaffected. The T2DM+I/R+VX-765 group exhibited noteworthy reductions in mitochondrial score, myocardial infarct size, and the release of CK-MB and cTnI when compared to the T2DM+I/R+DMSO control group. These findings underscore the pivotal role of caspase-1-mediated cell pyroptosis in the inflammatory and injurious progression of I/R injury in T2DM rats. Inhibition of caspase-1 activation through VX-765 emerges as a viable approach for safeguarding ischemic/reperfused myocardium.

Under normal physiological conditions, the mitochondrial respiratory chain minimally converts oxygen into reactive oxygen species (ROS). However, metabolic alterations in type 2 diabetes (T2DM) individuals, such as adipocyte hypertrophy, hypoxia, chronic hyperglycemia, and insulin resistance, activate the advanced glycation end product (AGE) and protein kinase C (PKC) pathways. These changes culminate in ROS accumulation within mitochondria, disrupting the balance between oxidation and antioxidation. This, in turn, triggers mitochondrial oxidative stress, impairs oxidative phosphorylation metabolism, and ultimately induces cell death 36 . Research indicates that ROS buildup and mitochondrial impairment serve as pivotal cues for the initiation of caspase-1-mediated cell pyroptosis. ROS can directly instigate NLRP3 inflammasome assembly and activation, fostering caspase-1-driven cell pyroptosis. Moreover, mitochondrial damage releases mtDNA, activating the caspase-1/GSDMD/IL-1β, IL-18 pathway. During myocardial ischemia/reperfusion (I/R), substantial ROS release due to myocardial damage fosters caspase-1-associated cell pyroptosis, exacerbating ischemic injury. This investigation identified T2DM and myocardial I/R as key instigators of ROS accumulation and mitochondrial damage in rat myocardial cells. Relative to the Nor+S group, T2DM+S and Nor+I/R group rats exhibited heightened ROS fluorescence intensity and mitochondrial scores in myocardial tissue. The caspase-1/GSDMD/IL-1β, IL-18 pathway activated within the myocardium of T2DM+S and Nor+I/R group rats. Subsequent to I/R treatment in T2DM rats, increased ROS production and aggravated mitochondrial damage in myocardial cells amplified caspase-1-dependent cell pyroptosis and the ensuing inflammatory response. This likely underpins the heightened myocardial injury observed in T2DM+I/R group rats in comparison to Nor+I/R group rats.

In our study, Fig.  5 F showed elevated pro-caspase levels in the DMSO-treated group without reaching statistical significance. This result can be attributed to several factors. First, the use of DMSO as a solvent may influence cellular physiology due to its biological activity, potentially interfering with intracellular signaling pathways and causing variability in pro-caspase levels 37 . This interference, while visually apparent, may not manifest as statistically significant differences. Second, biological variability among experimental animals could contribute to inconsistent results. Individual differences in the animal model may affect parameter variations between treatment groups, leading to high variability within groups 38 . Third, the sensitivity and specificity of the detection method used in this study might have limitations, especially in detecting subtle changes, which could result in non-significant statistical outcomes despite apparent differences. Finally, the biological mechanisms underlying the observed elevated pro-caspase levels in the DMSO-treated group require further investigation. DMSO might indirectly increase pro-caspase levels by affecting inflammatory pathways or apoptosis mechanisms 39 . Further studies are needed to elucidate these potential mechanisms and confirm the specific effects of DMSO on caspase activity and cell apoptosis.

N-acetylcysteine (NAC) is a well-established antioxidant renowned for its efficacy in neutralizing diverse reactive oxygen species (ROS), encompassing hydrogen peroxide, hydroxyl radicals, and hypochlorous acid. Its clinical utility in treating various ailments has garnered recognition over several decades. Earlier investigations have evidenced that continuous pre-ischemia infusion or infusion within 5 min before reperfusion of NAC notably mitigates ischemia/reperfusion (I/R)-induced myocardial injury, surpassing the efficacy of an equivalent dose of NAC administered through rapid intravenous injection 30 min before ischemia. In this study, we adopted continuous tail vein infusion of NAC and substantiated its efficacy via ROS immunofluorescence. The T2DM+I/R+NAC group exhibited considerably diminished relative ROS fluorescence intensity within rat myocardium in comparison to the T2DM+I/R+NS group, signifying abated oxidative stress-induced damage. Moreover, NAC administration significantly ameliorated I/R-triggered myocardial injury, as evidenced by marked reductions in myocardial infarct size and the release of cardiac biomarkers CK-MB and cTnI. Additionally, NAC treatment markedly downregulated the expression of NLRP3, pro-caspase-1, caspase-1 p10, and GSDMD. This correlated with diminished release of IL-1β and IL-18, hinting at a potential association between elevated cell death-associated molecules and heightened ROS production due to oxidative stress. NAC treatment partially hindered ROS-mediated activation of caspase-1-driven cell death, potentially contributing to the attenuation of myocardial and reperfusion injury. Collectively, these outcomes underscore NAC infusion as a promising therapeutic avenue for curtailing myocardial injury in the context of I/R.

This study possesses several limitations. Firstly, it solely investigates the role of cell necroptosis in animal models, omitting an examination of conventional cellular-level morphological shifts associated with necroptosis. Secondly, it solely examines short-term alterations in myocardial cell ultrastructure and necroptosis-related protein activation within T2DM rats. This study refrains from conducting extended observations and comparisons, which could offer deeper insights into the impact of T2DM-induced cell necroptosis on cardiac health and its underlying mechanisms. Thirdly, due to model constraints, the assessment of rats' cardiac function during the CPB process lacks ultrasound evaluation. Such an approach would have enhanced comprehension of cardiac functional changes and their correlation with cell necroptosis. Thirdly, the study does not comprehensively analyze necroptosis-related pathways, thus limiting our grasp of pathway interactions and mutual constraints. Lastly, although pentobarbital is commonly used in extracorporeal circulation myocardial protection studies due to its effectiveness in maintaining stable anesthesia and providing both anesthetic and analgesic effects in rodents, it does not fully replicate the complex, multi-agent anesthesia regimens (such as fentanyl, midazolam, sevoflurane) used in clinical cardiopulmonary bypass (CPB) procedures. It is important to adopt anesthesia protocols that closely mimic clinical conditions to enhance the translational potential of preclinical findings. Therefore, future studies should aim to incorporate anesthetic regimens that are more aligned with clinical practices.

To conclude, this investigation establishes that T2DM heightens myocardial necroptosis during CPB, concomitant with the activation of caspase-1-dependent cell pyroptosis. The elevation of cell death-related molecules potentially links to heightened ROS production arising from oxidative stress. NAC, functioning as an antioxidant, exhibits partial restraint over caspase-1-dependent cell death activation mediated by ROS. This intervention consequently ameliorates myocardial injury induced by I/R (Fig.  8 ). The findings underscore the therapeutic potential of targeting necroptosis-related pathways to safeguard against myocardial injury during CPB in T2DM patients.

Mechanistic pathway of myocardial cell pyroptosis under T2DM hyperglycemia conditions. Hyperglycemia conditions associated with T2DM exacerbate myocardial ischemia–reperfusion injury (MI-RI) occurring during CPB, leading to mitochondrial dysfunction. The dysfunction of mitochondria results in an increase in reactive oxygen species (ROS) and the release of mitochondrial DNA (mtDNA). These mitochondrial-derived signals activate the NLRP3 inflammasome, a critical component in the pyroptosis pathway. The activation of NLRP3 subsequently leads to the conversion of pro-caspase-1 into active caspase-1. Activated caspase-1 cleaves gasdermin D (GSDMD), producing its N-terminal (N) and C-terminal (C) domains. The N-terminal domain of GSDMD forms pores in the cell membrane, facilitating pyroptosis. Concurrently, caspase-1 processes pro-inflammatory cytokines, pro-IL-1β and pro-IL-18, into their mature forms, IL-1β and IL-18. The formation of GSDMD-N pores culminates in pyroptosis, characterized by cell swelling and lysis, thereby releasing IL-1β and IL-18 into the extracellular space and promoting inflammation. T2DM, Type 2 Diabetes Mellitus; MI-RI, Myocardial Ischemia–Reperfusion Injury; CPB, Cardiopulmonary Bypass; ROS, Reactive Oxygen Species; mtDNA, Mitochondrial DNA; NLRP3, NOD-like receptor family pyrin domain-containing 3; GSDMD, Gasdermin D; IL-1β, Interleukin-1 beta; IL-18, Interleukin-18.

Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

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Acknowledgements

This paper was supported by Zunyi science and technology plan project [zunshikeheHZ-2023-227], and was partially supported by National Natural Science Foundation of China (NSFC) under grant No. 81860062.

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These authors contributed equally: Wenjing Zhou and Yingya Yang.

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Department of Anesthesiology, Affiliated Hospital of Zunyi Medical University, 149 Dalian Street, Zunyi, 563000, Guizhou, People’s Republic of China

Wenjing Zhou, Yingya Yang, Yiman Chen & Haiying Wang

Guizhou Key Laboratory of Anesthesia and Organ Protection, Affiliated Hospital of Zunyi Medical University, Zunyi, 563003, Guizhou, People’s Republic of China

Wenjing Zhou, Zhouheng Feng, Yu Zhang & Tian Yu

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Conception and design: Haiying Wang, Wenjing Zhou Collection and assembly of data: Wenjing Zhou, Yingya Yang, Zhouheng Feng Data analysis and interpretation: Wenjing Zhou, Yingya Yang, Yiman Chen Manuscript writing: Wenjing Zhou, Yu Zhang Final approval of manuscript: All authors.

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Zhou, W., Yang, Y., Feng, Z. et al. Inhibition of Caspase-1-dependent pyroptosis alleviates myocardial ischemia/reperfusion injury during cardiopulmonary bypass (CPB) in type 2 diabetic rats. Sci Rep 14 , 19420 (2024). https://doi.org/10.1038/s41598-024-70477-5

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Experimental Models to Study Diabetes Mellitus and Its Complications: Limitations and New Opportunities

Beatriz martín-carro.

1 Bone and Mineral Research Unit, Instituto de Investigación Sanitaria del Principado de Asturias (ISPA), Hospital Universitario Central de Asturias, 33011 Oviedo, Spain

2 Redes de Investigación Cooperativa Orientadas a Resultados en Salud (RICORS), RICORS2040 (Kidney Disease), Instituto de Salud Carlos III, 28029 Madrid, Spain

Javier Donate-Correa

3 Research Unit, Hospital Universitario Nuestra Señora de Candelaria, 38010 Santa Cruz de Tenerife, Spain

Sara Fernández-Villabrille

Julia martín-vírgala, sara panizo, natalia carrillo-lópez, laura martínez-arias, juan f. navarro-gonzález.

4 Nephrology Service, Hospital Universitario Nuestra Señora de Candelaria, 38010 Santa Cruz de Tenerife, Spain

Manuel Naves-Díaz

José l. fernández-martín, cristina alonso-montes, jorge b. cannata-andía.

5 Department of Medicine, Universidad de Oviedo, 33006 Oviedo, Spain

Associated Data

Not applicable.

Preclinical biomedical models are a fundamental tool to improve the knowledge and management of diseases, particularly in diabetes mellitus (DM) since, currently, the pathophysiological and molecular mechanisms involved in its development are not fully clarified, and there is no treatment to cure DM. This review will focus on the features, advantages and limitations of some of the most used DM models in rats, such as the spontaneous models: Bio-Breeding Diabetes-Prone (BB-DP) and LEW.1AR1- iddm , as representative models of type 1 DM (DM-1); the Zucker diabetic fatty (ZDF) and Goto-kakizaki (GK) rats, as representative models of type 2 DM (DM-2); and other models induced by surgical, dietary and pharmacological—alloxan and streptozotocin—procedures. Given the variety of DM models in rats, as well as the non-uniformity in the protocols and the absence of all the manifestation of the long-term multifactorial complications of DM in humans, the researchers must choose the one that best suits the final objectives of the study. These circumstances, added to the fact that most of the experimental research in the literature is focused on the study of the early phase of DM, makes it necessary to develop long-term studies closer to DM in humans. In this review, a recently published rat DM model induced by streptozotocin injection with chronic exogenous administration of insulin to reduce hyperglycaemia has also been included in an attempt to mimic the chronic phase of DM in humans.

1. Concept, Classification and the Role of Experimental Models in the Study of Diabetes Mellitus

Diabetes mellitus (DM) is a major global health problem not only because of its high and growing prevalence, which has tripled in the last 20 years, but also because of the number of premature deaths it causes. In the year 2021, worldwide records indicated that approximately 537 million adults were living with DM, and more than one in ten of global deaths from all causes (12%; 6.7 million adults) were related to complications derived from the disease [ 1 ]. The prevalence of DM in 2021 was approximately 10.5%, an increase of 3,9% compared to 2010. Moreover, DM generates a great economic impact on health systems, around 11.5% of the global health spending according to the World Health Organization [ 1 ].

The DM is a multifactorial disease triggered by a combination of genetic, epigenetic and environmental factors. The increase in life expectancy and unhealthy lifestyle habits, such as a sedentary lifestyle and the consumption of foods rich in saturated fats and added sugars, are risk factors for the development of obesity and associated comorbidities, such as metabolic syndrome, also called insulin resistance syndrome. This scenario is considered a predictor of DM [ 1 ]. In fact, the coexistence of diabetes and obesity, known as “diabesity”, shows an alarming rise [ 2 ].

Although the disorders associated with DM are diverse, the common feature to all of them is hyperglycaemia due to a relative or complete insulin resistance and/or deficiency. This hormone, which is produced in the pancreatic β-cell in the islets of Langerhans, is essential in the control of the glucose homeostasis by facilitating the glucose uptake and metabolism in peripheral tissues [ 3 ].

Chronic hyperglycaemia leads to a variety of complications, such as neuropathy, retinopathy and nephropathy [ 3 ]. The latter, known as diabetic kidney disease (DKD), is one of the most frequent long-term complications associated to DM, and its prevalence has increased in recent years in parallel to the substantial rise in the obese and diabetic population. The DKD, with different degrees of renal impairment, occurs in approximately 40% of diabetic patients, and today, it is the main cause of chronic kidney disease (CKD) that needs renal replacement therapy [ 4 , 5 ].

The main factors involved in the progression of DKD are uncontrolled hyperglycaemia, dyslipidaemia, hemodynamic (glomerular hypertension), inflammatory and profibrotic changes [ 6 ]. DM courses silently in early stages [ 7 , 8 ], but in the long-term, it damages several vital organs such as kidneys, blood vessels, heart and bones [ 1 , 3 , 9 , 10 ]. The high prevalence of the DKD, its lethal complications together with the still uncomplete knowledge of its pathogenesis [ 4 ], makes necessary the use of preclinical models to better understand the disease.

DM can be classified into four general categories: type 1 DM (DM-1), type 2 DM (DM-2), gestational DM and a group of specific types of DM due to other causes including monogenic DM syndromes, diseases of the exocrine pancreas (such as cystic fibrosis and pancreatitis) and drug- or chemical-induced DM (such as post-transplantation DM). DM-1 and DM-2 are the most common forms of the disease. DM-1, also called insulin-dependent DM, is present in 5–10% of patients and is due to autoimmune pancreatic β-cell destruction by T-cells and macrophages, usually leading to a widespread and irreversible insulin deficiency. Although DM-1 can appear at all ages, it is most often diagnosed in children and young people. DM-2, also called insulin-independent DM, is present in 90–95% of patients. DM-2 is due to a progressive loss of β-cell insulin secretion which leads to the inadequate response of the body to the action of insulin, known as insulin resistance [ 3 ].

Preclinical biomedical models of DM are fundamental to improving the knowledge and management of the disease. For research purposes, the spontaneous, induced and transgenic models of DM, currently using rodents, are the most used. However, unfortunately there is no animal model that presents all the phenotypic and/or genotypic alterations of DM in humans. Most of the studies of DM performed in rodent models are focused on the development of strategies for the prevention and early treatment of DM. However, in the experimental field, there is a gap in the knowledge of the long-term complications of DM. One of the main reasons is the difficulties of maintaining animal experiments long enough to be comparable with what happens in advanced stages of DM in humans. Therefore, there is an urgent need to carry out long-term studies to investigate the maintained effect of hyperglycaemia and its impact on the target organs of the disease [ 11 ].

The scientific and regulatory aspects of the use of experimental animals are subject to strict ethical guidelines around the world based on the principles of the 3Rs (replacement, refinement and reduction), which aim to improve both the quality of science and animal welfare when the use of animals is unavoidable [ 12 ]. In recent years, the trend has been towards a reduction in the use of experimental animals, improving alternative models such as 3D, computational and mathematical models of diseases, and every day important progress are made in this field. In relation to DM, there are some studies that have used this approach to study gestational DM [ 13 ] or to identify molecular markers to assess the glucose response [ 14 ]. However, research with animal models remains a fundamental tool to better understand biological processes and human diseases and to develop therapies, especially for systemic diseases such as DM.

2. Available Models for the Study of DM-1 and DM-2

2.1. the spontaneous dm rat models.

Spontaneous autoimmune DM has been observed in several rodent strains [ 15 ]. These rodent models have been widely used for the study of the pathogenesis of the insulitis of DM-1. The major rodent model of spontaneous DM-1 is the Bio-Breeding (BB) rat, which includes both the T-lymphopenic diabetes-prone (BB-DP) stock and the non-lymphopenic diabetes-resistant stock [ 16 ]. Other examples of strains of inbred rats include the LEW.1AR1- iddm rats, as representative models of DM-1, and Zucker diabetic fatty (ZDF) and Goto-kakizaki (GK) rats, as representative models of DM-2.

2.1.1. The Bio-Breeding Diabetic Rats

The Bio-Breeding diabetic-prone rats (BB-DP rats) were discovered in the 1970s at Bio-Breeding Laboratories in Canada. They originated from a spontaneous mutation in an outbred colony of Wistar rats affecting the major histocompatibility complex (MHC). The development frequency of DM-1 occurs in males and females in the same proportion, and between 50 and 90 days after birth, the rats show severe pancreatic insulitis, leading to a hypoinsulinemia state. The first manifestation of the disease is glycosuria at 8–16 weeks, and 90% of the rats develop overt DM-1 with hyperglycaemia, weight loss, polyuria, polydipsia and very severe ketoacidosis that requires exogenous insulin administration in order to survive [ 17 , 18 ]. Bio-Breeding diabetes-resistant (BB-DR) rats do not develop DM, and they are used as controls.

Even though the features of the BB-DP rats are similar to DM-1 in humans, an important limitation of this model is that DM is accompanied by a T-cell decrease, a disorder that does not occur in humans or in other animal models that makes it a questioned model. In addition, some promising antidiabetic drugs, such as anti-CD3 antidiabetic therapy, have shown the side effect of a decrease in the T-lymphocyte population, a finding that makes it unable to use this model for the study of this type of drugs [ 19 , 20 , 21 , 22 , 23 ]. Despite the mentioned limitations, the BB-DP rats have been widely used for to study the pathophysiology of DM and islet transplantation [ 24 ].

2.1.2. The LEW.1AR1- iddm Rats

The LEW.1 AR1- iddm rats were originated from the congenic strain LEW.1AR1 by a spontaneous mutation which also affects a gene associated with MHC. DM-1 occurs in males and females in the same proportion, showing intense pancreatic insulitis that causes subsequent hypoinsulinemia. The LEW.1AR1- iddm rats develop a prediabetic state for approximately one week [ 25 ], and by week 8 of life, they present many of the signs and symptoms of DM-1 such as hypoinsulinemia, weight loss, hyperglycaemia, polydipsia, polyuria, glycosuria and ketoacidosis. They have a long life expectancy, a fact that makes them an ideal model for long-term studies [ 17 , 24 , 26 ].

2.1.3. The Zucker Diabetic Fatty Rats

The Zucker fatty (ZF) rats are obese rats due to a mutation in the leptin receptor gene making them hyperphagic. They develop hyperlipidaemia and hyperinsulinemia; however, they maintain normal blood glucose levels and rarely progress to mild hyperglycaemia [ 27 ]. These alterations are also observed in the prediabetic state in humans, where obesity plays an important role as a risk factor for the development of DM-2. After selective crosses between ZF rats, Zucker diabetic fatty (ZDF) rats emerged which, unlike ZF, develop advanced insulin resistance and, progressively, hyperglycaemia that, at week 10, reaches values above 500 mg/dL.

The development of DM-2 occurs spontaneously, more frequently in male rats. Despite the genetic origin of the disease differing between these rats and humans, they develop similar complications as those observed in advanced stages of the human disease such as glomerular lesions, expansion of the mesangial matrix and tubulointerstitial fibrosis, among others. This model has been used to study the alterations associated to advanced stages of DM-2 [ 28 , 29 , 30 ].

2.1.4. The Goto-kakizaki Rats

The Goto-kakizaki (GK) rats constitute a very popular DM-2 model that, unlike the previous model, does not present obesity nor hyperlipidaemia. They result from selective inbreeding between Wistar rats with impaired glucose tolerance. They develop hyperglycaemia, hypoinsulinemia and peripheral insulin resistance at 12 weeks of age. The exposure of the foetus of the pregnant rat to a hyperglycaemic environment seems to affect the normal development of β-cells. Thus, at birth, the rats have a reduced number of pancreatic islets. Additionally, in these rats, exercise can reduce the increase of glycemia. Therefore, this model shares some environmental factors of the human DM, such as the hyperglycaemia “in utero” and the effect of the physical activity, making it an attractive model for studies related to the prevention and treatment of DM-2. This model develops retinal, kidney and peripheral nerves abnormalities, which is useful for studying the complications associated with the disease. A factor that limits the choice of this model for research purposes is the low rate of effective pregnancies and the decreased number of rats obtained per litter [ 30 , 31 , 32 ].

2.2. The Surgical Induced DM Rat Models

This model is obtained by ligation of the pancreatic ducts or by partial or total removal of the pancreas. They are not used frequently due to the traumatic nature of the technique, though it is used in research related to pancreas transplantation [ 33 , 34 , 35 ].

2.3. The Diet-Induced DM Rat Models

The dietary models are useful for studying the prodromal period of the diabetic syndrome, and they are considered more a model of obesity than of DM. As it is difficult to induce DM in rats just by feeding them only with hypercaloric diets, the use of this model often requires the combination with other techniques, such as pharmacological (streptozotocin, alloxan) or partial nephrectomy, to accelerate kidney damage an reduce the time of establishment and progression of the disease [ 11 ].

Different dietary interventions, such as the consumption of the Mediterranean diet [ 36 ], caloric restriction [ 37 , 38 ], intermittent fasting [ 39 ] and the therapeutic potential of various dietary supplements [ 40 ], have shown antioxidant, anti-inflammatory and metabolic profile improvement effects, constituting non-pharmacological complementary therapeutic strategies for the prevention and treatment of obesity and DM. Table 1 lists some therapeutic dietary strategies carried out in diet-induced DM-2 rat models.

Different dietary interventions strategies in diet-induced DM-2 rat models.

DM: Diabetes mellitus; DM-2; type 2 DM; SD: Sprague Dawley; STZ: Streptozotocin; HFD: High fat diet; Glc: glucose; NA: nicotinamide.

2.4. The Chemical-Induced DM Rat Models

Several chemical compounds have shown to be able to induce DM in animal models, and the two most widely used diabetogenic agents are alloxan [ 47 ] and streptozotocin (STZ) [ 48 , 49 ]. Both are cytotoxic glucose analogues that bind to pancreatic β-cell GLUT-2 transporters causing irreversible damage, leading to hyperglycaemia, β-cell necrosis and weight loss, without causing damage to other organs. These diabetogenic agents are very unstable, so the preparations must be prepared at the time they are injected (half-life: alloxan, 1–2 min; STZ, 1 h).

The main advantage of the chemically induced models is that they are simple and relatively cheap. In addition, following different protocols of the time of induction, route of administration and dose, it is possible to induce DM-1 or DM-2 [ 50 ]. The main disadvantages of these models are (a) that the human DM is rarely caused by a toxic substance; (b) the possibility that these compounds can cause toxicity in the liver and tubular cells where GLUT-2 is expressed; and (c) that a single dose can cause mortality due to ketosis associated with acute damage [ 51 , 52 ].

2.4.1. The Alloxan Model

Alloxan is a uric acid derivative that can selectively inhibit glucose-induced pancreatic insulin secretion by inhibiting glucokinase inducing insulin-dependent DM by promoting the formation of reactive oxygen species that cause selective β-cell necrosis. The diabetogenic dose range is very narrow, and even a mild overdose can cause systemic toxicity, especially to the kidney, although the damage is reversible in the surviving animals. It can be administered intraperitoneally (i.p.), intravenously (i.v.) and subcutaneously (s.c.), and the most frequent dose in rats is 45–65 mg/Kg i.v. [ 47 ].

2.4.2. The Streptozotocin Model

Streptozotocin (STZ) or [2-deoxy-2-(3-(methyl-3-nitrosoureido)-D-glucopyranose] is a nitrosourea analogue attached to a glucosamine moiety, isolated from Streptomyces achromogenes . The mechanism of action also involves the binding of the GLUT-2 transporter before entering in the β-cells and the nucleus where it causes alkylation and, consequently, fragmentation of the DNA promoting pancreatic β-cell necrosis, resulting in an insulin-dependent state due to insulin deprivation. Its sensitivity depends on the species, strain, sex (males are more susceptible), age and nutritional status of the animal.

The STZ can be administered i.p., i.v. and s.c., either as a single dose, between 35 and 65 mg/Kg (the most frequently used), or multiple doses, between 20 and 40 mg/Kg, during several days [ 49 , 53 ]. Adult rats are usually used to establish DM-1 by multiple doses (20–40 mg/Kg) during several days or a single dose of 40–200 mg/Kg. To establish DM-2, neonatal rats with a single dose (35–65 mg/Kg i.p.) or adult rats are used in which nicotinamide or fructose are added as antioxidants to protect the animals from the cytotoxic action of STZ obtaining a partial destruction of the pancreas. The use of STZ is preferred than alloxan in rats.

With the use of STZ or alloxan, the metabolic result is a DM-2 hyperglycaemia state which does not include other epigenetic/environmental factors, such as obesity, which play an important role in DM-2 in humans. To bring these models closer to the DM-2 in humans, the combination of these toxins with diets rich in fat/sugars can be used to better resemble the state of poor nutrition/overweight/obesity currently found in the DM-2 in humans [ 54 , 55 ].

3. Are the Current Rat Models to Study the Human Diabetic Kidney Disease Enough?

The diabetic kidney disease (DKD) is a very important complication of the DM. In the human DKD, clinical and/or biochemical data, shown in Table 2 , are useful to determine the evolutionary course of the disease, avoiding invasive techniques [ 56 , 57 ].

Evolutionary course of human DKD in different stages.

DKD: Diabetic kidney disease; GBM: Glomerular basement membrane; UACR: Urine Albumin-Creatinine Ratio; BP: Blood pressure.

In animal models of DM, the beginning and progress of the signs and symptoms of the Table 2 are variable. Due to the multifactorial aetiology and complex pathogenesis of the human DM, there is no animal model that mimics all the structural and functional changes observed in humans [ 58 , 59 ]. These limitations led the Animal Models of Diabetic Complications Consortium (AMDCC) to propose three criteria to be met by a murine model to be considered as an acceptable DKD, listed in Table 3 [ 60 ].

Criteria to be met by a murine model of desirable DKD, according to the AMDCC.

DKD: Diabetic kidney disease; AMDCC: Animal Models of Diabetic Complications Consortium; GFR: glomerular filtration rate; GBM: Glomerular basement membrane.

The three criteria are not usually met in the same animal model; however, many of them are close to the changes observed in the human DKD. As it is unlikely that a single animal model will develop all the multifactorial complications of the DM in humans, it is advisable to use the different experimental models according to the main objectives of the planned study.

Thus, the first step is to choose the most suitable animal model to answer the research questions of the study [ 24 ].

4. Use of Rat Models for the Study of Antidiabetic Drugs

The very promising antidiabetic therapy with anti-CD3 antibodies induces a decrease in the lymphocyte population, a fact which makes it unable to use BB-DP rats due to their lymphopenia derived from the loss of GTPase function. This limitation is not present in the LEW.1AR1- iddm rat model which better resembles the pathophysiological characteristics of DM-1 in humans. In addition, LEW.1AR1- iddm rats develop DM rapidly, without a prolonged prodromal phase, a circumstance that, added to a longer life expectancy, makes it possible to use this strain to analyse the effect of therapies before developing age-related changes.

The new DM therapeutic approaches go far beyond only lowering blood glucose levels; they aim to act on key metabolic steps, such as sodium-glucose cotransporter-2 inhibitors (SGLT2i) and glucagon-like peptide-1 receptor agonists (GLP-1RAs) [ 61 ], which in turn shows clear benefits on the cardiorenal health of the DM [ 10 ]. Thus, to study the efficacy of SGLT2i and GLP-1RA, as well as their mechanisms of action, experimental models that develop cardiorenal complications because of the diabetic condition are necessary. Below is Table 4 , which compiles different models of DM in rats used in studies with SGLT2i and/or GLP-1RA.

The main characteristics of the rat DM models used in studies with SGLT2i and/or GLP-1RA.

DM: Diabetes mellitus; GK: Goto-kakizaki; SD: Sprague Dawley; STZ: Streptozotocin; HFD: High fat diet; Glc: glucose; SGLT2i: sodium-glucose cotransporter-2 inhibitors; GLP-1RA: Glucagon-like peptide-1 receptor agonist. Note: Only SGLT2i or GLP-1RA are detailed, although many of the referenced studies look at more drugs (monotherapy or multitherapy).

Most of the studies included in Table 4 concentrated on the general beneficial effects of the drugs used secondary to the damage associated with short-term hyperglycaemia. It is necessary to design long-term hyperglycaemia experimental models, closer to human DM, to further study their impact in the complications of DM.

5. Similarity of the Histological Finding in the Experimental and Human DKD

The histologic findings of the human DKD include glomerular hypertrophy, glomerular basement membrane thickening with absence of immune deposits, mesangial matrix expansion, loss of podocytes, glomerular capillary walls thickening, nodular sclerosis (±Kimmelstiel Wilson nodules), arteriolar hyalinosis and tubulointerstitial fibrosis [ 77 ].

The absence of a uniform classification has led the Renal Pathology Society to develop a consensus classification of the glomerular histological lesions present in the DKD, listed in Table 5 [ 58 ].

Consensus classification of glomerular histological lesions present in the DKD.

DKD: Diabetic kidney disease; OM: Optical microscopy; GBM: glomerular basement membrane.

Table 6 lists the kidney histological findings found in different DM rat models.

Histological findings at kidney level found in different models of DM in rats.

DM: Diabetes mellitus; STZ: streptozotocin; NA: nicotinamide; Glc: glucose; H&E: haematoxylin and eosin; PAS: Periodic acid–Schiff; TEM: transmission electron microscopy; ↑: Increased; GBM: glomerular basement membrane.

The non-uniformity in terms of the time of onset and severity of glomerular histological lesions found in the different studies in rats may be explained by the differences in the animal models/species of DM used and time of evolution of the DM, among others. Furthermore, compared to what happens in the DM in humans, the histological abnormalities are less than those observed in humans [ 59 , 82 ].

The DM model induced by STZ injection has been widely used to study the development and evolution of DKD. However, there is still no consensus regarding the age, dose of STZ used, the time to develop DKD, the parameters to consider a success the establishment of DKD and the end points of the experiments. Table 7 lists the details of the protocols used in studies with male Wistar rats and STZ.

Examples of protocols used in the induction of DKD by STZ.

DKD: Diabetic kidney disease; STZ: streptozotocin; DM: Diabetes mellitus; Glc: Glucose; UACR: Urine Albumin-Creatinine; BUN: Blood urea nitrogen; ↑: Increased.

The human DKD is a long-term complication of DM-1 and DM-2 that occurs in patients receiving insulin. However, none of the models previously described includes the use of insulin to correct or minimize the negative effects of hyperglycaemia. This circumstance has been acknowledged as a limitation to the study by several authors, in which potential biomarkers of platelet activation were analysed as instruments for the evaluation of thromboembolic risk in a model of long-term DM (28 weeks) induced by STZ (single dose of 60 mg/dL) [ 85 ].

To shed some light in this area, we recently developed a long-term rat model of DM in which we used long-term insulin administration in order to improve the control of the hyperglycaemia [ 86 ].

6. Experimental Model of DM and Partial Correction of the Hyperglycaemia Using Insulin

The model attempted to assess the effects of long-term hyperglycaemia in a chemically induced DM-1 model in which the blood glucose level was partially corrected during 24 weeks by the administration of exogenous insulin. Briefly, the experimental model of DM consisted in two groups of 4-month-old male Wistar rats (425 ± 43 g) and controls in which DM was obtained using STZ (55 mg/Kg). Rats were considered diabetics when weight loss, polyuria, hyperglycaemia, hyperglycosuria and elevated HbA1c levels were achieved. After the 24 weeks, the rats showed microalbuminuria (UACR > 30 mg/g) and hyperfiltration. Histologically, the kidneys showed structural changes, such as increased diameter of the proximal tubules, thickening of the glomerular basement membrane and denuded foot processes of the podocytes, with no changes in kidney fibrosis. The changes observed in this model allowed us to classify them as class I diabetic nephropathy, according to the classification of the Society of Renal Pathology [ 58 ]. In addition, the diabetic rats showed higher serum and urinary levels of advance glycation end-products (AGEs) and of their soluble receptors in urine (sRAGE) but lower soluble serum Klotho. A higher degree of fibrosis was observed in the heart. The control rats did not show any kind of changes in the kidney nor in the heart. In summary, despite a partial control of the hyperglycaemia using long-term administration of insulin, the diabetic rats showed kidney and heart important alterations.

7. Conclusions

Experimental models of DM are an essential biomedical research tool to better understand and improve the pathogenesis and management of the DM. However, unfortunately, so far there are no animal models that clearly resemble the disorders observed in the human DM. Furthermore, most investigations are centred in the early phase of DM. More studies are needed to mimic the long-term complications associated with the disease and the long-term effects of the treatment in the different organs damaged by DM. As a stimulus for hope, this review includes a summary of a recent long-term model of DM (24 weeks) induced by streptozotocin in which exogenous insulin was administered that can help to better understand some aspects of the pathogenesis and management of DM.

Funding Statement

This research was funded by Fondo Europeo de Desarrollo Regional (FEDER), Plan de Ciencia, Tecnología e Innovación 2013–2017 and 2018–2022 of the Principado de Asturias, grant numbers GRUPIN 14-028, IDI-2018-000-152 and IDI/2021/000080. Instituto de Salud Carlos III, Red Cooperativa en Salud REDinREN and RICORS2040, grant numbers RD12/0021/1023, RD16/0009/0017, RD21/0005/0013 and RD21/0005/0019. Instituto de Salud Carlos III and co-funded by the European Regional Development Fund/European Social Fund “A way to make Europe”/“Investing in your future”, grant numbers: PI17/00384, PI19/00532, PI20/00753 and PI20/00633. B.M.-C. and S.F.-V. were supported by a graduate fellowship from the Gobierno del Principado de Asturias (“Severo Ochoa” program): BP19-057, BP20-081, J.M.-V. by a graduate fellowship from the Ministerio de Ciencia, Innovación y Universidades (FPU program): FPU2019-00483, S.P. was supported by Fundación para la Investigación Biosanitaria de Asturias (FINBA), C.A.-M. by RICORS2040 (Kidney Disease) and N.C.-L. by IDI-2018-000152 and IDI-2021-000080.

Author Contributions

Conceptualization, J.B.C.-A., J.L.F.-M., B.M.-C., C.A.-M., M.N.-D., S.P. and N.C.-L.; resources, J.D.-C., J.F.N.-G., M.N.-D., S.P., C.A.-M., N.C.-L., J.L.F.-M. and J.B.C.-A.; writing—original draft preparation, B.M.-C., J.D.-C., S.F.-V., J.M.-V., L.M.-A. and J.B.C.-A.; writing—review and editing, B.M.-C., J.D.-C., J.F.N.-G., S.F.-V., J.M.-V., L.M.-A., M.N.-D., S.P., C.A.-M., N.C.-L., J.L.F.-M. and J.B.C.-A.; supervision, J.B.C.-A., J.L.F.-M., C.A.-M., M.N.-D., N.C.-L. and S.P.; funding acquisition, M.N.-D., S.P., C.A.-M., N.C.-L., J.L.F.-M. and J.B.C.-A. All authors have read and agreed to the published version of the manuscript.

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[Mechanism of tea polyphenols improving the sarcopenia in the aged type 2 diabetes model rats via mitochondrial quality control]

Affiliations.

• 1 School of Public Health, Shanxi Medical University, Key Laboratory of Coal Environmental Pathogenicity and Prevention, Taiyuan 030001, China.
• 2 Shanxi Provincial Center for Disease Control and Prevention, Taiyuan 030012, China.
• PMID: 39155220
• DOI: 10.19813/j.cnki.weishengyanjiu.2024.04.004

Objective: To explore whether tea polyphenols(TP) improve sarcopenia in the aged type 2 diabetes(T2DM)model rats via mitochondrial quality control(MQC).

Methods: A total of 55 2-month-old male SD rats were randomly divided into the control group(n=10), the aged model group(aged, n=10) and the aging T2DM model group(n=35). The aging T2DM model group rats were fed with high-sugar and high-fat diet and intraperitoneally injected with 50 mg/kg D-galactose daily. After 4 weeks, the aging T2DM model group rats were given a single intraperitoneal injection of 30 mg/kg streptozotocin(STZ). After STZ injection for 2 weeks, fasting blood glucose(FBG) ≥ 16.7 mmol/L was defined as successful T2DM model. When the model was successfully induced, the 30 model rats were randomly divided into aged T2DM group(Mod), 300 mg/kg TP teatment group(TP) and 3 mg/kg rosiglitazone treatment group(RSG) according to FBG, with 10 rats in each group. Each group was treated with 50 mg/kg D-galactose to induce senescence and fed with high glucose and fat for 8 weeks. Western blot was used to detect the expression of P53 protein in gastnemius muscle tissue of the model group at the end of the experiment, which was higher than that of the control group, indicating that the aging T2DM model was successfully established. FBG was detected by the blood glucose meter, gastnemius muscle relative weights was calculated, the microstructure of mitochondria of gastnemius muscle was observed by transmission electron microscope(TEM), the expression of mitochondrial biosynthesis-related proteins PGC-1α, mitochondrial dynamics-related proteins(OPA1, DRP1) and mitochondrial autophagy-related proteins(P62, LC3) in gastnemius muscle were detected by western blot.

Results: Compared with the control group, the level of FBG and the expression of P53 in the Mod group were increased(P&lt;0.01). The gastnemius muscle relative weights, the expression level of PGC-1α, OPA1 and the ratio of LC3II/LC3I were decreased(P&lt;0.01). The expression level of P62 and DRP1 were significantly increased(P&lt;0.01). The number of mitochondria decreased, the volume decreased and a large number of vacuolization, and there were no obvious autophagolysosomes and fission and fusion. After 8 weeks, compared with the Mod group, the number of mitochondria in the gastrocnemius of TP and RSG groups, vacuolization, fission and fusion were improved, and the autophagolysosomes was significantly increased. The expression levels of P53, DRP1 and P62, the level of FBG in the TP group were significantly decreased(P&lt;0.01, P&lt;0.05). The expression levels of OPA1 and PGC-1α, the ratios of LC3II/LC3I and gastnemius muscle relative weights were significantly increased(P&lt;0.05, P&lt;0.01).

Conclusion: TP can improve the sarcopenia in the aged T2DM model rats, and its mechanism is related to the regulation of mitochondrial quality control.

Keywords: aged type 2 diabetes; mitochondrial quality control; sarcopenia; tea polyphenols.

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Effect of 28 days treatment of baricitinib on mechanical allodynia, osteopenia, and loss of nerve fibers in an experimental model of type-1 diabetes mellitus

• Published: 19 August 2024

Cite this article

• Rosa I. Acosta-González 1 ,
• Angélica Y. Hernández-Jiménez 1 ,
• Laura Y. Ramírez-Quintanilla 1 ,
• Héctor F. Torres-Rodríguez 1 ,
• Virginia M. Vargas Muñoz 1 &
• Juan M. Jiménez-Andrade   ORCID: orcid.org/0000-0002-2703-9736 1  

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Type-1 diabetes mellitus (T1DM) is associated with numerous health problems, including peripheral neuropathy, osteoporosis, and bone denervation, all of which diminish quality of life. However, there are relatively few therapies to treat these T1DM-related complications. Recent studies have shown that Janus kinase (JAK) inhibitors reverse aging- and rheumatoid arthritis-induced bone loss and reduce pain associated with peripheral nerve injuries, and rheumatoid arthritis. Thus, we assessed whether a JAK1/JAK2 inhibitor, baricitinib, ameliorates mechanical pain sensitivity (a measure of peripheral neuropathy), osteoporosis, and bone denervation in the femur of mice with T1DM.

Female ICR mice (13 weeks old) received five daily administrations of streptozotocin ( ip , 50 mg/kg) to induce T1DM. At thirty-one weeks of age, mice were treated with baricitinib ( po ; 40 mg/kg/ bid ; for 28 days) or vehicle. Mechanical sensitivity was evaluated at 30, 33, and 35 weeks of age on the plantar surface of the right hind paw. At the end of the treatment, mice were sacrificed, and lower extremities were harvested for microcomputed tomography and immunohistochemistry analyses.

Mice with T1DM exhibited greater blood glucose levels, hind paw mechanical hypersensitivity, trabecular bone loss, and decreased density of calcitonin gene-related peptide-positive and tyrosine hydroxylase-positive axons within the marrow of the femoral neck compared to control mice. Baricitinib treatment significantly reduced mechanical hypersensitivity and ameliorated sensory and sympathetic denervation at the femoral neck, but it did not reverse trabecular bone loss.

Conclusions

Our findings suggest that baricitinib may represent a new therapeutic alternative to treat T1DM-induced peripheral neuropathy and bone denervation.

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Data availability

The data used in this study are available from the corresponding author upon reasonable request.

Abbreviations

Adaptor protein-2 associated kinase

Analysis of variance

bis in die, twice daily

Bone mineral density

Trabecular bone volume per tissue volume

Diabetes mellitus

Immunohistochemistry

Janus kinase

Phosphate-buffered saline

Standard error of the mean

Suppressor of cytokine signaling

Streptozotocin

• Type-1 diabetes mellitus

Trabecular thickness

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This research was funded by the National Council of Humanities Science and Technology of Mexico, project CF-2023-I-24 (RIAG).

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Rosa I. Acosta-González, Angélica Y. Hernández-Jiménez, Laura Y. Ramírez-Quintanilla, Héctor F. Torres-Rodríguez, Virginia M. Vargas Muñoz & Juan M. Jiménez-Andrade

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All authors have made a substantial contribution to the concept and design of the article. Conceptualization of ideas: RIAG, JMJA. Methodology: RIAG, JMJA, AYHJ, LYRQ, VMVM. Investigation: LYRQ, VMVM, HFTR. Formal analysis: JMJA, AYHJ, HFTR, LYRQ. Writing original draft: RIAG, AYHJ, HFTR, VMVM. Writing - review & editing: JMJA. Funding acquisition: RIAG, JMJA.

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Acosta-González, R.I., Hernández-Jiménez, A.Y., Ramírez-Quintanilla, L.Y. et al. Effect of 28 days treatment of baricitinib on mechanical allodynia, osteopenia, and loss of nerve fibers in an experimental model of type-1 diabetes mellitus. Pharmacol. Rep (2024). https://doi.org/10.1007/s43440-024-00634-0

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Received : 21 May 2024

Revised : 30 July 2024

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Published : 19 August 2024

DOI : https://doi.org/10.1007/s43440-024-00634-0

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