AdipoRon ameliorates vascular endothelial injury in type 2 diabetes mellitus and is associated with suppression of the NLRP3-Caspase-1-GSDMD pyroptosis axis

Abstract

Background

AdipoRon is a bioactive synthetic agonist of the adiponectin receptor. It has been demonstrated to improve glucose tolerance and mitigate insulin resistance. However, the long-term effects on vascular endothelial pyroptosis in type 2 diabetes mellitus (T2DM) remain poorly understood. This study aims to investigate whether AdipoRon exerts a protective effect against vascular endothelial pyroptosis in T2DM.

Methods

The expression levels of NLRP3 inflammasome-related proteins were measured by western blotting. Vascular endothelial pyroptosis was assessed using multiple approaches, including biochemical analysis, western blotting, flow cytometry analysis, tube formation assay, and immunofluorescent test, with each method detecting distinct relevant indexes.

Results

In high glucose (HG)-challenged human umbilical vein endothelial cells (HUVECs) and aortae of T2DM mice, the expression levels of NLRP3, ASC, pro-caspase-1, cleaved-caspase-1, GSDMD, and p-NF-κB p65 were significantly upregulated, with a concurrent reduction in Nrf2 protein level. Moreover, Oxidative stress-related indices, such as ROS, MDA, and GSH-Px, were increased, while SOD levels were decreased in both models. In HG-treated HUVECs, the levels of LDH and ET-1 were markedly elevated, the release of NO was suppressed, and tube formation ability was severely disrupted compared with the control group. Interestingly, AdipoRon treatment successfully reversed all these HG-induced aberrant alterations in vitro and in vivo.

Conclusions

AdipoRon is a promising candidate compound for protecting against T2DM. Its endothelial protective effects are closely associated with the suppression of the NLRP3-Caspase-1-GSDMD axis-mediated pyroptosis. These findings provide novel insights into the development of a new therapeutic strategy for the clinical management of T2DM.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12872-026-05969-5.

Introduction

Diabetes mellitus (DM) represents a complex chronic metabolic disorder characterized by systemic insulin resistance and dysregulated glucose homeostasis. DM is clinically categorized into three subtypes: type 1 diabetes mellitus (T1DM), gestational diabetes mellitus (GDM), and type 2 diabetes mellitus (T2DM), according to the classification criteria established by the American Diabetes Association [1, 2]. T2DM is the predominant form among all diabetic subtypes, accounting for approximately 90% of total DM cases globally. The development of T2DM is strongly associated with modifiable lifestyle factors, including unhealthy dietary patterns, obesity, and physical inactivity. Without effective long-term management, chronic T2DM raises the risk of severe macrovascular and microvascular complications, such as diabetic retinopathy, heart disease, kidney failure, and strokes [3–5]. Vascular endothelial dysfunction, which is closely intertwined with insulin resistance and sustained hyperglycemia, serves as a pivotal precursor to cardiovascular disease (CVD). Torre et al. [6] demonstrated that vascular endothelial dysfunction contributes to impairments in both physiological function and subcellular structure of vascular tissue in patients with diabetes mellitus. Furthermore, the decrease of endothelial repair capacity and angiogenic potential induced by this dysfunction are recognized as critical pathogenic factors contributing to the development of CVD in diabetes. Thus, elucidating the molecular mechanisms underlying hyperglycemia-induced vascular endothelial dysfunction in T2DM and developing targeted therapeutic strategies for endothelial function modulation hold substantial clinical significance. Such endeavors may yield novel approaches for preventing and managing T2DM-related vascular complications.

Pyroptosis, a type of programmed cell death, is characterized by cell swelling with large bubbles, proinflammatory cytokine release, and rapid membrane rupture [7]. Accumulating evidence has demonstrated that pyroptosis, particularly that mediated by the NOD-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome, serves a critical role in the pathogenesis and progression of T2DM and its related complications [8]. More importantly, Luo et al. have reported that the impaired vascular function in T2DM rat models is associated with NLRP3 inflammasome-mediated pyroptosis [9]. However, the upstream and downstream regulatory mechanisms of NLRP3 inflammasome-mediated vascular endothelial cell pyroptosis during the progression of T2DM have not been fully understood.

Adiponectin is a key member of the adipokine family secreted by adipocytes [10]. Recent studies have shown that it exerts multiple beneficial effects on metabolic syndrome due to its antifibrotic, antioxidant, and anti-inflammatory properties [10]. Emerging evidence from animal experiments has demonstrated that supplementation of exogenous adiponectin ameliorates insulin resistance and enhances glucose tolerance in murine models [11, 12]. Consequently, adiponectin has emerged as a promising therapeutic target for the management of T2DM. AdipoRon, a novel orally bioavailable adiponectin receptor agonist, has been developed and exerts pharmacological effects analogous to those of adiponectin. Consistent with the biological actions of adiponectin, AdipoRon enhances insulin sensitivity, improves glucose tolerance, and modulates lipid metabolism in both cultured cells and murine models of T2DM [13]. Moreover, AdipoRon administration has been shown to ameliorate metabolic homeostasis and extend lifespan in T2DM mice [14]. Despite extensive investigations into the efficacy of AdipoRon across various pathophysiological conditions, its impacts on vascular function, particularly its regulatory role in vascular endothelial cell pyroptosis, remain unexplored.

In the present study, we established two experimental models: a high glucose (HG)-challenged human umbilical vein endothelial cell (HUVEC) in vitro model, and an in vivo T2DM murine model induced by high-fat diet (HFD) feeding combined with streptozotocin (STZ) injection. The objective of this study was to elucidate the regulatory effects of AdipoRon on NLRP3-Caspase-1-GSDMD axis-driven vascular endothelial pyroptosis. These findings may provide novel therapeutic insights for the clinical management of T2DM.

Methods

Cell culture and treatment

Immortalized HUVECs were purchased from Pricella Biotechnology (Wuhan, China). The culture conditions were maintained in a 5% CO₂ and 95% air incubator at 37 °C. All parts of the experiment in HUVECs were conducted at 3‑6 passages. After incubated with various concentrations of AdipoRon (0, 0.5, 1, 10, and 100 µg/mL; MedChemExpress, Shanghai, China; cat.no. HY-15848) for 12 h, HUVECs were exposed to HG (33.3 mM) in DMEM for 24 h, 48–72 h. Those cultured in DMEM (containing 5.5 mM glucose) was served as the control. Ac-YVAD-CMK is a specific irreversible caspase-1 inhibitor to block caspase-1-dependent canonical pyroptosis. In the in vitro HUVEC model, Ac-YVAD-CMK was used as a positive control to verify the causal role of caspase-1-dependent pyroptosis in HG-induced endothelial injury. To explore the role of pyroptosis in HG-treated cells, HUVECs were pretreated with 50 µM Ac-YVAD-CMK (MedChemExpress; cat.no. HY-16990) for 12 h prior to HG stimulation. All cell studies were performed in triplicate, and each experiment was repeated three times.

Cell viability measurement

HUVECs (5000 cells per well) were seeded into a 96-well plate and subjected to different treatments. Subsequently, 10 µL of CCK-8 solution (Beyotime Biotechnology, Shanghai, China) was added to the medium and mixed thoroughly. After incubation for 2 h, HUVECs were treated with 10 µL of stop buffer. The cell viability was determined by measuring the absorbance at 450 nm.

Flow cytometry analysis for reactive oxygen species (ROS) production

ROS levels in HUVECs were quantified using a ROS detection kit (Beyotime). In brief, HUVECs cells (1 × 10⁶) were loaded with fluorescent probe 2’,7’-dichlorodihydrofluorescein diacetate (DCFH-DA) and incubated at 37 °C for 30 min. Following three consecutive washes with phosphate-buffered saline (PBS) to remove unbound probe, the 2’,7’-dichlorofluorescein (an oxidative product of DCFH-DA) fluorescence was measured by a flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA).

Detection of tube formation

HUVECs were seeded at a density of approximately 5,000 cells per well into Matrigel-precoated 24-well plates (Matrigel; BD Biosciences). The cells were then incubated in DMEM with 10% FBS for 24 h at 37 °C. After that, tube formation capacity was visualized using a light microscope (OLYMPUS Corporation, Tokyo, Japan), and the total tube length was quantified via ImageJ software (National Institutes of Health, Bethesda, MD, USA).

T2DM model in mice

A total of 30 male C57BL/6J mice (weighing 18–22 g; 4–6 weeks) were purchased from Vital River Laboratory Animal Technology (Pinghu, China). The mice were kept in specific pathogen-free facilities that strictly followed standard laboratory protocols. Environmental conditions were carefully managed, including a 12-hour light/dark cycle, humidity levels between 40% and 55%, and temperatures ranging from 22 to 25 °C. Throughout the study, the animals had free access to food and water to ensure their optimal health and welfare. The experiment was conducted in compliance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Experimental Animal Ethics Committee of Jinhua Food and Drug Inspection and Testing Research Institute (approval no. AL-JSYJ202416).

Following acclimatization for 1 week in laboratory, mice were randomly assigned to 5 groups using a simple randomization method with a random number table, as follows: the control group, T2DM model group, T2DM model + AdipoRon low dose (AdipoRon-L) group, T2DM model + AdipoRon high dose (AdipoRon-H) group, and T2DM model + MCC950 (MCC950) group. Each group included 6 mice. Mice in the T2DM model groups were fed HFD (1% egg, 1% cholate, 2.5% cholesterol, 10% fat, 20% sucrose, 30% bean sprout and 35.5% chow diet) lasting 8 weeks, and subsequently received a single intraperitoneal injection of STZ (100 mg/kg; Macklin Biochemical Technology Co., Ltd, Shanghai, China; cat.no. S817944) to induce T2DM. The mice in the control group were fed a normal chow diet throughout the experiment. At the end of 8 weeks, they received an intraperitoneal injection of an equivalent volume of 0.1 M citrate buffer (pH 4.5) instead of STZ. The fasting blood glucose (FBG) was measured 3 days later, with FBG levels more than 16.7 mM presenting the successful establishment of T2DM model. During model establishment, concerted efforts were made to minimize animal suffering and none of the mice died or were excluded due to poor health status. All mice included in the final statistical analysis successfully established stable diabetic models, with a modeling success rate of 100%. MCC950 is a highly selective and well-validated in vivo inhibitor of the NLRP3 inflammasome, which specifically suppresses the assembly and activation of the NLRP3 inflammasome at the upstream level of the pyroptotic signaling axis. MCC950 was employed in the in vivo T2DM mouse model to confirm the upstream regulatory role of NLRP3 inflammasome in diabetes-associated vascular endothelial pyroptosis, complementing the in vitro mechanistic evidence obtained with the caspase-1 inhibitor Ac-YVAD-CMK. Subsequently, mice in the AdipoRon-L, AdipoRon-H and MCC950 groups were intraperitoneally injected with AdipoRon (5 mg/kg), AdipoRon (20 mg/kg) and MCC950 (10 mg/kg; MedChemExpress; cat.no. HY-12815) respectively, lasting for another 8 weeks. Afterward, all mice were anesthetized by intraperitoneal injection of 20 mg/kg pentobarbital sodium and euthanized by cervical dislocation. FBG levels were analyzed with a blood glucose meter (Roche Diagnostics, Basel, Switzerland) once a week from blood collected from the tail vein of the mice. With a mouse insulin ELISA kit (Abbkine, Wuhan, China), fasting serum insulin (FINS) was determined in mice. Homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the following formula: HOMA-IR = (FBS × FINS)/22.5.

Hematoxylin and eosin (HE) staining

Aortae from mice were fixed in 4% paraformaldehyde, dehydrated in alcohol, and then embedded in paraffin wax. Paraffin sections of 4 μm thickness were prepared, stained with hematoxylin and eosin (Beyotime), sealed with neutral gum. Pathological changes of aortae were then observed under a light microscope (OLYMPUS Corporation).

Western blotting

Total proteins were extracted from HUVECs and artery blood vessels using ice-cold RIPA lysis buffer (Biosharp Life Sciences, Hefei, China). Protein concentrations were quantified with a BCA Protein Assay Kit (Biosharp Life Sciences). Equal amounts of protein samples (40 µg per lane) were separated by 10% SDS-PAGE, transferred onto PVDF membranes, and then blocked with 5% nonfat milk at room temperature. The membranes were incubated with primary antibodies overnight at 4 °C. The primary antibodies included NLRP3 (1:1,000; Affinity Biosciences, Changzhou, China), IL-1β (1:2,000; Proteintech, Manchester, UK), Nrf2 (1:7,000; Proteintech), ASC (1:2,000; Abcam, Cambridge, UK), cleaved-caspase-1 (1:1,000; Affinity Biosciences), pro-caspase-1 (1:1,000; Abcam), GSDMD (1:1,000; Bioss Biotechnology, Beijing, China), p-NF-κB p65 (1:1,000; Proteintech), and GAPDH (1:50,000; Proteintech). Subsequently, the membranes were probed with the secondary antibodies (1:10,000; Abbkine Scientific) at room temperature for 1.5 h. GAPDH was selected as the loading control, and its protein expression abundance remained stable in HG culture environment in our experimental system. Finally, protein bands were visualized using an enhanced chemiluminescence detection kit (Abbkine Scientific), and signal intensities were analyzed with ImageJ software (National Institutes of Health).

Biochemistry analysis

In accordance with the manufacturers’ instructions, commercially available assay kits were used to quantify the concentrations of caspase-1, lactate dehydrogenase (LDH), endothelin-1 (ET-1), and nitric oxide (NO) in HUVECs, as well as the activities of superoxide dismutase (SOD), malondialdehyde (MDA), and glutathione peroxidase (GSH-Px) in arterial tissues. The kits for caspase-1, LDH, NO, SOD, MDA, and GSH-Px were purchased from Abbkine Scientific, while the ET-1 assay kit was obtained from LuoChangShuo Biotech (Xiamen, China).

Immunofluorescent test

Mouse arterial tissues were fixed in 4% paraformaldehyde for 24 h, dehydrated through a graded ethanol series, embedded in paraffin, and sectioned into 5-µm slices. HUVECs were seeded on glass coverslips, fixed in 4% paraformaldehyde, and processed for paraffin embedding. After dewaxing and rehydration, antigen retrieval was performed on both arterial tissue sections and HUVECs, followed by non-specific binding blocking with goat serum. The samples were then incubated with a primary antibody against GSDMD (1:50 dilution; Abcam), and subsequently conjugated with Alexa Fluor 568- or 488-labeled secondary antibodies (1:200 dilution; Invitrogen, Carlsbad, CA, USA). After counterstaining nuclei with DAPI, fluorescence images of the samples were acquired using an OLYMPUS fluorescence microscope.

Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay

Vascular endothelial apoptosis was assessed using a one-step TUNEL apoptosis assay kit (Abbkine Scientific), following the manufacturer’s standardized protocols. Fluorescence images of the stained samples were acquired using an OLYMPUS fluorescence microscope.

Statistical analysis

Data were analyzed using SPSS 22.0. Intergroup differences were evaluated by two-way analysis of variance or one-way analysis of variance followed by Tukey’s post-hoc test. Linear trend analysis was performed to evaluate the monotonic dose-response relationship of AdipoRon in in vitro experiments. Data are expressed as mean ± standard deviation, with statistical significance set at P < 0.05.

Results

Effects of different concentrations of AdipoRon on HUVEC cell viability and NLRP3 inflammasome

To characterize the effects of AdipoRon on HG-induced HUVEC injury, cell viability was first assessed at various time points and concentrations of AdipoRon. As shown in Fig. 1A, HG exposure significantly reduced HUVEC viability at 24 h, 48 h, and 72 h, as compared with control group (P < 0.001). AdipoRon treatment remarkably reversed HG-induced cytotoxicity (P < 0.05). ROS production was then evaluated using flow cytometry. Compared with the control group, HG stimulation markedly elevated intracellular ROS levels (Fig. 1B, P < 0.001). While AdipoRon administration suppressed ROS accumulation in HG-stimulated HUVECs (Fig. 1B, P < 0.001), indicating its antioxidant capacity in HG-exposed HUVECs. To further explore the effect of AdipoRon on HG-induced NLRP3 inflammasome, several key markers of NLRP3 inflammasome were examined using Western blotting. As illustrated in Fig. 1C, HG exposure significantly upregulated the protein expression of IL-1β, NLRP3, ASC, pro-caspase-1, and cleaved caspase-1 (P < 0.001). Notably, the levels of these proteins were significantly reversed after co-treatment with AdipoRon (P < 0.05). Linear trend analysis confirmed a significant monotonic dose-response pattern for AdipoRon in reducing oxidative stress and inhibiting NLRP3 inflammasome activation (P < 0.05). Additionally, since AdipoRon at concentrations of 10 µg/mL or 100 µg/mL did not show significant differences in the protective effects against HG-treated cell injury, 10 µg/mL of AdipoRon was selected for the succeeding experiments.

Fig. 1.

Effect of AdipoRon on HG-induced HUVEC injury and NLRP3 inflammasome activation. After treatment of HUVECs with HG containing various concentrations of AdipoRon (0, 0.5, 1, 10, and 100 µg/mL), A cell viability was measured by CCK-8 assay; B ROS production was assessed via flow cytometry analysis; C the protein levels of IL-1β, NLRP3, ASC, pro-caspase-1 and cleaved-caspase-1 were measured by western blotting. Horizontal lines denote statistical comparisons between the two connected groups. Asterisks indicate the level of significance: ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

AdipoRon inhibits NLRP3-Caspase-1-GSDMD axis-mediated pyroptosis in HG-treated HUVECs

The activated NLRP3 inflammasome drives ASC to mediate the proteolytic cleavage of pro-caspase-1, generating the enzymatically active cleaved caspase-1. This activated caspase-1 not only facilitates the maturation and secretion of IL-1β, but also cleaves GSDMD. The resulting N-terminal fragment of GSDMD oligomerizes and inserts into the plasma membrane, forming transmembrane pores. These pores disrupt cellular integrity, culminating in cell swelling, membrane rupture, and the execution of pyroptosis. In this experiment, we treated HG-exposed HUVECs with a specific inhibitor of caspase-1 (Ac-YVAD-CMK). This intervention served as a positive control to validate the role of AdipoRon in NLRP3 inflammasome-mediated pyroptosis, given that targeted inhibition of this active protease can abrogate pyroptotic signaling cascades mediated by NLRP3 inflammasome activation. As shown in Fig. 2A-B, treatment with AdipoRon markedly suppressed HG-induced decrease in cell viability and increase in ROS production (P < 0.001), which was similar to the effect of Ac-YVAD-CMK. Results of western blotting showed that HG treatment significantly upregulated the expression of NLRP3 inflammasome components (NLRP3, ASC), pro-inflammatory mediators (mature IL-1β), pyroptosis executor (GSDMD), and pro-inflammatory signaling (p-NF-κB p65), promoted the cleavage of pro-caspase-1 into its active (cleaved) form (Fig. 2C, P < 0.001). Both AdipoRon and Ac-YVAD-CMK partially inhibited the HG-induced upregulation of these proteins (P < 0.05). The antioxidant transcription factor Nrf2 (a negative regulator of NLRP3) showed an opposite expression pattern, with HG suppressing Nrf2 levels and AdipoRon/Ac-YVAD-CMK restoring Nrf2 expression (P < 0.001). The results of caspase-1 activity in Fig. 2D further validated the changes in caspase-1 protein expression detected by using western blotting (P < 0.001). LDH serves as a cellular injury marker, while NO and ET-1 are core molecular biomarkers reflecting endothelial function. HG significantly increased the release of LDH and ET-1 and reduced NO production, which was reversed by treatment with AdipoRon (Fig. 2E-F, P < 0.01). Immunofluorescence staining results showed that HG triggered robust GSDMD accumulation in HUVECs, which was partially abolished by AdipoRon (Fig. 2G, P < 0.001). As shown in Fig. 2H, tube formation was markedly disrupted in the HG group when compared with the control group (P < 0.001). Notably, both AdipoRon and Ac-YVAD-CMK treatment elevated the capillary-like tube formation (P < 0.01 vs. HG group). Finally, the apoptosis of HUVECs was evaluated using TUNEL staining. The results showed that HG significantly enhanced TUNEL positivity in HUVECs, which was attenuated by AdipoRon or Ac-YVAD-CMK (Fig. 2I, P < 0.001).

Fig. 2.

AdipoRon inhibits NLRP3-Caspase-1-GSDMD axis-mediated pyroptosis in HG-treated HUVECs. After treatment of with HG containing AdipoRon (10 µg/mL) or Ac-YVAD-CMK (50 µM), A cell viability was measured by CCK-8 assay; B ROS production was assessed via flow cytometry analysis; C the protein levels of IL-1β, NLRP3, ASC, Nrf2, pro-caspase-1, cleaved-caspase-1, GSDMD and p-NF-κB p65 were measured by western blotting; D caspase-1 activity, E LDH concentration, F ET-1 and NO concentrations were assessed using biochemistry analysis; G immunofluorescent test was performed to calculate GSDMD expression; H tube length of HUVECs was measured through tube formation assay; I the apoptosis of HUVECs was assessed by TUNEL assay. Scale bar = 100 μm. Horizontal lines denote statistical comparisons between the two connected groups. Asterisks indicate the level of significance: ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

AdipoRon inhibits NLRP3-Caspase-1-GSDMD axis-mediated pyroptosis in T2DM mice

The effects of AdipoRon on NLRP3 inflammasome-mediated pyroptosis were further validated in T2DM mice. In the T2DM groups, FBG levels exhibited a continuous upward trend and remained elevated until week 8 (Fig. 3A). Treatment with AdipoRon (low/high dose) or MCC950 from the end of the 8th week significantly reduced FBG levels, with AdipoRon-H exerting a more pronounced hypoglycemic effect (Fig. 3A). HOMA-IR is a key index reflecting insulin sensitivity. As presented in Fig. 3B, mice with T2DM exhibited a relatively high HOMA-IR compared with those of control (P < 0.001). After treatment with AdipoRon or MCC950, HOMA-IR was significantly decreased in T2DM mice (P < 0.001). Meanwhile, the high dose of AdipoRon exerted a stronger inhibitive effect on HOMA-IR than the low dose of AdipoRon (P < 0.05). HE staining results (Fig. 3C) showed that the aortic intima, media, and adventitia in the control group were exhibited a regular arrangement. In T2DM mice, thickening of the vascular wall was observed, with a remarkable increase in the thickness of the aortic media and adventitia. Vascular smooth muscle cells (VSMCs) displayed disorganized arrangement and hyperplasia; the intima exhibited irregular morphology accompanied by extensive endothelial cell detachment and damage. Notably, massive inflammatory cell infiltration was observed in all layers of the vascular wall, indicating the presence of an inflammatory response. In the AdipoRon-L group, the pathological changes of the aortic vessels were not obviously improved. Compared with the T2DM group, the AdipoRon-H group exhibited a reduction in vascular wall thickness and partial improvement in the arrangement of VSMCs; however, the intima remained irregular, accompanied by mild endothelial cell detachment and damage. In the MCC950 group, vascular wall thickness was reduced, and the intima, media, and adventitia of the aorta exhibited relatively regular organization. Western blotting was performed to measure the levels of NLRP3-Caspase-1-GSDMD pathway-related proteins in mouse arterial tissues. As manifested in Fig. 3D, compared with the control group, T2DM mice exhibited a notable increase in the protein levels of IL-1β, NLRP3, ASC, pro-caspase-1, cleaved-caspase-1, GSDMD, and p-NF-κB p65, while a significant decrease in Nrf2 protein level (P < 0.001), which were reversed by administration with AdipoRon-H or MCC950 (P < 0.05). Immunofluorescence staining showed that GSDMD expression was significantly elevated in arterial tissues of T2DM mice (Fig. 3E-F, P < 0.001), which was inhibited when co-treated with AdipoRon (particularly for AdipoRon-H) or MCC950 (P < 0.01). Then the core biomarkers of oxidative stress, such as MDA, SOD, and GSH-Px, which are directly involved in NLRP3 inflammasome-mediated pyroptosis were analyzed. The results showed that MDA content was increased (Fig. 3G, P < 0.001), and the levels of SOD and GSH-Px were decreased in aortae of T2DM mice (Fig. 3H-I, P < 0.001). Notably, the administration of AdipoRon-H or MCC950 significantly reversed the production of these oxidative stress markers (Fig. 3G-I, P < 0.001). Furthermore, TUNEL staining assay was performed to assess arterial endothelial cell apoptosis. The results demonstrated that the percentage of TUNEL-positive cells was markedly increased in T2DM mice, whereas this increase was significantly reversed by AdipoRon-H or MCC950 administration (Fig. 3J, P < 0.01).

Fig. 3.

AdipoRon inhibits NLRP3-Caspase-1-GSDMD axis-mediated pyroptosis in T2DM mice. After administration with AdipoRon-L (5 mg/kg), AdipoRon-H (20 mg/kg) or MCC950 (10 mg/kg) in T2DM mice, A fasting blood glucose levels were measured; B homeostasis model assessment of insulin resistance (HOMA-IR) was calculated using the following formula: HOMA-IR = (fasting blood glucose × fasting serum insulin)/22.5; C HE staining was performed to the pathological changes in artery blood vessels; D the protein levels of IL-1β, NLRP3, ASC, Nrf2, pro-caspase-1, cleaved-caspase-1, GSDMD and p-NF-κB p65 were measured by western blotting; E-F immunofluorescent test was performed to calculate GSDMD expression; G-I the levels of MDA, SOD and GSH-Px were assessed using biochemistry analysis; J TUNEL staining assay was performed to assess arterial endothelial cell apoptosis. Scale bar = 100 μm. Horizontal lines denote statistical comparisons between the two connected groups. Asterisks indicate the level of significance: ^*P < 0.05, ^**P < 0.01, ^***P < 0.001

Discussion

T2DM represents a major global public health challenge. As reported in the 9th Edition of the International Diabetes Federation (IDF) Diabetes Atlas, the global prevalence of diabetes is projected to approach one billion individuals by 2030—representing a 25% increase relative to the 2020 estimate—and to further surge by 51% to a new peak by 2045 [15]. Notably, in 2021 alone, diabetes was responsible for an estimated 79.2 million disability-adjusted life years, a metric encompassing both years of life lost and years lived with disability [16]. However, approximately one-third of diabetes cases are misdiagnosed, which may result in an underestimation of the actual disease burden imposed by T2DM. Consequently, the clinical management of T2DM and its associated complications remains a key focus for researchers, clinicians, and healthcare providers worldwide. In the present study, we observed that activation of the NLRP3-Caspase-1-GSDMD axis was concurrently associated with endothelial pyroptosis and dysfunction in HG-challenged HUVECs and a T2DM mouse model. AdipoRon‑mediated vascular protection was accompanied by suppression of this pyroptosis‑related signaling axis.

NLRP3 acts as a key cytoplasmic pattern recognition receptor. The NLRP3 inflammasome has three core components: the sensor NLRP3, the adaptor protein ASC, and the effector pro-caspase-1. In T2DM, multiple metabolites and pathogenic factors, including glucose, mitochondrial ROS, fatty acids, amino acid homocysteine, and ceramide, have been shown to induce NLRP3 inflammasome activation [17–20]. Accumulating evidence from recent studies indicates a strong correlation between NLRP3 inflammasome activation and the pathological progression of T2DM [21, 22]. In this study, we first established an HG-challenged HUVEC model to mimic T2DM in vitro, and further explored the effects of HG on NLRP3 inflammasome activation and vascular endothelial cell injury. Our results showed that HG treatment significantly reduced HUVEC viability and NO concentration, while elevated the levels of LDH and ET-1. Furthermore, the NLRP3 inflammasome was found to be activated by HG treatment, as evidenced by the elevated protein levels of NLRP3, ASC, and pro-caspase-1. These findings suggest that NLRP3 inflammasome activation is closely associated with vascular endothelial cell injury during the progression of T2DM.

NLRP3 inflammasome-mediated pyroptosis plays a pivotal role in the progression of T2DM and its associated complications, including diabetic cardiomyopathy and diabetic nephropathy [23–25]. In a recent study by Li and colleagues, NLRP3 inflammasome activation was shown to induce pyroptosis in pancreatic β cells [26]. Within the canonical pyroptotic pathway, assembly of the NLRP3 inflammasome facilitates caspase-1 activation; this process in turn promotes the extracellular release of the proinflammatory mediator IL-1β and the cleavage of GSDMD, thereby contributing to the amplification of inflammatory responses [27, 28]. More importantly, numerous studies have confirmed the crucial role of NLRP3/caspase-1/GSDMD axis in the regulation of pyroptosis [29–31]. We therefore hypothesized that NLRP3/caspase-1/GSDMD axis-mediated pyroptosis may contribute to vascular endothelial cell injury during the progression of T2DM. Herein, we found that in addition to activating the NLRP3 inflammasome, HG treatment of HUVECs further elevated the levels of pro-caspase-1, cleaved-caspase-1, and GSDMD. These results indicated that the NLRP3/caspase-1/GSDMD axis induced HUVEC pyroptosis under HG exposure. Furthermore, tube formation capacity was markedly impaired in the HG group compared with the control group. This finding provides functional evidence that pyroptosis contributes to vascular endothelial cell injury. Interestingly, activation of the NLRP3 inflammasome and expression of GSDMD were inhibited following treatment with AdipoRon. Moreover, similar to Ac-YVAD-CMK, AdipoRon also inhibited caspase-1 activity. To further validate the effect of AdipoRon on NLRP3 inflammasome in the animal model and explore whether NLRP3 inflammasome acts as the upstream regulatory factor of NLRP3-Caspase-1-GSDMD axis, T2DM mice were administrated with MCC950, the specific inhibitor of NLRP3 inflammasome. As expected, AdipoRon also exerted an inhibitory effect on NLRP3-Caspase-1-GSDMD axis-mediated pyroptosis in T2DM mice in vivo. These results indicated that the endothelial protective effects of AdipoRon occurred in parallel with the suppression of the NLRP3-Caspase-1-GSDMD signaling axis and pyroptosis. Our findings supported a significant association between these events. We further clarified that the vascular protective effects of AdipoRon and MCC950 in T2DM mice involved both direct and indirect mechanisms. In vitro experiments in HG‑treated HUVECs confirmed that AdipoRon directly suppressed endothelial pyroptosis and dysfunction independent of systemic metabolic changes. In T2DM mice, these compounds not only directly inhibited the endothelial NLRP3‑Caspase‑1‑GSDMD axis to reduce pyroptosis, but also improved systemic glucose homeostasis and insulin resistance, which secondarily alleviated hyperglycemia‑induced endothelial injury. Thus, the vascular benefits reflected a synergistic combination of direct endothelial protection and indirect metabolic improvement. Additionally, Nrf2 serves as a key target in regulating antioxidant responses and can enhance insulin sensitivity via both its upstream and downstream targets [32]. A previous report has demonstrated that the NF-κB pathway triggers GSDMD-mediated pyroptosis in tubular cells of mice with diabetic nephropathy. Moreover, overproduction of ROS can act on the NF-κB pathway, thereby promoting the secretion of IL-1β [33]. Thus, we proposed that ROS, Nrf2, and NF-κB p65 were also involved in the modulation of pyroptosis of HG-challenged HUVECs. In our in vitro and in vivo experiments, HG exposure markedly elevated the levels of ROS and p-NF-κB p65, and inhibited Nrf2 protein expression. However, treatment with AdipoRon significantly reversed their expression profiles. The above findings implied that AdipoRon may also exert potential antioxidant activity in T2DM.

Conclusion

In summary, the present study clarifies the regulatory mechanism by which AdipoRon alleviates vascular endothelial injury during the progression of T2DM. Specifically, the vascular protective effects of AdipoRon are closely associated with the attenuation of NLRP3-Caspase-1-GSDMD axis‑mediated vascular endothelial pyroptosis. Collectively, our findings provide a valuable theoretical basis for the management of vascular endothelial injury in T2DM progression.

Authors’ contributions

Conceptualization, Yi Yang; Methodology, Xiaoming Chen and Yi Yang; Software, Xiaoming Chen and Yi Yang; Validation, Xiaoming Chen and Yi Yang; Formal Analysis, Xiaoming Chen and Yi Yang; Investigation, Xiaoming Chen and Yi Yang; Resources, Xiaoming Chen and Yi Yang; Data Curation, Xiaoming Chen and Yi Yang; Writing-Original Draft Preparation, Xiaoming Chen; Writing-Review & Editing, Xiaoming Chen and Yi Yang; Visualization, Xiaoming Chen and Yi Yang; Supervision, Xiaoming Chen and Yi Yang; Project Administration, Xiaoming Chen and Yi Yang; Funding acquisition, Xiaoming Chen and Yi Yang.

Funding

This work was supported by Zhejiang Provincial Natural Science Foundation (Project No: LTGY23H070001), Key Projects of Jinhua Science and Technology Bureau (Project No: 2024-3-042), and Key Projects of Jinhua Science and Technology Bureau (Project No: 2022-3-137).

Data availability

The data are available from the corresponding author on reasonable request.

Declarations

Ethics approval and consent to participate

Ethical approvals were granted by the Experimental Animal Ethics Committee of Jinhua Food and Drug Inspection and Testing Research Institute (approval no. AL-JSYJ202416).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.