Targeting Pyroptosis: New Insights into the Treatment of Diabetic Microvascular Complications

Pyroptosis is an inflammatory form of programmed cell death that is dependent on inflammatory caspases, leading to the cleavage of gasdermin D (GSDMD) and increased secretion of interleukin (IL)-1β and IL-18. Recent studies have reported that hyperglycemia-induced cellular stress stimulates pyroptosis, and different signaling pathways have been shown to play crucial roles in regulating pyroptosis. This review summarized and discussed the molecular mechanisms, regulation, and cellular effects of pyroptosis in diabetic microvascular complications, such as diabetic nephropathy, diabetic retinopathy, and diabetic cardiomyopathy. In addition, this review aimed to provide new insights into identifying better treatments for diabetic microvascular complications.


Introduction
Diabetes is a low-grade inflammatory disease that seriously affects the quality of life of 463 million adults aged 20-79 years [1]. Diabetic microvascular complications (DMCs), such as diabetic nephropathy (DN), diabetic retinopathy (DR), and diabetic cardiomyopathy (DC), are the main contributors to the morbidity and mortality associated with diabetes. However, the pathogenesis and pathophysiology of these DMCs are complex. Chronic inflammatory response induced by hyperglycemia is a common mechanisms in DMCs. Further investigation of novel molecular mechanisms is required to develop new therapeutic approaches for DMCs.
Pyroptosis is a type of programmed cell death and its morphological characteristics are mainly caused by cell membrane rupture, chromatin condensation, nuclear fragmentation, and substantial release of cellular contents, including interleukin (IL)-1β, IL-18, and lactate dehydrogenase [2]. Pyroptosis is initially considered an innate immune and inflammatory response to invasion by external pathogens. Appropriate cell pyroptosis eliminates pathogens when they invade, but subsequent investigations suggest that pyroptosis is a double-edged sword, and that excess pyroptosis leads to a constant inflammatory response [3]. Recent studies have reported that a series of chronic inflammatory diseases, such as cardiovascular disease [4], rheumatoid arthritis [5], and nervous system diseases [6], are associated with pyroptosis, and that blocking excessive pyroptosis is a promising strategy for delaying disease development. Evidence suggests that diabetes is closely associated with pyroptosis [7]. is review provided an overview of the role of pyroptosis in DMCs and the effects of inhibition of the pyroptosis pathway in DMCs.

Pyroptosis and Pyroptosis-Related Mechanisms
tZychlinsky et al. first discovered the phenomenon of pyroptosis in 1992, which was initially considered apoptosis in macrophages infected by Shigella flexneri [8]. Studies have shown that pyroptosis is caused by caspase-1-regulated etiology. Scholars called this the canonical pyroptosis pathway and named it "pyroptosis" in 2001 to contrast with other types of cell death, meaning "fire" [9]. ereafter, a noncanonical pyroptosis pathway was observed [10]. In 2015, Kayagaki and Shi demonstrated that gasdermin D (GSDMD) was the ultimate executor of pyroptosis [11,12]. GSDMD is a gasdermin family member that can be cleaved into GSDMD-N fragments by inflammatory caspases, including caspase-1 and caspase-4/ caspase-5/caspase-11 (caspase-4/caspase-5 in humans and caspase-11 in mice) [13]. e GSDMD-N fragment of ringshaped oligomers anchored on the cell membrane forms pores to induce cell death [14].
Inflammation is a complex process that reflects local and systemic responses to different immunological and nonimmunological stimuli. Pyroptosis, which is essentially an inflammatory reaction, has provided a new perspective for research on the pathophysiological mechanisms of DMCs. Pyroptosis mechanisms include canonical and noncanonical pathways.
e difference between the two pathways is primarily due to their triggering factors. e two pathways are independently discussed below, and the details can be found in Figures 1(a) and 1(b).

Pyroptosis in Diabetic Microvascular Diseases
Recently, although limited, studies have attempted to explore the relationship between pyroptosis and DMCs and their potential mechanisms. Studies on pyroptosis and its associated diseases are summarized in this review.

Pyroptosis in Diabetic
Nephropathy. DN is one of the most prevalent and serious microvascular complications associated with diabetes. Its early pathological characteristics include basement membrane thickening, increased mesangial matrix production, and extracellular matrix accumulation, with the subsequent development of glomerulosclerosis and tubulointerstitial fibrosis, eventually leading to proteinuria and irreversible renal damage. e early clinical diagnosis of DN is based on canonical biochemical markers, such as glomerular filtration rate, urinary microalbumin, urinary microalbumin to urinary creatinine ratio, serum creatinine, urinary cystatin C, and serum β2 microglobulin. In addition, some biomarkers related to the pathogenesis of DN, such as kidney injury molecule 1 (Kim-1), neutrophil gelatinase-associated lipocalin (NGAL), tissue inhibitor of metalloproteinases-2, insulin-like growth factorbinding protein 7 (IGFBP-7), vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), monocyte chemoattractant protein-1(MCP-1), and inflammatory cytokines, such as tumor necrosis factor (TNF)α, MCP-1, and ILs (IL-1α, IL-1β, IL-18, IL-10), have also attracted significant attention [24,25]. However, the pathogenesis of DN remains unclear, and treatment strategies are limited. Recent studies have found that pyroptosis is involved in pathophysiological processes and may be a potential therapeutic target.
3.1.1. Glomerular Endothelial Cells. Glomerular endothelial cells (GECs) are located within the glomerulus and are the first layer to be involved in glomerular filtration. Recent findings suggest that both cell loss and inflammation in GECs are important causes of DN [26], and that pyroptosis may be a pivotal link between them. Activation of NLPR3 inflammasomes has been observed in the glomeruli of patients with DN and in animal models [27]. An in vitro study has reported that LPS directly induces pyroptosis in vascular endothelial cells [28]. Because low-grade inflammation and hyperglycemia form a vicious circle that promotes the development of DN toward end-stage renal disease, inhibiting pyroptosis may be an ideal strategy to block it. Our previous study showed that high glucose levels could induce pyroptosis in GECs, which was alleviated by a caspase-1 inhibitor or sodium butyrate [29]. Another study reported that hirudin ameliorated DN by inhibiting GSDMD-mediated pyroptosis in GECs [30].

Glomerular Mesangial
Cell. Glomerular mesangial cells (GMCs) play a pivotal role in maintaining the basic structure of the glomerulus. Increasing the mesangial matrix and GMCs proliferation promotes DN development. Inflammation is an important factor in GMC proliferation [46]. Previous studies have shown that high glucose level significantly induces the expression of pyroptosis markers, including NLRP3, caspase-1, pro-caspase-1, IL-1β, and pro-IL-1β, in GMCs [47,48], whereas inhibiting NLRP3 with MCC950 suppresses NLRP3/caspase-1/IL-1β activation and decreases renal fibrosis [32]. Nuclear enriched abundant transcript 1 and its target gene miR-34c are also found to regulate pyroptosis by mediating NLRP3 on GMCs in DN [49]. Furthermore, naringin and ginsenoside compound K have been reported to exert potential effects against pyroptosis in GMCs [50,51].

Renal Tubular Epithelial Cells.
Renal tubular epithelial cells (RTECs) are more vulnerable to death because they are inevitably stimulated by various pathogenic factors, such as toxins, hypoxia, and metabolic disorders. In patients with diabetes, the expression of IL-18, an inflammatory cytokine released during pyroptosis in renal tubular cells, increases significantly (approximately 83%) [52], suggesting the potential involvement of pyroptosis in RTEC damage. e expression of NLRP3, caspase-1, and IL-1β increases significantly in both STZ-induced DN rats and high glucosetreated RTECs [53]. Inhibition of caspase-1 or GSDMD knockdown ameliorates RTEC pyroptosis and reduces kidney damage in vivo and in vitro [54,55]. Moreover, upregulation of miR-23c inhibits RTEC pyroptosis by modulating MALAT1/ELAVL [53]. e TLR4/NF-κB signaling pathway also modulates GSDMD-mediated pyroptosis in RTECs [56]. Recent studies have reported that antisense noncoding RNA in the INK4 locus/miR-497/ TXNIP and KCNQ1OT1/miR-506-3p are involved in the regulation of high glucose-activated HK2 cell pyroptosis [57,58]. Moreover, circACTR2 regulates high glucoseinduced pyroptosis, inflammation, and fibrosis in proximal tubular cells [59]. Specific inhibitors of pyroptosis in RTEC have not been well studied, and existing evidence suggests a potential function for an A1 adenosine receptor agonist [35] or hirudin [30]. In summary, the canonical pyroptosis pathway is associated with intrinsic damage to RTECs and plays an essential role in DN development. RTEC pyroptosis inhibition may be a novel target for ameliorating albuminuria and renal fibrosis.

Pyroptosis in Diabetic
Retinopathy. DR is a hallmark complication of diabetes and a leading cause of vision loss in adults. Loss of retinal pericytes is one of the earliest changes associated with DR, and it has been postulated to initiate or trigger microaneurysm formation, abnormal leakage, edema, and ischemia, provoking proliferative neovascularization in the retina. Although the pathophysiological mechanisms of DR are complex, vascular endothelial damage, increased vascular permeability, and neovascularization are the most common phenomena.
Hemoglobin A1c (HbA1c) is the only validated systemic biomarker for DR progression. However, only 6.6% of the variation in the risk of DR is explained by HbA1c levels [60]. In addition, there are some biomarkers for the pathogenesis of DR, including VEGF, pigment epithelium-derived factor, platelet-derived growth factor subunit B, photoreceptorsecreted retinol-binding protein 3, forkhead box protein O1, NRF2, atypical protein kinase C, and inflammatory cytokines, such as TNF-α, IL1β, IL-6, IL-8, chemokines, C-C motif ligand-2, intercellular cell adhesion molecule-1, and vascular cell adhesion molecule-1 [61]. Furthermore, long noncoding RNAs (lncRNAs) and circular RNAs (circRNAs) in whole blood can serve as novel non-invasive biomarkers for proliferative DR [62,63]. However, there remains an urgent need to identify novel biomarkers for DR screening and detection.
Hyperglycemia-induced cell pyroptosis in the retina has been well demonstrated [64]. e density of corneal endothelial cells reduces and the expression of NLRP3, caspase-1, and IL-1β increases in patients with DR [65]. A similar event has been reported in STZ-induced diabetic rats [66]. Further studies have demonstrated that inhibiting caspase-1 [67] or silencing NLRP3 can reduce the secretion of caspase-1 and IL-1β, improve retinal layer thickness, and ameliorate blood-retinal barrier permeability [68], thus effectively retarding the progression of DR [69]. Moreover, the reactive oxygen species (ROS)/TXNIP/NLRP3 pathway is responsible for high glucose-induced retinal vascular permeability and pyroptosis in human retinal microvascular endothelial cells [69]. Further investigations have suggested that miR-590-3p induces the upregulation of NLRP1/ NADPH oxidase 4, which participates in the above modulation [70]. Furthermore, miR-214 and KCNQ1OT1 are involved in the caspase-1 signaling pathway [65]. In addition, high glucose levels can induce pyroptosis in retinal pigment epithelial cells through the METTL3/miR-25-3p/ PTEN/Akt signaling cascade or by regulating CircZNF532/ miR-20b-5p/signal transducer and activator of transcription 3 [71,72].
Although inhibition of caspase-1 or NLRP3 has been suggested to delay DR progression, a series of issues remain to be solved. First, current studies have mainly focused on the NLRP3-caspase-1 signaling pathway in DR. erefore, the role of the noncanonical pyroptosis pathway in this disease remains unknown. Moreover, as inflammasome activation does not always trigger pyroptosis, other signaling pathways involved in pyroptosis should be investigated. Second, the regulatory mechanism of pyroptosis is currently focused on noncoding RNA, and other regulatory mechanisms are yet to be defined. ird, GSDMD is the ultimate executor protein in pyroptosis, and its function in DR requires further in-depth study. In addition, although studies have indicated the influence of resolvin D1, minocycline, caspase 1, and NLRP3 inhibitors (such as Mcc950), or H3 relaxin [73] on blocking the NLRP3-caspase-1 signaling pathway [65][66][67]69], these have not been applied clinically, and more inhibitors are waiting to be screened.

Pyroptosis in Diabetic
Cardiomyopathy. DC is the main cardiovascular complication that occurs in approximately 60% of patients with well-controlled diabetes, resulting in systolic and diastolic dysfunctions, which are independent risk factors for any vascular disease or hypertension [74]. Increasing evidence suggests that hyperglycemia, lipotoxicity, and mitochondrial uncoupling contribute to cardiac inflammation, which plays an important role in the pathogenesis and progression of DC [75]. In this process, cytoplasmic calcium is increased, triggering mitochondrial changes, the production of ROS is increased, and activating ROS levels lead to oxidative damage in DC, among which oxidative stress and chronic inflammation are critical [75].
A broad spectrum of cardiovascular biomarkers that have been described in patients with DC includes brain natriuretic peptide (BNP), cardiac troponins (T, N, and I), and matrix metalloproteinases (MMPs), particularly MMP-9. Some biomarkers related to the pathogenesis of DR have been reported, such as cardiotrophin-1, IGFBP7, TGF-β, activin A, ROS-induced inflammatory cytokines (TNF-α, IL-6), galectin-3, suppression of tumorigenicity 2 (sST2), lncRNAs, and microRNAs [76]. However, the predictive roles of these biomarkers in patients with DM remain unclear owing to limited evidence [77]. We hope to identify novel biomarkers for DC screening.
Due to the undefined pathophysiology of DC, several studies have attempted to investigate the role of pyroptosis in DC. In early 2014, Luo et al. [78] reported that the expressions of NLRP3, ASC, caspase 1, and IL-1β increased significantly in cardiomyocytes of STZ-treated diabetic rats and that NLRP3 silencing or inhibition of caspase-1 reduced their expression, alleviated left ventricular dysfunction, and ultimately reversed myocardial remodeling in DC. Similar findings were observed in a DC model developed from C57BL/6J mice [79,80]. Apart from the caspase-1 modulated pathway, noncoding RNAs are also involved in this process. Upregulation of microRNA-30d directly modulates the downregulation of forkhead box class O 3a in STZtreated rats or high glucose-induced cardiomyocytes [81]. e study has observed that knockdown of the long noncoding RNA KCNQ1OT1 inhibits high glucose-induced pyroptosis by upregulating miR-214-3p and reducing caspase-1 expression in AC16 cells and primary cardiomyocytes [82]. Similarly, another study suggested that lncRNA-MALAT1 targeted miR-141-3p to promote HGinduced H9C2 cardiomyocyte pyroptosis [83]. Moreover, activation of the transforming growth TGF-β1/Smads pathway in cardiac fibroblasts is repressed by KCNQ1OT1 knockdown [80]. A further study reported that hsa_-circ_0076631, a caspase-1 related circRNA that suppresses miR-214-3p, increased in high-glucose-treated cardiomyocytes or serum from patients with diabetic [84]. Other noncoding RNAs, including AIM2 [85], miR-9 [86], GAS5 [87], and MIAT [88], are also potential modulators of pyroptosis in DC. A recent study demonstrated that the circRNAs circ_0071269 might promote the development of DC through the miR-145/GSDMA axis [89]. In addition, overexpression of mitochondrial aldehyde dehydrogenase 2 can reduce the high glucose-induced occurrence of pyroptosis in H9C2 cardiac cells [90].
Many drugs have been demonstrated to have some effects against pyroptosis (e.g., metformin [91], exendin-4 [92], pyrroloquinoline quinone (PQQ) [93], and skimming) [94]. A natural coumarin derivative has been found to protect against experimental DC by inhibiting pyroptosis in cardiomyocytes [94]. Empagliflozin has also been confirmed to alleviate the activation of NLRP3 and subsequent cardiomyocyte pyroptosis in the diabetic heart [95]. Overall, these studies indicate that the role of pyroptosis in DC and inhibition of the pyroptosis pathway might have an advantage in their ability to protect DC.

Conclusions and Prospects
In conclusion, the pathophysiological mechanisms underlying DMCs are complex and unclear. However, pyroptosis, a type of programmed cell death that triggers inflammatory reactions, provides a novel perspective for determining the mechanisms and therapeutic targets of DMCs (Table 1). ese experiments have demonstrated the involvement of pyroptosis in the pathophysiological process of DMCs in vitro and in vivo. Based on an enhanced understanding of these existing results, we further explore how pyroptosis and inflammasome are activated, including some novel studies related to pyroptosis regulation and its relationship with DMCs, to provide a promising avenue for DMC prevention and treatment. ree new insights have been discovered regarding the regulation of pyroptosis in DMCs: (1) e important causes of DMCs are oxidative stress and inflammation resulting in pyroptosis. Based on the antioxidative stress and antiinflammatory strategies, we determined effective drugs or smallmolecule compounds against pyroptosis to improve DMCs and further explored the regulation of pyroptosis and its potential mechanisms, which will open up a new direction for the prevention and treatment of DMCs.
(2) e pro-inflammatory effect of pyroptosis is a key factor in the development of DMCs. Some studies have found that several upstream regulatory signaling proteins, noncoding RNAs, and circRNAs play a role in the upregulation of pyroptosis. Furthermore, autophagy plays a protective role in the inhibition of NLPR3 overactivation [96]. erefore, further clarification of the regulatory mechanism of pyroptosis and targeting these pathways will also be an effective means to prevent and treat DMCs.
(3) e inflammatory response is an important factor in the development of DMCs. Pyroptosis maintains or expands inflammation by tightly controlling the inflammatory response via the release of IL-1β and IL-18. IL-1β and IL-18 are two essential proinflammatory cytokines that are upregulated in tissue-resident cells of patients and animals with diabetes and are further induced by positive feedback to pyroptosis to form inflammatory storms [97]. Based on the perspective of the upregulation of different pro-inflammatory factors triggered by metabolic and hemodynamic disorders, controlling the chronic inflammatory microenvironment of diabetes will provide potential insights into the prevention and treatment of DMCs.
Nevertheless, whether some promising pyroptosis biomarkers can be used as novel targets for diagnosing and treating DMCs requires further investigation, particularly in animal experiments and clinical studies. Further investigations into the exact molecular and regulatory mechanisms of pyroptosis are necessary for DMCs. Furthermore, GSDMD, as the ultimate pyroptosis protein, should be investigated for its function in DMCs and to determine whether inhibiting GSDMD is a more valuable therapeutic target for these complications. Moreover, inhibition or activation of the pyroptosis pathway affects various aspects of physiology and can have different effects. It is necessary to consider the overall body balance when studying drugs that target the pyroptosis pathway in DMCs.

Data Availability
No data were used to support this study.

Conflicts of Interest
All authors declare no conflicts of interest.

Authors' Contributions
J. G. researched the documents and wrote the manuscript. K. G. and M. G. revised the manuscript. Y. X. and YH. X. designed the study and reviewed the manuscript. W. H. and X. L. revised the manuscript for important intellectual content. All authors approved the final version of the manuscript. Junling Gu, Kang Geng, and Man Guo contributed equally to this work.