The prevalence of diabetes is increasing year by year, which has imposed economic burden on patients and caused their suffering. Cardiovascular complications are the main causes of death. In vivo, vascular endothelial cells are damaged and vascular diastolic function is reduced as blood glucose concentration increases. These effects lead to endothelial cell dysfunction, which in turn leads to vascular complications in diabetic patients. These complications can be reduced by controlling the blood glucose levels of diabetic patients. ncRNA can regulate endothelial cell pyroptosis and is involved in a variety of cardiovascular diseases[28, 29]. However, the role of piRNA in endothelial cell pyroptosis is unclear. This experiment used high glucose concentrations to establish a cell pyroptosis model and then explored the role and possible mechanism of piRNA-823 in endothelial cell pyroptosis.
Pyroptosis is a new form of pro-inflammatory programmed cell death. NLRP3 inflammasomes play an important role in cell pyroptosis and are multiprotein complexes composed of the receptor proteins NLRP3, ASC, and caspase-1, which lead to the excessive activation of the complexes under cell stress or in the presence of tissue damage or infectious pathogens. The activation of the downstream target caspase-1 promotes the secretion of inflammatory factors, such as IL-1β, and the occurrence of cell pyroptosis. Yang et al.[30] showed that in a high-glucose environment, NLRP3 inflammasomes are overactivated, the secretion of inflammatory factors IL-1β and IL-18 increases, and the inhibited activation of NLRP3 inflammasomes can inhibit cell pyroptosis. Gu et al.[31] found that the amounts of released LDH, IL-1β, and IL-18 increased in endothelial cells treated with glucose at high concentrations, and the expression levels of GSDMD and caspase-1 proteins increased. Our study found that high glucose concentrations significantly increase the expression of pyroptosis-related proteins and promote the secretion of inflammatory factors, indicating that high-glucose treatment significantly promotes pyroptosis in endothelial cells.
ncRNA is involved in the occurrence and development of a variety of cardiovascular diseases by regulating cell pyroptosis. In myocardial I/R, miR-132 expression is significantly upregulated, and the PGC-1α/NRF2 signaling pathway is activated by targeting Sirt1 and promotes pyroptosis, thereby aggravating myocardial I/R injury[32]. lncRNA KLF3-AS1 competitively binds to miR-138-5p to regulate the expression of Sirt1, thereby inhibiting cell pyroptosis and inhibiting the process of myocardial infarction[33]. In addition, in diabetic nephropathy, miR-497 expression is reduced, and miR-497 overexpression inhibits caspase-1-dependent cell pyroptosis[34]. miR-130a mitigates cell damage by regulating cell pyroptosis caused by the TNF-α/SOD1/ROS axis[35]. We also found observed in our previous studies that miRNA-223-3p promotes cardiomyocyte pyroptosis by downregulating the release of inflammasome factor SPI1 (PU.1)[36].
piRNA is a class of small-molecule ncRNAs. Little research into the role of piRNA in cardiovascular diseases has been conducted. piRNA is abnormally expressed in cardiovascular diseases and may be involved in the occurrence and development of diabetes and heart-related diseases, but the molecular mechanisms and signaling pathways involved in piRNA function have not been fully elucidated. One study analyzed piRNA expression profiling of islet cells in a rat model of type 2 diabetes mellitus was analyzed by using a gene chip technology and found that the expression levels of DQ732700 and DQ746748 significantly increased; these effects led to glucose-induced insulin secretion defects, but the specific mechanism is unclear[37]. Gao et al.[38] found that a type of piRNA (CHAPIR) is abundantly expressed in myocardial hypertrophy and can promote myocardial pathological hypertrophy and cardiac remodeling by targeting the m6A methylation of METTL3-mediated Parp10 mRNA transcripts. These studies provided novel insights into the potential value of piRNA in the clinical diagnosis, prognosis, and treatment of diabetes and heart diseases.
piRNA-823 is an important piRNA that is abnormally expressed in a variety of tumors and is involved in the development of multiple tumors. The expression of piRNA-823 in gastric and kidney cancer tissues is reduced, and the upregulation of piRNA-823 inhibits the growth of gastric cancer cells[39]. However, the expression of piRNA-823 in colon cancer tissues significantly increased and may have promoted the proliferation, invasion, and anti-apoptosis activity of colorectal cancer cells by regulating the G6PD/HIF-1α pathway[23, 40]. The expression of piRNA-823 was upregulated in MM tissues and cell lines, and the disruption of piRNA-823 expression inhibited tumor cell proliferation, induced apoptosis, and inhibited tumor development[24, 41]. Extracellular vesicles (EVs) carry a variety of RNA molecules and play a crucial role in the connection between tumors and surrounding stromal cells, including endothelial cells. Li et al.[41] found that piRNA-823 is mainly present in the peripheral blood of patients with MM and MM-derived EVs. In mice, when MM-derived EVs are cocultured with endothelial cells, piRNA-823 in EVs transfer to endothelial cells and promote the growth of transplanted tumors. The transfection of piRNA-823 mimics or MM-derived EVs significantly promotes endothelial cell proliferation, invasion, and tubule formation, possibly by enhancing the expression of VEGF, IL-6, and ICAM-1 and inhibiting apoptosis. These results suggest that piRNA-823 plays different roles in different tumors and piRNA-823 can be used as a biomarker and therapeutic target for diseases. The results of the present study showed that high glucose concentrations induced pyroptosis in endothelial cells while reducing the expression level of piRNA-823. Therefore, we speculated that piRNA-823 is involved in high glucose concentration–induced endothelial cell pyroptosis. The transfection of piRNA-823 mimics reduced the expression levels of NLRP3, GSDMD, caspase-1, and ASC proteins and inhibited the secretion of LDH and inflammatory factors IL-1β and IL-18 in the supernatant of the culture medium. piRNA-823 can inhibit high glucose concentration–induced endothelial cell pyroptosis. To further explore the specific mechanism of the piRNA-823 inhibition of high glucose concentration–induced epithelial cell pyroptosis, we performed high-throughput sequencing analysis. Gene chip expression analysis indicated that LCN2 is the most tightly bound to piRNA-823 and has the most stable structure.
LCN2 is a secreted protein of neutrophil and expressed in the kidneys, brain, lungs, liver, adipocytes, neutrophils, macrophages, endothelial cells, smooth muscle cells, cardiomyocytes[42],[43]. White adipose tissues are the main sources of LCN2. In a low expression state, LCN2 expression is significantly increased when epithelial cells are stimulated by infection, inflammation, or ischemia and is involved in the body's inflammatory response, lipid metabolism, and iron transport[26, 27]. LCN2 plays a key role in cardiovascular remodeling and unstable atherosclerotic plaque formation[44],[45]. LCN2 not only is involved in the development of hypertension but also promotes the occurrence of aneurysms through mechanisms, such as inflammatory response[42, 46]. At present, LCN2-mediated oxidative stress, chronic inflammatory response, and fibrosis play an important role in the occurrence and development of cardiovascular diseases. Our study showed that increasing the expression of piRNA-823 mimics reduces the degree of pyroptosis under high-glucose culture conditions. LCN2 recombinant plasmids transfected into cells promotes pyroptosis. This result was confirmed by changes in cell morphology observed through scanning electron microscopy.
In summary, the expression of piRNA-823 decreased in high glucose conccentration–induced endothelial cells, and increasing the expression of piRNA-823 inhibited high glucose concentration–induced endothelial cell pyroptosis. The mechanism was to reverse hyperglycemia-induced HUVEC pyroptosis by targeting LCN2. This mechanism serves as a basis for the further study of the roles of piRNA in endothelial cell pyroptosis and in the prevention and treatment of cardiovascular diseases caused by diabetes.