LncRNA NEXN-AS1-NEXN Pathway Mediated The Inhibition Effects On Pyroptosis Of Atorvastatin Calcium In HUVECs

Background and aimsAtherosclerosis(AS)is the leading cause of many cardiovascular diseases,and its incidence has increased in recent years.Regarding the etiology of AS,the theory of inflammatory response is one of the most important viewpoints.Besides,many molecules and pathways have been reported to participate in the occurrence and development of AS,including long non-coding RNAs(lncRNAs).We have confirmed that multiple IncRNAs,including newly identified lncRNA NEXN-AS1,can inhibit AS through different pathways.Ho we ver,their mechanism is unclear.In addition,reports showed that the patterns of cell death were also closely related to the development of AS.It's said that endothelial cells pyroptosis can promote the recruitment of monocytes to the inner membrane of vascular,exposure to hyperlipidemia,and differentiate into macrophages which at last promote AS and plaque formation.Pyroptosis is a kind of programmed cell death that is different from apoptosis and necrosis.In recent years,with the clearance of its effector gasdermin(GSDMD),pyroptosis has attracted wide attention.A variety of non-pathagenic factors can inducecells pyroptosis bycanonical inflammasome pathways,which is characterized by the activation of NLRP3 inflammasome and then activated caspase1 to cause a series of cascades,and at last causing the formation of non-selective GSDMD pore,from which inflammatory factors IL-1? and IL-18released,and cells swell and rupture.Atorvastatin calcium which are widely used to lower blood cholesterol levels,have demonstrated anti-inflammatory,anti-atherosclerosis,and stabilized plaques in many clinical trials.Also,some evidence suggests that statins may impact cell apoptosis,although no study has reported the influence of statins on pyroptosis.Therefore,this study aimed on the effects of atorvastatin calcium on lncRNA NEXN-AS1-NEXN pathway and pyroptosis.Materials and methods1.Atorvastatin calcium was used to treat human umbilical vein vascular endothelial cells(HUVECs);Q-PCR and WB were performed to detect the expression levels of NEXN-AS1,NEXN and pyroptosis related molecules such as caspase-1,NLRP3,GSDMD,IL-1? and IL-18 in canonical inflammasome pathways.2.Lentivirus short hairpin RNAs(shRNAs)targeting NEXN-AS1(sh NEXN-AS1)and siRNAs against NEXN(siNEXN)were used to rescue the effects of atorvastatin calcium on pyroptosis.Results1.Atorvastatin calcium up-regulated the expression levels of NEXN-AS1 and NEXN in HUVECs.Q-PCR showed that the lncRNA level of NEXN-AS]and the mRNA level of NEXN were significantly increased in a concentration-or time-dependent manner by atorvastatin calcium in HUVECs(p<0.05).WB showed that NEXN protein levels were also up-regulated by atorvastatin.2.Atorvastatin calcium inhibited the expression of pyroptosis key molecules such as NLRP3,caspase-1,GSDMD,IL-1? and IL-18 in HUVECs.Q-PCR results showed that atorvastatin calcium can significantly reduce mRNA levels of caspase-1,NLRP3,GSDMD,IL-1? and IL-18 in a dose-or time-dependent manner.WB showed that atorvastatin calcium reduced NLRP3,caspase-1 mature body,GSDMD full-length fragment,C-terminus of GSDMD,and active IL-1? and IL-18.The differences were statistically significant(p<0.05),but had no significant effects on inactive precursors of caspase-1,IL-1?(p>0.05).3.LncRNA NEXN-AS1 mediated atorvastatin calcium to inhibit pyroptosis of HUVECs through up-regulation of NEXN.Knockdown of NEXN-AS1 resulted in thatl)firstly,NEXN-AS1 and NEXN expression were both down-regulated(p<0.05),while expression levels of caspase-1,NLRP3,GSDMD,IL-1? and IL-18 were up-regulated(p<0.05);2)secondly,the upregulation of NEXN-AS1 and NEXN and the inhibition of pyroptosis caused by atorvastatin were mostly offseted(p<0.05).It indicated that NEXN-AS1 mediatesthe inhition effects on pyroptosis of atorvastatin calcium by upregulating NEXN.4.NEXN mediated atorvastatin calcium to induce pyroptosis of HUVECs.1)Interfering with siNEXN,lncRNANEXN-AS1 did not change significantly(p>0.05),and the expression level of NEXN was significantly reduced(p<0.05).2)When siNEXN and atorvastatin acted synergistically,lncRNA NEXN-AS1 had no difference compared with atorvastatin acted alone(p>0.05),while the expression of NEXN and key molecules of pyroptosis almost recovered to the normal levels(p<0.05),indicating that NEXN mediated atorvastatin calcium to induce pyrotosis of HUVECs.ConclusionsWe were the first to report that lncRNA NEXN-AS1-NEXN pathway mediated the inhibition effects of atorvastatin,which provide a new insight in the mechanism of how atorvastatin proceding non-lipid-lower effects against the development of atherosclerosis and provided a new direction for non-coding RNA researches and new ideas for further clarifying the role of pyroptosis in atherosclerosis.