The Mechanism Of Sirt1 Regulating H₂O₂-induced Pyroptosis Of Human Aortic Endothelial Cells By Deacetylating Of NF-κB （p65）

BackgroundAtherosclerosis is a chronic inflammatory disease that may lead to cardiovascular disease,stroke and ischemic gangrene.Vascular endothelial dysfunction is the initial stage of atherosclerosis,and oxidative stress is an important pathogenesis of endothelial dysfunction.Various incentives for the formation of atherosclerosis,including hypercholesterolemia,hyperglycemia,hypertension and smoking,cause excessive accumulation of reactive oxygen species（ROS）,which results in the imbalance between oxidation and antioxidant defense systems,resulting in acute oxidative stress response,endothelial damage,and ultimately endothelial dysfunction and various oxidative stress-related diseases.Therefore,intervention of oxidative stress is an effective prevention and treatment strategy for atherosclerosis endothelial injury.Recently,it has been found that pyroptosis plays an important role in oxidative stress mediated vascular endothelial dysfunction.Pyroptosis is an inflammatory form of cell death characterized by dependence on caspase-1 and the release of a large number of proinflammatory factors.When pyroptosis occurs,pores form on the surface of cell membrane,accompanied by swelling,rupture and release of contents,which induces inflammatory response.NLRP3 inflammasome has been studied most extensively in classical pyroptosis pathways.NLRP3 binds pro-caspase-1 to form inflammasome complex via ASC,and pro-caspase-1 is cleaved to form cleaved caspase-1.Cleaved caspase-1 sliced gastermin-D（GSDMD）,pro-IL-1β and pro-IL-18 to form GSDMD with N-terminal,matured IL-1β and IL-18.N-GSDMD forms perforated channels on the surface of cell membrane to promote the inward flow of H2 O and the outflow of cell contents,which induce and expand the inflammatory response,leading to cell death.Therefore,pyroptosis is an important ways of endothelial cell death,and revealing its mechanism has important clinical significance for the prevention and treatment of atherosclerosis.Nuclear factor κappa B（NF-κB）is a series of related protein complexes,which play transcriptional regulatory roles as heterologous and homologous dimers and are widely involved in the occurrence of inflammatory diseases.It has been found that p65,subunit of NF-κB,regulates NLRP3 expression in the pathogenesis of atherosclerosis.We investigated that whether NF-κB（p65）is involved in endothelial cells pyroptosis.There are complex and highly regulated mechanisms for NF-κB activity,in which acetylation of p65 subunit plays an important role in regulating its transcriptional activity.To investigate the mechanism of p65 mediated pyroptosis,we found that SIRT1,an NAD+ dependent histone deacetylase,is a key regulator of oxidative stress.SIRT1 regulates key metabolic processes including gene silencing,stress resistance,apoptosis,aging,and inflammation through deacetylation of a variety of substrates.We investigated that whether SIRT1 affects NF-κB transcriptional activity by regulating p65 acetylation level and inhibiting the entry of p65 into the nucleus during oxidative stress response of endothelial cells.This may help us to better study the pathogenesis of pyroptosis in atherosclerosis.Micro RNAs（miRNAs）are the most widely studied non-coding RNAs.Mi RNAs can negatively regulate gene expression by binding to target m RNA and inducing its degradation or inhibiting its translation.Mi R-200a-3p is widely expressed in all tissues,and studies have shown that miR-200a-3p has a regulatory effect on a variety of diseases in the cardiovascular system.In order to explore the upstream regulatory mechanism affecting SIRT1 expression and explore the technical means to intervene SIRT1 regulation of pyroptosis,bioanalysis predicted that miR-200a-3p may have a target with SIRT1,and miR-200a-3p may affect the occurrence of pyroptosis by regulating SIRT1 expression.In this study,taking human aortic endothelial cells（HAECs）as the research object,we aimed to study the regulation of NF-κB（p65）,SIRT1 and miR-200a-3p on pyroptosis and its mechanism in HAECs oxidative stress model.These results may provide a new theoretical basis for the role of p65,SIRT1 and miR-200a-3p in oxidative stress response,and may provide a new strategy for clinical prevention and treatment of atherosclerosis.ObjectsThe aim of this study was to investigate the generation of pyroptosis in H₂O₂-mediated oxidative stress model of HAECs,to clarify the change trend and the role of SIRT1 in oxidative stress response of HAECs,exploring the relationship between miR-200a-3p and SIRT1,and whether miR-200a-3p regulated pyroptosis by SIRT1 in HAECs.In this study,we established the H₂O₂-induced oxidative stress injury model of HAECs,and NF-κB（p65）/NLRP3 was used as the entry point to explore the mechanism of SIRT1 in regulating of pyroptosis,aimed to explore the technical means to interfere with SIRT1 regulation of pyroptosis in order to inhibit oxidative stress-induced vascular endothelial dysfunction,and provide experimental basis and theoretical basis.Methods1.Establishment of oxidative stress-induced pyroptosis model of HAECs.Explore the effects of H₂O₂ on HAECs damage at different concentrations（0.2m M,0.4m M,0.6m M）and at different times（2h,4h,8h）.CCK-8 activity detection kit was used to detect the survival rate of cells,and DCFH-DA probe was used to detect the intracellular ROS content of HAECs at different time points.LDH activity assay and PI staining were used to analyze the effects of H₂O₂ on HAECs injury degree and survival rate.To detect the effect of H₂O₂ on pyroptosis at different concentrations and at different times.Western blot was used to detect the expression levels of NLRP3,ASC,procaspase-1,cleaved caspase-1,GSDMD-N,IL-1β,and IL-18 in H₂O₂-induced HAECs.To clarify the effects of NLRP3 inflammasome intervention on H₂O₂-induced HAECs injury.Transient transfection was used to construct model of NLRP3 downexpression and ASC downexpression,and caspase-1 inhibitor VX-765 was used to detect the expression level of pyroptosis related proteins in H₂O₂-induced HAECs.2.To analyze the role of NF-κB（p65）in H₂O₂-induced HAECs pyroptosis: Western blot was used to detect the expression level of p65 after H₂O₂-induced HAECs.Transient transfection was used to construct a p65 downexpression of HAECs,and LDH activity was used to detect PI staining to analyze the effect of p65 on cell damage and survival rate in H₂O₂-induced HAECs.After downexpression of p65,the m RNA expression levels of NLRP3 and IL-1β were detected by q PCR,confirming that p65 regulates pyroptosis through NLRP3 and IL-1β.Western blot was used to detect the effects of p65 on the expression levels of NLRP3,ASC,cleaved caspase-1,GSDMD-N,IL-1β and IL-18 in H₂O₂-induced HAECs.Western blot was used to detect the expression of p65 in the nucleus and cytoplasm of H₂O₂-induced HAECs.3.To analyze the mechanism of SIRT1 regulating NF-κB（p65）activity by H₂O₂ and its effect on pyroptosis: PCR and Western blot were used to detect the expression level of SIRT1 in H₂O₂-induced HAECs.The overexpressed SIRT1 model was established by transient transfection.The effects of SIRT1 on H₂O₂-induced HAECs damage and survival rate were analyzed by detecting LDH activity and PI staining.Western blot was used to detect the effects of SIRT1 on the expression level of pyroptosis-related proteins in HAECs.The effect of H₂O₂-induced expression levels of ac-p65 and p65 in HAECs,and the direct interaction between SIRT1 and p65 was verified by protein immunoprecipitation.Western blot was used to detect the effect of SIRT1 on the expression level of p65 in the nucleus and cytoplasm.The expression of SIRT1 on p65 in the nucleus and cytoplasm was detected by immunofluorescence staining,and the changes of nuclear translocation of p65 were analyzed.4.To analyze whether H₂O₂ participates in pyroptosis by regulating miR-200a-3p to induce SIRT1 downregulation.1)Bioanalysis predicted that miR-200a-3p targeted inhibition of SIRT1.PCR was used to detect the expression of miR-200a-3p after H₂O₂-induced HAECs.2)Transient transfection was used to construct miR-200a-3p downexpression model,and the effects of miR-200a-3p on H₂O₂-induced HAECs injury and survival rate were detected by LDH activity and PI staining.Western blot was used to detect the effect of miR-200a-3p on the expression of pyroptosis associated protein in HAECs.3)Overexpression and downexpression of miR-200a-3p models were constructed,and PCR and Western blot were used to detect whether miR-200a-3p has regulatory effects on SIRT1 expression level.Double luciferase reporter gene assay was used to construct SIRT1 wild-type and mutant-type reporter plasmids and co-transfected with miR-200a-3p mimics or negative control.The activity of luciferase reporter gene was detected to verify the existence of direct binding sites between miR-200a-3p and SIRT1.Co-transfection models with downexpression of miR-200a-3p and SIRT1 were constructed.Western blot was used to detect the expression changes of pyroptosis related proteins in H₂O₂-induced HAECs to verify whether miR-200a-3p targets SIRT1 to regulate the occurrence of pyroptosis.Results1.Intracellular ROS levels increased,LDH activity increased in cell culture medium,and the expression levels of pyroptosis related protein NLRP3,pro-caspase-1,cleaved caspase-1,ASC,IL-1β,and IL-18 were increased in H₂O₂-induced HAECs injury model.These results suggested that oxidative stress induced pyroptosis.The expression of pyroptosis related proteins GSDMD-N and IL-1β was decreased by NLRP3 and ASC intervention and caspase-1 inhibitor,suggesting that NLRP3 inflammasome mediated pyroptosis plays an important role in oxidative stress induced vascular endothelial injury.2.H₂O₂-induced increased protein expression ratio of ac-p65/p65 in HAECs,and the expression level of p65 in both nucleus and cytoplasm was increased,which was more obvious in nucleus,suggesting that H₂O₂ promoted nuclear translocation of p65.Inhibition of p65 inhibited the expression of NLRP3 and IL-1β in HAECs,and inhibited pyroptosis and reduced the damage of HAECs caused by H₂O₂.3.The expression of SIRT1 in H₂O₂-induced HAECs was decreased.Overexpression of SIRT1 improved cell activity and survival rate,and the expression of pyroptosis related proteins decreased,suggesting that SIRT1 played a protective role in H₂O₂-induced HAECs pyroptosis.The direct interaction between SIRT1 and p65 was verified by protein immunoprecipitation.SIRT1 also significantly reduced the protein expression ratio of ac-p65/p65 and inhibited the level of p65 in the nucleus.Immunofluorescence staining showed that SIRT1 inhibited the nuclear translocation of p65.4.Bioinformatics predicted that miR-200a-3p could directly target SIRT1.The expression level of miR-200a-3p was significantly increased in H₂O₂-induced HAECs,and miR-200a-3p reduced cell activity and survival rate,and promoted the expression of pyroptosis related proteins.The double luciferase reporter gene assay proved that there were targeted binding sites between miR-200a-3p and SIRT1.After miR-200a-3p was inhibited,the expression level of SIRT1 increased,which proved that miR-200a-3p regulated the expression of SIRT1.Inhibition of SIRT1 reversed the protective effect of downexpression of miR-200a-3p on cells,suggesting that miR-200a-3p regulates the occurrence of HAECs pyroptosis by targeting SIRT1.Conclusions1.NLRP3 inflammasome was activated by H₂O₂ via NF-κB（p65）,which promoted HAECs pyroptosis and aggravated cell damage.2.SIRT1 was down-regulated in H₂O₂-induced HAECs injury model,which promoted the acetylation of p65 and promoted the nuclear translocation of p65 to induce HAECs pyroptosis.3.Mi R-200a-3p was significantly increased in H₂O₂-induced HAECs injury model and regulated SIRT1,and inhibition of miR-200a-3p reduced HAECs pyroptosis and protected cells from oxidative stress damage.Innovation and significanceIn this study,we explored the mechanism of SIRT1 regulation of pyroptosis and the technical means of intervention of SIRT1 regulation of pyroptosis by establishing H₂O₂-induced HAECs oxidative stress injury model and taking NF-κB（p65）/ NLRP3 as the entry point,providing experimental basis and theoretical basis for inhibiting oxidative stress-induced vascular endothelial dysfunction.