| Part 1 The role of NLRP3 inflammasome and pyroptosis in myocardial reperfusion-induced microvascular dysfunctionPurpose:To investigate the role of NLRP3 inflammasome and pyroptosis in cardiac microvascular endothelial cells and myocardial tissue.MethodsIn vitro:using cardiac microvascular endothelial cells line establish hypoxic/re-oxygenation model to simulate the ischemia-reperfusion.Pretreatment with MCC950 or VX-765 before re-oxygenation,thereafter,cells were randomly divided into the following groups:control(normoxia)group,H/R group,H/R+MCC950 group,H/R+VX-765 group.Quantitative Real-time PCR(RT-PCR)was used to detect the levels of NLRP3 and IL-1?;Western blot was performed to detect the expression of NLRP3,caspase-1,caspase-4,IL-1? and GSDMD in each groups,to observe the effects of MCC950 pretreatment on the expression of NLRP3,caspase-1,IL-1? and GSDMD in CMECs,to detect the effects of VX-765 pretreatment on the expression of caspase-4 and GSDMD;immunofluorescence was used to observe the changes of NLRP3,caspase-1 and GSDMD expression in each group;the toxicity of CMECs was analyzed using LDH Cytotoxicity Assay Kit.In vivo:C57BL/6J adult male(6-8weeks,18-22 g)were used in the study.Mouse ischemia-reperfusion model was established.The slipknot was released for 1 h,6 h and 12 h after occlusion for 45 min and selection of the optimal time was used in subsequent experiments.MCC950(15 mg/kg),a potent,selective,small-molecule inhibitor of NLRP3 or VX-765(30 mg/kg),small-molecule inhibitor of caspase-4 was intraperitoneally administrated before ligation of the left anterior descending(LAD),fifteen minutes before reperfusion,an additional dose of MCC950(15 mg/kg)or dose of VX-765(30 mg/kg)was immediately administrated in the peritoneal in a randomized.Thereafter,animals were randomly divided into the following groups:Sham group,I/R group,I/R+GAS group,MCC950+I/R group,VX-765+1/R group Western blot analysis was performed to observe the changes of NLRP3 and caspse-4 inflammasome-associated protein expression in different groups;immunofluorescence was used to detect the levels of NLRP3 inflammasome associated protein expression;immunohistochemistry was used to observe the inflammatory cells infiltration in infarct and ischemic areaResults:1.RT-PCR results showed that the levels of NLRP3 and IL-1? were higher in hypoxia re-oxygenation compared with control group and peaked at 2 h after hypoxia re-oxygenation in CMECs(P<0.05,n=3).2.Western blot analysis revealed that the expression of NLRP3 and IL-1? was higher in hypoxia re-oxygenation group compared with control group and peaked at 2 h after hypoxia re-oxygenation(P<0.05,n=3).Therefore,2 h of hypoxia/2 h of re-oxygenation was used in subsequent experiments.Compared to control group,the expression of NLRP3,caspase-1,caspase-4,IL-1? and GSDMD was higher in H/R group(P<0.05,n=3).These results indicated that H/R could activate NLRP3 inflammasome in CMECs and induce CMECs pyroptosis.Compared to H/R group,pretreated with different concentrations MCC950(1,10,100 ?M),especially at 1 ?M,remarkably decreased the expression of NLRP3,caspase-1 and GSDMD in CMECs(P<0.05,n=3).In comparison with H/R group,pretreated with VX-765(10?M)before re-oxygenation significantly decreased the expression of caspase-4 and GSDMD in CMECs(P<0.05,n=3).These results demonstrated that MCC950 and VX-765 could prevent H/R-induced CMECs pyroptosis,suggesting that NLRP3/caspase-1 and caspase-4 pathways might be involved in H/R-induced CMECs pyroptosis.In vivo,the expression of NLRP3,caspase-1,caspase-4,IL-1? and GSDMD was higher in I/R group compared with sham group and peaked at 6 h after reperfusion(P<0.05,n=3).These results indicated that I/R could induce myocardial tissue pyroptosis,suggesting that NLRP3/caspase-1 and caspase-4 pathways might be involved in I/R-induced myocardial tissue pyroptosis.3.Immunofluorescence results revealed that the expression of NLRP3,caspase-1 and GSDMD in CMECs was increased in H/R group compared with control group.In comparison with H/R group,the caspase-1 and GSDMD protein levels in CMECs were decreased in pretreatment with MCC950(1 uM)group.In vivo,I/R group remarkably increased NLRP3 and caspase-1 expression compared to sham group(P<0.05,n=3).However,NLRP3 and caspase-1 activation were significantly decreased in MCC950 group in comparison to I/R group(P<0.05,n=3),These results suggest that NLRP3 inflammasome activation and pyroptosis may be involved in the pathophysiological process of myocardial ischemia-reperfusion injury.4.Immunohistochemical staining indicated that the number of F4/80 and GSDMD positive cells in I/R group was higher than sham group(P<0.05,n=3),Compared to I/R group,the number of F4/80 positive cells was decreased in pretreatment with MCC950 group(P<0,05,n=3).The number of GSDMD positive cells was lower in administration with MCC950 or VX-765 group in comparison to I/R group(P<0.05,n=3).The results indicated that MCC950 or VX-765 could prevent myocardial tissue pyroptosis induced by MI/R.5.LDH Cytotoxicity Assay showed that H/R increased LDH release in CMECs compared to control group(P<0.05,n=3).Administration with MCC950 or VX-765 inhibited LDH release in CMECs compared to H/R group(P<0.05,n=3).Conclusions:NLRP3 and caspase-4 inflammasome activation and pyroptosis in microvascular endothelial cells and heart.However,NLRP3 inhibition by MCC950 and caspase-4 inhibition by VX-765 prevent NLRP3 and caspase-4 inflammasome activation and pyroptosis in microvascular endothelial cells and heart induced by myocardial ischemia reperfusion,which protect the myocardial reperfusion-induced microvascular injury and dysfunction.Part 2 The role and mechanism of Beclinl overexpression negatively regulating NLRP3 in myocardial reperfusion-induced microvascular dysfunctionPurpose:To investigate the regulation mechanism of beclinl overexpression on NLRP3 activation induced by myocardial ischemia reperfusion.MethodsIn vivo:WT and beclin1overexpression C57BL/6J adult male were used to establish myocardial ischemia-reperfusion model.Animals were randomly divided into the following groups:WT group,BECNl-Tg group,WT-I/R group,BECNl-Tg-I/R group.TTC and Evan's blue dye double staining methods were used to mensurate the myocardial infarct size after myocardial ischemia reperfusion;immunohistochemistry was used to observe the inflammatory cells infiltration in ischemic area.ELISA was used to observe the levels of IL-1? in serum of mice.Western blot was performed to detect the effect of beclinl overexpression and to observe effect of beclinl overexpression on the expression of NLRP3 and IL-1? in heart;immunofluorescence was used to observe the levels of NLRP3 expression in each group.In vitro:Using cardiac microvascular endothelial cells line establish hypoxic/re-oxygenation model.Pretreatment of CMECs with autophagy inhibitor 3-MA and NLRP3 inhibitor MCC950 before re-oxygenation,subsequently,cells were randomly divided into the following groups:control group,H/R group,H/R+MCC950 group,H/R+3-MA group.Western blot was used to observe the expression of beclinl,LC3,NLRP3 and IL-1? in each group.Construction of beclinl overexpression lentivirus(Len-becn1)and control lentivirus(Len-GFP)were transfected into CMECs.After transfection for 48 h,the transfection effect was observed by fluorescence microscope.Subsequently,cells were randomly divided into the following groups:normoxia+Len-GFP group,normoxia+Len-becn1 group,H/R+Len-GFP group,H/R+Len-becn1 group.Western blot and immunofluorescence were used to observe the expression of NLRP3 in each group.Results:1.TTC and Evan's blue dye double staining showed that the infarct size was higher in WT-I/R group compared with the BECN1-Tg-I/R group(P<0.05,n=5).2.Immunohistochemical staining indicated that the number of F4/80 positive cells in the BECN1-Tg-I/R group was lower than that in the WT-I/R group(P<0.05,n=5).3.ELISA results revealed that the serum levels of IL-1? was increased in I/R group compared with the sham group(P<0.05,n=5).Compared to WT-I/R group,the serum levels of IL-1? was decreased in BECN1-Tg-I/R group(P<0.05,n=5)4.Western blot analysis indicated that the expression of NLRP3 was decreased in BECN1-Tg group(P<0.05,n=3).Compared to WT-I/R group,the levels of NLRP3 and IL-1? were lower in BECN1-Tg group(P<0.05,n=3).The results implied that beclinl might be involved in the regulation of inflammatory response induced by myocardial ischemia-reperfusion.In vitro,compared with the control group,the expression of NLRP3,IL-1?,beclin1,LC3 was increased in H/R group in CMECs(P<0.05,n=3).After pretreatment of CMECs with autophagy inhibitor 3-MA,the expression of autophagy-related proteins beclin1 and LC3? was decreased,while the expression of NLRP3 and IL-1? was further increased(P<0.05,n=3).The results implied that inhibition of autophagy could significantly activate NLRP3 in CMECs.However,after pretreatment of CMECs with NLRP3 inhibitor MCC950,the expression of autophagy-related proteins beclin1 and LC3II was further increased,and the expression of NLRP3 and IL-1(3 was further decreased(P<0.05,n=3).The results implied that inhibition of NLRP3 expression could promote autophagy in CMECs.To further explore the regulation of beclin1 on NRLP3,we observe the changes of NLRP3 expression in Len-GFP group and Len-becn1 group in CMECs.Compared to Len-GFP group,the expression of NLRP3 in CMECs was decreased in Len-becn1 group(P<0.05,n=3).These results indicated that beclin1 overexpression could inhibit the activation of NLRP3 in CMECs.Conclusions:Beclinl overexpression inhibits the inflammatory response induced by myocardial ischemia-reperfusion by regulating the activation of NLRP3,which protects cardiac microvascular endothelium injury during myocardial ischemia reperfusion.Part 3 Beclinl Overexpression inhibits NLRP3 activation by regulating TNFAIP3 in myocardial reperfusion-induced microvascular dysfunctionPurpose:To investigate the effect of TNFAIP3 on NLRP3 activation and the regulation of beclin1 overexpression on TNFAIP3 during myocardial reperfusion-induced microvascular dysfunction.MethodsIn vitro:Using cardiac microvascular endothelial cells line establish hypoxic/reoxygenation model.Pretreatment of CMECs with autophagy inhibitor 3-MA and NLRP3 inhibitor MCC950 before re-oxygenation,subsequently,cells were randomly divided into the following groups:control group,H/R group,H/R+MCC950 group,H/R+3-MA group.Western blot and immunofluorescence were performed to observe the expression of TNFAIP3 and NLRP3 in each group.Construction of beclin1 overexpression lentivirus(Len-becn1)and control lentivirus(Len-GFP)were transfected into CMECs.Subsequently,cells were randomly divided into the following groups:normoxia+Len-GFP group,normoxia+Len-becn1 group,H/R+Len-GFP group,H/R+Len-becn1 group.Western blot and immunofluorescence were used to observe the expression of TNFAIP3 in each group.In vivo:WT and beclin1 overexpression C57BL/6J adult male were used to establish myocardial ischemia-reperfusion model.Animals were randomly divided into the following groups:WT group,BECN1-Tg group,WT-I/R group,BECN1-Tg-I/R group.Western blot was used to observe the expression of TNFAIP3 in each group.Results:1.Western blot analysis showed that the expression of TNFAIP3 in CMECs was decreased in H/R group compared with control group(P<0.05,n=3).After pretreatment of CMECs with NLRP3 inhibitor MCC950,NLRP3 expression was decreased in CMECs,while the expression of TNFAIP3 was increased compared to H/R group(P<0.05,n=3).The results implied that inhibition of NLRP3 could activate TNFAIP3 in CMECs.However,after pretreatment of CMECs with autophagy inhibitor 3-MA,the expression of TNFAIP3 was further reduced,while NLRP3 inflammasome-associated protein expression were increased(P<0.05,n=3).These results suggested that autophagy might be involved in the regulation of TNFAIP3 in CMECs.To further explore the regulation of beclin1 on TNFAIP3,we observe the changes of TNFAIP3 expression in Len-GFP group and Len-becn1 group in CMECs.Compared to Len-GFP group,the expression of TNFAIP3 in CMECs was elevated in Len-becn1 group(P<0.05,n=3).These results indicated that beclin1 overexpression inhibited H/R-induced NLRP3 activation possibly via promoting TNFAIP3 expression in CMECs.In vivo:Western blot analysis showed that the expression of TNFAIP3 was up-regulated while the NLRP3 expression was decreased in BECN1-Tg group compared to WT group(P<0.05,n=3).Compared to WT-I/R group,the TNFAIP3 expression was increased while the expression of NLRP3 was decreased in BECN1-Tg-I/R group(P<0.05,n=3).The results were consistent with in vitro.2.Immunofluorescence results revealed that the expression of TNFAIP3 in CMECs was decreased in H/R group,while the NLRP3 expression was increased compared with control group.Pretreatment of CMECs with NLRP3 inhibitor MCC950,the expression of NLRP3 was decreased in CMECs,while the TNFAIP3 expression was elevated compared to H/R group,However,after pretreatment of CMECs with autophagy inhibitor 3-MA,the TNFAIP3 expression was further reduced,while the expression of NLRP3 was further up-regulated.Conclusions:Myocardial ischemia-reperfusion induces NLRP3 activation by inhibiting TNFAIP3 expression;whereas beclin1 overexpression inhibits NLRP3 activation induced by myocardial reperfusion by promoting TNFAIP3 expression. |
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