| Part one:Postn and NLRP3 inflammasome mediated pyroptosis participates in myocardial ischemia/reperfusion injury in ratsObjective To identify the key genes of myocardial ischemia/reperfusion injury in rats by bioinformatics method,and to determine the optimal time point of reperfusion by observe the expression of the genes at 2,6 and 24h after myocardial ischemia/reperfusion injury in rats.To observe myocardial pyroptosis induced by NLRP3 inflammasomes and myocardial tissue injury of myocardial ischemia/reperfusion in rats.Methods(1)The suitable gene expression profiles were selected from GEO database,and the differentially expressed genes were identified by GEO2R online analysis tool.The cross genes were analyzed online by Draw Venn Diagram,and the protein-protein interaction network of the differential genes was constructed online by the interaction gene retrieval tool String.Module and GO analyses were performed using MCODE plug-ins Cytoscape 3.6 and BINGO,respectively.(2)Healthy male SD rats with a body weight of 250～300g were randomly divided into 4 groups(n=4)by random number table method:Sham group,threading without ligation;Ischemia/reperfusion for 2h group(2h group),the left anterior descending coronary artery was lapped for 30min and the perfusion was resumed for 2h;Ischemia/reperfusion for 6h group(6h group),the left anterior descending coronary artery was lapped for 30min and the perfusion was resumed for 6h;Ischemia/reperfusion for 24h group(24h group),the left anterior descending coronary artery was lapped for 30min and the perfusion was resumed for 24h.RT-qPCR was used to detect Postn mRNA expression and Western blot was used to detect Postn protein expression of damaged myocardial tissue.(3)Healthy male SD rats with a body weight of 250-300g were randomly divided into two groups(n=5):Sham group,threading without ligation;Ischemia/reperfusion group(MIRI group),the left anterior descending coronary artery was lapped for 30min and the perfusion was resumed for a best time point.Evans Blue-TTC staining was used to measure the ischemic and infarcted area of heart tissue and HE staining was used to observe the pathological changes of heart tissue.ELISA was used to detect the expression of IL-1β and IL-18 in peripheral blood,and TUNEL staining was used to detect the apoptosis of myocardial tissue.Immunohistochemical staining was used to detect the expression of Postn,NLRP3,Caspase-1,ASC,IL-18 and IL-1β in heart tissue,Western blot and RT-qPCR were also used to detect the expression of them.Results(1)The GSE4105 dataset was screened out from GEO database and divided into three cases.126 overlapping differentially expressed genes were identified through data analysis,and the 126 genes were constructed into protein-protein interaction network.Finally,three key genes were obtained from three important modules,including Sgca,Cd47 and Postn.Postn is the key gene in the most important module.(2)Compared with Sham group,Postn protein and mRNA expression in myocardial tissue of 6h and 24h groups were significantly increased(P<0.05).Compared with 2h group,Postn protein expression in 24h group was significantly increased(P<0.05),the expression of Postn mRNA in 6h group and 24h group were significantly up-regulated(P<0.05).Compared with 6h group,the protein and mRNA expression of Postn in myocardial tissue of 24h group were significantly increased(P<0.05).(3)Compared with Sham group,the percentage of myocardial ischemia risk area(75.7±3.6%VS 2.3±0.6%)and infarct area(42.7±2.1%VS 0%)in MIRI group were significantly increased,the expressions of serum IL-1β and IL-18 in MIRI group were significantly increased,and the apoptosis rate of myocardial cells in MIRI group was also significantly increased.The protein and mRNA expression levels of NLRP3,Caspase-1,ASC,IL-18 andIL-1β in myocardial tissue were also significantly increased in MIRI group(P<0.05).Conclusion Postn is a key gene in myocardial ischemia/reperfusion injury in rats.Postn and NLRP3 inflammasome mediated pyroptosis participates in myocardial ischemia/reperfusion injury in rats.Part two:Postn can activates NLRP3 inflammasome-mediated pyroptosis and exacerbates hypoxia/reoxygenation injury of H9C2 cardiomyocytesObjective We used hypoxia/reoxygenation injury model of H9C2 rat cardiomyocytes to simulate myocardial ischemia/reperfusion injury in vitro.Then we investigated the effects of Postn on NLRP3 inflammasome-mediated pyroptosis and hypoxia/reoxygenation injury of H9C2 cardiomyocytes by knockdown or overexpression of Postn.Methods(1)H9C2 rat cardiomyocytes were divided into 4 groups by random number table method:Blank group,conventional culture.Hypoxia 4h/reoxygenation 4h group[H/R(4/4)group],cells were cultured in sugar-free medium for 4h under the condition of hypoxia,and then cultured in a constant temperature cell incubator containing 5%CO2 at 37℃for 4h after replacing the conventional medium.In the hypoxia 4h/reoxygenation 8h group[H/R(4/8)group],cells were cultured in sugar-free medium for 4h under hypoxia conditions,and then cultured in a constant temperature cell incubator containing 5%CO2 at 37℃ for 8h after replacing the conventional medium.In the hypoxia 4h/reoxygenation 16h group[H/R(4/16)group],cells were cultured in sugar-free medium for 4h under the condition of hypoxia,and then continued to be cultured for 16h in a constant temperature cell incubator containing 5%CO2 after replacing the conventional medium.The activity of cardiomyocytes was detected by CCK-8 assay,the expressions of IL-18 and IL-1β in the medium supernatant were detected by ELISA,and the expressions of Postn and pyroptosis related proteins(NLRP3,ASC,Caspase-1,IL-18,IL-1β)in cardiomyocytes were determined by Western blot.RT-qPCR was used to detect the mRNA expressions of Postn and pyroptosis related molecules in cardiomyocytes.(2)H9C2 rat cardiocytes were divided into 4 groups by random number table method:siNC/NC+Con group,transfected with control siRNA or empty plasmid,and then cultured.H/R group,the cells were hypoxic for 4h and then reoxygenated for 8h at the same time.SiNC/NC+H/R group,after transfection with control siRNA or empty plasmid,the cells were hypoxic for 4h and reoxygenated for 8h.SiPostn/Postn+H/R group,after transfection with Postn siRNA or Postn overexpression plasmid,the cells were hypoxic for 4h and reoxygenated for 8h.The activity of cardiomyocytes was detected by CCK-8,the expressions of IL-18 and IL-1β in the medium supernatant were detected by ELISA,the expressions of pyroptosis-related molecules(NLRP3,ASC,Caspase-1,IL-18,IL-1β)in cardiomyocytes were detected by RT-qPCR and Western blot,and myocardial cell pyroptosis rate was detected by flow cytometry.Results(1)Compared with Blank group,the cell viability of H/R(4/4),H/R(4/8)and H/R(4/16)groups were significantly decreased(P<0.05).Compared with H/R(4/4)group,the cell viability of H/R(4/8)and H/R(4/16)groups were significantly decreased(P<0.05).(2)Compared with Blank group,the levels of IL-18 and IL-1β in H/R(4/4),H/R(4/8)and H/R(4/16)groups were significantly increased(P<0.05).Compared with H/R(4/4)group,the levels of IL-18 and IL-1β in H/R(4/8)group were significantly increased(P<0.05);Compared with H/R(4/8)group,the levels of IL-18 and IL-1β in H/R(4/16)group were significantly decreased(P<0.05).(3)Compared with Blank group,the expression of Postn protein and mRNA in H/R(4/4),H/R(4/8)and H/R(4/16)groups were significantly increased(P<0.05).Compared with H/R(4/4)group,the expression of Postn protein and mRNA in H/R(4/8)and H/R(4/16)groups were also significantly increased(P<0.05).(4)Compared with Blank group,the protein and mRNA expressions of NLRP3,ASC,Caspase-1,IL-18 and IL-1β in H/R(4/4),H/R(4/8)and H/R(4/16)groups were significantly increased(P<0.05).Compared with H/R(4/4)group,the protein and mRNA expressions of NLRP3,ASC,Caspase-1,IL-18 and IL-1β in H/R(4/8)group were significantly increased,and the protein expressions of ASC,Caspase-1,IL-18,IL-1β and the mRNA expressions of NLRP3,Caspase-1,IL-18,IL-1β in H/R(4/16)group were significantly increased(P<0.05).Compared with H/R(4/8)group,the protein expressions of NLRP3,ASC,Caspase-1 and IL18 in H/R(4/16)group were significantly decreased(P<0.05),and the mRNA expressions of NLRP3,ASC,Caspase-1 and IL-1β were significantly decreased(P<0.05).(5)Compared with siNC+Con group,the cell viability of cardiomyocytes in H/R,siNC+H/R and siPostn+H/R groups were significantly decreased,and the levels of IL-18 and IL-1β in the medium superfine were significantly increased(P<0.05).The mRNA and protein expressions of NLRP3,ASC,Caspase-1,IL-18 and IL-1β and the pyroptosis rates in the three groups were significantly increased than siNC+Con group(P<0.05).Compared with the siNC+H/R group,the cell viability of cardiomyocytes in the siPostn+H/R group was significantly increased,the levels of IL-18 and IL-1β in the medium supernatant were significantly decreased,the mRNA and protein expressions of NLRP3,ASC,Caspase-1,IL-18 and IL-1βwere significantly decreased,and the pyroptosis rate was also significantly decreased(P<0.05).(6)Compared with NC+Con group,the cell viability of cardiomyocytes in H/R,NC+H/R and Postn+H/R groups were significantly decreased,the levels of IL-18 and IL-1βin medium supernatant were significantly increased,the mRNA and protein expressions of NLRP3,ASC,Caspase-1,IL-18 and IL-1β were significantly increased,and the pyroptosis rates were significantly increased(P<0.05).Compared with H/R group and NC+H/R group,the cell viability of myocardial cells in Postn+H/R group was significantly decreased,levels of IL-18 and IL-1β in the medium supernatant were significantly increased,the mRNA and protein expressions of NLRP3,ASC,Caspase-1,IL-18 and IL-1β were significantly increased,and the pyroptosis rate of myocardial cells was significantly increased(P<0.05).Conclusion Postn can activates NLRP3 inflammasome-mediated pyroptosis and exacerbates hypoxia/reoxygenation injury of H9C2 cardiomyocytes.Part three:Postn involved in hypoxia/reoxygenation injury of H9C2 cardiomyocytes by regulating NLRP3 inflammasome mediated pyroptosisObjective To investigate whether Postn is involved in hypoxia/reoxygenation injury of H9C2 cardiomyocytes by regulating NLRP3 inflammasome-mediated pyroptosis.Methods H9C2 rat cardiomyocytes were divided into 4 groups by random number table method:siNC+NC or NC group,transfected with control siRNA and empty plasmid or omly transfected with empty plasmid only.H/R group,the cells were hypoxic for 4h and then reoxygenated for 8h at the same time.Postn+H/R group,after transfection with Postn overexpressed plasmid,the cells were hypoxic for 4h and then reoxygenated for 8h.Postn+H/R+siNLRP3 or Postn+H/R+NLRP3 group:After transfection with Postn overexpressed plasmid and NLRP3 siRNA or NLRP3 overexpressed plasmid,the cells were hypoxic for 4h and reoxygenated for 8h.CCK-8 was used to detect the activity of cardiomyocytes,RT-qPCR and Western blot were used to detect the expression of pyroptosis related molecules(NLRP3,ASC,Caspase-1,IL-18,IL-1β)in cardiomyocytes,and flow cytometry was used to detect pyroptosis rate of cardiomyocytes.Results(1)Compared with siNC+NC group,the cell viability of cardiomyocytes in H/R group and Postn+H/R group were significantly decreased,the mRNA and protein expressions of NLRP3,ASC,Caspase-1,IL-18 and IL-1β were significantly increased,and the rate of pyroptosis was significantly increased(P<0.05).Compared with H/R group,the activity of cardiomyocytes of Postn+H/R group was significantly decreased,the mRNA and protein expressions of NLRP3,ASC,Caspase-1,IL-18 and IL-1β were significantly increased,and the rate of pyroptosis was significantly increased(P<0.05).Compared with H/R group,the cell viability of Postn+H/R+siNLRP3 group was significantly increased,the mRNA and protein expressions of NLRP3,ASC,Caspase-1,IL-18 and IL-1β were significantly decreased,and the rate of pyroptosis was ignificantly decreased(P<0.05).Compared with Postn+H/R group,the cell viability of Postn+H/R+siNLRP3 group was significantly increased,the mRNA and protein expressions of NLRP3,ASC,Caspase-1,IL18 and IL-1β were significantly decreased,and the pyroptosis rate was significantly decreased(P<0.05).(2)Compared with NC group,the cell viability of cardiomyocytes in H/R group,Postn+H/R group and Postn+H/R+NLRP3 group were significantly decreased,the mRNA and protein expressions of NLRP3,ASC,caspase-1,IL-18 and IL-1β were significantly increased,and the pyroptosis rate was significantly increased(P<0.05).Compared with H/R group,the activity of myocytes in Postn+H/R group and Postn+H/R+NLRP3 group were significantly decreased,the mRNA and protein expressions of NLRP3,ASC,caspase-1,IL-18 and IL-1β were significantly increased,and the rate of pyroptosis was significantly increased(P<0.05).Compared with Postn+H/R group,the activity of myocytes in Postn+H/R+NLRP3 group was significantly decreased,the mRNA and protein expressions of NLRP3,ASC,caspase-1,IL-18 and IL-1β were significantly increased,and the rate of pyroptosis was significantly increased(P<0.05).Conclusion Postn involved in hypoxia/reoxygenation injury of H9C2 cardiomyocytes by regulating NLRP3 inflammasome-mediated pyroptosis. |
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