| Objective:Myocardial samples from patients with clinical atrial fibrillation are used to detect necroptosis in myocardial fibrotic tissues in atrial fibrillation.Mouse primary cardiac fibroblasts(CFs)are extracted and treated with RIP1 and RIP3 small interfering RNA and overexpression plasmids to investigate the mechanism of necroptosis in CFs regulated by RIP1 and RIP3.To observe the therapeutic effects of ginsenoside Rgl on myocardial fibrosis and necroptosis in myocardial tissue using a mouse model of myocardial fibrosis.Finally,the inhibitory effect of ginsenoside Rgl on necroptosis of CFs through the regulation of RIP1 and RIP3 is investigated.Methods:1.Detection of fibrosis and necroptosis in myocardial tissues of patients with atrial fibrillation:myocardial tissues of clinical patients were examined for fibrosis by HE,Masson and Sirius red staining;central myocardial fibrosis-related proteins COL1,αSMA and necroptosis-related proteins RIP1,RIP3,MLKL and RIP3 were also detected using qRT-PCR,WB and immunohistochemistry.RIP3,MLKL,and P-MLKL levels.Necroptosis in myocardial fibrotic tissue in atrial fibrillation was observed by comparing patients with clinical atrial fibrillation(AF group)and patients with sinus rhythm(SR group).2.Role of RIP1 and RIP3 in necroptosis of CFs:primary CFs in mice were extracted(Control group)and utilized with necroptosis inducers to construct a necroptosis model of CFs(Model group);then small interfering RNAs of RIP1 and RIP3 were used to silence the expression of RIP1 and RIP3 in cells(RIP1-siRNA group and RIP3-siRNA group),necroptosis in CFs was detected by flow assay,and COL1,α-SMA,RIP1,RIP3,MLKL,P-MLKL mRNA and protein expression were detected using qRT-PCR,WB,and immunofluorescence,and the levels of IL-1β and IL-6 in the cell supernatant were measured by ELISA,to initially explore CFs necroptosis;RIP1 and RIP3 were overexpressed in CFs(RIP 1-Vector group and RIP3-Vector group)by RIP1 and RIP3 overexpression plasmids,and COL1,α-SMA,RIP1,RIP3,MLKL,P-MLKL were detected by qRT-PCR and WB mRNA and protein expression to explore the regulatory effects of RIP1 and RIP3 on necroptosis in CFs;silencing RIP3 while overexpressing RIP1 in CFs cells(RIP1-Vector+RIP3-siRNA group)and silencing RIP1 while overexpressing RIP3(RIP3-Vector+RIP1-siRNA group).The above indexes were continued to be tested to further investigate the specific regulatory mechanism of necroptosis of CFs by RIP1 and RIP3.3.The effect of ginsenoside Rgl on necroptosis in myocardial tissue in ISO-induced myocardial fibrosis model in mice:Isoproterenol(ISO)was used to construct a mouse myocardial fibrosis model(Model group),and ginsenoside Rg1(Rg1 group)was given to intervene and compare with the control group(Control group)for myocardial tissue HE,Masson and Sirius red staining was performed to assess myocardial tissue fibrosis;the levels of serum inflammatory factors IL-1β and IL-6 were measured;the central myocardial fibrosis-related proteins COL1,α-SMA and necroptosis-related proteins RIP1,RIP3,MLKL,P-were detected in mouse myocardial tissue using qRT-PCR,WB and immunohistochemistry.MLKL.mRNA and protein expressions of The therapeutic effect of Rgl on myocardial fibrosis and the inhibitory effect of programmed still were clarified by comparing with the intervention effect of ginsenoside Rg1.4.The effects of ginsenoside Rgl on necroptosis of CFs by RIP1 and RIP3:primary mouse cells were extracted(Control group),necroptosis inducer induced necroptosis of CFs(Model group),and ginsenoside Rg1 was given to intervene and necroptosis of CFs was detected by flow-through,using qRT-PCR,WB,immunofluorescence COL 1,α-SMA,RIP1,RIP3,MLKL,P-MLKL mRNA and protein expression,and the levels of IL-1β and IL-6 in the cell supernatant by ELISA,to initially investigate the inhibitory effect of ginsenoside Rgl on necroptosis in CFs;overexpression of RIP1 and RIP3 in CFs cells(RIP1-Vector group and RIP3-Vector group),followed by the administration of ginsenoside Rg1 intervention to two groups of cells(RIP1-Vector+Rg1 group and RIP1-Vector+Rg1 group),respectively.qRT-PCR and WB were used to detect COL1,α-SMA,RIP1,RIP3,MLKL,P-MLKL mRNA and protein expression to clarify the regulatory role of Rgl on RIP1 and RIP3 in necroptosis of CFs.Results:1.Detection of fibrosis and necroptosis in myocardial tissues of patients with atrial fibrillation:compared with the SR group,pathological indices of HE,Masson and Sirius red staining were significantly higher in the AF group(P<0.05);the in mRNA of COL1,α-SMA,RIP1,RIP3,MLKL,P-MLKL were significantly higher(P<0.05),and the in mRNA of COL 1,α-SMA,RIP1,RIP3,MLKL,P-MLKL protein expression were significantly elevated in WB and immunohistochemical results(P<0.05).2.Role of RIP1 and RIP3 in necroptosis of CFs:compared with the Control group,the necroptosis rate of cells in the Model group was increased(P<0.05),and the mRNA and protein levels of COL1,α-SMA,RIP1,RIP3,MLKL,and P-MLKL in cells were significantly increased(P<0.05),and immunofluorescence showed the same trend(P<0.05),and the levels of cell supernatant IL-1β and IL-6 were significantly increased(P<0.05);compared with the Model group,there was no significant change in the necroptosis rate in the RIP1-siRNA group,and the mRNA and protein levels of RIP1 were significantly decreased(P<0.05),and immunofluorescence showed the same trend(P<0.05),and COL1,α-SMA,RIP3,MLKL,and P-MLKL showed no significant changes in mRNA and protein levels(P>0.05),and the levels of cellular supernatant IL-1β and IL-6 also showed no significant changes(P>0.05);compared with the Model group,the cellular COL1,α-SMA,RIP3,MLKL,and P-MLKL in the RIP3-siRNA group mRNA and protein levels were significantly lower(P<0.05),and immunohistochemistry showed the same trend(P<0.05),but the above results were not significantly changed for RIP1(P>0.05),and the levels of cell supernatant IL-1βand IL-6 were also significantly lower(P<0.05);compared with the Control group,in the RIP 1-Vector group,the levels of cellular COL1,mRNA and protein levels of αSMA,RIP3,MLKL,and P-MLKL were significantly higher in the RIP3-Vector group compared with the Control group(P<0.05),and the mRNA and protein levels of RIP 1 There was no significant change in the above results(P>0.05);compared to the Control group,the mRNA and protein levels of RIP1 were significantly elevated in the RIP1Vector+RIP3-siRNA group(P<0.05),and the mRNA and protein levels of COL1,αSMA,RIP3,MLKL,P-MLKL were not significantly changed(P>0.05);the mRNA and protein levels of COL1,α-SMA,RIP3,MLKL,P-MLKL were significantly higher in the RIP3-Vector+RIP1-siRNA group compared to the Control group(P<0.05),and there was no significant change in the above results for RIP1(P>0.05).3.The effect of ginsenoside Rgl on necroptosis in myocardial tissue in ISO-induced myocardial fibrosis model in mice:compared with the Control group,the levels of serum IL-1β and IL-6 were significantly elevated in the Model group mice(P<0.05),and the mRNA and The levels of serum IL-1β and IL-6 were significantly increased in the Rg1 group compared with the Model group(P<0.05),and the mRNA and protein levels of COL1,α-SMA,RIP 1,RIP3,MLKL,and P-MLKL in myocardial tissues were all significantly increased(P<0.05),and immunohistochemistry showed the same trend(P<0.05).were significantly reduced(P<0.05),and immunohistochemistry showed the same trend(P<0.05).4.The effects of ginsenoside Rgl on necroptosis of CFs by RIP1 and RIP3:compared with the Control group,the necroptosis rate of cells in the Model group was increased(P<0.05),and the mRNA and protein levels of COL1,α-SMA,RIP1,RIP3,MLKL,and P-MLKL in cells were significantly increased(P<0.05).Immunofluorescence showed the same trend(P<0.05),and the levels of cell supernatant IL-1β and IL-6 were significantly increased(P<0.05);compared with the Model group,the necroptosis rate of cells in the Rg1 group was reduced(P<0.05),and the mRNA and protein levels of COL 1,α-SMA,RIP 1,RIP3,MLKL,and P-MLKL in cells were significantly decreased(P<0.05),immunofluorescence showed the same trend(P<0.05),and the levels of cell supernatant IL-1β and IL-6 were significantly increased(P<0.05);compared with the Control group,in the RIP 1-Vector group compared with the Control group,the levels of cellular COL1,α-SMA,RIP3,MLKL,and P-MLKL mRNA and protein levels were significantly increased in the RIP3-Vector group compared with the Control group(P<0.05),and the above results were not significantly changed for RIP1(P>0.05);compared with RIP 1-Vector,the mRNA and protein levels of COL1,α-SMA,RIP1,RIP3,MLKL,and P-MLKL were significantly lower in the RIP1-Vector+Rg1 group(P<0.05);compared with RIP3-Vector,the mRNA and protein levels of The mRNA and protein levels of COL1,α-SMA,RIP3,MLKL,and P-MLKL were significantly lower(P<0.05),and there was no significant change in the above results for RIP1(P>0.05).Conclusion:1.Necroptosis of cardiac fibroblasts leads to myocardial fibrosis in atrial fibrillation.2.RIP3 can induce necroptosis of cardiac fibroblasts independently of RIP1,but RIP1induced necroptosis of cardiac fibroblasts requires RIP3 as a downstream.3.Ginsenoside Rgl attenuates ISO-induced myocardial fibrosis and attenuates necroptosis of myocardial tissue during fibrosis.4.Ginsenoside Rg1 could attenuate necroptosis of cardiac fibroblasts and further attenuate myocardial fibrosis by inhibiting the expression of RIP1 and RIP3. |
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