| Objective: Astragalosides IV(ASIV)is an effective active substance extracted from Astragalus membranaceus,which has many pharmacological effects such as antiinflammatory,antioxidant and anti-apoptotic.Relevant studies have shown that it has multiple protective effects on cardiovascular diseases,but the specific mechanism is still unclear.This study was based on the cardiomyocyte hypertrophy induced by Angiotensin II(Ang II),to clarify the protective effect of ASIV on cardiomyocyte hypertrophy and explore its related mechanisms.Methods:(1)H9c2 cardiomyocytes were cultured and intervened with different concentrations of Ang II and ASIV.Cell Counting Kit 8(CCK8)was used to detect the viability of cardiomyocytes and determine the optimal intervention concentration and time.(2)The experiment was divided into six groups: control group,ASIV group,Ang Ⅱ group,AngⅡ+ASIV 25 μmol/L group,Ang Ⅱ+ASIV 50 μmol/L group,AngⅡ+ASIV 100 μmol/L group.Laser scanning confocal microscopy was used to observe cell morphology and calculate cell area;reactive oxygen species(ROS)level was detected by ROS detection kit;real-time fluorescence quantitative polymerase chain reaction(RT-qPCR)was used to detect the expression of atrial natriuretic peptide(ANP),β-myosin heavy chain(β-MHC),nuclear factor erythroid 2 related factor 2(Nrf2),heme oxygenase 1(HO-1)mRNA;Western blot(WB)was used to detectthe expression of ANP,Nrf2,HO-1 protein.(3)H9c2 cardiomyocytes were transfected with Nrf2-negative control shRNA(NC)and different sequences Nrf2 shRNA(shRNA-1,shRNA-2,shRNA-3),and the research was divided into control group,NC group,shRNA-1 group,shRNA-2 group,shRNA-3 group.The expression of green fluorescent protein(GFP)was observed under fluorescence microscope to detect transfection efficiency.The expression of Nrf2 mRNA and protein was detected by RT-qPCR and WB respectively.(4)After transfected,the research was divided into eight groups: NC+Control group、NC+AngⅡgroup、NC+ASIV group、NC+AngⅡ+ASIV group、shRNA+Control group、shRNA+AngⅡgroup、shRNA+ASIV group、shRNA+AngⅡ+ASIV group.Laser scanning confocal microscopy was used to observe cell morphology and calculate cell area;ROS level was detected by ROS detection kit;the expression of ANP、β-MHC、Nrf2 and HO-1 mRNA were detected by RT-qPCR;the expression of ANP、Nrf2and HO-1 protein were detected by WB.Results:(1)AngⅡreduced the relative cellular activity of cardiomyocytes depending on concentration and time.Just choosing 1μmol/L and 24 hours as the best intervention concentration and time in the further experiments.ASIV had no significant effect on the relative activity of H9c2 cardiomyocytes(P>0.05).ASIV could inhibit the decrease of cardiomyocytes relative cellular activity induced by Ang IIwith significant difference(P<0.05),and selecting concentration gradient of 25,50,100 μmol/L.(2)The area of Ang II group H9c2 cardiomyocytes was significantly larger and the expression of ANP、β-MHC mRNA and ANP protein was increased than the control group(P<0.05),and ASIV reversed the enlargement of cardiomyocyte induced by AngⅡand reduced the expression of ANP、β-MHC mRNA and ANP protein in a concentration-dependent manner with significant difference(P<0.05).(3)In AngⅡgroup,the lever of ROS increased significantly compared with control group and the expression of Nrf2,HO-1 mRNA and protein decreased(P<0.05),whereas ASIV could reduce the production induced by AngⅡ and up-regulate the expression of Nrf2,HO-1 mRNA and protein(P<0.05).(4)After H9c2 cardiomyocytes were transfected with Nrf2-negative control shRNA(NC)and different sequences Nrf2 shRNA(shRNA-1,shRNA-2,shRNA-3),there was no statistical significance between the NC group and the control group,but the expression of Nrf2 mRNA and protein decreased significantly in shRNA group(P<0.05).(5)The intervention after transfected showed that AngⅡ could enlarge the area of H9c2 cardiomyocytes,up-regulate the expression of ANP、β-MHC mRNA and ANP protein,increase the lever of ROS with significant difference(P<0.05);ASIV could alleviate the enlargement area of H9c2 cardiomyocytes induced by AngⅡ,down-regulate the expression of ANP、β-MHC mRNA and ANP protein,decrease the leverof ROS(P<0.05),however,the protective effect of ASIV was abolished with shRNA transfected.After shRNA transfected,the expression of Nrf2,HO-1 mRNA and protein reduced significantly compared with the H9c2 cardiomyocytes transfected with NC(P<0.05).Conclusion:(1)ASIV inhibits cardiomyocytes hypertrophy induced by AngⅡ.(2)ASIV alleviates the oxidative stress injury in H9c2 cardiomyocytes induced by AngⅡ.(3)ASIV resists cardiomyocytes hypertrophy through inhibiting oxidative stress via activating Nrf2/HO-1signaling pathway. |
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