Yiqi Huoxue Recipe Inhibits The Hypertrophy Of Cardiomyocytes Induced By Angiotensin Ⅱ Through Sigma-1 Receptor

Myocardial hypertrophy is an important factor in initiating the pathological process of heart failure and promoting the occurrence and development of heart failure.Sigma-1 receptor(Sig-1R)concentrated on mitochondrial-associated endoplasmic reticulum has anti-hypertrophic and cardioprotective effects,and can alleviate myocardial hypertrophy and dysfunction.On the one hand,Sig-1R can regulate intracellular Ca2 concentration and inhibit intracellular Ca2+ overload through inositol 1,4,5-triphate receptor type 2(IP3R2)and ryanodine receptor type 2(RyR2).On the other hand,intracellular Ca2+ transport from sarcoplasmic reticulum to mitochondria can help mitochondrial ATP production and prevent cardiomyocyte hypertrophy and apoptosis.Therefore,Sig-1R plays an important role in cardiomyocyte hypertrophy and apoptosis,which is worth exploring and studying.The method of invigorating Qi and activating blood circulation,which is an important way to treat heart failure in traditional Chinese medicine,can improve heart failure by improving energy metabolism,reducing apoptotic rate of cardiomyocytes,inhibiting cardiomyocyte hypertrophy and delaying ventricular remodeling.In view of the important role of Sig-1R in cardiovascular system and the mechanism of Yiqi Huoxue Recipe in the treatment of heart failure,we hypothesize that Yiqi Huoxue Recipe can regulate Ca2+ transport,maintain Ca2+homeostasis,improve mitochondrial energy metabolism and ATP production,and inhibit the occurrence of cardiomyocyte hypertrophy and apoptosis by regulating Sig-1R.Objective:1.In cultured H9c2 cells,the mechanism of Sig-1R in cardiomyocyte hypertrophy was studied by small interfering ribonucleic acid(siRNA).2.To explore the mechanism of Yiqi Huoxue Recipe in inhibiting cardiomyocyte hypertrophy and apoptosis,and whether Sig-1R regulates Ca2+ transport,improves mitochondrial energy metabolism and cell apoptosis.Method:1.In cultured H9c2 cells,angiotensin Ⅱ(Ang Ⅱ)was used to stimulate cardiomyocyte hypertrophy.Cell area was photographed and measured by inverted microscope with ghostpen-ring peptide staining.The effects of different concentrations of Ang Ⅱ on cardiomyocyte hypertrophy were observed.2.In cultured H9c2 cells,Sig-1R siRNA was transfected by liposome,and the expression of Sig-IR was inhibited at the level of mRNA.At the same time,Yiqi Huoxue Decoction was used to intervene.Cells were divided into control group[Negative control-siRNA(NC siRNA)];model group(Ang II 10-7mol/L+NC siRNA);Yiqi Huoxue recipe group(Ang Ⅱ 10-7mo;/L+Yiqi Huoxue recipe 0.1g/L+NC siRNA);Yiqi Huoxue recipe + Sig-1R group(Ang Ⅱ 10-7mol/L+Yiqi Huoxue recipe 0.1g+Sig-1R gene silencing);Sig-1R group(Sig-1R gene silencing).3.The expression of Sig-1R and IP3R2 was detected by Western Blot to determine the role of Sig-1R in myocardial hypertrophy and the effect of Yiqi Huoxue Recipe on the expression of Sig-1R.The hypertrophy and apoptosis of myocardial cells were determined by ghostpen cyclic peptide staining and Tunel staining.The intracellular Ca2+ level and mitochondrial membrane potential were detected by Western Blot.The results of ATP showed that Yiqi Huoxue Recipe could regulate Cad+and protect the structure and function of mitochondria.Result:1.Ang Ⅱ at different concentrations stimulated H9c2 cells for 48 hours.With the increase of Ang Ⅱ concentration,the area of H9c2 cells increased.When Ang Ⅱ concentration was 10-7 Hol/L,the area of H9c2 cells increased significantly compared with the control group(P<0.05);when Ang Ⅱ concentration was 10-6mol/L,the area of H9c2 cells began to decrease;when Ang Ⅱ concentration was 10-5mol/L,the area of H9c2 cells was lower than that of the control group(P>0.05).2.Compared with the control group,the expression of Sig-1R in cardiac myocytes of the model group tended to decrease,but there was no significant difference between the two groups.Compared with the model group,the expression of Sig-1R in the Yiqi Huoxue Formula group tended to increase,with no statistical difference.Compared with the control group,the expression of Sig-1R in the Yiqi Huoxue Formula+Sig-1R siRNA group and Sig-IR siRNA group decreased significantly(P<0.01,P<0.001).Compared with Sig-1R siRNA group,the expression of Sig-1R mRNA in Yiqi Huoxue Fang+Sig-1R siRNA group tended to increase,but there was no statistical difference.Compared with Yiqi Huoxue Fang+Sig-1R siRNA group,the expression of Sig-1R mRNA in Yiqi Huoxue Fang+Sig-1R siRNA group decreased significantly(P<0.001).Compared with the control group,the protein expression level of Sig-1R in the model group was significantly lower(P<0.01);compared with the model group,the protein expression level of Sig-1R in the Yiqi Huoxue recipe group was higher(P<0.01);the protein expression level of Sig-1R in the Yiqi Huoxue recipe+Sig-1R siRNA group was significantly lower than that in the Yiqi Huoxue recipe group(P<0.001);the protein expression level of Sig-1R in the Yiqi Huoxue recipe+sig-1R siRNA group and sig-1R siRNA group was significantly lower(P<0.001);Compared with the control group,the expression level of Sig-1R protein was significantly lower in both groups(P<0.001).The expression of calcium channel protein IP3R2 increased after Ang Ⅱinduction,and was significantly inhibited by Yiqi Huoxue Decoction(P<0.01).3.Compared with the control group,the cell area of the model group increased significantly(P<0.01),and decreased significantly(P<0.01)after the intervention of Yiqi Huoxue Formula.After the inhibition of Sig-1R expression by Sig-1R siRNA,the cell area of the model group decreased,but there was no statistical difference.Compared with the control group,the apoptotic rate of model group was significantly higher(P<0.01),and the apoptotic rate of Sig-IR siRNA group was more than that of control group(P<0.05),but the apoptotic rate was less than that of model group(P>0.05).Compared with model group,the apoptotic rate of model group decreased significantly(P<0.01).After Sig-1R siRNA transfection inhibited the expression of Sig-1R,Yiqi Huoxue Decoction decreased significantly(P<0.01).The effect of Xuefang on apoptosis was reduced,but there was still significant difference compared with model group(P<0.05).4.The content of Ca2+ in the cytoplasm of model group increased significantly P<0.01);the content of Ca2+ in the cytoplasm of model group decreased(P<0.05);the content of Ca2+ in the cytoplasm of Sig-1R siRNA group also increased,but the difference was not significant.After Ang II stimulation,the level of mitochondrial depolarization decreased significantly compared with the control group(P<0.05).After adding Yiqi Huoxue recipe,the level of mitochondrial depolarization improved,but there was no significant difference.After adding Sig-1R siRNA intervention,the level of mitochondrial depolarization decreased significantly compared with the control group P<0.05),but compared with Yiqi Huoxue recipe,the level of mitochondrial depolarization did not change significantly.In addition,compared with the control group,ATP content in model group and Sig-1R siRNA group decreased(P<0.05);after giving Yiqi Huoxue recipe,ATP content increased(P<0.05),and after increasing Sig-1R siRNA intervention,ATP content in cells decreased(P=0.09>0.05).Conclusion:1.Ang Ⅱ 10-7mol/L stimulates H9c2 cells for 48h,which can lead to cardiomyocyte hypertrophy.2.Ang Ⅱ induced H9c2 cells hypertrophy,which was related to Sig-1R.Yiqi Huoxue Recipe inhibited Ang Ⅱ induced H9c2 cells hypertrophy through Sig-1R.3.Yiqi Huoxue Decoction can inhibit the apoptosis of cardiomyocyte induced by Ang Ⅱ,and its mechanism is related to Sig-1R and its signaling pathway.At the same time,the decrease of Sig-1R level can induce cardiomyocyte apoptosis.4.Ang Ⅱ has obvious mitochondrial damage in the process of stimulating cardiomyocyte hypertrophy.Yiqi Huoxue Fang has certain protective effect on mitochondria,and its mechanism is related to Sig-1R.