| Objective:1.To establish the pressure overload heart failure(HF)to explore the possible mechanism of miR-214/221,endoplasmic reticulum stress and autophagy.2.To evaluate the effect of benefiting qi,warming yang,activating blood and inducing diuresis on prevention and treatment.of HF,and to explain its mechanism of regulating miR-214/221-endoplasmic reticulum stress-autophagy to prevent and treat HF.Methods:1.miR-214/221-endoplasmic reticulum stress-autophagy axis in HFC57BL/6J wild-type mice were randomly divided into six groups:sham operation group(Sham group),heart failure model group(HF group),model+inhibiting miR-221 group(HF+anti-miR221 group),model+inhibiting miR-214 group(HF+anti-miR214 group),model+4-PBA group(HF+4-PBA group),model+3-MA group(HF+3-MA group).Inhibition of miR-214/221 was followed by tail vein injection of lentiviral miR-221,miR-214.The tail vein was injected with lentiviral miR-214 and miR-221 to transfect cardiomyocvtes to inhibit miR-214 and miR-221.Continuous gastric perfusion for 4 weeks,including 3-MA(15 mg/kg/day)and 4-PBA(100 mg/kg/day)to inhibit autophagy and endoplasmic reticulum stress,respectively.The other groups were given the same volume of pure Water.The relevant indicators were tested:(1)Changes in miR-214/221,endoplasmic reticulum stress,and autophagy during HFDetection of expression levels of miR-214/221 in each group by qPCR.The homogenate of each group was homogenized,and the expression levels of endoplasmic reticulum stress-related protein XBP1,XBP1s,autophagy-related proteins LC3B ?/?,Beclin-1,BNIP3 and LAMP1 were detected by western blotting(WB).The expression level of the endoplasmic reticulum stress protein GRP78 and the positive expression of autophagy-related protein LC3B were detected by immunohistochemical staining;the changes of autophagosomes were observed by transmission electron microscopy.(2)Inhibition of endoplasmic reticulum stress on autophagy and cardiac function in HFCardiac function was detected by echocardiography.The myocardial hypertrophy related index:HE staining of heart sections were evaluated.The fibrotic lesions were visualized by MASSON staining of heart tissue.Quantitative analysis of apoptotic myocaedium using TUNEL staining.The homogenate of each group was homogenized,and the expression levels of endoplasmic reticulum stress-related protein XBP1,XBP1s,autophagy-related proteins LC3B ?/?,Beclin-1,BNIP3 and LAMP1 were detected by WB.The expression level of the positive expression of autophagy-related protein LC3B were detected by immunohistochemical staining;the changes of autophagosomes were observed by transmission electron microscopy.(3)Inhibition of miR-214 and inhibition of miR-221 on endoplasmic reticulum stress-autophagy axis and cardiac function in HF,respectively.Detection of expression levels of miR-214/221 in each group by qPCR.Cardiac function was detected by echocardiography.The myocardial hypertrophy related index:HE staining of heart sections were evaluated.The fibrotic lesions were visualized by MASSON staining of heart tissue.Quantitative analysis of apoptotic myocaedium using TUNEL staining.The homogenate of each group was homogenized,and the expression levels of endoplasmic reticulum stress-related protein XBP1,XBP1s,autophagy-related proteins LC3B ?/?,Beclin-1,BNIP3 and LAMP1 were detected by WB.The expression level of the positive expression of autophagy-related protein LC3B were detected by immunohistochemical staining;the changes of autophagosomes were observed by transmission electron microscopy.2.The effect of benefiting qi,warming yang,activating blood and inducing diuresis method on miR-214/221-endoplasmic reticulum stress-autophagy axis in HFC57BL/6J mice were randomly divided into six groups:sham group,model(HF)group,Xinyang tablets low dose(340 mg/kg)group,Xinyang tablets medium dose(680 mg/kg),Xinyang tablets high-dose(1360mg/kg)group and Perindopril group.Each group was administered by continuous int.ragastric administration for 6 weeks.The relevant indicators were detected after 6 weeks.(1)The effect of benefiting qi,warming yang,activating blood and inducing diuresis method on heart function,myocardial cell hypertrophy and fibrosis and cardiomyocyte apoptosis of HF.Using cardiac ultrasound to detect cardiac function in mice,evaluate the cardiac function of each group of mice.The myocardial cross-sectional area was observed by HE staining,and the myocardial hypertrophy indexes of each group were evaluated.The fibrotic lesions were displayed by MASSON staining,and the levels of interstitial fibrosis were evaluated.Apoptotic cardiomyocytes were detected by TUNEL staining.(2)Exploring the mechanism of benefiting qi,warming yang,activating blood and inducing diuresis method in treating heart failureDetection of expression levels of miR-214/221 in each group by qPCR.The homogenate of each group was homogenized,and the expression levels of endoplasmic reticulum stress-related protein XBP1,XBP1s,autophagy-related proteins LC3B ?/?,Beclin-1,BNIP3 and LAMP1 were detected by western blotting.Result:1.MiR-214/221-endoplasmic reticulum stress-autophagy axis in heart failure(1)Changes in miR-214/221,endoplasmic reticulum stress,and autophagy during HFCompared with Sham group,LVEF and LVFS of HF were significantly decreased,LVIDd,LVIDs,LVEDV,LVESV,LVmass were significantly increased(P?0.05),and the relative area of single cardiomyocytes in HF group was significantly increased.The relative area of myocardial fibrosis increased significantly(P?0.05),and the degree of cardiomyocyte apoptosis in the HF group increased significantly.Compared with Sham group,the expression of endoplasmic reticulum stress-related protein,autophagy-related protein and miR-214/221 expression in HF group were significantly increased(P<0.05).(2)Endoplasmic reticulum stress-autophagy axis in HFCardiac color ultrasonography showed a significant improvement in cardiac function in the HF+4-PBA group and the HF+3-MA group compared with the HF group(P?0.05).HE staining showed that the relative area of single cardiomyocytes in HF+4-PBA group and HF+3-MA group was significantly decreased(P?0.05).The results of MASSON staining showed that compared with the HF group,the relative myocardial fibrosis area was significantly lower in the HF+4-PBA group and the HF+3-MA group(P?0.05).TUNEL staining showed that the apoptotic cardiomyocytes of the HF+4-PBA group and the HF+3-MA group were reduced compared with the HF group.WB results showed that,compared with HF group,the expression of autophagy-related proteins LC3B,Beclinl,BNIP3 and LAMP1 in HF+4-PBA group was significantly decreased(P?0.05).Immunohistochemical staining showed that the LC3B positive expression region of the HF+4-PBA group was also reduced compared with the HF group.Electron microscopy results also showed that inhibition of endoplasmic reticulum stress reduced the number of autophagosomes compared to the HF group.(3)Effect of miR-214/221 on endoplasmic reticulum stress-autophagy axis,cardiac function and ventricular remodeling in HFCardiac color ultrasonography,HE staining,MASSON staining and TUNEL staining showed that compared with HF group,cardiac function,myocardial cell hypertrophy and myocardial fibrosis were significantly improved in HF+anti-miR214 group and HF+anti-miR-221 group(P?0.05).),apoptotic cardiomyocytes are reduced.The WB results showed that compared with the HF group,the endoplasmic reticulum stress-related proteins XBP1 and XBP1S in the HF+anti-miR214 group and HF+anti-miR-221 group were significantly decreased,and autophagy-related proteins LC3B,Beclinl,BNIP3,and LAMP1 were significantly decreased.Significantly decreased expression(P<0.05).Immunohistochemical staining showed that compared with the HF group,the endoplasmic reticulum stress-related protein GRP78 and autophagy-related protein LC3B positive expression in the HF+anti-miR214 group and HF+anti-miR-221 group were also decreased.Electron microscopy results also showed that the number of autophagosomes in the HF+anti-miR214 group and the HF+anti-miR-221 group was reduced compared with the HF group.(3)Compared with HF model group,the relative area of single cardiomyocytes were significantly reduced in HF+anti-miR221 group,HF+anti-miR214 group,HF+4-PBA group and HF+3-MA group in HE staining,(P?0.05).MASSON staining showed that,compared with the model group,the relative myocardial fibrosis area was significantly lower in the HF+anti-miR221 group,HF+anti-miR214 group,HF+4-PBA group and HF+3-MA group(P?0.05).2.The regulation effect of benefiting qi,warming yang,activating blood and inducing diuresis method on miR-214/221-endoplasmic reticulum stress-autophagy axis in HF.The results showed that compared with the HF group,heart function,cardiomyocyte hypertrophy,myocardial fibrosis,and cardiomyocyte apoptosis were improved in the low,middle,and high dose groups of Xinyang tablets,and the perindopril group(P<0.05).The low,medium and high doses of Xinyang tablets and perindopril group can effectively inhibit the expression of endoplasmic reticulum stress-related proteins,and also effectively inhibit the expression of autophagy-related proteins and decrease the expression of miR-214 and miR221(P?0.05).Conclusion:1.MiR-214/221-endoplasmic reticulum stress-autophagy axis plays an important role in HF,inhibiting miR-214/221-endoplasmic reticulum stress-autophagy axis can improve heart function and ventricular weight of HF Structure.2.Benefiting qi,warming yang,activating blood and inducing diuresis method can effectively prevent HF caused by pressure load by inhibiting miR-214/221 and endoplasmic reticulum stress-mediated excessive autophagy. |
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