| Background:At present,the treatment of heart failure has entered the plateau phase,and it is necessary to thoroughly study the pathogenesis of heart failure and find out the corresponding treatment methods.Myocardial mitochondria is the main site of cardiac energy metabolism,whose dysfunction is an important factor leading to cardiac dysfunction and heart failure.Mitochondria are highly dynamic organelles.Continuous biogenesis,fusion,fission and mitophagy,contribute to the balance of mitochondria’s morphology,quantity and quality,which is called mitochondrial quality control.Mitochondrial quality control is the cornerstone of normal mitochondrial function and plays an important role in the pathological process of heart failure.Mitochondrial quality control is a complex,multi-level,multi-faceted,and influential mechanism,which coincides with the multi-link,multi-target,and network-based therapeutic concepts of traditional Chinese medicine.Therefore,the mechanism of traditional Chinese medicine treatment on myocardial mitochondrial dysfunction in heart failure is studied.It may provide new ideas and new directions for the treatment of heart failure.Objective:Using mitochondrial quality control as the starting point,to explore the effect of Xin Fu Kang oral liquid on myocardial mitochondrial biogenesis,mitochondrial dynamics(fusion/fission),and mitophagy in rats with heart failure.To explain the pharmacological mechanism of Xin Fu Kang oral liquid in the treatment of heart failure.Methods:1.A model of heart failure after myocardial infarction was established by ligation of the left anterior descending coronary artery in rats.2.Detection of cardiac function in rats:All rats were examined by echocardiography before and after surgery and after 8 weeks of drug treatment.Left ventricular internal dimension diastole(LVIDd)and left ventricular internal dimension systole(LVIDs)were measured using a color Doppler ultrasound imaging system.Left ventricular ejection fraction(EF)and left ventricular short axis shortening(FS)were calculated.3.Morphological observation of rat heart:HE staining and Masson staining were used to observe the myocardial structure and myocardial fibrosis in rats.The ultrastructure of rat myocardium was observed by transmission electron microscope.4.The cardiac mitochondrial biogenesis related regulatory proteins SIRT1/SIRT3 and PGC-la were detected by qPCR and western blot.5.Western blot was used to detect myocardial mitochondrial dynamics related proteins Mfnl,Mfn2,Opal,Drp1,Fisl and Mff.6.Co-localization of myocardial mitophagy-related proteins parkin,p62,LC3 and mitochondrial inner membrane marker protein COX IV was detected by frozen section-immunofluorescent double-labeling method.The results were analyzed by IPP image analysis software and expressed by Pearson’s correlation.qPCR and Western blot were used to detect myocardial mitophagy-related proteins pink1,parkin,p62 and LC3.Results:1.Effect of Xin Fu Kang oral liquid on cardiac function in rats with heart failureAn animal model of heart failure after myocardial infarction was established.After 8 weeks of drug intervention,heart function was measured in each group.After analyzing the data,the LVIDd and LVIDs in the model group increased compared to the sham group and the difference was statistically significant(p<0.01);both EF and FS decreased,and the difference was statistically significant(p<0.01).Compared with the model group,LVIDs and LVIDs decreased in Captopril group,Xin Fu Kang large-dose(XFK-L)group,Xin Fu Kang middle-dose(XFK-M)group and Xin Fu Kang small-dose(XFK-S)group.Among them,the difference of LVIDs reduction in Captopril group was statistically significant(p<0.05).The other differences were not statistically significant(p>0.05).In addition,compared with the model group,the EF and FS in Captopril group,XFK-L group,XFK-M group and XFK-S group increased.Among them,the difference of EF in Captopril group,XFK-L group,XFK-M group and XFK-S group was statistically significant(p<0.01,p<0.05),and the difference of FS in Captopril group was statistically significant(p<0.05).The other differences were not statistically significant(p>0.05).2.Effect of Xin Fu Kang oral liquid on myocardial pathological structure and myocardial ultrastructure in rats with heart failure(1)The results of HE staining in each group were as follows:In the sham group,the morphological structure of the myocardium was normal and the structure was clear.The muscle fibers were arranged neatly,densely,and uniformly colored.The nucleus was clear and uniform in size,with no inflammatory cell infiltration.In the model group,the range of myocardial infarction was larger,myocardial cells were significantly decreased,and myocardial cells in the infarct margin were hypertrophied.The structure was ambiguous,the muscle fibers were arranged in disorder,and the myofilament were dissolved and broken.Nuclear fragmentation and nuclear dissolution occurred,with a large number of inflammatory cell infiltration.Captopril group,XFK-L group,XFK-M group and XFK-S group:myocardial tissue lesions were alleviated compared with the model group,myocardial cell necrosis,infarct range was reduced,and myocardial cell hypertrophy was reduced.Myocardium fibers were loosely arranged,the size of the nucleus was not uniform,the myofilament was partially broken,and some inflammatory cells infiltrated.(2)The results of Masson staining in each group are as follows:In the sham group,the myocardial tissue was normal,and the myocardial cells were arranged neatly and tightly,with a small amount of collagen fibers scattered in the cell gap.In the model group,a large number of necrotic myocardial cells in the infarct lesion were replaced by collagen fibers.In the infarct margin,the cells are loosely arranged and the cell gap is filled with collagen fibers.Captopril group,XFK-L group,XFK-M group and XFK-S group:Collagen fibers in the infarct lesion and infarct marginal area were significantly reduced compared with the model group.Myocardial cells were arranged more closely and less collagen fiber existed in cell gap.(3)The ultrastructure of myocardium in each group was observed as follows:In the sham group,the myocardial structure was normal,the myocardial fibers were arranged neatly,the transverse stripes were clear,the sarcomeres were intact,the number of mitochondria was enough,the mitochondrion was normal in morphology,the structure was complete,and the mitochondrial membrane structure was clear.The mitochondrial ridges were clear and tightly packed,with no swelling or vacuolization.Autophagosomes were occasionally found.In the model group,the myocardial structure was seriously damaged,and the myocardial fibers were arranged loosely,disordered,irregularly shaped,and fractured.The sarcomere was not complete,the transverse stripes were not clear,the ridges inside the mitochondria were disordered,the membrane structure was destroyed,mitochondria were swollen,the autophagosomes were obvious,vacuolation was evident,and the number of mitochondria was reduced.Captopril group,XFK-L group,XFK-M group and XFK-S group:myocardium fibers were arranged more neatly,sarcomeres were regular,transverse stripes were slightly blurred,mitochondria showed different degrees of swelling and membrane damage,and mitochondrial ridges were more disorderly.Autophagy-related unilateral membrane structures and myeloid bodies were observed around the mitochondria.3.Effect of Xin Fu Kang oral liquid on SIRT1/SIRT3,PGC-1α in rats with heart failureCompared with the sham group,SIRT1 mRNA levels in the model group increased significantly,and SIRT3 and PGC-1α mRNA levels decreased significantly(p<0.05).Compared with the model group,the mRNA levels of SIRT1 in Captopril group,XFK-L group,XFK-M group and XFK-S group had a decrease in levels.Among them,the differences in Captopril group,XFK-L group,XFK-M group and XFK-S group were statistically significant(p<0.05).In addition,compared with the model group,mRNA levels of SIRT3 and PGC-la in Captopril group,XFK-L group,XFK-M group and XFK-S group increased to varying degrees.Among them,the differences in XFK-L group,XFK-M group and XFK-S group were statistically significant(p<0.05).The difference of mRNA level of PGC-la in XFK-M group was statistically significant(p<0.05).The other differences were not statistically significant(p>0.05).Compared with the sham group,the protein expression levels of SIRT1,SIRT3,and PGC-1α in the model group were significantly lower and the differences were statistically significant(p<0.05,p<0.01,p<0.01);Compared with the model group,the protein expression levels of SIRT1,SIRT3,and PGC-1α in Captopril group,XFK-L group,XFK-M group and XFK-S group increased to varying degrees.Among them,there was a statistically significant increase in the expression of SIRT1 in Captopril group(p<0.05).The differences of SIRT3 in XFK-L group,XFK-M group and XFK-S group were statistically significant(p<0.05).The difference of PGC-1α in the XFK-L group was statistical significance(p<0.05),other differences were not statistically significant(p>0.05).4.Effect of Xin Fu Kang oral liquid on myocardial mitochondrial dynamics in heart failure ratsCompared with the sham group,the protein expression levels of Mfnl,Mfn2,and Opal in the model group were significantly lower,and the differences were statistically significant(p<0.01).Compared with the model group,the protein expression levels of Mfn1,Mfn2,and Opal in Captopril group,XFK-L group,XFK-M group and XFK-S group increased to varying degrees.Among them,there was a statistically significant difference in the expression of Mfnl in Captopril group(p<0.05),and there were differences in the protein expression levels of Mfn2 in Captopril group,XFK-L group,XFK-M group and XFK-S group(p<0.05).The differences in protein expression levels of Opal in Captopril group,XFK-M group and XFK-S group were statistically significant(p<0.05),and the other differences were not statistically significant(p>0.05).Compared with the sham group,the protein expression levels of Drp1,Fis1,and Mff in the model group were significantly higher and the differences were statistically significant(p<0.01,p<0.05,p<0.01).Compared with the model group,protein expression levels of Drpl,Fis1,and Mff in Captopril group,XFK-L group,XFK-M group and XFK-S group were decreased to different degrees.Among them,the differences of protein expression levels of Fis1 in XFK-M group and XFK-S group were statistically significant(p<0.05).The difference of protein expression levels of Mff in Captopril group,XFK-L group,XFK-M group and XFK-S group was statistically significant(p<0.05,p<0.01,p<0.01,p<0.05).The other differences were not statistically significant(p>0.05).5.Effect of Xin Fu Kang oral liquid on mitophagy in rats with heart failure(1)Co-localization of myocardial mitophagy-related protein fluorescenceCompared with the sham group,the amount of co-localization of parkin and COX IV in the model group increased significantly,and the differences was statistically significant(p<0.05).Compared with the model group,the amount of co-localization in Captopril group,XFK-L group,XFK-M group and XFK-S group had a decrease,among which,the difference of XFK-L group was statistically significant(p<0.05).There were no statistically significant differences in the other groups(p>0.05).Compared with the sham group,the co-localization of p62 and COX IV in the model group increased significantly,and the difference was statistically significant(p<0.05).Compared with the model group,the amount of co-localization in Captopril group,XFK-L group,XFK-M group and XFK-S group had a tendency to decrease,but the difference was not statistically significant(p>0.05).Compared with the sham group,the co-localization of LC3 and COX IV in the model group increased significantly,and the difference was statistically significant(p<0.05).Compared with the model group,the amount of co-localization in Captopril group,XFK-L group,XFK-M group and XFK-S group had a decreasing trend.Among them,the amount of co-localization in Captopril group was slightly decreased,with no statistically significant difference(p>0.05).The amount of co-localization in XFK-L group,XFK-M group and XFK-S group was significantly lower,even slightly lower than the sham group,and the differences were statistically significant compared with the model group(p<0.05,p<0.05,p<0.01).(2)mRNA and protein expression of pink1,parkin,p62,and LC3 in rat myocardial tissueCompared with the sham group,the mRNA level of pinkl in the model group decreased,and the mRNA levels of parkin and LC3 increased.The differences were statistically significant(p<0.01).The mRNA level of p62 also increased significantly,and the difference was statistically significant(p<0.05).Compared with the model group,the mRNA levels of pink1 in Captopril group,XFK-L group,XFK-M group and XFK-S group increased to different extents,among which the difference in XFK-S group was statistically significant(p<0.05).In addition,compared with the model group,the mRNA levels of parkin,p62,and LC3 in Captopril group,XFK-L group,XFK-M group and XFK-S group decreased to varying degrees.Among them,the mRNA levels of parkin and LC3 in Captopril group decreased statistically significant(p<0.05,p<0.01).The mRNA levels of parkin and LC3 in XFK-M group and XFK-S group decreased statistically significant(p<0.01).The other differences were not statistically significant(p>0.05).Compared with the sham group,the expression levels of pinkl,parkin and p62 in the model group decreased,and the difference was statistically significant(p<0.01).The ratio of LC3II/LC3I was significantly higher and the difference was statistically significant(p<0.5).Compared with the model group,the expression levels of pinkl,parkin,and p62 proteins increased to varying degrees.Among them,the expression levels of parkin and p62 in Captopril group increased,and the differences were statistically significant(p<0.05).The expression levels of parkin and p62 protein in XFK-M group increased significantly,and the differences were statistically significant(p<0.01,p<0.05).In addition,compared with the model group,the LC3Ⅱ/LC3Ⅰ ratios in Captopril group,XFK-L group,XFK-M group and XFK-S group had different degrees of reduction.Among them,the ratio of LC3Ⅱ/LC3Ⅰ decreased significantly in XFK-M group and XFK-S group,and the difference was statistically significant(p<0.05).The other differences were not statistically significant(p>0.05).Conclusion:1.Xin Fu Kang oral liquid and Captopril can reduce LVIDd,LVIDs,increase EF,FS,relieve left ventricular remodeling and promote cardiac pumping function.2.Xin Fu Kang oral liquid and Captopril can reduce myocardial pathological structural damage and myocardial fibrosis in rats with heart failure,and have obvious improvement on myocardial ultrastructure.3.Xin Fu Kang oral liquid and Captopril can significantly upregulate the expression of SIRT1,PGC-1α,and SIRT3,thereby reducing the damage of myocardial mitochondrial biosynthesis in rats with heart failure and playing a role in promoting myocardial energy metabolism.4.Xin Fu Kang oral liquid and Captopril can regulate myocardial mitochondrial dynamics in heart failure rats,specifically by up-regulating mitochondrial fusion proteins Mfn1,Mfn2,and Opal,down-regulating mitochondrial fission proteins Drpl,Fisl and Mff,promoting mitochondrial fusion,inhibiting mitochondrial fission,thus restoring the balance of mitochondrial dynamics.5.Xin Fu Kang oral liquid and Captopril can inhibit excessive mitophagy in rats with heart failure via pinkl/parkin pathway.6.Xin Fu Kang oral liquid and Captopril can affect myocardial mitochondrial quality control in rats with heart failure by regulating mitochondrial biogenesis,mitochondrial dynamics,and mitophagy,so that it can promote myocardial energy metabolism and improve cardiac function. |
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