| Purpose:In this study,rats with high-fat myocardial ischemia-reperfusion injury(MIRI)were used as the research object to investigate the effects of Yimai Granule on mitochondrial autophagy and mitochondrial fusion and fission.From the perspective of mitochondrial autophagy and mitochondrial dynamics,the effect and molecular mechanism of Yimai Granule in preventing and treating MIRI were revealed.Material and method:100 rats were randomly divided into 5 groups including the sham operation group,the model group,the high,medium and low dose Yimai Granule group,with 20 rats in each group.The sham-operated rats were conventional fed,and the other groups were fed with high-fat diet for 2 weeks.The other conditions were the same.Rats in the sham operation group and the model group were given normal saline 10 ml/kg each time.The high,medium and low dose groups of Yimai Granule were given 10 ml/kg Chinese herbal medicine decoction each time,every group was administered twice a day by gavage.The administration was continued for 1 week before ischemia-reperfusion model establishment.After myocardial ischemia in each group for 30 minutes and reperfusion for 2 hours,the left anterior descending coronary artery was ligated in situ,and 2 ml Evans blue was rapidly perfused through the carotid artery.After the dye was distributed to the whole heart tissue(except for the anterior descending perfusion area),the heart was quickly removed,washed with saline,frozen at-20℃,and then the myocardial infarct size was measured.This study was divided into three parts to investigate the preventive effect and molecular mechanism of Yimai Granule on myocardial injury in rats with high-fat combined with MIRI.1.Effect of Yimai Granule on e NOS and NO in rats with high-fat combined with MIRI The levels of serum lipid(TC,TG,LDL-C,HDL-C)in rats were detected by automatic biochemical analyzer.The myocardial infarct size of rats after ischemia-reperfusion was determined by TTC staining.The morphology and inflammatory infiltration of myocardial cells in each group were observed by HE staining.The serum levels of CK,CK-MB,LDH and HBD in each group were detected by colorimetry.The serum NO of each group was determined by nitrate reductase chemical colorimetry.The content of e NOS protein in myocardial tissue was detected by Western blot.2.Effect of Yimai Granule Regulating PINK1-Parkin Pathway on Myocardial Mitochondrial Autophagy in Rats with high-fat and MIRI The mitochondria of myocardial tissue was extracted from each group.The mitochondrial membrane potential was determined by JC-1 method.Western blot was used to detect the expression levels of Cytc and autophagy marker proteins LC3II/I and Beclin1,and the relative expression levels of mitochondrial autophagy-related proteins PINK1,Parkin and P62.3.Effect of Yimai Granule on myocardial mitochondrial fusion and fission-associated protein in rats with high-fat combined with MIRI Western blot analysis was used to detect the relative expression levels of mitochondrial fusion proteins(OPA1,Mfn1,Mfn2 proteins),mitochondrial fission proteins(Drp1,Fis1 protein)and mitochondrial biosynthesis-related proteins(PGC-1α protein)in rats cardiomyocytes.Results: 1.Compared with the sham-operated rats,the serum levels of TC,TG,and LDL-C in the model group were significantly increased(p<0.01),and the HDL-C levels were significantly decreased(p<0.01).The serum levels of TC,TG and LDL-C in the high-dose group were significantly lower(p<0.01),and the HDL-C level was significantly increased(p<0.01).The serum levels of TG and LDL-C in medium-dose group were significantly decreased(p<0.01)or(p<0.05),and HDL-C levels were significantly increased(p<0.01)or(p<0.05).The serum TG was significantly decreased(p<0.01)and HDL-C(p<0.05)level was significantly increased of Yimai Granule in low dose group.Compared with the high-dose group of Yimai Granule,the serum levels of TC and LDL-C in the medium and low dose groups of Yimai Granule were significantly increased(p<0.01)or(p<0.05),serum HDL-C level in the low-dose group was significantly decreased(p<0.01)or(p<0.05),and the high dose of Yimai Granule was better than the medium and low doses.2.There was no infarction in the sham operation group.Compared with the sham-operated group,the percentage of myocardial infarct size in the model group was significantly increased,and the infarct area accounted for 17.89% of the total area of the left ventricle.After high,medium and low dose intervention of Yimai Granule,the percentage of myocardial infarct size in rats was significantly lower than that in sham operation group(p<0.01).The effect of Yimai Granule in the high and medium dose groups was significantly better than that in the low-dose group.3.Compared with the sham-operated rats,the serum levels of HBDH,CK-MB,CK and LDH in the model group rats were significantly increased(p<0.01).After Yimai Granule intervention,the serum levels of HBDH,CK-MB,CK and LDH in the high,medium and low dose group were significantly decreased(p<0.01);Compared with the high dose group of Yimai Granule,the serum levels of CK and LDH in medium dose group of Yimai Granule were significantly increased(p<0.01).The serum levels of CK-MB、CK and LDH in the low dose group of Yimai Granule were significantly increased(p<0.01).The high dose Yimai Granule was better than the medium and low dose.4.Myocardial tissue structure was clear in the sham-operated group,and the myocardial fibers were well arranged.No pathological changes such as myocardial fibrosis,necrosis,swelling and inflammatory cell infiltration were observed.Compared with the sham-operated rats,the myocardium of the model group showed extensive myocardial fibrosis,necrosis,interstitial edema,telangiectasia and inflammatory cell infiltration.Compared with the model group,the myocardial tissue of the high and medium dose group of Yimai Granule showed mild muscle fiber degeneration and edema,and the infiltration of inflammatory cells was also relieved.No other abnormalities were observed.5.Compared with the sham-operated rats,the serum NO level of the model group was significantly decreased(p<0.01).After Yimai Granule intervention,serum NO levels in the high,medium and low dose groups of Yimai Granule were significantly increased(p<0.01).6.Compared with the sham-operated rats,the relative expression level of e NOS protein in the model group was significantly decreased(p<0.05).The relative expression level of e NOS protein in the high-dose Yimai Granule group was significantly increased(p<0.05),the relative expression levels of e NOS protein in the other four groups of rats showed an upward trend,which was no statistical difference.7.Compared with the sham-operated rats,the mitochondrial membrane potential of the model group was significantly lower(p<0.01).Compared with the model group,the mitochondrial membrane potential of the high-dose Yimai Granule group was significantly increased. (p<0.01)In the medium dose and low dose groups of Yimai Granule,the mitochondrial membrane potential of rats increased,which was no statistical difference.8.Compared with the sham-operated rats,the relative expression level of Cytc,LC3II/I and Beclin1 protein in the model group were significantly increased(p<0.01).Compared with the model group,the relative expression level of Cytc,LC3II/I and Beclin1 protein in the high and medium dose group were significantly decreased(p<0.01)or(p<0.05).The relative expression level of Cytc and Beclin1 protein in the low dose group of Yimai Granule was significantly decreased(p<0.05),and LC3II/I had a decreasing trend,which was no statistical difference.9.Compared with the sham-operated rats,the relative expression level of PINK1 and Parkin protein in the model group was significantly increased,and the relative expression level of P62 protein was significantly decreased(p<0.01)or(p<0.05).Compared with the model group,the relative expression levels of PINK1 and Parkin protein in the high and medium dose group were significantly decreased,and the relative expression level of P62 protein was significantly increased(p<0.01)or(p<0.05).The relative expression level of PINK1 protein in the low dose group of Yimai Granule was significantly decreased,and the relative expression level of P62 protein was significantly increased(p<0.01).Parkin had a downward trend and was no statistical difference.10.Compared with the sham-operated rats,the expression level of PGC-1α protein in the model group was decreased(p<0.01).Compared with the model group,the relative expression level of PGC-1α protein in the high,medium and low dose groups of Yimai Granule were significantly increased(p<0.01)or(p<0.05).11.Compared with sham-operated rats,the expression level of OPA1 and Mfn1 protein in the model group was decreased,and the expression level of Mfn2 protein was increased(p<0.01)or(p<0.05).Compared with the model group,the expression level of Mfn2 protein was decreased and the expression levels of Mfn1 and OPA1 protein were increased in the high dose group of Yimai Granule.The expression levels of Mfn1 and OPA1 protein in the medium and low dose groups of Yimai Granule were increased(p<0.01)or(p<0.05),and the expression level of Mfn2 protein was decreased,which was was no statistical difference. 12.Compared with the sham-operated rats,the expression levels of Drp1 and Fis1 protein in the model group were increased(p<0.01).Compared with the model group,the expression levels of Drp1 and Fis1 protein in the high and medium dose group were decreased(p<0.01)or(p<0.05).The expression level of Fis1 protein in the low dose group was decreased(p<0.01)or(p<0.05).Conclusion: 1.Yimai Granule can correct dyslipidemia and reduce myocardial damage in high-fat combined with MIRI rats.2.The protective effect of Yimai Granule on high-fat combined with MIRI rats may be related to mitochondrial quality control.3.Yimai Granule may increase mitochondrial membrane potential by regulating mitochondrial autophagy via PINK1/Parkin pathway.4.Yimai Granule may regulate the mitochondrial dynamic balance by regulating mitochondrial fusion protein,fission protein and mitochondrial synthesis related protein,to protect the myocardium of high-fat combined with MIRI rats. |
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