Study On The Effect Of Yiqi Huoxue Recipe On Cytoskeleton Related Proteins And AMPK Regulatory Pathways In Ischemic Myocardium

Background:According to the 2017 China Cardiovascular Disease Report,the mortality rate of cardiovascular disease(CVD)accounts for more than 40 percent of the total deaths among the population.Due to the impact of aging and other factors,the CVD burden in China has been increasing.Incidence and mortality are on the rise.It has been found that after myocardial ischemia and hypoxia,myocardial cytoskeleton system can be damaged in different degrees,the changes of cytoskeleton-related proteins and their microstructure may also become the standard to assess myocardial ischemia injury.AMPK can regulate cell energy metabolism and cytoskeletal remodeling.Therefore,it is clear that AMPK regulates myocardial cytoskeleton and plays an important role in preventing and treating myocardial ischemia.Purpose:Revealing the changes of myocardial cytoskeleton and its key regulatory pathways after myocardial ischemia and hypoxia can provide theoretical basis and experimental basis for the prevention and treatment of myocardial ischemia by Yiqi huoxue decoction.Methods:The study was divided into two parts:animal experiment and cell ExperimentAnimal Experiment:An animal model of acute myocardial infarction was established by ligating the anterior descending branch of the left coronary artery and its accompanying vessels(LAD).According to the quantity of Q wave in 24 hours after operation,the animals were randomly divided into 4 groups:Model Group(M),Yiqi huoxue decoction(Y),metoprolol group(MT)and Sham Operation Group(S),and time points of 3 days,7 days and 28 days.The left ventricular LVEF,LVFS,LVIDd and LVIDs were calculated by M-mode curve,and the pathological changes of myocardial tissue were observed by HE staining.The ultrastructural changes of myocardial cells after myocardial infarction were observed by transmission electron microscope,and the expressions of microtubules,microfilaments,gap junction proteins and proteins related to AMPK signal pathway were observed by Western blot and Immunofluorescence.To reveal the changes of myocardial cytoskeleton protein after myocardial ischemia and clarify the relationship between AMPK and cytoskeleton protein.Cell Experiment:hypoxia induced H9c2 cardiomyocytes were divided into 5 groups:normal Group(C)and hypoxia model Group(M),100? g/ml Yiqi huoxue decoction group(Y1),200? g/ml Yiqi huoxue decoction group(Y2),400? g/ml Yiqi huoxue decoction group 4).The cell viability was monitored by real time marker free cell function analyzer,the levels of LDH,MDA and SOD in supernatant were measured,and the cytoskeleton was stained by Phalloidin staining.To evaluate the effects of Yiqi huoxue decoction of different concentrations on the cyto skeleton structure and function of H9c2 cardiomyocytes after hypoxia.In order to further explore the interaction of AMPK pathway with microtubule,microfilament ultrastructure and related proteins of cytoskeleton,cardiomyocytes were divided into 5 groups after adding AMPK inhibitor Compound C:Normal Group(C)and hypoxia model Group(M),100?g/ml Yiqi huoxue decoction group(Y1),100? g/ml Yiqi huoxue decoction group(Y1)+AMPK inhibitor Compound C group(Y1+CC),AMPK inhibitor Compound C group(CC).The expression of Tubulin,Tubulin,MAP4,F-actin,MLCK,MLC-2,p-MLC-2 and p-AMPK were detected by Western Blot.Results:1.Pathological results of myocardial tissue in rats with myocardial infarction in each group.In S group,the myocardial cells were arranged regularly,the structure was clear.In M group,the wall of infarcted area became thinner and the number of myocardial cells decreased significantly,inflammatory cells infiltrated,and there were hyperemia and inflammatory cells in the infarcted border area.The myocardial cells in Y group and MT group were found to be disorganized and partly necrotic,and inflammatory cells were found in the infarct border area.The myocardial cells were still swollen irregularly,but the intercellular space decreased and the inflammatory fibers decreased.The infarct area became thinner in M group 28 days after MI and was replaced by fibroblasts and collagenous fiber bundles.The arrangement of myocardial cells was disordered,the infiltration of inflammatory cells was decreased,and the cells in myocardial tissue were swollen seriously,the intercellular space was large,the edge was irregular and the nucleus was scattered In YQHX group and MT group,the structure of myocardial cells was disordered,some inflammatory cells and the shape of myocardial cells tended to be regular,the intercellular space and diameter decreased.2.The ultrastructure of myocardial cells in each group after myocardial infarction was observed by electron microscope.In S group,the Z and M lines of myocardial ultrastructure were clearly discernible,the thick and thin myofilaments were arranged in order,the myofibrillar fibers were arranged perpendicular to the cell axis,and the mitochondria showed uniform matrix granules and closely arranged mitochondrial Cristae 3 days after myocardial infarction on the 7th and 28th day after myocardial infarction,the structure of myocardial cells in M group was seriously damaged,and the myofibrils,Z lines and m lines were completely disordered or even absent myofilaments were broken,thick myofilaments accumulated,mitochondria swelled abnormally and lost their complete morphological structure,there were many vacuoles and mitochondria cristae were unclear in Y group,the Z lines were arranged in wave shape at 7 days after myocardial infarction,the thick and thin myofilaments had fused without obvious dividing line.The mitochondria were aggregated,but the membrane structure of mitochondria in M group was still intact.After 28 days after Mi,the myofilaments in S group were more regular and linear.2.Expression of tubulin and microfilament-associated protein in the infarct border area of rats in each group.Western Blot showed that the expression levels of microtubulin and MLCK?Cx43?p-MLC-2 were significantly lower in the myocardium of M group than those of S group,and the expression of p-AMPK?MLC-2 were still significantly increased(p<0.01).The level of ?-tubulin?MAP4?MLCK?Cx43?p-MLC-2 in MT group and Y group was significantly higher than that in M group(p<0.05).However,the levels of p-AMPK?MLC-2 were significantly decreased(p<0.05).The result of immunofluorescence showed the same trend as that of WB.3.Protective Effects of Yiqi huoxue decoction on H9c2 cardiomyocytes after hypoxia.Compared with Group C,the cell activity decreased significantly after 12h and 24h of Hypoxia(p0.0001).Compared with M group,different concentrations of YQHX at 12h and 24h after hypoxia could protect cardiomyocytes from hypoxia-induced Injury The myocardial cell activity in Y 1,Y2 and Y4 groups were significantly increased(p<0.05,p<0.01).On the basis of exploring the anoxic time in the earlier experiment,the anoxic time of 12h was selected as the model of the follow-up experiment.Compared with Group C,LDH and MDA in supernatant of group M were significantly released(P<0.01),while LDH and MDA in supernatant of group Y1,Y2 and Y4 were significantly decreased(P<0.01).In addition,SOD content in supernatant of M group was significantly decreased(P<0.01),and the release of SOD in H9c2 cells was significantly increased after Y1 treatment(P<0.01).The cytoskeleton was stained with FITC-labeled phalloidin.It was found that the skeleton structure of normal cardiomyocytes in C group was surrounded by nucleus,arranged in a grid,and stained evenly.After hypoxia for 12 hours,the myocardial cytoskeleton of M group was damaged,the structure was blurred,the lattice structure was seriously damaged,and the fluorescence intensity was lower than that of C group.Some areas were not stained and vacuoles were formed.The skeletal structure damage was improved in each drug-administered group,and the fluorescence intensity was higher than that in the hypoxic injury group alone.The abnormal staining and nuclear fragmentation were alleviated,and the improvement was more obvious in the Y1-administered group,indicating that Yiqi huoxue decoction can enhance the cytoskeletal structure stability.The apoptosis rate of cardiomyocytes in each group was detected by Hoechst 33258 staining.Compared with C group,the nucleus of group M was irregular after 12h hypoxia injury,and it was divided into broken sequins and apoptotic bodies.In contrast,Y1 and Y2 reduce nuclear enrichment and reduce cell debris.When the AMPK inhibitor compound C was administered,the dense staining and fragmentation of the nucleus increased significantly,and the apoptotic bodies increased.Compared with the Y1 and Y2 groups,Y1CC and Y2CC were effective in alleviating the increase of debris.4.Yiqi huoxue decoction protects H9c2 cardiomyocyte cytoskeleton tubulin by hypoxia after AMPK/MAP4 pathway.The results showed that compared with group C,group M in hypoxia reduced the expression of ?-tubulin and ?-tubulin(p<0.01).At the same time,compared with the M group,Y1 treatment of H9c2 cardiomyocytes significantly enhanced the expression of?-tubulin and ?-tubulin(P<0.01).Based on the changes of cytoskeletal tubulin at 12h in the early hypoxia,the experiment was performed with Yiqi huoxue decoction at a concentration of 100 ?g/ml.To further evaluate the relationship between AMPK and tubulin,the AMPK inhibitor CC group was compared and found that compared with the C group,the p-AMPKa(Thr172)was significantly increased and the expression of MAP4 was significantly decreased in the model group(P<0.01),Y1 treatment of H9c2 cardiomyocytes significantly increased the expression of p-AMPKa,up-regulated the expression of MAP4(P<0.01),CC can significantly eliminate the protective effect of Y1 on tubulin.5.The protective effect of Yiqi huoxue decoction on H9c2 myocardial cytoskeleton microfilament protein after hypoxia through AMPK/MLCK pathway.The results showed that compared with group C,MLCK level and p-MLC-2 level were significantly decreased in group M(P<0.01),while the expression of MLCK and p-MLC-2 was inhibited by CC,indicating that Yiqi huoxue decoction.The effect of myosin in H9c2 myocardial cytoskeletal damage after hypoxia is associated with AMPK phosphorylation.6.Effect of Yiqi huoxue decoction on HIF-1? protein expression in H9c2 cardiomyocytes after hypoxia.Compared with group C,the expression of HIF-1? protein in the cytoplasm and nucleus of M9H2 cells was significantly increased(P<0.01).After administration,HIF-1? in the cytoplasm and nucleus of Y1 group was observed.The protein expression level can be returned to the normal group level to some extent(P<0.01).Conclusion:1.Yiqi huoxue decoction can protect the ultrastructure of myocardial cells after myocardial infarction,improve the left ventricular function and reduce the degree of ventricular remodeling after myocardial infarction.2.Yiqi huoxue decoction can promote the expression of myocardial cytoskeleton microtubule-associated protein MAP4,MLCK,p-MLC-2 after ischemia and hypoxia,which indicates that Yiqi huoxue decoction has the effect of protecting myocardial cytoskeleton.3.Compound C can inhibit the up-regulation of p-AMPK,microtubule protein MAP4 and microfilament protein MLCK,p-MLC-2 by Yiqi huoxue decoction.It is suggested that Yiqi huoxue decoction may protect the structure and function of microtubules and microfilaments in myocardial cytoskeleton through AMPK/MAP4 and AMPK/MLCK pathway AMPK may serve as a bridge to regulate the interaction network between cytoskeleton microtubulin and microfilament myosin remodeling.