Effect Of Yiqi Huoxue Recipe On Nerve Remodeling And ERK1/2 Signal Pathway After Myocardial Infarction

BACKGROUNDThe inhomogeneity of the over-regeneration and distribution of the sympathetic nerve after myocardial infarction,which is called Nerve Remodeling,can lead to irreversible cardiac function damage.The previous study showed that the Yiqi Huoxue decoction(YQHX)can effectively improve the left ventricular function after myocardial infarction and reduce the incidence of heart failure.The purpose of this study was to observe the protective effects of YQHX on myocardial tissue and hypoxic myocardial cells,and to explore possible mechanism of action.ObjectiveTo observe the changes of myocardial injury and nerve remodeling in rats with myocardial infarction,to prove the protective effect of YQHX decoction on heart,and to explore the molecular mechanism of YQHX decoction based on ERK1/2signal pathway.MethodsThe study was performed both in vivo and in vitro.1.Animal experiments:The rat MI model was conducted by left anterior descending coronary aitery ligation.The 8-week-old male SD rats were randomly divided into 4 groups:Sham group(Sham,S),Model group(Model,M),YQHX group(YQHX,Y)and Metoprolol group(Metoprolol,Meto).The changes of corresponding indications were observed at 3rd day,7th days and 28th days after myocardial infarction.The tissues of the marginal area of infarction,the survival area of the distal end and the left stellate ganglion were taken to observe the changes of the corresponding indexes.The left ventricular function was observed by echocardiography,the pathological changes of myocardium were observed by HE staining,the distribution of TH positive sympathetic nerve fibers and the expression of ANP factor were observed by immunofluorescence technique.The content of NPY in plasma was detected by enzyme-linked immunosorbent assay(Elisa),and the expression of nerve remodeling related factor NGF,GAP43,Sema-3A,TH protein,NPY protein and myocardial hypertrophy index ANP and β-MHC protein were detected by WB.2.Cell experiment:H9c2 cardiomyocyte hypoxia model was used.According to the results of the previous research,the optimal antioxidant dose of 200μg/ml and 12 hours was selected as the time point of mild to moderate cell injury.Cardiomyocytes were divided into three groups:①Control group(C);②Hypoxia model group(M);③Hypoxia model with 200μg/ml YQHX decoction water extract group(Y).The changes of LDH in cell culture were detected by lactic dehydrogenase kit,and the contents of MDA and SOD in cells were detected by antioxidant kit.In order to further explore the regulatory mechanism of YQHX on anoxic cardiomyocytes,we used PD98059,an inhibitor of ERK1/2 signaling pathway.The experiment was divided into 5 groups:Group①②③is the same as before;④Hypoxia model with PD98059 group(PD);(5)Hypoxia model with 200μg/ml YQHX decoction water extract and PD98059 group(Y+PD),we observed the apoptosis of cardiomyocytes before and after hoechst 33258 staining,and detected the expression of the protein of ERK1/2,p-ERK1/2,c-FOS,c-MYC,NGF and Sema-3A in anoxic cardiomyocytes.Results1 Effect of YQHX decoction on the results of echocardiography in ratsOn the 3rd day,7th day and 28th day,the cardiac function of group M was damaged.After using Y and Meto,group Y could significantly up-regulate the value of EF,FS,and the curative effect was determined.the curative effect of Meto group was better than that of subacute phase and chronic phase.2 Effect of YQHX decoction on myocardial HE staining results3 days after myocardial infarction:there was no significant change in group S.In group M,the morphological changes,swelling,uneven staining of cytoplasm,pyknosis,broken cardiomyocytes,structural destruction,disordered arrangement and inflammatory cell infiltration were observed.The pathological changes in group Y and Meto were slightly lighter than those in the model group.7 days and 28 days after myocardial infarction:pathological changes were alleviated in Y and Meto groups.3 Effect of YQHX decoction on the distribution of TH positive sympathetic nerve fibers stained with immunofluorescenceIn the marginal area of the infarction,the group S positive nerve fibers were rare.The density of the positive nerve fibers in the group M was significantly increased,the distribution was disordered,the morphology was large,and the cluster was collected.With the increase of time,the nerve form and distribution tended to normalize,and the density of TH-positive nerve fibers in the group Y and the group Meto was significantly lower than that of the group M.The density of TH-positive nerve fibers was slightly decreased in the distal area.4 Effect of YQHX decoction on dynamic expression of nerve remodeling related Factor NGF,GAP43,Sema-3A and TH proteinIn terms of protein expression,the group M of NGF and TH were highly expressed on the 7th day in the marginal area and on the 3rd day in non-infarction area,GAP43 was high expressed in different sites at 3rd day of acute phase.Increased significantly compared with group S(P<0.05);The lowest value of Sema-3A was found in group M at 7/28th day,which was significantly lower than that in group S(P<0.05).From the perspective of drug efficacy,the expression of GAP43,TH and NGF in group Y was significantly lower than that in group M,and Sema-3A was increased(P<0.05).There was no significant difference between group Meto and group Y(P>0.05),but the curative effect was better in group Meto on the 3rd day and 7th day,while the mean value of group Y gradually tended to group S with time going on,and the difference was significant at 28 days compared with group M(P<0.05).5 Effect of YQHX decoction on the expressions of relevant indicators of hypertrophy ANP and β-MHC in rats with MI in the margin of infarctionThe positive expression site of ANP was brown in the cytoplasm of cardiac myocytes.On the 3rd day,the structure of cardiac myocytes in group S was neat and clear,and the ANP positive expression in group M showed large brown granules in nucleus and cytoplasm,which aggregated into pieces.There were a few light or dark brown granules in group Y.The positive area of group Meto was similar to that of group Y.On the 7th day and 28th day,the positive area of group Meto was similar to that of group Y.Compared with group M,the color of group Meto was lighter and the positive expression area was smaller than that of group Y.At the margin of infarction,the expression of ANP and β-MHC protein increased significantly in group M compared with group S(P<0.05),but decreased significantly in group Y and Meto compared with group M(P<0.05),and decreased significantly in group Meto compared with group Y(P>0.05).On the 28th day,group Meto was significantly higher than group Y(P>0.05).6 Effect of YQHX decoction on LDH,MDA,SOD of anoxic cardiomyocytesAfter 12 hours of hypoxia,YQHX decoction could significantly decrease the release of LDH and the content of MDA,increase the activity of SOD(P<0.05).7 Observe the effect of YQHX decoction on cardiomyocyte injury induced by hypoxia before and after Hoechst33258 staining.Before Hoechst33258 staining,the morphology of the cells in group C was fusiform,the boundary was clear,the morphology was complete and the distribution was uniform,the cardiomyocytes in group M were seriously damaged,the cells became round,the boundary was blurred,the gap between cells became larger,some of them were in the shape of clusters,and some of the cells floated.In group Y,the anoxic injury was alleviated,the aggregation was alleviated,and the distribution was uniform,and the morphology of the cells was between fusiform and round.The injury in group PD was lighter than that in group Y,most of the cells were fusiform,the gap was smaller,the number of floating cells was less.group Y+PD was similar to group PD.After Hoechst33258 staining,the morphology of the cells in group C was complete and the nuclear staining was uniform.After hypoxia treatment,the nucleus of group M was irregular pyknosis and condensation,showing dense and dense staining,and the nucleus split into fragments,and the intact cells were rare under microscope.Compared with group M,group Y,PD and Y+PD could reduce nuclear condensation and cell fragments.8 Effect of YQHX decoction on the expression of ERK1/2 signaling pathway protein and nerve remodeling related factor NGF,Sema-3A protein in cardiomyocytes induced by hypoxiaERK:There was no significant change in the expression of ERK(P>0.05);p-ERK:group M was significantly higher than group C(P<0.05),ERK1/2 pathway inhibitor group PD(PD98059)and group Y were significantly lower than that of group M(P<0.05),Group Y+PD was significantly lower than that in group Y(P<0.05).The expression trend of c-FOS and c-MYC downstream proteins in ERK1/2pathway was similar to that in p-ERK,but the expression of c-FOS and c-MYC in group Y+PD was significantly lower than that in group PD(P<0.05).NGF:Compared with group C,group M was significantly increased,and that in group Y and PD was significantly lower than that in group M(P<0.05),group PD and Y+PD was significantly lower than that in group Y(P<0.05).Compared with group PD,group Y+PD was significantly lower(P<0.05).The expression of Sema-3A is contrary to that of NGF.Conclusion1 YQHX decoction can significantly improve the injury of myocardial tissue induced by MI and myocardial cells induced by hypoxia.2 YQHX decoction can significantly improve the excessive regeneration and distribution density of nerve fibers after MI,and regulate the expression of related factors of nerve remodeling.The 7th day as the subacute phase,is the critical point of the efficacy of traditional Chinese medicine and western medicine.3 The protective effect of water extract of YQHX decoction on H9c2 of anoxic cardiomyocytes was achieved by inhibiting the phosphorylation pathway of ERK induced by hypoxia,and its effect was consistent with that of ERK1/2pathway inhibitor.However,its activation of proteins downstream of the pathway is not only through the ERK1/2pathway,but may also involve other related pathways or targets.4 The water extract of YQHX decoction may partly affect the expression of NGF and Sema-3A in anoxic cardiomyocytes through ERK1/2 signal pathway.It is suggested that YQHX decoction may protect myocardial cells from hypoxia by inhibiting the expression of nerve remodeling related factors in ERK1/2signaling pathway.