| BackgroundAfter myocardial infarction,calcium overload of myocardial cells caused by ischemia and hypoxia is one of the key factors causing myocardial ischemic injury.Cellular calcium overload causes excessive contraction of cardiomyocytes,and the cytoskeletal components are deformed beyond the normal contraction,resulting in irreversible damage,and causing adjacent cardiomyocytes to destroy each other and expand the scope of necrosis,resulting in a variety of pathological changes after myocardial infarction,including Acute cardiac insufficiency,arrhythmia,heart rupture,etc.Therefore,it is necessary to explore the regulation mechanism of intracellular calcium homeostasis after myocardial infarction,and to elucidate the mechanism of Yiqi Huoxue Recipe in regulating myocardial calcium homeostasis after myocardial infarction and the target of Chinese medicine,which may be important for the prevention and treatment of myocardial cell damage after myocardial ischemia.Theoretical and practical significance.Severe ventricular arrhythmia after myocardial infarction is an important cause of morbidity and mortality,so it is also the treatment focus of reducing mortality and improving the quality of life of patients after myocardial infarction.PurposeThis study clarified the protective effect of Yiqi Huoxue Recipe on the heart after myocardial infarction by observing the changes in cardiac function,tissue and subcellular structure,myocardial calcium homeostasis regulation-related proteins,etc.Prevention and treatment of arrhythmia after myocardial infarction.MethodExperimental research is divided into animal experiments and acute myocardial cell separation experimentsAnimal experiment:Healthy male SD rats underwent left anterior descending coronary artery(LAD)ligation to replicate the rat model of acute myocardial infarction.According to the electrocardiogram at 24 hours after surgery,the animals were randomly divided into 4 groups:model group(Model,M),Yiqi Huoxue Recipe group(YQHX,Y),Metoprolol group(Metoprolol,MT),and only threading without ligation Sham operation group(Sham,S).After 4 weeks,cardiac ultrasound,HE staining,Masson staining,transmission electron microscopy was performed,serum BNP content was detected by Elisa,Western blot were used to detect the expression of calcium homeostasis-regulated proteins,gap junction protein Cx43 and its phosphorylated protein in the marginal area of the infarct;in addition,in vivo cardiac electrical stimulation was used to detect each group Susceptibility to arrhythmia.Acute cardiomyocyte separation experiment:At 28 days,the cells in the marginal zone of myocardial infarction in each group were acutely isolated to study the contraction and relaxation of cardiomyocytes,calcium transients,and calcium leakage of the sarcoplasmic reticulum in each group under continuous pulsation.Changes in calcium transient amplitude,etc.Result1.Evaluation of changes in rat cardiac function.Compared with the sham-operated group,the model group suffered severely impaired cardiac function.After intervention with Yiqi Huoxue Recipe and Metoprolol,the cardiac function improved significantly.2.Changes in myocardial tissue morphology and cell ultrastructure after myocardial infarction.HE staining results of myocardial cells showed that the cells in the sham-operated group were neatly arranged,the structure of myocardial fibers was clear,no obvious collagen fibers were seen,and there was no damage to the cell junctions;the model group had obvious myocardial fiber breakage and large gaps in the myocardial cells,Irregular borders,irregular arrangement of myocardial cells,infiltration of inflammatory cells and significant collagen fiber hyperplasia;some inflammatory cell infiltrations were found in the myocardial tissues of the infarcted marginal zone of rats in Yiqi Huoxue group and Metoprolol group,and the cell shape tended to be regular.There are some fiber and collagen tissue hyperplasia.Compared with the sham operation group,the average diameter,perimeter,and cross-sectional area of cardiomyocytes in the model group were significantly increased.Compared with the model group,the parameters of the dual-use drug group were reduced,and the change trend was close to the sham operation group.Masson staining displayed that,in the sham group,myocardial cells were red stained,normal in size,neatly arranged,and the structure was clear,the interstitium was blue stained,and a small amount of collagen fibers could be seen;in the model group,the myocardial cell structure is disordered and the gap is widened.The infarct tissue is extensively blue-stained with collagen fibers and diffusely distributed.A large number of interstitial fiber bundles separate the myocardial cells into strips or islands.More collagen fibers are seen around the large blood vessels.The pathological changes in the dual-use drug group were all reduced compared with the model group,and the blue staining area in the infarct area was reduced compared with the model group.In terms of the ultrastructure of myocardial cells,the myocardial fibers of the sham-operated group are neatly arranged and have complete sarcomere,the Z line and the M line are clearly distinguishable,the mitochondria are arranged in the middle of the myofibrils,they are evenly distributed,the mitochondrial ridge structure is clear,and the matrix particles The distribution is even;the myocardial cell structure in the marginal zone of myocardial infarction in the model group is severely damaged,there is obvious rupture of myofilament,the Z line and M line are unclear,the mitochondrial arrangement is disordered,the shape is irregular,the original structure is lost,and the mitochondrial crest is blurred,part of the mitochondria are vacuolated,but some have a tendency to repair;the internal structure of the cardiomyocytes in the Yiqi Huoxue Recipe group is relatively intact,the broken myofilaments tend to be linearly arranged,there is still a gap between the mitochondria and the myofilaments,and the broken muscles can be seen filled with mitochondria near the silk,the overall arrangement of mitochondria is more regular,the internal structure is clearer,and the matrix particles are more evenly distributed.3.Changes of myocardial ventricular fibrillation threshold in myocardial infarction rats.After 28 days of myocardial infarction,the ventricular fibrillation threshold of the model group was significantly lower than that of the sham-operated rats.The ventricular fibrillation threshold of the myocardial infarction rats in the Yiqi Huoxue Recipe group and the metoprolol group increased,and the susceptibility to ventricular arrhythmia reduce.4.Changes of Myocardial Cell Contraction and Calcium Transient in the Infarct Marginal Zone of Myocardial Infarction Rats.Compared with the sham operation group,the myocardial cell systolic function of the model group was reduced,and the change of diastolic function was significant(p<0.01).Both Yiqi Huoxue Recipe and metoprolol could improve the systolic function damage of myocardial cells after myocardial infarction,and Compared with the model group,Yiqi Huoxue Recipe can significantly increase the maximum contraction rate,maximum relaxation rate,50%contraction time,50%relaxation time of myocardial cells in the marginal zone of myocardial infarction(p<0.01),and increase the contraction amplitude of myocardial cells(p<0.01),Reducing the peak time of contraction of myocardial cells(p<0.01),Metoprolol's improvement of cardiomyocyte systolic function is mainly manifested in enhancing cardiomyocyte diastolic function(p<0.01)and the increase of diastolic sarcomere length(p<0.01),and shorten the cell contraction peak time(p<0.01).In terms of calcium transient-related indicators,compared with the sham operation group,the calcium transient amplitude and the calcium ion elimination time constant of the model group were increased(p<0.05).Compared with the model group,the calcium transient amplitude of the dual-use drug group was The downward trend but the difference was not obvious.The calcium ion elimination time constant of the Yiqi Huoxue Recipe group decreased significantly(p<0.01).The Metoprolol group showed a downward trend but the difference was not significant.5.Changes of sarcoplasmic reticulum calcium leakage of myocardial cells in the marginal zone of rat myocardial infarction and the expression of calcium homeostasis-regulated proteins in the marginal zone of infarction.The results of sarcoplasmic reticulum calcium leakage test showed that,compared with the sham operation group,the model group had a higher myocardial cell sarcoplasmic reticulum calcium leakage(p<0.01).Compared with the model group,the Yiqi Huoxue Recipe group and the metoprolol group myocardium Cellular calcium leakage decreased significantly(p<0.05).Westem Blot results showed that compared with sham operation,the expression of RYR2 and SERCA2? protein in the marginal zone of myocardial infarction in the model group was significantly reduced(p<0.01),and the expression of RYR2 was significantly increased after intervention of Yiqi Huoxue Recipe and metoprolol(p<0.05);Calcium homeostasis on the mitochondria regulates the expression of the related protein MICU1:Compared with the sham-operated group,the expression of MICU1 was reduced in the model group(p<0.05),and the Yiqihuoxue Recipe group was higher than the model group,but there was no statistical difference.Compared with the model group,the expression of MICU1 protein in the metoprolol group was increased(p<0.05).6.Gap connexin and its phosphorylation in the marginal zone of rat myocardial infarction.Compared with the sham operation group,the expression of Cx43 in the model group was significantly decreased(p<0.05).After treatment with Yiqi Huoxue Recipe and Metoprolol,the expression level of Cx43 increased(p<0.05);compared with the sham operation group In the model group,the Cx43 phosphorylation level of myocardial tissue decreased significantly(p<0.01).After treatment with Yiqi Huoxue Recipe and Metoprolol,the expression level of Cx43 increased(p<0.01).Conclusion1.Yiqi Huoxue Recipe can significantly improve the structural and functional damage of myocardial tissue due to ischemia and hypoxia.2.Yiqi Huoxue Recipe can reduce the susceptibility of ventricular fibrillation after myocardial infarction.3.Yiqi Huoxue Recipe has a significant improvement effect on the myocardial infarction marginal myocardial cell's systolic and contractile capacity,this effect is produced by affecting the myocardial cell calcium transient.4.Yiqi Huoxue Recipe has a regulating effect on the important Ca2+ regulatory protein in the sarcoplasmic reticulum and mitochondrial after myocardial infarction,which is consistent with the susceptibility of arrhythmia and the improvement of cardiac function.5 Yiqi Huoxue Recipe can increase the gap junction protein and phosphorylation level of myocardial cells in the marginal zone of infarction after myocardial infarction.This effect may be one of the reasons for improving overall cardiac function and reducing the susceptibility to arrhythmia. |
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