| ObjectivesBased on the successful establishment of myocardial ischemia model rats,the research was aimed to investigate the effect on cardiac structure and function,myocardial cell mitochondria and related signal pathways in experimental rats of the modification by the supplementing qi and activating blood formula.Expound the formula’s mechanism of action of the heart damage by regulating mitochondrial dynamics related proteins,which has influence on the TGF-β1/Smad3 signaling pathways,to reduce myocardial fibrosis and other pathological process.MethodsConstructed by the model of myocardial ischemia through ligaturing the left anterior descending coronary artery of SPF grade SD rats.The successful build model rats were randomly divided into three groups,including model group,the supplementing qi and activating blood group,perindopril group,and only were punctured but with no ligation in sham operation group.Intragastric administration of corresponding drugs was given after operation from the second day.Take the 7th day and the 28th day as the observation time point.The changes of cardiac structure and function were detected by the small animal ultrasound system,the pathological changes of myocardium and mitochondria were observed by the HE staining and the transmission electron microscopy,the severity of myocardial fibrosis were observed by the Masson staining.The contents of ATP and AMP in mitochondria were detected by high performance liquid chromatography(HPLC),the expressed levels of Mfn2,Drp1 AMPK,TGF-β1 and Smad3 gene and protein and phosphorylation AMPK were detected by the Wesrern Blotting,immune real-time fluorescent quantitative PCR method in the infarction marginal zone of myocardial rats.Investigated the mechanism by regulating mitochondrial dynamics related proteins Mfn2 and Drp1 to promote mitochondrial fusion and inhibit the excessive fission,to promote the expression of AMPK protein,and to enhance the inhibition of TGF-β1/Smad3 signaling pathways.In order to explore the supplementing qi and activating blood formula on MI rats and the possible mechanism.ResultsThe experiment 1 The results of the HE staining:in the 7th and 28th day groups,The size of cardiomyocytes was normal,arranged neatly,the structure was clear,the nucleus was normal,no lipofuscin deposition,no significant changes in cell interstitial space in sham group;the number of cardiomyocytes was significantly decreased,the arrangement of myocardial cells was disturbed,and a large number of inflammatory cells were infiltrated in the model group;the drug group was significantly improved compared with the model group.The results of echocardiography:In the 7th and 28th day groups,compared with sham operation group,in model group rats,the LVIDs,LVIDd,ESV and EDV significantly increased(P<0.01),the EF and FS significantly decreased(P<0.01),indifferences were statistically significant.Compared with the model group,in the the supplementing qi and activating blood group and perindopril group,the LVIDs,LVIDd,ESV and EDV significantly decreased(P<0.01 or P<0.05),the EF and FS significantly increased(P<0.01 or P<0.05),indifferences were statistically significant.The experiment 2 The results of the transmission electron microscopy;in the 7th and 28th day groups,In the sham operation group,the structure of myocardium and mitochondria was normal,the distribution of myocardial fibers was regular,the size of morphology was normal,the structure of mitochondria was complete,and the cristae were clear;In the model group,the myocardial fibers were broken,arranged in disorder,nuclear dissolved and disappeared,mitochondria swelled and cristae disappeared;After treatment,the myocardial fibers arranged more orderly,the interstitial space became smaller,the size and morphology of mitochondria were significantly higher than that of the model group,mild edema,most of the cristae were normal,and some of them disappeared.The results of ATP and AMP detection of myocardial energy products:In the 7th and 28th day groups,compared with sham operation group,in model group rats.the ATP significantly decreased(P<0.01),the AMP significantly increased(P<0.01),indifferences were statistically significant.Compared with the model group,in the the supplementing qi and activating blood group and perindopril group,the ATP significantly increased(P<0.01),the AMP significantly decreased(P<0.01),indifferences were statistically significant;The results of mitochondrial Mfn2 and Drpl detection in myocardial cells:In the 7th and 28th day groups,compared with sham operation group,in model group rats,the Mfn2 gene and protein significantly decreased(P<0.01),the Drpl gene and protein significantly increased(P<0.01),indifferences were statistically significant.Compared with the model group,in the the supplementing qi and activating blood group and perindopril group,the Mfn2 gene and protein significantly increased(P<0.01 or P<0.05),the Drpl gene and protein significantly decreased(P<0.01 or P<0.05),indifferences were statistically significant.The experiment 3 The results of the Masson staining:in the 7th and 28th day groups,compared with the sham operation group,the blue area increased,collagen production increased,myocardial fibrosis increased in the model group,compared with the model group,the blue area decreased,the amount of collagen decreased,and the fibrosis decreased in the drug group.Detection results of AMPK,p-AMPK,TGF-1 and Smad3 in the marginal zone of myocardial infarction:in the 7th and 28th day groups,compared with sham operation group,in model group rats,the AMPK gene,protein and phosphorylated AMPK significantly decreased(P<0.01 or P<0.05),the TGF-(31 and Smad3 gene and protein significantly increased(P<0.01),indifferences were statistically significant.Compared with the model group,in the the supplementing qi and activating blood group and perindopril group,the AMPK gene,protein and phosphorylated AMPK significantly increased(P<0.01 or P<0.05),the TGF-β1 and Smad3 gene and protein significantly decreased(P<0.01 or P<0.05),indifferences were statistically significant.Conclusions1.This experiment applied successful build myocardial ischemia rat model,validation of the supplementing qi and activating blood formula can repair the myocardial cell structure and enhance the myocardial function,reduce the size of the infarction area,etc,effectively relieve myocardial ischemia injury of myocardial cells and ventricular remodeling2.The supplementing qi and activating blood formula can reduce the damage of mitochondria of myocardial cells by promoting the fusion and inhibit excessive fission,improve the structure and function of mitochondria,and promote energy synthesis,to reduce the myocardial ischemia injury,which may be one of the targets of the formula.3.The supplementing qi and activating blood formula can increase AMPK,phosphorylated AMPK expression and inhibit TGF-β1/Smad3 signaling pathway to alleviate myocardial fibrosis and ventricular remodeling.4.Supported by the experimental results and related literature theory,it is suggested that:the supplementing qi and activating blood formula can act on AMPK/TGF-β signal pathway by regulating the mitochondrial dynamics related proteins Mfn2 and Drpl. |
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