| Objective:Heart failure(HF)is the terminal stage of many cardiovascular diseases.The incidence of HF after myocardial infarction is gradually increasing.It is currently one of the most common and important causes of HF in the world.Therefore,this study chose HF caused by myocardial infarction as the target disease for research.When in a state of heart failure,the activity of mitochondrial respiratory chain enzymes in cardiomyocytes changes,oxidative stress increases,and the stable state of mitochondria is also destroyed.Therefore,the treatment and improvement of mitochondrial function is one of the new hot spots for the treatment of heart failure in recent years.Qishen Granules(QSG)is an effective traditional Chinese medicine compound developed by the research group to combat HF after myocardial infarction,and has been researched for more than ten years.Previous studies have found that QSG can enhance the overall myocardial energy supply of HF rats after myocardial infarction.In this study,a chronic HF model induced by myocardial infarction was established by ligation of the left coronary artery in rats,to observe the effect of QSG on the cardiac function of HF rats,and combined with transcriptomics technology to explore the mechanism of QSG in the treatment of heart failure after myocardial infarction.To clarify whether it can improve the cardiac function of rats by improving the mitochondrial function of cardiomyocytes,aiming to provide a theoretical basis for the clinical treatment of HF by QSG.Methods:1.This experiment uses left anterior descending(LAD)to prepare animal models of heart failure caused by acute myocardial ischemia in rats.Heart failure rats are divided into sham operation group,model group,There are three groups in the QSG group.21 days after LAD ligation,the cardiac function of the rats in each group was evaluated by echocardiography,and the arrangement of myocardial cells in each group was detected and evaluated by the hematoxylineosin staining(HE).2.Analyze the mRNA expression of rat myocardium in the sham operation group,model group and QSG group by high-throughput sequencing technology;through GO function enrichment analysis and pathway annotations in the KEGG database,select candidates for the therapeutic effect of QSG on heart failure Genes and signaling pathways.Real-time fluorescent quantitative PCR(RT-qPCR)technology was used to verify the mRNA expression levels of some target genes screened by transcriptomics.3.Take rat myocardial tissue and observe the ultrastructure of myocardial mitochondria with transmission electron microscope;culture H9C2 rat cardiomyocytes in vitro to establish hypoxia,hypoglycemia and reoxygenation and reoxygenation models,and the kit detects both in vitro and in vivo conditions The levels of adenosine triphosphate(ATP),glutathione peroxidase(GSH-Px)and inducible nitric oxide synthase(iNOS)in rat cardiomyocytes,observe the QSG pair The effect of heart failure on myocardial tissue respiratory function and energy metabolism in rats;4.Westernblot experiments detect the mitochondrial dynamics protein fusion mitochondrial fusion factor 1(mitofusinl,Mfn1),mitochondrial fusion factor 2(mitofusin2,Mfn2)Optic nerve atrophy related protein 1(opticatrophyl,Opal)mitochondrial fission protein 1(fission1,Fisl)and dynamin-related protein 1(Drpl)levels to observe the effect of QSG on the mitochondrial dynamics of heart failure rats’ myocardial cells;Western blot experiments to detect mitochondrial proliferator-activated receptor coactivator-a(peroxisome proliferatoractivated receptor coactivator-1α,PGC-1α)in rat myocardial tissue and cell injury models as the core and downstream nuclear respiratory factor-1(nuclear respiratory factor 1,NRF-1)and mitochondrial transcription factor A(TFAM)typical mitochondrial biogenesis-related protein expression.Results:1.The results of cardiac ultrasound showed that 21 days after LAD ligation,the ejection fraction and short axis shortening rate of the myocardium in the model group were significantly reduced,and the reduced myocardial contractility resulted in insufficient pumping power,suggesting that the heart failure model was successfully established.The EF value of the QSG group increased significantly(P<0.01),suggesting that QSG can effectively inhibit the decline of heart function in rats caused by heart failure.The results of HE staining of myocardial tissue showed that compared with the rats in the LAD ligation model group,the myocardial cells of the QSG group were arranged more neatly,the cell spacing was reduced,and the structure was clear.2.Transcriptomics technology was used to analyze the mRNA expression in the sham operation group,model group and heart tissues of QSG rats.In the comparison between the model group and the sham operation group,40 genes were up-regulated and 39 genes were down-regulated;in the comparison between the QSG group and the model group,38 genes in the QSG group were up-regulated and 29 genes were down-regulated.Among the genes that can be coregulated by the model group and the QSG group,the 27 differential genes that QSG can significantly call back,and then proceed to follow-up analysis.By enriching the KEGG pathway of differential genes,9 pathways were screened out(P<0.01),mainly related to oxidative phosphorylation,cardiac muscle contraction,metabolic pathway ways,etc.;KEGG pathway enriched The results of the collection indicate that the potential drug targets of QSG are closely related to the mitochondrial function of cells;the GO biological process analysis takes the first 16 according to the number of enrichment.The cellular component(CC)involves mitochondria and the mitochondria.Respiratory chain,extracellular matrix components,cytoplasm,etc.;biological process(BP)mainly involves drug response,aging,muscle tissue development,response to oxygen transport levels,response to oxidative stress,etc.;molecular functions(molecular function,MF)mainly involves ATPase activity,cytochrome C oxidase activity,NADH dehydrogenase/ubiquinone activity and other processes.3.Observation of the ultrastructure of myocardial mitochondria under a transmission electron microscope found that compared with the model group,most of the mitochondria in the QSG group had complete morphology and structure,and they were neatly arranged,indicating that QSG promotes the recovery of mitochondria;JC-1 staining results show that QSG is significant Increased the mitochondrial membrane potential of H9C2 cardiomyocytes damaged by hypoxia and hypoglycemia model;ROS results showed that the excitation level of ROS in the QSG group was lower than that of the model group;GSH-PX,iNOS,in the QSG group in vitro and in vivo experiments The expression level of ATP increased(P<0.01,P<0.01,P<0.01).4.In animal experiments,Westen blots showed that compared with the model group,the expression of mitochondrial fusion proteins Mfn1,Mfn2,and Opal in the myocardial tissue of the QSG group was significantly increased(P<0.01,P<0.01,P<0.01),The expression of mitochondrial fission proteins Fisl and Drp1 in the QSG group was significantly reduced(P<0.05,P<0.05),and the expression levels of proteins related to mitochondrial biogenesis pathway PGC-1α,NRF-1 and TFAM in the QSG group were significantly increased(P<0.01,P<0.01,P<0.01).In the vitro experiment,compared with the hypoxia and hypoglycemia model group,the expression levels of the classical pathway proteins PGC-1α,NRF-1 and TFAM in the QSG group increased(P<0.05,P<0.05,P<0.05).The expression of the corresponding myocardial mitochondrial fusion proteins Mfn1 and Mfn2 increased significantly(P<0.05,P<0.05).Although the expression of Opal protein increased but not statistically significant,the fission proteins Fisl and Drpl decreased significantly(P<0.01,P<0.05).<0.05);Compared with the model group,there is no significant difference in mitochondrial biogenesis-related proteins in the cell group co-treated with PGC-1α inhibitor(SR-18292)and QSG,indicating that this pathway is related to mitochondrial dynamics close.Combined with the results of animal experiments,it shows that QSG can regulate the dynamic balance of mitochondria through the PGC-1α/NRF-1/TFAM pathway to improve mitochondrial function.Conclusions:1.QSG can significantly improve the heart function and tissue morphology of rats with heart failure after myocardial infarction.2.Observation under the electron microscope found that QSG can significantly improve the ultrastructure of cardiomyocytes mitochondria;increase the clearance of ROS,increase the membrane potential of mitochondria,increase the content of GSH-PX and ATP,and reduce the content of iNOS,which proves that QSG is significantly improved The morphology and function of mitochondria in the state of heart failure.3.Combining animal experiments and cell experiments,it is confirmed in vitro and in vivo that QSG regulates the expression of NRF-1 and TFAM related pathway proteins of mitochondrial biogenesis by using PGC-1α as the core to improve mitochondrial fusion and reduce mitochondria The division of mitochondria regulates the function of mitochondria,affects energy metabolism,and promotes the recovery of normal function of the heart in rats with heart failure.This study provides a scientific basis for the traditional Chinese medicine compound QSG to regulate mitochondrial dysfunction and inhibit heart failure. |
PDF Full Text Request