| Purpose:This study established atherosclerosis（AS）animal model of Rabbits in New Zealand.and ox-LDL-induced cardiomyocyte lipid injury model,to observe the effect of Huayu Qutan recipe on mitochondrial energy metabolism of AS rabbits and ox-LDL-induced lipid injury cardiomyocytes,and to explore the possible effect and mechanism of Huayu Qutan recipe on mitochondrial energy metabolism disorders of AS cardiomyocytes With the energy metabolism of mitochondrial respiratory chain as the main entry point,the effect and mechanism of Huayu Qutan recipe and c AMP-PKA pathway on energy metabolism of atherosclerotic myocardium were discussed,so as to provide objective experimental basis for treating cardiovascular diseases with phlegm and blood stasis.Material and method:1 Animal experiments in vivoAnimal grouping and modeling:According to the preliminary research results of the research group,the effect of huayu Qutan Recipe is the best when the dosage is high,so the high dose is used in this study.A total of 45 healthy SPF male New Zealand white rabbits were randomly divided into the control group,the model group,and the Huayu Qutan recipe group,Each group of 15 only.All groups were fed with normal diet for 2 weeks and control group was fed with normal diet and injected with normal saline solution by ear vein;other groups were fed with high fat diet and injected with bovine serum albumin（BSA）by ear vein solution for 8 weeks.1.1 intervening method:The group of Huayu Qutan Recipe was given the corresponding drug treatment by intragastulation with the dose of 16g·kg-1·d-1and the volume of 8m L·kg-1·d-1for continuous 4 weeks,while the control group and the model group were given the same volume of normal saline.Sampling and index detection:The rabbits in each group were given 10%chloral hydrate by ear vein slowly.After complete anesthesia,the abdominal cavity was opened in a sterile state,and the heart tissue of the rabbits was separated.The 4%paraformaldehyde was fixed and stored at room temperature for hematoxylin-eosin（HE）and Masson staining to observe the myocardial histomorphological changes.The tissue was frozen in-80℃ultra low temperature refrigerator,Then PCR array was used to detect the expression of energy metabolism-related genes in myocardial mitochondria of rabbits.Immunohistochemical observation was used to detect the myocardial tissue of each group,and Western Blot was used to detect the protein expression levels of CREB and NDUFS4 in myocardial mitochondria of rabbits in each group.2 Cell experiments in vitro2.1 Huayu Qutan Recipe preparation of drug-containing serum and cell groupingThe H9C2 cardiomyocyte lipid damage model was established by 50ug/m L ox-LDL,and the H9C2 cardiomyocytes were divided into blank group,model group,TCM group,TCM with inhibitor group.2.2 Cell culture and screening of intervention conditionsH9C2 cardiomyocytes were resuscitated,and conventional subculture was carried out to the third generation.cck-8 method was used to screen the optimal concentration of intervention cells with drug-containing serum.2.3 Indicator DetectionThe mitochondrial complexⅠactivity and mitochondrial ATP level of H9C2 cardiomyocytes in each group were measured by multifunctional microplate analyzer.The expression content of c AMP in H9C2 myocardial cells was detected by ELISA.The m RNA expressions of PKA,CREB and NDUFS4 in H9C2 myocardial cells were detected by Realtime RT-PCR.Western Blot was used to detect the levels of camp-PKA pathway related protein p-PKA,p-CREB and NDUFS4 in H9C2 cardiomyocytes.Results:1 Animal experiment in Vivo1.1 staining results showed:Compared with normal group,the myocardial cells in model group were disordered,hypertrophy and swelling.Some cardiomyocytes were atrophic and necrotic,vacuoles appeared around the nucleus.Even the nucleus is missing.Mild hyperplasia of the intercellular fibrous tissue of myocardium and obvious blood stasis phenomenon;Compared with the model group,the myocardial cells in Huayu Qutan recipe group were clearly defined,neatly arranged,and slightly swollen.There was no obvious atrophy and necrosis of myocardial cells,vacuoles around the nucleus,and no obvious proliferation of fibrous tissue.1.2 PCR array results showed:Compared with the normal group,39 of 84 genes related to mitochondrial energy metabolism in the model group were down-regulated with significant statistical difference（P<0.05）.Compared with the model group,the expression of 40 genes in the Huayu Qutan recipe group was significantly up-regulated after drug treatment,and the expression trend was reversed with statistical difference（P<0.05）.1.3 Compared with the normal control group,the expression of NDUFS4 protein in the myocardial tissue of the model group was significantly decreased（p<0.01）.Compared with model group,the expression of NDUFS4 protein in myocardial tissue of rabbit was significantly increased in huayu Qutan Recipe group（p<0.01）.1.4 Compared with the normal group,The expression levels of protein CREB and NDUFS4 in model group were significantly decreased（p<0.01）.while compared with the model group,the expression levels of protein CREB and NDUFS4 in huayu Qutan Recipe group significantly increased（p<0.01）.2 Cell experiments in vitro2.1 Compared with the blank group,the mitochondrial respiratory chain enzyme complex I activity of H9C2 cardiomyocytes in model group was significantly decrease（p<0.01）;Compared with model group,the mitochondrial respiratory chain enzyme complex I activity of H9C2 cardiomyocytes in TCM group was significantly increased（p<0.01）;Compared with the TCM group,the mitochondrial respiratory chain enzyme complex I activity of H9C2 cardiomyocytes in the TCM with inhibitor group wassignificantly decreased（p<0.05）2.2 Compared with the blank group,ATP level of H9C2 cardiomyocytes in model group was significantly decreased（p<0.01）;Compared with model group,ATP level of H9C2cardiomyocytes in TCM group was significantly increased（p<0.01）;Compared with the TCM group,the ATP level of H9C2 cardiomyocytes in the TCM with inhibitor group was significantly decreased（p<0.05）.2.3 Compared with blank group,c AMP content of H9C2 cardiomyocytes in model group was significantly decreased（p<0.01）;Compared with model group,c AMP content of H9C2cardiomyocytes in TCM group was significantly increased（p<0.01）;Compared with the TCM group,c AMP content of H9C2 cardiomyocytes in the TCM with inhibitor group wasdecreased but there was no statistical difference.2.4 The m RNA expression of PKA,CREB and NDUFS4 in H9C2 cardiomyocytes was significantly down-regulated in model group compared with blank group（p<0.01orp<0.05）;Compared with the model group,the m RNA expression of CREB NDUFS4 was significantly up-regulated in TCM group（p<0.05）;The m RNA expression level of PKA NDUFS4 in H9C2 cardiomyocytes in TCM with inhibitor group was significantlydown-regulated compared with that TCM group（p<0.05）.2.5 Compared with blank group,the expression levels of c AMP-PKA pathway related protein p-PKA,p-CREB and NDUFS4 in H9C2 cardiomyocytes in model group were significantly down-regulated（p<0.01）;Compared with model group,the protein expression levels ofp-PKA,p-CREB and NDUFS4 in H9C2 cardiomyocytes in TCM group were significantly up-regulated（p<0.01or p<0.05）;Compared with the TCM group,the protein expression levels of p-PKA,p-CREB and NDUFS4 in H9C2 cardiomyocytes in the TCM with inhibitor group were significantly down-regulated（p<0.01or p<0.05）Conclusion:1.Huayu Qutan Recipe can improve myocardial cell injury in AS rabbits.2.Huayu Qutan Recipe interferes with energy metabolism disorder of AS rabbitcardiomyocytes by regulating the expression of mitochondrial respiratory chain enzyme complex I related protein.3.Huayu Qutan Recipe regulates the activity of mitochondrial respiratory chain enzyme complex I through the c AMP-PKA pathway,and affects the energy metabolism ofcardiomyocytes,thus improving myocardial injury. |
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