| Coronary atherosclerotic heart disease(CHD)has become a major public health problem.According to the China Cardiovascular Health and Disease Report 2021,the number of cardiovascular diseases in China is about 330 million,of which 11.39 million are coronary heart disease patients.The prevalence and mortality of acute myocardial infarction(AMI)are both on the rise.At present,coronary heart disease is commonly treated with revascularization and routine secondary prevention,and most patients can benefit from it.However,some patients have complicated conditions,such as severe coronary artery lesions,failure to open vessels,angina pectoris after PCI,stent thrombosis,etc.,all of which affect the efficacy of patients to varying degrees.TCM is effective in treating coronary heart disease by means of invigorating qi,warming Yang,promoting blood circulation and dredging collaterals.It can improve clinical symptoms,reduce inflammation and improve patients’ quality of life.Yiqi Huoxue Formula is an effective prescription summarized by the research group combined with years of clinical practice and famous traditional Chinese medicine experience.The drug composition includes turmeric,Astragalus,codonopsis,Rhizome,Rhizoma medica,cinnamon,Gualou,rhubarb,asarum and licorice,which can benefit Qi and huoxue,disperse cold and relieve pain,Tongyang and reduce turbidity,and treat coronary heart disease syndrome of Qi deficiency and blood stasis.Therefore,clarifying the mechanism of Yiqi Huoxue Formula in treating myocardial injury of coronary heart disease is a key problem to be solved urgently in clinic.This study was derived from the National Natural Science Foundation of China(81973836),"Mechanism of repairing mitochondrial damage in myocardial Infarction by invigorating Qi and Activating Blood Circulation based on miR-126-5p/Bcl-2/mPTP regulatory Pathway and Dynamic visualization Technology".By analyzing the distribution of the syndromes of acute myocardial infarction,the main clinical syndromes were determined.The hypoxia and reoxygenation model of H9C2 cardiomyocytes was constructed using the three-qi incubator to simulate myocardial injury.The overexpression and inhibition of miR-126-5p were achieved by cell transfection,and the effects of Yiqi Huoxue Formula and its main effective components astragaloside Ⅳ and curcumin on myocardial injury were observed.Based on the regulatory pathway of miR-126-5p/Bcl-2/mPTP,the mechanism of action was discussed to clarify the way of improving mitochondrial energy metabolism of cardiomyocytes with Yiqi Huoxue Formula,providing certain basic value for the clinical treatment of coronary heart disease and myocardial infarction.ObjectiveThis paper includes clinical syndrome analysis and cell experiment,aiming to explore the mechanism of repairing myocardial cell mitochondrial damage by Yiqi Huoxue Formula based on miR-126-5p/Bcl-2/mPTP regulatory pathway.Specifically,they are:1.Analyze the distribution characteristics of TCM syndromes and medication rules of patients with acute myocardial infarction of coronary heart disease in Guang’anmen Hospital,China Academy of Chinese Medical Sciences.2.To observe the effect of Yiqi Huoxue Formula on H9C2 cardiomyocyte injury induced by hypoxia/reoxygenation,and construct miR-126-5p overexpression and underexpression H9C2 cardiomyocytes through cell transfection.To further explore the mechanism of Yiqi Huoxue Formula and its main active components astragaloside Ⅳand curcumin in improving myocardial cell mitochondrial damage based on miR-1265p/Bcl-2/mPTP regulatory pathway.Meanwhile,mitochondrial pressure and glycolysis rate were measured by seahorse XFe 24,and the mechanism of improving mitochondrial energy metabolism and inhibiting myocardial cell apoptosis based on miR-126-5p was clarified.Method1.A total of 272 patients diagnosed with acute myocardial infarction in the cardiovascular Department of Guang ’anmen Hospital,China Academy of Chinese Medical Sciences from January 2018 to December 2022 were included in the part of syndrome analysis.Clinical data of patients were collected,and statistical analysis was performed using Excel and SPSS 28.0.2.Cell experiment2.1 The hypoxia and reoxygenation model of H9C2 cardiomyocytes was constructed in a three-gas incubator,and the optimal concentration of drugs on H9C2 cardiomyocytes was screened by CCK-8 method.The experiment was conducted according to cell grouping.After the intervention,the LDH release level of myocardial supernatant and ROS content of myocardial cells were observed.Tunel was used to detect the apoptosis rate of cardiomyocytes.The open level of mitochondrial mPTP was detected by flow cytometry.The expression levels of miR-126-5p,Bcl-2,Caspase-3 and Caspase-9 were detected by qRT-PCR.2.2 Overexpression and inhibition of miR-126-5p was achieved through cell transfection.The experiment was conducted according to cell grouping.After the intervention,the LDH release level of myocardial supernatant and ROS content of myocardial cells were observed.Myocardial cell apoptosis was detected by Tunel.The open level of mitochondrial mPTP was detected by flow cytometry.The expression levels of miR-126-5p,Bcl-2,Caspase-3 and Caspase-9 were detected by qRT-PCR.Western Blot was used to detect the release level of Cyt C and the expression levels of Bcl-2,Caspase-3 and Caspase-9 proteins.2.3 Cell culture was performed according to cell groups.Seahorse XFe24 instrument was used to determine mitochondrial pressure and glycolysis rate,and changes in mitochondrial energy metabolism of cardiomyocytes were observed.Results1.Part of syndrome analysis:Clinical syndromes of 272 patients with acute myocardial infarction in Guang ’anmen Hospital,China Academy of Chinese Medical Sciences were analyzed,and it was found that:(1)Gender and age can affect the occurrence and prognosis of acute myocardial infarction.(2)Distribution law of TCM syndromic elements:blood stasis(86.03%),phlegm turbidness(51.47%),heat accumulation(34.56%)were the main evidence,followed by qi stagnation(9.56%)and cold coagulation(6.62%).The main deficiency syndrome was Qi deficiency(61.03%),followed by Yin deficiency(33.09%)and Yang deficiency(19.12%).(3)Distribution law of TCM syndrome:Qi deficiency and blood stasis syndrome(52.21%)>phlegm-stasis interassociation syndrome(38.24%)>Qi and Yin deficiency syndrome(29.41%)>Phlegm-heat internal disturbance syndrome(20.59%)>Yin and Yang deficiency syndrome(8.82%)>Qi stagnation and blood stasis syndrome(8.09%)>cold coagulation heart pulse syndrome(6.62%),among which the main syndrome was Qi deficiency and blood stasis syndrome.(4)The use frequency of traditional Chinese medicine in AMI patients was analyzed.There were 23 kinds of traditional Chinese medicine with frequency>60,which were Radix Astragalus,pinellia japonica,Ligusticum ligusticum,Red peony root,poria trichosanthes,sauteed jujube kernel,angelica sinensis,Allium macrostemon,Trichosanthes Trichosanthes,Radix antiphala,Radix coptidis,Radix erythricae,Radix salvia miltiorrhiza,Radix rehmanniae,raw keel,raw oyster and honeysuckle.2.Cell experiment2.1 To observe the effects of Yiqi Huoxue Formula on the injury of H9C2 cardiomyocytes,it is found that:(1)The optimal hypoxia time and reoxygenation time of H9C2 cardiomyocytes were determined to be 24h and 12h.(2)The optimal low concentration of Yiqi Huoxue Formula was determined to be 5μg/ml,the optimal moderate concentration was 10μg/ml,and the optimal high concentration was 20μg/ml,of which the medium concentration was the best;The optimal concentration of curcumin was 50μg/ml.The optimal concentration of astragaloside Ⅳ was 50μg/ml.The optimal concentration of trimetazidine hydrochloride tablets was 10μg/ml.(3)Compared with blank group,LDH content of H9C2 cardiomyocyte supernatant in model group was significantly increased(P<0.01);Compared with model group,LDH content in Yiqi Huoxue Formula groups,curcumin group,astragaloside Ⅳ group and trimetazidine group was significantly decreased(P<0.01).(4)Compared with blank group,ROS content of H9C2 cardiomyocytes in model group was significantly increased(P<0.01);Compared with model group,ROS content in Yiqi Huoxue Formula groups,curcumin group,astragaloside Ⅳ group and trimetazidine group was significantly decreased(P<0.01).(5)Compared with blank group,the apoptosis rate of H9C2 myocardial cells in model group was significantly increased(P=0.03<0.05);Compared with the model,the apoptosis rate of Yiqi Huoxue Formula groups,curcumin group,astragaloside Ⅳgroup and trimetazidine group decreased significantly(P<0.01).(6)Compared with blank group,the mPTP openness of H9C2 cardiomyocytes in model group was significantly increased(P<0.01);Compared with model group,the open level of mPTP in Yiqi Huoxue Formula groups,curcumin group,astragaloside Ⅳgroup and trimetazidine group was significantly decreased(P<0.01).(7)The expression levels of miR-126-5p(P<0.01)and Bcl-2 gene could be upregulated in all dosage groups of Yiqi Huoxue Formula,curcumin,astragaloside Ⅳ and trimetazidine(P<0.05);Down-regulated Caspase-3(P<0.05)and Caspase-9 gene expression levels(P<0.05).2.2 By observing the effects of Yiqi Huoxue Formula on the injury of H9C2 cardiomyocytes based on the regulatory pathway of miR-126-5p/Bcl-2/mPTP,it is found that:(1)Compared with miR-126-5p low-expression cardiomyocytes under different treatment conditions,the LDH of cardiomyocytes in miR-126-5p overexpression group decreased significantly;ROS release content decreased significantly;The apoptosis rate of miR-126-5p overexpressed cardiomyocytes was lower,CalceinAM level was higher but mPTP openness was lower.Overexpression of miR-126-5p can reduce the Cyt C release level of H9C2 cardiomyocytes.(2)Overexpression of miR-126-5p can up-regulate the expression level of miR126-5p in cardiomyocytes;Up-regulated expression of Bcl-2 gene and protein;Downregulated gene and protein expression levels of Caspase-3 and Caspase-9.2.3 The mitochondrial pressure and glycolysis rate of H9C2 cardiomyocytes were measured by seahorse,and the changes of mitochondrial energy metabolism of cardiomyocytes were observed.(1)Mitochondrial pressure results:Compared with normal cardiomyocytes,miR-126-5p overexpressed cardiomyocytes and miR-126-5p underexpressed cardiomyocytes blank group,the OCR value of H9C2 cardiomyocytes in each model group was significantly decreased.Compared with model group,Yiqi Huoxue Formula groups,curcumin group,astragaloside Ⅳgroup and trimetazidine group could increase cell basic oxygen consumption to different degrees.Improve proton leakage levels;Increase non-mitochondrial oxygen consumption and ATP production capacity;Increase the maximum respiratory capacity and reserve respiratory capacity of cells.(2)glycolysis rate:Compared with normal cardiomyocytes,miR-126-5p overexpressed cardiomyocytes and miR-126-5p underexpressed cardiomyocytes blank group,the ECAR value of H9C2 cardiomyocytes in all model groups was significantly decreased(P<0.01).Compared with model group,Yiqi Huoxue Formula low-dose group,astragaloside Ⅳgroup and trimetazidine group could increase the level of cellular basal glycolysis to varying degrees.The overall ECAR value of cardiac cells with low miR-126-5p expression was low.ConclusionGender and age can affect the occurrence and prognosis of acute myocardial infarction.The syndrome of AMI was mixed with deficiency and accumulation,in which the deficiency syndrome was mainly Qi deficiency,and the empirical evidence was mainly blood stasis,phlegm turbidness and heat accumulation.The main pathogenesis is Qi deficiency and blood stasis,and the main clinical method is qi tonifying and blood activating.The commonly used Chinese medicines include astragalus,red peony root,ligusticum Chuanxiong,pinellia fascia,poria cocos,etc.,which can guide the clinical treatment based on syndrome differentiation.To a certain extent,Yiqi Huoxue Formula and its main active components astragaloside Ⅳ and curcumin can improve myocardial injury after hypoxia,reduce oxidative stress response,reduce mitochondrial membrane permeability,reduce the release of CytC apoptotic factor,and inhibit myocardial cell apoptosis.Its mechanism may be related to up-regulation of miR-126-5p expression level,up-regulation of Bcl2,down-regulation of Caspase-3 and Caspase-9 gene and protein expression levels,improvement of mitochondrial pressure and glycolysis rate,and improvement of mitochondrial energy metabolism level. |
