Protective Effect Of Huang Lian-4 Decoction Extract On Hypoxia/Reoxygenation Injury Of H9c2 Cardiomyocytes

Objective:By observing the protective effect of extracts of Mongolian medicine Huang lian-4 Decoction on H/R injury of H9c2 myocardial cells,this study provides a reference for the clinical treatment of myocardial ischemia reperfusion injury by Huang lian-4 decoction.Method:1.The transitional components in the blood of mice after the intragastric administration of Huang lian-4 decoction were identified,and the drug-containing serum samples of mice were detected by liquid-mass spectrometry,and the changes of drug-containing serum at different time points were compared.Molecular docking method was used to analyze the binding ability of blood active ingredients to disease targets Bax and Bcl-2.2.H9c2 cardiomyocytes were treated with extracts of Huang lian-4 decoction at different concentrations for 12 h,24h and 48 h,respectively.The cell viability was detected by CCK-8,and the optimal dosage concentration and time were selected.3.Anoxia/reoxygenation models were established for H9c2 cardiomyocytes by anoxia/reoxygenation methods under different conditions,and CCK-8 was used to detect cell viability and screen out the best modeling conditions.4.H9c2 cardiomyocytes were modeled under the optimal conditions,and then the optimal concentration of Huang lian-4 decoction extract was given,and then the supernatant of the cells was extracted.The myocardial enzymes LDH and CK-MB and the anti-inflammatory factors IL-6 and TNF-α were detected by ELisa.5.H9c2 cardiomyocytes were modeled under optimal modeling conditions,and the optimal concentration of Huang lian-4 decoction extract was given,and then RNA was extracted.The gene expressions of Bax and Bcl-2 in cardiomyocytes were detected by RT-q PCR.Results:1.A variety of alkaloids,phenylpropanes,iridoids,terpenoids and other compounds were found in the blood active components of Huang lian-4 decoction.Among them,swertiamarin,Sinomenine,Gentiopicrin,Ammothamnine(oxymatrine)were selected as the top serum active ingredients,Bax and Bcl-2,which were related to MIRI through literature review The target has good binding ability.This study indirectly verified the protective effect of Huang lian-4 decoction on cardiomyocyte H/R.2.The results of different concentrations of the drug on H9c2 cardiomyocytes showed that(50-400)μg/m L,the cell growth rate of Huang lian-4 decoction extract was increased,indicating that the drug could enhance the activity of H9c2 cardiomyocytes to a certain extent.Among them,the cell growth rate increased significantly after treatment with 150μg/m L for 48 h,and the efficacy was relatively stable.Therefore,150μg/m L concentration of drug was selected for follow-up experiment in this study,and the drug action time was 48 h.3.The results of CCK-8 data showed that compared with the normal group,the apoptosis rate of 3h/ 3h hypoxia group was significantly different;The apoptosis rate was stable.4.Elisa data showed that compared with the normal group,the expression levels of IL-6,TNF-α,CK-MB and LDH in the supernatant of cell culture in the model group were significantly increased(P<0.01).Compared with model group,the expression levels of IL-6 and LDH in cell culture supernatant in treatment group were significantly decreased(P<0.05).The expression level of CK-MB was significantly decreased(P <0.01).The expression level of TNF-α was decreased,but there was no significant difference(P >0.05).5.RT-q PCR data showed that Bax expression level in the model group was significantly higher than that in the normal group(P<0.01).Compared with model group,Bax m RNA expression level in treatment group was significantly decreased(P<0.01).Compared with normal group,the expression level of Bcl-2 in model group was significantly decreased(P<0.01).Compared with model group,the m RNA expression level of Bcl-2 in treatment group was significantly increased(P <0.01).Conclusion:The extracts of Huang lian-4 decoction can protect H9c2 myocardial cells from hypoxia/reoxygenation injury,and its protective mechanism may be related to reducing myocardial enzymes,reducing inflammatory factors and inhibiting cell apoptosis.