Study On The Protective Effect Of Ramulus Cinnamomi-Radix Glycyrrhizae Herb Pair On H9c2 Myocardial Cell Injury

Objective:According to the statistics of the World Health Organization,coronary heart disease has become one of the diseases with high prevalence and fatality rate,which has caused heavy economic burden to the society.It has become a major public problem threatening people’s health.The decrease of myocardial oxygen supply is a common pathological state of heart diseases.How to reduce the hypoxia injury of myocardial cells is the key to the treatment of cardiovascular diseases.According to the"Multi-component-multi-target-multi-pathway"mechanism,traditional Chinese medicine has unique advantages in the treatment of myocardial anoxia,which is increasingly prominent in the treatment of cardiovascular diseases.Ramulus Cinnamomi-Radix Glycyrrhizae（RC-RG）herb pair was commonly used in clinical compatibility and was a classic herb pair for the treatment of cardiovascular diseases,but the potential active ingredients and therapeutic mechanism of"RC-RG"for the treatment of myocardial anoxia were still unclear.In this study,the difference of active ingredients before and after herb pair compatibility was studied through fingerprint.The extracts before and after herb pair compatibility were applied to the H9c2 hypoxic injury model to study the difference of treating MIRI.The mechanism of"RC-RG"herb pair on treating myocardial hypoxia was predicted through network pharmacology and verified by cell experiment.Molecular docking method was used to predict the active components of"RC-RG"in the treatment of myocardial hypoxia,and H9c2 cell experiments were conducted to verify the mechanism of active components.This study attempted to identify the effect of active small molecule component and target protein to clarify the action mechanism of active components.Methods:A High Performance Liquid Chromatography method（HPLC）was constructed for the determination of the active components of RC-RG decoction.The fingerprint method was used to analyze the effects of the decocting method and the compatibility on the components of RC-RG decoction to determine the appropriate decocting method for the herb pair.The hypoxia injury model of H9c2 cardiomyocytes was established by Co Cl₂.The extracts before and after the drug combination were applied to the injury model.The cell viability,Lactate Dehydrogenase（LDH）,Reactive Oxygen Species（ROS）,Superoxide Dismutase（SOD）,Propidium Iodide（PI）/Calcein-AM and Mitochondrial Membrane Potential（MMP）were detected to compared the difference of effects of RC-RG before and after the combination.The network pharmacology was used to predict the mechanism of"RC-RG"in the treatment of myocardial hypoxia and the Western Blot was used to determine the effect of"RC-RG"on the expression of phosphatidylinositol 3-kinase/protein kinase B（PI3K/Akt）pathway related proteins and apoptosis related proteins,in order to verify the network pharmacology speculation.Finally,the binding strength between the active ingredient and PI3K/Akt pathway protein was studied by molecular docking method to screen out the potential active ingredient.The screened components were applied to the H9c2 cell hypoxia model,and the therapeutic effect and mechanism of the selected components on myocardial hypoxia were studied by measuring relevant indexes.Results:The HPLC method was established for the analysis of the active components of“RC-RG”.The results of methods verification showed good adaptability,precision,accuracy and so on.The analysis results showed that the extraction efficiency of active ingredient of the co-decoction of RC-RG significantly was higher than those of the single decoction.The extraction efficiency of liquiritin,isoliquiritin,coumarin,cinnamic alcohol,cinnamic acid,cinnamic aldehyde and glycyrrhizic acid was higher when Ramulus Cinnamomi was extracted without peeling and under reflux.The results of applying the extracts before and after the combination of the two drugs to the hypoxia model indicated that the extracts could improve the cell survival rate,reduce the leakage amount of LDH in the culture medium,improve the level of SOD,reduce the ratio of PI/Calcein,increase the level of MMP and reduce hypoxia-induced apoptosis.However,the treatment effect of the combination of RC-RG on hypoxia was better than RC or RG alone.The mechanism of action of"RC-RG"in the treatment of myocardial hypoxia was analyzed by network pharmacological.101 active ingredients were screened out and 181 targets were common targets between RC-RG and myocardial hypoxia and were predicted to be potential targets for the treatment of myocardial hypoxia.PPI network analysis and KEGG pathway analysis predicted that Akt1,TNF and IL6 were the potential targets which may be related to PI3K/Akt signaling pathway.Western Blot results showed that,compared with the model group,"RC-RG"could protect myocardial cells by reducing the expression of apoptotic protein Caspase family proteins and increasing the ratio of p-PI3K/PI3K and p-Akt/Akt.The ratio of p-PI3K/PI3K and p-Akt/Akt in the"RC-RG"+PI3K inhibitor LY294002 group was significantly decreased compared with the"RC-RG"group.According to the comprehensive energy of molecular docking and literature review,four active components of coumarin,cinnamic acid,cinnamic aldehyde and glycyrrhizic acid were screened out,and the effects of these four components were further studied through cell experiments.Cellular experiments showed that these four components could improve the survival rate of cells damaged by hypoxia,reduce the leakage of LDH,increase the expression of SOD,reduce the ratio of dead cells to living cells,increase the level of MMP.Coumarin,cinnamic acid and cinnamic aldehyde could increase the ratio of p-PI3K/PI3K and p-Akt/Akt,while glycyrrhizic acid could decrease the ratio of p-PI3K/PI3K and increase the ratio of p-Akt/Akt.Conclusion:The compatibility of"RC-RG"pair could increase the dissolution of the active components,while the effect of the compatible extract on myocardial hypoxia was better than that of the single decoction.It has been verified by network pharmacology and cell experiments that"RC-RG"could regulate PI3K/Akt pathway in the treatment of myocardial hypoxia.Molecular docking and cell experiment results showed that the active components of coumarin,cinnamic acid,cinnamic aldehyde and glycyrrhizic acid in the herb pair played a role in the treatment of myocardial hypoxia through promoting the phosphorylation of Akt.In this study,various methods were used to explore the small molecule active components and action mechanism of"RC-RG"in the treatment of myocardial hypoxia,and the correlation between the main component activities and pharmacological effects was clarified,which provided a theoretical basis for clinical treatment of myocardial hypoxia.