Protective Effect Of Huanglian-4 Decoction On Myocardial Cell Injury Induced By MIRI

Objective:To analyze the composition and metabolism of blood in the body after oral administration of Coptis chinensis and to select a blood component for cell experiments to explore its mechanism of action.The MIRI model of SD rats was established,and the protective effect of myocardial damage induced by MIRI was pretreated with Huanglian-4 decoction based on berberine,further clarifying the protective mechanism of Huanglian-4 decoction in protecting myocardial ischemia-reperfusion injury.Methods:1.According to Mongolian medicine theory,the clinical dose of Coptis chinensis(5g/60kg)was administered to healthy volunteers.After oral administration,ultra-high performance liquid chromatography and high resolution mass spectrometry LTQ-Orbitrap were used to analyze the metabolism of Coptis chinensis in human blood and urine Happening.2.Rat myocardial H9C2 cells were stimulated with COCL2(cobalt chloride)as a cell model of myocardial ischemia/reperfusion injury.Cell viability,ROS detection,apoptosis detection,and cell calcium channel were measured after intervention with berberine.Effects and detection of mitochondrial membrane potential.3.The SD rat MIRI model was established by ligation of the LAD method;randomly divided into sham operation group,model group,Huanglian-4 Decoction component low dose(1.3g/kg)medium dose(2.6g/kg)High dose(5.2g/kg),15 days;Salvia miltiorrhiza group(1.5g/kg),as a ginseng.Experiments were performed using proteomics TMT technique,Western Blot,ELISA,HE staining and other detection methods.Results:1.A total of 14 and 30 components including prototypes and metabolites were identified in human plasma and urine after oral administration of Coptis powder.Among them,the three prototype components(M20,M26,and M28)were compared with standard samples of coptisine,berberine,and palmatine.The metabolic pathway of Coptis chinensis is mainly coupled with glucuronic acid and sulphuric acid to form phase Ⅱ metabolites,and glucuronide conjugates may be the main form of coptis in human blood and urine.2.Berberine can significantly reduce the apoptosis of H9C2 induced by CoC12,protect against oxidative damage,and increase the survival rate of H9C2 cells.However,it did not cause mitochondrial membrane potential changes and changes in cell calcium channels.3.The high-dose group Huanglian-4 Decoction can significantly down-regulate the levels of IL-6 and TNF-alpha,and the effect of Huanglian-4 Decoction is higher than that of Salvia miltiorrhiza group(P<0.05).It can also significantly up-regulate the levels of the oxidative factors GSH-Px and SOD,and down-regulate the levels of MDA without significant differences with the Salvia miltiorrhiza group.At the same time,it significantly down-regulated the level of apoptotic protein BAX,and the effect was similar to that of Salvia miltiorrhiza.Upregulation of BCL-2 protein levels,and the effect of Huanglian-4 Decoction was significantly higher than that of Salvia miltiorrhiza group.After treatment,3827 proteins were found,and 138 differential proteins with significant changes in expression after the MIRI model in rats and after administration were screened.Through cluster analysis of differential proteins,GO enrichment,and KEGG pathway analysis,it was found that "WNT-related pathway"and "ROS oxidative stress signaling pathway" may participate in the pathophysiological process of myocardial ischemia-reperfusion.Conclusions:Mongolian Medicine Huanglian-4 Decoction has protective effect on myocardial injury caused by MIRI,especially in large dose group of Huanglian-4 Decoction.Its mechanism inhibits IL-6,TNF-alpha and down-regulation of oxidative factor MDA,up-regulates GSH-Px and SOD levels and down-regulates the level of pro-apoptotic protein BAX,and up-regulates the inhibition of apoptosis protein BCL-2 protein levels.After using TMT technology,3827 proteins were found,and 138 differential proteins with significant changes in expression after the MIRI model in rats and after administration were screened.Through cluster analysis of differential proteins,GO enrichment and KEGG pathway analysis,it was found that the"WNT-related pathway" and "ROS oxidative stress signaling pathway" may participate in the pathophysiological process of myocardial ischemia-reperfusion.Therefore,Huanglian-4 Decoction may protect myocardial ischemia-reperfusion injury through intervention of WNT-related pathway and ROS oxidative stress signal pathway.In addition to these two signaling pathways,the functional mechanisms of differentially expressed proteins need further study.At the same time,we found that 14 and 30 components including prototypes and metabolites were identified in human blood and urine after oral administration of the main drug of Huanglian-4 decoction.Among them,three prototype components(M20,M26 and M28)were successfully identified as coptisine,berberine and palmatine.The metabolic pathway of Huanglian chinensis is mainly coupled with glucuronic acid and sulphuric acid to form phase Ⅱmetabolites,and glucuronide conjugates may be the main form of Huanglian in human blood and urine.The berberine,one of the prototype components of blood,was selected for cell experiments and it was found that berberine protects rat cardiomyocytes H9C2 from MIRI by inhibiting apoptosis and regulating oxidative stress.The in vivo metabolism of Huanglian chinensis in this experiment and the cellular test of the erythropoietin,the main ingredient of the blood into the blood,were selected as the main single-drug berberine in Huanglian-4 decoction.Therefore,it is necessary to further select other single-taste drugs and other blood components for comprehensive and systematic research in later experiments.Through the exploration of this research topic,it provides a new research idea and new research method for the research of Mongolian medicine compound preparations in search of effective substances.