Studies On The Protective Effect And Its Underlying Mechanism Of Different Ingredients Extracted From Gualou-Xiebai-Banxia Decoction On Cardiomyocytes Induced By Ischemia-hypoxia

Objective: Compared the protection of each part of GXBD extraction to cardiomyocytes impaired by ischemia-hypoxia and screened the effective fractions,and then did the research on the possible cell signaling pathways, so that provide scientific basis for the treatment of myocardial ischemia used GXBD.Methods:(1)Drug preparation: Used 50% ethanol to extract Gualou Xiebai Banxia decoction and obtained the total extraction.Then concentrate the extractions into a certain concentration. At last used petroleum ether,chloroform,ethyl acetate and n-butanol to extract successively, and then decoct the dregs of decoction. Recycle the solvent of each extract part by stress recovery, and then dry them by vacuum, obtained petroleum ether part(SM1),chloroform part(SM2),ethyl acetate part(SM3),n-butanol part(SM4), water soluble part(SM5) and dregs of decoctation part(SM6)finally.(2)Cell process: Used newborn Wistar rats and adopted the method of trypsin digestive to extract cardiomyocytes. Then cultured in the serum-free DMEM medium and hypoxia gas to establish the cell damage model.(3) Selection of effective fractions: Each fraction of GXBD extraction was added to culture model cells. Determined the effective fractions and concentration of drugs by cytological observations,MTT method and the level of CK and CK-MB.(4) Studies of the signal pathway: Detected the contents of BK in cell supernatant by ELISA and Ado by HPLC.Results:(1) The rate of cell survival: MTT results showed that SM4 had the best protection to cardiomyocytes damaged by ischemia-hypoxia, and SM5, SM6 also had good protection, and the lowest active concentrations of the three parts were 100 ?g·m L-1(P<0.05).(2) The level of CK and CK-MB: Biochemical kit results showed that the model group was higher than the control group(P<0.01),but SM4, SM5 and SM6 groups were lower than the model group,and the lowest active concentrations of the three parts were 100 ?g·m L-1(P<0.05 or P<0.01).(3)The contents of BK: ELISA results showed that the expression of BK in cells of model group was lower than the control group(P<0.01). While the expression of SM4, SM5 and SM6 groups were all higher than the model group.(4)The contents of Ado: HPLC results showed that the expression of BK in cells of model group was lower than the control group(P<0.01). While the expression of SM4, SM5 and SM6 groups were not significantly higher than the model group(P>0.05).Conclusion: This study extracted and separated GXBD of different fractions.The SM4, SM5 and SM6 had good protection on cardiomyocytes damaged by ischemia-hypoxia, and the lowest active concentrations of the three parts were 100 ?g·m L-1.And its action mechanism may be associated with activated myocardial BK in release,but not with Ado.