Effects Of Monotherapy Danggong-3 On The Activity Of Hypoxia-Reoxygenated Myocytes In Rats

Objective To investigate the effects of Monotherapy Danggong-3 on the Activity of Hypoxia-Reoxygenated Myocytes in Rats.Methods DG-3 compound drug was extracted to obtain the total drug extract,cultured H9c2 cardiomyocytes in vitro,given cell DG-3 drug intervention.Compound danshen drop pill（FF）was used as the positive control group.Co Cl₂solution was used as anoxic agent.Thiazole blue（MTT）was used to detect the cell survival rate,to verify the toxic effect of the drug and to screen the drug concentration,to optimize the H/R conditions,and to simulate the MIRI in vitro（Myocardial Ischemia-Reperusion Injury）.Cardiomyocytes were randomly divided into 6 groups:blank control group（control）,hypoxia/reoxygenation injury group（H/R）,DG-3 low,medium and high dose groups（H/R+low,medium and high corresponding concentrations dose DG-3）,FF dose group（H/R+FF）,the cell survival rate was detected by MTT method,the kit was used to detect the level of CAT（Catalase）in cell homogenate,and LDH（Lactate Dehydrogenase）in cell supernatant as well as the amount of CK（Creatine Kinase）released,Rt-q PCR（Real-time Quantitative）was used to detect the levels of Bax,Bcl-2,and caspase-3 mRNA.Results 1.The MTT result shows that the DG-3 drug concentration is in the range of0.05-0.4 mg/m L,the cell survival rate has no significant change,and there is no statistical significance between the groups（P>0.05）.Within 0.8-6.4 mg/m L,the cell survival rate decreased（P<0.05）,confirming that the low,medium and high drug concentrations of DG-3 were 0.1,0.2 and 0.4 mg/m L,respectively. 2.According to the survival rate of cells,the best condition for determining the H/R model is that the concentration of Co Cl₂is 1000umol/L,the hypoxia time is 21 h,and the reoxygenation time is 2 h.3.Control comparison,H/R cell survival rate decreased（P<0.05）,CAT level in cell homogenate was reduced（P<0.05）,the release of LDH and CK in cell supernatant was increased（P<0.05）,the mRNA levels of Bax and caspase-3 increased（P<0.05）,and the mRNA levels of Bcl-2 decreased（P<0.05）.Compared with H/R ratio,DG-3medium and high dose groups can increase cell survival rate（P<0.05）,reduce LDH and CK release（P<0.05）,increase CAT activity（P<0.05）,and the low,medium and high dose groups can reduce the mRNA expression level of Bax and caspase-3（P<0.05）,and increase the mRNA expression level of Bcl-2（P<0.05）,and the effect can show concentration dependence.Conclusion DG-3 total extract can inhibit cardiomyocyte apoptosis arising from Co Cl₂induced H/R,the protective mechanism may be related to DG-3 ability to reduce the Bax、caspase-3 mRNA level and increase the Bcl-2 mRNA level.