The Effect Of Wenyangtongmai Prescription On Hypoxia/reoxygenation Injury Of H9C2 Cardiomyocytes By Endoplasmic Reticulum Stress Pathway

ObjectiveTo investigate whether the serum containing Wenyangtongmai Prescription could inhibit cardiomyocyte apoptosis and protect H9C2 cardiomyocytes from hypoxia/reoxygenation injury of induced through endoplasmic reticulum stress-related pathway.Methods33 SD rats were divided into four groups: blank control group（15 rats）;low-,medium-and high-dose Wenyangtongmai prescription groups（6 rats in each group）.The rats were given normal saline and Wenyangtongmai Prescription with different doses（3.24g/kg,6.48g/kg,12.96g/kg）.The rats were intragastrically administered for 3 days.The blank serum and the serum containing Wenyangtongmai Prescription were prepared respectively by common carotid artery blood collection method.The H9C2 cells were divided into 5 groups: blank serum control group,model group,and Wenyangtongmai Prescription groups with low-,medium-and high-dose.Establish hypoxia/reoxygenation model on H9C2 cardiomyocytes:Chemical anoxic agent（CoCl₂）were used to establish the an hypoxic model on H9C2 cardiomyocytes for 22 h,and then Fresh medium were used to establish the reoxygenation model for 2h.The optimum serum dosage（20%）and the optimal concentration of CoCl₂（600μM）during hypoxia/reoxygenation were assessed by Tetramethylazozolium salt method（MTT）.The blank serum control group and model group were treated with 20% blank serum,and Wenyangtongmai Prescription groups with low-,medium-and high-dose were treated with 20% Wenyangtongmai Prescription Serum with low-,medium-and high-dose respectively.The cell viability of each group were assessed by MTT assay;The enzyme activity of lactate dehydrogenase（LDH）in supernatant and the cysteine protease 3（Caspase3）were determined by Colorimetry;Morphological changes of apoptosis were observed by Hoechst33342 fluorescence staining;Flow cytometry was used to detect the apoptosis rate of each group;And the expressions of GRP78,Caspase12,CHOP,Bcl-2 and Bax were detected by Western Blot.ResultsCompared with the blank serum control group,the cell viability of the model group was significantly reduced,the levels of Caspase3 and LDH of the model group were significantly increased（P<0.01）,the number of apoptotic cells showing by apoptotic morphology was significantly increased,and the apoptosis rates showing by Flow cytometry was significantly increased（P<0.01）;and the expression of GRP78,Caspase12,CHOP and Bax were remarkably increased while Bcl-2 was remarkably decreased（P<0.01）.Compared with the model group,the cell viability of Wenyangtongmai groups with low-,mediumand high-dose were significantly increased to varying degrees,the levels of Caspase3 and LDH were significantly decreased to varying degrees（P<0.01）,and the rates of apoptosis were decreased to varying degrees（P<0.05）,the expression of GRP78,Caspase12 and Bax were decreased to varying degrees（P<0.05）,the expression of CHOP were decreased to varying degrees（Wenyangtongmai groups with medium-and high-dose P<0.01）,the expression of Bcl-2 were increased to varying degrees（Wenyangtongmai groups with medium-and high-dose P<0.01）.The therapeutic effect of Wenyangtongmai groups with high-dose is better than Wenyangtongmai groups with low-and medium-dose.ConclusionsWenyangtongmai Prescription could protect H9C2 cells from hypoxia/reoxygenation injury,and inhibit cardiomyocyte apoptosis.Its mechanism may be related to inhibit the endoplasmic reticulum stress-mediated apoptosis.Its protective effect and anti-apoptotic effect were dose-dependent and the best effect was obtained by the high dose of Wenyangtongmai Prescription group.