Study On The Protective Effect Of Huoluoxiaoling On Endothelial Cells

ObjectiveCardiovascular disease accounts for China’s urban and rural residents died first, cell apoptosis involved in the pathological process of many cardiovascular diseases, such as arrhythmia, atherosclerosis, and endoplasmic reticulum stress is one of the mechanisms of apoptosis. More and more researches showed that the traditional Chinese medicine can protect the cell apoptosis by regulating the endoplasmic reticulum stress. The previous research of our research group found Huoluoxiaoling Dan (HLXLD)could inhibit the occurrence and development of atherosclerosis in rabbits fed with high lipid, and the improvement of type 2 diabetic rats blood glucose and glucose tolerance, also reduced vascular smooth muscle cell phenotype conversion. This experiment is mainly to explore the specific mechanism of action. First using hydrogen peroxide induced apoptosis, explore HLXLD’S effect on apoptosis, and then through the test the protein detection related endoplasmic reticulum stress, explore the mechanism of HLXLD inhibiting apoptosis, so as to provide new experimental evidence and research ideas for prevention and treatment of cardiovascular disease.Methods1.Establishing of cells apoptosis model induced by hydrogen peroxide (H2O2) and determining the HLXLD’s active effect concentration range and the protective effect of concentration. Cells were divided into normal group, model group, HLXLD’s the concentration gradient group, and medicine added before H2O2, through the detection of cell viability determining HLXLD’s concentration.2.To investigate the role of HLXLD on cell apoptosis. Cells were divided into normal group, model group, HLXLD’s the concentration gradient group (80μg/ml、40μg/ml、 20μg/ml), and quercetin group, medicine adding before H2O2. Cell viability, cell morphology and apoptosis were observed. The expression of apoptosis protein caspase-9 and caspase-3 were detected by western blot.3.To explore the active effect mechanism of HLXLD regulating of endoplasmic reticulum stress to inhibit apoptosis. Cells were divided into normal group, model group, HLXLD’s the concentration gradient group (80μg/ml、40μg/ml、20μg/ml), and quercetin group, medicine adding before H2O2. Western blot was employed to detect the expression of the endoplasmic reticulum stress related protein GRP78, XBP1.Results1. Determination the concentration of H2O2 and HLXLDAfter H2O2 acting on cells, cell survival rate decreased with the increase of the concentration of H2O2. Compared with the normal group, after 500μM H2O2 treated cells, the cell viability significantly reduced (P<0.01) were 65.0 ± 4.7%, and determined the concentration to induce the cell apoptosis. HLXLD serum had inhibitory effect on endothelial cell proliferation (P<0.01), while the concentration were 20μg/ml、40μg/ml、80μg/ml of HLXLD’s water decocted liquid, the cell viability without obvious effect, but the dose dependence, so with the three concentrations as subsequent experimental concentration.2.HLXLD can inhibit the cell apoptosis induced by H2O2After H2O2 acting on cells and cell morphology occurred obvious change, compared with the normal group, the cell shrinking, and peripheral cell gap became larger, the apoptosis rate increased (P<0.01). HLXLD pretreatment significantly protected cells, with improving the morphology of cell shrinkage, decreasing apoptosis rate (P<0.01). In the detection of Western Blot, compared with the normal group, model group caspase-3, caspase-9 protein high expressed (P<0.01). Compared with the model group,80μg/mll could significantly reduce the expression of caspase-9 and caspase-3, and the expression of the two decreased in a dose-dependent.3.HLXLD regulated endoplasmic reticulum stress inhibiting cell apoptosis induced by H2O2hi the detection of GRP78 and XBP1 with Western Blot, compared with the normal group, the model group GRP78, XBP1 protein upexpression (P<0.01). Compared with the model group, HLXLD could significantly reduce the expression of GRP78 and XBP1 (P<0.01).Conclusions1.HLXLD could decrease the rate of endothelial cell injury and cell apoptosis, and down regulated the expression of caspase-3, caspase-9.2.HLXLD’s protective effect on endothelial cell may related to endoplasmic reticulum stress, through lowered the endoplasmic reticulum stress protein GRP78, XBP1 expression.