The Experimental Research On Protective Effect And Mechanism Of Sofren On H9C2 Cell Ischemia-reperfusion Injury

Purpose:The aim of the study is to discuss on the protective effect of salidroside on H9C2 cell with myocardial ischemia-reperfusion injury, and is also to explore its mechanism and to provide a scientific and objective basis for myocardial ischemia-reperfusion injury by using Chinese medicine.Methods:H9C2 cell line inoculate to cell culture bottle, then take logarithmic phase cell to detect index of correlation. Dividing cells into four groups:normal control、I/R、T/R+S、I/R+S+LY294002.1. normal control:H9C2 cells were cultivated in cell incubator(37℃、21%O2、5%CO2)for 24h;2.1/R:10% DMEM were placed to simulation of oxygen liquid and cell were placed in three gas incubator(95%N2 and 5%CO2) for 16h,next, H9C2 were cultured in 10% DMEM medium and incubator 8h to simulate reperfusion;3.I/R+S:at the placing simulation of oxygen liquid time, put in 20ul 200ug/ml salidroside solution, other operations like the I/R;4.I/R+S+LY294002:on I/R+S basis, join LY294002,the LY294002 in the previous 30 minutes. other operation slike the I/R.After completed those experiments, detecting the protective of effect of salidroside on cell multiplication by MTT method, detecting ROS level,the LDH content of cell inside and outside to assess degree of injury of myocardial cell. Detecting change of AKT and Pakt,GSK3-β and p-GSK3-β.The levels of pAKT and p-GSK3-b protein wee detected by western-bloting.Results:1.The OD value determined by MTT method compared between groups:①I/R、I/R+S、I/R+S +LY294002 compared with CON group:The OD values obviously low(p<0.05);②I/R compared with I/R+S,The OD valuesobviously low(p<0.05);③I/R+S compared with I/R+S+LY294002,The OD valuesobviously high(p<0.05);④I/R compared with I/R+S+LY294002(p>0.05),there have no statistical difference.2. Comparison between the LDH content groups:①I/Rcompared with I/R+S, the extra cellar of LDH levels increased obviously(p<0.05);the inside cell of LDH content below obviously(p<0.05)②I/R+S compared withI/R+S+LY294002,the extra cellar of LDH levels below obviously(p<0.05); the inside cell of LDH content higher obviously(p<0.05)③I/Rcompared withI/R+S+LY294002, the outside cell of LDH content have no obviously difference(p>0.05), the inside cell of LDH content slightly lower.3. ROS levels between groups:①I/Rcompared with I/R+S, The content of ROS increased significantly(p<0.05);②I/R+S compared withI/R+S+LY294002,The content of ROS lower significantly(p<0.05).③I/Rcompared with I/R+S+LY294002(p>0.05), there have no statistical difference.4. AKT phosphorylation(pakt) protein expression compared between groups:①I/R compared with CON group, pakt phosphorylation protein expression slightly increased,But the difference have statistically significant(p<0.05)②I/Rcompared with I/R+S,the I/R+S group obviously increase the phosphorylation evel(p<0.05);③I/R+S compared withI/R+S+LY294002,the I/R+S group obviously increase the phosphorylation level(p<0.05).6. GSK3-βphosphorylation(p-GSK3-β) protein expression compared between groups:①I/R compared with CON group, pgsk3-β phosphorylation protein expression slightly increased, the difference have statistically ;ignificant(p<0.05)②I/Rcompared with I/R+S,the I/R+S group obviously increase the phosphorylation evel(p<0.05);③I/R+S compared with I/R+S+LY294002,the I/R+S group obviously increase the )hosphorylation level(p<0.05).Conclusions:Salidroside can inhibit cell apoptosis,lowering the release level of ROS, Play a role of )rotection of H9C2 cells in ischemia-reperfusion injury, This protective effect may be related to the activation of PI3K/AKT/GSK3-β signaling pathways.