| Aims:To do the research on the effect of Qinghuobaiduyin on Hsp70/p-Akt pathway and apoptosis related factor Caspase-3in intestinal epithelia after burn injury both in vivo and in vitro,to investigate the protective mechanism of these factors to intestinal epithelia after burn injury.Methods:Select136healthy SD rats, to divide them into three groups randomly:normal group(control group),burned-only group(burned group),burned-QHBDY treatment group(treatment group).According to the treatment dosage of QHBDY,the treatment group were divided into three groups,they were0.5ml/100g, lml/100g,1.5ml/100g groups.The intestinal tissues were collected from burned group and treatment group at six hours,twelve hours,twenty-four hours and forty-eight hours to do the pathology experiments. Intestinal tissues were collected to investigate the apoptosis rate by TUNEL, and the expression level of Hsp70,Caspase-3were measured by real time PCR and immunohistochemistry. To construct Hsp70-pZsGreenl-C1expression vector,and transfect this vector into EEC-18cell. Real time PCR was used to identify the Hsp70mRNA expression level and Western blot was used to identify the Hsp70protein and p-Akt protein relative expression level at the time point of twelve hours,twenty-four hours,forty-eight hours and seventy-two hours after transfection.FCM was used to identify the influence of Hsp70on cell apoptosis rate after transfection, Caspase-3spectrophotometry testing kit was used to identify the influence of Hsp70on Caspase-3activity.For the research at cell level, firstly we do the research to choose the serum concentration and drug concentration and treatment time,according to the cell livability,we chose the best serum concentration and drug concentration and treatment time.We chose three serum concentrations and one time points to treat IEC-18cell,we used FCM to analysis the effect of burn serum and (or) drug on cell apoptosis rate, Caspase-3spectrophotometry testing kit was used to identify the influence of burn serum and (or) drug on Caspase-3activity. Western blot was used to analyze the protein expression of Hsp70under the treatment of burn serum together with drug.Results:The apoptosis rates of intestinal epithelial cell of burned SD rats were increased. The apoptosis rates of lml/100g and1.5ml/100g groups were statistically different from the burned group(P<0.05) at the time point of six hours,twelve hours,twenty-four hours and forty-eight hours. The expression of Hsp70in the intestinal tissue of burned SD rats was increased both at the mRNA level and protein level, the expression of Caspase-3in the intestinal tissue of burned SD rats was increased at the protein level.After QHBDY was given, the Hsp70and Caspase-3relative expression of1.5ml/100g group were statistically different from the burned group(P<0.05). We have successfully constructed Hsp70-pZsGreenl-Cl expression vector,after transfection, Western blot results shows that after the Hsp70expression was increased,meanwhile, the p-Akt expression was increased accordingly.The relative activity of Caspase-3was decreased,and the cell apoptosis rate was decreased also.The MTT data shows that burn serum and drug have concentration effect and time effect on EEC-18cell livability. To treat IEC-18cell together with burn serum and QHBDY,compared to burn serum group,the Caspase-3activity was decreased in drug treatment group,also the cell apoptosis was decreased,statistical difference was exist between the two group (P<0.05).Western blot result shows that Hsp70expression was higher in drug treatment group than in burn serm group.Conclusions:after burn injury, the expression of Hsp70was increased both at the mRNA level and protein level,it may play a protective role. QHBDY could elevate the expression level of Hsp70in severely burned rats, it could decrease the expression of Caspase-3, also it could decrease the apoptosis rate,so HQBDY may play an anti-apoptotic role in animal model. Hsp70protein could increase the expression of p-Akt, Hsp70could decrease the relative activity of Caspase-3and decrease the cell apoptosis rate also. QHBDY may have a protective effect on IEC-18cell that treated with burn serum,it could increase the expression of Hsp70,decrease Caspase-3activity and cell apoptosis rate.There are... |
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