Study For Effect And Mechanism Of Huayujiedufa On PC12 Cell Injury Model Induced By Thrombin And Hypoxia Via ERK1/2 Signaling Pathway

Objective: Stady on thrombin with hypoxia of PC12 cells damage the optimal reaction time point, the establishment of in vitro hypoxia combined with thrombin induced PC12 cell injury model simulated acute ischemic stroke when thrombin combined with hypoxic damage of cells within the environment. Brain cell damage of acute ischemic stroke caused by molecular mechanism and Huayu Jiedu Fang on thrombin combined with hypoxia injury intervention effect and Ras/Raf/ tear mitogen activated protein kinase / extracellular signal regulating kinase signal transduction pathway in the mechanism of action.Methods:With 3 gas incubator and serum glucose free medium with different concentrations of thrombin(0, 50, 100, 150 and 200U/ml) cause cell damage, 1H, 6h, 12 h, 24 h cultured in each group under the hypoxic condition, by methyl thiazolyl tetrazolium(MTT) analysis of cell survival rate, degree of TUNEL method was used to detect cell apoptosis, thrombin with hypoxia on PC12 cell injury model was the best condition was screened, thrombin with hypoxia in vitro cell model was established. The PC12 cells were randomly divided into blank group, model group, Jieduhuayufang group and PD98059 group(ERK1 / 2 inhibitor group), using fluorescent real-time quantitative PCR(real time PCR) method for detection of MEK1, PAR-1 and ERK1 / 2 m RNA expression, immunofluorescence staining was used to detect ERK, p-ERK protein.Results:1. Cell survival rate was detected with MTT The toxic effect of thrombin on PC12 cells was dependent on the time and concentration of thrombin. Thrombin on PC12 cells with the direct toxic effects, low concentrations of thrombin for shorter periods may has certain protective effect on cells, with the extension of time, thrombin to cytotoxic damage increased. The detection of cell viability by MTT showed that 50 U/ml low concentration of thrombin might have a protective effect on the cell. Cell viability decreased with increasing time of hypoxia time prolonged and the concentration of thrombin, especially to hypoxia 12 h after the start cell damage is more obvious, 150 U / ml thrombin group at 12 h and simple hypoxia group(control group) compared to the cell survival rate decreased significantly(P < 0.01). The apoptotic rate increased significantly(P < 0.05) and 200 U / ml thrombin(12h) and 150 U / ml(24h), 200 U / ml(24h) the role of thrombin group after 24 h compared with the control group, the differences were statistically significant(P < 0.05). 2. Apoptosis of PC12 cells was detected by TUNEL fluorescent staining method. The nucleus of the normal control group showed blue fluorescence, with a small amount of green fluorescence, the shape of the nucleus was more regular oval, and the chromatin distribution was uniform, no obvious apoptotic cells were found. With the prolonged hypoxia time and the increase of the concentration of thrombin, the number of green fluorescent cells increased, and the 1H and 6h groups showed irregular morphology. 50 U / ml(12h) and 100 u / ml(12h) thrombin concentration and the number of green fluorescent cells further increased when the thrombin concentration reached 150U/ml, significantly increased the number of green fluorescent cells and nuclei pyknosis, fragmentation, cell death number increase. After the hypoxia of 24 h, most of the cells died, and the number of blue fluorescent cells decreased significantly. 3. PCR Real-Time method for detection of PAR-1, MEK1, ERK1 and m RNA ERK2 expression in PC12 cells. The expression of PAR-1, MEK1, ERK1 and m RNA in the model group was significantly higher than that in the blank group(p<0.01); Jiedu Huayu fang group, the plasma prothrombin(PAR-1), MEK1, ERK1 and ERK2 m RNA expression compared with the model group, the expression of m RNA decreased(P < 0.01), and ERK1 / 2 inhibitor(PD98059 blocked agent group) to compare the m RNA expression difference was not statistically significant(P > 0.05). 4. The expression of ERK1, ERK2 and p-ERK1/2 protein in PC12 cells was detected by immunofluorescence assay. Compared with the blank group, model group p-ERK1 / 2 and ERK1 / 2 increased significantly(P < 0.01); the ERK1 / 2 inhibitor(PD98059 blocked agent group) Jiedu Huayu group can inhibit thrombin with hypoxia induced p-ERK1 / 2 and ERK1 / 2 expression increased(P < 0.01), the Jiedu Huayu decoction group and ERK1 / 2 inhibition agent group, there was no significant difference(P > 0.05).Conclusion: 1. PC12 cells were induced by thrombin combined with ischemia and hypoxia, and the in vitro cell model of acute ischemic stroke(TIH) was established. The model is established on PC12 cells into neuron OGD model based on, in thrombin with ischemia hypoxia for 1h, 6h, 12 h, 24 h after the application of MTT and TUNEL method was used to detect cell survival rate and apoptosis rate. The results showed that, increased with the time prolonged and the concentration of thrombin, cell injury was gradually aggravated, suggesting that the model was successfully established. Two experimental results also showed that the model can cause damage to PC12 cells, successfully simulate the process of AIS, and provide an effective and reliable method for the study of nerve cell injury. 2. After thrombin with ischemia hypoxia injury of nerve cells, prothrombin(PAR-1) and ERK1/2 pathway of m RNA and protein expression significantly enhanced, suggesting that thrombin can aggravate acute ischemic stroke on the cell damage, which may mediate the inflammatory reaction induced by the ERK1/2 signaling pathway, the principle of thrombin may is the role of molecular targets. Inhibition of prothrombin and its receptor mediated inflammatory response by blocking the signaling pathway. It has a significant effect on inhibiting the expression of prothrombin and its receptor and blocking the related signaling pathway, which has a protective effect on PC12 cell injury, but its specific molecular target is not yet clear.