The Correlation Between MLCK Expression And Mapk/erk Pathway In Rats’ Hippocampal Neurons Induced By High Glucose And The Protection Mechanism Of Tsg On Neurons

Objective: 1. To establish the primary culture model of rat hippocampal neurons and determine the optimal concentration and time of action.2. To detect the expression of ERK, p-ERK and MLCK in hippocampal neurons under the action of high glucose. 3. To investigate the correlation between MLCK and MAPK/ERK signaling pathway in cultured hippocampal neurons under high glucose environment. 4. To make a preliminary study on the mechanism of the decrease of MLCK expression induced by two styrene glycosides after high glucose. Methods:1. Primary culture and purity identification of the hippocampal neurons: culture the hippocampal neurons of SD newborn rats by Trypsin digestion, observe the cell morphology in Inverted microscope and identify the purity by NSE immunohistochemistry. 2. Explore the optimum concentration of high glucose:Primary cultured hippocampal neuron cells, when the cells culture for fifth days,divided them into 25 m M-24 h group, 25mM-48 h group, 25mM-72 h group,45hmM-24 h group, 45mM-48 h group, 45mM-72 h group; After each corresponding sugar action time, detected the OD of of each group by CCK-8, to get the optimum effect of high glucose concentration. 3. Explore the optimum time of high glucose:Primary cultured hippocampal neuron cells and divided the cells into 25 mM group,45mM-24 h group,45mM-48 h group, 45mM-72 h group; After each corresponding sugar action time, stained the cells by immunohistochemistry, then observed the expression of ERK, p-ERK and MLCK with laser confocal microscopy.4. The expression of ERK, p-ERK and MLCK in hippocampal neurons after the effect of high glucose: Primary cultured hippocampal neurons, then the cells were dividedinto the control group and the high glucose group; After reaching the high glucose effect of 48 h, collecting the protein and detected the concentration by BCA; Then detected the expression of MLCK and p-ERK by western blot. 5. Discussion on the correlation between p-ERK and MLCK: Primary cultured hippocampal neurons for fifth days, then the cells were divided into high glucose group, ERK inhibitor group(PD group) and MLCK inhibitor group(ML-7 group); At the same time of the high glucose effecting, the ERK inhibitor group injected PD98059 inhibitor to effect for 24 h and the MLCK inhibitor group injected MLCK inhibitor to effect for 8h; After reaching the high glucose effect of 48 h, collecting the protein and detected the concentration by BCA, then detected the expression of MLCK and p-ERK by western blot. 6. Protective effects of TSG on the cells of hippocampal neurons: Primary cultured hippocampal neurons for fifth days, then the cells were divided into high glucose group, TSG-L group(TSG concentration were 10 mol/L), TSG-M group(TSG concentration were 50 mol/L) and TSG-H group(TSG concentration were 100 mol/L);At the same time of high glucose effecting, adding the corresponding concentration of TSG effected for 24h; After reaching the high glucose effect of 48 h, collecting the protein and detected the concentration by BCA; Then detected the expression of MLCK and p-ERK by western blot. Result: 1. Primary culture and identification of hippocampal neurons: Successfully cultured primary hippocampal neurons by Trypsin digestion. Culture until the 7th day, cells in the inverted microscope appear as:mature neuronal morphology, larger cell volume, Aggregation of survival, obvious halo, developed neurite extended outwardly, mutually connected to form a dense network. The identification of the hippocampal neurons purity was more than 90%.2. Study on the optimum concentration of high glucose: The CCK-8 results showed that the survival rate of 45mM-24 h group, 45mM-48 h group and 45mM-72 h group was >80%, which belonged to lever 1 cells, which could be used in the following experiments. 3. Study on the optimum time of high glucose: Under the confocal laser scanning we found, with increasing sugar concentration, in the hippocampal neurons,the fluorescence intensity of ERK was relatively constant(P > 0.05), on the contray,the fluorescence intensity of p-ERK and MLCK were significantly increased(P <0.05); Compared with the 45mM-24 h group, the 45mM-48 h and 45mM-72 h group increased significantly(P<0.05), but there’s no difference between 45mM-48 h group and 45mM-72 h group(P>0.05). 4. The expression of p-ERK and MLCK in hippocampal neurons after the effect of high glucose: The western blot results showed that, compared with the control group, the expression of p-ERK and MLCK in the hippocampal neurons of the high glucose group was significantly increased(P<0.01),and there’s no significant change in the expression of ERK(P>0.05). 5.Discussion on the relationship between p-ERK and MLCK under the effect of high glucose: Western Blot results showed that, compared with the high glucose group, the MLCK expression of ML-7 group declined obviously(P<0.01), but the p-ERK level did not change significantly(P >0.05); The MLCK and p-ERK expression levels were both decreased in the PD group(P<0.01), and they were positively correlated(r=0.878, P<0.01). 6. Protective effects of TSG on the cells of cultured hippocampal neurons: Western blot results showed that TSG-M group, TSG-H group MLCK expression and p-ERK expression were both reduced(P<0.05), MLCK expression was positively correlated with the level of p-ERK(r=0.829, P< 0.01); The expression of MLCK and p-ERK in TSG-L group was decreased but the difference was not statistically significant(P>0.05). Conclusion: 1. High glucose can increasing the expression of MLCK and p-ERK in hippocampal neurons, but the expression of ERK was not changed. 2. Under the effect of high glucose, the expression of MLCK in hippocampal neurons is regulated by the MAPK/ERK signaling pathway. 3. TSG can regulate the expression of MLCK through MAPK/ERK pathway, by which protect the hippocampus neurons cells.