| Objective Primary culture the hippocampal neurons of SD newborn rats, using Immunofluorescence double staining technology (IF) and Confocal laser scanning microscope (CLSM) to observe the levels of myosin light chain kinase (MLCK) and the phosphorylation ERK(p-ERK) of Hippocampal neurons in high glucose environment, to clarify the the relevance of MLCK and p-ERK,the effect of Tetrahydroxystilbene Glucoside(TSG) on MLCK and MAPK/ERK pathway. 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 time of high glucose:set up the experiment groups:25mM group,45mM-24h group(high glucose 24h),45mM-48h group(high glucose 48h) and 45mM-72h group(high glucose 72h),25mM was the control group conventional neuronal cells in culture,using IF and CLSM to observe the fluorescence intensity of MLCK, ERK and the p-ERK in hippocampal neurons, using IPP6.0(image pro Plus6.0) image analysis software for processing data of fluorescence images and SPSS 17.0 software was used for statistical analysis of data, analyze the level of expression of MLCK, ERK and p-ERK, to determine the optimum glucose action time of increased expression of MLCK started with high glucose; 3.The relevance of MLCK and p-ERK:according to the experimental result of method 2,the high glucose concentration was established as 45mM, culture hippocampal neuron cells in 48h and grouping:GT control group(high glucose into control group), ML-7 group (MLCK inhibitor 20μmol/L role 8h), PD group(ERK inhibitor 30μmol/L role 24h), using IF and CLSM to observe the levels of MLCK and the p-ERK in hippocampal neurons; 4.The levels of MLCK and p-ERK in hippocampal neurons started with TSG:according to the different dose of TSG, culture hippocampal neuron cells in 24h and grouping:TSG-L(10μmol/L), TSG-M group(50μ mol/L) and TSG-H group(100μmol/L),using IF and CLSM to observe the levels of MLCK and the p-ERK in hippocampal neurons. Result 1. 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.With the extension of time of culturing hippocampal neurons, the levels of MLCK and p-ERK of 45mM group gradually stronger than the 25mM group, they were positively correlated (r=0.881, P<0.01),but ERK was unchanged(P>0.05). The levels of MLCK and p-ERK of 45mM-48h group and 45mM-72h group were changed with 25mM group significantly (P<0.01),but the two groups were no difference on MLCK and p-ERK levels, so 48h was the most suitable high glucose time; 3.Compared with GT group, started with the MLCK inhibitor ML-7 in hippocampal neurons, the level of MLCK decreased (P<0.01), but the p-ERK level unchanged(P>0.05); Started with the ERK inhibitor PD98059, the levels of MLCK and p-ERK were both decreased(P<0.01), they were positively correlated(r=0.933,P<0.01); 4.Compared with GT group, started with TSG in hippocampal neurons, the levels of MLCK and p-ERK were both decreased, they have dose-dependent manner. The levels of MLCK and p-ERK were positively correlated (r=0.861,.P<0.01). Compared with GT group, the levels of MLCK and p-ERK in TSG-H group decreased significantly (P<0.01), they were positively correlated (r=0.861, P<0.01). Conclusion 1.Increase the glucose concentration of medium can cause the increased expression of MLCK and p-ERK levels in hippocampus neuron, but the ERK unchanged; 2.P-ERK can affect the level of MLCK,the level of p-ERK decreased, thus affecting the level of MLCK; 3.TSG may affect the level of p-ERK, through the MAPK/ERK pathway to regulate the expression of MLCK. |
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