Stilbene Glucoside Mediated MAPK/JNK Signaling Pathway Regulation Of Expression Of MLCK And Apoptosis Of Hippocampal Neurons In Rat

Objective: to detect the expression of hippocampal nerve cell apoptosis induced by high glucose and intracellular MLCK,JNK and p-JNK;to investigate the correlation between MLCK and MAPK/JNK signaling pathway;preliminary study on protective effect of two styrene glycoside on hippocampal neurons in rats.Methods: neonatal rat hippocampal neurons of SD rats were cultured in 1.primary 25mM: sugar concentration in cultured hippocampal neurons of sugar concentration,light microscope and morphological changes of neural cells,cultured for seventh days,using NSE immunohistochemical staining with nerve cells,the cell purity was identified;2.nerve cell apoptosis in hippocampus of water Flat detection: the primary cultured hippocampal neurons,25 mM concentrations of glucose to fifth days after nerve cell groups:(1)control group: the 25 mM concentration of sugar maintenance medium to culture 48h;(2)high glucose control group: sugar concentration by 45 mM cultured in high glucose 48h;(3)ML-7 group: join the MLC in the high glucose 45 mM.At the same time K inhibitor ML-7 8h;(4)SP group: adding JNK inhibitor SP600125 12 h 45mM in high glucose at the same time;(5)two styrene glycosides low,medium and high concentration intervention group: two stilbene glucoside in 24 h treated with 45 mM high glucose effects.At the same time were added to 10 ?mol /L,50?mol /L and 100?mol /L;the effect of high glucose on 48 h after harvest Apoptosis rate of each group by flow cytometry.3.the effect of high glucose,the expression of JNK,p-JNK and MLCK in hippocampus neurons: hippocampal neurons to fifth days,the nerve cells were divided into control group and high glucose group,high glucose 48 h after extraction of cell protein,BCA protein,the expression of Western Blot detected in hippocampus neurons,JNK,p-JNK and MLCK,and the Statistical analysis.Study on correlation between p-JNK 4.and MLCK: the primary cultured hippocampal neurons to fifth days,the nerve cells were divided into high glucose group,ML-7 group and SP group,high glucose 48 h after the extraction of cell protein,BCA protein,the expression of Western Blot detected in hippocampus neurons of p-JNK and MLCK,and statistical analysis.5.effects of two stilbene glucoside role on high glucose injured hippocampal cells: the primary cultured hippocampal neurons to fifth days,the nerve cells were divided into 5 groups,respectively: control group,high glucose group and two styrene glycosides low,medium and high concentration group,high glucose 48 h after the extraction of cell protein,BCA protein Western,Blot.The expression of JNK,p-JNK and MLCK measurement of hippocampal nerve cells in each group.Results: the primary culture and identification of 1.hippocampal neural cells: the primary cultured hippocampal neurons,8h after inoculation of adherent growth is good,seventh days to mature by NSE immunohistochemical staining.The cell purity was over 85%,to meet the requirements of following experiments.Detection of 2.hippocampal neuronal apoptosis level: flow cytometry showed that the apoptosis rate of nerve cells in high glucose group was much higher than that of control group was 12%;compared with high glucose group,ML-7 group and SP group of nerve cell apoptosis rate were decreased(P < 0.01),the two Houhai horse intervention stilbene glucoside the apoptosis rate of nerve cells decreased high concentrations Low concentration significantly(P < 0.05);3.the effect of high glucose,the expression of JNK,p-JNK and MLCK in hippocampus neurons: Western Blot results showed that compared with the control group,the expression of JNK was not changed after the effect of high glucose(P > 0.05),while p-JNK and MLCK expression was significantly increased(P < 0.01),and the two have positive correlation(r=0.9025,P < 0.01);4.the effect of high glucose,MLCK and p-JNK to discuss the correlation of nerve cells in the hippocampus: Western Blot test results show that: compared with the high glucose group,adding MLCK inhibitor ML-7 expression of hippocampal neurons of MLCK decreased significantly(P < 0.01),while p-JNK showed no significant change(P > 0.05);adding JNK inhibitor SP600125 after p-The expression of JNK was significantly decreased(P < 0.01),while the expression of MLCK also decreased(P < 0.01),and the expression of two changes have a positive correlation(P < 0.01,r=0.8924).The protective effect of 5.two styrene glycoside on hippocampal neural cells: Western Blot results showed that: compared with high glucose group,the expression of two stilbene glucoside in low concentration group of MLCK and the change of p-JNK had no significant difference(P > 0.05),decreased expression of two stilbene glucoside concentration groups MLCK and p-JNK(P < 0.05),two stilbene glucoside the high concentration of group MLCK and p-The expression of JNK decreased significantly(P < 0.01),with obvious dose dependence,and MLCK and p-JNK expression had a positive correlation(P < 0.05,r=0.9539).Conclusion: 1.high glucose can induce the apoptosis of hippocampal neurons.2.sugar concentration increased,increased MLCK and p-JNK expression in hippocampal nerve cells,and the expression of MLCK by JNK signal pathway.3.two stilbene glucoside intervention can inhibit the phosphorylation of JNK,down-regulation of MLCK expression,thus to protect the hippocampal neurons in sugar concentration.