The Protection Effects And Mechanism Of Puerarin On RSC96 Cells Damage Induced By High Glucose

Objective: diabetic peripheral neuropathy(DPN)is one of the most common complic-ations of diabetes mellitus,mainly for multiple,symmetric end neuropathy,its patho-genesis is not clear.More and more Chinese medicine research in this field get more attention,this study aimed to investigate the protection effects and mechanism of Puerarin on RSC96 cells damage induced by high glucose.Methods: Observing normal Schwann cell morphological structure and using PCR and Immunocytochemical method for identification of the Schwann cell;Selecting the 3-5 generation of RSC96 Schwann cell line for MTT colorimetric assay,which were divided into 4 groups: blank group(with normal 1640 med ium,no cells),control group(normal 1640 culture medium + cells),model gro up(cultured cells 24 h in in high glucose with the concentration of 200 mM+1640 medium),Puerarin intervention group(the concentration of the interventio n group is 0.01,0.1,1 mmol/L puerarin +1640 medium,cultured cells for 3 h),each group sets 5 holes,the experiment was repeated 3 times;The change of mitochondrial membrane potential were observed by JC-1 staining;The Immuno histochemical staining was used to detect the expression of NGF in the cells;T he expression of NGF ?caspase-3?AKt and pAKt protein in the cells were de tected by Western Blot.Results: The cultured RSC96 Schwann cell line which was used in this experi ment,growed and proliferated in good condition,the cells showed long spindle shape with protrusions.the expression and positioning of S-100 protein is pos itive and accurate in cells,which was,identified by immunocytochemistry and PCR method.MTT assay results showed that the 24-72 h of Schwann cells cultured af ter vigorous growth,72 h slowed down;compared with the control group,the o ptical density of glucose in the model group 24 h values(optical density,OD)decreased significantly,there was statistical significance(P<0.05);compared with the high glucose group,the OD value of Puerarin in each group were higherthan the high glucose group,and showed a concentration dependent,with statis tical significance(P<0.05).The results of JC-1 staining: normal Schwann cells showed green fluorescence,cell apoptosis is gradually turned into orange fluorescence.Compared with the control group,the number of cells in high glucose group was significantly reduced,and the cytoplasm appears orange fluorescence;compared with the model group,puerarin group,with its concentration(0.01,0.1,1 mmol/L)graduall y increased from low to high,showing the orange fluorescent cells decreased g radually,and the green fluorescence increased consequently.Immunocytochemistry results:Compared with the normal group,the positive expression of NGF in model group and each treatment group was significant ly decreased(P<0.05);compared with the model group,the expression of NG F protein in each treatment group increased significantly(P<0.05);no signific ant difference between the treatment group(P>0.05).Western Blot results: compared with normal group,the expression of NGF protein in high glucose group was significantly reduced;compared with the model group,NGF protein expression in puerarin groups increased,especially i n the high concentration group(1 mmol/L)it had significant difference(P<0.05).Compared with the normal group,the expression of caspase-3 protein in th e model group increased significantly(P<0.05);but compared with the model g roup,the expression of caspase-3 protein in the intervention group of puerarin was obviously decreased,which in high concentration group(1 mmol/L)show ed significant differences(P<0.05).Compared with the control group,the expres sion of pAkt decreased in the high glucose group of 200 mM,there was a si gnificant difference(P<0.05);but in puerarin intervention group with high conc entration,the expression of pAkt protein was increased(P<0.01),then PI3 K inhi bitor(LY2940002)treatment group,the expression of pAkt protein was less than it in puerarin group.Conclusion: high glucose can inhibit the proliferation of Schwann cells,and negatively correlated with the concentration of glucose;Puerarin can inhibit damage of Schwann cell induced by high glucose,promote the expression of NGF protein in RSC96 cells,inhibit the activity of Caspase-3;the puerarin can pro mote the phosphorylation of Akt of RSC cells in high glucose environment,an d this effect was blocked by inhibitors of P13K(LY294002),It's said that this mechanism of puerarin is achieved through the activation of the PI3K/AKt signaling pathway.