Study On Protective Efficacy And Mechanism Of The Insulin Against Paraquat-induced By Using PC12 Cells

Objective:Protective efficacy and mechanism of the insulin against paraquat-induced by using PC12 cells,were investigated in vivo and in vitro studies.1) To observe dopamine DA D2R protein expression on PC 12 cells and its effects of insulin on the survival rate,morphology,and dopamine contents of PQ-induced PC12 ceils.2) To investigate the mechanism that insulin may protect the dopaminergic neuron against paraqua-induce PC12 cells.3) To observe the curative effect on the PD rat models with both insulin and BMSCs interfered.Methods:1) Immunoprecipitate Western blotting method was performed to observe DA D2R protein expression on PC 12 cells, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-zolium bromide(MTT) assay was performed to analyze the alteration of viability and cells morphology in PC 12 cells were exposed to the different PQ and insulin concentrations.2) Based on the PartⅠ, Immunoprecipitate Western Blotting method was performed to observe the expression of INR Tyr^1162/1163 and Akt^Ser473 in the group of normal PC12 cells,insulin interfered PC 12 cells,and PQ-induced and PQ combined insulin interfered in PC 12 cells respectively.3)①From BMSCs cultivating pints of view,Adherent method was used to raise and repeatedly purify BMSCs.In addition flow cytornetry was exploited to detect expression of CD29,CD34,CD44 and CD45,which all the markers existed on the cell's surface of the BMSCs in the third generation;②The rotation test was taken to detect the efficacy rat models of whose striaturn were half destructed by 6-OHDA and induced hemiparkinsonian.Morever high-performance liquid chromatography(HPLC) was adopted for the detection of the DA contents in the brains of PD rat models.③The succeed PD rat models were randomizely divided to three groups,which were employed as injected BMSCs,insulin associates BMSCs and normal saline group,respectively. Four weeks later,the rotating frequency was calculated and the cell's survival was tested by the immunofluorescent method.The DA level in the brain(substantia nigra and striatum) was detected by HPLC.Results:1)①DA D2R protein in quality was expression on PC 12 cells.②Normal PC12 cells bodies showed fusiform shape and the synapsis were integrity.The cells exposed to the 600μM concentration of PQ became ball-like,vacuolar degeneration,and the synapse shorten or disappeared.But the cell's morphology showed a little alteration between the normal PC12 and the insulin groups, except to the cells with different morphology of the fusiform shape or abnomalism,not round shape and the synapsis longer again.③With the increase of the concentration of PQ,the viability of the cells decreased.Insulin increased the viability of the cell which was exposed to the 600μM concentrations of PQ.Insulin elevated dopamine concentration both in the normal PC12 and the cells were exposed to 600μM concentrations of PQ,but there was not the statistical significance(P＞0.05).2) The insulin groups showed the expression in different phosphorylation degree on of INR Tyr^1162/1163 and Akt^Ser473.①CD29,CD34,CD44 and CD45 BMSCs in the third generation were expressed 97.1%,0.0%,99.0%,0.5%.②The rotating frequencies in the insulin group combined BMSCs was much more decreased in comparson with the BMSCs(P＜0.05) and the normal saline group(P＜0.01).③the cell'survival was to observe.Brdu positive cells in the group interfered by insulin combined BMSCs were much more than the BMSCs group(P＜0.01).④DA concentration in the group of insulin combined BMSCs were increased compared to the other 2 groups,however it did not have the statistical significance(P＞0.05).Conclusion:1) Insulin could protect the dopaminergic neuron against PQ-induced PC 12 cells.2) The increased expression on the phosphorylation degree of INR Tyr^1162/1163 and Akt^Ser473 in the insulin groups showed might be one of the clues to prove insulin may have the protective efficacy to PC 12 cells. 3) The curative effect of the insulin combined BMSCs interfered the rat model was better than BMSCs treated group alone.It is essential to provide further evidence in vivo on insulin protect dopaminergic neurons.