| Objective: To observe the function of microglia in different concentrations of insulin and evaluate the role of PI3k/Akt in the changing of microglial function.Methods: BV2 cell and PC12 cell were used to represent microglia and neurons respectively in vitro. BV2 cells were divided as control group and inhibitor group, depending on whether the inhibitor of PI3K/AKt(LY294002) was used or not. Different concentrations of insulin(0 ng/ml,50ng/ml, 100ng/ml,150ng/ml and 200 ng/ml)were added to the supernatant of BV2 cells for 20 h.Then the supernatant was collected to culture PC12 cells respectively for 1h,after that H2O2 was put into the supernatant of PC12 cells. The viability of PC12 cell was determined by MTT assay to reflect the function of microglia in different concentrations of insulin, and make clear the effect of PI3k/Akt in metergasis of microglia. The levels of IL-1β, NGF secreted by BV2 cell were quantified using enzyme-linked immune sorbent assay( ELISA).Results: 1. When the concentrations of insulin were 0ng/ml, 50ng/ml, 100ng/ml, 150ng/ml and 200 ng/ml, the viabilities of PC12 cell in control group were 47.58±0.58%, 58.23±1.07%, 73.67±0.58%, 71.87±1.34% and 44.56±1.31% respectively. The difference was statistically significant by one-way analysis of variance, there were significant differences between every two subgroups(P <0.05), except for the subgroups between the concentrations of insulin being 100 ng/ml and 150 ng/ml. The viabilities of PC12 cell were 48.91±1.21%, 55.31±2.34%, 70.87±1.03%, 51.64±1.43% and 39.01±3.20% respectively in inhibitor group. Compared with control group in the same concentration of insulin(except 0ng/ml), the viabilities of PC12 cell were significantly decreased in inhibitor group(P <0.05).2. As the concentrations of insulin were 0 ng/ml, 50ng/ml, 100ng/ml, 150ng/ml, and 200 ng/ml, the corresponding levels of NGF in control group were 537.01±5.91 ng/L, 564.49±10.18 ng/L,588.23±6.52 ng/L,586.07±15.16 ng/L and 517.25±4.33 ng/L. The difference was statistically significant by One-Way ANOVA, further, the difference was statistically significant between every two subgroups(P<0.05), except for the subgroups between the concentrations of insulin being 100 ng/ml and 150 ng/ml. The levels of NGF in inhibitor group respectively were 522.19±10.13 ng/L, 541.39±5.87 ng/L, 568.49±3.52 ng/L, 528.17±1.43 ng/L and 492.73±10.27 ng/L. Compared with control group in the same concentration of insulin(except 0ng/ml), the levels of NGF in inhibitor group were significantly decreased(P<0.05).3. The contents of IL-1β in control group respectively were16.14±0.15 ng/L, 15.24±0.52 ng/L, 11.73±0.40 ng/L, 13.67±0.56 ng/L and 19.30±0.52 ng/L, when the concentrations of insulin were 0ng/ml, 50ng/ml, 100ng/ml, 150ng/ml and 200 ng /ml. The difference was significant using One-Way ANOVA, there were significant differences comparing any other two subgroups(P<0.05). The levels of IL-1β in inhibitor group respectively were 16.38±0.11ng/L, 15.03±0.56ng/L, 11.67±0.50ng/L, 13.58±0.57 ng/L and 19.38±1.46 ng/L. Compared with control group in the same concentration of insulin, the levels of IL-1β in inhibitor group had no statistical differences(P>0.05). Conclusions: 1. Microglia presented double-edged effect in different concentrations of insulin, it showed protective effect when the ranges of insulin concentration were from 50ng/ml to150 ng/ml, while it presented pathogenic role when the concentration of insulin was more than 200 ng/ml. The transformations of the secretion of NGF and IL-1β might be due to the diversification of microglia function. 2. PI3K/AKt plays an important role in microglial protective effect by regulating the secretion of NGF. |
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