| Objective:By establishing a cellular oxygen glucose deprivation model and treating PC 12 cells with PI3K signaling pathway inhibitor LY294002,we examined the apoptosis and PI3K protein expression changes in PC 12 cells under different temperatures to elucidate the mechanism of cell protection by low temperature through PI3K/AKT signaling pathway.Methods:PC 12 cells were cultured,and after cell passaging culture,PC 12 cell oxygen glucose deprivation model was established by using sodium hyposulfite(Na2S2O4),combined with sugar-free DMEM,and the degree of PC 12 cell injury was evaluated by CCK-8 cell counting method,IRAKI,TNF-α inflammatory factors to determine whether the model construction was successful.The PC 12 cells cultured in vitro were divided into 9 groups,3 control groups,3 OGD/R groups and 3 LY294002 groups.The control group,OGD/R group and LY294002 group were put into 3 temperature segments for culture:①normothermic group(37℃ incubator for 24h),②sub-low temperature group(32℃ incubator for 24h)and③deep low temperature group(27℃ incubator for 24h).After measuring cell activity by CCK-8 assay for cell viability followed by DnnexinV/PI double-staining method to detect apoptosis,the expression of PI3K and p-PI3k proteins in the three groups of cell specimens were detected by Western-Blot technique,and the results were analyzed by SPSS17.0.Results:(1)CCK-8 method to determine the cell activity of OGD/R group with different time treatments:with the 0thh of OGD/R group as the control group,the cell activity decreased at 3h,and the difference between the two groups was not statistically significant(P>0.05);after 6h,it decreased significantly compared with the control group,and the difference between the two groups was statistically significant(P<0.05);with the extension of OGD/R time,the cell activity of 12h and 24h(2)CCK-8 method to determine the activity of PC 12 cells at different temperatures:37℃ control group cell viability as 100%,for the OGD/R group and LY294002 group within the comparison,37℃ and 32℃ LY294002 group and OGD/R group cell activity differences are not statistically significant(P>0.05);to 27℃ control group activity was 100%,for intra-group comparison between OGD/R group and LY294002 group,the cell activity of LY294002 group decreased,the difference was statistically significant(P<0.05).The cell activity of 37℃normothermia group,32℃ subcooling group and 27℃ deep low temperature group were compared between groups,and the cell activity of 32℃ subcooling group and 27℃ deep low temperature group were increased with the control of 37℃normothermia group,and the difference was statistically significant(P<0.05)(3)The cellular ischemic and hypoxic damage of OGD/R group was verified by PCR amplification method with IRAK1 and TNF-α inflammatory factors as indicators.IRAK1:After 0h culture as control,the expression of IRAK1 increased with time after 6h treatment of cells OGD/R,and the difference was statistically significant(P<0.05);with the prolongation of OGD/R time,the mRNA expression of IRAK1 continued to increase at 12h and 24h,and the difference was statistically significant(P<0.05).TNF-α:After 0h culture as control,the expression of IRAK1 increased with time,and the difference was statistically significant(P<0.05).As a control,the mRNA expression level of TNF-α increased with time after OGD/R treatment of cells for 6h,and the difference was statistically significant(P<0.05).With the prolongation of OGD/R time,the expression of TNF-α at 12h versus 24h,increased with time,and the difference was statistically significant(P<0.05).(4)Apoptosis analysis by DnnexinV/PI double-staining method:Under the three temperatures of 37℃normothermia group,32℃ sub-low temperature group and 27℃ deep low temperature,comparing the OGD/R group with LY294002 group,the apoptosis rate of LY294002 group was significantly increased,and the differences were all statistically significant(P<0.05).Comparing apoptosis in 37℃ room temperature group,32℃ sub-low temperature group and 27℃ deep low temperature group between groups,apoptosis was decreased in 32℃ sub-low temperature group and 27℃ deep low temperature group with 37℃ room temperature group as the control group,and the difference was statistically significant(P<0.05)(5)Western-Blot measurement of p-P I3K/PI3K protein expression:comparing the OGD/R group with LY294002 group p-PI3K/PI3K ratio was compared,and the p-PI3K/PI3K was significantly decreased in the LY294002 group with the OGD/R group as the control,and the difference was statistically significant(P<0.05);the expression of p-PI3K/PI3K was compared within the group for the three groups of cells in the 37℃normothermia group,32℃ sub-cold group,and 27℃ deep cold group.With the 37℃normothermia group as the control,the p-PI3K/PI3K ratio of cells in the 32℃ subcold group and 27℃ deep low temperature group were decreased,and the difference was statistically significant(P<0.05).Conclusion:(1)PC12 cells cultured at three temperatures of 37℃,32℃ and 27℃ without any intervention conditions,the cell viability was the highest in the 37℃ group.(2)After OGD/R treatment,PC12 cells cultured at 37℃,32℃ and 27℃,the cell viability of the 27℃ group was the highest.(3)PC 12 cells treated with OGD/R for 6h showed significantly higher expression of IRAK1 and TNF-α inflammatory factors,which could mimic the ischemic stroke model.(4)The addition of inhibitor LY294002 decreased PI3K protein expression and increased apoptosis rate.(5)Among the three temperatures of 37℃,32℃ and 27℃ in this experiment,the highest expression of phosphorylated PI3K signal and the lowest apoptosis rate were observed in the 27℃ cell group. |
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