Effects Of Low Temperature Combined With Sodium Hydrogen Sulfide On Apoptosis And Expression Of GSK-3β And Bcl-2 In PC12 Cells By Oxidative Stress Injury

Objectives:To replicate the model of oxidative stress in PC 12 cells injured by H2O2,to investigate the protective effect of low temperature combined with hydrogen sulfide on the oxidative stress injury of nerve cells,and the possible mechanism of low temperature combined with H2S against oxidative stress injury in nerve cells.Methods:PC12 cells were cultured routinely,the cells adherent 80%-90%and with good growth status were selected,PC 12 cells were damaged 12 hours by H2O2 at the end concentration of 100μmol/L,then we got the model of oxidative stress in neurons.RT-QPCR methods were used to detect the expression of IRAK1 and TNF-a two indexes of PC 12 cells before and after oxidative stress injury,and obtain the target cells of this experiment;Experimental group(n=3),the normal blank control group(37℃,5%CO2,24h);Low temperature intervention group:the target cells were cultured at 37℃,32℃,27℃,5%CO2 incubator,24h;Low temperature combined H2S intervention group:using NaHS as H2S donor,adding NaHS to culture medium,adjusting the final concentration of 100μmol/L,respectively,cultured at 37℃,32℃,27℃,5%CO2 incubator,24 hours;NaHS control group:37℃ 24 hours.Then the proliferation of cells was detected by CCK-8;The morphological changes of PC 12 nuclei after oxidative stress injury by H2O2 for 12 hours were observed by fluorescence microscopy and Hoechst33258 fluorescence staining;FITC and PI double staining and FCM were used to detect apoptosis;The protein and mRNA contents of Bcl-2 and GSK-3β in different intervention groups were detected by electrophoresis、Western Blot and RT-QPCR techniques,the results was analyzed by SPSS17.0.Results:1.PC12 cells grew well at 27-37℃ range;2.H2O2 oxidative stress model is feasible and effective;3.Low temperature can reduce the down-regulation of Bcl-2 protein and mRNA expression by H202,and this effect can be further enhanced by H2S combined with low temperature;4.Mild hypothermia can alleviate the up regulation of H2O2 on Gsk-3β protein,but there was no significant difference between the deep hypothermia group and the mild hypothermia group,H2S combined with low temperature can further reduce the up regulation of H2O2 on GSK-3β protein expression;Hypothermia can alleviate the up regulation of H2O2 on GSK-3βmRNA,H2S combined with hypothermia can further alleviate this effect.Conclusions:(1).The model of PC 12 cell in vitro is simple and convenient,and it can be easily repeated,can simulate the process of low temperature after oxidative stress injury,It is a good model of neuron hypothermia in vitro.(2).After PC12 oxidative stress,the expression of Bcl-2 protein and mRNA was down regulated,and the expression of GSK-3β protein and mRNA was up-regulated.(3).At the temperature range of 27℃-37℃,with the decrease of temperature,the expression of Bcl-2 protein and mRNA increased gradually,indicating that low temperature can promote the transcription、expression of Bcl-2 protein,and alleviate apoptosis,H2S can strengthen this effect.(4).At the temperature range of 27℃-37℃℃,with the decrease of temperature,the expression of GSK-3 beta protein and mRNA decreased gradually,H2S could enhance this effect.(5).The effect of low temperature combined with H2S on the expression of GSK-3 β-and Bcl-2 proteins and mRNA may be one of the mechanisms of brain protection at low temperature.