Protective Study Of Low Temperature On PC12 Cells With Oxy-Gen Glucose Deprivation/Reperfusion Injury

Objective:Establish PC 12 cells cultured in vitro with oxygen glucose depriva-tion/reperfusion (oxygen glucose deprivation and reperfusion, OGD/R) injury model, To study the effects of low temperature on cultured PC 12 cells of oxygen glu-cose deprivation/reperfusion injury protective effects and related mechanisms.Methods:PC 12 cell strain were used for cell culture and passage. After two generations of the cells, the PC 12 cells which were cultured for fourth days were used for the experiment. Application of oxygen scavenger sodium dithionite (Na2S2O4)4), United sugarless DMEM (Dulbecco minimum essential medium Toure Boke minimal essential medium) to establish a method of PC 12 cells OGD/R (oxygen glucose dep-rivation and reperfusion) damage model, MTT (Thiazolyl Blue tetrazolium Bromide MTT) staining and detection of LDH (lactate dehydrogenase, LDH) release to evaluate the extent of PC 12 cells damage. Cultured PC 12 cells were divided into six groups:normal control group, OGD/R normothermia group (OGD/R), OGD/R 32 ℃ group (OGD/R+32℃), OGD/R 32℃ administration group (OGD/R+32 ℃+LY294002 group), OGD/R 27℃ group (OGD/R+27℃), OGD/R 27 ℃ administration group (OGD/R+27 ℃+LY294002 group), Flow cytometry was used to detect the apoptosis of the cells in each group, The protein content of p-Akt was detected by Western-Blot technique, and the results were statistically analyzed by SPSS 17.0 software.Results:(1) MTT analysis:In the normal control group, the cell viability was 100%, and the percentage of PC 12 cells in the OGD/R model group decreased to 21.17% in the control group, and the difference was statistically significant between the two groups (P< 0.05)., Compared with the normal control group, the percentage of PC 12 cells in 27℃ group and 32℃ group were 37.17% and 34.12% respectively. the difference was statistically significant (P< 0.05).OGD/R+27 ℃+LY294002 group and OGD/R+32 ℃+LY294002 group and OGD/R room temperature group, the difference was not statistically significant (P> 0.05). (2) LDH release:Nor-mal control group PC 12 cells only a small amount of LDH release,the LDH release of PC 12 cells in OGD/R model group was significantly increased, which was about 2 times of the normal control group, and the difference was significant (P< 0.05) com-pared with the control group. Compared with the OGD/R group, the LDH release of PC 12 cells in 27℃OGD/R group and 32℃ OGD/R group were decreased, Compared with OGD/R room temperature group, OGD/R+27℃+LY294002+OGD/R+32℃ +LY294002 group, the difference was not statistically significant. (3) The protein expression of p-Akt in low temperature group was higher than that in normal tem-perature group, and the higher the temperature was, the higher the expression level was, the difference was statistically significant (P< 0.05). The expression of p-Akt protein in OGD/R+32℃+LY294002 group was significantly lower than that of OGD/R+32 ℃, the difference was statistically significant (P< 0.05). The expression of p-Akt protein in OGD/R+27℃+LY294002 group was significantly lower than that of OGD/R+27 ℃, the difference was statistically significant (P< 0.05).Conclusions:Hypothermia has a protective effect on PC 12 damage in OGD/R cells, which may related with the PI3K/Akt signaling pathway.