Effect of lipopolysaccharide on C6 glioma cells injuried by H_2O_2

Objective To observe the effect of lipopolysaccharide(LPS) on malignant glioma C6 cells injuried by hydrogen peroxide(H2O2),investigate the mechanism maybe involved.Methods The experiment was divided into four groups: control group,100 μmol·L-1 LPS group,1 000 μmol·L-1 H2O2 group and LPS combined with H2O2 treatment group.After C6 cells were treated by drugs for 6 h,the LDH release rate was detected by ultraviolet spectrophotometry with LDH detecting kit,the apoptotic rate of C6 cells was determined by TUNEL,the Bcl-2,Bax,Caspase-3 mRNA levels were detected by RT-PCR and Bcl-2,the Bax,Caspase-3,Cyt-C,and CLIC4 protein levels were detected by Western blotting.Results Compared with control group,the apoptotic rate and LDH release rate increased in LPS group(P0.05),the expression levels of Bcl-2,Bax mRNA did not change(P0.05),the expression levels of Caspase-3 mRNA was higher than that in control group(P0.05),but the Bcl-2,Bax,CLIC4 and cytosolic Cyt-C protein expression levels did not change(P0.05),the expression level of Caspase-3 protein increased(P0.05).Compared with control group,the apoptotic rate and LDH release rate increased in 1 000 μmol·L-1 H2O2 treatment group(P0.05),the expression of Bcl-2 mRNA decreased compared with control group(P0.05),the expression levels of Bax and Caspase-3 mRNA increased compared with control group(P0.05);but the expression level of Bcl-2 protein decreased,the Bax,Caspase-3,CLIC4 and cytosolic Cyt-C protein expression levels increased obviously(P0.05).When compared with H2O2 group and LPS group,the apoptotic rate and LDH release rate increased in LPS combined with H2O2 treatment group(P0.05),the expression level of Bcl-2 mRNA decreased,the expression levels of Bax and Caspase-3 mRNA increased(P0.05);the expression level of Bcl-2 protein decreased,the Bax,Caspase-3 and cytosolic Cyt-C protein expression levels increased obviously compared with control group(P0.05). and the CLIC4 protein expression did not change(P0.05).Conclusion LPS can activate Caspase-3 signal pathway,induce the early apoptotic event of C6 cells.Meanwhile,the LPS combined with H2O2 can aggravate the injury of C6 cells induced by H2O2 through the overactivation of apoptosis relative Bcl-2/Bax and Caspase-3 pathway.The function of mitochondrial chloride intracellular channel CLIC4 seems uneffective.