内毒素对过氧化氢引起的神经胶质瘤C6细胞损伤的影响

Effect of lipopolysaccharide on C6 glioma cells injuried by H_2O_2

目的:观察内毒素(LPS)对过氧化氢(H2O2)诱导的神经胶质瘤C6细胞损伤的影响,探讨其相关机制。方法:C6细胞按处理方法不同分为空白对照组、100μmol.L-1LPS组、1 000μmol.L-1H2O2组及1 000μmol.L-1H2O2与100μmol.L-1LPS联合应用组。药物作用C6细胞6 h,取培养液上清与细胞沉淀,利用LDH检测试剂盒,采用紫外分光光度法检测乳酸脱氢酶(LDH)释放率;原位末端标记技术(TUNEL)检测细胞凋亡率;应用RT-PCR检测相关基因Bcl-2、Bax、Caspase-3 mRNA表达;Western blotting检测Bcl-2、Bax、Caspase-3、细胞色素C(Cyt-C)及CLIC4蛋白水平。结果:与空白对照组比较,100μmol.L-1LPS组C6细胞凋亡率及LDH释放率增加(P<0.05),在mRNA水平上Bcl-2、Bax表达率未见明显改变(P>0.05),而Caspase-3表达率升高(P<0.05);在蛋白水平上,与空白对照组比较,Bcl-2、Bax、CLIC4及胞浆Cyt-C蛋白水平未见明显变化(P>0.05),而Caspase-3蛋白水平高于空白对照组(P<0.05)。与空白对照组比较,1 000μmol.L-1H2O2组C6细胞凋亡率及LDH释放率增加(P<0.05);在mRNA水平上Bcl-2表达率低于空白对照组(P<0.05),Bax、Caspase-3表达率高于空白对照组(P<0.05);在蛋白水平上,Bcl-2蛋白表达低于空白对照组(P<0.05),Bax、Caspase-3、CLIC4及胞浆Cyt-C蛋白水平明显高于空白对照组(P<0.05)。与H2O2组和LPS组比较,LPS与H2O2联合作用组细胞凋亡率及LDH释放率明显增加(P<0.05),在mRNA水平上Bcl-2表达率下降(P<0.05),Bax、Caspase-3表达率升高(P<0.05);在蛋白水平上,Bcl-2蛋白表达低于对照组(P<0.05),Bax、Caspase-3及胞浆Cyt-C蛋白水平明显高于对照组(P<0.05),而CLIC4蛋白水平变化不明显(P>0.05)。结论:单独应用LPS能够激活Caspase-3信号通路,引起C6细胞早期凋亡,同时LPS能促进H2O2引起的C6细胞损伤,其机制可能与凋亡相关的Bcl-2/Bax及Caspase-3途径过度激活有关。线粒体氯通道蛋白CLIC4在此过程中作用不明显。

Objective To observe the effect of lipopolysaccharide （LPS） on malignant glioma C6 cells injuried by hydrogen peroxide （H2 O2 ）, investigate the mechanism maybe involved. Methods The experiment was divided into four groups： control group, 100 μmol · L^-1 LPS group, 1 000μmol · 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 speetrophotometry with I.DH 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 （P 〈 0.05）, the expression levels of Bcl-2, Bax mRNA did not change （P〈0.05）, the expression levels of Caspase-3 mRNA was higher than that in control group （P〈0.05）, but the Bcl-2, Bax, CLIC4 and eytosolic Cyt-C protein expression levels did not change （P〉0.05）, the expression level of Caspase-3 protein increased （P〈0.05）. Compared with control group, the apoptotic rate and LDH release rate increased in 1 000 μmol · L^-1 H2O2 treatment group （P〈0.05）, the expression of Bcl-2 mRNA decreased compared with control group （P〈0.05）, the expression levels of Bax and Caspase-3 mRNA increased compared with control group （P〈0.05）; but the expression level of Bcl-2 protein decreased, the Bax, Caspase-3, CLIC4 and eytosolie Cyt-C protein expression levels increased obviously （P〈0.05）. When compared with H2O2 group and LPS group, the apoptotic rate and LDH release rate increased in I.PS combined with H2O2 treatment group （P〈 0. 05）, the expression level of Bcl-2 mRNA decreased, the expression levels of Bax and Caspase-3 mRNA increased （P〈0.05）; 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 （P〈0. 05）. and the CLIC4 protein expression did not change （P〉0.05）. Conclusion LPS can activate Caspase-3 signal pathway, induce the early apoptotie event of C6 cells. Meanwhile, the LPS combined with H202 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.