二硫苏糖醇通过激活calpain-2/caspase-12信号通路诱导BRL-3A细胞凋亡

Dithiothreitol induces apoptosis of BRL-3A cells by activation of calpain-2/caspase-12 signaling pathway

目的:探讨二硫苏糖醇(DTT)诱导大鼠正常肝细胞株BRL-3A发生内质网应激时细胞内葡萄糖调节蛋白78(GRP78)、钙蛋白酶2 (calpain-2)、caspase-12及caspase-3的表达变化及对细胞凋亡的影响。方法:采用2. 5 mmol/L DTT分别处理BRL-3A细胞12 h和24 h,应用real-time PCR检测细胞内GRP78、calpain-2、caspase-12及caspase-3的mRNA水平;采用细胞免疫荧光检测细胞内GRP78、calpain-2、caspase-12及caspase-3的蛋白表达;应用Western blot检测cleaved caspase-12及cleaved caspase-3的表达变化;采用流式细胞术检测细胞凋亡情况。结果:BRL-3A细胞经DTT处理12 h及24 h后,GRP78、calpain-2及caspase-12的mRNA表达较正常对照组显著升高(P <0. 01),而caspase-3的mRNA水平与正常对照组比较无显著变化;细胞免疫荧光及Western blot检测发现,DTT处理细胞12 h及24 h后,BRL-3A细胞内GRP78、calpain-2、caspase-12及caspase-3的蛋白表达均较正常对照组显著增高,同时,cleaved caspase-12及cleaved caspase-3的表达也较正常对照组明显增多(P <0. 05);流式细胞术检测发现,经DTT处理后BRL-3A细胞的凋亡率较正常对照组显著增加(P <0. 05)。结论:二硫苏糖醇诱导BRL-3A细胞凋亡可能与calpain-2/caspase-12信号通路激活有关。

AIM:To investigate the expression changes of glucose-regulated protein 78(GRP78),calpain-2,caspase-12 and caspase-3 in dithiothreitol(DTT)-induced endoplasmic reticulum stress in rat normal liver cell line BRL-3A,and to explore their effects on apoptosis of BRL-3A cells.METHODS:BRL-3A cells were treated with 2.5 mmol/L DTT to induce endoplasmic reticulum stress.The mRNA expression of GRP78,calpain-2,caspase-12 and caspase-3 was detected by real-time PCR.The protein expression of GRP78,calpain-2,caspase-12 and caspase-3 was detected by cell immunofluorescence technique.The protein levels of cleaved caspase-12 and cleaved caspase-3 were determined by Western blot.The apoptosis of BRL-3A cells was analyzed by flow cytometry.RESULTS:After treatment with DTT for 12 h and 24 h,the mRNA expression of GRP78,calpain-2 and caspase-12 in the BRL-3A cells was obviously increased compared with normal control group(P<0.01),and no significant change of caspase-3 mRNA after treatment with DTT for 12 h and 24 h was observed.The results of immunofluorescence technique and Western blot showed that the protein levels of GRP78,calpain-2,caspase-12,cleaved caspase-12,caspase-3 and cleaved caspase-3 were obviously elevated after treatment with DTT for 12 h and 24 h(P<0.05).In addition,the increased apoptotic rate was also found in the BRL-3A cells treated with DTT for 12 h and 24 h(P<0.05).CONCLUSION:The activation of calpain-2/caspase-12 signaling pathway may be involved in apoptosis of BRL-3A cells induced by DTT.