钙蛋白酶2及自噬相关蛋白5对内质网应激诱导的肝细胞凋亡的影响

Effects of calpain-2 and autophagy-related protein 5 on hepatocyte apoptosis induced by endoplasmic reticulum stress

目的:探讨二硫苏糖醇(DTT)诱导大鼠正常肝细胞BRL-3A发生内质网应激(ERS)过程中钙蛋白酶2(calpain-2)及自噬相关蛋白5(Atg5)对肝细胞凋亡的影响。方法:采用2.0 mmol/L DTT处理BRL-3A细胞0、6、12和24 h,诱导细胞发生ERS;实时无标记细胞分析仪(RTCA)检测DTT对BRL-3A细胞增殖的影响;流式细胞术检测细胞凋亡及细胞周期;real-time PCR检测calpain-2和Atg5的m RNA表达;Western blot检测calpain-2、Atg5、Atg7、Atg12和微管相关蛋白1轻链3(LC3)蛋白水平的变化;免疫共沉淀(Co-IP)方法研究calpain-2与Atg5之间的相互作用。结果:不同浓度的DTT处理细胞后,细胞增殖受到显著抑制;流式细胞术检测凋亡发现,DTT处理BRL-3A细胞6、12和24 h后,细胞凋亡较0 h组显著增多(P<0.05)。流式细胞术检测细胞周期发现,DTT处理细胞后,细胞被阻滞在G1期(P<0.05)。DTT处理细胞6、12和24 h后,细胞中calpain-2和Atg5的m RNA表达水平较0 h组显著上调(P<0.05),calpain-2、Atg12和Atg7的蛋白水平也较0 h组显著升高,且LC3-II/LC3-I比值明显增大,而Atg5的表达较0 h组显著降低(P<0.05)。Co-IP发现,calpain-2抗体可以将Atg5蛋白从细胞裂解物中沉淀下来,Atg5抗体也可以将calpain-2从细胞裂解物中沉淀下来,证实calpain-2与Atg5之间存在相互作用。结论:calpain-2可能通过与Atg5之间的相互作用参与ERS诱导的肝细胞凋亡过程。

AIM:To investigate the effects of calpain-2 and autophagy-related protein 5(Atg5)on apoptosis of BRL-3 A rat normal liver cells during endoplasmic reticulum stress(ERS) induced by dithiothreitol(DTT).METHODS:BRL-3 A cells were treated with DTT at 2.0 mmol/L for 0,6,12 and 24 h to induce ERS.Real-time cell analysis(RTCA)was used to measure the effect of DTT on BRL-3 A cell proliferation.Apoptosis and cell cycle distribution were analyzed by flow cytometry.The mRNA expression of calpain-2 and Atg5 was detected by real-time PCR.The protein levels of calpain-2,Atg5,Atg7,Atg12 and microtubule-associated protein 1 light chain 3(LC3)were determined by Western blot.The interaction between calpain-2 and Atg5 was investigated by co-immunoprecipitation(Co-IP).RESULTS:The proliferation of BRL-3 A cells treated with DTT was significantly inhibited.The apoptosis of BRL-3 A cells was significantly increased after DTT treatment for 6,12 and 24 h as compared with 0 h group(P<0.05).The cell cycle was arrested in G1 phase after DTT treatment(P<0.05).After DTT treatment for 6,12 and 24 h,the mRNA expression of calpain-2 and Atg5 in the BRL-3 A cells was significantly increased as compared with 0 h group(P<0.05).The protein levels of calpain-2,Atg12 and Atg7 in the cells treated with DTT for 6,12 and 24 h were significantly higher than those in0 h group,and the ratio of LC3-II/LC3-I was also significantly higher than that in 0 h group,while Atg5 expression was significantly lower than that in 0 h group(P<0.05).The results of Co-IP found that the anti-calpain-2 antibody precipitated Atg5 protein from the cell lysates,and the anti-Atg5 antibody also precipitated calpain-2 from the cell lysates,which confirmed the interaction between calpain-2 and Atg5.CONCLUSION:Calpain-2 may participate in ERS-induced hepatocyte apoptosis by interacting with Atg5.