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
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摘要
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.
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.
引文
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[2]Guo B,Zhang W,Tume RK,et al.Disorder of endoplasmic reticulum calcium channel components is associated with the increased apoptotic potential in pale,soft,exudative pork[J].Meat Sci,2016,115:34-40.
[3]Gessner DK,Schlegel G,Ringseis R,et al.Up-regulation of endoplasmic reticulum stress induced genes of the unfolded protein response in the liver of periparturient dairy cows[J].BMC Vet Res,2014,10:46.
[4]Lin S,Zhang J,Chen H,et al.Involvement of endoplasmic reticulum stress in capsaicin-induced apoptosis of human pancreatic cancer cells[J].Evid Based Complement Alternat Med,2013,2013:629750.
[5]Zhang XQ,Xu CF,Yu CH,et al.Role of endoplasmic reticulum stress in the pathogenesis of nonalcoholic fatty liver disease[J].World J Gastroenterol,2014,20(7):1768-1776.
[6]Hammerich L,Tacke F.Eat more carrots?Dampening cell death in ethanol-induced liver fibrosis byβ-carotene[J].Hepatobiliary Surg Nutr,2013,2(5):248-251.
[7]孙丽娜,周东方,周俊英,等.内质网应激在大鼠酒精性肝病肝细胞凋亡中的作用[J].中华肝脏病杂志,2012,20(1):35-39.
[8]de la Cadena SG,Hernández-Fonseca K,Camacho-Arroyo I,et al.Glucose deprivation induces reticulum stress by the PERK pathway and caspase-7-and calpain-mediated caspase-12 activation[J].Apoptosis,2014,19(3):414-427.
[9]Tao YY,Wang QL,Shen L,et al.Salvianolic acid B inhibits hepatic stellate cell activation through transforming growth factor beta-1 signal transduction pathway in vivo and in vitro[J].Exp Biol Med(Maywood),2013,238(11):1284-1296.
[10]Cho EA,Kim EJ,Kwak SJ,et al.c AMP signaling inhibits radiation-induced ATM phosphorylation leading to the augmentation of apoptosis in human lung cancer cells[J].Mol Cancer,2014,13:36.
[11]Oruno C,Carbonneau S,Stewart RA,et al.Interdependence of Bad and Puma during ionizing-radiation-induced apoptosis[J].PLo S One,2014,9(2):e88151.
[12]Hsu WL,Chen CL,Huang SW,et al.The untranslated regions of classic swine fever virus RNA trigger apoptosis[J].PLo S One,2014,9(2):e88863.
[13]Jiang ZQ,Ma YX,Li MH,et al.5-Hydroxymethylfurfural protects against ER stress-induced apoptosis in Gal N/TNF-α-injured L02 hepatocytes through regulating the PERK-e IF2αsignaling pathway[J].Chin J Nat Med,2015,13(12):896-905.
[14]吴锦波,叶小汉,冼绍祥,等.比索洛尔联合培哚普利对阿霉素诱导的心力衰竭大鼠心肌内质网应激的影响[J].中国病理生理杂志,2016,32(11):1939-1944.
[15]陈肖燕,邓春玉,邝素娟,等.毒胡萝卜素诱导大鼠冠状动脉平滑肌细胞内质网应激模型的建立[J].中国病理生理杂志,2017,33(1):128-132.
[16]林帆,陈亚红,廖程程,等.硫化氢对尼古丁诱导的人支气管上皮细胞内质网应激及细胞凋亡的影响[J].中华医学杂志,2015,95(28):2297-2301.
[17]Wu H,Ye M,Yang J,et al.Endoplasmic reticulum stress-induced apoptosis:a possible role in myocardial ischemia-reperfusion injury[J].Int J Cardiol,2016,208:65-66.
[18]Fan J,Long H,Li Y,et al.Edaravone protects against glutamate-induced PERK/EIF2α/ATF4 integrated stress response and activation of caspase-12[J].Brain Res,2013,1519:1-8.
[19]Roberson EC,Tully JE,Guala AS,et al.Influenza induces endoplasmic reticulum stress,caspase-12-dependent apoptosis,and c-Jun N-terminal kinase-mediated transforming growth factor-βrelease in lung epithelial cells[J].Am J Respir Cell Mol Biol,2012,46(5):573-581.
[20]谢汝佳,韩冰,杨婷,等.钙中性蛋白酶2及Bax在肝纤维化大鼠肝组织中的表达变化及意义[J].中国病理生理杂志,2013,29(9):1603-1608.
[21]Pal S,Ghosh M,Ghosh S,et al.Atorvastatin induced hepatic oxidative stress and apoptotic damage via MAPKs,mitochondria,calpain and caspase12 dependent pathways[J].Food Chem Toxicol,2015,83:36-47.