XIAP在H_2O_2诱导牙周膜细胞凋亡的机制研究
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摘要
目的探讨XIAP在H_2O_2诱导牙周膜细胞凋亡的作用机制。方法不同浓度的H_2O_2刺激牙周膜细胞,Western blot检测XIAP和凋亡相关蛋白的表达;过表达及沉默XIAP后,给予250μM H_2O_2作用72 h,流式细胞术检测细胞凋亡,Western blot检测Akt、JNK2和GSK3β,免疫荧光检测Akt、p-Akt,激光共聚焦显微镜检测c-jun、Ap-1在细胞中的定位和表达。结果 250μM H_2O_2作用72 h后,XIAP蛋白显著下降(P<0.05);随着作用时间的增加,细胞内Caspase-3、Caspase-8、PTEN蛋白水平显著升高(P<0.05);250μM的H_2O_2作用过表达XIAP与PDLC 72 h后,过表达XIAP细胞内JNK2、Akt、p-Akt及GSK3β蛋白显著高于PDLC;过表达组细胞凋亡率显著下降,c-jun以及AP-1蛋白的表达显著上升(P<0.05);沉默XIAP后,细胞凋亡率显著上升,JNK2、Akt、p-Akt及GSK3β蛋白表达水平显著下降(P<0.05)。结论 H_2O_2通过诱导XIAP表达水平的下降,抑制XIAP与凋亡执行蛋白Caspase-3的结合,致使牙周膜细胞凋亡的发生;AKT、JNK2、GSK3β蛋白在H_2O_2诱导牙周膜细胞凋亡的机制中起着重要作用。
Objective To investigate the mechanism of XIAP in the apoptosis of periodontal ligament cells induced by H_2O_2.Methods The periodontal membrane cells were stimulated by H_2O_2 at different concentrations.Western blotting was used to detect the expression of XIAP and apoptosis-related proteins.Upon over-expression of XIAP protein and gene silencing,respectively,the apoptosis rate was detected by flow cytometry double staining technique.The expressions of Akt,JNK2 and GSK3β signaling pathway proteins were detected by Western blotting.The protein expressions of Akt and p-Akt were detected by immunofluorescence.Focusing microscopy was used to determine the localization and expression of c-jun and Ap-1 in cells.Results The level of XIAP protein was decreased with the induction of 250 μM H_2O_2 for 72 h.Cells were treated with 250 μM H_2O_2,and the contents of Caspase-3,Caspase-8 and PTEN in the cells were increased with the increase of time.After over-expression of XIAP,the expressions of JNK2,Akt,p-Akt and GSK3β proteins in cells did not change significantly.H_2O_2 was simultaneously applied to cells overexpressing XIAP and normal cells for 72 h,and the expressions of JNK2,Akt,p-Akt and GSK3β in overexpressing XIAP cells were higher than those in normal cells.The over-expression group treated with H_2O_2 showed a significant decrease in apoptosis rate and the expressions of c-jun and AP-1 proteins were increased.After gene silencing,the apoptotic rate was increased and the expression levels of JNK2,Akt,p-Akt and GSK3β proteins were lower than those in the control group.Conclusion H_2O_2 induces apoptosis of periodontal ligament cells by decreasing XIAP expression and inhibiting the binding of XIAP to the apoptosis-executing protein Caspase-3.Akt,JNK2,and GSK3β proteins play an important role in the mechanism of H_2O_2-induced apoptosis of periodontal ligament cells.XIAP might regulate these three signaling pathways to maintain the expression levels of these three proteins at normal levels to achieve anti-apoptotic effects.XIAP activates the expression of C-jun and AP-1,indicating that XIAP might positively regulate JNK2 by phosphorylating C-jun.
Objective To investigate the mechanism of XIAP in the apoptosis of periodontal ligament cells induced by H_2O_2.Methods The periodontal membrane cells were stimulated by H_2O_2 at different concentrations.Western blotting was used to detect the expression of XIAP and apoptosis-related proteins.Upon over-expression of XIAP protein and gene silencing,respectively,the apoptosis rate was detected by flow cytometry double staining technique.The expressions of Akt,JNK2 and GSK3β signaling pathway proteins were detected by Western blotting.The protein expressions of Akt and p-Akt were detected by immunofluorescence.Focusing microscopy was used to determine the localization and expression of c-jun and Ap-1 in cells.Results The level of XIAP protein was decreased with the induction of 250 μM H_2O_2 for 72 h.Cells were treated with 250 μM H_2O_2,and the contents of Caspase-3,Caspase-8 and PTEN in the cells were increased with the increase of time.After over-expression of XIAP,the expressions of JNK2,Akt,p-Akt and GSK3β proteins in cells did not change significantly.H_2O_2 was simultaneously applied to cells overexpressing XIAP and normal cells for 72 h,and the expressions of JNK2,Akt,p-Akt and GSK3β in overexpressing XIAP cells were higher than those in normal cells.The over-expression group treated with H_2O_2 showed a significant decrease in apoptosis rate and the expressions of c-jun and AP-1 proteins were increased.After gene silencing,the apoptotic rate was increased and the expression levels of JNK2,Akt,p-Akt and GSK3β proteins were lower than those in the control group.Conclusion H_2O_2 induces apoptosis of periodontal ligament cells by decreasing XIAP expression and inhibiting the binding of XIAP to the apoptosis-executing protein Caspase-3.Akt,JNK2,and GSK3β proteins play an important role in the mechanism of H_2O_2-induced apoptosis of periodontal ligament cells.XIAP might regulate these three signaling pathways to maintain the expression levels of these three proteins at normal levels to achieve anti-apoptotic effects.XIAP activates the expression of C-jun and AP-1,indicating that XIAP might positively regulate JNK2 by phosphorylating C-jun.
引文
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[8] Schimmer A D,Welsh K,Pinilla C,et al.Small-molecule antagonists of apoptosis suppressor XIAP exhibit broad antitumor activity[J].Cancer Cell,2004,5(1):25-35.
[9] Takahashi R,Deveraux Q,Tamm I,et al.A single BIR domain of XIAP sufficient for inhibiting caspases[J].J Biol Chem,1998,273(14):7787-7790.
[10] 叶闻远,欧阳学农,余宗阳.非小细胞肺癌XIAP和Smac的表达与临床病理特征及预后的关系[J].中国肿瘤临床,2014,41(7):444-448.
[11]Zhou F,Xu J,Ding G,et al.Overexpressions of CK2beta and XIAP are associated with poor prognosis of patients with cholangiocarcinoma[J].Pathol Oncol Res,2014,20(1):73-79.
[12]Mohamed M S,Bishr M K,Almutairi F M,et al.Inhibitors of apoptosis:clinical implications in cancer[J].Apoptosis,2017,22(12):1487-1509.
[13]Van Themsche C,Chaudhry P,Leblanc V,et al.XIAP gene expression and function is regulated by autocrine and paracrine TGF-beta signaling[J].Mol Cancer,2010,9:216.
[2] 王振冉.X连锁凋亡抑制蛋白与肿瘤关系的研究进展[J].中国医药指南,2017,15(10):34-35.
[3] Schimmer A D,Dalili S,Batey R A,et al.Targeting XIAP for the treatment of malignancy[J].Cell Death Differ,2006,13(2):179-188.
[4] Cummins J M,Kohli M,Rago C,et al.X-linked inhibitor of apoptosis protein (XIAP) is a nonredundant modulator of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-mediated apoptosis in human cancer cells[J].Cancer Res,2004,64(9):3006-3008.
[5] 龚芳,祁小飞,张日.凋亡抑制蛋白XIAP在白血病中的研究进展[J].临床血液学杂志,2017,30(1):73-75.
[6] Qian H,Huang T,Chen Y,et al.X-linked inhibitor of apoptosis protein inhibitor Embelin induces apoptosis via PI3K/Akt pathway and inhibits invasion in osteosarcoma cells[J].J Cancer Res Ther,2018,14(Supple 1):648-655.
[7] 李隽,胥春,郝轶,等.人牙周膜细胞原代培养及鉴定[J].上海交通大学学报:医学版,2008,28(11):1373-1376.
[8] Schimmer A D,Welsh K,Pinilla C,et al.Small-molecule antagonists of apoptosis suppressor XIAP exhibit broad antitumor activity[J].Cancer Cell,2004,5(1):25-35.
[9] Takahashi R,Deveraux Q,Tamm I,et al.A single BIR domain of XIAP sufficient for inhibiting caspases[J].J Biol Chem,1998,273(14):7787-7790.
[10] 叶闻远,欧阳学农,余宗阳.非小细胞肺癌XIAP和Smac的表达与临床病理特征及预后的关系[J].中国肿瘤临床,2014,41(7):444-448.
[11]Zhou F,Xu J,Ding G,et al.Overexpressions of CK2beta and XIAP are associated with poor prognosis of patients with cholangiocarcinoma[J].Pathol Oncol Res,2014,20(1):73-79.
[12]Mohamed M S,Bishr M K,Almutairi F M,et al.Inhibitors of apoptosis:clinical implications in cancer[J].Apoptosis,2017,22(12):1487-1509.
[13]Van Themsche C,Chaudhry P,Leblanc V,et al.XIAP gene expression and function is regulated by autocrine and paracrine TGF-beta signaling[J].Mol Cancer,2010,9:216.