HDAC抑制剂N2E诱导人肝星状细胞LX-2凋亡作用的研究
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摘要
目的研究N2E诱导人肝星状细胞LX-2细胞凋亡,初步探讨其抗肝纤维化的可能性。方法使用细胞周期法检测N2E对LX-2的增殖抑制作用;通过AnnexinV-APC/PI双染检测法来验证N2E对LX-2的凋亡诱导作用;通过线粒体膜电位和Caspase-3分光光度计法进一步验证N2E对LX-2的凋亡诱导作用;肝星状细胞LX-2经N2E作用24 h后,采用Western blot检测HDAC3、Caspase-3、Cleaved-Caspase-3、TGF-β1以及α-SMA的表达水平。结果 N2E能明显抑制人肝星状细胞LX-2的增殖生长,诱导细胞凋亡。高浓度(10μmol/L)能明显下调线粒体膜电位,差异具有统计学意义(P<0.01)。N2E能明显提升LX-2中Caspase-3的活性,且强于SAHA;N2E显著下调LX-2细胞系中HDAC3、TGF-β1以及α-SMA的蛋白表达,同时能够下调Caspase-3酶原蛋白,促进活化状态Cleaved-Caspase-3蛋白表达。结论 N2E能够显著地诱导人肝星状细胞LX-2凋亡和下调纤维化蛋白的表达,可能具有潜在的抗肝纤维化作用。
Objective To investigate the apoptosis of human hepatic stellate(LX-2) cells induced by HDAC inhibitor N2 E,and explore the possibility of anti-hepatic fibrosis by N2 E. Methods Cell cycle method was used to detect the inhibitory effect of N2 E on the proliferation of LX-2 cells,and the proapoptosis effect of N2 E on LX-2 cells was detected by AnnexinV-APC/PI double dyeing method. The apoptosis-inducing effect of N2 E on LX-2 cells was further verified by mitochondrial membrane potential and Caspase-3 spectrophotometry. Western blot was used to detect the expression of HDAC3,Caspase-3,Cleaved-Caspase-3,TGF-β1 and α-SMA. Results N2 E obviously inhibited the proliferation and growth of LX-2 cells,and induced LX-2 cell apoptosis. The mitochondrial membrane potential was significantly reduced when treated with high concentration(10 μmol/L) N2 E(P<0.01). N2 E significantly enhanced the activity of Caspase-3 in LX-2 cells,which was stronger than SAHA. N2 E markedly reduced the protein expression of HDAC3,TGF-β1 and α-SMA in LX-2 cells. N2 E decreased the Caspase-3 zymogen protein and promoted the expression of active cleaved-caspase-3 protein. Conclusion N2 E can induce the apoptosis of human hepatic stellate LX-2 cells and reduce the expression of fibrosis protein,which may have potential anti-hepatic fibrosis effect.
Objective To investigate the apoptosis of human hepatic stellate(LX-2) cells induced by HDAC inhibitor N2 E,and explore the possibility of anti-hepatic fibrosis by N2 E. Methods Cell cycle method was used to detect the inhibitory effect of N2 E on the proliferation of LX-2 cells,and the proapoptosis effect of N2 E on LX-2 cells was detected by AnnexinV-APC/PI double dyeing method. The apoptosis-inducing effect of N2 E on LX-2 cells was further verified by mitochondrial membrane potential and Caspase-3 spectrophotometry. Western blot was used to detect the expression of HDAC3,Caspase-3,Cleaved-Caspase-3,TGF-β1 and α-SMA. Results N2 E obviously inhibited the proliferation and growth of LX-2 cells,and induced LX-2 cell apoptosis. The mitochondrial membrane potential was significantly reduced when treated with high concentration(10 μmol/L) N2 E(P<0.01). N2 E significantly enhanced the activity of Caspase-3 in LX-2 cells,which was stronger than SAHA. N2 E markedly reduced the protein expression of HDAC3,TGF-β1 and α-SMA in LX-2 cells. N2 E decreased the Caspase-3 zymogen protein and promoted the expression of active cleaved-caspase-3 protein. Conclusion N2 E can induce the apoptosis of human hepatic stellate LX-2 cells and reduce the expression of fibrosis protein,which may have potential anti-hepatic fibrosis effect.
引文
[1] S贾继东,魏来,庄辉.中国肝脏病学现状及发展方向[J].中国病毒病杂志,2011,21(1):1-4.
[2] 李永贵,聂伟,吴忠文.浅谈肝病范畴[J].中医药导报,2005,11(8):4-6.
[3] 王辰,王建安.内科学[M].第3版.北京:人民卫生出版社,2015:117-119.
[4] GEERTS A.History,heterogeneity,developmental biology,and functions of quiescent hepatic stellate cells[J].Semin Liver Dis,2001,21(3):311-335.
[5] SEKI E,BRENNER D A.Recent advancement of molecular mechanisms of liver fibrosis[J].J Hepatobiliary Pancreat Sci,2015,22(7):512-518.
[6] TSUKAMOTO H,ZHU Nianling,ASAHINA K,et al.Epigenetic cell fate regulation of hepatic stellate cells[J].Hepatol Res,2011,41(7):675-682.
[7] LI Xing,WU Xiaoqin,XU Tao,et al.Role of histone deace-tylases(HDACs) in progression and reversal of liver fibrosis[J].Toxicol Appl Pharmacol,2016,306(2):58-68.
[8] LAN Qin,HAN Yuanping.Epigenetic repression of matrix metallopro-teinases in myofibroblastic hepatic stellate cells through histone deacetylases 4:implication in tissue fibrosis[J].Am J Pathol,2010,177(4):1915-1928.
[9] SANDERS Y Y,HAGOOD J S,LIU H,et al.Histone deacetylase inhibition promotes fibroblast apoptosis and ameliorates pulmonary fibrosis in mice[J].Eur Respir J,2014,43(5):1448-1458.
[10] ZHANG Song,HUANG Weibin,LI Xia,et al.Synthesis,biological evaluation,and computer-aided drug designing of new derivatives of hyperactive suberoylanilide hydroxamic acid histone deacetylase inhibitors[J].Chem Biol Drug Des,2015,86(4):795-804.
[11] FRIEDMAN S L.Hepatic fibrosis--Overview[J].Toxicology,2008,254(3):120-129.
[12] PORTER A G,JANICKE R U.Emerging roles of caspase-3 in apoptosis[J].Cell Death Differ,1999,6(2):99-104.
[13] RODRIGUEZ-AGUILERA J R,GUERRERO-HERNANDEZ C,PEREZ-MOLINA R,et al.Epigenetic effects of an adenosine derivative in a wistar rat model of liver cirrhosis[J].J Cell Biochem,2018,119(1):401-413.
[2] 李永贵,聂伟,吴忠文.浅谈肝病范畴[J].中医药导报,2005,11(8):4-6.
[3] 王辰,王建安.内科学[M].第3版.北京:人民卫生出版社,2015:117-119.
[4] GEERTS A.History,heterogeneity,developmental biology,and functions of quiescent hepatic stellate cells[J].Semin Liver Dis,2001,21(3):311-335.
[5] SEKI E,BRENNER D A.Recent advancement of molecular mechanisms of liver fibrosis[J].J Hepatobiliary Pancreat Sci,2015,22(7):512-518.
[6] TSUKAMOTO H,ZHU Nianling,ASAHINA K,et al.Epigenetic cell fate regulation of hepatic stellate cells[J].Hepatol Res,2011,41(7):675-682.
[7] LI Xing,WU Xiaoqin,XU Tao,et al.Role of histone deace-tylases(HDACs) in progression and reversal of liver fibrosis[J].Toxicol Appl Pharmacol,2016,306(2):58-68.
[8] LAN Qin,HAN Yuanping.Epigenetic repression of matrix metallopro-teinases in myofibroblastic hepatic stellate cells through histone deacetylases 4:implication in tissue fibrosis[J].Am J Pathol,2010,177(4):1915-1928.
[9] SANDERS Y Y,HAGOOD J S,LIU H,et al.Histone deacetylase inhibition promotes fibroblast apoptosis and ameliorates pulmonary fibrosis in mice[J].Eur Respir J,2014,43(5):1448-1458.
[10] ZHANG Song,HUANG Weibin,LI Xia,et al.Synthesis,biological evaluation,and computer-aided drug designing of new derivatives of hyperactive suberoylanilide hydroxamic acid histone deacetylase inhibitors[J].Chem Biol Drug Des,2015,86(4):795-804.
[11] FRIEDMAN S L.Hepatic fibrosis--Overview[J].Toxicology,2008,254(3):120-129.
[12] PORTER A G,JANICKE R U.Emerging roles of caspase-3 in apoptosis[J].Cell Death Differ,1999,6(2):99-104.
[13] RODRIGUEZ-AGUILERA J R,GUERRERO-HERNANDEZ C,PEREZ-MOLINA R,et al.Epigenetic effects of an adenosine derivative in a wistar rat model of liver cirrhosis[J].J Cell Biochem,2018,119(1):401-413.