基于Cleaved caspase-9研究丹酚酸B对肝纤维化细胞凋亡的影响
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摘要
目的从凋亡角度,观察丹酚酸B对体内外肝纤维化细胞的影响及对凋亡蛋白cleaved caspase-9的影响。方法采用二乙基亚硝胺诱导小鼠肝纤维化模型,共造模12周;体外采用(25、50、100μmol·L~(-1))丹酚酸B作用于经TGF-β1刺激的HSC-T6细胞。HE染色检测病理学改变,并确定纤维化病变区域; Hoechst 33258荧光染色法观察纤维化区域细胞凋亡情况; Annexin V-FITC/PI检测凋亡率; Western blot法检测纤维化组织和HSC-T6细胞中cleaved caspase-9的表达。结果 HE染色结果提示12周为小鼠肝纤维化时期,丹酚酸B低、高剂量组未形成假小叶,纤维化程度均轻于模型组; Hoechst 33258染色显示,与模型组相比,丹酚酸B低、高剂量能够明显促进纤维化区域细胞凋亡; Annexin V-FITC/PI结果显示,丹酚酸B促进TGF-β1刺激的HSC-T6凋亡;Western blot结果提示,与模型组比较,丹酚酸B组cleaved caspase-9表达水平明显升高;与TGF-β1组比较,丹酚酸B50μmol·L~(-1)组cleaved caspase-9表达明显增加。结论丹酚酸B能够明显促进体内外肝纤维化细胞凋亡,其促凋亡进机制可能与上调cleaved caspase-9有关。
Aim To investigate the effect of salvianolic acid B( Sal B) on liver fibrotic cells in vivo and in vitro from the perspective of apoptosis,as well as the effect on cleaved caspase-9. Methods Diethylnitrosamine( DEN) was used to induce liver fibrosis in mice for 12 weeks. The pathological changes were detected by HE staining,and the fibrotic lesion area was determined.The cell apoptosis in the fibrotic area was observed by Hoechst 33258 fluorescence staining. The expression of cleaved caspase-9 in fibrotic tissues was detected by Western blot. The apoptotic rate of each group was detected by double standard method AnnexinV-FITC/PI,and the expression of apoptotic protein cleaved caspase-9 in HSC-T6 was detected by Western blot.Results HE staining suggested that 12 weeks were the period of liver fibrosis in mice. No pseudoplobular structure was formed in group with low and high dose of Sal B,and the degree of fibrosis was lower than that in model group. In the fibrotic lesion area,the fluorescence staining of Hoechst 33258 showed that apoptotic cells significantly increased in group with low Sal B and high dose compared with model group. The results of the AnnexinV-FITC/PI method showed that TGF-β1 inhibited the apoptosis of HSC-T6,and Sal B promoted the apoptosis of HSC-T6 after TGF-β1 intervention and showed a concentration dependence( P < 0. 01). Western blot results showed that in fibrotic liver tissues,Sal B increased the expression of cleaved caspase-9 in HSC-T6 cells compared with model group. Compared with TGF-β1 group,Sal B increased cleaved caspase-9 protein expression( P < 0. 01). Conclusions Sal B can significantly promote apoptosis of liver fibrotic cells in vitro and in vivo,and its pro-apoptosis mechanism may be related to the up-regulation of cleaved caspase-9.
Aim To investigate the effect of salvianolic acid B( Sal B) on liver fibrotic cells in vivo and in vitro from the perspective of apoptosis,as well as the effect on cleaved caspase-9. Methods Diethylnitrosamine( DEN) was used to induce liver fibrosis in mice for 12 weeks. The pathological changes were detected by HE staining,and the fibrotic lesion area was determined.The cell apoptosis in the fibrotic area was observed by Hoechst 33258 fluorescence staining. The expression of cleaved caspase-9 in fibrotic tissues was detected by Western blot. The apoptotic rate of each group was detected by double standard method AnnexinV-FITC/PI,and the expression of apoptotic protein cleaved caspase-9 in HSC-T6 was detected by Western blot.Results HE staining suggested that 12 weeks were the period of liver fibrosis in mice. No pseudoplobular structure was formed in group with low and high dose of Sal B,and the degree of fibrosis was lower than that in model group. In the fibrotic lesion area,the fluorescence staining of Hoechst 33258 showed that apoptotic cells significantly increased in group with low Sal B and high dose compared with model group. The results of the AnnexinV-FITC/PI method showed that TGF-β1 inhibited the apoptosis of HSC-T6,and Sal B promoted the apoptosis of HSC-T6 after TGF-β1 intervention and showed a concentration dependence( P < 0. 01). Western blot results showed that in fibrotic liver tissues,Sal B increased the expression of cleaved caspase-9 in HSC-T6 cells compared with model group. Compared with TGF-β1 group,Sal B increased cleaved caspase-9 protein expression( P < 0. 01). Conclusions Sal B can significantly promote apoptosis of liver fibrotic cells in vitro and in vivo,and its pro-apoptosis mechanism may be related to the up-regulation of cleaved caspase-9.
引文
[1]Ralf W,Frank T.Liver fibrosis:from pathogenesis to novel therapies[J].Dig Dis,2016,34(4):410-22.
[2]蔡卫民.抗肝纤维化治疗研究的若干问题[J].临床肝胆病杂志,2011,27(3):225-32.[2]Cai W M.Several problems in the study of antihepatic fibrosis therapy[J].J Clin Hepatol,2011,27(3):225-32.
[3]杨涛,费振海,钟兴明.Caspase家族与细胞凋亡的研究进展[J].浙江医学,2018,40(18):2083-7,2091.[3]Yang T,Fei Z H,Zhong X M.Research progress of Caspase family and apoptosis[J].Zhejiang Med J,2018,40(18):2083-7,2091.
[4]Bahman A A,Abaza M S I,Khoushiash S I,et al.Sequence-dependent effect of sorafenib in combination with natural phenolic compounds on hepatic cancer cells and the possible mechanism of action[J].Int J Mol Med,2018,42(3):1695-715.
[5]赵先,王婧雯,陆杨,等.丹酚酸B药理作用的研究进展[J].西北药学杂志,2015,30(1):107-10.[5]Zhao X,Wang J W,Lu Y,et al.Advances in pharmacological action of salvianolic acid B[J].Northwest Pharm J,2015,30(1):107-10.
[6]马滢,方萌,伍超,等.丹酚酸B调控pSmad3C/pS-mad3L发挥抗肝纤维化-肝细胞癌作用[J].中国药理学通报,2018,34(1):44-50.[6]Ma Y,Fang M,Wu C,et al.Salvianolic acid B regulates pS-mad3C/pSmad3L to play an anti-fibrosis-hepatocellular carcinoma(HCC)role[J].Chin Pharmacol Bull,2018,34(1):44-50.
[7]王晓玲,刘平,崔云华,等.丹酚酸B抑制体外培养的原代大鼠肝星状细胞的增殖[J].中国中医基础医学杂志,2007,13(2):126-7.[7]Wang X L,Liu P,Cui Y H,et al.Salvianolic acid B inhibited the proliferation of primary rat hepatic stellate cells cultured in vitro[J].J Basic Chin Med,2007,13(2):126-7.
[8]卢明芹,潘陈为,李骥,陈永平.丹酚酸B对肝纤维化大鼠TGF-β1、MMP-2和TIMP-2表达的影响[J].世界华人消化杂志,2007,15(36):3847-51.[8]Lu M Q,Pan C W,Li J,Chen Y P.Effect of salvianolic acid B on expression of TGF-β1,MMP-2 and TIMP-2 in liver fibrosis rats[J].World Chin J Digestol,2007,15(36):3847-51.
[9]Tao Y Y,Wang Q L,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 Bio Med(Maywood),2013,238(11):1284-96.
[10]Kumar M,Sarin S K.Is cirrhosis of the liver reversible[J]?lndian J Pediatr,2007,74(4):393-9.
[11]Lei X F,Fu W,Kim-Kaneyama J R,et al.Hic-5 deficiency attenuates theactivation of hepatic stellate cells and liver fibrosis through upregulation of Smad7 in mice[J].J Hepatol,2016,64(1):110-7.
[12]卢晓晔,钟雪云.Caspases与细胞凋亡(综述)[J].暨南大学学报(自然科学与医学版),2000,21(6):121-4.[12]Lu X Y,Zhong X Y.Caspases and apoptosis(review)[J].J Jinan Univ(Natural SciMed Ed),2000,21(6):121-4.
[2]蔡卫民.抗肝纤维化治疗研究的若干问题[J].临床肝胆病杂志,2011,27(3):225-32.[2]Cai W M.Several problems in the study of antihepatic fibrosis therapy[J].J Clin Hepatol,2011,27(3):225-32.
[3]杨涛,费振海,钟兴明.Caspase家族与细胞凋亡的研究进展[J].浙江医学,2018,40(18):2083-7,2091.[3]Yang T,Fei Z H,Zhong X M.Research progress of Caspase family and apoptosis[J].Zhejiang Med J,2018,40(18):2083-7,2091.
[4]Bahman A A,Abaza M S I,Khoushiash S I,et al.Sequence-dependent effect of sorafenib in combination with natural phenolic compounds on hepatic cancer cells and the possible mechanism of action[J].Int J Mol Med,2018,42(3):1695-715.
[5]赵先,王婧雯,陆杨,等.丹酚酸B药理作用的研究进展[J].西北药学杂志,2015,30(1):107-10.[5]Zhao X,Wang J W,Lu Y,et al.Advances in pharmacological action of salvianolic acid B[J].Northwest Pharm J,2015,30(1):107-10.
[6]马滢,方萌,伍超,等.丹酚酸B调控pSmad3C/pS-mad3L发挥抗肝纤维化-肝细胞癌作用[J].中国药理学通报,2018,34(1):44-50.[6]Ma Y,Fang M,Wu C,et al.Salvianolic acid B regulates pS-mad3C/pSmad3L to play an anti-fibrosis-hepatocellular carcinoma(HCC)role[J].Chin Pharmacol Bull,2018,34(1):44-50.
[7]王晓玲,刘平,崔云华,等.丹酚酸B抑制体外培养的原代大鼠肝星状细胞的增殖[J].中国中医基础医学杂志,2007,13(2):126-7.[7]Wang X L,Liu P,Cui Y H,et al.Salvianolic acid B inhibited the proliferation of primary rat hepatic stellate cells cultured in vitro[J].J Basic Chin Med,2007,13(2):126-7.
[8]卢明芹,潘陈为,李骥,陈永平.丹酚酸B对肝纤维化大鼠TGF-β1、MMP-2和TIMP-2表达的影响[J].世界华人消化杂志,2007,15(36):3847-51.[8]Lu M Q,Pan C W,Li J,Chen Y P.Effect of salvianolic acid B on expression of TGF-β1,MMP-2 and TIMP-2 in liver fibrosis rats[J].World Chin J Digestol,2007,15(36):3847-51.
[9]Tao Y Y,Wang Q L,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 Bio Med(Maywood),2013,238(11):1284-96.
[10]Kumar M,Sarin S K.Is cirrhosis of the liver reversible[J]?lndian J Pediatr,2007,74(4):393-9.
[11]Lei X F,Fu W,Kim-Kaneyama J R,et al.Hic-5 deficiency attenuates theactivation of hepatic stellate cells and liver fibrosis through upregulation of Smad7 in mice[J].J Hepatol,2016,64(1):110-7.
[12]卢晓晔,钟雪云.Caspases与细胞凋亡(综述)[J].暨南大学学报(自然科学与医学版),2000,21(6):121-4.[12]Lu X Y,Zhong X Y.Caspases and apoptosis(review)[J].J Jinan Univ(Natural SciMed Ed),2000,21(6):121-4.