丹酚酸B调控pSmad3C/pSmad3L发挥抗肝纤维化-肝细胞癌作用
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摘要
目的观察丹酚酸B(salvianolic acid B,Sal B)对二乙基亚硝胺(diethylnitrosamine,DEN)诱导小鼠肝纤维化-肝细胞癌进程的影响,并探讨Sal B经由p Smad3C/p21介导的抑癌信号、p Smd3L/PAI-1/c-Myc介导的促肝纤维化-肝癌信号转换机制。方法昆明种♂小鼠100只,随机分组,DEN诱导小鼠肝纤维化-肝细胞癌模型,不同剂量Sal B(15、30mg·kg~(-1)·d~(-1),ig)及阳性药秋水仙碱(0.2 mg·kg~(-1)·d~(-1),ig)干预。于造模第12周、第16周分批处死小鼠,肝脏活检,苏木精-伊红(HE)染色、Van Gieson(VG)染色观察肝组织病理学特征,Western blot法检测肝组织中p Smad3C、p S-mad3L、p21、纤溶酶原激活物抑制剂-1(plasminogen activator inhibitor 1,PAI-1)及c-Myc蛋白表达。结果正常组肝脏表面光滑、质地柔软,模型组第12周时肝脏表面粗糙、质地变硬,第16周时肝脏表面可见弥漫性结节、质地坚硬;而丹酚酸B干预组以上病变明显改善。HE及VG染色显示,正常组肝组织结构正常,模型组第12周时肝组织炎细胞浸润、胶原纤维增生,形成假小叶结构;第16周时肝小叶结构紊乱,细胞核变大、分裂相增多、异型性明显;Sal B干预组肝组织病变程度明显减轻。Western blot结果显示,正常组肝组织中p Smad3C、p Smad3L、PAI-1表达均较少,p21、c-Myc几乎不表达;模型组第12周时肝组织中p Smad3C无明显变化,p S-mad3L、PAI-1、p21表达增多,第16周时p Smad3C、p Smad3L、p21、PAI-1、c-Myc表达皆有增加;而Sal B干预组第12周时p Smad3C、p21表达明显增加,p Smad3L、PAI-1蛋白水平明显降低,第16周时p Smad3C表达明显增加,p21几乎无变化,p Smad3L、PAI-1、c-Myc表达明显降低。结论Sal B延缓DEN诱导的小鼠肝纤维化-肝细胞癌进程,其机制可能与调控p Smad3C/p21、p Smad3L/PAI-1/c-Myc信号转换有关。
AimTo observe the effect of Salvianolicaid B(Sal B)on the progression of hepatic fibrosiscarcinoma in mice induced by diethylnitrosamine(DEN)and investigate the mechanism of Sal B involved in the shift between p Smad3C/p21-mediated tumor suppressive signaling and p Smad3L/PAI-1/cMyc-mediatedpro-fibrogenic/oncogenicsignaling.MethodsA total of 100 male Kunming mice were randomly grouped,DEN-induced hepatic fibrosis-carcinoma model of mice was established,which was intragastrically treated by Sal B with two dosages(15,30 mg·kg~(-1))and colchicine with one dosage(0.2mg·kg~(-1)),respectively.The mice were sacrificed at12th week or 16th week after the start of DEN administration.Pathological changes of livers in each group were assessed by liver biopsy,hematoxylin-eosin(HE)staining and Van Gieson(VG)staining.The protein expressions of p Smad3C,p Smad3L,p21,plasminogen activator inhibitor-1(PAI-1)and c-Myc in liver tissues were assayed by Western blot.Results In the normal control group,the surface of mouse liver was smooth and soft,and the structure of the hepatic lobule was intact.In the DEN alone group,at 12th week,the surface of mouse liver was rough and hard,the hepatic lobule was encysted or separated by collagen bundles,and pseudolobules emerged.At 16th week,the surface of mouse liver in the DEN alone group was rough with some nodules.HE and VG staining showed that the hepatocytes of nodules with obvious atypia and hyperchromatic nuclei were verified.However,these pathological changes were evidently improved in Sal B treatment groups compared with the DEN group,which was proved by reductive cirrhotic nodules and alleviative fibrosis at 12th week,and decreasing cancerous nodes and ameliorative differentiation via Sal B treatment at 16th week.Western blot results showed that the protein expression of p S-mad3C,p Smad3L,PAI-1 were less,and c-Myc expression was scarcely found in normal group;in DEN alone group,at 12th week,the protein expression of p Smad3C had no significant change,while the protein levels of p Smad3L,PAI-1,p21 were up-regulated,and at 16th week,the protein expressions of p S-mad3C,p Smad3L,p21,PAI-1 and c-Myc increased.In Sal B treatment group,the expressions of p21 and p Smad3C increased significantly,p Smad3L and PAI-1protein levels markedly decreased at 12th week,the expression of p Smad3C increased obviously,p21 was almost unchanged,and the expression of p Smad3L,PAI-1 and c-Myc were significantly reduced at 16th week.Conclusions Sal B could delay the progression of hepatic fibrosis-carcinoma in mice induced by DEN,and the mechanism may involve mediating the shift of p Smad3C/p21 and p Smad3L/PAI-1/c-Myc signaling.
AimTo observe the effect of Salvianolicaid B(Sal B)on the progression of hepatic fibrosiscarcinoma in mice induced by diethylnitrosamine(DEN)and investigate the mechanism of Sal B involved in the shift between p Smad3C/p21-mediated tumor suppressive signaling and p Smad3L/PAI-1/cMyc-mediatedpro-fibrogenic/oncogenicsignaling.MethodsA total of 100 male Kunming mice were randomly grouped,DEN-induced hepatic fibrosis-carcinoma model of mice was established,which was intragastrically treated by Sal B with two dosages(15,30 mg·kg~(-1))and colchicine with one dosage(0.2mg·kg~(-1)),respectively.The mice were sacrificed at12th week or 16th week after the start of DEN administration.Pathological changes of livers in each group were assessed by liver biopsy,hematoxylin-eosin(HE)staining and Van Gieson(VG)staining.The protein expressions of p Smad3C,p Smad3L,p21,plasminogen activator inhibitor-1(PAI-1)and c-Myc in liver tissues were assayed by Western blot.Results In the normal control group,the surface of mouse liver was smooth and soft,and the structure of the hepatic lobule was intact.In the DEN alone group,at 12th week,the surface of mouse liver was rough and hard,the hepatic lobule was encysted or separated by collagen bundles,and pseudolobules emerged.At 16th week,the surface of mouse liver in the DEN alone group was rough with some nodules.HE and VG staining showed that the hepatocytes of nodules with obvious atypia and hyperchromatic nuclei were verified.However,these pathological changes were evidently improved in Sal B treatment groups compared with the DEN group,which was proved by reductive cirrhotic nodules and alleviative fibrosis at 12th week,and decreasing cancerous nodes and ameliorative differentiation via Sal B treatment at 16th week.Western blot results showed that the protein expression of p S-mad3C,p Smad3L,PAI-1 were less,and c-Myc expression was scarcely found in normal group;in DEN alone group,at 12th week,the protein expression of p Smad3C had no significant change,while the protein levels of p Smad3L,PAI-1,p21 were up-regulated,and at 16th week,the protein expressions of p S-mad3C,p Smad3L,p21,PAI-1 and c-Myc increased.In Sal B treatment group,the expressions of p21 and p Smad3C increased significantly,p Smad3L and PAI-1protein levels markedly decreased at 12th week,the expression of p Smad3C increased obviously,p21 was almost unchanged,and the expression of p Smad3L,PAI-1 and c-Myc were significantly reduced at 16th week.Conclusions Sal B could delay the progression of hepatic fibrosis-carcinoma in mice induced by DEN,and the mechanism may involve mediating the shift of p Smad3C/p21 and p Smad3L/PAI-1/c-Myc signaling.
引文
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[2]Wang Q L,Wu Q,Tao Y Y,et al.Salvianolic acid B modulates the expression of drug-metabolizing enzymes in Hep G2 cells[J].Hepatobiliary Pancreat Dis Int,2011,10(5):502-8.
[3]李艳,张端莲,王兰,等.Sal B对肝癌细胞株Hep G2的促凋亡作用[J].中国组织化学与细胞化学杂志,2012,21(1):9[3]Li Y,Zhang R L,Wang L,et al.Sal B promotes apoptosis of human liver cancer cell line Hep G2[J].Chin J Histochem Cytochem,2012,21(1):9-13.
[4]Yoshida K,Murata M,Yamaguchi T,Matsuzaki K.TGF-β/Smad signaling during hepatic fibro-carcinogenesis[J].Int J Oncol,2014,45(4):1363-71.
[5]Hu X P,Rui W J,Wu C,et al.Compound Astragalus and Salvia miltiorrhiza extracts suppress hepatocarcinogenesis by modulating transforming growth factor-β/Smad signaling[J].J Gastroenterol Hepatol,2014,29(6):1284-91.
[6]吴佳俊,江宇峰,伍超,等.Smad3 WT、Smad3 EPSM、Smad33S-A 3种质粒稳转Hep G2细胞株的建立与功能研究[J].中国药理学通报,2016,32(6):825-31.[6]Wu J J,Jiang Y F,Wu C,et al.Construction and functional study of three plasmids including Smad3 WT,Smad3 EPSM and Smad3 3S-A stably transfected Hep G2 cell lines[J].Chin Pharmacol Bull,2016,32(6):825-31.
[7]Lee J S,Chu I S,Mikaelyan A,et al.Application of comparative functional genomics to identify best-fit mouse models to study human cancer[J].Nat Genet,2004,36(12):1306-11.
[8]Tolba R,Kraus T,Liedtke C,et al.Diethylnitrosamine(DEN)-induced carcinogenic liver injury in mice[J].Lab Anim,2015,49(1 Suppl):59-69.
[9]Sharma S,Gao P,Steele V E.Quantitative morphometry of respiratory tract epithelial cells as a tool for testing chemopreventive agent efficacy[J].Anticancer Res,2010,30(3):737-42.
[10]Cheng T O.Cardiovascular effects of Danshen[J].Int J Cardiol,2007,121(1):9-22.
[11]Liu C,Gaca M D,Swenson E S,et al.Smads 2 and 3 are differentially activated by transforming growth factor-beta(TGF-beta)in quiescent and activated hepatic stellate cells.Constitutive nuclear localization of Smads in activated cells is TGF-beta-independent[J].J Biol Chem,2003,278(13):11721-8.
[12]刘成海,刘平,胡义扬,朱大元.丹酚酸B盐对转化生长因子-β1刺激肝星状细胞活化与胞内信号转导的作用[J].中华医学杂志,2002,82(18):1267-72.[12]Liu C H,Liu P,Hu Y Y,Zhu D Y.Effects of salvianolic acid-B on TGF-β1stimulated hepatic stellate cell activation and its intracellula[J].Natl Med J China,2002,82(18):1267-72.
[13]Yoshida K,Matsuzaki K,Mori S,et al.Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury[J].Am J Pathol,2005,166(4):1029-39.
[14]Urukawa F,Matsuzaki K,Mori S,et al.p38 MAPK mediates fibrogenic signal through Smad3 phosphorylation in rat myofibroblasts[J].Hepatology,2003,38(4):879-89.
[15]Matsuzaki K,Kitano C,Murata M,et al.Smad2 and Smad3phosphorylated at both linker and COOH-terminal regions transmit malignant TGF-beta signal in later stages of human colorectal cancer[J].Cancer Res,2009,69(13):5321-30.
[16]芮文娟,何淑芳,黄先甲,等.DEN诱导大鼠肝纤维化-肝癌过程中p Smad3L和PAI-1蛋白的表达[J].中国药理学通报,2012,28(10):1393-7.[16]Rui W J,He S F,Huang X J,et al.Expression of p Smad3L and PAI-1 during the process of DEN-induced hepatic fibrosis-carcinoma in rats[J].Chin Pharmacol Bull,2012,28(10):1393-7.
[2]Wang Q L,Wu Q,Tao Y Y,et al.Salvianolic acid B modulates the expression of drug-metabolizing enzymes in Hep G2 cells[J].Hepatobiliary Pancreat Dis Int,2011,10(5):502-8.
[3]李艳,张端莲,王兰,等.Sal B对肝癌细胞株Hep G2的促凋亡作用[J].中国组织化学与细胞化学杂志,2012,21(1):9[3]Li Y,Zhang R L,Wang L,et al.Sal B promotes apoptosis of human liver cancer cell line Hep G2[J].Chin J Histochem Cytochem,2012,21(1):9-13.
[4]Yoshida K,Murata M,Yamaguchi T,Matsuzaki K.TGF-β/Smad signaling during hepatic fibro-carcinogenesis[J].Int J Oncol,2014,45(4):1363-71.
[5]Hu X P,Rui W J,Wu C,et al.Compound Astragalus and Salvia miltiorrhiza extracts suppress hepatocarcinogenesis by modulating transforming growth factor-β/Smad signaling[J].J Gastroenterol Hepatol,2014,29(6):1284-91.
[6]吴佳俊,江宇峰,伍超,等.Smad3 WT、Smad3 EPSM、Smad33S-A 3种质粒稳转Hep G2细胞株的建立与功能研究[J].中国药理学通报,2016,32(6):825-31.[6]Wu J J,Jiang Y F,Wu C,et al.Construction and functional study of three plasmids including Smad3 WT,Smad3 EPSM and Smad3 3S-A stably transfected Hep G2 cell lines[J].Chin Pharmacol Bull,2016,32(6):825-31.
[7]Lee J S,Chu I S,Mikaelyan A,et al.Application of comparative functional genomics to identify best-fit mouse models to study human cancer[J].Nat Genet,2004,36(12):1306-11.
[8]Tolba R,Kraus T,Liedtke C,et al.Diethylnitrosamine(DEN)-induced carcinogenic liver injury in mice[J].Lab Anim,2015,49(1 Suppl):59-69.
[9]Sharma S,Gao P,Steele V E.Quantitative morphometry of respiratory tract epithelial cells as a tool for testing chemopreventive agent efficacy[J].Anticancer Res,2010,30(3):737-42.
[10]Cheng T O.Cardiovascular effects of Danshen[J].Int J Cardiol,2007,121(1):9-22.
[11]Liu C,Gaca M D,Swenson E S,et al.Smads 2 and 3 are differentially activated by transforming growth factor-beta(TGF-beta)in quiescent and activated hepatic stellate cells.Constitutive nuclear localization of Smads in activated cells is TGF-beta-independent[J].J Biol Chem,2003,278(13):11721-8.
[12]刘成海,刘平,胡义扬,朱大元.丹酚酸B盐对转化生长因子-β1刺激肝星状细胞活化与胞内信号转导的作用[J].中华医学杂志,2002,82(18):1267-72.[12]Liu C H,Liu P,Hu Y Y,Zhu D Y.Effects of salvianolic acid-B on TGF-β1stimulated hepatic stellate cell activation and its intracellula[J].Natl Med J China,2002,82(18):1267-72.
[13]Yoshida K,Matsuzaki K,Mori S,et al.Transforming growth factor-beta and platelet-derived growth factor signal via c-Jun N-terminal kinase-dependent Smad2/3 phosphorylation in rat hepatic stellate cells after acute liver injury[J].Am J Pathol,2005,166(4):1029-39.
[14]Urukawa F,Matsuzaki K,Mori S,et al.p38 MAPK mediates fibrogenic signal through Smad3 phosphorylation in rat myofibroblasts[J].Hepatology,2003,38(4):879-89.
[15]Matsuzaki K,Kitano C,Murata M,et al.Smad2 and Smad3phosphorylated at both linker and COOH-terminal regions transmit malignant TGF-beta signal in later stages of human colorectal cancer[J].Cancer Res,2009,69(13):5321-30.
[16]芮文娟,何淑芳,黄先甲,等.DEN诱导大鼠肝纤维化-肝癌过程中p Smad3L和PAI-1蛋白的表达[J].中国药理学通报,2012,28(10):1393-7.[16]Rui W J,He S F,Huang X J,et al.Expression of p Smad3L and PAI-1 during the process of DEN-induced hepatic fibrosis-carcinoma in rats[J].Chin Pharmacol Bull,2012,28(10):1393-7.