【作者】 王华；

【导师】 魏伟；

【作者基本信息】 安徽医科大学 ， 药理学， 2005， 博士

【摘要】 肝纤维化是一种严重危害人类健康的疾病,其共同的病理改变是细胞外基质（extracellular matrix,ECM）的过度沉积,肝星状细胞（hepatic stellate cell,HSC）的表型激活是肝纤维化形成过程的关键,其中活化的HSC大量增殖和胶原的过度生成是导致ECM大量沉积的重要原因。目前治疗肝纤维化手段仍不尽人意,深入了解肝纤维化分子作用机制,寻找新的抗纤维化药物具有重要理论和实际意义。白芍总苷（Total glucosides of paeony,TGP）是祖国传统中药白芍根中提取的有效部位,本课题组以往的研究发现,TGP具有抗炎、抗氧化、免疫调节、抗肝损伤和免疫性肝纤维化等作用。本研究采用经典的CCl₄诱导的肝纤维化模型,首先从整体上考察了TGP的抗化学性肝纤维化作用,然后选用HSC-T6细胞株,研究了TGP体外抑制血清和细胞因子血小板衍生生长因子BB（platelet-derived growth factor,PDGF-BB）、转化生长因子β1（transforming growth factor β1,TGF-β1）刺激HSC-T6细胞增殖和胶原合成的抑制作用的量效和时效关系,最后采用Western-blot技术从蛋白表达水平观察了TGP对PDGF-BB、TGF-β1刺激后的G蛋白表达改变情况,进而对TGP抑制HSC-T6细胞增殖和胶原合成与G蛋白-AC-cAMP通路的关系作了初步探讨。 1.TGP对CCl₄诱导化学性肝纤维化大鼠的保护作用 TGP两个剂量(60、120mg·kg^-1)对CCl₄诱导化学性肝纤维化大鼠具有明显的保护作用。结果表明,给予TGP(60、120mg·kg^-1)和Col(0.1mg·kg^-1)ig给更多还原

【Abstract】 Hepatic fibrosis is a kind of severe disease with high morbidity and mortality, and its common pathological feature is over-aggradation of extracellular matrix （ECM）. The activation of hepatic stellate cell （HSC） is critical step in the development of liver fibrosis. Proliferation and fibrogenesis of activated HSC are attributed to over-aggradation of ECM. There have been no satisfactory drugs so far in the treatment of hepatic fibrosis. It is hoped that understanding the molecular pathophysiology of hepatic fibrosis will lead to novel therapeutic strategies and antifibrogenic drugs. Total glucosides of paeony （TGP） is an active compound extracted from roots of paeonia lactiflora Pall. Our previous study showed that TGP has anti-inflammatory, anti-oxidative, immunomodulatory, anti-hepatic injury and anti-immunological hepatofibrotic effects. In the present study, anti-chemical hepatofibrotic effects of TGP on CCl₄-induced hepatic fibrosis model were observed in vivo. The dose-effect and time-effect relationships of TGP on the proliferation and fibrogenesis in HSC-T6 cell line stimulated with mixed sera, platelet-derived growth factor BB （PDGF-BB） or transforming growth factor β1 （TGF-β1） were investigated in vitro. Lastly, the effects of TGP on the changes of the expressions of G-Protein stimulated by PDGF-BB or TGF-Bl were observed by the method of Western-blot analysis. The relationshipbetween the inhibitory effect of TGP on HSC-T6 proliferation or fibrogenesis and G-Protein-AC-cAMP signal transduction pathways was also investigated.1. Protective effects of TGP on chemical hepatic fibrosis induced by CCLjin ratsTGP at two doses of 60 and ^Omg-kg"1 had obvious protective effects on CCU-induced hepatic fibrosis in rats. The results showed that the serum ALT> AST remarkably decreased and A/G ratio also be slightly elevated by TGP （60 and 120mg-kg’’, ig） treatment, which indicate that TGP has protective effect on hepatic cells directly. Pathological examination showed that TGP could remarkably alleviate the hepatic fibrosis. TGP not only decreased the Hyp content of liver homogenates, but also decreased the elevated level of HA, LN, PCIII, CIV in serum, which indicate that TGP decrease the ECM production of hepatic fibrosis in rats.TGP also ameliorated the oxidative stress state of hepatic fibrosis rats, decreased the production of MDA and NO and enhanced the activities of antioxidative enzyme including SOD and GSH-px. TGP had anti-inflammatory effect on hepatic fibrosis in rats, which not only reduced the TNF-a and IL-lp in serum, but also decreased TNF-a and IL-ip production of Kupffer cells （KCs） in vivo or in vitro. TGP antagonized the production of TGF-pl and PDGF, inhibited the proliferation and fibrogenesis of HSC in vivo. The results mentioned above suggest that TGP ameliorate oxidative state of liver, inhibite the production of inflammatory cytokines via KCs, and inhibite the production of TGF-pi and PDGF, which may be part of the mechanisms of TGP anti-hepatofibrotic effects.2. The effect of TGP on HSC-T6 proliferation and production of collagen in different conditioned stimulusIn In vitro models for proliferation and collagen production of a cell strain named HSC-T6 were established, stimulated with cytokines PDGF-BB （lOjig-L’1） , TGF-pi（2ug?L"1） and combined blood serum （10%FCS+3% rat serum） respectively, which were measured by 3H-TdR and 3H-Proline incorporation. The results ofconcentration-inhibition and time-effect relationships of TGP on the proliferation and collagen production in vitro showed as followings: （1） TGP （15²40 mg’L"1） had no obvious cytotoxicity on HSC-T6 in vitro. （2）TGP（15²40mg?L’1） had inhibitory effect on HSC-T6 stimulated by PDGF-BB （lOjag’L’1）, which had concentration- and time-dependence. （3） TGP （15 ²40mg?L"1） had inhibitory effect on collagen production of HSC-T6 stimulated by TGF-(31 （2[ig#L"1）, which had concentration- and time-dependence. （4 ）TGP （15²40mg?L’1） had inhibitory effect on HSC-T6 stimulated by combined blood serum, which had concentration-dependence. The results stated above suggest that TGP （15²40mg?L"]） concentration- and time-dependently inhibit the proliferation and collagen production of HSC-T6 stimulated by various kinds of stimulation factors.3. Changes of G-protein-AC-cAMP signal transduction pathways in HSC-T6 induced by PDGF-BB and the effect of TGPThe changes of the expression of G-protein on HSC-T6 cell membrane induced by PDGF-BB （lOug’L"1） were detected by Western-blot analysis. The results showed that PDGF-BB remarkably increased the expression of Gai2, but had no evident effect on expression of Gail n Gai3 and Gas. The level of cAMP in cells was detected by RIA, PDGF-BB decreased the level of cAMP concentration-dependently. Furthermore, the tendency of cAMP was closely related with the proliferation of HSC-T6. The expression of Gi2 was remarkably inhibited by TGP, which also increased the level of cAMP, and then inhibited the proliferation of HSC-T6. The results above indicate that TGP may inhibit the proliferation of HSC-T6 induced by PDGF-BB via G-protein-AC-cAMP pathway.4. Changes of G-protein-AC-cAMP signal transduction pathways in HSC-T6 induced by TGF-pi and the effect of TGPThe changes of the expression of G-protein on HSC-T6 cell membrane induced by TGF-pi （2ug?L’]） were detected by Western-blot analysis. The results showed that更多还原