【作者】 刘浩；

【导师】 魏伟；

【作者基本信息】 安徽医科大学 ， 药理学， 2008， 博士

【摘要】 肝纤维化是指由多种慢性疾病引起的肝脏持续创伤修复反应而导致的细胞外基质（extracellular matrix,ECM）过度沉积。严重的肝纤维化导致肝硬化并引起肝功能衰竭,门静脉高压甚至需要肝脏移植治疗。长期以来广泛持续的肝纤维化被认为是不可逆转的,而越来越多的研究证据表明,肝纤维化甚至肝硬化都有逆转的可能。对肝纤维化细胞及分子机制的研究表明,多种细胞参与了肝纤维化的形成,其中激活的肝星状细胞（hepatic stellate cells,HSCs）被认为是促进ECM过度沉积的主要细胞,而多种细胞因子参与了肝星状细胞的激活,其中转化生长因子β₁（transforming growth factor,TGF-β₁）被认为是最关键的因素之一。其可激活HSCs和调节ECM的合成和降解平衡。以干扰HSCs中TGF-β₁及其细胞信号转导通路为靶点的抗肝纤维化治疗已成为当今研究的热点之一。而在肝纤维化及其胶原合成过程中,除TGF-β₁及其细胞信号转导通路外,尚有其他信号途径参与。前列腺素E₂（prostaglandin E₂,PGE₂）、PGE受体-2（PGE receptor-2,EP₂）及环磷酸腺苷（cyclic adenosine monophosphate,cAMP）所构成的信号转导途径,即PGE₂-EP₂-cAMP通路在多种免疫性疾病中发挥重要作用,越来越多的研究表明PGE₂-EP₂-cAMP信号转导途径参与了肝纤维化的调节,通过调节其信号转导可能为肝纤维化的治疗提高新的靶点。尽管对抗肝纤维化药物的研究不断深入,而目前结果仍不令人满意。黄芪（Radix Astragali）作为一种宝贵的中药具有悠久的应用历史,黄芪总皂苷为从荚膜黄芪（Astragalus membranaceus）根中提取的有效部位,多年来大量的研究表明黄芪总皂苷具有抗炎、衰老、抗氧化、抗心肌损伤及免疫调节的作用且无明显的毒副作用。本课题组以往的研究表明,黄芪总皂苷可显著的抑制四氯化碳引起的肝脏损伤及肝纤维化的形成。黄芪甲苷（AstragalosideⅣ,ASG-Ⅳ）是一种环波罗烷型三萜糖苷,从中草药荚膜黄芪中提取纯化而得,目前对AGS-Ⅳ对免疫性肝纤维化的影响尚不清楚。基于以往对黄芪在保护肝脏中的应用及本课题组对黄芪总皂苷抗肝纤维化作用的研究基础,本研究探讨了对单体AGS-Ⅳ抗猪血清诱导的大鼠免疫性肝纤维化的体内作用,从病理学及血清学角度对其抗肝纤维化作用进行分析,并观察了AGS-Ⅳ对大鼠血清TGF-β₁水平及肝脏组织的影响。体外研究观察了AGS-Ⅳ对HSCs增殖、凋亡及胶原合成的影响及对TGF-β₁/Smads信号转导的调节。在肝纤维化的形成及TGF-β₁刺激的HSCs胶原合成中,PGE₂-EP₂-cAMP信号通路与TGF-β₁刺激HSCs胶原合成的有着怎样的关系呢?而AGS-Ⅳ是否会通过对PGE₂-EP₂-cAMP信号通路的影响而发挥其抗肝纤维化作用呢?为此,本实验通过对猪血清诱导大鼠肝纤维化形成过程中PGE₂水平及EP₂表达的改变进行观察,并使用选择性环氧化酶-2（cyclooxygenase-2,COX-2）抑制剂降低PGE₂水平,分析EP₂表达的改变,深入的观察了PGE₂-EP₂-cAMP在猪血清诱导大鼠肝纤维化形成过程的作用;在体外观察了PGE₂-EP₂-cAMP对TGF-β₁刺激HSCs胶原合成中的影响,并从PGE₂-EP₂-cAMP信号通路角度研究了AGS-Ⅳ抗肝纤维化的机制。目的探讨AGS-Ⅳ抗猪血清诱导大鼠免疫性肝纤维化作用;探讨AGS-Ⅳ对体外培养HSCs增殖、凋亡及胶原合成的影响及对Smad3和Smad7蛋白表达的影响;探讨PGE₂-EP₂-cAMP信号通路在肝纤维化形成中的作用及AGS-Ⅳ对其影响。方法猪血清连续8周腹腔注射（0.5 ml,每周2次）诱导大鼠免疫性肝纤维化,同时给予AGS-Ⅳ(2.0,4.0 mg·kg^-1)灌胃治疗。检测大鼠肝脏组织病理及血清学指标改变,ELISA法检测大鼠血清TGF-β₁水平,免疫组化法检测大鼠肝脏组织TGF-β₁表达。[³H]标记胸腺嘧啶核苷酸（[³H]-thymidine,[³H]-TdR）掺入法检测血小板源性生长因子（PDGF-BB）刺激的HSCs增殖,流式细胞仪分析HSCs凋亡。TGF-β₁刺激的HSCs胶原合成采用[³H】标记脯氨酸（[³H]-proline,[³H]-Pro）掺入法检测,westernblot法检测Smads蛋白表达。选择性COX-2抑制剂塞来昔布（celecoxib）15mg·kg^-1灌胃,以检测POE₂及EP₂蛋白的变化及其在大鼠肝纤维化形成中的作用。检测PGE₂（5,500nM）和EP₂ agonist ONO-AE1-259-01（1,100nM）对TGF-β₁诱导HSCs胶原合成的影响及细胞内cAMP水平的改变;分析AGS-Ⅳ对HSCs中EP₂表达及胞内cAMP水平的影响。结果1.AGS-Ⅳ对猪血清诱导肝纤维化的保护作用AGS-Ⅳ两个剂量2.0 mg·kg^-1和4.0mg·kg^-1对猪血清诱导的免疫性肝纤维化大鼠均具有良好的保护作用。模型组透明质酸（hyaluronic acid,HA）,层连蛋白（laminin,LN）,Ⅲ型前胶原（procollagen typeⅢ,PCⅢ）及Ⅳ型胶原（CollagenⅣ,CⅣ）等纤维化血清学标记物水平较正常组明显升高,给予AGS-Ⅳ治疗后可使其显著降低。肝纤维化的一个重要指标肝组织羟脯氨酸（hydroxyproline,Hyp）含量的检测结果显示,AGS-Ⅳ2.0和4.0mg·kg^-1可显著降低猪血清刺激后升高的Hyp值。组织病理学结果表明AGS-Ⅳ可明显的抑制猪血清诱导的肝纤维化,改善肝组织结构。AGS-Ⅳ还可以缓解肝纤维化大鼠肝脏的氧化应激状态,降低肝脏丙二醛（malondiadehyde,MDA）的合成,增加超氧化物歧化酶（super oxide dismutase,SOD）、谷胱甘肽过氧化物酶（glutathione peroxidase,GSH-px）等抗氧化酶的活性。鉴于TGF-β₁在肝纤维化中的重要作用,实验中观察了AGS-Ⅳ对猪血清诱导肝纤维化大鼠血清及肝脏组织中TGF-β₁表达的影响。免疫组织化学结果显示AGS-Ⅳ治疗后大鼠肝组织中TGF-β₁的表达显著降低,血清检测结果显示,AGS-Ⅳ可降低模型中升高的血清TGF-β₁水平。2.AGS-Ⅳ对HSCs增殖、凋亡及胶原合成的影响和对TGF-β₁/Smads信号转导的影响体外细胞实验观察了不同浓度AGS-Ⅳ(1.5、3.0、6.0、12.0、24.0 mg·L^-1)对HSCs增殖、凋亡及胶原合成的影响。结果显示,AGS-Ⅳ呈浓度依赖性的抑制PDGF-BB刺激的HSCs增殖。并可增加sub-G₁ DNA含量,诱导HSCs凋亡,且增加caspase-3的活性。AGS-Ⅳ可显著抑制TGF-β₁诱导的HSCs胶原合成。为进一步研究AGS-Ⅳ对Smads信号转导的调节作用,实验中采用western blot法分析了Smad3和Smads7蛋白的表达,结果显示,TGF-β₁刺激HSCs后,Smad3表达水平显著升高,在给予AGS-Ⅳ处理后,随浓度增加(6.0、12.0、24.0 mg·L^-1),可降低Smad3的表达水平。同时,AGS-Ⅳ可使TGF-β₁刺激后升高的Smad7的表达水平进一步升高。3.PGE₂-EP₂-cAMP信号转导在HSCs胶原合成中的作用及AGS-Ⅳ对其影响为检测PGE₂水平降低及EP₂表达改变对猪血清诱导肝纤维化大鼠的影响,实验中给予选择性COX-2抑制剂塞来昔布处理大鼠。病理学结果显示,塞来昔布可显著增加猪血清诱导的肝纤维化,值得注意的是,除了加重纤维化的形成外,塞来昔布还可使猪血清诱导肝纤维化大鼠肝脏出现炎性坏死。而其他各组均无肝组织坏死出现。而塞来昔布本身并不引起肝纤维化。实验中检测了血清肿瘤坏死因子-α（tumor necrosis factor-α,TNF-α）,白介素-1β（interleukin-1β,IL-1β）两种重要的炎性因子,猪血清及塞来昔布单独刺激对血清TNF-α和IL-1β水平无明显的影响,而塞来昔布可使猪血清诱导肝纤维化大鼠血清TNF-α和IL-1β显著升高。血清PGE₂水平的检测结果显示,猪血清刺激2周后,大鼠血清PGE₂水平显著升高,随着猪血清的持续刺激,血清PGE₂水平开始下降,至第8周时显著低于正常对照组。第8周时,塞来昔布处理后各组大鼠血清PGE₂水平均低于对照组,塞来昔布可使猪血清免疫后大鼠血清PGE₂的水平进一步降低,western blot结果显示,猪血清刺激8周后分离培养的大鼠HSCs中EP₂表达水平显著低于正常对照组大鼠HSCs。体外实验研究了PGE₂及EP₂受体激动剂ONO-AE1-259-01对HSCs胶原合成的影响,PGE₂（5,500nM）及ONO-AE1-259-01（1,100 nM）对TGF-β₁刺激的胶原合成具有显著的抑制作用,并可升高细胞内cAMP水平,实验中进一步观察了AGS-Ⅳ对PGE₂-EP₂-cAMP通路的影响,结果显示,AGS-Ⅳ可减少猪血清诱导肝纤维化大鼠血清PGE₂水平的降低,增加EP₂在HSCs中的表达,升高细胞内cAMP浓度。结论1.AGS-Ⅳ对猪血清诱导大鼠肝纤维化具有良好的保护作用AGS-Ⅳ两个剂量对猪血清诱导肝纤维化大鼠肝细胞具有良好的保护作用,并改善肝组织病理改变,阻止肝纤维化的形成。AGS-Ⅳ可改善猪血清诱导肝纤维化大鼠肝脏氧化应激状态,并可以抑制模型组大鼠HSCs的胶原合成及增殖。2.AGS-Ⅳ可抑制HSCs的增殖、诱导HSCs凋亡,并通过调节TGFβ/Smads信号通路抑制其胶原合成AGS-Ⅳ1.5～24.0 mg·L^-1对PDGF-BB刺激的HSCs增殖具有浓度依赖性的抑制作用;AGS-Ⅳ具有诱导HSCs凋亡的作用,其机制可能是与增加caspase-3活性有关;AGS-Ⅳ1.5～24.0 mg·L^-1对TGF-β1诱导的HSCs胶原合成具有浓度依赖性抑制作用。AGS-Ⅳ可降低Smad3表达降低,而升高抑制型Smad7的表达,提示AGS-Ⅳ可能是通过TGFβ₁/Smad信号转导的调节对HSCs胶原合成发挥抑制作用。3.PGE₂-EP₂-cAMP通路在猪血清诱导大鼠肝纤维化形成及HSCs胶原合成中发挥重要作用猪血清诱导肝纤维化大鼠中,血清PGE₂水平呈下降趋势,EP₂表达降低,给予COX-2抑制剂celecoxib处理后,血清PGE₂进一步降低,EP₂蛋白表达降低,而纤维化程度增加,表明PGE₂-EP₂在猪血清诱导大鼠肝纤维化中可能发挥保护作用,体外研究表明,PGE₂和选择性的EP₂受体激动剂均可抑制TGF-β₁诱导的HSCs胶原合成,增加细胞内cAMP浓度,提示PGE₂-EP₂-cAMP信号转导参与了TGF-β₁诱导HSCs胶原合成。4.AGS-Ⅳ抗猪血清诱导大鼠肝纤维化作用及及抑制HSCs胶原合成的作用可能是与其抑制血清PGE₂水平的降低、增加EP₂蛋白表达及升高胞内cAMP水平有关。体内研究结果说明AGS-Ⅳ的抗猪血清诱导大鼠肝纤维化作用可能与其对抑制猪血清免疫大鼠后引起的血清POE₂水平降低,增加HSCs中EP₂的表达有关。而体外给予AGS-Ⅳ处理后,可增加HSCs中EP₂的表达,升高细胞内cAMP浓度,提示AGS-Ⅳ对TGF-β₁诱导HSCs胶原合成的抑制作用可能与其对PGE₂-EP₂-cAMP通路的调节有关。总之,本课题研究了AGS-Ⅳ对猪血清诱导肝纤维化大鼠的保护作用;探讨了AGS-Ⅳ对PDGF-BB刺激HSCs增殖、凋亡及TGF-β₁诱导胶原合成的影响,及AGS-Ⅳ对TGF-β/Smad信号转导的影响;探讨了PGE₂-EP₂-cAMP在猪血清诱导大鼠肝纤维化中的作用及其在TGF-β₁诱导HSCs胶原合成中的作用。对PGE₂-EP₂-cAMP通路的调节可能是AGS-Ⅳ抑制TGF-β₁诱导HSCs胶原合成的重要机制之一。更多还原

【Abstract】 Liver.fibrosis results from chronic damage to the liver in conjunction with the accumulation of extracellular matrix（ECM） proteins,which is a characteristic of most types of chronic liver diseases.Advanced liver fibrosis results in cirrhosis,liver failure,and portal hypertension and often requires liver transplantation.Extensive and persistent hepatic fibrosis has for a long time been considered irreversible.However, accumulating evidence suggests that liver fibrosis is reversible and recovery from cirrhosis may be possible.Hepatic stellate cells（HSCs） are major sources of the excess ECM deposited in fibrotic liver.Transforming growth factorβ₁（TGF-β₁） is considered to play a generic role in the development of tissue fibrosis,as it stimulates the synthesis and deposition.Smad pathways are central mediators of signals from the receptors for TGF-β₁ to the nucleus.However,growing biochemical and developmental evidence supports that PGE₂-EP₂-cAMP signal transduction also participates in TGF-β₁ signalling.Despite efforts to develop antifibrotic agents,no drugs have been approved as antifibrotic agents in humans.Radix Astragali is one of the valuable medical herbs that have been widely used in East Asia for a long time.Astragalosides is the major active component extracted from the root of Astragalus membranaceus.The anti-inflammatory,anti-aging, anti-oxidative,anti-myocardial injury and immunoregulatory activities without evident toxic or side effects of astragalosides have been extensively proved for many years.Previous studies from our laboratory demonstrated that crude astragalosides fraction can significantly inhibit the progression of hepatic fibrosis induced by Carbon tetrachloride（CCl₄）.AstragalosideⅣ（AGS-Ⅳ）,a new glycoside of cycloartane-type triterpene, 3-O-β-Dxylopyranosyl-6-O-β-D-glucopyranosylcycloastragenol was purified from the Chinese medical herb Astragalus membranaceus（Fisch） Bge.Up to now,whether AGS-Ⅳhas an exact antifibrotic effect in immunological hepatic fibrosis and the mechanism are not known.In the present study,we examined the in vivo effect of AGS-Ⅳon liver fibrosis induced by administration of porcine serum（PS） and observed the changes of transforming growth factor-β₁ in serum and liver tissue in rats treated with AGS-Ⅳ.In vitro effects of AGS-Ⅳon primary rat HSCs proliferation,apoptosis and collagen synthesis were detected,the effects of ASG.-Ⅳon the expressions of Smad3 and Smad7 were investigated.Further more The relationship between the PGE₂-EP₂-cAMP signal transduction and HSCs fibrogenesis and the effects of AGS-Ⅳwere also evaluated.AIMTo investigate the anti-fibrositic effects of AGS-Ⅳon immunological hepatic fibrosis in rats induced by PS;to investigate the in vitro effects of AGS-Ⅳon the proliferation,apoptosis and collagen synthesis in HSCs and the effects on the expressions of Smad3 and Smad7;to investigate the role of PGE₂-EP₂-cAMP signal transduction in fibrogenesis and the effects of AGS-Ⅳ.METHODSLiver fibrosis was induced by PS injection（0.5 ml,twice a week） for 8 weeks in Wistar rats.At the same time,AGS-Ⅳ(2.0,4.0mg·kg^-1) was administered intragastrically.Histopathology was examined by hematoxylin-eosin（HE） and Masson’s staining.Serum marker and TGF-β₁ levels were detected by radioimmunoassay and enzyme-linked immunosorbent assay（ELISA）,respectively. The expression of TGFβ₁ in liver tissue was evaluated by immunohistochemistry. Effect of AGS-Ⅳon platelet-derived growth factor-B subunit homodimer（PDGF-BB） stimulated proliferation was detected by[³H]-thymidine incorporation assays. Flow-cytometric analysis was performed to examine the apoptosis-inducing effect. Collagen synthesis stimulated by TGF-β₁ was detected by[³H]-proline incorporation assays.Western blotting analysis was performed to detect the expressions of Smads protein.COX-2 inhibitor,celecoxib 15 mg·kg^-1 body weight daily were given by gastric gavage to evaluated the changes and the effects of PGE₂ and EP₂ on PS induced liver fibrosis.Detected the effects of PGE₂（5,500nM） and EP₂ agonist ONO-AE1-259-01（1,100 nM） on TGF-β₁ stimulated collagen synthesis and the changes of intracellular cAMP level.RESULTS1.Protective effect of AGS-Ⅳon immunological hepatic fibrosis in rats induced by porcine-serumAGS-Ⅳat two dose of 2.0 and 4.0 mg·kg^-1 had obvious protective effects on PS-induced immunological hepatic fibrosis.Serum levels of hyaluronic acid（HA）, laminin（LN）,procollagen typeⅢ（PCⅢ） and collagenⅣ（CⅣ）,the surrogate markers of liver fibrogenesis,increased significantly in hepatic fibrotic rats in model group and were suppressed by treatment with AGS-Ⅳ.Hyp contents in liver tissue were carried out as an index of liver fibrosis,the increased was effectively reduced by the administration with AGS-Ⅳ(2.0 and 4.0mg·kg^-1).Histological results showed that hepatic fibrosis caused by PS was significantly inhibited by AGS-Ⅳ.AGS-Ⅳimproved alterations in the liver structure.AGS-Ⅳalso ameliorated the oxidative stress state of hepatic fibrosis rats, decreased the production of malondiadehyde（MDA） and enhanced the activities of anti-oxidative enzyme including super oxide dismutase（SOD） and glutathione peroxidase（GSH-px）.Because TGF-β₁ plays an important role in hepatic fibrosis,we further examined whether the PS induced TGF-β₁ expression was affected by treatment with AGS-Ⅳ.In rats treated with AGS-Ⅳ,the positive staining TGF-β₁ in HSCs and in fibroblasts was markedly reduced than in model group,serum levels of TGF-β₁ were increased markedly in model group and were significantly decreased by treatment with AGS-Ⅳ(2.0,4.0mg·kg^-1).2.Effect of AGS-Ⅳon HSCs proliferation,apoptosis,collagen synthesis and on TGF-β₁/SmadsThe in vitro study was carried out to investigate the effects of AGS-Ⅳon the proliferation,apoptosis and collagen synthesis in HSCs.Platelet-derived growth factor-B subunit homodimer（PDGF-BB） stimulated proliferation was detected by [³H]-thymidine incorporation assays.Flow-cytometric analysis was performed to examine the apoptosis-inducing effect of AGS-Ⅳon HSCs.TGF-β₁ stimulated collagen synthesis was detected by[³H]-proline incorporation assays.Results revealed that AGS-Ⅳ(1.5、3.0、6.0、12.0、24.0 mg·L^-1) suppressed the proliferation of HSCs in a concentration-dependent manner,and increased sub-G₁ DNA content and increased the activity of caspase-3.Meanwhile,TGF-β₁ stimulated collagen synthesis was significantly inhibited by AGS-Ⅳ.The results suggest that AGS-Ⅳsignificantly inhibited HSCs proliferation,induced apoptosis in HSCs via caspase-3 activation.To determine the effects of AGS-Ⅳon Smads expressions,western blotting analysis was applied to detect its protein levels,The level of Smad3 was increased stimulated by TGF-β₁,while treated with AGS-Ⅳ,it declined gradually with an increase in the concentration from 6.0 to 24.0mg·L^-1.Meanwhile,AGS-Ⅳfuther increased the expression of Smad7 stimulated by TGF-β₁.3.Effects of AGS-Ⅳon PGE₂-EP₂-cAMP signal transduction in liver fibrosis in rat and in HSCs In order to examine the effects of decreased prostaglandin E₂（PGE₂） level on PS-induced hepatic fibrosis,rats were treated with selective cyclooxygenase-2 （COX-2） inhibitor,celecoxib.Histological results showed that fibrosis was significantly increased by co-administration of celecoxib.Most notably,besides hepatic fibrosis formation,livers in PS plus celecoxib treated group showed hepatocellular necrosis,no obvious hepatocellular necrosis was found in other three groups.Celecoxib alone without PS injection did not result in any hepatic fibrosis and hepatocellular necrosis.Plasma levels of tumor necrosis factor-α（TNF-α） and interleukin-1β（IL-1β）,the principal pro-inflammatory cytokines were measured, neither PS nor celecoxib alone induced notable change in the serum levels of TNF-αand IL-1β.However,co-treatment with PS and celecoxib resulted in a marked increase in plasma TNF-αand IL-1βlevel.In rats treated with PS alone,plasma level of PGE₂ was increased after 2 weeks PS injection followed by a slight decrease, at week 8,it was significantly lower than that in normal control group.At week 8,the level of PGE₂ in plasma and EP₂ expression in HSCs were significantly lower in all groups of rats receiving celecoxib,when compared with normal control.The plasma level of PGE₂ and EP₂ expression in rats treated with PS was further decreased by celecoxib administration at week 8.In vitro studies investigated the effects of PGE₂ and EP₂ agonist on collegen synthesis in HSCs,results shown that,PGE₂（5,500nM） and EP₂ agonist ONO-AE1-259-01（1,100 nM） could induce inhibitory effect on TGF-β₁ stimulated HSCs collagen synthesis via increase the intracellular cAMP level. We investigated the effects of AGS-Ⅳon the expression of EP₂ and intracellular cAMP level.Results showed that AGS-Ⅳ(6.0、12.0、24.0 mg·L^-1) could significantly increased the expression of EP₂ and cAMP level in HSCs.CONCLUSION1.AGS-Ⅳhas an anti-hepatic fibrosis effects in PS-induced rat models via ameliorated the oxidative stress,decreased the TGF-β₁ levels in plasma and the expression in liver tissue; 2.AGS-Ⅳmay achieve its inhibit effects on HSCs by inhibiting the proliferation, inducing apoptosis in HSCs via caspase-3 activation,and modulating TGF-β₁/Smads signaling response and consequently suppressing the collagen productions deposition;3.PGE₂-EP₂-cAMP signal pathway had important role in liver fibrosis induced by PS injection in rats and in collegen synthesis in HSCs.Increaseing EP₂ expression and intracellular cAMP level may inhibit collagen synthesis in HSCs. 4.Anti-fibrosis effects of AGS-Ⅳmay be associated with its effects of increaseing PGE₂ level in plasma in rats,increasing EP₂ expression and cAMP level in HSCs.更多还原