【作者】 孙妩弋；

【导师】 魏伟；

【作者基本信息】 安徽医科大学 ， 药理学， 2008， 博士

【摘要】 肝纤维化是肝组织对慢性损伤的修复反应,是多种类型细胞、氧化应激、细胞因子和生长因子等一系列复杂作用的结果,以细胞外基质（extracellular matrix, ECM）成分的过度增生与异常沉积为主要特征。肝纤维化是慢性肝病重要的病理特征,也是肝硬化发生的前奏和必经的中间环节,是临床治疗慢性肝病的关键环节。近年来国内外取得广泛共识的是:肝星状细胞（hepatic stellate cell,HSC）是肝脏ECM的主要来源,是肝纤维化形成的细胞学基础,它在ECM代谢和各种细胞介质的产生过程中处于中心地位,HSC的表型激活和过度增殖是肝纤维化形成过程的关键。抗肝纤维化的治疗,国内外虽陆续有过一些报道,但因种种原因尚未找到十分理想的药物,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。芍芪多苷（extract from Paeonia lactiflora and Astragalus membranaceus, SQDG）是采用科学的浸提和纯化技术,由白芍和黄芪混合提取制成的质量可控的天然药物的有效部位,主要含芍药苷、黄芪甲苷等成分。本课题组前期研究发现,SQDG对四氯化碳诱导的化学性肝损伤和卡介苗加脂多糖诱导的免疫性肝损伤具有保护作用,且效果优于白芍、黄芪分别单独提取后再混合制剂（白芍总苷+黄芪总皂苷）及单独使用白芍总苷或黄芪总皂苷。本实验在以往研究基础上,采用猪血清诱导的免疫性肝纤维化模型,首先从整体水平考察了SQDG对大鼠免疫性肝纤维化的作用;体外选用HSC-T6细胞株,从细胞和分子水平进一步探讨SQDG中有效活性成分芍药苷（paeoniflorin,Pae）抑制HSC增殖的分子机制,观察Pae对重组大鼠血小板衍生生长因子-BB（recombinant rat platelet derived growth factor-BB, rrPDGF-BB）刺激HSC-T6增殖的影响;探讨G蛋白偶联的信号转导通路与ERK1/2信号通路在rrPDGF-BB刺激HSC-T6增殖中的作用及相互关系,寻找其抑制HSC增殖的作用靶点。目的:采用猪血清诱导的免疫性肝纤维化模型,从病理形态学、转氨酶、血清纤维化标志物、脂质过氧化等方面,明确SQDG对大鼠免疫性肝纤维化的治疗作用;以肝纤维化过程中的关键细胞-HSC为突破口,观察SQDG中主要有效成分Pae对rrPDGF-BB刺激HSC-T6增殖的影响以及环氧合酶-2（cyclooxygenase-2,COX-2）在HSC-T6增殖中的作用;探讨G蛋白偶联的信号转导通路与ERK1/2信号通路在rrPDGF-BB刺激HSC-T6增殖中的作用,探讨Pae对rrPDGF-BB刺激的HSC-T6 G蛋白的表达及ERK1/2通路活化的影响,并进一步研究两条信号通路之间的相互关系,部分阐明Pae抑制HSC增殖的分子机制。方法:大鼠腹腔注射猪血清建立免疫性肝纤维化模型,设立正常对照组、模型组、SQDG给药组(42.5, 85, 170mg·kg^-1)和阳性对照秋水仙碱组(Col 0.1 mg?kg^-1)。HE染色和Masson染色对肝脏组织作病理检查。分光光度法检测血清中转氨酶活性和肝匀浆中丙二醛（MDA）含量、超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）活性、羟脯氨酸（Hyp）含量;放免法检测血清中透明质酸（HA）、Ⅲ型前胶原（PCⅢ）、IV型胶原（CIV）和层粘蛋白（LN）、前列腺素E2（PGE2）水平;MTT法检测HSC增殖情况;放射性免疫法测定HSC-T6 cAMP水平;采用Western blot技术检测HSC-T6中COX-2、Gαs、Gαi^-1、Gαi-2、Gαi-3蛋白表达和ERK1/2磷酸化水平的变化。结果:1. SQDG对猪血清诱导的免疫性肝纤维化大鼠的保护作用SQDG对猪血清诱导的免疫性肝纤维化大鼠具有明显的保护作用。结果表明,SQDG(85, 170mg·kg^-1)能显著降低猪血清诱导的免疫性肝纤维化大鼠升高的肝、脾指数,对升高的血清转氨酶有降低趋势,但无显著性意义;病理组织学检查发现,SQDG可降低猪血清诱导的肝纤维化大鼠的纤维化程度,与模型组相比,纤维沉积、肝小叶的破坏等均有所减轻。SQDG还可显著降低纤维化大鼠肝组织Hyp含量,降低血清中HA、LN、PCIII和CIV含量,提示SQDG可以减少纤维化大鼠ECM的生成。进一步研究发现,SQDG显著降低肝纤维化大鼠肝匀浆中MDA的含量,升高抗氧化酶SOD、GSH-Px活性,改善肝脏氧化状态,还能显著降低肝纤维化大鼠血清中升高的炎性细胞因子PGE2的产生。体外分离大鼠HSC检测其增殖情况,结果表明SQDG可以显著抑制HSC的增殖,SQDG还可以明显抑制模型组肝脏升高的PDGFR-β的表达。提示:抑制氧化应激和致纤维化炎性细胞因子的产生,抑制PDGFR-β的表达从而抑制HSC增殖可能是SQDG抗肝纤维化的部分机制。2. SQDG对免疫性肝纤维化大鼠MAPK相关蛋白磷酸化和G蛋白表达的影响在猪血清诱导的免疫性肝纤维化模型大鼠中,肝组织ERK1/2、p38和JNK磷酸化程度增加,肝组织中Gαs的表达明显降低,Gαi-2和Gαi-3的表达明显增加,而Gαi^-1的表达没有明显变化。给予SQDG可以明显抑制免疫性肝纤维化大鼠肝组织ERK1/2、p38和JNK磷酸化程度,SQDG还可以明显促进Gαs的表达,抑制Gαi-2和Gαi-3的表达。提示影响MAPK和G蛋白通路的变化可能是SQDG发挥抗肝纤维化作用的重要机制之一。3. Pae对rrPDGF-BB刺激HSC-T6增殖的影响体外建立rrPDGF-BB诱导的HSC-T6增殖模型,结果表明Pae在12.5～200mg?L^-1浓度范围内明显抑制HSC-T6增殖。进一步观察COX-2在rrPDGF-BB刺激HSC-T6增殖中的作用,结果表明rrPDGF-BB刺激可引起COX-2表达量持续增加,选择性的COX-2抑制剂NS-398可以明显抑制rrPDGF-BB诱导的HSC-T6增殖。Pae(50, 100 mg·L^-1)可以明显抑制rrPDGF-BB引起的HSC-T6 COX-2表达增加。结果提示抑制COX-2的表达可能是Pae抑制HSC-T6增殖的作用机制之一。4. Pae对rrPDGF-BB刺激的HSC-T6 ERK1/2信号通路活化的影响Western-blot检测发现,rrPDGF-BB刺激HSC-T6可引起ERK1/2的迅速磷酸化, Pae(25, 50, 100 mg·L^-1)可以抑制rrPDGF-BB引起的HSC-T6 ERK1/2的激活,降低p-ERK1/2水平。提示抑制rrPDGF-BB刺激的HSC-T6 ERK1/2信号通路的活化是Pae抑制其增殖的主要机制之一。5. rrPDGF-BB刺激下HSC-T6 G蛋白-AC-cAMP通路的改变及Pae的作用应用Western-blot方法,检测rrPDGF-BB(50μg·L^-1)刺激HSC-T6中G蛋白-AC-cAMP通路的改变。结果发现,rrPDGF-BB可以明显促进Gαi^-1和Gαi-2蛋白表达水平,但对Gαi-3和Gαs表达无明显影响。同时,rrPDGF-BB可以降低细胞内cAMP水平和PKA活性,促进HSC-T6增殖。Pae(50, 100mg?L^-1)可明显抑制rrPDGF-BB引起的Gαi^-1、Gαi-2的表达升高,提高细胞内cAMP水平。且相关性与回归分析结果表明Pae抑制rrPDGF-BB刺激的HSC-T6增殖反应与其提高HSC-T6内cAMP水平密切相关。提示下调HSC-T6 Gαi蛋白的表达是Pae抑制rrPDGF-BB刺激的HSC-T6过度增殖的重要机制之一。6. rrPDGF-BB刺激下HSC-T6 ERK1/2信号通路与Gαi介导信号通路间的关系采用Gi特异性的抑制剂PT作用于HSC-T6,结果表明PT可抑制rrPDGF-BB诱导的HSC-T6的增殖及p-ERK1/2的表达,提示PT敏感的G蛋白对ERK1/2的激活具有调节作用。用MEK1/2特异性的抑制剂U0126抑制ERK1/2的激活,观察其对rrPDGF-BB刺激下HSC-T6 PT敏感的G蛋白通路的影响。结果发现,U0126对Gαi^-1和Gαi-2蛋白表达水平没有明显影响。rrPDGF-BB刺激的HSC-T6细胞内cAMP水平和PKA活性明显下降,在rrPDGF-BB刺激下HSC-T6中加入U0126后,对降低的cAMP水平和PKA活性没有明显的影响。提示ERK1/2可能存在于PT敏感的Gi蛋白通路的下游发挥作用。Pae可能通过下调Gαi表达,进而抑制ERK1/2的激活,发挥抑制rrPDGF-BB刺激HSC-T6异常增殖的作用。结论:1. SQDG具有明显的抗肝纤维化作用,其机制与改善肝纤维化大鼠肝脏的氧应激状态、抑制炎性细胞因子的生成、抑制HSC增殖等有关。2. rrPDGF-BB刺激可引起COX-2表达量持续增加,选择性的COX-2抑制剂NS-398可以明显抑制rrPDGF-BB诱导的HSC-T6增殖。SQDG主要成分Pae可以明显抑制rrPDGF-BB引起的HSC-T6 COX-2表达增加,抑制COX-2的表达可能是Pae抑制HSC-T6增殖的作用机制之一。3.rrPDGF-BB刺激HSC-T6可引起ERK1/2的迅速磷酸化。Pae(25, 50, 100 mg·L^-1)可以抑制rrPDGF-BB引起的HSC-T6 ERK1/2的激活,降低p-ERK1/2水平。MEK抑制剂U0126可以抑制rrPDGF-BB引起的HSC-T6 COX-2表达增加。提示Pae可能通过抑制rrPDGF-BB刺激的HSC-T6 ERK1/2信号通路的活化,减少COX-2的表达发挥其抑制HSC-T6增殖的作用。4. rrPDGF-BB可以明显促进Gαi^-1和Gαi-2蛋白表达水平,降低细胞内cAMP水平和PKA活性,Pae可明显抑制rrPDGF-BB引起的Gαi^-1、Gαi-2的表达升高,且Pae抑制rrPDGF-BB刺激的HSC-T6增殖反应与其提高HSC-T6细胞内cAMP水平密切相关。Pae可能通过下调HSC-T6 Gαi蛋白偶联的信号通路抑制rrPDGF-BB刺激的HSC-T6过度增殖。5. Gi特异性的抑制剂PT可抑制rrPDGF-BB诱导的HSC-T6的增殖及p-ERK1/2的表达,MEK1/2特异性的抑制剂U0126对Gαi^-1和Gαi-2蛋白表达水平及细胞内降低的cAMP水平、PKA活性没有明显的影响。提示在HSC上,PT敏感的Gi蛋白可促进ERK1/2的激活。Pae可能通过下调Gαi的表达,抑制ERK1/2的激活,降低p-ERK1/2水平,发挥其抑制rrPDGF-BB刺激HSC-T6过度增殖的作用。更多还原

【Abstract】 Hepatic fibrosis can be classified as a wound healing response to a variety of chronic stimuli. It is characterized by an excessive deposition of extracellular matrix proteins （ECM） of which type I collagen predominates. This excess deposition of extracellular matrix proteins disrupts the normal architecture of the liver that alters the normal function of the organ, resulting in pathophysiological damage to the organ. Hepatic stellate cell （HSC） are presently regarded as one of the key cell types involved in the progression of liver fibrosis. The activation of HSC to a proliferative, myofibroblastic phenotype plays a key role in hepatic fibrogenesis, since these cells are the principal cellular source of the excess collagen synthesis during hepatic fibrosis. Efforts have been made to search for effective anti-fibrotic agents. However, no effective antifibrotic therapies are available until now. It is hoped that understanding the molecular pathophysiology of hepatic fibrosis will lead to novel therapeutic strategies and anti-fibrotic drugs.SQDG is standardized extract of the Chinese herb prescription composed of Paeonia lactiflora and Astragalus membranaceus. SQDG was mainly composed of paeoniflorin and astragaloside IV etc. Our previous studies have shown that SQDG has protective effects on carbon tetrachloride （CCl4）-induced liver injury and Bacillus Calmette-Guérin （BCG） plus lipopolysaccharide （LPS） induced liver injury. To further evaluate the antifibrotic activity of SQDG, the present study was designed to investigate the effects of SQDG on porcine serum-induced liver fibrosis rats in vivo. Furthermore, the actions of SQDG on markers of oxidative stress and fibrogenesis were investigated. In addition, the effects of paeoniflorin （Pae） on the proliferation of HSC-T6 stimulated with recombinant rat platelet derived growth factor-BB （rrPDGF-BB） were evaluated in vitro. The effect and relationship between G protein-AC-cAMP signal pathway and extracellular signal-regulated protein kinase （ERK） pathways in HSC-T6 stimulated with rrPDGF-BB was also investigated. Meanwhile, the effects of Pae on the signal transduction protein were observed by Western-blot analysis.OBJECTIVE The animal model of porcine serum-induced liver fibrosis was used to evaluate the protective effects of SQDG according to the changes of histopathological examination, serum transaminase activities, serum fibrotic markers and lipid peroxidation. Effects of Pae on the proliferation of HSC-T6 stimulated with rrPDGF-BB and expression of cyclooxygenase-2 （COX-2） were observed. The effect and relationship between G protein-AC-cAMP signal pathway and ERK1/2 pathways in HSC-T6 stimulated with rrPDGF-BB were also investigated. To confirm the mechanisms of Pae, the effects of Pae on the signal transduction proteins were measured meanwhile. METHODS Rats were intraperitoneally injected with 0.5 ml of porcine serum twice a week to establish immunological liver fibrosis model. The rats were randomly divided into normal control group, liver fibrosis model group, SQDG (42.5, 85, 170mg·kg^-1) treated group and colchicine （0.1mg/kg） treated group. HE stain and Masson stain were used to examine the histopathological change. The activities of transaminase in serum, malondiadehyde （MDA） content, superoxide dismutase （SOD） and glutathione peroxidase （GSH-Px） activities, hydroxyproline （Hyp） content in liver homogenate were assayed by spectrophotometry. The level of hyaluronic acid （HA）, procollagen III （PCIII）, collagen type IV （CIV）, laminin （LN） and prostaglandin E2 （PGE2） in serum were determined by radioimmunoassay. The proliferation of HSC was measured by MTT assay. The level of cAMP in HSC-T6 was determined by radioimmunoassay. The expression of COX-2, G protein and ERK1/2 of HSC-T6 were detected by Western blot analysis.RESULTS1. Protective effects of SQDG on immunological hepatic fibrosis induced by porcine serum in ratsSQDG at doses of 85 and 170mg·kg^-1 had obvious protective effects on porcine serum-induced hepatic fibrosis in rats. SQDG treatment prevented the increase of liver and spleen indices. The results showed that the serum ALT and AST decreased by SQDG treatment, but had no significant difference compared with model group. Pathological examination showed that SQDG could remarkably alleviate the hepatic fibrosis. SQDG not only decreased the Hyp content in liver homogenates, but also decreased the elevated level of HA, LN, PCIII, CIV in serum, which indicate that SQDG decrease the ECM production of hepatic fibrosis rats.SQDG also ameliorated the oxidative stress state of hepatic fibrosis rats, decreased the production of MDA and enhanced the activities of antioxidative enzyme including SOD and GSH-Px. SQDG had anti-inflammatory effect on hepatic fibrosis rats, which evidenced by inhibiting the production of PGE2 in serum. Furthermore, SQDG significantly inhibited the proliferation of isolated rat HSC. Western bolt showed that SQDG could significantly decrease the elevated expression of PDGFR-βin liver tissue of fibrotic rats. The results mentioned above suggest that SQDG ameliorate oxidative state of liver, inhibite the production of inflammatory cytokines, and inhibite the proliferation of HSC, which may be part of the mechanisms of SQDG anti-fibrotic effects.2. Effects of SQDG on the expression of MAPK and G proteins in immunological liver fibrosis ratsIn the liver tissue of porcine serum induced liver fibrosis rat, the phosphorylation of ERK1/2, p38 and JNK were significantly increased. The expression of Gαs decreased, Gαi-2 and Gαi-3 increased significantly, but the expression of Gαi^-1 had no significant change compared with normal rats. Treatment with SQDG could remarkably alleviated above changes. These results indicated that SQDG probabley through regulating MAPK and ERK1/2 signal transduction to exert its anti-fibrotic effects.3. Effect of Pae on the proliferation of HSC-T6 stimulated with rrPDGF-BBHSC-T6 stimulated with rrPDGF-BB (50μg?L^-1) was used as in vitro model to evaluate the antiproliferative effect of Pae. Pae at concentration of 12.5～200mg?L^-1 could significantly inhibit the proliferation of HSC-T6 stimulated with rrPDGF-BB. Results showed that rrPDGF-BB induced the expression of COX-2 in HSC-T6, and NS-398 （a selective inhibitor of COX-2） had inhibitory effect on the proliferation of HSC-T6 stimulated by rrPDGF-BB. Meanwhile, Pae(50, 100 mg?L^-1) significantly decreased the expression of COX-2 in HSC-T6. This suggested that decrease the expression of COX-2 is one of mechanisims of Pae in inhibiting the proliferation of HSC-T6. 4. Effect of Pae on the activation of ERK1/2 in HSC-T6 stimulated with rrPDGF-BBThe results of Western blot showed that rrPDGF-BB induced the rapid phosphorylation of ERK1/2 in HSC-T6. Addition of Pae(25, 50, 100 mg·L^-1) obviously decreased the level of phosphorylated ERK1/2. Thus, inhibiting the activation of ERK1/2 stimulated by rrPDGF-BB is one of the important mechanisms of the antiproliferative effects of Pae.5. Changes of G protein-AC-cAMP pathways in HSC-T6 stimulated with rrPDGF-BB and the effect of PaeThe changes of the expression of G-protein in HSC-T6 stimulated with rrPDGF-BB (50μg·L^-1) were detected by Western-blot. The results showed that the expression of Gαi^-1 and Gαi-2 were remarkably increased in HSC-T6 stimulated by rrPDGF-BB, but the expression of Gαi-3 and Gαs had no significant change. Meanwhile, the level of cAMP and the activity of PKA in HSC-T6 were obviously decreased after addition of rrPDGF-BB. The expression of Gαi^-1 and Gαi-2 were remarkably inhibited by Pae(50, 100 mg·L^-1), which also increased the level of cAMP in cells, and then inhibited the proliferation of HSC-T6. The correlation analysis demonstrated that the effect of Pae on inhibiting the proliferation of HSC-T6 is correlated intimately with its effect on increasing cAMP level in HSC-T6. The results above indicated that Pae probably inhibit the proliferation of HSC-T6 induced by rrPDGF-BB via Gi-AC-cAMP pathway.6. Relationship between ERK1/2 and Gi protein mediated signal transduction in HSC-T6 stimulated with rrPDGF-BBPertussis toxin （PT）, inhibiting the role of Gi protein, is used to detect the effect of PT sensitive G protein on the activation of ERK1/2 in HSC-T6 stimulated with rrPDGF-BB. The results showed that PT significantly decreased the phosphorylation of ERK1/2 and inhibited the proliferation of HSC-T6. These results suggested that PT sensitive Gi protein could regulate the activation of ERK1/2. U0126, a specific inhibitor of MEK, is used to inhibit the activation of ERK1/2 and further explore the effect of ERK1/2 on Gi protein mediated signal transduction in HSC-T6 stimulated with rrPDGF-BB. The results showed that U0126 had no obvious effects not only on the expression of Gαi^-1 and Gαi-2, but also on the level of cAMP and the activity of PKA in HSC-T6 stimulated by rrPDGF-BB. These data indicate that PT sensitive Gi protein and PKA located upstream of ERK1/2. Pae probably through downregulating Gi-AC-cAMP pathway to inhibit the activation of ERK1/2, thus inhibit the proliferation of HSC-T6 stimulated by rrPDGF-BB.CONCLUSIONS1. SQDG has protective effect on liver fibrosis rats induced by porcine serum. The mechanisms of its anti-fibrotic effects may be associated with its action of ameliorating the oxidative stress in liver, inhibiting the production of inflammatory cytokines and inhibiting the proliferation of HSC and so on.2. The expression of COX-2 was constantly increased in HSC-T6 after stimulating by rrPDGF-BB. NS-398 （a selective inhibitor of COX-2） had significant inhibitory effect on the proliferation of HSC-T6 stimulated by rrPDGF-BB. Meanwhile, Pae significantly decreased the expression of COX-2 in HSC-T6. This suggested that decrease the expression of COX-2 is one of mechanisms of Pae in inhibiting the proliferation of HSC-T6.3. rrPDGF-BB induced the rapid phosphorylation of ERK1/2 in HSC-T6. Addition of Pae (25, 50, 100 mg·L^-1) obviously decreased the level of phosphorylated ERK1/2. U0126 （a specific inhibitor of MEK） significantly inhibited the expression of COX-2 in HSC-T6 stimulated by rrPDGF-BB. Thus, inhibiting the activation of ERK1/2 stimulated by rrPDGF-BB, then inhibit the expression of COX-2 in HSC-T6 is one of the important mechanisms of the antiproliferative effects of Pae.4. The expression of Gαi^-1 and Gαi-2 were remarkably increased in HSC-T6 stimulated by rrPDGF-BB. Meanwhile, the level of cAMP and the activity of PKA in HSC-T6 were obviously decreased after addition of rrPDGF-BB. Pae significantly inhibited the expression of Gαi^-1 and Gαi-2, which also increased the level of cAMP in cells, and then inhibited the proliferation of HSC-T6. The effect of Pae on inhibiting the proliferation of HSC-T6 is correlated intimately with its effect on increasing cAMP level in HSC-T6. The results suggested that Pae probably inhibit the proliferation of HSC-T6 induced by rrPDGF-BB via Gi-AC-cAMP pathway.5. PT significantly decreased the phosphorylation of ERK1/2 and inhibited the proliferation of HSC-T6. On the other hand, U0126 had no obvious effect not only on the expression of Gαi^-1 and Gαi-2, but also on the level of cAMP and the activity of PKA in HSC-T6 stimulated by rrPDGF-BB. These data indicated that PT sensitive Gi protein and PKA located upstream of ERK1/2. Pae probably through downregulating Gi-AC-cAMP pathway to inhibit the activation of ERK1/2, thus inhibit the proliferation of HSC-T6 stimulated by rrPDGF-BB.更多还原