【作者】 储德勇；

【导师】 沈继龙；

【作者基本信息】 安徽医科大学 ， 药理学， 2007， 博士

【摘要】 血吸虫病是人类主要热带病之一。血吸虫病主要死于肝虫卵肉芽肿以及继发性的肝纤维化。有研究显示,杀虫治疗后未成熟虫卵仍可继续发育,血吸虫虫卵肉芽肿反应仍在继续,而且,一旦肝纤维化达到一定程度,即使去除了肝纤维化诱因,纤维化仍将继续发展。因此,如何在肉芽肿形成阶段和肝纤维化早期,用药物干预以预防其发展,是杀虫治疗后第二个控制疾病发展的关键。然而至今无高效低毒的药物用于血吸虫性肝病的治疗。目前已证实,肝星状细胞(hepatic stellate cells,HSCs)是分泌细胞外基质(extracellular matrix,ECM),引起肝纤维化最主要的细胞,转移生长因子β1(transforming growth factor beta1,TGFβ1)是其最强的促ECM分泌因子。但TGFβ1对血吸虫病肝纤维化的影响,目前仍有争议。芍药苷(paeoniflorin,PAE)是从白芍中提取的主要有效成分。以往的研究发现,芍药苷具有免疫调节、抗炎等作用,但其在日本血吸虫虫卵肉芽肿和肝纤维化中的作用,国内外未见报道。本课题用血吸虫尾蚴感染小鼠,制成血吸虫病肝肉芽肿和纤维化模型,考察PAE在体内对肝肉芽肿和纤维化以及TGFβ1的影响,然后在体外考察血吸虫可溶性虫卵抗原(soluble egg antigen,SEA)对小鼠腹腔巨噬细胞(peritoneal macrophages,PMs)分泌TGFβ1的影响,以及TGFβ1和PAE对HSCs增殖和分泌胶原的影响。最后从基因和蛋白表达水平探讨PAE对TGFβ1-Smads信号转导通路的作用,以期了解PAE抗肝纤维化的部分机制。目的:研究PAE对小鼠日本血吸虫病肝纤维化TGFβ1-Smads信号转导通路的影响方法:用血吸虫尾蚴经皮肤感染小鼠,然后在感染后不同时间,分先后或同时给予吡喹酮和不同剂量PAE,用组织芯片HE染色和免疫组化技术了解PAE在杀虫不同时期对肝肉芽肿、肝纤维化形成和TGFβ1表达的影响;用SEA刺激PMs,分别用反转录多聚酶链反应(reverse transcriptase polymerase chain reaction,RT-PCR)、Western blotting以及酶联免疫分析法(enzyme-linked immunosorbent assay,ELISA)分析SEA和PAE对PMs产生TGFβ1的影响;用原位灌流-密度梯度离心法提取正常健康小鼠HSCs,经SEA刺激的腹腔巨噬细胞条件培养基(stimulated peritoneal macrophage-conditioned medium,Stimulated-PMCM)刺激HSCs后,用四甲基偶氮唑盐(methylthiazolyl tetrazolium,MTT)法和ELISA分别观察PMCM和PAE对HSCs增殖和分泌胶原的影响。我们选择稀释度为1:2的PMCM为最适浓度的巨噬细胞条件培养基(optimum macrophage-conditioned medium,OPMCM),然后,在OPMCM中加入抗TGFβ1单抗,以了解TGFβ1对HSCs增殖和胶原分泌的影响。最后,用RT-PCR和Western blotting分析PAE对HSCs中的Smads信号蛋白以及前胶原转录和合成的影响。结果:1.早期给予PAE可预防血吸虫病小鼠肝肉芽肿和肝纤维化的形成,降低肝内TGFβ1表达感染血吸虫尾蚴后第12 d(在杀虫治疗前第30 d)开始给予PAE(30、120 mg/kg),能显著降低肝肉芽肿大小和肝纤维化程度,以及降低TGFβ1在肝组织中的表达,而在感染后第42 d(在杀虫治疗同时)或第72 d(在杀虫治疗30 d后)开始给予PAE则无上述作用。说明血吸虫感染后,早期给予PAE能预防肝肉芽肿和肝纤维化;同时也说明,肝纤维化可能与TGFβ1的表达有关。2. SEA促进PMs分泌TGFβ1,而PAE抑制TGFβ1的分泌ELISA、RT-PCR和Western blotting都显示SEA(0、2.5、5、10、20、40 mg/L)能刺激PMs分泌TGFβ1,且SEA在10 mg/L时能刺激PMs产生最大量的TGFβ1;同时,RT-PCR和Western blotting显示,PAE(0、7.5、15、30、60、120 mg/L)能浓度依赖性地抑制PMs产生TGFβ1。提示PAE抗肝纤维化的机制可能与其抑制PMs分泌TGFβ1有关。3. PMCM及TGFβ1促进HSCs增殖和胶原分泌,而PAE抑制HSCs增殖及前胶原、胶原的转录、合成和分泌MTT法和ELISA显示,不同稀释度的PMCM(1:32、1:16、1:8、1:4、1:2、1:0)对HSCs的增殖和I型胶原(collagen type I, Col I)、III型胶原(collagen type III, Col III)的分泌都具有促进作用。但稀释度为1:2和1:0的PMCM具有更大的效应。用抗TGFβ1单抗中和后的OPMCM其促进HSCs增殖和胶原分泌的作用明显下降,但与对照仍有差别,提示TGFβ1在HSCs增殖和胶原分泌中发挥一定的促进作用。同时,MTT法、ELISA、RT-PCR和Western blotting显示PAE(7.5、15、30、60、120 mg/L)能浓度依赖性地抑制HSCs增殖及I型α1前胶原(procollagen, type I, alpha1,Col Iα1)、III型α1前胶原(procollagen, type III, alpha1,Col IIIα1)、Col I和Col III的转录、合成和分泌。4. PAE在转录和合成水平抑制HSCs中Smad3的表达RT-PCR和Western blotting显示PAE对TGFβ1-Smads信号转导通路中的Smad2、Smad4和Smad7无明显作用,而对Smad3具有浓度依赖型性的抑制。提示,抑制Smad3转录和合成可能是PAE抗血吸虫病肝纤维化的机制之一。结论:1. TGFβ1可促进小鼠日本血吸虫病肝纤维化;2.早期给予PAE可预防小鼠日本血吸虫病肝肉芽肿、肝纤维化的形成和TGFβ1表达;3. SEA可刺激PMs分泌TGFβ1,后者可促进HSCs增殖和胶原分泌;4. PAE预防血吸虫病肝纤维化可能与下述机制有关a)抑制PMs分泌TGFβ1b)抑制HSCs的增殖c)通过抑制Smad3 mRNA的表达抑制HSCs产生胶原总之,在血吸虫病肝纤维化中,SEA能刺激巨噬细胞产生TGFβ1,后者可使HSCs增殖和分泌胶原。PAE通过抑制巨噬细胞分泌TGFβ1、HSCs的增殖、Smad3的转录和合成而最终抑制胶原的产生,发挥其抗纤维化的作用。以上研究结果将为PAE开发成治疗血吸虫病肝纤维化中药新药提供了实验依据。更多还原

【Abstract】 Schistosomiasis is one of the major tropical diseases of mankind. The primary cause of death in schistosomiasis is the formation of liver egg granulomas and secondary hepatic fibrosis. Studies have showed that granuloma inflammatory reaction and fibrosis in liver tissue would continue to be aggravated, even though efficacious schistosomicides were given. Moreover, once fibrosis has reached a certain level, it will continue to develop, although the inducements are eradicated. So the intervention and control of such aggravation during or before the formation of granuloma or at the early stage of fibrosis become another key therapeutic strategy after efficacious treatment of praziquantel. However, so far, no drug of anti-fibrosis with low toxicity and high efficiency has been applied to prevent or reverse the hepatic fibrosis in schistosomiasis. Hepatic stellate cells (HSCs) play a key role in hepatic fibrosis through secreting extracelluar matrix (ECM). Transforming groeth factor beta1 (TGFβ1) is the most potent fibrogenetic cytokine and can stimulate HSCs activation through paracrine or/and autocrine by mediating TGFβ1-Smads signalling. But the effect of TGFβ1 on hepatic fibrosis of schistosomiasis is controversial at present.Paeony (Paeoniae radix) root is one of the well-known herbs in China and other Asian countries. Paeoniflorin (PAE, C23H28O11) is known to be one of the principal bioactive components of paeony root. PAE has been reported to have immunoregulatory and anti-inflammatory effects. Preparations of many traditional Chinese herbs used in anti-hepatic fibrosis contain paeony root. However, the mechanism by which it elicits anti-hepatic fibrosis in schistosomiasis japonica has not been elucidated.The present study was first designed to investigate the effects of PAE on expression of TGFβ1 in liver tissue, hepatic granuloma and fibrosis of schistosomiasis in mouse induced by S. japonicum cercarie. Then the effect of schistosomal soluble egg antigen (SEA) and PAE on the secretion of TGFβ1 from mouse peritoneal macrophages (PMs) and the effects of TGFβ1 and PAE on the proliferation of HSCs and secretion of collagens were investigated in vitro. Finally, we explored the effects of PAE on TGFβ1-Smads signalling at gene and protein levels in order to realize the partial mechanisms of PAE on hepatic fibrosis of S. japonicum.AIM To study on the effects of PAE on TGFβ1-Smads signalling in hepatic fibrosis of schistosomiasis japonica in mouseMETHODS We first infected mouse via skin with cercarie of S. japonicum, then PAE was orally administrated before and after praziquantel treatmentand and both therapeutics were given simultaneously, with different dose of PAE and at different time after infection. The effects of PAE on formation of hepatic granuloma and fibrosis and expression of TGFβ1 in liver tissue at different time after infection were analyzed by tissue microarray, in combination with immunohistochemistry.We stimulated mouse PMs with SEA, then the effects of SEA and PAE on the production of TGFβ1 from PMs were evaluated by reverse transcriptase polymerase chain reaction (RT-PCR), Western blotting, and enzyme-linked immunosorbent assay (ELISA); The HSCs isolated from health mouse by in situ perfusion and density gradient centrifugation were stimulated by mouse peritoneal macrophage-conditioned medium (PMCM), then the effects of PMCM and PAE on the proliferation of HSCs and collagens secretion from HSCs were detected by methylthiazolyl tetrazolium (MTT) assay and ELISA, respectively. We chose dilution 1:2 as the optimum macrophage-conditioned medium (OPMCM). Then the effects of TGFβ1 on the proliferation of HSCs and collagens secretion from HSCs were detected with OPMCM and anti-TGFβ1 antibodies. Finally, the effects of PAE on Smads and procollagens were assayed by RT-PCR and Western blotting.RESULTS1. PAE early administrated could inhibit formation of hepatic granuloma and fibrosis and expression of TGFβ1 in liver tissue of mouse in schistosomiasis PAE (30、120 mg/kg) administrated at second week (just four weeks before schistosomicides were given) after cercarie attack could obviously decrease the area of hepatic granuloma and the degree of fibrosis, and downregulate the expression of TGFβ1 in liver tissue. However, PAE administrated at sixth week (just at the same time when schistosomicides were given) or at tenth week (just after four weeks when schistosomicides were given) after cercarie attack had no effect on the area of hepatic granuloma or the degree of fibrosis, or the expression of TGFβ1 in liver tissue, suggesting that PAE early given could prevent hepatic granuloma and fibrosis of S. japonicum, which might be related to TGFβ1.2. SEA could promote TGFβ1 secretion from PMs, and PAE could depress TGFβ1 secretionTGFβ1 could be secreted from PMs stimulted by SEA (0、2.5、5、10、20、40 mg/L) and SEA at 10 mg/L could induce the highest production level of TGFβ1 from PMs. Meanwhile, the production of TGFβ1 from PMs could be depressed significantly by PAE (0、7.5、15、30、60、120 mg/L) in a concentration-dependent manner, suggesting that the mechanism by which PAE inhibited the formation of hepatic granuloma and fibrosis might be relate to depressed the secretion of TGFβ1 from PMs.3. PMCM and TGFβ1 could promote the proliferation of HSCs and the secretion of collagens, but PAE could depress the proliferation and secretion at gene and protein levelsPMCM (1:32、1:16、1:8、1:4、1:2、1:0) could promote the proliferation of HSCs and the secretion of collagen type I (Col I) and collagen type III (Col III), moreover PMCM at dilution 1:2 and 1:0 had more significant effects on the proliferation and secretion. But both proliferation and secretion were partly inhibited in the presence of anti-TGFβ1 antibody, suggesting that TGFβ1 could play an important role in the proliferation of HSCs and the sectetion of collagens. Meanwhile our results showed that PAE (0、7.5、15、30、60、120 mg/L) could inhibit the proliferation of HSCs and the production of procollagen, type I, alpha1 (Col Iα1), procollagen, type III, alpha1 (Col IIIα1), Col I and Col III at gene and protein levels.4. PAE could downregulate the Smad3 mRNA transcription and expression levelsPAE (0、7.5、15、30、60、120 mg/L) could downregulte the Smad3 mRNA transcription and expression levels in a concentration-dependent manner, but had no effects on the transcription and expression levels of Smad2, Smad4 and Smad7 mRNA, suggesting that the mechanism by which PAE inhibited the formation of hepatic fibrosis might be relate to downregulating the transcription and expression levels of Smad3 in HSCs.CONCLUSIONS1. TGFβ1 could promote hepatic fibrosis of S. japonicum in mouse;2. PAE early administrated could inhibit formation of hepatic granuloma and fibrosis and expression of TGFβ1 in liver tissue of mouse in schistosomiasis;3. SEA could stimulate PMs to secrete TGFβ1, and the TGFβ1 could promote HSCs proliferation and collagens secretion;4. The anti-fibrotic role of PAE in schistosomiasis might be associated with:a) inhibition of the secretion of TGFβ1 from PMsb) inhibiton of the proliferation of HSCsc) downregulation of Smad3 mRNA transcription and expression which might be clsosely associated with inhibiton of collagens production from HSCs.In conclusion, TGFβ1 could be secreted by PMs stimulated by SEA, and the TGFβ1 could promote the proliferation of HSCs and the secretion of collagens in schistosomiasis. PAE could inhibit the production of collagens by depressing the secretion of TGFβ1 from PMs, reducing the proliferation of HSCs, downregulating the transcription and expression of Smad3 mRNA. Our results might provide the experimental basis for PAE as a natural therapeutic agent to prevent hepatic fibrosis of schistosomiasis japonica.更多还原