【作者】 王剑锋；

【导师】 孙世发；

【作者基本信息】 南京中医药大学 ， 方剂学， 2013， 博士

【摘要】 研究目的与意义肝纤维化是多种慢性肝病的共同病理基础,是肝硬化必经的早期可逆阶段,及时干预,可以不发展到肝硬化阶段,如不及时治疗可进展为肝硬化,肝硬化难以逆转。因此,探讨肝纤维化的发病机制、开发有效的治疗肝纤维化的药物对预防慢性肝病的进一步发展十分重要,对早期逆转肝纤维化具有重要的临床价值。近年来,我国的中医药在肝纤维化防治研究中显示出较为光明的发展前景,目前已经报导一大批中药制剂具有抗肝纤维化作用,但针对这些中药制剂的研究很多局限于动物实验和初步临床观察,缺少分子水平作用机制的系统研究,不利于中医药现代化发展的需要,因此,急需深入地研究、探索更为安全有效的抗肝纤维化的复方中药。本课题组前期临床观察中,根据肝纤维化具有“肝郁脾虚络瘀”的病机特点,采用四逆散加味方治疗肝纤维化患者120例,证实四逆散加味方能显著改善肝功能、降低肝纤维化指标、改善肝组织结构,提示其有明显的抗纤维化作用,但其详细的分子机制仍不清,目前国内外鲜见有报道对其深入研究。因此,有必要结合“肝主疏泄”理论深入研究肝纤维化发生、发展的机制,并对四逆散加味、四逆散拆方配伍,治疗肝纤维化的机制进行系统研究。四逆散是疏肝理气的基础方,在临床中虽有良好的疏肝解郁功效,但也有其不足,结合多年临床治病经验体会,发现运用四逆散加味治疗效果明显优于四逆散。本研究以肝主疏泄为理论依据,在前期临床和实验研究得出四逆散加味对肝纤维化有确切疗效的基础上,深入研究四逆散拆方及其加味方抗肝纤维化的作用机制。观察不同剂量的四逆散加味、四逆散配伍组方对免疫损伤性肝纤维化大鼠模型的影响,探索四逆散加味及其配伍抗肝纤维化作用的分子机制,为该方的临床应用和新药开发提供实验依据。研究方法本论文分为理论研究和实验研究两部分。理论研究部分是在系统整理中医学对肝纤维化认识古今相关文献的基础上,结合现代医学对肝纤维化的研究进展,重点探讨了肝纤维化的中医病因病机,肝纤维化的中医治疗研究现状,肝纤维化的免疫学发病机制,四逆散加味方的组方配伍思想,为四逆散加味抗肝纤维化提供理论依据。实验研究部分首先采用猪血清诱导免疫损伤性肝纤维化模型,并采用血清生化、光镜观察及免疫印迹、RT-PCR、免疫组化等分子生物学技术检测观察了四逆散、四逆散加味方治疗肝纤维化的效用；四逆散、四逆散加味方对肝组织中TGFβ1信号通路、Rhoa信号通路的调控,为四逆散加味方的临床应用及新药开发提供实验依据。研究结论及意义本研究以肝主疏泄理论为指导,采用四逆散加味方对肝纤维化大鼠模型进行治疗,得到如下结论：1.肝主疏泄理论是防治肝纤维化的重要理论,以该理论为指导的四逆散加味方对大鼠肝纤维化模型具有良好的治疗作用。2.四逆散加味方具有良好的保护肝细胞,抗炎、降酶改善肝功能的作用。四逆散加味中剂量组、四逆散加味预防组对肝纤维化模型大鼠血清谷丙转氨酶(ALT)、谷草转氨酶(AST)、GGT、ALP活性有显著降低作用,且能明显降低肝纤维化模型大鼠血清HA、Ⅲ型胶原的含量,体现了四逆散加味方“疏肝理气,健脾益气,软坚散结”功效和“肝主疏泄”理论的科学性。3.四逆散加味方能降低大鼠组肝组织中TGFβ1、TGFRmRNA和蛋白表达量、HA蛋白表达量,其机制可能通过多种途径,减少TGF-β1产生及TGFR表达以减少肝星状细胞活化,对TGFβ1信号通路产生调控作用,减少ECM的生成和HA的产生,而起到抗肝纤维化和保护肝细胞损伤的作用。4.四逆散加味方能下调肝纤维化模型大鼠组肝组织中Rhoam基因和蛋白表达量、a-SMA蛋白表达量,且以四逆散加味中剂量组、四逆散加味预防组Rhoam基因和蛋白表达量、a-SMA蛋白表达量下调更为显著。证实四逆散加味治疗肝纤维化的可能作用机制是通过下调RhoA基因和蛋白表达,对RhoA/ROCK通路的产生调控作用,减少a-SMA蛋白表达,抑制肝星状细胞的活化与迁移,而起到保护肝细胞,进而发挥抗肝纤维化的作用。更多还原

【Abstract】 Purpose and significance of studyHepatic fibrosis is a common pathological basis of many chronic liver diseases, early stage cirrhosis of the liver is reversible, cirrhosis can not develop liver cirrhosis stage if it is intervened timely, otherwise it may progress to cirrhosis, liver cirrhosis is hard to reverse. Therefore, it’s significant for preventing the further development of chronic liver disease to explore the pathogenesis of liver fibrosis and develop some effective drugs for liver fibrosis, and it has an important clinical value for reversing the early hepatic fibrosis. In recent years, our traditional Chinese medicine has shown a relatively bright development prospects in the study on prevention and cure of liver fibrosis. And it’s reported that a large number of traditional Chinese medicine preparations have the effect of anti-liver fibrosis. But many of the studies on the Chinese native medicine preparation are confined to preliminary clinical observation and animal experiment, and lack in the system research about molecular mechanism of action, the situation is not conducive to the needs of the development of modernization of Chinese medicine. So we need the further researches badly and explore more safe and effective compound Chinese medicine anti-liver fibrosis. Our previous clinical observation,"according to liver fibrosis with liver qi stagnation, spleen deficiency of Collaterals Disease machine has the characteristic of, Sinisan Jiawei prescription treatment in120cases of liver fibrosis, confirmed four inverse scattered prescription can significantly improve liver function, reduce the indexes of liver fibrosis, improve the liver tissue structure of the obvious anti-fibrosis effect. But the precise molecular mechanism is still not clear, and at present there few reports for its further study domestic and overseas. Therefore, it is necessary to study deeply the mechanism of occurrence and development of liver fibrosis, and the mechanism of treating liver fibrosis about Sinisan Jiawei flavored, four inverse scattered compatibility. Sinisan Jiawei is a basic prescription in clinical liver qi, it has the good efficacy of Shugan Jieyu, but it also has its shortcomings. Mang years’clinical experience in treating proves that the treatment effect of using Sinisan Jiawei is better than Sinisan. In the clinical and experimental research derived from Sinisan Jiawei Decoction on liver fibrosis has exact efficacy, and based on the theory of the liver controlling dispersion, this study study deeply the mechanism of Sinisan Jiawei disassembled prescription and prescription of anti-liver fibrosis. Observing the effects of different doses of Sinisan Jiawei, the influence of Sinisan’s compatibility formula on the immune damage and hepatic fibrosis in rats model, and exploring the Sinisan compatibility and its molecular mechanism of liver fibrosis resistant, provide experimental basis for clinical application and development of new drugsThe method of studyThis paper is divided into two parts, theoretical study and experimental study. The part of theory research combining the system basis of traditional Chinese medicine on hepatic fibrosis of understanding of ancient and modern literature with modern medicine research progress of hepatic fibrosis, focus on the traditional Chinese medicine etiology and pathogenesis of liver fibrosis, liver fibrosis treatment of traditional Chinese medicine research status, the immunological pathogenesis of liver fibrosis, the thought of Sinisan jiawei compatibility prescription, and all these provide the theory basis for the Sinisan Jiawei modified against liver fibrosis.Experimental research part, first of all, using pig serum induced immune damage liver fibrosis model, and using light microscopy and immunoblotting, RT-PCR, immunohistochemistry and molecular biology techniques and so on monitored the effectiveness of Sinisan, Sinisan Jiawei on liver fibrosis. Sinisan jiawei on TGF-β1and RhoA related signal’s regulation in rat liver tissue of hepatic fibrosis provides experimental basis for clinical application and development of new medicines and f Sinisan powder prescription.The conclusion and significance of studyThis topic regarding the liver controlling dispersion theory as a guide, and using Sinisan Jiawei on treatment of liver fibrosis rat model get the following conclusions:1. The theory of liver controlling dispersion is the important theory of prevention and treatment of liver fibrosis, under the guidance of the theory, Sinisan Jiawei prescription has a good therapeutic effect on rat liver fibrosis model.2. Sinisan Jiawei prescription has good functions of protecting liver cells, anti-inflammatory, reducing enzymes to improve liver. The dose group of Sinisan Jiawei, the prevention group of Sinisan Jiawei has significant effect to reduce liver fibrosis model rats serum alanine aminotransferase (ALT), glutamic-oxalacetic transaminease (AST), GGT and ALP activity, and can significantly reduce liver fibrosis models of rats serum HA, III collagen type, these embody Sinisan Jiawei prescription’s effect of "soothing-liver regulating qi, invigorating spleen and supplementing qi, soften hard lumps and dispel nodes" and the scientific of the theory of "liver controlling dispersion". 3. Sinisan Jiawei prescription can reduce the rat liver tissue’s expression amount of TGF beta1, TGFRmRNA, HA protein and protein expression, and through various channels, its mechanism may reduce TGF-beta1and TGFR expression in order to decrease hepatic stellate cell activation, control TGF beta1signaling pathways, reduce the generation of the ECM and reduce the generation of HA, so that it can play the part of anti-liver fibrosis and protecting the liver cell damage.4. Sinisan Jiawei prescription can reduce the expression amount of Rhoam gene and protein, a-SMA protein of liver tissue in rat liver fibrosis model, and more significant is the reducing of amount about Sinisan Jiawei dose group, Sinisan Jiawei prevention Rhoam gene and protein expression, and a-SMA protein expression. These confirm that Sinisan Jiawei effect mechanism of the treatment of liver fibrosis may be through the approaches such as cutting RhoA gene and protein expression, having the controlling effect on RhoA/ROCK pathway, reducing alpha SMA protein expression, restraining the hepatic stellate cell activation and migration, and so forth, thereby protect liver cell, and then play the role of anti liver fibrosis.更多还原