基于浊毒理论的肝复健方抗大鼠肝纤维化作用及机制研究
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摘要
肝纤维化(Hepatic fibrosis,HF)是指各种致病因素所引起肝脏内细胞外基质(extracellular matrix,ECM)的合成大于降解，致使ECM在肝脏内过度沉积，它是大多数慢性肝病所共有的病理特征，也是慢性肝炎进一步向肝硬化、肝癌发展的重要环节。肝纤维化是可被逆转的阶段，如能阻断和逆转肝纤维化的发生，就可阻止严重后果的发生。
肝纤维化的形成是一个多因素、多环节、多阶段的复杂过程，其形成不是静止的，而是不断变化的动态过程。各种细胞因子形成的复杂调节网络，不断影响着肝纤维化的形成和转归。从细胞因子及其信号转导角度研究药物的作用机制，将不断深化肝纤维化的治疗学研究。瘦素（leptin）与肝纤维化的形成之间有着密切的关系，与瘦素受体结合后，激活相应的信号转导通路，促进肝纤维化的发生发展。
目前临床上仍然缺乏确切特效的抗纤维化药物与方法。中医药具有多层次、多环节、多途径、多靶点的作用特点，在治疗肝纤维化方面取得了显著的成果。李佃贵教授提出浊毒内蕴，肝络瘀滞是肝纤维化的主要病机之一，并从浊毒论治肝纤维化，在此指导下确立肝复健方，取得显著疗效。既往临床及实验研究表明，肝复健方具有明显的阻断病毒性乙型肝炎慢性化、减轻肝纤维化及保护肝功能的作用。本课题继续在腹腔注射猪血清复制免疫性肝纤维化模型的基础上，观察肝复健方对大鼠肝组织病理形态学、肝纤四项、MMP-1、瘦素(leptin)及其受体（OB-Rb)、JAK2、STAT3的表达情况，探讨其对瘦素调控肝纤维化大鼠JAK2/STAT3信号转导通路的影响，旨在对本方抗HF的药效和可能的作用机制进一步的研究，为肝纤维化的预防与治疗提供理论依据。本实验分为以下五部分：第一部分浊毒理论与肝纤维化
目的：探讨浊毒理论与肝纤维化的关系，明确其在肝纤维化中的重要地位，提出肝纤维化治疗的新观点与新方法。
方法：通过温习、回顾、整理中医文献，并结合临床实践经验，提出浊毒理论，提出肝纤维化的新病机，系统总结了肝纤维化浊毒证证治规律。
结果：提出了浊毒理论，明确了浊毒的概念、临床表现及致病特点。探讨了浊毒与肝纤维化的关系，总结出了肝纤维化浊毒证证治规律，从临床上验证了从浊毒治疗肝纤维化的可行性。
结论：浊毒理论是中医学术体系的新的组成部分，是研究浊毒致病的病因、病机、诊断、治疗等理法方药的学科。浊毒与肝纤维化关系密切，是其发生发展的关键环节。
第二部分肝复健方对肝纤维化大鼠病理形态学和血清标志物的影响
目的：观察肝复健方对猪血清诱导的肝纤维化大鼠病理形态学及血清HA、LN、PCⅢ、Ⅳ型胶原含量的影响，探讨其抗纤维化的作用机制。
方法：清洁级SD大鼠60只，雄性，体重200±20g。适应性喂养一周后随机分为正常对照组（Normal group）、模型组（Model group）、秋水仙碱组（Colchicine）、肝复健方低剂量组（GFJL group）、肝复健方中剂量组（GFJM group）、肝复健方高剂量组（GFJH group）六组，每组10只。每周二、周五上午8点，采用无菌未灭活的猪血清腹腔注射进行造模，连续8周。造模成功后正常对照组与模型组给予生理盐水（10ml/kg），日1次灌胃。GFJL组给予含生药1.4g/ml水煎剂灌胃；GFJM组给予含生药2.8g/ml水煎剂灌胃；GFJH组给予含生药4.2g/ml水煎剂灌胃（灌胃液体量为10ml/kg，1次/d）；秋水仙碱组给予0.154mg/kg·d灌胃，均至12周末。实验期间观察大鼠的一般情况。12周末采用腹主动脉取血，分离血清，用放射免疫分析法测定各组大鼠血清中HA、LN、PCIII、CIV水平。留取大鼠肝组织，采用苏木精-伊红染色（HE染色）和胶原纤维特殊染色法（Masson染色）观察肝组织病理形态学改变。
结果：
1一般情况
正常对照组一般情况良好，精神状态好，活泼好动，大鼠食欲佳，进食量正常，皮毛光滑，体重逐渐增加。模型组大鼠精神萎靡、不喜活动，进食较正常组少，皮毛杂乱、黯淡无光泽、尿黄，个别大鼠鼻有出血，体重增长变慢。秋水仙碱及中药治疗各组大鼠精神状态、活动、进食量尚可，被毛光泽，体重有所增加，无特殊异常状态出现。
2肝脏大体观察
正常组大鼠肝脏质地较软，表面光滑，略呈深红色，有光泽，无任何斑点、突起，边缘锐利。模型组大鼠肝脏质地硬，表面欠光滑，部分标本表面粗糙，凹凸不平，颜色暗红，边缘钝。秋水仙碱及中药治疗组大鼠肝脏质较脆，表面较光滑，颜色基本正常，基本无斑点、突起等。
3肝复健方对肝纤维化大鼠肝组织病理的影响
正常组肝小叶结构清晰完整，肝细胞围绕中央静脉呈放射状排列，无变性坏死，未见纤维组织增生及炎性细胞浸润。模型组肝小叶结构破坏，肝细胞索排列紊乱，肝细胞变性，部分肝细胞坏死，伴有中性粒细胞及淋巴细胞浸润，纤维组织大量增生，个别动物可见假小叶形成。秋水仙碱组与中药治疗各组肝脏损伤程度均较模型组轻，肝小叶结构破坏减轻，肝脏胶原纤维增生减轻，肝细胞水肿好转，变性情况明显改善，炎细胞浸润减少。肝复健方高剂量组肝脏结构改善最为明显。
4肝复健方对肝纤维化大鼠血清HA、LN、PCⅢ、CIV的影响
与正常对照组相比，模型组大鼠血清HA、LN、PCⅢ、CIV水平升高，差异有统计学意义(P<0.05)。与模型对照组相比，12周末各药物干预组大鼠血清HA、LN、PCⅢ、CIV水平下降(P<0.05)。肝复健方高剂量组血清HA、LN、PCⅢ、CIV水平显著性降低，优于秋水仙碱组、中剂量组及低剂量组（P＜0.05）；秋水仙碱组、肝复健中剂量组之间比较，差异无统计学意义（P>0.05）；秋水仙碱组与肝复健方低剂量组比较，血清HA、LN、PCⅢ、CIV水平降低，差异有统计学意义(P<0.05)。
结论：肝复健方能够改善大鼠的一般状况，显著降低大鼠血清HA、LN、PCⅢ、Ⅳ型胶原含量，促进ECM的降解，抑制纤维组织增生，改善肝纤维化大鼠的病理组织结构，恢复肝细胞损伤，达到抗肝纤维化的作用。
第三部分肝复健方对肝纤维化大鼠瘦素及瘦素受体表达的影响
目的：观察肝复健方对血清瘦素、肝组织瘦素及瘦素受体的影响，进一步明确瘦素在肝纤维化形成过程中的生物学效应以及化浊解毒法抗肝纤维化的深层作用机制。
方法：放射免疫分析法检测血清瘦素(Leptin)水平，逆转录-多聚酶链反应(RT-PCR)技术检测肝组织瘦素(leptin)及瘦素受体(OB-Rb)的表达。
结果：
1肝复健方对大鼠血清Leptin水平的影响
与正常对照组相比,模型对照组、各药物组血清瘦素水平明显升高,差异有统计学意义(P<0.05)；与模型对照组相比，秋水仙碱组和肝复健方各剂量组血清瘦素水平明显下降，差异有统计学意义（P＜0.05）。各药物治疗组比较，肝复健方高剂量组血清瘦素水平显著性降低，优于秋水仙碱组、中剂量组及低剂量组（P＜0.05），秋水仙碱组与中剂量组比较，无显著性差异(P﹥0.05)。秋水仙碱组与低剂量组比较，血清瘦素水平下降，差异有统计学意义（P＜0.05）。
2肝复健方对大鼠肝组织Leptin、OB-RbmRNA表达的影响
与正常对照组相比，模型对照组、秋水仙碱组和肝复健方各剂量组Leptin、OB-RbmRNA的表达明显增强，差异有统计学意义（P＜0.05）。与模型对照组相比，秋水仙碱组和肝复健方各剂量组Leptin、OB-RbmRNA表达明显减弱，差异有统计学意义（P＜0.05）。各药物治疗组比较，肝复健方高剂量组Leptin、OB-RbmRNA表达显著性降低，优于秋水仙碱组、中剂量组及低剂量组（P＜0.05），秋水仙碱组与中剂量组比较Leptin、OB-RbmRNA的表达有下降趋势，但均无显著性差异(P﹥0.05)。秋水仙碱组与低剂量组比较，Leptin、OB-RbmRNA的表达减弱，差异有统计学意义（P＜0.05）。
结论：瘦素是内生浊毒的物质基础之一，肝复健方能够有效降低肝纤维化模型大鼠瘦素及瘦素受体的表达，减少两者的结合，进而抑制下游信号转导通路，从而达到抗肝纤维化的作用。
第四部分肝复健方对大鼠肝组织MMP-1表达的影响
目的：观察肝复健方对HF大鼠肝脏MMP-1表达的影响，从分子水平探讨肝复健方抗肝纤维化的深层作用机理。
方法：逆转录-多聚酶链反应(RT-PCR)技术检测肝组织MMP-1的表达。
结果：与正常对照组相比，模型对照组、秋水仙碱组和肝复健方各剂量组MMP-1mRNA的表达均明显减弱，差异有统计学意义（P＜0.05）。与模型对照组相比，秋水仙碱组和肝复健方各剂量组MMP-1mRNA表达明显增强，差异有统计学意义（P＜0.05）。各药物治疗组比较，肝复健方高剂量组MMP-1mRNA的表达显著性增强，优于秋水仙碱组、中剂量组及低剂量组（P＜0.05），秋水仙碱组与中剂量组比较MMP-1mRNA的表达有增强趋势，但均无显著性差异(P﹥0.05)。秋水仙碱组与低剂量组比较，MMP-1mRNA的表达增强，差异有统计学意义（P＜0.05）。
结论：肝复健方可以显著提高MMP-1mRNA的表达水平，通过抑制HSC合成和分泌瘦素，促进MMP-1的表达，从而使细胞外基质降解，阻碍肝纤维化进程。第五部分肝复健方对肝纤维化大鼠肝组织JAK2/STAT3信号转导通路的影响
目的：观察肝复健方对HF大鼠肝脏JAK2、STAT3蛋白表达的影响，以进一步探讨肝复健方抗肝纤维化的深层作用机理，为临床防治肝纤维化提供理论依据
方法：Western blot检测HF大鼠肝组织JAK2、STAT3的表达。
结果：
1肝复健方对大鼠肝组织JAK2蛋白表达的影响
(1)所有实验组大鼠肝组织中JAK2的表达量均高于正常对照组(P<0.05)。(2)肝复健方各剂量组及秋水仙碱组与模型组相比JAK2的蛋白表达量显著性降低(P<0.05)。(3)肝复健方中剂量组与秋水仙碱组相比JAK2蛋白表达量均无显著性差异(P﹥0.05)；秋水仙碱组与肝复健方低剂量组相比JAK2蛋白的表达量有显著性降低(P<0.05)，肝复健方高剂量组与秋水仙碱组相比，JAK2蛋白的表达量有显著性降低(P<0.05)。(4)肝复健方药不同剂量组之间比较，高剂量组JAK2蛋白的表达量优于中剂量组，中剂量组优于低剂量组，差异有统计学意义（P＜0.05）。
2肝复健方对大鼠肝组织STAT3蛋白表达的影响
(1)所有实验组大鼠肝组织中STAT3的表达量均高于正常对照组(P<0.05)。(2)肝复健方各剂量组及秋水仙碱组与模型组相比STAT3的蛋白表达量有显著性降低(P<0.05)。(3)肝复健方中剂量组与秋水仙碱组相比STAT3蛋白表达量均无显著性差异(P﹥0.05)；秋水仙碱组与肝复健方低剂量组相比STAT3蛋白的表达量有显著性降低(P<0.05)，肝复健方高剂量组与秋水仙碱组相比，STAT3蛋白的表达量有显著性降低(P<0.05)。(4)肝复健方药不同剂量组之间比较，高剂量组STAT3蛋白的表达量优于中剂量组，中剂量组优于低剂量组，差异有统计学意义（P＜0.05）。
结论：肝复健方能够下调肝纤维化大鼠肝组织JAK2、STAT3的表达，进而抑制HSC活化、增殖，降低胶原的表达和分泌，最终起到抗肝纤维化的作用。瘦素调控的JAK2/STAT3信号转导通路是肝复健方抗肝纤维化的新的作用途径和作用靶点。
Hepatic fibrosis is a pathologic process that caused by various riskfactors. Its essence is excessive deposition of extracellular matrix (ECM)components in liver, which occurs due to an imbalance between theproduction and degradation of matrix. It is the most common pathologicalfeatures of chronic liver disease, further to the important link of thedevelopment of hcc.HF can be reversed. It can prevent serious consequencesif hepatic fibrosis blocked and reversed.
Hepatic fibrosis is a complex process of multiple factors, multiple links,multiple stage. Its forming process is not static, but constantly changing.Complex regulatory network composed of various cytokines has influencedthe formation and prognosis of Hepatic fibrosis. It is advantageous in HFtreatment from the cell factor and its signal pathways to study mechanism ofdrug intervention.Leptin is considered a key factor to promote the formationof HF.By binding with leptin receptor, it activates signal transduction pathwaycorresponding, promotes the occurrence and development of HF.JAK/STATsignal transduction pathway is the focus of the current study.
At present clinical still lacks highly effective drugs and methods to treatHF.The characteristics of Traditional Chinese Medicine (TCM) treatment aremultiple positions, multiple links and multiple targets. There are muchsuperiority in the prevention and treatment on HF.After many years clinicalpractices and experiment research, combining with current disease pattern anddisease spectrum changes, professor Li dian-gui finds Zhuodu accumulationand liver collateral stasis is one of the main pathogenesis of HF.He applies thistheory to treat Hepatic Fibrosis patients and obtains the good curative effect.Previous clinical and experimental researches show that, Ganfujian formulabased on zhuodu theory could block viral hepatitis B chronically and reduce liver fibrosis and liver function. We still used the pig’s serum to set thehepatic fibrosis rats’ model by intraperitoneal injection. we observed theeffect of Ganfujian formula on the levels of serum HA,LN,PCⅢ,CIV andPathomorphology in hepatic fibrosis rats. We observed the expression ofleptin, OB-Rb, JAK2, STAT3and MMP-1in rats with HF, and then exploredthe effect of the JAK2/STAT3signal transduction pathway regulated by leptinin rats with HF.Investigated on possible Mechanism of the resistance to HF,so that provided theoretical and experimental basis for treating and preventingHF.This experiment was separated into five parts:
Part one ZhuoDu theory and Hepatic Fibrosis
Objective:The aim was to explore the relationship between the Zhuodutheory and HF, clarify its important role in HF, and propose a new idea andnew method for the treatment of HF.
Methods: Reviewed the TCM literature combined with clinical practiceexperience, presented Zhuodu theory,put forward that the new pathogenesis ofHF was zhuodu and summarized the rule of syndrome and treatment ofhepatic fibrosis with zhuodu.
Results:We presented Zhuodu theory, defined the concept,etiology,clinical manifestations and pathogenic characteristics ofzhuodu,discussed the relationship between zhuodu and Hepatic Fibrosis,summarized the rule of syndrome and treatment of hepatic fibrosis withzhuodu. Feasibility was verified in treating hepatic fibrosis using Zhuodutheory in clinical.
Conclusion: Zhuodu theory is part of the new academic system ofTCM.It is the discipline that studying the etiology,pathogenesis,diagnosis,treatment and prescription of Zhuodu.Zhuodu closely correlateswith HF and is a key link in its occurrence and development.
Part two Effect of Ganfujian formula on the levels of serum markersand Pathomorphology in hepatic fibrosis rats
Objective: The aim was to observe the effect of Ganfujian formula onthe levels of serum HA,LN,PCⅢ,CIV and Pathomorphology in hepatic fibrosis rats. Investigate the mechanism of anti-fibrosis.
Methods: Total60clean male SD rats weighed200±20grams wererandomly divided into six groups: the normal group, the model group,colchicine group,Ganfujian formula of low dose group(GFJL),Ganfujianformula of medium dose group(GFJM) and Ganfujian formula of high dosegroup(GFJH)with10rats for each group. In addition to the normal group,pig′sserum(0.5ml per mouse)was established in rats by intraperitoneal injection foreight weeks. The time was fixed at9am on Tuesday and Friday morning. Thedrugs were given through stomach-perfusion to GFJL group(1.4g/ml),GFJMgroup(2.8g/ml),GFJH group(4.2g/ml) and colchicine treatmentgroup(0.154mg/kg·d) after the model was successful. Gastric lavage liquidamount is10ml/kg,1times/d.The rats in the normal control and model groupreceived intragastric administration of0.9%saline(10ml/kg).Observed rats’ordinary circumstances. At the12th weekend,We draw blood from abdominalaorta. The level of HA, LN, PCIII, CIV was measured withradioimmunoassay (RIA).The specimens were taken and the degree of hepaticfibrosis was judged by routine haematoxylin-eosin staining and Massonstaining.
Results:
1The ecumenic state of rats
The normal control group were generally good, with good mental state,good appetite, smooth hair, and gradual weight gain. The rats in model groupwere apathetic, lazy, anorexia.The hair were pell-mell and the urine wasyellow. There was blood in some rats’ noses. Weight growth is slow.The stateof the other treatment groups was better than rats in model group.
2Gross findings
The liver of normal group was soft, fresh, and smooth with no stain andprominency.The colour was deep red. The liver of model group was hard, bigand rough with prominency.The brink was blunt. The colour was dark red.The liver of colchicine and TCM treatment group was not swollen obviously.The liver was crisp, smooth with almost no stain and prominency.The color was normal by and large.
3Effect of Ganfujian formula to pathomorphology in hepatic fibrosis rats
Under the light microscope，the hepatic lobules of normal group werearranged in order and ranged radially around the central vein. There was notdegeneration and necrosis in liver tissue. There was not collagen proliferation.The Structure of model control rats was destroyed. Most of hepatocytes werehydropic degeneration, ballooning degeneration and necrosis. There wereneutrophilic granulocytes and lymphocytes around central vein and portal area.The fibrous tissue was in proliferation.Pseudolobule proliferation could beseen in individual animal. The degree of hepatocyte injury in all medicineintervention groups was less serious than model group. The destructions werelightened, proliferations of collagen fibers were also lightened, Swelling ofliver cells and degeneration alleviated, infiltrating cells decreased. Theseimprovements were most significantly in high dose group.
4Effect of Ganfujian formula on the level of serum HA, LN, PCIII, CIV
Compared with the normal control group, the levels of serum HA, LN,PCIII, CIV all increased in model group, Ganfujian group and colchicinesgroup(P <0.05). Compared with the model control group, the levels of serumHA, LN, PCIII, CIV could be decreased from all medicine intervention groupsignificantly(P<0.05). The level of Ganfujian formula of high dose groupdecreased most significantly, better than the colchicine group, middle dosegroup and low dose group (P<0.05).The difference between the colchicinetreatment group and GFJM group was not significant (P>0.05).The level ofserum HA, LN, PCIII, CIV in colchicine treatment group was significantlyreduced compared with GFJL group (P<0.05).
Conclusion: Ganfujian formula can improve the HF rat’s ordinarycircumstances and obviously reduce the levels of serum HA,LN,PCⅢ,andCIV in hepatic fibrosis rats. It can resist hepatic fibrosis in inhibiting theproliferation of fibrous tissue, promoting the degradation of ECM, improvingthe pathological structure of liver fibrosis in rats, and recovering the liver celldamage. Part three Effect of Ganfujian formula on leptin, OB-Rb in HF rats
Objective: The aim was to explore Ganfujian formula’s effect onexpression of leptin, OB-Rb in HF rats. To further clarify the biological effectof leptin in the process of forming HF as well as the deep mechanism ofHuazhuo Jiedu method against HF.
Methods: The Leptin level was measured with radioimmunoassay(RIA),leptin and OB-RbmRNA expression were determined using RT-PCR.
Results:
1Effect of Ganfujian formula on the level of serum Leptin
Compared with the normal control group, the levels of serum Leptin allincreased in model group,Ganfujian group and colchicines group(P<0.05).Compared with the model control group, the levels of serum Leptin could bedecreased from all medicine intervention group significantly(P<0.05). Theserum leptin level of Ganfujian formula of high dose group decreased mostsignificantly, better than the colchicine group, middle dose group and lowdose group(P<0.05).The difference between the colchicine treatment groupand GFJM group was not significant(P>0.05).The level of serum leptin incolchicine treatment group was significantly reduced compared with GFJLgroup (P<0.05).
2Effect of Ganfujian formula on Leptin, OB-RbmRNA in HF rats
Compared with the normal control group, the expression of Leptin,OB-RbmRNA in other groups was stronger(P<0.05).Compared with the modelcontrol group, the expression of Leptin, OB-RbmRNA in Ganfujian groupsand colchicine group was significantly lighter(P<0.05).We compare betweeneach drug treatment group. The expression of Leptin,OB-RbmRNA in GFJHgroup decreased most significantly, better than the colchicine group, middledose group and low dose group(P<0.05).The difference between thecolchicine treatment group and GFJM group was not significant(P>0.05).Theexpression of Leptin,OB-RbmRNA in colchicine treatment group wassignificantly reduced compared with GFJL group (P<0.05).
3Effect of Ganfujian formula on OB-RbmRNA in HF rats
Compared with the normal control group, the expression of OB-RbmRNAin other groups was stronger(P<0.05).Compared with the model control group,the expression of OB-RbmRNA in Ganfujian groups and colchicine group wassignificantly lighter(P<0.05).We compare between each drug treatment group.The expression of OB-RbmRNA in GFJH group decreased most significantly,better than the colchicine group, middle dose group and low dosegroup(P<0.05).The difference between the colchicine treatment group andGFJM group was not significant(P>0.05).The expression of LeptinmRNA incolchicine treatment group was significantly reduced compared with GFJLgroup (P<0.05).
Conclusion: Leptin is one of the material basis of Zhuodu. Ganfujianformula can effectively reduce the expression of leptin and leptin receptor inHF rats, reduce the combination of the two, and then inhibit the downstreamsignal transduction pathway, so as to achieve the anti-fibrotic effect.
Part four Effect of Ganfujian formula on MMP-1in HF rats
Objective: The aim was to explore Ganfujian formula’s effect onexpression of MMP-1in HF rats. To further clarify the deep mechanism ofGanfujian formula against HF.
Methods: The MMP-1mRNA expression were determined by usingimmunohistochemical technique and RT-PCR.
Results: Compared with the normal control group, the expression ofMMP-1mRNA in other groups was lighter(P<0.05).Compared with the modelcontrol group, the expression of MMP-1mRNA in Ganfujian groups andcolchicine group was significantly stronger(P<0.05).We compare betweeneach drug treatment group. The expression of MMP-1mRNA in GFJH groupincreased most significantly, better than the colchicine group, middle dosegroup and low dose group(P<0.05).The expression of MMP-1mRNA incolchicine treatment group was stronger than that of GFJM group, but therewas no difference between two groups(P>0.05).The expression ofMMP-1mRNA in colchicine treatment group significantly increased compared with GFJL group (P<0.05).
Conclusion: Ganfujian formula can significantly increase the expressionof MMP-1mRNA.It can inhibit the synthesis and secretion of leptin by HSC,promote the expression of MMP-1, degradate extracellular matrix, so thathinder the progression of hepatic fibrosis.Part five Effect of Ganfujian formula on JAK2/STAT3signaltransduction pathway in HF rats
Objective: The aim was to explore Ganfujian formula’s effect onexpression of JAK2, STAT3protein in HF rats. To further explore theunderlying mechanism of Ganfujian formula of anti hepatic fibrosis, andprovide a theoretical basis for the clinical prevention and treatment of HF.
Methods: Western blotting was used for detecting the protein expressionlevel of JAK2, STAT3.
Results:
1Effect of Ganfujian formula on JAK2protein of the hepatic fibrosis rats
Compared with normal group, the expression of JAK2in other groupswas stronger(P<0.05).Compared with model group, the expression of JAK2inGanfujian groups and colchicine group was significantly lighter(P<0.05).Thedifference between the colchicine treatment group and GFJM group was notsignificant(P>0.05).The expression of JAK2in colchicine treatment groupwas significantly reduced compared with GFJL group(P<0.05).The expressionof JAK2in GFJH group was significantly reduced compared with colchicinetreatment group(P<0.05).The expression of JAK2in GFJH group was lighterthan that of GFJM group. The GFJM group was superior to GFJL group. Thedifference was statistically significant(P>0.05).
2Effect of Ganfujian formula on STAT3protein of the hepatic fibrosis rats
Compared with normal group, the expression of STAT3in other groupswas stronger(P<0.05).Compared with model group, the expression of STAT3in Ganfujian groups and colchicine group was significantlylighter(P<0.05).The difference between the colchicine treatment group andGFJM group was not significant(P>0.05).The expression of STAT3in colchicine treatment group was significantly reduced compared with GFJLgroup(P<0.05).The expression of STAT3in GFJH group was significantlyreduced compared with colchicine treatment group(P<0.05).The expression ofSTAT3in GFJH group was lighter than that of GFJM group. The GFJM groupwas superior to GFJL group. The difference was statisticallysignificant(P>0.05).
Conclusion: Ganfujian formula could inhibit the expression of JAK2,STAT3.It can inhibit the activation, proliferation of HSC, reduce theexpression and secretion of collagen, and ultimately resist hepatic fibrosis.JAK2/STAT3signal transduction pathway regulated by leptin is the newfunction way and target for Ganfujian formula anti HF.
肝纤维化的形成是一个多因素、多环节、多阶段的复杂过程，其形成不是静止的，而是不断变化的动态过程。各种细胞因子形成的复杂调节网络，不断影响着肝纤维化的形成和转归。从细胞因子及其信号转导角度研究药物的作用机制，将不断深化肝纤维化的治疗学研究。瘦素（leptin）与肝纤维化的形成之间有着密切的关系，与瘦素受体结合后，激活相应的信号转导通路，促进肝纤维化的发生发展。
目前临床上仍然缺乏确切特效的抗纤维化药物与方法。中医药具有多层次、多环节、多途径、多靶点的作用特点，在治疗肝纤维化方面取得了显著的成果。李佃贵教授提出浊毒内蕴，肝络瘀滞是肝纤维化的主要病机之一，并从浊毒论治肝纤维化，在此指导下确立肝复健方，取得显著疗效。既往临床及实验研究表明，肝复健方具有明显的阻断病毒性乙型肝炎慢性化、减轻肝纤维化及保护肝功能的作用。本课题继续在腹腔注射猪血清复制免疫性肝纤维化模型的基础上，观察肝复健方对大鼠肝组织病理形态学、肝纤四项、MMP-1、瘦素(leptin)及其受体（OB-Rb)、JAK2、STAT3的表达情况，探讨其对瘦素调控肝纤维化大鼠JAK2/STAT3信号转导通路的影响，旨在对本方抗HF的药效和可能的作用机制进一步的研究，为肝纤维化的预防与治疗提供理论依据。本实验分为以下五部分：第一部分浊毒理论与肝纤维化
目的：探讨浊毒理论与肝纤维化的关系，明确其在肝纤维化中的重要地位，提出肝纤维化治疗的新观点与新方法。
方法：通过温习、回顾、整理中医文献，并结合临床实践经验，提出浊毒理论，提出肝纤维化的新病机，系统总结了肝纤维化浊毒证证治规律。
结果：提出了浊毒理论，明确了浊毒的概念、临床表现及致病特点。探讨了浊毒与肝纤维化的关系，总结出了肝纤维化浊毒证证治规律，从临床上验证了从浊毒治疗肝纤维化的可行性。
结论：浊毒理论是中医学术体系的新的组成部分，是研究浊毒致病的病因、病机、诊断、治疗等理法方药的学科。浊毒与肝纤维化关系密切，是其发生发展的关键环节。
第二部分肝复健方对肝纤维化大鼠病理形态学和血清标志物的影响
目的：观察肝复健方对猪血清诱导的肝纤维化大鼠病理形态学及血清HA、LN、PCⅢ、Ⅳ型胶原含量的影响，探讨其抗纤维化的作用机制。
方法：清洁级SD大鼠60只，雄性，体重200±20g。适应性喂养一周后随机分为正常对照组（Normal group）、模型组（Model group）、秋水仙碱组（Colchicine）、肝复健方低剂量组（GFJL group）、肝复健方中剂量组（GFJM group）、肝复健方高剂量组（GFJH group）六组，每组10只。每周二、周五上午8点，采用无菌未灭活的猪血清腹腔注射进行造模，连续8周。造模成功后正常对照组与模型组给予生理盐水（10ml/kg），日1次灌胃。GFJL组给予含生药1.4g/ml水煎剂灌胃；GFJM组给予含生药2.8g/ml水煎剂灌胃；GFJH组给予含生药4.2g/ml水煎剂灌胃（灌胃液体量为10ml/kg，1次/d）；秋水仙碱组给予0.154mg/kg·d灌胃，均至12周末。实验期间观察大鼠的一般情况。12周末采用腹主动脉取血，分离血清，用放射免疫分析法测定各组大鼠血清中HA、LN、PCIII、CIV水平。留取大鼠肝组织，采用苏木精-伊红染色（HE染色）和胶原纤维特殊染色法（Masson染色）观察肝组织病理形态学改变。
结果：
1一般情况
正常对照组一般情况良好，精神状态好，活泼好动，大鼠食欲佳，进食量正常，皮毛光滑，体重逐渐增加。模型组大鼠精神萎靡、不喜活动，进食较正常组少，皮毛杂乱、黯淡无光泽、尿黄，个别大鼠鼻有出血，体重增长变慢。秋水仙碱及中药治疗各组大鼠精神状态、活动、进食量尚可，被毛光泽，体重有所增加，无特殊异常状态出现。
2肝脏大体观察
正常组大鼠肝脏质地较软，表面光滑，略呈深红色，有光泽，无任何斑点、突起，边缘锐利。模型组大鼠肝脏质地硬，表面欠光滑，部分标本表面粗糙，凹凸不平，颜色暗红，边缘钝。秋水仙碱及中药治疗组大鼠肝脏质较脆，表面较光滑，颜色基本正常，基本无斑点、突起等。
3肝复健方对肝纤维化大鼠肝组织病理的影响
正常组肝小叶结构清晰完整，肝细胞围绕中央静脉呈放射状排列，无变性坏死，未见纤维组织增生及炎性细胞浸润。模型组肝小叶结构破坏，肝细胞索排列紊乱，肝细胞变性，部分肝细胞坏死，伴有中性粒细胞及淋巴细胞浸润，纤维组织大量增生，个别动物可见假小叶形成。秋水仙碱组与中药治疗各组肝脏损伤程度均较模型组轻，肝小叶结构破坏减轻，肝脏胶原纤维增生减轻，肝细胞水肿好转，变性情况明显改善，炎细胞浸润减少。肝复健方高剂量组肝脏结构改善最为明显。
4肝复健方对肝纤维化大鼠血清HA、LN、PCⅢ、CIV的影响
与正常对照组相比，模型组大鼠血清HA、LN、PCⅢ、CIV水平升高，差异有统计学意义(P<0.05)。与模型对照组相比，12周末各药物干预组大鼠血清HA、LN、PCⅢ、CIV水平下降(P<0.05)。肝复健方高剂量组血清HA、LN、PCⅢ、CIV水平显著性降低，优于秋水仙碱组、中剂量组及低剂量组（P＜0.05）；秋水仙碱组、肝复健中剂量组之间比较，差异无统计学意义（P>0.05）；秋水仙碱组与肝复健方低剂量组比较，血清HA、LN、PCⅢ、CIV水平降低，差异有统计学意义(P<0.05)。
结论：肝复健方能够改善大鼠的一般状况，显著降低大鼠血清HA、LN、PCⅢ、Ⅳ型胶原含量，促进ECM的降解，抑制纤维组织增生，改善肝纤维化大鼠的病理组织结构，恢复肝细胞损伤，达到抗肝纤维化的作用。
第三部分肝复健方对肝纤维化大鼠瘦素及瘦素受体表达的影响
目的：观察肝复健方对血清瘦素、肝组织瘦素及瘦素受体的影响，进一步明确瘦素在肝纤维化形成过程中的生物学效应以及化浊解毒法抗肝纤维化的深层作用机制。
方法：放射免疫分析法检测血清瘦素(Leptin)水平，逆转录-多聚酶链反应(RT-PCR)技术检测肝组织瘦素(leptin)及瘦素受体(OB-Rb)的表达。
结果：
1肝复健方对大鼠血清Leptin水平的影响
与正常对照组相比,模型对照组、各药物组血清瘦素水平明显升高,差异有统计学意义(P<0.05)；与模型对照组相比，秋水仙碱组和肝复健方各剂量组血清瘦素水平明显下降，差异有统计学意义（P＜0.05）。各药物治疗组比较，肝复健方高剂量组血清瘦素水平显著性降低，优于秋水仙碱组、中剂量组及低剂量组（P＜0.05），秋水仙碱组与中剂量组比较，无显著性差异(P﹥0.05)。秋水仙碱组与低剂量组比较，血清瘦素水平下降，差异有统计学意义（P＜0.05）。
2肝复健方对大鼠肝组织Leptin、OB-RbmRNA表达的影响
与正常对照组相比，模型对照组、秋水仙碱组和肝复健方各剂量组Leptin、OB-RbmRNA的表达明显增强，差异有统计学意义（P＜0.05）。与模型对照组相比，秋水仙碱组和肝复健方各剂量组Leptin、OB-RbmRNA表达明显减弱，差异有统计学意义（P＜0.05）。各药物治疗组比较，肝复健方高剂量组Leptin、OB-RbmRNA表达显著性降低，优于秋水仙碱组、中剂量组及低剂量组（P＜0.05），秋水仙碱组与中剂量组比较Leptin、OB-RbmRNA的表达有下降趋势，但均无显著性差异(P﹥0.05)。秋水仙碱组与低剂量组比较，Leptin、OB-RbmRNA的表达减弱，差异有统计学意义（P＜0.05）。
结论：瘦素是内生浊毒的物质基础之一，肝复健方能够有效降低肝纤维化模型大鼠瘦素及瘦素受体的表达，减少两者的结合，进而抑制下游信号转导通路，从而达到抗肝纤维化的作用。
第四部分肝复健方对大鼠肝组织MMP-1表达的影响
目的：观察肝复健方对HF大鼠肝脏MMP-1表达的影响，从分子水平探讨肝复健方抗肝纤维化的深层作用机理。
方法：逆转录-多聚酶链反应(RT-PCR)技术检测肝组织MMP-1的表达。
结果：与正常对照组相比，模型对照组、秋水仙碱组和肝复健方各剂量组MMP-1mRNA的表达均明显减弱，差异有统计学意义（P＜0.05）。与模型对照组相比，秋水仙碱组和肝复健方各剂量组MMP-1mRNA表达明显增强，差异有统计学意义（P＜0.05）。各药物治疗组比较，肝复健方高剂量组MMP-1mRNA的表达显著性增强，优于秋水仙碱组、中剂量组及低剂量组（P＜0.05），秋水仙碱组与中剂量组比较MMP-1mRNA的表达有增强趋势，但均无显著性差异(P﹥0.05)。秋水仙碱组与低剂量组比较，MMP-1mRNA的表达增强，差异有统计学意义（P＜0.05）。
结论：肝复健方可以显著提高MMP-1mRNA的表达水平，通过抑制HSC合成和分泌瘦素，促进MMP-1的表达，从而使细胞外基质降解，阻碍肝纤维化进程。第五部分肝复健方对肝纤维化大鼠肝组织JAK2/STAT3信号转导通路的影响
目的：观察肝复健方对HF大鼠肝脏JAK2、STAT3蛋白表达的影响，以进一步探讨肝复健方抗肝纤维化的深层作用机理，为临床防治肝纤维化提供理论依据
方法：Western blot检测HF大鼠肝组织JAK2、STAT3的表达。
结果：
1肝复健方对大鼠肝组织JAK2蛋白表达的影响
(1)所有实验组大鼠肝组织中JAK2的表达量均高于正常对照组(P<0.05)。(2)肝复健方各剂量组及秋水仙碱组与模型组相比JAK2的蛋白表达量显著性降低(P<0.05)。(3)肝复健方中剂量组与秋水仙碱组相比JAK2蛋白表达量均无显著性差异(P﹥0.05)；秋水仙碱组与肝复健方低剂量组相比JAK2蛋白的表达量有显著性降低(P<0.05)，肝复健方高剂量组与秋水仙碱组相比，JAK2蛋白的表达量有显著性降低(P<0.05)。(4)肝复健方药不同剂量组之间比较，高剂量组JAK2蛋白的表达量优于中剂量组，中剂量组优于低剂量组，差异有统计学意义（P＜0.05）。
2肝复健方对大鼠肝组织STAT3蛋白表达的影响
(1)所有实验组大鼠肝组织中STAT3的表达量均高于正常对照组(P<0.05)。(2)肝复健方各剂量组及秋水仙碱组与模型组相比STAT3的蛋白表达量有显著性降低(P<0.05)。(3)肝复健方中剂量组与秋水仙碱组相比STAT3蛋白表达量均无显著性差异(P﹥0.05)；秋水仙碱组与肝复健方低剂量组相比STAT3蛋白的表达量有显著性降低(P<0.05)，肝复健方高剂量组与秋水仙碱组相比，STAT3蛋白的表达量有显著性降低(P<0.05)。(4)肝复健方药不同剂量组之间比较，高剂量组STAT3蛋白的表达量优于中剂量组，中剂量组优于低剂量组，差异有统计学意义（P＜0.05）。
结论：肝复健方能够下调肝纤维化大鼠肝组织JAK2、STAT3的表达，进而抑制HSC活化、增殖，降低胶原的表达和分泌，最终起到抗肝纤维化的作用。瘦素调控的JAK2/STAT3信号转导通路是肝复健方抗肝纤维化的新的作用途径和作用靶点。
Hepatic fibrosis is a pathologic process that caused by various riskfactors. Its essence is excessive deposition of extracellular matrix (ECM)components in liver, which occurs due to an imbalance between theproduction and degradation of matrix. It is the most common pathologicalfeatures of chronic liver disease, further to the important link of thedevelopment of hcc.HF can be reversed. It can prevent serious consequencesif hepatic fibrosis blocked and reversed.
Hepatic fibrosis is a complex process of multiple factors, multiple links,multiple stage. Its forming process is not static, but constantly changing.Complex regulatory network composed of various cytokines has influencedthe formation and prognosis of Hepatic fibrosis. It is advantageous in HFtreatment from the cell factor and its signal pathways to study mechanism ofdrug intervention.Leptin is considered a key factor to promote the formationof HF.By binding with leptin receptor, it activates signal transduction pathwaycorresponding, promotes the occurrence and development of HF.JAK/STATsignal transduction pathway is the focus of the current study.
At present clinical still lacks highly effective drugs and methods to treatHF.The characteristics of Traditional Chinese Medicine (TCM) treatment aremultiple positions, multiple links and multiple targets. There are muchsuperiority in the prevention and treatment on HF.After many years clinicalpractices and experiment research, combining with current disease pattern anddisease spectrum changes, professor Li dian-gui finds Zhuodu accumulationand liver collateral stasis is one of the main pathogenesis of HF.He applies thistheory to treat Hepatic Fibrosis patients and obtains the good curative effect.Previous clinical and experimental researches show that, Ganfujian formulabased on zhuodu theory could block viral hepatitis B chronically and reduce liver fibrosis and liver function. We still used the pig’s serum to set thehepatic fibrosis rats’ model by intraperitoneal injection. we observed theeffect of Ganfujian formula on the levels of serum HA,LN,PCⅢ,CIV andPathomorphology in hepatic fibrosis rats. We observed the expression ofleptin, OB-Rb, JAK2, STAT3and MMP-1in rats with HF, and then exploredthe effect of the JAK2/STAT3signal transduction pathway regulated by leptinin rats with HF.Investigated on possible Mechanism of the resistance to HF,so that provided theoretical and experimental basis for treating and preventingHF.This experiment was separated into five parts:
Part one ZhuoDu theory and Hepatic Fibrosis
Objective:The aim was to explore the relationship between the Zhuodutheory and HF, clarify its important role in HF, and propose a new idea andnew method for the treatment of HF.
Methods: Reviewed the TCM literature combined with clinical practiceexperience, presented Zhuodu theory,put forward that the new pathogenesis ofHF was zhuodu and summarized the rule of syndrome and treatment ofhepatic fibrosis with zhuodu.
Results:We presented Zhuodu theory, defined the concept,etiology,clinical manifestations and pathogenic characteristics ofzhuodu,discussed the relationship between zhuodu and Hepatic Fibrosis,summarized the rule of syndrome and treatment of hepatic fibrosis withzhuodu. Feasibility was verified in treating hepatic fibrosis using Zhuodutheory in clinical.
Conclusion: Zhuodu theory is part of the new academic system ofTCM.It is the discipline that studying the etiology,pathogenesis,diagnosis,treatment and prescription of Zhuodu.Zhuodu closely correlateswith HF and is a key link in its occurrence and development.
Part two Effect of Ganfujian formula on the levels of serum markersand Pathomorphology in hepatic fibrosis rats
Objective: The aim was to observe the effect of Ganfujian formula onthe levels of serum HA,LN,PCⅢ,CIV and Pathomorphology in hepatic fibrosis rats. Investigate the mechanism of anti-fibrosis.
Methods: Total60clean male SD rats weighed200±20grams wererandomly divided into six groups: the normal group, the model group,colchicine group,Ganfujian formula of low dose group(GFJL),Ganfujianformula of medium dose group(GFJM) and Ganfujian formula of high dosegroup(GFJH)with10rats for each group. In addition to the normal group,pig′sserum(0.5ml per mouse)was established in rats by intraperitoneal injection foreight weeks. The time was fixed at9am on Tuesday and Friday morning. Thedrugs were given through stomach-perfusion to GFJL group(1.4g/ml),GFJMgroup(2.8g/ml),GFJH group(4.2g/ml) and colchicine treatmentgroup(0.154mg/kg·d) after the model was successful. Gastric lavage liquidamount is10ml/kg,1times/d.The rats in the normal control and model groupreceived intragastric administration of0.9%saline(10ml/kg).Observed rats’ordinary circumstances. At the12th weekend,We draw blood from abdominalaorta. The level of HA, LN, PCIII, CIV was measured withradioimmunoassay (RIA).The specimens were taken and the degree of hepaticfibrosis was judged by routine haematoxylin-eosin staining and Massonstaining.
Results:
1The ecumenic state of rats
The normal control group were generally good, with good mental state,good appetite, smooth hair, and gradual weight gain. The rats in model groupwere apathetic, lazy, anorexia.The hair were pell-mell and the urine wasyellow. There was blood in some rats’ noses. Weight growth is slow.The stateof the other treatment groups was better than rats in model group.
2Gross findings
The liver of normal group was soft, fresh, and smooth with no stain andprominency.The colour was deep red. The liver of model group was hard, bigand rough with prominency.The brink was blunt. The colour was dark red.The liver of colchicine and TCM treatment group was not swollen obviously.The liver was crisp, smooth with almost no stain and prominency.The color was normal by and large.
3Effect of Ganfujian formula to pathomorphology in hepatic fibrosis rats
Under the light microscope，the hepatic lobules of normal group werearranged in order and ranged radially around the central vein. There was notdegeneration and necrosis in liver tissue. There was not collagen proliferation.The Structure of model control rats was destroyed. Most of hepatocytes werehydropic degeneration, ballooning degeneration and necrosis. There wereneutrophilic granulocytes and lymphocytes around central vein and portal area.The fibrous tissue was in proliferation.Pseudolobule proliferation could beseen in individual animal. The degree of hepatocyte injury in all medicineintervention groups was less serious than model group. The destructions werelightened, proliferations of collagen fibers were also lightened, Swelling ofliver cells and degeneration alleviated, infiltrating cells decreased. Theseimprovements were most significantly in high dose group.
4Effect of Ganfujian formula on the level of serum HA, LN, PCIII, CIV
Compared with the normal control group, the levels of serum HA, LN,PCIII, CIV all increased in model group, Ganfujian group and colchicinesgroup(P <0.05). Compared with the model control group, the levels of serumHA, LN, PCIII, CIV could be decreased from all medicine intervention groupsignificantly(P<0.05). The level of Ganfujian formula of high dose groupdecreased most significantly, better than the colchicine group, middle dosegroup and low dose group (P<0.05).The difference between the colchicinetreatment group and GFJM group was not significant (P>0.05).The level ofserum HA, LN, PCIII, CIV in colchicine treatment group was significantlyreduced compared with GFJL group (P<0.05).
Conclusion: Ganfujian formula can improve the HF rat’s ordinarycircumstances and obviously reduce the levels of serum HA,LN,PCⅢ,andCIV in hepatic fibrosis rats. It can resist hepatic fibrosis in inhibiting theproliferation of fibrous tissue, promoting the degradation of ECM, improvingthe pathological structure of liver fibrosis in rats, and recovering the liver celldamage. Part three Effect of Ganfujian formula on leptin, OB-Rb in HF rats
Objective: The aim was to explore Ganfujian formula’s effect onexpression of leptin, OB-Rb in HF rats. To further clarify the biological effectof leptin in the process of forming HF as well as the deep mechanism ofHuazhuo Jiedu method against HF.
Methods: The Leptin level was measured with radioimmunoassay(RIA),leptin and OB-RbmRNA expression were determined using RT-PCR.
Results:
1Effect of Ganfujian formula on the level of serum Leptin
Compared with the normal control group, the levels of serum Leptin allincreased in model group,Ganfujian group and colchicines group(P<0.05).Compared with the model control group, the levels of serum Leptin could bedecreased from all medicine intervention group significantly(P<0.05). Theserum leptin level of Ganfujian formula of high dose group decreased mostsignificantly, better than the colchicine group, middle dose group and lowdose group(P<0.05).The difference between the colchicine treatment groupand GFJM group was not significant(P>0.05).The level of serum leptin incolchicine treatment group was significantly reduced compared with GFJLgroup (P<0.05).
2Effect of Ganfujian formula on Leptin, OB-RbmRNA in HF rats
Compared with the normal control group, the expression of Leptin,OB-RbmRNA in other groups was stronger(P<0.05).Compared with the modelcontrol group, the expression of Leptin, OB-RbmRNA in Ganfujian groupsand colchicine group was significantly lighter(P<0.05).We compare betweeneach drug treatment group. The expression of Leptin,OB-RbmRNA in GFJHgroup decreased most significantly, better than the colchicine group, middledose group and low dose group(P<0.05).The difference between thecolchicine treatment group and GFJM group was not significant(P>0.05).Theexpression of Leptin,OB-RbmRNA in colchicine treatment group wassignificantly reduced compared with GFJL group (P<0.05).
3Effect of Ganfujian formula on OB-RbmRNA in HF rats
Compared with the normal control group, the expression of OB-RbmRNAin other groups was stronger(P<0.05).Compared with the model control group,the expression of OB-RbmRNA in Ganfujian groups and colchicine group wassignificantly lighter(P<0.05).We compare between each drug treatment group.The expression of OB-RbmRNA in GFJH group decreased most significantly,better than the colchicine group, middle dose group and low dosegroup(P<0.05).The difference between the colchicine treatment group andGFJM group was not significant(P>0.05).The expression of LeptinmRNA incolchicine treatment group was significantly reduced compared with GFJLgroup (P<0.05).
Conclusion: Leptin is one of the material basis of Zhuodu. Ganfujianformula can effectively reduce the expression of leptin and leptin receptor inHF rats, reduce the combination of the two, and then inhibit the downstreamsignal transduction pathway, so as to achieve the anti-fibrotic effect.
Part four Effect of Ganfujian formula on MMP-1in HF rats
Objective: The aim was to explore Ganfujian formula’s effect onexpression of MMP-1in HF rats. To further clarify the deep mechanism ofGanfujian formula against HF.
Methods: The MMP-1mRNA expression were determined by usingimmunohistochemical technique and RT-PCR.
Results: Compared with the normal control group, the expression ofMMP-1mRNA in other groups was lighter(P<0.05).Compared with the modelcontrol group, the expression of MMP-1mRNA in Ganfujian groups andcolchicine group was significantly stronger(P<0.05).We compare betweeneach drug treatment group. The expression of MMP-1mRNA in GFJH groupincreased most significantly, better than the colchicine group, middle dosegroup and low dose group(P<0.05).The expression of MMP-1mRNA incolchicine treatment group was stronger than that of GFJM group, but therewas no difference between two groups(P>0.05).The expression ofMMP-1mRNA in colchicine treatment group significantly increased compared with GFJL group (P<0.05).
Conclusion: Ganfujian formula can significantly increase the expressionof MMP-1mRNA.It can inhibit the synthesis and secretion of leptin by HSC,promote the expression of MMP-1, degradate extracellular matrix, so thathinder the progression of hepatic fibrosis.Part five Effect of Ganfujian formula on JAK2/STAT3signaltransduction pathway in HF rats
Objective: The aim was to explore Ganfujian formula’s effect onexpression of JAK2, STAT3protein in HF rats. To further explore theunderlying mechanism of Ganfujian formula of anti hepatic fibrosis, andprovide a theoretical basis for the clinical prevention and treatment of HF.
Methods: Western blotting was used for detecting the protein expressionlevel of JAK2, STAT3.
Results:
1Effect of Ganfujian formula on JAK2protein of the hepatic fibrosis rats
Compared with normal group, the expression of JAK2in other groupswas stronger(P<0.05).Compared with model group, the expression of JAK2inGanfujian groups and colchicine group was significantly lighter(P<0.05).Thedifference between the colchicine treatment group and GFJM group was notsignificant(P>0.05).The expression of JAK2in colchicine treatment groupwas significantly reduced compared with GFJL group(P<0.05).The expressionof JAK2in GFJH group was significantly reduced compared with colchicinetreatment group(P<0.05).The expression of JAK2in GFJH group was lighterthan that of GFJM group. The GFJM group was superior to GFJL group. Thedifference was statistically significant(P>0.05).
2Effect of Ganfujian formula on STAT3protein of the hepatic fibrosis rats
Compared with normal group, the expression of STAT3in other groupswas stronger(P<0.05).Compared with model group, the expression of STAT3in Ganfujian groups and colchicine group was significantlylighter(P<0.05).The difference between the colchicine treatment group andGFJM group was not significant(P>0.05).The expression of STAT3in colchicine treatment group was significantly reduced compared with GFJLgroup(P<0.05).The expression of STAT3in GFJH group was significantlyreduced compared with colchicine treatment group(P<0.05).The expression ofSTAT3in GFJH group was lighter than that of GFJM group. The GFJM groupwas superior to GFJL group. The difference was statisticallysignificant(P>0.05).
Conclusion: Ganfujian formula could inhibit the expression of JAK2,STAT3.It can inhibit the activation, proliferation of HSC, reduce theexpression and secretion of collagen, and ultimately resist hepatic fibrosis.JAK2/STAT3signal transduction pathway regulated by leptin is the newfunction way and target for Ganfujian formula anti HF.
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