【作者】 贺松其；

【导师】 吕志平；

【作者基本信息】 第一军医大学 ， 中西医结合临床， 2007， 博士

【摘要】 科研工作来源:国家自然基金资助项目（30672769）;广东省中医药局资助项目（1050128）。研究背景:肝纤维化（hepatic fibrosis,HF）主要是由肝脏内细胞外基质（extracellular matrix,ECM）的合成大于降解,致使ECM在肝脏内过度沉积所致。它是大多数慢性肝病所共有的病理特征,也是慢性肝炎、肝硬化等进一步发展、恶化的重要中间环节。据统计,我国患慢性乙型肝炎的病人超过3000万,每年死于肝炎后肝硬化者有40万人,而治愈肝硬化的唯一途径是阻断肝纤维化。迄今为止,全世界包括我国在内的国家尚没有批准一个抗纤维化西药,中医药抗肝纤维化的实验和临床研究已凸现其特有的优势,其中尤以复方中药抗肝纤维化的多靶点治疗作用更为引人注目。我们在全面认识慢性肝病肝纤维化病因病机的基础上,研制出抗肝纤维化新方保肝宁,它寓疏肝解郁、益气健脾;活血化瘀、软坚散结;兼清热利湿解毒等重要治法于一体,应用于临床,疗效卓著。在国家自然科学基金、广东省自然科学基金的资助下,我们开展了保肝宁抗肝纤维化的信号转导机制研究。保肝宁抑制HSC增殖作用可能与NF-κB信号转导通路有关,保肝宁能明显降低HSC细胞NF-κB的表达及IκB的磷酸化。瘦素（Leptin）是新发现的促肝纤维化的细胞因子,目前对瘦素促肝纤维化的分子机制研究,国外刚刚起步,国内相关文献报道更加少有。因此,我们认为系统的研究瘦素及其信号转导机制,并在此基础上探讨中药保肝宁对瘦素、瘦素受体及受体后介导的信号转导通路的影响,有助于进一步阐明其抗肝纤维化的作用途径和作用靶点,为保肝宁防治和逆转肝纤维化提供更加充分的理论依据,同时,对于深化和完善肝纤维化的机制研究具有十分重要的理论意义。目的和意义:探讨保肝宁抗肝纤维化的分子机制,为保肝宁应用于临床提供充分的理论依据,为开发抗肝纤维化新药保肝宁奠定坚实的理论基础。研究方法:以复合因素诱导的肝纤维化模型大鼠为研究对象,观察保肝宁（方药由黄芪、桃仁、丹参、黄芩、鳖甲等组成）对模型大鼠瘦素及其信号转导通路的影响。50只Wistar大鼠随机分为5组,即正常对照组（A组）、模型组（B组）、秋水仙碱组（C组）、保肝宁大剂量组（D组）、保肝宁中剂量组（E组）。除A组外,其余大鼠于实验第1d每只皮下注射40%（体积分数）CCl₄花生油0.5ml·100g^-1;以后每3日注射0.3ml·100g^-1。除A组外,各组均以普通饲料喂养,并以5%乙醇为饮料。A组注射等量花生油,进食普通饲料并自由饮水。各组从造模第2天开始,每日给相应药物灌胃,C组每日灌服秋水仙碱0.011mg·100g^-1,D、E组分别每日灌服保肝宁3.54g·100g^-1、1.77g·100g^-1,B组及A组灌以等量蒸馏水作对照。第6厨末,处死动物,取血和肝组织备用。取新鲜肝组织梯度置于-70℃中保存,用于蛋白印迹实验（Western blotting analysis）;一部分肝组织固定于10%中性福尔马林溶液,肝组织常规脱水,石蜡包埋,切片厚5μm,做光镜观察和免疫组化用;血清谷丙转氨酶（ALT）和谷草转氨酶（AST）活性;总胆固醇（TC）、甘油三脂（TG）采用全自动生化分析仪检测;放免法测定血清瘦素（Leptin）水平;光学显微镜下观察肝组织病理学变化;免疫组织化学技术检测瘦素（Leptin）及瘦素受体（OB-Rb）等的表达水平;应用蛋白印迹试验（Western blotting analysis）检测瘦素受体（OB-Rb）,JAK2（Janus激酶,Januskinase）和转录活化因子（STAT3）蛋白的表达水平以及它们的磷酸化水平。统计学处理:计量资料用平均值±标准差（x±s）表示,数据处理采用SPSS14.0统计软件包进行单因素方差分析（one-way ANOVA）。研究内容和过程:1.观察保肝宁对模型大鼠的症状、体征及肝、脾指数的影响模型组大鼠精神不振、消瘦、厌食,饮水减少,大便稀溏,小便色黄而少;肝脏表面较正常组欠光滑,颜色浅,边缘钝,质地硬,部分标本表面凹凸不平,呈小结节状;脾脏明显肿大,呈暗红色。各药物组大鼠精神状态、毛色泽、饮食饮水、排便等一般情况均明显改善,以保肝宁各组改善情况为佳。在增加体重方面,与模型组比较,秋水仙碱、保肝宁大、中剂量均能显著增加大鼠体重（P＜0.05、P＜0.01、P＜0.01）;各药物组比较差异无显著性意义（p＞0.05）。各药物组大鼠肝脏表面较光滑,颜色较红;脾脏与正常组大鼠相比较无明显差异。与正常组比较,模型组、各药物组肝指数均明显增加,差异有非常显著性意义（P均＜0.01）,模型组脾脏指数亦明显升高,差异有非常显著性意义（P均＜0.01）;与模型组比较,各药物组均能显著降低模型大鼠肝、脾指数,差异有显著性意义（P均＜0.01）;其中,保肝宁大剂量组降低肝指数的作用明显优于秋水仙碱组,差异有显著性意义（p＜0.05）。2.观察保肝宁对肝纤维化大鼠肝组织形态学的影响光镜观察显示:模型组正常肝小叶结构破坏,肝索排列紊乱,可见肝细胞点状坏死,肝细胞空泡变性散在分布,间质增生明显,胶原纤维构成的纤维间隔形成,形成较多完整的假小叶。各药物组肝小叶样结构明显改善,胶原纤维间隔较少,肝细胞坏死、炎性细胞浸润少见,但极少数大鼠仍有不完全性假小叶。3.保肝宁对模型大鼠血清肝功能、血脂的影响与正常组比较,模型组血清ALT、AST、AST/ALT比值均明显升高（P均＜0.01）,各药物组ALT、AST水平亦明显升高（P均＜0.01）;与模型组比较,各药物组血清ALT、AST活性明显下降,差异有非常显著性意义（P均＜0.01）,保肝宁中剂量组AST/ALT比值也明显下降,差异有显著性意义（P＜0.05）;保肝宁大剂量组降低AST作用明显优于秋水仙碱（P＜0.05）。与正常组比较,模型组及各药物组血清TC、TG水平明显下降,差异有非常显著性意义（P＜0.01）;与模型组比较,保肝宁大、中剂量组血清TC、TG水平明显升高,差异有非常显著性意义（P均＜0.01）。其中,保肝宁大剂量升高TC作用明显明显优于秋水仙碱组（P＜0.05）,保肝宁大、中剂量组升高TG作用均明显优于秋水仙碱组（P均＜0.05）。4.保肝宁对模型大鼠血清瘦素（Leptin）水平的影响与正常组比较,模型组及各药物组血清Leptin水平明显升高,差异有非常显著性意义（P＜0.01）;与模型组比较,各药物组则明显下降（P＜0.01、P＜0.01、P＜0.05）;各药物组降低Leptin作用,差异无显著性意义（P＞0.05）。5.保肝宁对肝纤维化大鼠肝组织瘦素（Leptin）及瘦素受体（OB-Rb）的影响（1）Leptin的表达:正常组肝细胞内呈阴性或极弱阳性表达,偶见于汇管区、小叶中央静脉周围及肝索Disse腔间隙中。模型组肝内Leptin表达明显增强,数目增多,呈棕黄色胞质型分布;小叶中央静脉周围、汇管区及肝小叶内均有大量条索状、星芒状瘦素阳性表达,高倍镜下,阳性表达主要分布于HSC胞质中。各药物组阳性染色程度较模型组明显减轻,阳性细胞数目减少。统计学显示,与正常组比较,模型组、各药物组Leptin表达明显增多,差异有非常显著性意义（P＜0.01）;与模型组比较,各药物组均可以降低Leptin的表达,差异有显著性意义（p＜0.01）;各药物组两两比较差异无显著性意义（P＞0.05）。（2）OB-Rb表达:正常组肝细胞内呈阴性或极弱阳性表达,偶见于汇管区、小叶中央静脉周围及肝索Disse腔间隙中。模型组肝内表达明显增强,数目增多,呈棕黄色胞质型分布;高倍镜下,阳性表达主要分布于非实质细胞（包括肝星状细胞、窦内皮细胞、枯否细胞等）胞质及胞膜上,部分可定位于核。统计学显示,与正常组比较,模型组、各药物组OB-Rb表达明显增多,差异有显著性意义（P均＜0.01）;与模型组比较,各药物组OB-Rb表达均无明显变化,差异无显著性意义（P均＞0.05）;各药物组OB-Rb的表达两两比较差异无显著性意义（P＞0.05）。6.保肝宁对肝纤维化大鼠肝组织瘦素受体（OB-Rb）及其JAK2-STAT3信号转导通路的影响（1）OB-Rb、JAK2、STAT3蛋白表达。用Western Blot法检测肝脏组织细胞内OB—-b、JAK2、STAT3蛋白表达情况,正常组蛋白表达量最少,保肝宁和秋水仙碱处理组蛋白条带颜色均较浅,模型组条带颜色最深。与正常组比较,模型组、保肝宁大、中剂量组和秋水仙碱组OB-Rb、JAK2、STAT3蛋白程度均增高;与模型组相比,各药物组的OB-Rb、JAK2、STAT3蛋白表达显著降低;保肝宁大、中剂量组和秋水仙碱组相比,OB-Rb、JAK2、STAT3蛋白显著降低。（2）JAK2、STAT3蛋白磷酸化水平。用Western Blot法检测肝脏组织细胞内JAK2、STAT3蛋白磷酸化水平,正常组蛋白磷酸化水平最低,保肝宁和秋水仙碱处理组蛋白条带颜色均较浅,模型组条带颜色最深。与正常组比较,模型组、各药物组P-JAK2、P-STAT3蛋白表达水平均增高;与模型组相比,各药物组的P-JAK2、P-STAT3蛋白表达显著降低;保肝宁大、中剂量组和秋水仙碱组相比,P-JAK2、P-STAT3蛋白水平显著降低。结论:保肝宁能有效的阻止模型大鼠肝纤维化进程,其作用机制为:1.保肝宁可以有效的改善肝纤维化模型大鼠的一般情况:如改善模型大鼠的精神状况,增加体重,改善食欲和饮水等症状,提高大鼠生存质量;显著降低模型大鼠肝、脾指数。2.形态学观察表明保肝宁能明显改善破坏的肝小叶结构,减轻纤维组织增生;明显减少胶原纤维间隔、肝细胞坏死以及炎性细胞的浸润等。3.保肝宁明显降低纤维化模型大鼠血清ALT、AST含量,TG、TC水平明显升高,提示保肝宁能有效减轻炎症反应,调节血脂水平,保护肝细胞,从而抑制早期肝纤维化的启动。4.保肝宁明显降低纤维化模型大鼠血清Leptin水平,有效的阻止了Leptin与OB-Rb结合而发挥的诱导HSC活化和增殖作用。5.保肝宁明显抑制纤维化模型大鼠肝组织Leptin的表达,明显减轻Leptin阳性染色程度,减少阳性细胞数目,提示Leptin是保肝宁抗肝纤维化的作用靶点之一。6.保肝宁有效抑制纤维化模型大鼠肝组织OB-Rb以及其信号转导分子JAK2、STAT3的蛋白表达,显著降低JAK2、STAT3蛋白磷酸化水平,提示保肝宁具有抑制OB-Rb及其信号转导作用,这可能是其抗肝纤维化的分子机制之一。综上所述,保肝宁具有综合抗肝纤维化的药理作用,其作用机制为多途径、多靶点。抑制Leptin的表达、抑制OB-Rb及其JAK2/STAT3信号转导通路,可能是其抗肝纤维化的分子机制之一。而这一机制与抑制炎症反应,调节血脂水平,保护肝细胞,减少细胞因子所产生的瀑布效应,从而抑制肝纤维化的启动密切相关。更多还原

【Abstract】 Source of the research work:This research work was funded by National Natural Sciences Fund and Guangdong Medicine Bureau of Guangdong province.Research background:Liver fibrosis results from chronic damage to the liver in conjunction with the accumulation of ECM proteins,which is a characteristic of most types of chronic liver diseases and also is the important middle key to chronic hepatitis and deteriorating hepatocirrhosis.According to statistics there are over 30000000 patients suffering from chronic B hepatitis in China.Each year about 400000 patients die of posthepatitic cirrhosis.And the only way to cure hepatocirrhosis is to prevent liver fibrosis.Until now there is no authorized anti-fibrosis drug in the world including China.The clinical and experimental studies on effectiveness of TCM against fibrosis have demonstrated its special advantages. The multi-target mechanisms on anti-fibrosis with compound prescriptions are especially extraordinary.Abstract:Aiming at the pathogenesis characteristics of liver stasis and spleen deficiency,blood stasis and damp wet of hepatic fibrosis among chronic liver diseases,we have invented a new formula,Baoganning,which has the functions to relieve stagnancy of the liver Qi,reinforce Qi and invigorate the spleen,promote blood circulation to eliminate stasis,soften and resolve hard masses,concurrently clear up toxic heat and dampness.Clinic and experimental research have confirmed the efficacy of Baoganning in treating liver fibrosis.The main mechanisms are to decrease the inflammation,suppress the proliferation and promote the apoptosis of hepatic stellate cells by inhibiting the expression of nuclear factorκB.Leptin is a newly discovered cytokine which promotes liver frbrosis.Currently research on the molecular mechanism of leptin in promoting liver fibrosis has just started overseas and in China there are few publications in this field.Research on the signal transduction mechanism is insufficient and the existing studies are limited to the exsomatized cells.To the author’s knowledge there is no open literature about vivo experiments.Therefore we believe research on signal transduction mechanism of leptin to stimulate the HSCs to proliferate in vivo and in vitro has great significance in understanding the mechanisms of liver fibrosis.Investigations on effects of Chinese medicine Baoganning on leptin,leptin-receptor and its signal transduction are helpful in finding the anti-fibrosis working principles.It will also provide the theoretical basis for Baoganning to prevent and treat the liver fibrosis.Objectives and purposes:to investigate the molecular mechanism of anti-liver-fibrosis treatment with Baoganning,thus providing a theoretical foundation for applying Baoganning to the clinic,and establishing a solid theoretical platform for developing the traditional Chinese medicine on anti-liver fibrosis.Methods:The fibrosis in the rats model was induced by multiple factors.And Baoganning consisting of Chinese herbs Huangqi,Taoren,Danshen,Huangqin,Biejia, etc was given to the rats.The effects of Baoganning on fibrosis were studied.Fifty Wistar rats were randomly divided into normal group（A）,model group（B）, Colchicine group（C）,high dose of Baoganning group（D）and medium dose of Baoganning group（E）.All rats except rats in group A were subcutaneously injected with 40%carbon tetrachloride in peanut oil with a dose of 0.5ml/100g according to the body weight once on the first day,then with a dose of 0.3ml/100g once every three days for six weeks.Meanwhile,they were fed with normal fodder.5%alcohol was used as drinking water.Rats in group A were subcutaneously injected with equal peanut oil and fed with normal fodder and water.Rats in group C,D and E were respectively given Colchicine（0.011mg/100g）,Baoganning（3.54g/100g）and Baoganning（1.77g/100g）per day for six weeks via gastrogarage.Rats in group A and B were served with distilled water at the same time.At the end of the sixth week, blood and tissue specimens were taken from all the rats.Some serum were used for analysis of aspartate transaminase（AST）and alanine transaminase（ALT）activities according to the direction of the kit and others for the analysis of total cholesterol （TC）,Triglyceride（TG）with an automatic biochemical machine and leptin（LP）by radioimmunoassay（RIA）.Some liver specimens were mixed in a 10%solution of formaldehyde in 0.1 mol/L phosphatobuffered saline,and embedded in paraffin. Five-micron thick sections were prepared to evaluate the degree of fibrosis under an optical microscope and analyze the expression of leptin（LP）,leptin receptor（OB-R） and signal transducer and activator transcription 3（STAT3）with immunohistochemistry（IH）methods and western blotting and analysis.As to statistical analysis,data were analyzed with the software of SPSS14.0.Quantitative data were presented as mean±SD and compared using one way ANOVA procedure.Results and Discussions:1.To observe the influence of Baoganning on symptoms,physical signs and indices（liver wet weight/100g body weight）of liver and spleen（spleen wet weight/100g body weight）in liver fibrosis rat model.Rats in model group displayed lassitude,thinness,anorexia and their stool was loose,the urine was yellow and less.The surface of livers in model group was not smooth,the colour was lightly red,the edge was blunt and the quality was hard.There were even some little tubercles on the liver surface.The spleens were obviously swollen.Compared with the rats in the model group,rats in the treatment groups were in better conditions.High and medium dosages of Baoganning and colchicines were effectual in improving weight（P＜0.01,P＜0.01,P＜0.05）,There was no major difference between high,medium dosages of Baoganning and colchicines（P＞0.05）,and all significantly decreased the indices of liver and spleen（P＜0.01 in both groups）.High dosages of Baoganning were better than colchicines（P＜0.05）.2.To observe the influence of Baoganning on liver morphology in liver fibrosis rat model.Under a microscope damaged lobules of liver degeneration,necrosis of liver cells, lots of inflammatory cells,collage fibers and even pseudolobules were observed for the model group,while for the treatment groups there were less damaged lobules of liver degeneration,necrosis of liver cells,inflammatory cells and collage fibers.But there were still incomplete pseudolobules in very few rats.3.The effects of Baoganning on serum hepatic function and lipid in liver fibrosis rat model.Compared with the normal group,levels of ALT,AST and AST/ALT in the model group went up sharply（P＜0.01）;Compared with the model group,levels of ALT and AST in treatment groups went down obviously（all P＜0.01）.The effects of a high dosage of Baoganning on decreasing the levels of AST were also more significant than colchicines（P＜0.05）;AST/ALT significantly was decreased with medium dosages of Baoganning（P＜0.05）.Compared with the normal group,levels of TC and TG were significantly decreased（P＜0.01）.Compared with the model group,levels of TC and TG with high and medium dosages of Baoganning went up significantly（P＜0.01）;The effects of high dosage Baoganning on increasing the levels of TC was greater than colchicines （P＜0.05）;Baoganning with high and middle dosages was also more effective than colchicines in increasing the levels of TG（P＜0.05）. 4.The influence of Baoganning on serum leptin in liver fibrosis rat model.Compared with the normal group,levels of leptin in the model group went up noticeably（P＜0.01）;Compared with the model group,levels of leptin went down obviously（P＜0.01,P＜0.01,P＜0.05）for both Baoganning and colchicines groups with high and middle dosages.5.The effects of Baoganning on the expression of Leptin and its signal pathway in liver tissue in fibrosis rat model.Only a mild positive staining of leptin was found at central vein and Disse’s areas but none at hepatocytes on sections of normal controls,whereas on sections of the model group,the positive staining was seen at interstitial cells,inflammatory cells, impaired hepatocytes as well as normal hepatocytes.Fibrous c septa were only slightly stained.In the treatment groups the positive staining cells decreased and the staining was evidently light.Normal liver tissue hardly expressed OB-Rb or only a mild positive staining was found at portal vein,central vein and portal tract.Whereas in the model group OB-Rb mostly was located in the portal tract,around sinusoid and fibrotic septa.Expression of OB-Rb exhibited as brown particles predominantly in cytoplasm or cell membrane.A few positive expressions were found in nucleus.Contrast to the model group,the positive staining cells and the staining degree in the treatment groups were not obviously changed.Normal liver tissue hardly expressed Stat3,only a mild positive staining was found at matrix in portal tract and at cytoplasm in interstitial cells,whereas in the model group Smad4 dominated.It was detected in interstitial cells at fibrous septa,portal tract, portal vein,central vein and around sinusoid,especially at fibrous septa.In the treatment groups the number of positive staining cells decreased,mainly expressed at portal tract,portal vein and central vein. Statistical data analysis indicated that the expression level of leptin,OB-Rb and Stat3 were increased significantly in the model group（P＜0.01）.Compared with the model group,the Immunostaining score of leptin in high and middle dosage Baoganning,colchicines groups was remarkably decreased（P＜0.01 in both groups）, Stat3 was also remarkably decreased（P＜0.01,p＜0.051 P＜0.05）.OB-Rb was not noticeably decreased（P＞0.05 in all treatment groups）.6.The effects of Baoganning on the expression of OB-Rb and JAK2-STAT3 signal pathway in liver tissue in fibrosis rat model.The expression of OB-Rb,JAK₂,STAT₃ and the activity of phosphor-JAK₂, phosphor-STAT₃ were detected by Western blotting analysis.All of these in normal liver tissue were barely detected,whereas in the model group expressed strongly. Compared with the model group,the expression of them in all treatment groupsConclusions:Baoganning can inhibit the process of liver fibrosis in rats model, the mechanisms are as follows.1.Baoganning can improve the life quality through improving the spirits, increasing the body weight,food and water intake and decreasing the indices of liver and spleen2.Morphological observation shows Baoganning can improve the damaged structure of heptocyte,decrease the fibrosis tissue.3.Baoganning can significantly decrease the levels of serum ALT,AST and increase the levels of TC and TG.4.Baoganning can reduce the expression of Leptin,OB-Rb and its signal transduction molecule JAK2 and STAT3 in the liver tissue,so the signal transduction pathway of leptin is inhibited by the EHAWE therapy.In conclusion,Baoganning has the complicated pharmacological action on preventing the liver fibrosis.It has multi-way and multi-target anti-fibrosis mechanisms.One of the molecular mechanisms is to inhibit the expression of leptin and its signal transduction which is concerned to inhibit the inflammation,protect the liver cell,decrease the waterfall action of the cytokine and inhibit the start of the fibrosis.更多还原