酒精性肝病的中医证候及中药抗酒精性肝纤维化的作用机制研究

【作者】 郑琪；

【导师】 李志红；

【作者基本信息】 北京中医药大学 ， 中医内科学， 2012， 硕士

【摘要】 目的：酒精性肝病(Alcoholic liver disease, ALD)是由于长期大量饮酒导致中毒性肝损伤及一系列病变,是临床的常见病。本研究运用流行病学的方法和原则,探讨酒精性肝病的中医证候特点的分布,及其与临床资料的相关性。通过大鼠酒精性肝纤维化模型,观察中药复方对酒精性肝纤维化的影响,探讨中药抗肝纤维化作用机制。方法：1.理论研究：一部分总结了中医对酒精性肝病病因病机的认识及中医药治疗酒精性肝病的临床研究,另一部分论述了酒精性肝纤维化的发病机制。2.临床研究：应用统计软件分析患者的临床信息,运用因子分析的方法,观察酒精性肝病的中医证候特点的分布情况,以及酒精性肝病的影响因素。3.实验研究：将雄性Wistar大鼠随机分为2组：空白组和模型组。采用“白酒、植物油、吡唑”混合液灌胃法制备酒精性肝纤维化动物模型,造模时间为16周。模型组造模结束后,随机分为5组,即模型组、模型自逆组、阳性药治疗组、高剂量慢肝消治疗组、低剂量慢肝消治疗组。治疗组治疗3周后,取血和肝组织,检测血清中谷草转氨酶、谷丙转氨酶、白蛋白、总蛋白、Ⅰ型胶原、层粘连蛋白、透明质酸、Ⅰ型前胶原氨基端前肽、Ⅰ型胶原交联羧基末端肽、Ⅲ型前胶原蛋白、纤维结合蛋白的含量,肝组织行HE染色、Mallory染色及PCNA、Ⅰ、Ⅳ型胶原、LN免疫组化染色。采用SPSS18.0软件对血清学指标检测结果和病理图像分析结果进行统计学分析。结果与结论：1.临床研究：本次收集的病例中,男性与女性的比例为6：1,年龄在35-44岁和45-54岁这两个年龄组,日饮酒量80-119g／d,饮酒年限在10-19年,GGT在50-89u／L时,酒精性肝病的患者居多。酒精性肝病的患者证侯分型以肝气郁滞、湿热内蕴、肝郁脾虚、痰湿内阻及痰瘀互结为主,其中肝郁脾虚及湿热内蕴所占的比例最多。年龄、饮酒年限、AST/ALT值对酒精性肝病中医证候特点的分布有影响,且有显著性差异(p<0.05)。而体重指数、日饮酒量对酒精性肝病中医证候的分布无影响。初步表明酒精性肝病的中医证候特点与肝脏的损伤情况密切相关。2.实验研究：模型组大鼠,血清中谷丙转氨酶、谷草转氨酶、Ⅰ型胶原、层粘连蛋白、透明质酸、Ⅰ型前胶原氨基端前肽、Ⅰ型胶原交联羧基末端肽、纤维结合蛋白的含量显著升高(p<0.05或p<0.01),血清中总蛋白、白蛋白、Ⅲ型前胶原蛋白无显著变化(P>0.05)。肝组织内大量胶原沉积,呈现典型肝纤维化病理特点。药物治疗组大鼠血清中谷丙转氨酶、谷草转氨酶、Ⅰ型胶原、透明质酸、Ⅰ型前胶原氨基端前肽、Ⅰ型胶原交联羧基末端肽的含量显著降低(p<0.05或p<0.01)。肝组织内胶原大量减少,肝小叶结构基本完整,未见假小叶。免疫组织化学结果表明,模型组大鼠肝组织PCNA免疫组化染色,可见大量增殖的细胞核,Ⅰ型胶原、Ⅳ型胶原及层粘连蛋白阳性反应颗粒显著增多,药物治疗后大鼠肝组织PCNA免疫组化染色,可见肝细胞核增殖减少,Ⅰ型胶原、Ⅳ型胶原及LN阳性反应颗粒显著减少。说明慢肝消有保护细胞膜的作用,能抑制细胞核的过度增殖。可以有效的作用于Ⅰ型胶原、Ⅳ型胶原、透明质酸、Ⅰ型前胶原氨基端前肽、Ⅰ型胶原交联羧基末端肽及层粘连蛋白等,从而抑制细胞外基质的沉积,达到抑制肝纤维化的目的。更多还原

【Abstract】 Objective: Alcoholic liver disease (ALD) is the toxic liver injury and a serious of diseases caused by drinking for long time, which is the clinical common disease. By means of the methods and doctrines of Clinical Epidemiological Research, we aim at the investigation of the characteristics of traditional Chinese medical syndrome distribution of alcoholic liver disease, and its correlation with clinical data. To explore the mechanism research of intervention target of traditional Chinese medicine compounds, we evaluated its effectiveness on alcoholic liver fibrosis by observing the rat models of alcoholic liver fibrosis.Methods:1. Theoretical study: Ⅰ summarized the cognition of the cause and mechanisms of alcoholic liver fibrosis by the angle of traditional Chinese medicine, and the clinical research on the Chinese medicine treatment of ALD. On the other hand, I expounded the pathogenesis of ALD by the angle of western medicine.2. Clinical investigation: We use statistical software to analyse clinical information; use factor analysis method to analyse and discuss the TCM Hou distribution of ALD, and the influencing factors.3. Experimental research: Divided the male Wistar into two groups randomly: the blank control group and model group. The rats of the model group were fed of the compound in the proportion of Distilled spirit、Vegetable oil、Qyrazole to make animal models of ALF, which cost used16weeks. When finished, the animal models were divided randomly into five groups:model group、model inverse group、positive drug treatment group、high dose Manganxiao treatment group and low dose Manganxiao treatment group. After three weeks in treatment group, we picked the serums respectively to detect the content of aspertate aminotransferase (AST), alanine transaminase (ALT), albumin, total protein, Ⅰ collagen type, laminin protein(LN), hyaluronic acid, Ⅰ type collagen amino end before before Ⅰ peptides, collagen type cross-linking end carboxyl peptide, Ⅲ type collagen protein and fiber combination before protein; liver tissues were embedded generally by olefins, sliced up and dyed by HE and Mallory. And finally, we took statistics that resulted from the serology test with the software SPSS18.0, and the pathological image to get the mean and make unit factor analysis of variance. Result and Conclusion:1. Clinical research: in the collection of cases, the male and female ratio is6:1at the age of35to44years and45to54years old. In the two age groups, Daily alcohol consumption is80-119g/d, drinking fixed number of year is10-19years. When GGT in50-89u/L, incidence of alcoholic liver disease is the highest. Syndrome type of alcoholic liver disease patients is mainly Stagnation and Spleen Deficiency, Dampness and Heat, phlegm and blood stasis, Phlegm and Dampness Resistance, and1iver and kidney deficiency. The proportion of Stagnation and Spleen Deficiency, Dampness and Heat is the most. Age, drinking fixed number of year, AST/ALT value to distribution characteristics of the alcoholic liver disease of traditional Chinese medical syndrome have influence, and have significant difference (p<0.05). And body mass index, daily alcohol consumption to syndromes of alcoholic liver disease had no effect on the distribution. Preliminary showed that the TCM syndrome of alcoholic liver disease is closely related with liver damage. Experimental study: content of glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminease(AST), I collagen type, Laminin protein, Hyaluronic acid, I before the collagen amino type before peptides, I collagen type cross-linking end carboxyl peptide, Fibronectin (FN) in serum of modelset rats is significantly rised(p<0.05or p<0.01). Total protein, albumin, III before collagen type of serum have no significant change (p>0.05). In the liver tissue, a lot of collagen is deposited, it has a characteristic of liver f ibrosis pathological characteristics, content of glutamic-pyruvic transaminase (ALT), glutamic-oxalacetic transaminease(AST), I collagen type, Laminin protein, Hyaluronic acid, I before the collagen amino type before peptides, I collagen type cross-linking end carboxyl peptide, Fibronectin (FN) in serum of Medication Group rats is significantly reduced (p<0.05or p<0.01). In the liver tissue, collagen is decreased greatly, the structure of the hepatic lobule is essential complete, has not seen the false flocculus. Immunohistochemistry results shows, the liver tissue of modelset rats after PCNA dyeing, we can see a large number proliferous nucleus, positive reaction particles of I collagen type and IV collagen type and Laminin protein(LN) is significantly increased.After medical treatment, the liver tissue after PCNA dyeing, we can see proliferous nucleus is reduced, positive reaction particles of I collagen type and IV collagen type and Laminin protein(LN) is significantly decreased. This shows that the Manganxiao can protect cytomembrane, it can inhibit excessive prol iferation of nucleus, i t can effective impact in I col lagen type, IV collagen type, Laminin protein, Hyaluronic acid, I before the collagen amino type before peptides, I collagen type cross-linking end carboxyl peptide Fibronectin (FN), so it can inhibit the deposit in the extracellular matrix, thereby it can inhibit Liver fibrosis.更多还原