木苏丸防治二甲基亚硝胺诱导的大鼠肝纤维化的实验研究

目的　观察木苏丸防治大鼠肝纤维化的疗效并探讨其作用机制。方法　采用 1%二甲基亚硝胺 (DMN) 10mg/ (kg·d)腹腔注射复制大鼠肝纤维化模型 ,木苏丸治疗组予木苏丸3.12 5 g/ (kg·d)灌胃 ,观察肝功能、血透明质酸 (HA)、超氧化物歧化酶 (SOD)、丙二醛 (MDA)含量变化及基质金属蛋白酶 - 2 (MMP - 2 )、金属蛋白酶组织抑制因子 - 1(TIMP - 1)、α -平滑肌肌动蛋白 (α -SMA)在肝组织中的表达 ;并观察肝脏显微结构改变。结果　木苏丸治疗组与模型对照组比较 ,碱性磷酸酶 (ALP)、MDA、HA明显下降 (P <0 .0 1和P <0 .0 5 ) ,血清白蛋白(ALB)、SOD明显增高 (P <0 .0 1) ,TIMP - 1表达下降 (P <0 .0 5 ) ,MMP - 2的表达无明显改变(P >0 .0 5 ) ;光镜下肝纤维化程度减轻。结论　木苏丸具有保肝、延缓肝纤维化形成的作用 ,其作用机制可能与其抗氧化及其下调TIMP - 1的表达有关     

Upregulation of TNF-α mRNA in hepatic fibrosis rats induced by dimethylnitrosamine

Objective：To observe the expression level of TNF-α mRNA in rats with hepatic fibrosis induced by dimethylnitrosamine （DMN） and to explore its relationship with collagen metabolism and its diagnostic value for hepatic fibrosis.Methods： Twenty-five male Wistar rats were randomly divided into normal control group （n=10） and model group （n=15）. Model rats were induced by DMN for 4 weeks and at final stage were executed. TNF-α mRNA were detected by RT-PCR and the inflammatory necrosis and collagen deposition in hepatic tissue were observed by HE stain and Sirius red stain. The liver functions were determined by automatic biochemical analytic device. The serum marks of liver fibrosis, such as HA, LN and Ⅳ-C were measured with ELISA and RIA. Results： In this study, the rat model of liver fibrosis induced by DMN was successfully constructed. RT-PCR reveals that TNF-α mRNA expression in control group is lower than that of model group. The liver functions of model group were impaired compared with those of the control group （P〈0.01）. Semi-quantitive analysis revealed that TNF-α/β-actin of normal rats was 0.39±0.12, while 0.93±0.05 of model rats. The concentration of HA （434.44±98.81 vs 252.9±26.59 ng/ml, P〈0.01）, LN （70.67±6.32 vs 37.90±5.97 ng/ml, P〈0.01） and Ⅳ-C （79.39±10.52 vs 21.40±4.17 ng/ml, P〈0.01） were significantly increased in the model group as well. Changes of the indexes were similar to the pathological damage of the liver. Conclusion： The results suggested that activation of TNF-α in liver tissues may be the common pathogenic mechanism of liver fibrosis. TNF-α may be a useful index for the diagnosis of hepatic fibrosis which worthies further investigation.     

Sister chromatid exchange response of human diploid fibroblasts and chinese hamster ovary cells to dimethylnitrosamine and benzo(a)pyrene

In the search for relevant assays for mutagenicity testing, considerable attention has been given to the use of mammalian cells in vitro and the incorporation of metabolic activation in the protocol. Chinese hamster ovary (CHO) cells are commonly chosen as the target cells for cytogenetic tests because of their excellent growth characteristics and long lifespan in culture. However, there may be cellular factors affecting the uptake, metabolism, and repair of damage which are not the same in all cell lines. The response of CHO cells and three human diploid fibroblast strains (IMR-90, WI-38, S-3299) to benzo(a)pyrene (BP) and dimethylnitrosamine (DMN) were compared using sister chromatid exchange (SCE) analysis as a measure of genetic damage. For both BP and DMN the human cells and the CHO cells showed dose-response slopes that were significantly different from zero, except CHO cells treated with BP for 1 hr and S-3299 cells treated with DMN. Whereas human and CHO cells showed similar dose-responses to BP and the three human cell strains had similar dose-responses to BP and DMN, the dose-response of the human cells to DMN was statistically less significant than that of CHO cells. Reducing the duration of chemical treatment in CHO cells had no effect on the slope of the dose-response curves for BP or DMN. The observed differences between human and CHO cells may reflect differences in the fate of metabolic intermediates of DMN.     

虫草多糖逆转DMN诱导大鼠肝纤维化的作用及机制研究

目的：研究虫草多糖抗肝纤维化作用的机制。方法： 将SD大鼠随机分为正常组、模型组、虫草多糖组。采用二甲基亚硝胺诱导的大鼠肝纤维化模型。经过虫草多糖治疗4周后,大鼠肝脏用丽春红胶原染色观察大鼠胶原纤维沉积的变化,观察大鼠血清肝功能的变化,盐酸水解法检测肝组织羟脯氨酸含量,Envision 两步法测定肝组织Ⅳ型胶原含量和金属蛋白酶组织抑制因子2（TIMP-2）含量；酶图法检测肝组织金属蛋白酶2（MMP-2）活性,同时检测肝组织超氧化物歧化酶（SOD）活性和丙二醛（MDA）含量的改变。结果：镜下检查可见模型组大鼠肝脏胶原纤维间隔形成；血清肝功能指标异常,MMP-2含量降低；组织Hyp含量,Ⅳ型胶原含量,I型胶原蛋白表达,TIMP-2,MDA含量,均较正常组均显著增加,SOD活性下降,而虫草多糖组的上述指标较模型组均有不同程度的显著下降,使SOD活性升高,MMP-2含量升高。结论：虫草多糖有显著的抗肝纤维化作用,其机制与促进胶原降解和抗脂质过氧化作用有关。     

黄芪甲苷抗二甲基亚硝胺诱导肝纤维化大鼠效应研究

目的探讨黄芪甲苷对二甲基亚硝胺(Dimethylnitrosamine,DMN)诱导肝纤维化大鼠模型的作用。方法选取27只雄性Wistar大鼠,随机分为正常组、模型组和黄芪甲苷组。模型组和黄芪甲苷组大鼠,采用腹腔注射DMN诱导肝纤维化模型,持续4周。于造模第3周开始,黄芪甲苷组在继续造模的同时予以黄芪甲苷干预,正常组和模型组给于等容量蒸馏水灌胃。4周末(即药物干预两周)处死全部大鼠,观察各组大鼠一般情况、肝组织病理学;检测各组大鼠血清肝功能水平、肝组织中羟脯氨酸(Hydroxypro鄄line,Hyp)含量;免疫组化法检测肝组织α-平滑肌肌动蛋白(α-SMA)蛋白表达情况。结果与正常组比较,模型组肝脏明显缩小,组织炎细胞浸润增加,胶原沉积明显增多,肝功能指标明显异常(P<0.01),提示纤维化形成。与模型组比较,黄芪甲苷可以显著改善肝脏病理,减轻肝脏胶原沉积及肝脏Hyp含量,降低血清ALT、AST、GGT水平及TBil含量,升高ALB含量,显著抑制肝组织α-SMA蛋白表达(P<0.05或P<0.01)。结论黄芪甲苷具有显著保肝降酶抗肝纤维化形成作用,其机制可能跟抑制肝星状细胞的活化相关。     

Loss of FGF21 in diabetic mouse during hepatocellular carcinogenetic transformation

Diabetes associated metabolic syndrome has been shown to be an independent risk factor for the development of hepatocellular carcinoma (HCC). Cirrhosis, in fact, was not always a prerequisite of HCC development and this might particularly apply to the metabolic abnormality associated HCC. This study was to investigate diabetes associated HCC and the potential role of FGF21 during carcinogenetic transformation of HCC. Dimethylnitrosamine (DEN) was used to induce HCC in the diabetic OVE26 mice. Pronounced damage characterized by steatohepatitis was found in the liver of diabetic mice. Steatohepatitis accompanied by constant cell proliferation and tumor cell growth were also found in the hepatic tissues of diabetic OVE26 mice when DEN being administrated. FGF21 protein level increased in liver tissues at an early stage along with steatohepatitis in diabetic OVE26 mice, but decreased in liver tissues later when HCC was developed. In addition, decreased FGF21 protein level was associated with cancerous hyper-proliferation and aberrant p53 and TGF-β/Smad signaling during HCC development. Loss of FGF21 may play an important role in HCC carcinogenetic transformation during metabolic liver injury in diabetic animals. The present finding calls attention to the need to control metabolic disorders associated with diabetes and may further develop a protective strategy against HCC.     

The hepatocarcinogenicity of diethylnitrosamine to rainbow trout and its enhancement by Aroclors 1242 and 1254

Rainbow trout (Salmo gairdneri) were fed diets containing 100 ppm Aroclor 1242 (AC42) or Aroclor 1254 (AC54) in combination with 1100 ppm diethylnitrosamine (DEN) for one year. The incidence of hepatocarcinomas was determined and compared with the incidence in trout fed 1100 ppm DEN alone. The two Aroclors dramatically enhanced tumor incidence from 10.2% in the positive controls (DEN alone) to 40.2% for AC42 and 21.6% for AC54. This is in contrast to previous results obtained when AC54 was fed concomitantly with aflatoxin B1 (AFB1), where a substantial inhibition of carcinogenesis was observed. The alteration of chemical carcinogenesis in trout by PCB, therefore, depends upon the carcinogen involved and is not a generalized effect.     

Relative extents of hydrogen-deuterium exchange of nitrosamines: relevance to biological isotope effect studies

Relative extents of base-catalyzed, hydrogen-deuterium exchange have been determined for a number of nitrosamines. Observed trends in the exchanges are discussed in terms of substitution, ring size and conformation. The relevance of the exchanges to deuterium isotope effects in carcinogenesis tests is discussed. Those compounds which give pronounced biological isotope effects undergo exchange only to a small extent. No biological isotope effect is found for compounds which undergo extensive exchange.     

茵陈蒿汤对二甲基亚硝胺与四氯化碳诱导的肝硬化大鼠模型凋亡相关基因影响的比较研究

目的：通过＂方-效-证＂相结合研究,明确茵陈蒿汤对二甲基亚硝胺（di methylnitrosamine,DMN）或四氯化碳（carbon tetrachloride,CCl4）两种造模方式所致的肝硬化模型大鼠的疗效特点。方法：分别采用DMN和CCl4建立大鼠肝硬化模型,以肝纤维化已经形成并向肝硬化发展的时期（CCl4造模8周后）及其肝硬化成型后（DMN造模4周后）作为干预治疗的切入点,横向比较茵陈蒿汤在不同模型中的药效。基因芯片技术检测肝脏基因表达情况。结果：在DMN模型中,随着造模时间的延长,模型大鼠血清丙氨酸氨基转移酶（alanineaminotransferase,ALT）、天冬氨酸氨基转移酶（aspartate aminotransferase,AST）、γ谷氨酰转移酶（gamma-glutamyl transferase,GGT）活性和总胆红素（total bilirubin,TBil）含量逐渐升高,均于4周时达到高峰（P〈0.01）;血清白蛋白（albumin,ALB）含量逐渐降低,4周时降至最低（P〈0.01）;与6周模型对照组比较,茵陈蒿汤组大鼠血清ALT、AST和GGT活性及TBil含量显著降低（P〈0.05或P〈0.01）,血清ALB含量显著升高（P〈0.05）。在CCl4模型中,茵陈蒿汤显著降低血清ALT、AST和GGT活性（P〈0.05）,但对血清TBil和ALB含量的改善没有显著作用。茵陈蒿汤能显著改善DMN大鼠肝脏病理改变,降低羟脯氨酸含量,但对CCl4模型则没有显著作用。基因芯片结果表明,在CCl4模型中,茵陈蒿汤显著抑制Fas、Bax和caspase-3的表达,促进Bcl-xL的表达,但不能抑制CCl4诱导的肝细胞凋亡,反而促使酪氨酸激酶受体上调了4.8倍。结论：茵陈蒿汤对DMN和CCl4两种大鼠肝硬化模型都表现出了疗效,但对DMN模型的干预尤其是降低肝组织羟脯氨酸含量、改善肝组织病理变化及调控凋亡基因等的作用显著优于CCl4模型。     

经典退黄三方抗二甲基亚硝胺诱导的大鼠肝纤维化的方证比较研究

目的：通过观察经典退黄三方茵陈蒿汤、茵陈五苓散和栀子柏皮汤对二甲基亚硝胺（dimeth—ylnitrosamine,DMN）诱导的大鼠肝纤维化模型的效应,探讨该模型的病机。方法：48只大鼠按体质量分层随机分为正常对照组、模型组、茵陈蒿汤组、茵陈五苓散组和栀子柏皮汤组,采用DMN腹腔注射4周的方法诱导大鼠肝纤维化模型。从造模第3周开始,各药物组在继续造模的同时予以相应的药物干预,正常对照组和模型组给以等量生理盐水。4周末结束实验,杀鼠取材,观察各组大鼠一般情况、肝组织病理学、羟脯氨酸（hydroxyproline,Hyp）含量及肝功能变化。结果：与正常对照组相比,模型组大鼠出现显著的肝损伤和肝纤维化,肝组织Hyp含量显著升高（P〈0．01）,肝功能显著异常（P〈0．01）。与模型组比较,茵陈蒿汤可显著改善肝功能（P〈0．05或P〈0．01）,显著改善肝组织病理改变,降低肝组织Hyp含量及肝脏胶原增生程度（P〈0．01）;茵陈五苓散可显著降低血清总胆红素含量（P〈0．01）,对肝组织病理影响不明显;而栀子柏皮汤显著改变肝脏病理,并对肝组织Hyp含量有降低作用。结论：茵陈蒿汤对DMN诱导的大鼠肝纤维化的治疗效果优于茵陈五苓散和栀子柏皮汤;DMN诱导的大鼠肝纤维化模型在其形成期的病机以“湿（瘀）热内蕴”为主,且湿（瘀）热并重,与茵陈蒿汤方证高度相关。