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。     

Phyllanthus amarus: ethnomedicinal uses, phytochemistry and pharmacology: a review

Ethnopharmacological relevance

Phyllanthus amarus Schum. &; Thonn. belongs to the family Euphorbiaceae is a small herb well known for its medicinal properties and widely used worldwide. P. amarus is an important plant of Indian Ayurvedic system of medicine which is used in the problems of stomach, genitourinary system, liver, kidney and spleen. It is bitter, astringent, stomachic, diuretic, febrifuge and antiseptic. The whole plant is used in gonorrhea, menorrhagia and other genital affections. It is useful in gastropathy, diarrhoea, dysentery, intermittent fevers, ophthalmopathy, scabies, ulcers and wounds.

Materials and methods

The present review covers a literature across from 1980 to 2011. Some information collected from traditional Ayurvedic texts and published literature on ethanomedicinal uses of Phyllanthus amarus in different countries worldwide.

Results

Phytochemical studies have shown the presence of many valuable compounds such as lignans, flavonoids, hydrolysable tannins (ellagitannins), polyphenols, triterpenes, sterols and alkaloids. The extracts and the compounds isolated from P. amarus show a wide spectrum of pharmacological activities including antiviral, antibacterial, antiplasmodial, anti-inflammatory, antimalarial, antimicrobial, anticancer, antidiabetic, hypolipidemic, antioxidant, hepatoprotective nephroprotective and diurectic properties.

Conclusion

The present review summarizes information concerning the morphology, ecology, ethnopharmacology, phytochemistry, biological activities, clinical applications and toxicological reports of P. amarus. This review aims at gathering the research work undertaken till date on this plant in order to provide sufficient baseline information for future works and commercial exploitation.     

Factors influencing the microsome- and mitochondria-catalyzed in vitro binding of diethylnitrosamine and N-nitrosopiperidine to deoxyribonucleic acid.

[¹⁴C]Diethylnitrosamine ([¹⁴C]DEN) and [¹⁴C]N-nitrosopiperidine ([¹⁴]NPiP) bind covalently to calf thymus DNA in an in vitro incubation system containing rat liver microsomes. The reaction is NADPH-dependent. Pretreatment of the animals with phenobarbital (PB) enchances the binding of both DEN and NPiP to DNA, whereas the binding of DEN to DNA decreases after 3-methylcholanthrene pretreatment. The PB effect, as observed from the binding of DEN to DNA. is more pronounced in young rats than in the older animals. Addition of cytosol to the incubation system enhances the binding of DEN 3- to 4-fold and the binding of NPiP 2- to 3-fold. Addition of mitochondria to the incubation system increases the binding of [¹⁴C]DEN only slighty. but increases the binding of NPiP more than 5-fold. Addition of mitochondria has no effect on the binding of [¹⁴C]dimethylnitrosamine ([¹⁴C]DMN). Mitochondria alone markedly catalyze the binding of NPiP to DNA. Addition of benzylamine. which is a substrate of mitochondrial monoamine oxidase as well as an inhibitor of DMN-demethylase, inhibits the binding of NPiP catalyzed by microsomes and microsomes plus mitochondria.     

熊胆对亚硝酸钠的清除作用及其对二甲基亚硝胺在体外合成的阻断作用

为了研究熊胆的防癌作用，进行了亚硝酸钠清除实验和对二甲基亚硝胺合成阻断实验。结果发现熊胆能够有效地清除亚硝酸钠，并阻断二甲基亚硝胺在体外合成。     

二甲基亚硝胺对大鼠肝细胞核膜流动性的影响

用荧光标记的方法观察了致癌物二甲基亚硝胺对大鼠肝细胞核膜流动性的影响。结果表明：二甲基亚硝胺能明显改变核膜的流动性，并呈现一定的剂量一效应关系。提示：二甲基亚硝胶能通过改变核膜流动性，削弱核膜对遗传物质的保护功能，从而增强其致癌作用。     

Differential effect of chronic ethanol consumption by the rat on microsomal oxidation of hepatocarcinogens and their activation to mutagens

The effect of chronic ethanol consumption by rats on hepatic microsomal activation of the hepatocarcinogens dimethylnitrosamine (DMN) and 2-acetylaminofluorene (2-AAF) was investigated. There was a marked increase in the rate of the oxidative demethylation of DMN and its activation to a mutagen by microsomes following ethanol intake. N- and C-hydroxylation of 2-AAF were measured at substrate concentrations ranging from 2 to 70 microM. The ratio of formation of N-hydroxy-2-acetylaminofluorene to C-hydroxy-2-acetylaminofluorenes increased with decreasing substrate concentration, suggesting enhanced carcinogenic potential of 2-AAF with diminishing levels of carcinogen. Kinetic analysis indicated that N-hydroxylation as well as 7-, 5- and 3-hydroxylation of 2-AAF do not follow Michaelis-Menten kinetics. In contrast to the marked inductive effect of ethanol consumption on the metabolic activation of DMN, only a minimal random effect on the N-hydroxylation of 2-AAF was demonstrable in two separate experiments. Furthermore, N-hydroxylation of 2-AAF by microsomes from control and ethanol-treated rats followed similar kinetics. While ethanol consumption enhanced the mutagenic activation of DMN by hepatic microsomes, no such effect of ethanol consumption on the conversion of 2-AAF to a mutagen was observed. The data indicate that chronic ethanol consumption does not have a general inductive effect on the microsomal activation of hepatocarcinogens.     

Increased mitochondrial stress and modulation of mitochondrial respiratory enzyme activities in acetaminophen-induced toxicity in mouse macrophage cells

Overdose of acetaminophen (APAP) causes tissue injury particularly in the liver. However, the precise mechanism of APAP toxicity is not clear. Glutathione (GSH) depletion and oxidative stress are believed to be the main cause of APAP toxicity. The role of macrophages in APAP-induced tissue injury is controversial. Using mouse macrophage J774.2 cells, we recently demonstrated that like in animal models, APAP reduces GSH pool and alters GSH metabolism by increasing the production of reactive oxygen species (ROS). In the present study, we show that APAP-induced cytotoxicity and apoptosis in macrophages are associated with increased mitochondrial metabolic and oxidative stress, alterations in the mitochondrial membrane potential and activities of the respiratory enzyme complexes. APAP treatment also altered ROS/NO production and inhibited the expression of COX-2 and iNOS in LPS-stimulated macrophages. Electron microscopic studies also confirmed morphological changes associated with apoptosis at the lower dose of APAP, while at the higher dose late apoptosis/necrotic changes were visible. These results suggest that mitochondrial metabolic and oxidative stress are the main causes of cytotoxicity and cell death in APAP treated macrophages. The study may have long term implications to better understand the role of macrophages in the toxicology and pharmacology of APAP.     

Antifibrotic effects of CGX,a traditional herbal formula,and its mechanisms in rats

Aim

CGX is a modification of a traditional herbal medicine for “liver cleaning,” which is used to treat various chronic liver disorders in oriental clinics. This study investigated the antifibrotic effects and associated mechanisms of CGX.

Materials and methods

Liver fibrosis was induced in rats by dimethylnitrosamine (DMN; 10 mg kg⁻¹, ip) injection on 3 consecutive days per week for 4 weeks. CGX (100 or 200 mg kg⁻¹, po) was administrated once a day for 4 weeks. Three cell lines (HepG2, RAW 264.7, and HSC-T6) were used to examine its mechanisms.

Results

CGX treatment dramatically ameliorated the change in liver and spleen weight and serum albumin (p < 0.01), aspartate transaminase (p < 0.01), alanine transaminase (p < 0.01), alkaline phosphatase (p < 0.01), and total bilirubin (p < 0.01) levels. Histopathologically, CGX administration decreased necrosis, inflammatory cell infiltration, and collagen accumulation. The antifibrotic effects of CGX were confirmed from hydroxyproline determination and the reduction in the numbers of activated hepatic stellate cells. In addition, antioxidant proteins, glutathione content, and glutathione peroxidase, catalase, and superoxide dismutase activities were maintained in the CGX-treated groups compared with the DMN group. CGX downregulated fibrosis-related genes (inducible nitric oxide synthase, tumor necrosis factor-alpha, transforming growth factor-beta, connective tissue growth factor, and platelet-derived growth factor-beta) and decreased the protein levels of profibrotic cytokines (transforming growth factor-beta and platelet-derived growth factor-beta) in liver tissues. In the cell line-based studies, CGX showed supportive effects, such as the protection of hepatocytes from CCl₄-toxicity, inhibition of NO production in RAW 264.7 cells, and inactivation of hepatic stellate cells.

Conclusion

These results demonstrated the antifibrotic effects of CGX and the corresponding mechanisms associated with sustaining the antioxidative system and inhibiting hepatic stellate cell activation via the downregulation of fibrogenic cytokines.     

华支睾吸虫与二甲基亚硝胺诱发动物肝癌的初步实验研究

本文报道用华支睾吸虫与二甲基亚硝胺诱发动物肝癌的实验结果。Ａ组每只金地鼠通过胃管人工感染华支睾吸虫囊蚴，３０ｄ后当鼠粪中查见华支睾吸由卵时开始自由饮服０．００２５％二甲基亚硝胺溶液１７周，结果１１只鼠中４只诱发肝癌，１只诱发肝胆管癌和５只肝硬变。Ｂ组每只鼠仅自由饮服０．００２５％二甲基亚硝胺溶液１７周，１５只鼠中３只诱发肝癌，５只肝硬变。Ｃ组每只鼠仅通过胃管人工感染华支睾吸虫囊蚴，不饮服二甲基亚硝胺，１２只鼠中仅６只发生肝硬变。空白对照的１５只鼠均未发现病变。结果显示金地鼠饮服二甲基亚硝胺可引起肝细胞和胆管细胞恶变，而华支睾吸虫在肝内的寄生，可促使肝癌的发生。