中草藥複方B預防二乙基亞硝胺及高油脂高膽固醇飼料所造成的非酒精性脂肪肝炎與肝臟纖維化

Abstract

非酒精性脂肪肝疾病是指非因飲酒過量而導致肝臟有超過5%的脂肪堆積在肝細胞中的慢性肝臟疾病，近年來國內成人患有非酒精性脂肪肝疾病的盛行率高達26-34%。而非酒精性脂肪肝疾病一般認為是不需要積極治療，但需要避免進一步演變成非酒精性脂肪肝炎。許多研究更顯示，非酒精性脂肪肝炎的患者有相當的比例會發展成肝纖維化、肝硬化甚至是肝癌。目前研究指出，給小鼠腹腔注射二乙基亞硝胺及餵食高油脂的食物，會藉由增加肝臟TNF-alpha與IL-6的表現造成肝臟發炎與纖維化的形成。本實驗的研究目的是想了解在我們研發的中草藥複方B，在給小鼠腹腔注射二乙基亞硝胺及餵食高油脂高膽固醇飲食造成的非酒精性脂肪肝炎與肝臟纖維化的動物模式中，是否具有預防的功效。 我們先前的研究發現，小鼠在高油脂高膽固醇飲食之下給予中草藥複方B具有預防脂肪肝的功效。而我們進一步地在這次動物實驗中評估複方B的抗肝臟發炎和纖維化的潛力。動物實驗使用3週大C57BL/6公鼠進行實驗，將之分為四組，控制組餵食正常飼料以及灌食滅菌水，另外三組實驗組皆餵食高油脂高膽固醇飼料及10%果糖水以及每2週腹腔注射一劑二乙基亞硝胺，其中B( 0 mg/kg)組每天灌食滅菌水，B(450mg/kg) and B(900mg/kg)組每天分別灌食相當於每公斤體重給予450與900毫克的複方B藥材乾重。飼養6週與12週實驗後，結果顯示灌食複方B不僅可顯著減少肝臟三酸甘油酯及總膽固醇含量，也能顯著降低血漿中AST和ALT活性。肝臟組織切片亦發現具有明顯改善脂肪油滴堆積、發炎反應及纖維化之情形。以RT-Q-PCR分析發炎細胞激素與免疫細胞入侵及肝纖維化指標之基因表現，結果顯示灌食複方B可減少TNF-alpha, IL-6, MCP-1, F4/80, AFP, alpha-SMA, TGF-beta 1以及Collagen type I的 mRNA 量。以免疫組織染色分析的結果也顯示灌食複方B可降低CD68及alpha-SMA的蛋白質表現。由這些結果顯示複方B在此動物模式下具有預防非酒精性脂肪肝、肝臟發炎與纖維化之效用。 我們也探討組合成B配方的3種中草藥單方萃取物(代號A14、A28及A50號)，是否對於油酸(Oleic acid, OA)所誘導的細胞三酸甘油酯以及發炎細胞激素基因表現存有抑制的功效。將HepG2 細胞同時處理油酸以及單方中草藥萃取物24小時，測量細胞內三酸甘油酯含量以及TNF-alpha與IL-6的mRNA表現，結果顯示A28與A50號單方中草藥萃取物可以降低油酸所增加的細胞三酸甘油酯以及發炎細胞激素的基因表現。 由動物實驗結果發現，B複方具有預防二乙基亞硝胺及餵食高油脂高膽固醇飼料造成的非酒精性脂肪肝、脂肪肝炎與肝臟纖維化之功效。後續的研究可以找出B複方的有效成分及作用的分子機制。

Nonalcoholic fatty liver disease (NAFLD) is a disease which is defined as more than 5% fat accumulation in hepatocytes of people who consumed limited amount of alcohol. The prevalence of NAFLD in Taiwan is about 26-34%. Non-alcoholic steatohepatitis (NASH) is more progressive form of NAFLD. Many studies suggest that NASH patients have a higher risk for liver fibrosis progression, the most common risk factor for cirrhosis and hepatocellular carcinoma. A recent study demonstrated that diethylnitrosamine (DEN) and high fat diet promote liver inflammation and tumorigenesis by enhancing TNF-alpha and IL-6 expression. Our previous study identified a CHM mixture-Mix B that prevents high fat/high cholesterol diet-induced NAFLD. The aim of this study is to test if the Chinese herbal medicines (CHM) mixture-Mix B can be used to prevent DEN and high fat/high cholesterol diet induced NASH and liver fibrosis. In this study, we evaluate if Mix B has anti-inflammatory and anti-fibrogenic potential in vivo. Three-week old male C57BL/6 mice were used in this study, they were divided into four groups. The control group was fed with chow diet and given distilled water by gavage. The three experimental groups were fed high fat/high cholesterol diet, 10% fructose water and intraperitoneally injected with DEN every 2 weeks, and the B( 0 mg/kg) was gavaged with normal saline, B(450mg/kg) and B(900mg/kg) were gavaged with 450 mg/kg and 900mg/kg Mix B, respectively, daily for 12 weeks. After 6 and 12 weeks of treatment, Mix B not only significantly lowered liver triacylglycerol (TG) and cholesterol but also significantly reduced plasma AST and ALT activities. Histological analyses showed that Mix B improved liver steatosis, inflammation and fibrosis significantly. Mix B also decreased mRNA levels of pro-inflammatory cytokines, macrophage infiltration and fibrosis marker genes, such as TNF-alpha,IL-6, MCP-1, F4/80, AFP, alpha-SMA, TGF-beta 1 and Collagen type I. Immunohistochemistry analyses showed that mice fed with Mix B expressed less CD68 and alpha-SMA in the liver. The results suggest that Mix B could inhibit liver fat accumulation, inflammation and fibrosis. We further tested if the three components of Mix B ( code number: A14、A28、A50) can reduce oleic acid-induced expression of proinflammatory cytokines in HepG2. Cells were treated with the CHMs and oleic acid for 24h, and cellular TG and mRNA levels of TNF-alpha and IL-6 were determined. Results show that the CHMs (code number: A28 and A50) prevented oleic acid-induced increase of cellular TG and expression of TNF-alpha and IL-6 mRNAs. Taken together, our results show that Mix B is effective in prevention of DEN and high fat/high cholesterol diet-induced NASH and liver fibrosis. Further studies on the identification and isolation of the functional component(s) in Mix B, and the underline molecular mechanisms are deserved.