探討黃連解毒湯與柴胡舒肝湯於HepG2細胞及三個非酒精性脂肪肝小鼠模式之作用

Studies on the effects of Hwang-Lian-Jiee-Dwu-Tong and Chir-Hwu-Shu-Gau-Tong in HepG2 cells and three mouse non-alcoholic fatty liver disease models

翁啟書(Ci-Shu Wong)

指導教授 ： 呂紹俊

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/碩士(2008年)

https://doi.org/10.6342/NTU.2008.02815

摘要

脂肪肝，肝細胞含脂肪重量達5%以上時稱之。近年來國內成年人口脂肪肝盛行率高達26-34%。脂肪肝可分為酒精性及非酒精性，酒精性脂肪肝起因於飲酒過量，非酒精性脂肪肝則多與肥胖、糖尿病及代謝疾病等相關。近來研究顯示，有相當比例非酒精性脂肪肝會發展為肝纖維化、肝硬化甚至是肝癌。然而目前臨床上尚無令人滿意的藥物，這使得治療非酒精性脂肪肝之藥物開發有相當的空間。
本實驗室與順天堂藥廠合作，將中醫書上記載具有保肝功能之中草藥篩選可降低細胞三酸甘油酯的中草藥，我們篩選出其中兩種複方—黃連解毒湯及柴胡舒肝湯，並探討其用於治療非酒精性脂肪肝之可能性。
首先我們證實在LXR agonist—T0901317存在與否，此兩複方皆可降低HepG2細胞中三酸甘油酯含量，而以RT-PCR及real-time RT-PCR偵測HepG2中脂質代謝相關基因的mRNA後，我們發現此兩複方皆會降低SREBP-1c、FAS及ACC之mRNA表現量，因此推論它們皆藉由抑制SREBP-1c進而影響FAS及ACC的轉錄。之後我們更以Western-blot發現此兩複方皆降低細胞中FAS蛋白質含量，顯示其具有抑制脂肪酸合成的功效。
接下來我們以十週大的C57BL/6小鼠進行動物實驗，將之分為五組，分別為T0901317、T0901317 +柴胡舒肝湯、lipogenic diet (35% cornstarch +35% sucrose)、lipogenic diet+柴胡舒肝湯及control組。飼養3週後，由肝組織切片觀察，T0901317組並無脂肪肝產生，但出現肝臟血管阻塞情況，而柴胡舒肝湯在此動物模式下可舒緩肝臟血管阻塞情況。然而lipogenic diet組肝組織的三酸甘油酯及總膽固醇含量相較於control組明顯上升，伴隨血漿中AST濃度被提升，並由肝組織切片發現以macrovesicular steatosis型式為主的脂肪肝。柴胡疏肝湯在此動物模式下可降低血漿中ALT活性，並發現其可降低肝臟被提升的FAS蛋白質含量，但尚無減輕肝臟脂肪堆積的情況。
之後我們續以ob/ob小鼠為動物模式，將之分為兩組，分別為黃連解毒湯及control組，飼養2週後，control組肝組織三酸甘油酯約為前一次C57BL/6小鼠實驗control組的三倍，而總膽固醇則約為兩倍，血漿中ALT及AST活性也同時較高，肝組織切片則觀察到較嚴重microvesicular steatosis型式的脂肪肝形成
，且有發炎現象產生。而黃連解毒湯在此動物模式下可明顯降低血漿中三酸甘油酯及AST活性，並減緩肝組織發炎現象，但無減輕肝臟脂肪堆積。
細胞實驗與動物實驗結果不甚一致，在論文中討論可能的原因及如何規劃更適合的動物模式。本篇論文所建立的研究脂肪肝之動物模式，將可成為本實驗室日後在脂肪肝研究上的參考。

關鍵字

非酒精性脂肪肝 ； 黃連解毒湯 ； 柴胡舒肝湯 ； HepG2細胞

並列摘要

Fatty liver, is called when the fat content of liver cell is more than 5%. The prevalent rate of fatty liver is around 26% to 34% of adult in Taiwan. Fatty liver can be divided into alcoholic and non-alcoholic types. Alcoholic fatty liver is caused by alcohol abuse, and non-alcoholic fatty liver disease (NAFLD) is usually associated with obesity, type II diabetes, metabolic syndrome, etc. Recent studies suggested that some NAFLD may develop into liver fibrosis, cirrhosis or even hepatocellular carcinoma. At present, there is no satisfactory medicine for treating NAFLD. Therefore, it makes developing of medicines for treatment of NAFLD more important.
Our lab has the opportunity to collaborate with Sun Ten pharmaceutical company to identify Chinese herbal medicines that have potential to be used to treat NAFLD. Two of the Chinese herbal medicines—Hwang Lian Jiee Dwu Tong (HLJDT) and Chir Hwu Shu Gau Tong (CHSGT) which we found could decrease HepG2 cellular triglyceride. Therefore, HLJDT and CHSGT were selected to study on their potential in treating NAFLD.
First, we found that in the present or absence of LXR agonist—T0901317, both HLJDT and CHSGT could decrease cellular triglyceride in HepG2. The mRNA levels of lipid metabolism related genes in HepG2 were analyzed by RT-PCR and real-time RT-PCR. The results showed that both of the two Chinese herbal medicines decreased SREBP-1c, FAS and ACC mRNA levels. The decrease of FAS in HepG2 was also shown by Western-bolt. The data suggest that HLJDT and CHSGT may decrease the transcription of FAS and ACC through suppressing SREBP-1c.
Animal experiment was carried out to test the effect of CHSGT on liver fat using 10-week old C57BL/6 mice. Mice were divided into five groups: control, T0901317 (by i.p.), T0901317 +CHSGT (by i.p. and gavage), lipogenic diet and lipogenic diet +CHSGT (by gavage). After three weeks of treatment, histological analysis showed that T0901317 group did not have hepatic steatosis but showed blood vessel congestion. Relief of blood vessel congestion was observed in the liver in CHSGT treated mice. When compared with control group, the contents of hepatic triglyceride and cholesterol and plasma AST in lipogenic diet group are apparently higher. Histological analysis showed that the liver of lipogenic diet group had hepatic macrosteatosis. And CHSGT could decrease the levels of plasma ALT, and reduced the elevated FAS protein in the liver, but unable to reduce hepatic steatosis.
Another animal experiment was carried out using 4-week old ob/ob mice. The ob/ob mice were divided into control and HLJDT (by gavage) groups. After two weeks of treatment, the hepatic triglyceride and cholesterol of control group were approximately three times and two times, respectively, as that of the control group in C57BL/6 mice. The levels of plasma AST and ALT were also higher in the control ob/ob mice. HE-stained liver specimens showed that the ob/ob mice had severe hepatic microsteatosis with inflammation. HLJDT could apparently decrease the level of plasma triglyceride and AST, and release inflammation, but unable to reduce hepatic triglyceride.
The results of the animal studies were not in accordance with cell culture studies; however, the reasons for the inconsistent results were discussed. In addition, the animal models we used may provide our lab some useful references to study NAFLD.

並列關鍵字

non-alcoholic fatty liver disease ； Hwang-Lian-Jiee-Dwu-Tong ； Chir-Hwu-Shu-Gau-Tong ； HepG2 cell

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