桑葚萃取物促進肝癌細胞凋亡與自噬作用並預防二甲基亞硝胺造成之肝癌形成

肝細胞癌俗稱肝癌，為台灣最常見的癌症之一，其常見危險因子除包括病毒性肝炎、黃麴毒素與過量的飲酒，這些危險因子會造成慢性肝炎，進而發展至纖維化、肝硬化與肝癌。為了降低肝癌的發生，研發天然的護肝食品是可不容緩的。桑葚已經被研究為一種機能性食品，其富含花青素、類黃酮化合物和酚酸，這些化合物具有抗發炎及抗氧化的特性，其中預防癌症這功能已被應用在胃癌、黑色素瘤與血癌之外，還能用來預防酒精與四氯化碳的肝損傷。本研究旨在桑葚萃取物是否能抑制肝癌細胞並且能預防肝癌形成，首先將肝癌細胞HepG2 以及 Hep3B處理桑葚多酚 (mulberry polyphenol extracts, MPE)之後，發現 MPE可以抑制肝癌細胞生長，為了釐清細胞死亡的方式，利用流式細胞儀分析肝癌細胞的細胞週期，結果顯示HepG2細胞會走向細胞凋亡，並且使用西方點墨法證實 MPE會促使HepG2細胞表現凋亡相關蛋白，而 MPE抑制 Hep3B細胞生長則並非透過凋亡，MPE可以抑制Hep3B細胞的Akt與mTOR磷酸化，並促進細胞自噬達到抑癌的效果。在動物實驗方面，使用二甲基亞硝胺 (Dimethylnitrosamine, DEN)作為誘導肝癌的毒化物，DEN不僅可以造成肝細胞突變，尚會讓肝細胞產生活性氧 (reative oxygen species, ROS)、造成肝細胞凋亡，還能導致肝臟分泌發炎因子導致癌化。本篇實驗將Wistar的大鼠以每週40 mg/kg的DEN腹腔注射後並餵食桑葚水萃物(mulberry water extracts, MWE)，於每月測量肝功能指數，結果顯示伴隨餵食MWE可以降低 DEN造成的肝功能指數增加。誘導十六週之後犧牲，以蘇木紫伊紅染色觀察病理切片，發現 DEN組相較於餵食 MWE組腫瘤數目多且體積都大，可見MWE能預防肝癌的生成。此外分析肝臟酵素亦發現MWE可以回復肝臟之抗氧化酵素的活性；MWE還可以降低發炎因子TNF-α、IL-1β與IL-6的表現、減緩肝臟的損傷，因此鑒於這些實驗結果，我們期許桑葚可以作為預防肝癌的保健食品。

關鍵字

桑葚；肝癌；細胞凋亡；細胞自噬；二甲基亞硝胺

並列摘要

Hepatocellular carcinoma (HCC) is one of the most common malignancies in Taiwan. The major risk factors include chronic infections with the hepatitic virus and exposure to dietary AFB1 or alcohol consumption. Those risks factors induce liver chronic inflammtation, fibrosis, cirrhosis, and hepatocellular carcinoma. Therefore, health supplement for liver protection is imperatively essential. Mulberry, the common fruit in Taiwan, contain many functional compositions such as anthocyanins, flavonoids and phenolic acids to remove free radicals and mitigate inflammation. Furthermore, Mulberry has been reported to not only against gastric cancer, melanoma and leukemia but also prevent liver injury induced by alcohol or CCl4 in previous researches. The aim of this study is to examine whether Mulberry could inhibit hepatocarcinogenesis. At first, we observe that mulberry polyphenol extracts (MPE) inhibit the cell growth of HepG2 cell and Hep3B cell. To investigate how MPE works, we analyze cell cycle by flow cytometry and western blotting. These data show MPE induce HepG2 cell apoptosis by observing increase subG1 cells and the elevated expression of caspase-3/8/9 whereas Hep3B cell does not. Instead of apoptosis, MPE cause Hep3B cells autophagy by inhibiting Akt and mTOR phosphorylation. In animal experiment, liver cancer is induced by a kind of mutantgen, diethylnitrosamine (DEN). DEN induces hepatocyte apoptosis and promotes inflammatory factors secretion to lead to hepotocarcinoma. After injecting DEN, the rats treated with mulberry water extracts (MWE) have less and smaller tumor than others without MWE. Moreover, MWE reduces the serum ALT as well as AST and recovers the low activity of antioxidant enzymes induced by DEN. Above of our data, we think the mulberry has a potential to be an outstanding health supplement to prevent hepatocarcinogenesis in the future.

並列關鍵字

mulberry ； hepatocellular carcinoma ； apoptosis ； autophagy ； dimethylnitrosamine

參考文獻

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桑葚萃取物促進肝癌細胞凋亡與自噬作用並預防二甲基亞硝胺造成之肝癌形成

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