Cucurbitacin is the main active constituent of Guadi (a traditional Chinese medicine), a natural tetracyclic triterpenoid, has reported to have hepatoprotective effects. Recent studies demonstrate Cucurbitacin has potent cytotoxic activities mediated by both apoptosis and autophagy. Ethanol, the major component of alcohol beverage, the alcoholic liver damage induced by Ethanol shows obvious rising trend, serious threat to human health. Ethanol induced liver injury is mainly induced by induced oxidative stress, inflammatory reaction, induction of apoptosis, etc. Recent report has shown Ethanol to be an effective autophagy regulator. Our Pre-Experiments found that Cu B has strong hepatocyte toxicity, which could be enhanced by Ethanol co-treament. Here, the effect of Ethanol on Cu B induced hepatotoxicity was investigated both in vitro and in vivo. Cu B dose-dependently decreased the cell viability of LO2 (normal hepatocytes) and HepG2 (hepatocellular carcinoma cells), which could be enhanced by Ethanol co-treatment,This phenomenon was not observed in MCF-7, A549 cells suggesting it might be liver specific. Ethanol also demonstrated similar enhanced toxic effect on Cu D and Cu E in LO2 cells suggesting that chemical structures might be of importance. JC-1 and DCFH2-DA staining found that Ethanol enhanced Cu B-induced mitochondria membrane potential (∆Ψ) depolarization without affecting intracellular reactive oxygen species (ROS) formation. Ethanol has no effects on Cu B indeced cell cycle arrest. Cu B-induced apoptosis was augmented by Ethanol as determined by Hoechst 33342 staining, Annexin V staining, and apoptotic-related proteins Bcl-2､Bax､Caspase expressions. Ethanol enhanced cell death was reversed by caspase inhibitor suggesting that the augmented cell death was apoptotic death. Furthermore, Ethanol inhibited Cu B-induced LO2 autophagy as determined by MDC staining, autophagic protein expression, transmission electron microscopy and immunofluorescence. The AKT/mTOR signaling pathway might be involved in the inhibition effect of Ethanol on Cu B induced autophagy. The animal experiment clearly demonstrated that Cu B is a potent hepatotoxic agent. Ethanol co-treatment dramatically increased Cu B-induced hepatotoxicity in BALB/c mice. In addition, Ethanol significantly decreased mice survival rate after Cu B administration. In conclusion, the present data suggested that Cu B has strong hepatotoxicity, Ethanol promotes Cu B-induced hepatotoxicity by inhibiting autophagy and augmenting apoptosis.

葫蘆素類是中藥瓜蒂中的主要活性成份,是一類天然的四環三萜類化合物｡ 據報導,葫蘆素類具有一定的肝臟保護作用｡最新研究顯示,葫蘆素類可同時誘導 細胞自噬與凋亡｡乙醇是全世界廣泛飲用的酒類主要成份,其誘導的酒精性肝損 傷發生率呈明顯上升趨勢,嚴重威脅人類健康｡酒精誘導的肝損傷主要通過誘導 氧化應激､誘導炎癥反應､誘導細胞凋亡等｡最近研究發現乙醇可有效地調節自噬 ｡我們預實驗發現葫蘆素 B 有較強的肝細胞毒性,乙醇能明顯增強其毒性｡故對乙 醇增強葫蘆素 B肝毒性作用與機制在體內外進行了研究｡MTT 結果顯示葫蘆素 B 可劑量依賴降低正常肝細胞 LO2及肝癌細胞HepG2的存活率,乙醇聯用可加強其 毒性｡但在 MCF-7 乳腺癌細胞以及 A549 肺癌細胞則無此增强作用,提示该作用可 能具有肝细胞特异性｡乙醇對與葫蘆素 B 結構類似葫蘆素 D 和葫蘆素 E 在 LO2 細胞上也顯示出毒性增強作用,提示化學結構也可能是重要因素｡螢光探針 JC-1 和 DCFH2-DA 研究發現,葫蘆素 B 可顯著降低 LO2 細胞線粒體膜電位,并增加細 胞內活性氧(ROS)生成｡乙醇可增強葫蘆素 B 導致細胞膜電位變化但不影響細胞 內 ROS 生成｡葫蘆素 B 誘導 LO2 細胞週期阻滯與 G2/M 期,乙醇對此無明顯影響｡ 通過 Hoechst 33342,Annexin V 染色,以及凋亡相關 Bcl-2､Bax､Caspase 家族､PARP 蛋白的表達顯示葫蘆素 B 可誘導 LO2 細胞凋亡,這些變化可被乙醇增強｡乙醇增 強細胞的死亡作用可被 Caspase 抑制劑逆轉,表明增強的細胞死亡可能是通過細 胞凋亡｡此外,通過 MDC 染色,自噬相關蛋白表達,以及電鏡､免疫螢光等實驗證實 了葫蘆素 B 可誘導 LO2 細胞自噬, 乙醇可抑制葫蘆素 B 誘導的自噬｡其 中,AKT/mTOR 信號通路可能參與了乙醇抑制葫蘆素 B 誘導的細胞自噬｡在 BALB/c 小鼠的整體動物實驗顯示,葫蘆素 B 具有較強的肝毒性,乙醇共同作用可 顯著增強葫蘆素 B 引起的小鼠肝毒性,更降低了小鼠的存活率｡ 綜上所述,本研究顯示葫蘆素 B 具有較強的肝毒性,乙醇可通過抑制自噬增加凋亡作用而增強其肝毒性｡