探討大蒜素對於酒精性脂肪肝小鼠體內腸道菌相、肝臟及大腦間之交互影響

Abstract

酗酒是造成酒精性脂肪肝的成因之一，並可能導致嚴重的肝臟疾病。近年來有研究顯示，腸道菌在肝臟疾病的致病及病程發展上扮演重要的角色，其機制可能包含對於菌相的直接影響，或因其副產物（例如：脂多醣）誘導發炎介質的釋出，造成體內系統性發炎及臟器的損壞，其中又以肝臟與腦部的影響最鉅。大蒜素（allicin）是新鮮大蒜萃取液中的有效成分，已有研究證實其具有抗菌、抗氧化、抗癌、降低血脂等功效。本研究的主要目的是探討大蒜素對於酒精性脂肪肝的護肝效果，及其對盲腸及糞便菌相所產生的影響，並進一步探討腸道菌相、肝臟及大腦間之交互影響。首先利用二烯丙基二硫化物（diallyl disulfide）及間氯過氧苯甲酸（m-chloroperbenzoic acid）進行反應，再經由開放式矽膠管柱及高效液相層析分析儀的純化，合成出純度高達95%且產率達3.86%的大蒜素。在動物模式中，給予C57BL/6雄性小鼠含有酒精的液態飼料（Lieber-DeCarli diet）誘導其酒精性脂肪肝的發生，並且以管餵方式每天給予大蒜素（5 or 20 mg/kg bw/day），四週以後進行犧牲取得其血液、臟器、尿液、糞便等檢體。分析結果顯示大蒜素可降低酒精性脂肪肝小鼠血漿中天門冬氨酸胺基轉移酶（AST）、丙胺酸轉胺酶（ALT）、肝臟重量與體重之相對重量、脂肪肝指數，以及肝臟中三酸甘油酯的堆積，並且提升其抗氧化相關因子的含量，例如：穀胱甘肽（GSH）、過氧化氫酶（CAT）、超氧化物歧化酶（SOD），同時降低肝臟微粒體細胞色素（CYP2E1）之表現量。此外大蒜素亦可抑制肝臟促發炎細胞激素如：腫瘤壞死因子α（TNF-α）、介白素1β（IL-1β）與介白素6（IL-6）之表現量，同時增進肝臟酒精脫氫酶（ADH）活性；以上結果顯示大蒜素可以減緩酒精誘導之肝臟氧化壓力及發炎現象。而由次世代基因定序結果顯示酒精與大蒜素之攝取會影響盲腸及糞便菌相多樣性，包括：數量及種類。整體而言，酒精傾向對於腸道菌造成不好的影響，但透過盲腸及糞便的分析結果顯示得知大蒜素可降低酒精引發肝臟三酸甘油酯升高之相關菌群；而正常飲食中介入大蒜素之組別和控制組相比，盲腸及糞便的菌相中 Lactobacillus sakei 顯著增加。在血漿中脂多醣（LPS）的含量分析，酒精攝取組之血漿 LPS 濃度顯著低於控制組，此結果顯示四週酒精的介入並未引起血液中 LPS 的濃度提升。此外酒精攝取組和控制組相比，其盲腸腸道菌相中格蘭氏陰性菌亦顯著較控制組低。在腸壁通透性方面，5 mg/kg bw/day 大蒜素的介入顯著改善因酒精引起的通透性改變。而在腸道菌─肝臟─腦部軸線，酒精攝取顯著地增加血漿及腦部中 TNF-α 濃度，而5 mg/kg bw/day 大蒜素的介入則顯著地降低此發炎現象。但由於酒精攝取組腸道菌中格蘭氏陰性菌及血漿 LPS 濃度和控制組相比均顯著降低，本研究推論酒精所造成之腦部發炎受到酒精本身的影響更勝於腸道格蘭氏陰性菌及血漿中LPS濃度改變。綜上所述，本研究之結論為大蒜素可能透過影響小鼠盲腸及糞便菌相而在酒精性脂肪肝疾病中具有肝臟保護之效果。

Excessive alcohol consumption can cause alcoholic fatty liver disease (AFLD). Recently, the scientists have found that gut microbiota may play a role in the pathogenesis or progression of certain liver diseases, including alcoholic liver disease, either through the direct effects of bacteria or their by-products (lipopolysaccharides, LPS) by triggering inflammatory mediators such as tumor necrosis factor α (TNF-α). These inflammatory mediators can lead to systemic inflammation and organ damage, particularly to the liver and brain. Allicin is a pharmacologically active substance found in the fresh aqueous extract of garlic (Allium sativum, 大蒜, dasuan), which has been investigated for its antimicrobial, antioxidant, anticancer and hypolipidemic activity. The objectives of the study were to investigate (1) hepatoprotective effects of allicin against AFLD; (2) influence of allicin on cecal and fecal microbiota; and (3) interaction of gut microbiota, liver and brain in AFLD. Allicin was extracted from diallyl disulfide and m-chloroperbenzoic acid, followed by purification of the open column with silica gel and HPLC, and it achieved 95% purity and 3.86% yield. Male C57BL/6 mice were fed a control diet or an alcohol-containing liquid diet (Lieber-DeCarli diet). The mice were divided into 5 groups: (1) control diet, (2) control diet + allicin 20 mg/kg bw/day, (3) alcohol diet, (4) alcohol diet + allicin 5 mg/kg bw/day, and (5) alcohol diet 20 mg/kg bw/day. Allicin was orally administered daily to the AFLD mice for 4 weeks. The results indicated that allicin promoted hepatoprotection by significantly reducing aspartate transaminase (AST) and alanine transaminase (ALT) levels in the plasma, which are key indicators of liver damage. Allicin reduced fat accumulation, increased glutathione and catalase levels, and decreased microsomal protein cytochrome P450 2E1 (CYP2E1) expression in the livers of the AFLD mice. Furthermore, allicin supplementation significantly decreased the levels of hepatic pro-inflammatory tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 and suppressed the expression of sterol regulatory element-binding protein-1 (SREBP-1). Additionally, it improved the hepatic alcohol dehydrogenase (ADH) activity. Collectively, these findings demonstrate that allicin attenuated liver oxidative stress and inflammation. In addition, next-generation sequencing (NGS) base DNA sequencing results revealed that alcohol and allicin influenced gut microbiota diversification in both cecum and feces. The impact of alcohol and allicin on gut microbiota in cecum and feces varied in type, ratio, and abundance. Overall, alcohol tended to induce bad effect on the gut microbiota. Allicin exhibited inhibitory effect on the gut microbiota both in cecum and feces that correlated with alcohol consumption. Supplementation with allicin in the control diet mice was enriched with Lactobacillus sakei in cecum and feces compared to control mice. Plasma LPS levels in alcohol diet mice were significantly lower than the mice intake with the control diet. Thus, chronic alcohol consumption for 4 weeks did not increase the LPS level in the plasma. In addition, alcohol-fed mice have reduced the gram-negative bacteria in the cecum compared to the control mice. 5 mg of allicin supplementation preserved the intestinal permeability compared in AFLD mice. To study the gut microbiota-liver-brain interaction, the TNF-α in plasma and the brain were evaluated. The TNF-α level was increased in both plasma and the brain. 5 mg of allicin supplementation reduced the brain inflammation by decreasing the TNF-α level. Because gram-negative bacteria and plasma LPS level in the AFLD group was lower than the control. Therefore, this study concluded that the cause of brain inflammation greatly more influenced by ethanol consumption than the LPS that derived from the gram-negative bacteria in the gut. In conclusion, allicin exhibited hepatoprotective effect against AFLD and it might be related to fecal and cecal microbiota in mice.