《Gut Microbes》的一項(xiàng)研究系統(tǒng)闡明了茯苓不溶性多糖（WIP）通過多靶點(diǎn)調(diào)控腸道微生物群落緩解酒精性肝脂肪變性（AHS）的機(jī)制，并首次揭示了腸道共生真菌——季也蒙邁耶氏酵母（Meyerozyma guilliermondii）通過生物合成前列腺素E2（PGE2）加重肝損傷的新病理機(jī)制。研究顯示，在酒精性肝脂肪變性模型中，茯苓不溶性多糖（WIP）口服干預(yù)能顯著改善肝臟炎癥損傷和脂肪堆積。其作用機(jī)制包括：1. 調(diào)節(jié)腸道細(xì)菌群落：提高厚壁菌門/變形菌門比值，顯著增加毛螺菌科（特別是瘤胃梭菌屬和未分類梭菌）豐度；2. 抑制真菌過度增殖：有效抑制乙醇誘導(dǎo)的腸道真菌過度生長；3. 激活保護(hù)性信號通路：通過激活PPAR-γ信號減輕結(jié)腸上皮炎癥，營造腸道低氧環(huán)境，從而抑制真菌和變形菌的過度生長。通過培養(yǎng)組學(xué)和ITS測序技術(shù)，研究團(tuán)隊(duì)在AHS小鼠糞便中發(fā)現(xiàn)共生真菌季也蒙邁耶氏酵母的異常增殖。將該菌株定植于無菌小鼠后，可顯著加重AHS表型。機(jī)制深入研究表明，該真菌能通過花生四烯酸的生物轉(zhuǎn)化生成PGE2，而肝臟中這種由腸道真菌誘導(dǎo)的PGE2產(chǎn)生被證實(shí)是慢性AHS的重要致病機(jī)制之一。該研究不僅證實(shí)了茯苓多糖通過協(xié)同調(diào)控腸道細(xì)菌和真菌群落發(fā)揮肝保護(hù)作用，也為開發(fā)針對腸道微生物譜系的酒精性肝病治療策略提供了新的理論依據(jù)和實(shí)踐方向。

研究背景

酒精性肝?。ˋLD）已成為全球最常見的慢性肝病之一。更重要的是，依據(jù)世界衛(wèi)生組織在2018年發(fā)布的《全球酒精與健康報告》，2016年因酒精導(dǎo)致的死亡占全球死亡總數(shù)的5.3%。近年來，越來越多的證據(jù)表明，腸道菌群失調(diào)與ALD的發(fā)生發(fā)展之間存在不可忽視且具有因果關(guān)系的關(guān)聯(lián)。長期飲酒會損傷腸道屏障完整性并導(dǎo)致腸道微生物組成改變。由于腸道細(xì)菌對乙醇高度敏感，酒精攝入會顯著影響腸道細(xì)菌群落。

腸道細(xì)菌、真菌和病毒之間的相互作用對維持腸道微生態(tài)平衡至關(guān)重要。多項(xiàng)研究顯示，厚壁菌門中的專性厭氧菌在抑制變形菌門潛在致病菌的擴(kuò)增以及限制共生真菌在腸道定植方面發(fā)揮重要作用。這些發(fā)現(xiàn)以及酒精誘導(dǎo)的細(xì)菌失調(diào)和真菌過度生長導(dǎo)致肝臟炎癥和脂肪沉積的證據(jù)，提示酒精導(dǎo)致的厚壁菌門減少可能是ALD發(fā)病機(jī)制的主要因素之一。因此，專門促進(jìn)厚壁菌門細(xì)菌生長的干預(yù)措施被寄予治療ALD的期望。早期研究已證實(shí)，能夠刺激乳酸桿菌和雙歧桿菌生長的益生元低聚果糖可改善小鼠的酒精性肝損傷。作為可食用且具藥用價值的真菌，茯苓（Wolfiporia cocos）的塊體在傳統(tǒng)中醫(yī)中因其利尿、鎮(zhèn)靜、補(bǔ)益等功效被廣泛使用。我們早期的研究表明，口服茯苓的水不溶性多糖（WIP）能夠增加ob/ob小鼠厚壁菌門中產(chǎn)丁酸菌的豐度，這提示其可能對ALD具有潛在益處。WIP是一種（1→3）?β?D?葡聚糖，平均分子量為4.486?×?10??Da，已通過核磁共振（NMR）和SEC?RI?MALLS等方法鑒定。目前尚未有研究探討茯苓對ALD的作用。

研究內(nèi)容

在本研究中，我們以酒精性肝脂肪變性小鼠模型為實(shí)驗(yàn)對象，展示了WIP對酒精誘導(dǎo)的肝脂肪堆積和炎癥的治療效果，證實(shí)了WIP對酒精導(dǎo)致的腸道菌群失調(diào)的改善作用，并揭示了共生酵母Meyerozyma guilliermondii 與ALD的關(guān)聯(lián)以及真菌誘導(dǎo)的PGE?在ALD發(fā)展中的貢獻(xiàn)。

研究結(jié)果

Figure 1. Oral treatment with WIP alleviates chronic ethanol feeding-induced hepatic injury and steatosis. (a) Experimental design. (b) The level of plasma alanine aminotransferase (ALT). (c) The level of plasma aspartate aminotransferase (AST). (d) The plasma levels of alkaline phosphatase (ALP). (e) The level of plasma lactate dehydrogenase (LDH). (f) Liver index. (g) The level of hepatic triglyceride (TG). (h) The level of hepatic total cholesterol (TC). (i) The expression of TNF-α in liver. (j) Representative picture of liver sections stained with oil-red. (k) Representative picture of liver sections with MCP-1 immunofluorescence staining. (b-h) N = 10 per group, (i) N = 5 per group, (j-k) N = 3 per group. Control: mice received isocaloric liquid diet instead of ethanol. Alcohol: mice fed with ethanol diet. WIP: mice fed with an ethanol diet supplemented with a water-insoluble polysaccharide from W. cocos. Data are presented as the mean ± standard error of the mean (SEM).

Figure 2. WIP treatment ameliorates the ethanol-induced gut dysbiosis. (a) OTU Venn diagram. (b) Weighted uniFrac-based principal coordinates analysis. (c) Shannon index. Bacterial taxonomic profiling of intestinal bacteria from different groups at the phylum (d) and family level (e). (f, g) Linear discriminant analysis (LDA) scores derived from LEfSe analysis. (h-k) Differentially abundant bacterial genera. The relative expression of occludin-1 (l) and ZO-1 (m) in colon. (n) The level of plasma lipopolysaccharide (LPS). (o) Total fungi in feces assessed by qPCR. The relative expression of ppar-γ (p) and nos2 (q) and colon TNF-α (r) and IL-Iβ (s). (a-k and n) N = 8 per group, (l-s) N = 5 per group. Control: mice received an isocaloric liquid diet instead of ethanol. Alcohol: mice fed with ethanol diet. WIP: mice fed with an ethanol diet supplemented with a water-insoluble polysaccharide from W. cocos. Data are presented as the mean ± standard error of the mean (SEM).

Figure 3. Identification of Meyerozyma guilliermondii as a casual fungus for AHS. (a) Fungi isolated from feces by aerobic culture-dependent approach. The number in the parentheses represented the strains obtained for each identified fungus. (b) Level of Meyerozyma guilliermondii (Mg) in fecal samples based on aerobic culture-dependent approach. (c) Abundance of Meyerozyma in Cecal contents based on ITS1 sequencing. (d) Experimental design. (e) The level of plasma alanine aminotransferase (ALT). (f) The level of plasma aspartate aminotransferase (AST). (g) The level of hepatic triglyceride (TG). (h) Representative picture of liver sections stained with oil-red. (i) The level of plasma triglyceride (TG). (j) The level of hepatic total cholesterol (TC). (k) The level of TNF-α in the liver. (l) The level of plasma β-glucan. (c) N = 4–5 per group, (e-g and i-l) N = 7–9 per group, (h) N = 3 per group. Ampho B: amphotericin B. Data are presented as the mean ± standard error of the mean (SEM).

Figure 4. Contribution of fungi-induced PGE2 to alcoholic hepatic steatosis. The level of PGE2 (a), the expression of EP2 (b), EP4 (c) and cxcl1 (d) in liver of ethanol-fed mice treated with WIP. The level of PGE2 (e), the expression of EP2 (f), EP4 (g) and cxcl1 (h) in liver of ethanol-fed mice treated amphotericin B (ampho B) or caspofungin. The level of PGE2 (i), the expression of EP2 (j), EP4 (k) and cxcl1 (l) in liver of the fungi-free mice treated with live M. guilliermondii (Mg). The level of ALT (m), hepatic TG (n), the expression of EP2 (o), EP4 (p) and cxcl1 (q) in liver after oral PGE2. (a and m-n) N = 9–10 per group, (b-d, f-h, j-l and o-q) N = 5 per group, (e and i) N = 7–9 per group. Control: mice received the isocaloric liquid diet instead of ethanol. Alcohol: mice fed with ethanol diet. WIP: mice fed with an ethanol diet supplemented with a water-insoluble polysaccharide from W. cocos. Ampho B: amphotericin B. Data are presented as the mean ± standard error of the mean (SEM).

研究結(jié)論

本研究表明，茯苓（Wolfporia cocos）來源的水不溶性多糖（WIP）可通過調(diào)節(jié)酒精性肝脂肪變性（AHS）小鼠的腸道微生物群，有效改善其肝臟炎性損傷與脂肪堆積?？诜IP能顯著提高厚壁菌門與變形菌門的比值，增加毛螺菌科細(xì)菌的豐度（，并抑制乙醇誘導(dǎo)的真菌過度生長。WIP處理可激活過氧化物酶體增殖物激活受體-γ（PPAR-γ）信號通路，減輕結(jié)腸上皮細(xì)胞的炎癥反應(yīng)，促進(jìn)腸道內(nèi)低氧環(huán)境形成，從而抑制腸道真菌與變形菌門細(xì)菌的過度生長。此外，通過培養(yǎng)法和內(nèi)轉(zhuǎn)錄間隔區(qū)（ITS）測序，我們發(fā)現(xiàn)酒精性肝脂肪變性小鼠糞便中共生真菌吉列蒙迪梅耶酵母菌（Meyerozyma guilliermondii）的數(shù)量大幅增加。將該酵母菌接種到無菌真菌小鼠體內(nèi)，會加劇酒精性肝脂肪變性的病理特征。研究還發(fā)現(xiàn)，吉列蒙迪梅耶酵母菌可通過花生四烯酸的生物轉(zhuǎn)化生成前列腺素E2（PGE2）。進(jìn)一步研究證實(shí)，腸道真菌（吉列蒙迪梅耶酵母菌）誘導(dǎo)肝臟產(chǎn)生PGE2，是慢性酒精性肝脂肪變性的致病機(jī)制之一。本研究證實(shí)，調(diào)控腸道微生物群（細(xì)菌和真菌）可作為緩解酒精性肝病的一種有效替代策略。

https://doi.org/10.1080/19490976.2020.1830693

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