Butyrate protects against diet-induced liver fibrosis and suppresses non-canonical TGF-β signaling in human stellate cells

Background & Aims: In obesity-associated non-alcoholic steatohepatitis (NASH), persistent hepatocellular damage and inflammation are key drivers of fibrosis, the main determinant of NASH-associated mortality. The short chain fatty acid butyrate can exert metabolic improvements and anti-inflammatory activities in NASH. However, its effects on NASH-associated liver fibrosis remains unclear. Methods: Putative antifibrotic effects of butyrate were studied in Ldlr-/-.Leiden mice fed an obesogenic NASH-inducing diet (HFD) containing 2.5% (w/w) butyrate for 38 weeks and compared to an HFD control group. Antifibrotic mechanisms of butyrate were further investigated in TGF-β-stimulated primary human hepatic stellate cells (HSC). Results: HFD-fed mice developed obesity, insulin resistance, increased levels of plasma leptin, adipose tissue inflammation, gut permeability, dysbiosis and NASH-associated fibrosis. Butyrate corrected hyperinsulinemia, lowered plasma leptin levels and attenuated adipose tissue inflammation, without affecting gut permeability or microbiota composition. Butyrate lowered plasma ALT and CK-18M30 and attenuated hepatic steatosis and inflammation. Butyrate inhibited fibrosis development as demonstrated by decreased hepatic collagen content and Sirius-red-positive area. In TGF-β-stimulated HSC, butyrate dose-dependently reduced collagen deposition and decreased procollagen1α1 and PAI-1 protein expression. Transcriptomic analysis and subsequent pathway and upstream regulator analysis revealed deactivation of specific non-canonical TGF-β signaling pathways RhoA/Rock and PI3K/AKT and other important pro-fibrotic regulators (e.g. YAP/TAZ and MYC) by butyrate, providing a potential rationale for its antifibrotic effects. Conclusions: Butyrate protects against the development of obesity and insulin resistance-associated NASH and liver fibrosis. These antifibrotic effects are at least in part attributable to a direct effect on collagen production in hepatic stellate cells, as a result of inhibiting non-canonical TGF-β signaling. Antifibrotic mechanisms of butyrate were investigated in TGF-β-stimulated primary human hepatic stellate cells (HSC). There were 3 HSC treatments, for 4 days: (i) Unstimulated Control, (ii) TGF-β1 (2 ng/mL), (iii) TGF-β1 (2 ng/mL) + Sodium Butyrate (1mM)

背景与目的：在肥胖相关非酒精性脂肪性肝炎（non-alcoholic steatohepatitis, NASH）中，持续的肝细胞损伤与炎症是纤维化的核心驱动因素，而纤维化是NASH相关死亡的主要决定因素。短链脂肪酸丁酸可在NASH中发挥代谢改善与抗炎作用，但其对NASH相关肝纤维化的作用尚不明确。
方法：本研究在喂食含2.5%（质量分数）丁酸的致肥胖NASH诱导高脂饮食（high fat diet, HFD）、造模38周的Ldlr基因敲除莱顿小鼠中探究丁酸的潜在抗纤维化作用，并与仅喂食HFD的对照组小鼠进行比较。此外，本研究还在转化生长因子β（transforming growth factor-β, TGF-β）刺激的原代人肝星状细胞（hepatic stellate cells, HSC）中进一步探究丁酸的抗纤维化机制。
结果：喂食HFD的小鼠出现肥胖、胰岛素抵抗，血浆瘦素水平升高、脂肪组织炎症、肠道通透性增加、菌群失调以及NASH相关纤维化。丁酸可纠正高胰岛素血症，降低血浆瘦素水平并减轻脂肪组织炎症，且不影响肠道通透性或菌群组成。丁酸可降低血浆谷丙转氨酶（alanine aminotransferase, ALT）与细胞角蛋白18 M30（CK-18M30）水平，同时减轻肝脏脂肪变性与炎症反应。丁酸可抑制纤维化进展，这一点可通过肝脏胶原含量降低与天狼星红（Sirius-red）染色阳性面积减少得到证实。在TGF-β刺激的HSC中，丁酸可呈剂量依赖性减少胶原沉积，并降低前胶原1α1（procollagen1α1）与纤溶酶原激活物抑制剂1（plasminogen activator inhibitor-1, PAI-1）的蛋白表达。转录组学分析及后续的通路与上游调控因子分析显示，丁酸可使特定的非经典TGF-β信号通路（RhoA/Rock与PI3K/AKT）以及其他重要促纤维化调控因子（如YAP/TAZ与MYC）失活，这为丁酸的抗纤维化作用提供了潜在的理论依据。
结论：丁酸可预防肥胖与胰岛素抵抗相关NASH及肝纤维化的发生发展。其抗纤维化作用至少部分归因于直接抑制肝星状细胞的胶原生成，这一作用通过阻断非经典TGF-β信号通路实现。本研究在TGF-β刺激的原代人肝星状细胞（HSC）中探究了丁酸的抗纤维化机制，共设置3组HSC处理方案，处理时长为4天：(i) 未刺激对照组，(ii) 转化生长因子β1（2 ng/mL）处理组，(iii) 转化生长因子β1（2 ng/mL）+丁酸钠（1 mM）联合处理组。