龙胆苦苷基于TLR4-TRIF信号通路调控酒精性脂肪肝的机制研究

Steatosis, the earliest response of the liver to alcohol abuse, is characterized by the accumulation of fat in hepatocytes. Excess fat accumulation in the liver could result from increased hepatic fatty acid synthesis and impaired hepatic fatty acid oxidation. Alcohol consumption increases fatty acid synthesis in hepatocytes via up-regulation of sterol regulatory element-binding protein 1 (SREBP-1), while inhibits fatty acid oxidation in hepatocytes mainly via inactivation of the peroxisome proliferator-activated receptor (PPAR)-α. Preventing the accumulation of fat within liver via SREBP-1 or degradation of accumulated fat via PPAR-α may block or delay the progression of fatty liver to steatohepatitis and fibrosis. Furthermore, alcohol renders the intestinal wall more permeable to endotoxin. Endotoxin in hepatic portal system then activates kupffer cells of the liver via the TLR4-TRIF signaling pathway. This activation initiates a cascade of events leading to increased transcription of inflammatory mediators. These changes are associated with fatty liver and mild inflammation of the liver. Gentiopicroside(GPS), a major bioactive herbal ingredient isolated from Gentiana manshurica Kitagawa (GM), exhibits analgesic activities, antibacterial and free radical scavenging activities. Previously we found that GPS can reverse alcoholic fatty liver via regulating TLR4/CD14, and GPS containing-GM extract possessed the ability to prevent alcohol-induced acute liver steatosis, possibly through blocking CYP2E1-mediated free radical scavenging effects and SREBP-1-regulated fatty acid synthesis. We hypothesize that GPS may reverse alcoholic steatosis via SREBP-1 and PPAR-α, as well block kupffer cell activation via TLR4-TRIF signaling pathways.

酒精性脂肪肝是酒精性肝病的前驱阶段，发生脂肪酸的氧化和合成失衡，导致肝脏脂肪蓄积。因此，抑制脂肪酸蓄积或促进脂肪酸的氧化可阻断或延迟脂肪肝发展至脂肪肝炎和肝纤维化，即，过氧化物酶增殖体激活受体-α（PPAR-α）激活与固醇调节元件结合蛋白1（SREBP-1）抑制是逆转酒精性脂肪肝的扳机点。同时，急慢性酒精摄入导致门脉循环内毒素（LPS）水平升高，激活枯否细胞内Toll样受体4（TLR4）-可诱导β干扰素的TIR结构域接头蛋白（TRIF）信号途径，释放炎症介质，导致肝损伤，并促进PPAR-α/SREBP-1介导的肝细胞脂肪酸合成及聚集。龙胆苦苷是清除自由基和保护肝脏的活性物质，前期研究发现龙胆苦苷通过下调TLR4-CD4干预急性酒精性脂肪肝。本课题着重研究龙胆苦苷以TLR4-TRIF为靶点，基于PPAR-α/SREBP-1特异性调节脂肪酸合成和蓄积，可以为酒精性脂肪肝的药物治疗开辟新的途径。

酒精性脂肪肝是酒精性肝病的前驱阶段，发生脂肪酸的氧化和合成失衡，导致肝脏脂肪蓄积。因此，抑制脂肪酸蓄积或促进脂肪酸的氧化可阻断或延迟酒精性脂肪肝发展至脂肪肝炎和肝纤维化。长期乙醇摄入对PPARα和Srebp1的作用通过AMPK介导实现，AMPK可在转录水平和转录后水平对Srebp1进行调节，因此，PPARα激活与Srebp1抑制是逆转酒精性脂肪肝的重点策略。本课题应用调控肝细胞LKB1/AMPK/PPARα/Srebp1并阻断巨噬细胞经LPS-TLR4/ATP-P2x7R-炎症小体信号途径激活的双靶向治疗，可改善脂肪蓄积及并发炎症，防止酒精性脂肪肝发展成为酒精性肝炎。本课题通过建立体内急性和慢性酒精性脂肪肝动物模型，研究龙胆苦苷在酒精性肝病动物模型中对脂肪肝发生的调控作用。龙胆苦苷显著下降了急性和慢性酒精性脂肪肝小鼠血清中转氨酶水平以及甘油三酯浓度。急性和慢性酒精摄入导致所升高的Srebp1和下降的PPARα表达，均给予龙胆苦苷后得到改善，并同时升高LKB1和AMPK活性。而且，龙胆苦苷阻断TLR4-P2x7R-炎症小体轴活化，从而抑制IL-1β生成。体内实验结果揭示阐明龙胆苦苷通过LKB1/AMPK/PPARα/Srebp1介导调控脂肪酸合成和氧化平衡以及基于LPS-TLR4/ATP-P2x7R-炎症小体介导改善酒精性肝病中发生的炎性反应。体外采用酒精刺激肝细胞及LPS联合ATP刺激巨噬细胞细胞，表明龙胆苦苷通过炎症小体调控降低酒精处理的HepG2肝细胞中的脂肪合成并促进脂肪氧化，而且龙胆苦苷阻断LPS与ATP激活巨噬细胞中TLR4-P2x7R信号转导，验证龙胆苦苷通过TLR4-P2x7R信号刺激巨噬细胞-巨噬细胞释放促炎症细胞因子-促炎症细胞因子损伤肝细胞-肝细胞的脂肪变性这一进程进行干预，并进一步阐明TLR4与炎症小体介导的炎症信号交互对话对肝脏脂肪蓄积的影响。总而言之，龙胆苦苷基于TLR4-P2x7R与PPARα/Srebp1交叉对话逆转酒精性脂肪肝的脂肪蓄积和炎性反应。