基于“肠—肝轴”探讨沙棘熊果酸改善酒精性肝损伤的效果及作用机理

It is well known that long-term excessive drinking can cause liver damage. Enterogenous endotoximia induced by impaired intestinal barrier function, inflammatory response and Kupper Cell activation were all related to hepatocellular injury. Ursolic acid (UA) was extracted from hippophae rhamnoides L. by preliminary experiment and was proved that it had hepatoprotective effect. Most researches on effective constituent in hippophae rhamnoides L. were focus on flavone and polysaccharide. In addition, these studies were limited to chemical liver damage. Enterocyte barrier function and intestinal microecology could affect inflammatory response and played an important role in alcoholic liver injury through current study. Therefore, UA might be related to hepatoprotective mechanism of alcoholic liver injury. This study was aim to establish “alcoholic liver injury animal model” and explore the effect and mechanism of UA on alcoholic liver injury based on “Gut-Liver Axis” through molecular biological technique and high throughput sequencing. TAK-242 and LY294002 were utilized for studying TLR4 signaling pathway and PI3K/Akt/FOXO4 signaling pathway respectively. In addition, ERIC PCR, DGGE finger-print analysis and 454 high throughput sequencing were used to analyze the molecular ecology changes of intestinal flora. This research achievement would provide a brand new strategy for controlling alcoholic liver injury and reliable evidence for developing edible and medicinal plants in the Inner Mongolia Autonomous Region.

长期大量酒精摄入可导致肝损伤。由IBF受损引发的肠源性内毒素血症及其介导的炎性反应与肝损伤密切相关。课题组前期已证实沙棘熊果酸(UA)具有保肝作用，而目前国内外有关沙棘有效成分保肝研究多见于黄酮和多糖，且局限于化学性肝损伤的研究。现已发现，肠上皮细胞屏障和肠道微生态可影响炎症反应进程，在ALD发生中起着重要作用，而UA具有显著的抗炎作用。因此，推测UA保肝作用机理可能与之相关。本研究拟建立大鼠ALD模型，以“肠—肝轴”为切入点，借助分子生物学及高通量测序技术，开展UA对ALD的保护作用机制研究。利用抑制剂干预，从PI3K/Akt/FOXO4和TLR4信号通路双管齐下，探讨UA改善ALD的分子机制。同时，利用ERIC、DGGE指纹图谱及454高通量测序技术，对比分析酒精和UA对肠道菌群分子生态学的影响。该研究为ALD防治提供新的思路和策略，也为内蒙古药食两用植物资源的开发利用提供理论依据。

本研究通过建立大鼠酒精性肝损伤模型，以沙棘熊果酸为干预物，一方面证实了熊果酸对酒精诱发的大鼠肝脏损伤具有明显的保护作用。它不仅可以抑制血清转氨酶升高和脂质代谢异常，还有效改善了肝组织脂肪变性、炎性细胞浸润、肠黏膜通透性增高，肠道微生态紊乱、修复了肠黏膜屏障，减少内毒素的产生和释放等损伤。另一方面，利用关键蛋白的抑制剂干预，从TLR4信号通路、PI3K/Akt/FOXO4信号通路以及肠道微生态角度多管齐下，证实了其保护作用机制与①下调TLR4蛋白过表达，调控TLR4信号转导通路，抑制NF-κB p65活化，减轻炎症损伤，从而达到改善酒精性肝损伤的作用。②调控PI3K/AKT信号通路活化，抑制下游蛋白FOXO4磷酸化，降低NF-κB p65活化，进而影响紧密连接蛋白的表达，减少促炎因子TNF-α、IL-1β等的释放，从而达到有效缓解肝细胞损伤的功效。③调节大鼠肠道菌群结构和数量的变化，修复肠道屏障功能，减少内毒素的产生和释放，最终达到有效改善酒精性肝损伤的目的。这一研究成果不仅为酒精性肝损伤的防治提供了新思路和策略，也为内蒙古药食两用植物资源的开发利用提供理论依据。