海兔素对酒精性肝损伤大鼠肝脏保护作用及机制

Protective Effect and Underlying Mechanism of Aplysin on Ethanol-Induced Liver Injury in Rats

本实验旨在观察海兔素对酒精性肝损伤大鼠的保护作用,并从内毒素介导的Kupffer细胞toll样受体4(Toll-like receptor 4,TLR4)信号通路角度探讨海兔素可能的保肝机制。雄性Wistar大鼠随机分为3组,包括正常对照组、酒精模型组和海兔素干预组。其中,酒精模型组和海兔素干预组大鼠分别灌胃给予8 g/(kg mb·d)乙醇2周后,再给予12 g/(kg mb·d)乙醇6周。海兔素干预组在给予乙醇前1h,灌胃给予海兔素150 mg/(kg mb·d),持续8周。末次灌胃后,禁食不禁水12 h,处死大鼠。采用苏木精-伊红染色进行肝组织病理学观察,采用透射电子显微镜进行肝组织超微结构观察,采用酶学实验检测肝脏损伤血清生物标志物水平,采用显色底物鲎试剂盒检测血清内毒素水平;采用门静脉胶原酶Ⅳ原位灌注及密度梯度离心获得大鼠原代Kupffer细胞。采用墨汁吞噬试验评价Kupffer细胞吞噬活性;采用逆转录聚合酶链反应测定Kupffer细胞中CD14、TLR4和NF-κB的mRNA表达水平;采用蛋白印迹实验测定Kupffer细胞中TLR4、MyD88、NF-κB p65和肿瘤坏死因子(tumor necrosis factor-α,TNF-α)的蛋白表达水平;采用酶联免疫吸附实验测定Kupffer细胞上清液中TNF-α和白细胞介素(interleukin-1β,IL-1β)含量。结果显示,海兔素补充可减轻乙醇引起的肝组织损伤,降低肝脏损伤血清生物标志物水平(P<0.05)。进一步研究发现,海兔素补充可减少血浆内毒素含量(P<0.05),有效恢复Kupffer细胞吞噬活性,显著降低Kupffer细胞中TLR4及其下游CD14、TLR4、MyD88、NF-κB p65等相关蛋白表达水平(P<0.05),并使TNF-α和IL-1β等炎性因子释放受到抑制(P<0.05)。结果表明,海兔素对酒精性肝损伤大鼠肝组织具有保护作用,其作用机制可能与海兔素抑制内毒素介导的MyD88依赖性TLR4信号通路活化有关。

This study aimed to explore the protective effect of aplysin on ethanol-induced liver injury in rats and to explore the possible underlying mechanism from the perspective of the Toll-like receptor 4(TLR4) signaling pathway. Male Wistar rats were randomly assigned into three groups: normal control, model control and aplysin-treated groups. The rats in the model control and aplysin-treated groups were given ethanol orally at 8 g/(kg·d) for two weeks, and then at 12 g/(kg·d) for another six weeks. The rats in the aplysin-treated group were administered with aplysin at 150 mg/(kg·d) via gavage one hour before ethanol for 8 weeks. After the last administration, all the animals were fasted with free access to water for 12 h and subsequently sacrificed;liver tissues were collected for histological and biochemical assessments;the levels of serum biomarkers for liver damage and endotoxin levels were detected with biochemical assay kits;primary rat Kupffer cells were cultured for assessment of their phagocytic activity with ink phagocytosis test. The mRNA expression levels of CD14, TLR4 and nuclear factor-kappa B(NF-κB) in Kupffer cells were assessed via reversed transcription polymerase chain reaction;the protein expression levels of TLR4, MyD88, NF-κB p65 and tumor necrosis factor-α(TNF-α) in Kupffer cells were assessed via western blotting;the levels of TNF-α and interleukin-1β(IL-1β) in Kupffer cells were measured by enzyme-linked immunosorbent assay. Aplysin relieved alcohol-induced hepatic histopathological changes, decreased plasma endotoxin levels and suppressed the elevation of liver damage biomarkers. Aplysin treatment effectively restored the phagocytic activity of Kupffer cells. Moreover, aplysin significantly decreased the expression levels of CD14, TLR4, MyD88, NF-κB p65 and TNF-α and the concentrations of TNF-α and IL-1β. These findings showed that aplysin exerted a potent hepatoprotective effect, which might be associated with the inhibition of the TLR4 signaling pathway.