OBJECTIVE: We explored the protective effects of Ampelopsis grossedentata extract (AGE) against chronic alcohol-induced hepatic injury (alcoholic liver disease, ALD), aiming to elucidate its underlying mechanisms.
METHODS: Firstly, UPLC-Q/TOF-MS analysis and network pharmacology were used to identify the constituents and elucidate the potential mechanisms of AGE against ALD. Secondly, C57BL/6 mice were pair-fed the Lieber-DeCarli diet containing either isocaloric maltodextrin or ethanol, AGE (150 and 300 mg/kg/d) and silymarin (200 mg/kg) were administered to chronic ethanol-fed mice for 7 weeks to evaluate the hepatoprotective effects. Serum biochemical parameters were determined, hepatic and ileum sections were used for histologic examination, and levels of inflammatory cytokines and oxidative stress in the liver were examined. The potential molecular mechanisms of AGE in improving ALD were demonstrated by RNA-seq, Western blotting analysis, and immunofluorescence staining.
RESULTS: Ten main constituents of AGE were identified using UPLC-Q/TOF-MS and 274 potential ALD-related targets were identified. The enriched KEGG pathways included Toll-like receptor signaling pathway, NF-κB signaling pathway, and necroptosis. Moreover, in vivo experimental studies demonstrated that AGE significantly reduced serum aminotransferase levels and improved pathological abnormalities after chronic ethanol intake. Meanwhile, AGE improved ALD in mice by down-regulating oxidative stress and inflammatory cytokines. Furthermore, AGE notably repaired damaged intestinal epithelial barrier and suppressed the production of gut-derived lipopolysaccharide by elevating intestinal tight junction protein expression. Subsequent RNA-seq and experimental validation indicated that AGE inhibited NF-κB nuclear translocation, suppressed IκB-α, RIPK3 and MLKL phosphorylation and alleviated hepatic necroptosis in mice.
CONCLUSIONS: In this study, we have demonstrated for the first time that AGE protects against alcoholic liver disease by regulating the gut-liver axis and inhibiting the TLR4/NF-κB/MLKL-mediated necroptosis pathway. Therefore, our present work provides important experimental evidence for AGE as a promising candidate for protection against ALD.
目的:我们探讨藤茶提取物(AGE)对慢性酒精性肝损伤(酒精性肝病,ALD),旨在阐明其潜在的机制。
方法:首先,UPLC-Q/TOF-MS分析和网络药理学用于鉴定成分并阐明AGE抗ALD的潜在机制。其次,C57BL/6小鼠配对喂养含有等热量麦芽糊精或乙醇的Lieber-DeCarli饮食,将AGE(150和300mg/kg/d)和水飞蓟素(200mg/kg)给予慢性乙醇喂养的小鼠7周以评估其肝脏保护作用。测定血清生化指标,肝和回肠切片用于组织学检查,并检测肝脏中炎性细胞因子和氧化应激水平。通过RNA-seq证明了AGE改善ALD的潜在分子机制,蛋白质印迹分析,和免疫荧光染色。
结果:使用UPLC-Q/TOF-MS鉴定了AGE的10种主要成分,并鉴定了274种潜在的ALD相关靶标。富集的KEGG通路包括Toll样受体信号通路,NF-κB信号通路,和坏死。此外,体内实验研究表明,AGE可显著降低慢性乙醇摄入后血清转氨酶水平,改善病理异常。同时,AGE通过下调氧化应激和炎性细胞因子改善小鼠ALD。此外,AGE显著修复受损的肠上皮屏障并通过提高肠紧密连接蛋白表达抑制肠源性脂多糖的产生。随后的RNA-seq和实验验证表明,AGE抑制NF-κB核易位,抑制IκB-α,RIPK3和MLKL磷酸化和减轻小鼠肝坏死。
结论:在这项研究中,我们首次证明AGE通过调节肠-肝轴和抑制TLR4/NF-κB/MLKL介导的坏死性凋亡途径,预防酒精性肝病.因此,我们目前的工作为AGE作为有希望的抗ALD候选物提供了重要的实验证据.