Supplementary Material for: Kaempferol ameliorated alcoholic hepatitis through improving intestinal barrier function by targeting miRNA-155 signaling

Introduction: To investigate the effect and mechanism of Kaempferol on alcoholic steatohepatitis. Methods: C57BL/6 N mice were utilized to establish Binge-on-Chronic alcohol exposure mice model. Kaempferol was given as the interventional drug to chronic alcohol-fed mice for six weeks to assess its effects. In vitro, intestinal epithelial Caco-2 cells were stimulated by alcohol, and miRNA-155 mimics were used to further study the effect of kaempferol to miRNA-155 signaling in intestinal epithelial cells. HE staining and oil red O staining were used to observe the liver and intestinal tissue damage in each group of mice, and ALT, AST, IL-1B and TNF-a were detected by kits; LPS expression was detected by ELISA kit, and the expression of IL-1B and TNF-a was assessed by qRT-PCR; activated inflammatory response of liver and colon tissue and the related signalling pathway activation. Results: Kaempferol treatment significantly improved pathological changes such as steatosis and vacuolated lesions in liver tissue of the alcohol diet model group, and reduced serum ALT and AST enzyme activities and liver tissue interleukin-1β and tumour necrosis factor-α mRNA expression levels. Kaempferol significantly reduced the expression of miRNA-155 in the intestinal tissue of alcohol-fed mice, significantly increased their SOCS1 protein expression, inhibited the activation of nuclear factor kappa-B and significantly increased the production of the intestinal tight junction proteins occludin and ZO-1. What's more, kaempferol significantly reduced serum LPS levels in ASH mice. In vitro experiments showed that compared with the control group, kaempferol significantly inhibited the expression level of miRNA-155 in Caco-2 cells under ethanol exposure, decreased the activation of nuclear factor kappa-B, led to an increase in the expression of SOCS1 protein, and increased the production level of occludin protein in Caco-2 cells under the effect of alcohol. In contrast, overexpression of miRNA-155 significantly decreased occludin and SOCS1 protein production and increased nuclear factor kappa-B activation levels in Caco-2 cells, and the administration of kaempferol significantly inhibited this effect. Conclusion: Kaempferol improved the stability of gut barrier function to ameliorate hepatic injury induced by alcohol intake through enhancing occludin protein expression, by targeting miR-155 to inhibit the excessive inflammatory response in the intestine.引言：本研究旨在探究山奈酚（Kaempferol）对酒精性脂肪性肝炎（alcoholic steatohepatitis, ASH）的作用及其潜在机制。方法：选用C57BL/6 N小鼠构建慢性饮酒联合单次暴饮小鼠模型。以山奈酚作为干预性药物，对慢性酒精喂养小鼠连续给药六周以评估其干预效果。体外实验中，采用酒精刺激肠上皮Caco-2细胞，并借助miRNA-155模拟物，进一步解析山奈酚对肠上皮细胞中miRNA-155信号通路的调控作用。实验采用苏木精-伊红（HE）染色、油红O染色观察各组小鼠肝脏与肠道组织的病理损伤；通过生化试剂盒检测血清谷丙转氨酶（ALT）、谷草转氨酶（AST）、白细胞介素-1β（IL-1β）及肿瘤坏死因子-α（TNF-α）水平；采用酶联免疫吸附试验（ELISA）试剂盒检测脂多糖（LPS）的表达情况，通过实时荧光定量聚合酶链反应（qRT-PCR）检测IL-1β与TNF-α的mRNA表达水平；同时分析肝脏与结肠组织的炎症激活状态及相关信号通路的活化程度。结果：与单纯酒精饮食模型组相比，山奈酚干预可显著改善模型小鼠肝脏组织的脂肪变性、空泡样病变等病理损伤，降低血清ALT、AST的酶活性，同时下调肝脏组织中IL-1β与TNF-α的mRNA表达水平。山奈酚可显著抑制酒精喂养小鼠肠道组织中miRNA-155的表达，上调细胞因子信号转导抑制因子1（SOCS1）蛋白的表达水平，抑制核因子κB（NF-κB）的活化，并显著提升肠道紧密连接蛋白occludin与ZO-1的表达量。此外，山奈酚可显著降低ASH模型小鼠的血清LPS水平。体外实验结果显示：相较于对照组，酒精刺激后的Caco-2细胞经山奈酚处理后，miRNA-155的表达水平显著下调，核因子κB的活化受到抑制，SOCS1蛋白表达升高，同时逆转了酒精诱导的occludin蛋白表达降低的现象。进一步实验发现，过表达miRNA-155可显著降低Caco-2细胞中occludin与SOCS1蛋白的表达，并升高核因子κB的活化水平，而山奈酚给药可显著阻断这一效应。结论：山奈酚可通过靶向miR-155抑制肠道过度炎症反应，上调紧密连接蛋白occludin的表达，从而维持肠道屏障功能稳定性，最终缓解酒精摄入诱导的肝损伤。