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**：本研究旨在探讨山奈酚（Kaempferol）对酒精性脂肪性肝炎（alcoholic steatohepatitis, ASH）的作用及其机制。
**方法**：选用C57BL/6 N小鼠构建慢性-暴饮酒暴露小鼠模型，对慢性饮酒造模小鼠给予山奈酚作为干预药物，连续给药6周以评估其干预效果。体外实验方面，采用酒精刺激肠上皮Caco-2细胞，并通过转染miR-155模拟物（miRNA-155 mimics），进一步探究山奈酚对肠上皮细胞中miR-155信号通路的调控效应。采用苏木精-伊红（HE）染色与油红O染色观察各组小鼠肝脏及肠道组织的病理损伤；通过试剂盒检测血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、白细胞介素-1β（IL-1β）与肿瘤坏死因子-α（TNF-α）的水平；采用酶联免疫吸附测定（ELISA）试剂盒检测脂多糖（LPS）的表达量，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；同时评估肝脏与结肠组织的炎症激活状态及相关信号通路的活化情况。
**结果**：山奈酚干预可显著改善酒精饮食模型组小鼠肝脏组织的脂肪变性、空泡样病变等病理损伤，同时降低血清ALT、AST的酶活性，以及肝脏组织中IL-1β与TNF-α的mRNA表达水平。山奈酚可显著降低饮酒小鼠肠道组织中miR-155的表达水平，显著上调细胞因子信号转导抑制因子1（SOCS1）的蛋白表达，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，与对照组相比，山奈酚可显著抑制乙醇刺激下Caco-2细胞中miR-155的表达水平，降低NF-κB的活化，上调SOCS1蛋白的表达，并提升酒精刺激下Caco-2细胞中occludin蛋白的表达量。反之，过表达miR-155可显著降低Caco-2细胞中occludin与SOCS1的蛋白表达量，并升高NF-κB的活化水平；而山奈酚干预可显著逆转这一效应。
**结论**：山奈酚可通过靶向调控miR-155，抑制肠道过度炎症反应，提升紧密连接蛋白occludin的表达，从而改善肠道屏障功能稳定性，最终缓解酒精摄入诱导的肝损伤。