背景:本研究的目的是探讨山奈酚对酒精性脂肪性肝炎的作用和机制。
方法:用C57BL/6N小鼠建立慢性酒精暴露模型。将山奈酚作为干预药物给予长期饮酒的小鼠6周,以评估其效果。体外,肠上皮Caco-2细胞受到酒精刺激,和miRNA-155模拟物用于进一步研究山奈酚对肠上皮细胞miRNA-155信号传导的影响。HE染色和油红O染色观察各组小鼠肝、肠组织损伤情况,ALT,AST,IL-1β,试剂盒检测TNF-α;ELISA试剂盒检测脂多糖(LPS)表达,并通过qRT-PCR评估IL-1β和TNF-α的表达;Westernblot用于评估肝脏和结肠组织的过度炎症反应及其相关信号通路的激活。
结果:山奈酚治疗可明显改善酒精饮食模型组肝组织脂肪变性和空泡性病变等病理变化,降低血清ALT和AST酶活性及肝组织白细胞介素-1β和肿瘤坏死因子-αmRNA表达水平。山奈酚显著降低酒精喂养小鼠肠组织中miRNA-155的表达,显着增加他们的细胞因子抑制信号1(SOCS1)蛋白表达,抑制核因子κB的激活,并显着增加肠紧密连接蛋白occludin和zonulaoccludes-1的产生。更重要的是,山奈酚显著降低酒精性脂肪性肝炎小鼠血清LPS水平。体外实验表明,与对照组相比,山奈酚显著抑制乙醇暴露下Caco-2细胞miRNA-155的表达,降低核因子κB的激活,导致SOCS1蛋白表达增加,并在酒精作用下增加了Caco-2细胞中occludin蛋白的产生水平。相比之下,在Caco-2细胞中,miRNA-155的过表达显著降低了occludin和SOCS1蛋白的产生,并增加了核因子κB的活化水平,山奈酚的给药显著抑制了这种作用。
结论:山奈酚通过增强occludin蛋白的表达,改善肠道屏障功能的稳定性,改善酒精摄入引起的肝损伤。通过靶向miR-155来抑制肠道中的过度炎症反应。
BACKGROUND: The aim of this study was 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 6 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-1β, and TNF-α were detected by kits; lipopolysaccharide (LPS) expression was detected by ELISA kit, and the expression of IL-1β and TNF-α was assessed by qRT-PCR; Western blot was utilized to assess the excessive inflammatory response of liver and colon tissue and the related signaling 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 tumor 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 cytokine suppressor signaling 1 (SOCS1) protein expression, inhibited the activation of nuclear factor kappa-B and significantly increased the production of the intestinal tight junction proteins occludin and zonula occludens-1. More importantly, kaempferol significantly reduced serum LPS levels in alcoholic steatohepatitis 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.
CONCLUSIONS: 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.