清毒汤保护肠屏障治疗慢加急性肝衰竭内毒素血症的机制研究  

Study on the mechanism of Qingdu Tang in protecting the intestinal barrier and treating endotoxemia in acute-on-chronic liver failure

作　　者：张笛 陈月 杨佳璇 师娟 高连印[1] ZHANG Di;CHEN Yue;YANG Jiaxuan;SHI Juan;GAO Lianyin(School of Traditional Chinese Medicine,Capital Medical University,Beijing 100069,China)

机构地区：[1]首都医科大学中医药学院,北京100069

出　　处：《环球中医药》2024年第11期2137-2142,共6页Global Traditional Chinese Medicine

基　　金：北京市自然科学基金面上项目(7222266)。

摘　　要：目的研究清毒汤对肠屏障的保护作用,探讨清毒汤保护肠屏障治疗慢加急性肝衰竭内毒素血症的机制。方法不同浓度的脂多糖(lipopolysaccharides,LPS)处理正常人肠上皮细胞(human intestinal epithelial cells-6,HIEC-6),通过细胞毒性法(cell counting kit-8,CCK-8)检测LPS对HIEC-6细胞存活率的影响。采用实时荧光定量PCR(quantitative real-time PCR,qRT-PCR)法检测紧密连接蛋白ZO-1、Occludin的mRNA表达情况,在体外构建肠屏障损伤的模型。通过蛋白免疫印迹法(western blot,WB)检测清毒汤对肠屏障紧密连接蛋白ZO-1、Occludin表达水平的影响。利用qRT-PCR法检测通路蛋白表皮生长因子受体(epidermal growth factor receptor,EGFR)、Zonulin及miRNA122a的mRNA表达情况。结果CCK-8法、qRT-PCR法的检测结果显示120μg/mL的LPS作为构建肠屏障损伤模型的浓度较为合适。qRT-PCR的结果表明,5%清毒汤含药血清能显著改善LPS对肠上皮细胞间紧密连接蛋白ZO-1、Occludin的mRNA的下调作用,因此后续实验中选取5%清毒汤作为实验条件。WB的结果表明清毒汤能明显改善LPS对肠上皮细胞间紧密连接蛋白ZO-1、Occludin的下调作用。qRT-PCR的结果表明,清毒汤能明显降低通路蛋白EGFR、Zonulin及miRNA122a的表达。结论清毒汤可能通过miR122a靶向EGFR调控Zonulin蛋白从而调节肠道紧密连接的表达,保护肠屏障,治疗慢加急性肝衰竭内毒素血症。Objective To study the protective effect of Qingdu Tang on the intestinal barrier,and to explore the mechanism of protecting the intestinal barrier by Qingdu Tang in the treatment of endotoxemia in slow plus acute liver failure.Methods HIEC-6 cells were treated with different concentrations of lipopolysaccharides(LPS),and the effect of LPS on the survival rate of HIEC-6 cells was detected by cytotoxicity assay(cell counting kit-8,CCK-8).The mRNA expression of tight junction proteins ZO-1 and Occludin was detected by quantitative real-time fluorescence PCR(qRT-PCR),and the model of intestinal barrier damage was constructed in vitro.The effect of Qingdu Tang on the expression levels of intestinal barrier tight junction proteins ZO-1 and Occludin were detected by protein immunoblotting(western blot,WB).The mRNA expressions of pathway proteins EGFR,Zonulin and miRNA122a were detected by qRT-PCR.Results The results of CCK-8 method and qRT-PCR showed that 120μg/mL LPS was a more appropriate concentration for constructing the intestinal barrier injury model.The results of qRT-PCR showed that 5%Qingdu Tang containing serum significantly improved the down-regulation of LPS on the mRNAs of ZO-1 and Occludin.The results of WB showed that the serum containing 5%Qingdu Tang significantly improved the down-regulation of LPS on ZO-1 and Occludin,which are tight junction proteins between intestinal epithelial cells.The result of qRT-PCR showed that Qingdu Tang significantly reduced the expression of the pathway proteins,such as EGFR,Zonulin,and miRNA-122a.Conclusion Qingdu Tang may target EGFR through miRNA-122a to regulate Zonulin protein thus regulating the expression of intestinal tight junction,protecting the intestinal barrier,and treating endotoxemia in slow plus acute liver failure.

关 键 词：清毒汤 人肠上皮细胞 肠屏障 慢加急性肝衰竭内毒素血症 miRNA122a 紧密连接 

分 类 号：R285.5[医药卫生—中药学]