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目的基于TLR5-NF-κB通路建立报告基因系统筛选四物汤中的有效成分。方法利用脂质体转染试剂Li-pofectin 2000将含有hTLR5基因和NF-κB Luc报告基因的质粒转染入人胚肾细胞(HEK293细胞),加入药物刺激后再检测细胞荧光素酶表达水平,筛选出能够激活TLR5受体及其下游NF-κB通路的有效成分。结果果糖、腺嘌呤能够显著激活此通路,且随果糖、腺嘌呤作用浓度的增加对细胞膜表面TLR5受体及其下游的NF-κB通路激活作用也随之增强,叶酸、东莨菪内酯、芍药苷、盐酸川芎嗪、β-谷甾醇、阿魏酸也能一定程度激活此通路。结论通过TLR5受体介导NF-κB通路激活剂筛选模型,筛选出四物汤内果糖、腺嘌呤、芍药苷、盐酸川芎嗪、叶酸、东莨菪内酯、β-谷甾醇、阿魏酸8种单体在一定浓度范围内能够激活TLR5受体及其下游的NF-κB通路。
OBJECTIVE: To establish a reporter gene system based on TLR5-NF-κB pathway to screen the active ingredients in Si Wu Tang. Methods The plasmids containing hTLR5 gene and NF-κB Luc reporter gene were transfected into human embryonic kidney cells (HEK293 cells) with Li-pofectin 2000, and the luciferase expression levels were detected by adding drugs. Screening out active ingredients that activate TLR5 receptor and its downstream NF-κB pathway. Results Fructose and adenine could significantly activate this pathway. With the increase of concentration of fructose and adenine, the activation of NF-κB pathway on TLR5 receptor and its downstream cell surface also increased. Folic acid, scopoletin, peony Glycosides, ligustrazine hydrochloride, β-sitosterol, ferulic acid can also activate this pathway to some extent. Conclusion TLR5 receptor-mediated NF-κB pathway activator screening model, screening out the Siwu Tang fructose, adenine, paeoniflorin, ligustrazine hydrochloride, folic acid, scopoletin, β-sitosterol, ferulic acid 8 Monomers in a certain concentration range can activate the TLR5 receptor and its downstream NF-κB pathway.