【摘要】 目的:阐明黄连解毒汤有效活性成分对炎症级联反应信号传导通路TLR4/NF-κB的阻断作用。方法:利用脂质体转染试剂Li-pofectin 2000将含有h TLR4(human toll-like receptor 4)基因和NF-κB-Luc报告基因的质粒转染入人胚肾细胞(HEK293细胞),加入不同浓度和配伍组成的药物刺激后再检测细胞荧光素酶表达水平,筛选出能够有效阻断TLR4/NF-κB信号传导通路的黄连解毒汤有效组分;进一步选择Caco-2细胞单层模型研究转运蛋白对黄连解毒汤有效活性成分的吸收和转运机理。结果:黄连解毒汤中小檗碱和黄芩苷活性单体能阻断TLR4介导的信号通路的传导,显著抑制NF-κB因子的生成;摄取实验中,黄芩苷和小檗碱配伍组能够显著提高小檗碱活性单体在Caco-2细胞中摄取量[(1.23±0.03)ng/μg],加入多药耐药相关蛋白(multidrug resistance-as-sociated protein 2,MRP2)抑制剂MK-571之后,小檗碱的摄取量有显著提高[(1.05±0.09)ng/μg],但是在加入P-糖蛋白(P-glycoprotein,P-gp)抑制剂维拉帕米后,虽然有黄芩苷存在,小檗碱的摄取量未见提高[(0.53±0.05)ng/μg]。结论:通过TLR4受体介导NF-κB通路抑制剂筛选模型,筛选出黄连解毒汤内小檗碱能够有效阻断TLR4/NF-κB信号通路的传导,在小檗碱的跨膜转运的过程中不存在肠道转运蛋白P-gp的作用,但存在MRP2的外排作用,这可能是因为黄连解毒汤中配伍黄芩苷可抑制MRP2蛋白活性,从而使Caco-2细胞中小檗碱被外排的量减少,增加小檗碱的吸收量。更多还原

【Abstract】 Objective: To clarify the regulation mechanism of effective fractions of Huanglian Jiedu Decoction( HLJDT) targeting TLR4/NF-κB signaling pathway on inflammatory cascade. Methods: Reconstructed plasmid containing NF-κB luciferase reporter gene and h TLR4 gene was constructed and transfected into human embryonic kidney( HEK 293) cells by liposomal transfection reagent Lipofectin2000. Related luciferase activities were detected after the treatment of different concentrations and combinations of drugs,the effect of active constituents in HLJDT was evaluated,and active constituents in HLJDT were screened which can block the TLR4/NF-κB signaling pathway. Further,the Caco-2 cell model was employed to study the mechanism of intracellular effective absorption and transport of the active ingredient( Berberine) in HLJDT Results: Berberine significantly blocked this signal pathway( P < 0. 001),results of cell uptake experiments showed that MRP2 inhibitor MK-571 enhanced the uptake of Berberine up to [( 1. 05 ± 0. 09) ng/μg],while P-gp inhibitor Verapamil did not have similar effect though baicalin existed[( 0. 53 ± 0. 05) ng/μg]. In the compatibility experiment,baicalin increased the uptake of berberine[( 1. 23 ± 0. 03)ng/μg]. The cell transfer experiment indicated MRP2 inhibitor increased the transport of Berberine from apical side to basolateral side,compatibility of baicalin promoted the transport of Berberine from AP to BL. Conclusion: A screening model for active ingredients in HLJDT based on dual-luciferase reporter gene assay is successfully established,and berberine in HLJDT could block TLR4 receptor-mediated NF-κB to some extent. The absorption of berberine is the passive diffusion. Berberine may be a substrate of MRP2,but not P-gp substrate. Its mechanism may be baicalin in HLJDT can decrease the efflux of Berberine from the Caco-2 cells,thus increase its uptake.更多还原