Effects of Huanglianjiedu Tang Drug Serum on the Expression of Toll-like Receptors and Downstream Signaling Components in RAW 264.7 Cells

ZHAO Bao-sheng; LIU Hong-bin; MA Yue-ying; SUI Feng; LI Lan-fang; Guo Shuying; HE Xi-rong; Huo Hai-ru; JIANG Ting-liang

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Effects of Huanglianjiedu Tang Drug Serum on the Expression of Toll-like Receptors and Downstream Signaling Components in RAW 264.7 Cells

更新时间：2024-01-04

- Effects of Huanglianjiedu Tang Drug Serum on the Expression of Toll-like Receptors and Downstream Signaling Components in RAW 264.7 Cells
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 13, Issue 5, Pages: 15-20(2007)
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- Published：2007
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ZHAO Bao-sheng, LIU Hong-bin, MA Yue-ying, et al. Effects of Huanglianjiedu Tang Drug Serum on the Expression of Toll-like Receptors and Downstream Signaling Components in RAW 264.7 Cells[J]. Chinese journal of experimental traditional medical formulae, 2007, 13(5): 15-20.
DOI：

ZHAO Bao-sheng, LIU Hong-bin, MA Yue-ying, et al. Effects of Huanglianjiedu Tang Drug Serum on the Expression of Toll-like Receptors and Downstream Signaling Components in RAW 264.7 Cells[J]. Chinese journal of experimental traditional medical formulae, 2007, 13(5): 15-20. DOI：

摘要

目的:研究黄连解毒汤含药血清对细菌脂多糖和双链RNA病毒核酸类似物聚肌胞刺激的小鼠巨噬细胞株RAW264.7 Toll样受体(TLR)及其下游主要信号转导通路的影响。方法:用脂多糖、聚肌胞分别刺激RAW264.7细胞

同时给予黄连解毒汤含药血清进行干预

24h后

取细胞培养上清液

ELISA法测定炎症因子TNF-α和IFN-β的含量

荧光定量PCR方法测定TLR3、TLR4和下游信号转导通路髓样分化因子88(MyD88)

肿瘤坏死因子受体相关因子-6(TRAF-6)

TOLL样受体相关分子(TRAM)和TOLL样受体相关的干扰素活化子(TRIF)mRNA的表达

Western blotting法分析TLR3、TLR4蛋白的表达

同时用细胞免疫荧光法分析MyD88

TRAF-6蛋白表达的强弱

在mRNA水平和蛋白水平探讨黄连解毒汤对Toll样受体的作用机理。结果:黄连解毒汤含药血清显著降低LPS诱导的TLR4、MyD88、TRAF-6、TRAM和TRlF的高表达(与单纯LPS刺激组比较

P<0.05或P<0.01);Poly(I:C)刺激后

黄连解毒汤含药血清仅对TRAM利TRIF的表达有明显的降低作用(P<0.01)。结论:黄连解毒汤能阻断TLR4高表达

阻断TLR4胞内信号转导的MyD88依赖和非依赖两条途径(以阻断非依赖途径为主)

抑制相关基因表达产物TNF-α、IFN-β过度分泌

具有TLR4拮抗剂样作用。黄连解毒汤也可直接作用于接头蛋白TRAM和TRIF

影响TLR3信号转录的MyD88非依赖性途径

抑制IFN-β的过度分泌。

Abstract

Objective:To investigate the influence of Huanglianjiedu Tang drug serum on the expression of TLRs and downstream signaling components in RAW264.7.Methods:RAW264.7 cell line was stimulated with Lipopolysaccharide and Poly(I:C)

respectively

then treated with the drug serum of Huanglianjiedu Tang at the same time

after 24 hours

collected the supernatant and measured the inflammatory factors such as TNF-α and IFN-β

observed the expression of TLR3

TLR4

MyD88

TRAF-6

TRAM and TRIF mRNA with real-time PCR

using western blotting method to analyze the expression of TLR3

TLR4 antibody

and using cell immumofluorescence method to analyze the expression of MyD88 and TRAF-6 antibody.Results:Huanglianjiedu Tang drug serum decreased the high expression of TLR4

MyD88

TRAF-6

TRAM and TRIF(vs LPS stimulated group

P<0.05 or P<0.01)

but it only weakened the expression of TRAM and TRIF when the cells were stimulated by Poly(I:C).Conclusion:Huanglianjiedu Tang blocked the high expression of TLR4

blocked both MyD88-dependent and MyD88-independent signaling pathways(mainly blocked the MyD88-independent signaling pathway)

inhibited the excess secrteion of related gene expression products such as TMF-α

IFN-β

showed TLR4-antagon-like actions.Huanglianjiedu Tang also effected the junction protein such as TRAM and TRIF directly

influenced the MyD88-independent signaling pathway of TLR3

as a result

it inhibited the excess secretion of IFN-β.

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Related Author

GUO Shu-ying

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JIANG Ting-liang

HUO Hai-ru~

Related Institution

Tang Center for Herbal Medicine Research,Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences

中国中医科学院中药研究所唐氏中药研究中心北京100700

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