Dahuang Lingxian Prescription Regulates Effect of TAK1 and TRAF6 Interaction and Co-localization on Inflammatory Response of Bile Duct Cells

Yi-rong GAN; Xiong-bin GUI; Yuan YU; Bin XU; Jian-ye DAI; Ming CHANG; Jin-mei CHEN; Cheng-ji LI

doi:10.13422/j.cnki.syfjx.20220640

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Dahuang Lingxian Prescription Regulates Effect of TAK1 and TRAF6 Interaction and Co-localization on Inflammatory Response of Bile Duct Cells

更新时间：2022-02-15

- Dahuang Lingxian Prescription Regulates Effect of TAK1 and TRAF6 Interaction and Co-localization on Inflammatory Response of Bile Duct Cells
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 28, Issue 6, Pages: 92-99(2022)
- 作者机构：
  
  1.广西中医药大学，南宁 530001
  2.广西中医药大学第一附属医院，南宁 530023
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20220640
  CLC： R2-0;R285;R33;R318.14
- Received：16 August 2021，
  
  Published Online：25 January 2022，
  
  Published：20 March 2022
- 稿件说明：
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甘苡榕,桂雄斌,俞渊等.大黄灵仙方调控TAK1与TRAF6相互作用及共定位对胆管细胞炎症反应的影响[J].中国实验方剂学杂志,2022,28(06):92-99.

GAN Yi-rong,GUI Xiong-bin,YU Yuan,et al.Dahuang Lingxian Prescription Regulates Effect of TAK1 and TRAF6 Interaction and Co-localization on Inflammatory Response of Bile Duct Cells[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(06):92-99.
甘苡榕,桂雄斌,俞渊等.大黄灵仙方调控TAK1与TRAF6相互作用及共定位对胆管细胞炎症反应的影响[J].中国实验方剂学杂志,2022,28(06):92-99. DOI： 10.13422/j.cnki.syfjx.20220640.

GAN Yi-rong,GUI Xiong-bin,YU Yuan,et al.Dahuang Lingxian Prescription Regulates Effect of TAK1 and TRAF6 Interaction and Co-localization on Inflammatory Response of Bile Duct Cells[J].Chinese Journal of Experimental Traditional Medical Formulae,2022,28(06):92-99. DOI： 10.13422/j.cnki.syfjx.20220640.

摘要

目的

观察大黄灵仙方（DHLX）对大鼠胆管上皮细胞的修复作用，探讨其作用机制是否通过调节转化生长因子-

（TGF-

）激活激酶1（TAK1）与肿瘤坏死因子受体相关因子6（TRAF6）的相互结合，调控核转录因子-

B（NF-

B）/丝裂原活化蛋白激酶（MAPK）信号通路活化而发挥作用。

方法

将20只SD大鼠随机分为正常组和DHLX组，分别予生理盐水及DHLX（320 mg·kg

-1

·d

-1

）灌胃8 d，制备正常血清及含DHLX血清；从正常SD大鼠中提取胆管上皮细胞，取胆管上皮细胞分为9组：正常组、模型组（20 mg·L

-1

）、脂多糖（LPS）+DHLX组（20 mg·L

-1

+10%含药血清）、LPS+PDTC组（20 mg·L

-1

+200 μmol·L

-1

）、LPS+SB203580组（20 mg·L

-1

+0.5 μmol·L

-1

）、LPS+PDTC+SB203580组（20 mg·L

-1

+200 μmol·L

-1

+0.5 μmol·L

-1

）、LPS+PDTC+DHLX组（20 mg·L

-1

+200 μmol·L

-1

+10%含药血清）、LPS+SB203580+DHLX组（20 mg·L

-1

+0.5 μmol·L

-1

+10%含药血清）、LPS+PDTC+SB203580+DHLX组（20 mg·L

-1

+200 μmol·L

-1

+0.5 μmol·L

-1

+10%含药血清）；显微镜下观察药物干预后各组细胞的形态学变化，采用酶联免疫吸附测定法（ELISA）检测各组细胞中白细胞介素（IL）-1

与IL-6的表达量；蛋白免疫印迹法（Western blot）检测各组细胞中TAK1与TRAF6蛋白表达水平，免疫共沉淀技术检测TAK1与TRAF6的相互作用，激光共聚焦显微镜观察TAK1与TRAF6在细胞中的分布及共定位情况。

结果

LPS作用后，细胞突触减少，胞体变圆变小，用药后各组细胞形态趋向正常；与正常组比较，模型组IL-1

和IL-6表达量明显上升（

0.05），TAK1表达量降低而TRAF6的表达量升高（

0.05），TAK1-TRAF6蛋白质复合物含量呈降低趋势；与模型组比较，LPS+DHLX组IL-1

和IL-6表达量明显降低（

0.05），TAK1表达量升高而TRAF6表达量降低（

0.05），TAK1-TRAF6蛋白质复合物含量显著增加（

0.01），两蛋白共定位于细胞质中；与LPS+DHLX组比较，其余各组IL-1

和IL-6表达量均明显降低（

0.05，

0.01），通路阻滞剂干预后各组TAK1-TRAF6蛋白质复合物含量明显降低（

0.05），通路阻滞剂联合DHLX干预后各组TAK1-TRAF6蛋白质复合物含量明显增加（

0.05），两蛋白在细胞质中存在共定位但不明显。

结论

在LPS诱导的胆管细胞炎症反应中，TAK1与TRAF6相互结合呈减弱趋势，大黄灵仙方可逆转此现象，其作用机制可能与促进TAK1与TARF6的相互结合从而抑制NF-

B/MAPK信号通路活化有关。

Abstract

Objective

To observe the repair effect of Dahuanglingxian prescription （DHLX） on bile duct epithelial cells of rats. To explore whether its mechanism of action is to adjust the mutual binding of transforming growth factor -

（TGF-

） activated kinase 1（TAK1） and tumor necrosis factor receptor-associated factor 6 （TRAF6）， and regulate the activation of the nuclear transcription factor -

B （NF-

B）/mitogen-activated protein kinase （MAPK） signaling pathway.

Method

The 20 SD rats were randomly divided into normal group and DHLX group， 10 rats in each group， were given saline and DHLX （320 mg·kg

-1

·d

-1

） for 8 days， to prepare normal serum and DHLX serum. Biliary epithelial cells were extracted from normal SD rats and divided into 9 groups： Normal group， model group （20 mg·L

-1

）， LPS+DHLX group （20 mg·L

-1

+10% DHLX）， LPS+PDTC group （20 mg·L

-1

+200 μmol·L

-1

）， LPS+SB203580 group （20 mg·L

-1

+0.5 μmol·L

-1

）， LPS+PDTC+SB203580 group （20 mg·L

-1

+200 μmol·L

-1

+0.5 μmol·L

-1

）， LPS+PDTC+DHLX group （20 mg·L

-1

+200 μmol·L

-1

+10% DHLX serum）， LPS+SB203580+DHLX group （20 mg·L

-1

+0.5 μmol·L

-1

+10% DHLX serum）， LPS+PDTC+SB203580 +DHLX group （20 mg·L

-1

+200 μmol·L

-1

+0.5 μmol·L

-1

+10% DHLX serum）. The microscopic observation of morphological changes in each group of cells after drug intervention. Enzyme-linked immunosorbent assay（ELISA） was used to detect the expression of （IL）-1

and IL-6 in each group of cells. Western blot detected the expression levels of TAK1 and TRAF6 proteins in each group of cells， Co-IP detected the interaction between TAK1 and TRAF6， and further observed the distribution and co-localization of TAK1 and TRAF6 using Laser confocal microscope.

Result

After the action of LPS， the cell synapses are reduced， the cell body becomes significantly rounded and smaller， but the cell morphology of each group tends to be normal after medication. Compared with normal group， the expression levels of IL-1

and IL-6 in model group were significantly increased （

0.05）， while the expression level of TAK1 was decreased while the expression level of TRAF6 was increased （

0.05）. The content of TAK1-TRAF6 protein complex showed a decreasing trend， and the two proteins co-located in the cytoplasm. Compared with model group， the expression levels of IL-1

and IL-6 in LPS+DHLX group were significantly decreased （

0.05）， the expression level of TAK1 was increased and the expression level of TRAF6 was decreased （

0.05）， the content of TAK1-TRAF6 protein complex was significantly increased （

0.01）， and the two proteins were significantly co-located in cytoplasm. Compared with LPS+DHLX group， the expression levels of IL-1

and IL-6 in other groups were significantly decreased （

0.05，

0.01）. TAK1-TRAF6 protein complex content in each group was significantly decreased after pathway blocker intervention （

0.05）， while TAK1-TRAF6 protein complex content in each group was significantly increased after pathway blocker combined with DHLX intervention

（P

0.05）. Co-localization of the TAK1-TRAF6 in cytoplasm was not obvious.

Conclusion

In the LPS-induced inflammatory response of bile duct cells， the binding of TAK1 and TRAF6 showed a weakening trend， but DHLX could reverse the phenomenon， we think the mechanism of action may be related to promoting the mutual binding of TAK1 and TARF6 to inhibit the activation of the NF-

B/MAPK signaling pathway.

关键词

Keywords

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