Mechanism of Duanteng Yimu Decoction on Expression of MEKK2 in Rheumatoid Arthritis Synovial Fibroblasts and Arthritis in CIA Mice

Qiang WANG; Long-yin HAN; Zhen-quan WEI; Nan LI; Dong-mei PAN; Min-ying LIU; Chang-song LIN

doi:10.13422/j.cnki.syfjx.20200502

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Mechanism of Duanteng Yimu Decoction on Expression of MEKK2 in Rheumatoid Arthritis Synovial Fibroblasts and Arthritis in CIA Mice

Pharmacology | 更新时间：2021-02-09

- Mechanism of Duanteng Yimu Decoction on Expression of MEKK2 in Rheumatoid Arthritis Synovial Fibroblasts and Arthritis in CIA Mice
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 26, Issue 7, Pages: 31-41(2020)
- 作者机构：
  
  1.广州中医药大学　第一临床医学院，广州　 510405
  2.暨南大学　中医学院，广州　 510405
  3.南方医科大学　中医药学院，广州　 510405
  4.广州中医药大学　第一附属医院，广州　 510405
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20200502
  CLC： R2-0; R22; R285.5; R289
- Received：27 July 2019，
  
  Published Online：20 November 2019，
  
  Published：05 April 2020
- 稿件说明：
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Qiang WANG, Long-yin HAN, Zhen-quan WEI, et al. Mechanism of Duanteng Yimu Decoction on Expression of MEKK2 in Rheumatoid Arthritis Synovial Fibroblasts and Arthritis in CIA Mice[J]. Chinese journal of experimental traditional medical formulae, 2020, 26(7): 31-41.
DOI：

Qiang WANG, Long-yin HAN, Zhen-quan WEI, et al. Mechanism of Duanteng Yimu Decoction on Expression of MEKK2 in Rheumatoid Arthritis Synovial Fibroblasts and Arthritis in CIA Mice[J]. Chinese journal of experimental traditional medical formulae, 2020, 26(7): 31-41. DOI： 10.13422/j.cnki.syfjx.20200502.

摘要

目的：

为了研究断藤益母汤(DTYMD)在类风湿关节炎中对丝裂原活化蛋白激酶(MAPK)上游激酶的调控作用，阐明DTYMD抗炎的分子机制。

方法：

培养成纤维样滑膜细胞，将细胞分为空白组，模型组，DTYMD高、中、低质量浓度组(1 000，800，600 mg·L

-1

)，甲氨蝶呤(MTX)组(20 μmol·L

-1

)组，采用蛋白免疫印迹法(Western blot)，实时荧光定量聚合酶链式反应(Real-time PCR)对丝分裂原活化蛋白激酶激酶激酶2(MEKK2)的蛋白和mRNA表达进行分析。将42只雄性DBA/1J小鼠随机分为6组，每组7只，分别为正常组，模型组，MTX组(2 mg·kg

-1

)，DTYMD低、中、高剂量组(6.25，12.5，25 mg·kg

-1

)。除正常组外，其他5组均采用二次免疫法构建胶原诱导型关节炎(CIA)模型。给药结束后，取小鼠后肢踝关节行苏木素-伊红(HE)染色并进行关节病理评分。

结果：

与模型组比较，DTYMD以浓度依赖方式抑制成纤维样滑膜细胞活性(

0.01)；与空白组比较，模型组细胞增殖率升高(

0.01)。与模型组比较，DTYMD高、中质量浓度组降低MEKK2蛋白及mRNA的表达(

0.05，

0.01)。与模型组比较，DTYMD不同剂量组均降低细胞基质金属蛋白酶-1(MMP-1)，肿瘤坏死因子-

(TNF-

)，白细胞介素-6(IL-6)的表达(

0.01)；与空白组比较，模型组MMP-1，IL-6，TNF-

表达明显上升(

0.05，

0.01)。在动物实验中，与模型组比较，DTYMD高、中剂量组可以降低CIA小鼠关节肿胀度(

0.05，

0.01)；与正常组比较，模型组小鼠二次免疫后关节明显肿胀(

0.05)。在CIA小鼠踝关节HE染色中，与空白组比较，模型组小鼠病理学评分升高(

0.01)；与模型组比较，DTYMD高、中剂量小鼠关节病理评分明显下降(

0.05，

0.01)。

结论：

DTYMD可能通过下调MEKK2对细胞因子IL-6，TNF-

，MMP-1负性调控，从而缓解类风湿关节炎的炎症反应。

Abstract

Objective:

Duanteng Yimu decoction(DTYMD)is effective in treatment of rheumatoid arthritis (RA) by relieving joint inflammation and down-regulating some inflammatory factors in a short period of time

but the mechanism is still unclear. We aimed to investigate upstream kinase of mitogen activated protein kinases(MAPK) and define the anti-inflammatory mechanism of DTYMD.

Method:

Fibroblasts-like synovial cells(FLSs) were divided into blank group

model group (IL-1

)

high-dose DTYMD group (1 000 mg·L

-1

)

medium-dose DTYMD group (800 mg·L

-1

)

low-dose DTYMD group (600 mg·L

-1

) and armour ammonia butterfly(MTX) group (20 μmol·L

-1

). The protein and mRNA expressions of mitogen-activated protein kinase kinase kinase 2 (MEKK2) were analyzed by real-time fluorescence quantitative PCR(Real-time PCR). Totally 42 male DBA/1J mice were randomly divided into 6 groups

with 7 mice in each group

namely normal group

model group and MTX group (2 mg·kg

-1

)

low-dose DTYMD group (6.25 mg·kg

-1

)

medium-dose DTYMD group (12.5 mg·kg

-1

)

and high-dose DTYMD group (25 mg·kg

-1

). Except for the normal group

the other five groups were included in collagen-induced arthritis(CIA) model by secondary immunoassay. After administration

the posterior limbs and ankle joints were stained with htoxylin-eosin(HE)

and the pathological scores of the joints were evaluated.

Result:

Compared with the model group

DTYMD inhibited the activity of FLSs in a concentration-dependent manner (

0.01). Compared with the blank control group

the cell proliferation rate of the model group increased (

0.01). Compared with the model group

high and middle-dose DTYMD groups could inhibit protein and mRNA expressions of MEKK2 (

0.01)

but there was no significant difference in low-dose group. However

the expression of DTYMD protein in high/medium/low-dose groups was significantly higher than that in blank group (

0.01)

but there was no significant difference in MTX group. Compared with the model group

the expressions of matrix metalloprotease-1 (MMP-1)

tumor necrosis factor-

(TNF-

) and interleukin(IL)-6 were negatively regulated in different DTYMD groups(

0.01)

and the expressions of MMP-1

IL-6

TNF-

in the model group were significantly higher than those in the blank group (

0.05

0.01). In the animal experiment

compared with the model group

high/middle-dose DTYMD groups could decrease the degree of joint swelling in CIA mice (

0.01)

but there was no significant difference in the low dose group

and the joint swelling in the model group was significantly higher than that in the blank group (

0.05). In HE staining of ankle joint of CIA mice

the pathological scores of high/small-dose DTYMD groups were significantly lower those of model group (

0.05

0.01)

and the pathological score of model group was higher than that of blank group (

0.01).

Conclusion:

DTYMD might down-regulate MEKK2 to negatively regulate inflammatory cytokines IL-6

TNF-

and MMP-1

thereby alleviating the inflammatory response in rheumatoid arthritis.

关键词

Keywords

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