Effect and Mechanism of Dihydroartemisinin on Rheumatoid Arthritis Animal Models

Cheng-cheng DU; Yu-qing TAN; Jian-ying SHEN; Mei HUANG; Fang-ji GE; Kun HONG; Jian-hui SUN; Jun LUO

doi:10.13422/j.cnki.syfjx.20191002

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Effect and Mechanism of Dihydroartemisinin on Rheumatoid Arthritis Animal Models

Pharmacology | 更新时间：2021-02-09

- Effect and Mechanism of Dihydroartemisinin on Rheumatoid Arthritis Animal Models
- Chinese Journal of Experimental Traditional Medical Formulae Vol. 25, Issue 10, Pages: 48-56(2019)
- 作者机构：
  
  1.贵州医科大学　基础医学院，贵州　 550025；
  2.中国中医科学院　中药研究所，北京　 100700
- 作者简介：
- 基金信息：
- DOI：10.13422/j.cnki.syfjx.20191002
  CLC： R2-0; R22; R285.5
- Received：21 September 2018，
  
  Published Online：12 February 2019，
  
  Published：20 May 2019
- 稿件说明：
移动端阅览
Cheng-cheng DU, Yu-qing TAN, Jian-ying SHEN, et al. Effect and Mechanism of Dihydroartemisinin on Rheumatoid Arthritis Animal Models[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(10): 48-56.
DOI：

Cheng-cheng DU, Yu-qing TAN, Jian-ying SHEN, et al. Effect and Mechanism of Dihydroartemisinin on Rheumatoid Arthritis Animal Models[J]. Chinese journal of experimental traditional medical formulae, 2019, 25(10): 48-56. DOI： 10.13422/j.cnki.syfjx.20191002.

摘要

目的：

研究双氢青蒿素(DHA)对佐剂性关节炎(AIA)和胶原诱导性关节炎(CIA)大鼠的保护作用，探讨DHA抗炎机制。

方法：

将Wistar大鼠随机分为8组，分别为AIA正常组，AIA模型组，AIA DHA组，AIA甲氨蝶呤组；CIA正常组，CIA模型组，CIA DHA组，CIA甲氨蝶呤组。佐剂性关节炎(AIA)的建立：除正常组外，各组大鼠右后足趾皮内注射0.1 mL弗式完全佐剂；胶原诱导性关节炎(CIA)的大鼠模型的建立：除正常组外，各组大鼠尾根部注射1 g·L

－1

二型胶原乳化剂0.2 mL，并在7 d后，在大鼠相同部位注射相同体积的胶原乳化剂加强免疫。造模成功后，并分别给予DHA(30 mg·kg

－1

·d

－1

)治疗后，并考察DHA抗AIA/CIA大鼠关节炎作用，包括关节炎指数(AI)，足趾肿胀度，以及DHA对AIA/CIA大鼠免疫器官指数，酶联免疫吸附测定(ELISA)检测血清中白细胞介素-6(IL-6)水平和AIA/CIA大鼠踝关节病理切片的影响。体外培养RAW264.7巨噬细胞系，分别以0.5，1，2，4，8 μmol·L

－1

DHA处理24 h，设立正常，采用噻唑蓝(MTT)比色法检测DHA对RAW264.7细胞活力的影响；设立正常组，脂多糖(LPS)组，LPS＋DHA(0.5，1，2 μmol·L

－1

)组，酶联免疫吸附测定(ELISA)检测细胞上清IL-6含量，蛋白免疫印迹法(Western blot)检测细胞核内核转录因子-

B p65(NF-

B p65)蛋白表达。

结果：

与正常组比较，模型组大鼠足肿胀度，AI，脾脏指数，IL-6水平显著增高(

0.05

0.01)，DHA可明显减轻AIA/CIA大鼠的足肿胀度(

0.05)，降低关节炎评分指数(

0.05)，降低AIA大鼠脾脏指数(

0.01)和胸腺指数(

0.05)以及AIA大鼠血清中IL-6水平(

0.05)；但是对CIA大鼠脾脏指数并没有降低作用，而对胸腺指数和IL-6水平虽有降低作用但不具有统计学意义。此外DHA能够明显改善AIA/CIA大鼠踝关节病变情况，并能降低病理评分(

0.01)。与正常组比较，DHA 4，8，16 μmol·L

－1

组对RAW264.7细胞活力明显抑制作用(

0.05

0.01)，DHA体外安全用药范围≤2 μmol·L

－1

。与正常组比较，LPS模型组IL-6含量明显高于正常组(

0.01)，与LPS模型组比较，DHA能够抑制IL-6的产生(

0.05)，Western blot结果显示，LPS能够上调 RAW264.7细胞核内NF-

B p65水平，而DHA能够抑制p65的入核。

结论：

DHA能够改善类风湿关节炎大鼠踝关节病变情况，其机制可能与抑制NF-

B信号通路有关。

Abstract

Objective:

To study the protective effect of dihydroartemisinin (DHA) on adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) in rats

in order to explore its possible mechanism.

Method:

Wistar rats were randomly divided into eight groups

namely AIA control group

AIA model group

AIA DHA group and AIA methotrexate group

CIA control group

CIA model group

CIA DHA group and CIA methotrexate group. To establish adjuvant-induced arthritis (AIA) model

rheumatoid arthritis rats were induced through intradermal injection with 0.1 mL Freund's complete adjuvant (FCA) into right postpedes except for the control group. To establish the model of collagen-induced arthritis (CIA)

except for control group

the caudal root of rats was immunized subcutaneously with 0.2 mL of emulsion containing 1 g·L

-1

of Collagen type Ⅱ (CⅡ). One week later

CⅡ emulsion was injected for the second time. After the rheumatoid arthritis model was successfully established and the administration with DHA (30 mg·kg

-1

·d

-1

)

the anti-inflammatory effect of DHA on AIA/CIA rats was observed

including the arthritis index (AI)

paw swelling degree and effect of DHA on immune organ index of AIA/CIA rats. Interleukin (IL)-6 levels in serum was detected by enzyme-linked immunosorbent assay (ELISA) and pathological sections of ankle joints of AIA/CIA rats. RAW264.7 macrophage cells were cultured

in vitro

and treated with DHA at various doses (0.5

16 μmol·L

-1

) for 24 h

and the cell viability was detected by methyl thiazolyl tetrazolium(MTT) assay. Lipopolysaccharides (LPS) group

LPS+ DHA groups (0.5

2 μmol·L

-1

) and control group were established. The level of IL-6 was detected by enzyme-linked immunosorbent assay(ELISA). The protein expression levels of nuclear transcription factor-

B p65 (NF-

B p65) was tested by Western blot.

Result:

Compared with control group

the paw swelling

spleen index and IL-6 levels of model group were significantly increased (

0.01

0.05). DHA could significantly reduce paw swelling (

0.05)

arthritis score index (

0.05)

and spleen index (

0.01)

thymus index (

0.05) and serum IL-6 level of AIA rats (

0.05). But the spleen index of CIA rats was not decreased

while the thymus index and IL-6 level were decreased but not statistically significant. In addition

DHA can significantly improve the pathological changes of ankle

and decrease the pathological score on AIA/CIA rats (

0.01). Compared with control group

DHA (4

16 μmol·L

-1

) groups had a remarkable effect on the cell viability (

0.05

0.01). The safe medication range of DHA was less than 2 μmol·L

- 1

. The level of IL-6 and the protein expression of NF-

B p65 in LPS group were higher than those of control group. Compared with LPS group

DHA (0.5 μmol·L

-1

) groups could significantly reduce the secretion of IL-6 (

0.05)

and inhibit the expression of NF-

B p65.

Conclusion:

DHA can alleviate the ankle joint lesion on rheumatoid arthritis rats. Its mechanism may be related to NF-

B signal pathway.

关键词

Keywords

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