基于TLR4/MAPKs/NF-κB炎症信号通路探讨三痹颗粒对Ⅱ型胶原性关节炎大鼠的治疗作用及机制研究

doi:10.11669/cpj.2020.07.005

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摘要目的探讨三痹颗粒对Ⅱ型胶原性关节炎大鼠的治疗作用以及对TLR4/MAPKs/NF-κB信号通路的影响。方法将60只Wistar大鼠随机分为空白对照组(CTL)、模型组、阳性对照组(地塞米松片,DXM)和受试药物低、中、高剂量组,每组各10只。建立胶原性关节炎(CIA)大鼠模型,各组于初次免疫2周后开始灌胃。连续灌服20 d,至免疫后35 d处死大鼠,观察大鼠状态、足肿胀度、关节炎指数(AI)、踝关节病变观察。采用ELISA法检测白介素(IL)-1β、IL-6、肿瘤坏死因子α(TNF-α)水平。采用qRT-PCR法和Western blot法检测Toll样受体4(TLR4)、核转录因子-κB(NF-κB)(p65)、p-NF-κB(p65)、p38、p-p38、细胞外信号调节激酶1/2(ERK1/2)、p-ERK1/2、c-Jun氨基末端激酶(JNK)、p-JNK mRNA和蛋白的表达水平。结果实验结束时,DXM组、受试药物高、中、低剂量组大鼠足踝关节肿胀程度和AI值均低于模型组(P<0.05)。经H&E染色和肉眼观察,受试药物可明显减轻足水肿以及滑膜组织病理学变化。DXM组和受试药物低、中、高剂量组大鼠外周血IL-1β、IL-6、TNF-α水平均明显低于模型组大鼠(P<0.05);而受试药物高剂量组大鼠外周血IL-1β、IL-6、TNF-α水平与DXM组大鼠比较未见统计学差异(P>0.05)。另外,模型组大鼠足踝关节滑膜组织TLR4、p-NF-κB(p65)、p-p38、p-ERK1/2、p-JNK mRNA和蛋白表达量明显高于CTL组(P<0.05);而且DXM组以及受试药物低、中、高剂量组较模型组明显降低(P<0.05);尤其以DXM组和受试药物高剂量组降低最为明显,与CTL组比较,无统计学差异(P>0.05)。结论三痹颗粒可能通过负性调控TLR4/MAPKs/NF-κB信号通路保护胶原性关节炎大鼠模型足踝关节炎症损伤。

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关键词 ：三痹颗粒, TLR4/MAPKs/NF-κB信号通路, 炎性因子, 类风湿性关节炎, CIA大鼠模型

Abstract：OBJECTIVE To discuss the effect of Sanbi granules on type Ⅱ collagen induced arthritis (CIA) rats by regulating the TLR4/MAPKs/NF-κB signal pathway. METHODS Sixty of Wistar rats were randomly divided into normal control group (CTL group, n=10), model group (n=10), positive control group (n=10) and low dose of Sanbi group (n=10), middle dose of Sanbi group (n=10), high dose of Sanbi group (n=10). The collagen induced arthritis (CIA) model of rats was adopted and treated for 20 days by intragastric administration from 2 weeks after primary immune. After exposure to sanbi for 35 d, the rats status, paw swelling, arthritis index (AI) and pathological change of synovial tissue were observed. The serum IL-1β, IL-6, tumor necrosis factor α (TNF-α) levels were detected by ELISA. And the expressions of Toll-like receptor 4 (TLR4), nuclear factor κB (NF-κB) (p65), p-NF-κB (p65), p38, p-p38, extracellular signal-regulated kinase 1/2 (ERK1/2), p-ERK1/2, c-Jun NH2-terminal kinase (JNK), p-JNK mRNA or proteins in synovial tissues were detected by qRT-PCR and Western blot. RESULTS At the end of experiment, compared with model group, the paw swelling degree and arthritis index (AI) of CIA rats in DXM group and low, middle, high dose of Sanbi groups were lower (P<0.05). Synovial tissue inflammatory of rats in high dose of Sanbi group changed obviously by H&E staining. The serum IL-1β, IL-6, TNF-α of CIA rats in DXM group and low, middle, high dose of Sanbi groups were lower than those in model group (P<0.05). And there was no difference of the serum IL-1β, IL-6, TNF-α between DXM group and high dose of Sanbi group (P>0.05). Besides, compared with CTL group, TLR4, p-NF-κB (p65), p-p38, p-ERK1/2, p-JNK mRNA and proteins in synovial tissues of CIA rats in model group, DXM group and low, middle, high dose of Sanbi groups were higher (P<0.05). And these mRNAs and proteins in DXM group and low, middle, high dose of Sanbi groups were lower than these in model group, particularly in DXM group and high dose of Sanbi group (P<0.05). CONCLUSION There are significant evidences that Sanbi granules could protect joint synovial tissues injury by down-regulation TLR4/MAPKs/NF-κB signal pathway on CIA rats.

Key words： Sanbi granule TLR4/MAPKs/NF-κB signal pathway inflammatory factor rheumatoid arthritis CIA rat model

收稿日期: 2020-03-06

PACS:

R965

基金资助:黑龙江省教育厅科研项目资助(11551463)

作者简介: 张春芳,女,主任医师,硕士生导师研究方向:中医药治疗类风湿关节炎机制研究 Tel:13904809839,(0592)2889597 E-mail:2810624579@qq.com

引用本文:

张春芳, 宋诚挚, 韩嘉琳, 赵丽华, 张万里, 马晓雪. 基于TLR4/MAPKs/NF-κB炎症信号通路探讨三痹颗粒对Ⅱ型胶原性关节炎大鼠的治疗作用及机制研究[J]. 中国药学杂志, 2020, 55(7): 519-526. ZHANG Chun-fang, SONG sincere, HAN Jia-lin, ZHAO Li-hua, ZHANG Wan-li, MA Xiao-xue. Effect of Sanbi Granules on TLR4/MAPKs/NF-κB Signal Pathway in Type Ⅱ Collagen Induced Arthritis Rats. Chinese Pharmaceutical Journal, 2020, 55(7): 519-526.

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