王少贤 李振彬

【摘 要】目的：觀察胶原诱导性关节炎（CIA）大鼠下丘脑视交叉上核（SCN）生物钟基因Bmal1、Clock的表达变化，以及雷公藤多苷的调节作用。方法：建立CIA大鼠模型，随机分为模型对照组、醋酸泼尼松组、雷公藤多苷组，并设空白对照组，每组8只。醋酸泼尼松组、雷公藤多苷组分别于20：00灌胃给药，雷公藤多苷用药量10.5 mg·kg-1·d-1，醋酸泼尼松片用药量1.5 mg·kg-1·d-1;空白对照组、模型对照组灌服生理盐水。连续给药6周。观测各组大鼠足爪、关节肿胀情况，ELISA法测定血清肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-1β、IL-6水平，qRT-PCR和Western-blot法检测SCN Bmal1、Clock基因和蛋白表达。结果：①实验结束，与空白对照组比较，CIA模型大鼠左后踝关节围长、足跖厚度、足趾容积、关节炎指数（AI）评分均明显增加（P < 0.05），血清TNF-α、IL-1β、IL-6水平明显升高（P < 0.05），下丘脑SCN Bmal1、Clock蛋白和基因表达均明显下降（P < 0.05）。②与模型对照组比较，醋酸泼尼松组、雷公藤多苷组均能明显改善大鼠足爪、关节肿胀程度，降低血清TNF-α、IL-1β、IL-6水平，增加SCN Bmal1、Clock蛋白和基因表达（P < 0.05），雷公藤多苷组对SCN Bmal1表达的调节作用优于醋酸泼尼松组（P < 0.05）。结论：关节炎的发病影响了血清炎性细胞因子和下丘脑SCN生物钟的表达;雷公藤多苷治疗CIA模型大鼠关节炎症的作用机制与调控下丘脑SCN核心钟基因表达和外周炎性细胞因子分泌水平相关。

【关键词】 胶原诱导性关节炎;雷公藤多苷;下丘脑视交叉上核;生物钟;Bmal1;Clock;大鼠

【ABSTRACT】Objective：To observe the expression of CLOCK and Bmall in suprachiasmatic nucleus（SCN）of hypothalamus in collagen induced arthritis（CIA）rats and the regulation of Tripterygium.Methods：The rat model of collagen induced arthritis was established and randomly divided into a model control group，a prednisone acetate group and a tripterygium glycosides group.There was a blank control group.There were 8 rats in each group.The prednisone acetate group and the tripterygium glycosides group were given intragastric administration at 20：00，respectively with dosage of 1.5 mg·kg-1·d-1 and 10.5 mg·kg-1·d-1，once a day.The blank control group and the model control group were given normal saline successively for 6 weeks.The paw and joint swelling of rats in each group were observed.ELISA was used to detected the levels of TNF-α，IL-1β and IL-6.The gene and protein expressions of CLOCK and Bmall in SCN were detected by qRT-PCR and Western-blot.Results：①At the end of the experiment，the left posterior ankle joint circumference，plantar thickness，toe volume，arthritis index（AI）scores of CIA model rats were significantly increased compared with the blank control group（P < 0.05）.Levels of TNF-α，IL-1β，IL-6 were significantly increased（P < 0.05），and the gene and protein expressions of CLOCK and Bmall in SCN significantly decreased（P < 0.05）.②Compared with the model control group，the swelling degree of joint and paw could be significantly improved，the levels of TNF-α，IL-1β and IL-6 decreased and the gene and protein expressions of CLOCK and Bmall in SCN increased in the prednisone acetate group and the tripterygium glycosides group.The regulatory effect of in the tripterygium glycosides group on Bmall expression in SCN was better than that in the prednisone acetate group（P < 0.05）.Conclusion：The pathogenesis of arthritis affects the expression of serum inflammatory cytokines and hypothalamic SCN circadian clock;the mechanism of Tripterygium wilfordii polyglycoside in treating CIA model rats is related to the regulation of hypothalamic SCN core clock gene expression and peripheral inflammatory cytokine secretion level.

【Keywords】 collagen induced arthritis;tripterygium glycosides;suprachiasmatic nucleus of hypothalamus;biological clock;Bmall;Clock;rats

类风湿关节炎（rheumatoid arthritis，RA）为慢性全身性免疫性疾病，其主要临床表现如关节僵硬、疼痛、肿胀等晨起明显，具有明显的昼夜节律特点;与RA相关的多种炎症因子[白细胞介素（IL）-1β、IL-6等]及抗炎因子（Cotisol）也呈现明显的昼夜变化[1-4]。研究发现，这种节律变化与生物钟基因表达变化密切相关[5-7]。

本研究拟观察雷公藤多苷对胶原诱导关节炎（CIA）大鼠下丘脑视交叉上核（SCN）生物钟基因Clock、Bmal1的表达，以及模型大鼠关节炎症和外周血炎性细胞因子的变化，旨在从生物钟基因角度探讨雷公藤多苷治疗RA的新机制。

1 材料与方法

1.1 实验动物 SPF级SD大鼠60只，6～7周龄，雄性，体质量（200±20）g，购自北京维通利华实验动物技术有限公司，实验动物许可证号SCXK（京）2016-0011。清洁级动物房饲养，室温（21±1）℃，相对湿度40%～60%，固定光照明、暗各12 h（明7：00～19：00，暗19：00～7：00），自由摄食、饮水。……