基于TLR4/NF-κB信号通路研究逍遥散对慢性心理应激加重胶原诱导性关节炎大鼠病情的调节作用

Study of Xiaoyaosan regulatory on collagen induced arthritis in rats aggravated by chronic psychological stress based on TLR4/NF-κB signaling pathway

目的:观察逍遥散对慢性心理应激加重胶原诱导性关节炎(CIA)大鼠病情的调节作用及可能机制。方法:40只Wistar大鼠适应性喂养1周后随机分为正常组(8只)与模型组(32只),正常组正常饲养4周,模型组大鼠在建立CIA动物模型基础上施加1周孤养与3周慢性不可预知温和应激(CUMS)建立类风湿关节炎伴发抑郁症(RAD)动物模型。通过比较第0天与第28天体质量、摄食量、糖水偏好率、旷场行为学变化及足趾容积、关节炎评分(AI)共同判断RAD模型大鼠是否制备成功。RAD模型大鼠全部成模并随机分为模型组、甲氨蝶呤组、逍遥散组、氟西汀组,每组8只。正常组与模型组每天灌胃0.9%氯化钠溶液,甲氨蝶呤组每周灌胃2次甲氨蝶呤水溶液(1 mg/kg),逍遥散组与氟西汀组除每周灌胃2次甲氨蝶呤水溶液(1 mg/kg)外,同时逍遥散组每天灌胃逍遥散水煎液(19.27 g/kg),氟西汀组每天灌胃氟西汀水溶液(2 mg/kg),给药持续6周。通过比较用药前后各组大鼠体质量、摄食量、糖水偏好率、旷场行为学及足趾容积、AI评分共同判断RAD大鼠抑郁与关节炎恢复程度;酶联免疫吸附法测定血清白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、白细胞介素-6(IL-6)水平;实时荧光定量PCR法测定海马IL-1β、TNF-α、IL-6、Toll样受体4(TLR4)、核转录因子-κB P65(NF-κB P65)mRNA表达;蛋白质印迹法(Western Blot)检测海马TLR4、NF-κB P65蛋白表达。结果:与正常组比较,模型组大鼠体质量增长缓慢,摄食量减少,糖水偏好率与旷场行为学异常,足趾容积与AI评分明显升高,兼具抑郁与关节炎外在表现,血清与海马组织中IL-1β、TNF-α、IL-6水平显著升高(P<0.01),海马组织中TLR4、NF-κB P65 mRNA与蛋白表达显著增加(P<0.01)。药物干预后,模型动物抑郁与关节炎表现得到有效改善,尤其加入抗抑郁药物逍遥散与氟西汀后效果更佳。同时,血清中IL-1β、TNF-α水平显著降低(P<0.05,P<0.01),海马组织IL-1β、TNF-α、IL-6 mRNA表达显著减少(P<0.05,P<0.01),海马组织中TLR4、NF-κB P65 mRNA与蛋白表达也显著减少(P<0.01)。结论:逍遥散可改善模型大鼠抑郁样行为同时减轻关节炎病情,其机制可能与调节TLR4/NF-κB信号通路,抑制炎症因子表达过多相关。

Objective:To observe the regulatory effect and possible mechanism of Xiaoyaosan on chronic psychological stress aggravated collagen induced arthritis(CIA)in rats.Methods:Forty Wistar rats were fed for 1 week and randomly divided into normal group(n=8)and model group(n=32).Normal group was fed normally for 4 weeks.On the basis of the established animal model of CIA,1 week of isolation and 3 weeks of chronic unpredictable mild stress(CUMS)were applied to establish the animal model of rheumatoid arthritis with depression(RAD)in the model group.The successful preparation of RAD model rats were determined by comparing the body weight,food intake,sugar water preference rate,open field behavioral changes,toe volume and arthritis index(AI)between day 0 and day 28.All RAD model rats were modeled,with a total of 32 rats,and randomly divided into model group,methotrexate group,Xiaoyaosan group and fluoxetine group,with 8 rats in each group.Normal group and model group were given normal saline,methotrexate group was given methotrexate solution(1 mg/kg)twice a week,Xiaoyaosan group and fluoxetine group were given methotrexate solution(1 mg/kg)twice a week,and Xiaoyaosan group was given Xiaoyaosan decoction(19.27 g·kg-1·d-1).Fluoxetine group was given fluoxetine solution(2 mg·kg-1·d-1).The experiment lasted for 6 weeks.Body weight,food intake,sugar water preference rate,open field behavior,toe volume and AI score were compared before and after treatment to evaluate the recovery degree of depression and arthritis in RAD model rats;the levels of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α),and interleukin-6(IL-6)in serum were measured by enzymelinked immunosorbent assay;the mRNA expressions of IL-1β,TNF-α,IL-6,toll like receptor 4(TLR4),nuclear factor-κB P65(NF-κB P65)in hippocampus were measured by Real-time fluorescence quantitative PCR;the protein expressions of TLR4 and NF-κB P65 in hippocampus were measured by Western Blot.Results:In the model group,body weight increased slowly,food intake decreased,sugar water preference rate and open field behavior were abnormal,toe volume and AI score were significantly increased,indicating that the animals in the model group had both depression and arthritis.The levels of IL-1β,TNF-αand IL-6in serum and hippocampus were increased(P<0.01),the mRNA and protein expressions of TLR4 and NF-κB P65 in hippocampus were increased(P<0.01).After drugs intervention,the manifestations of depression and arthritis in the model animals were effectively improved,especially after the addition of antidepressants Xiaoyaosan and fluoxetine.At the same time,the levels of IL-1βand TNF-αin serum were significantly decreased(P<0.05,P<0.01),and the mRNA expressions of IL-1β,TNF-αand IL-6 in hippocampus were distinctly decreased(P<0.05,P<0.01).The mRNA and protein expressions of TLR4 and NF-κB P65in hippocampus were also apparently decreased(P<0.01).Conclusion:Xiaoyaosan can alleviate arthritis and improve depressionlike behavior in model rats,and its mechanism may be related to regulating TLR4/NF-κB signaling pathway and inhibiting overexpression of inflammatory factors.