复方芪芎颗粒通过TLR4/MCP-1通路抑制类风湿关节炎大鼠软骨炎症的机制

Compound astragalus ligustici granule inhibits cartilage inflammation in rats with rheumatoid arthritis via TLR4/MCP-1 pathway

目的 基于Toll样受体4(TLR4)/单核细胞趋化因子-1(MCP-1)通路观察复方芪芎颗粒对类风湿关节炎大鼠软骨炎症的影响,探讨其治疗类风湿关节炎的机制。方法 取50只雄性SD大鼠,随机选择8只大鼠作为空白组,其余大鼠建立胶原诱导关节炎(CIA)模型。造模第14天,将造模成功的30只大鼠随机分为模型组8只、TAK-242组7只、复方芪芎组7只、复方芪芎+TAK-242组8只。从第15天开始,复方芪芎组按0.03 g/mL剂量给予复方芪芎颗粒液灌胃,1次/d;TAK-242组给予TAK-242 3 mg/(kg·d)腹腔注射,2次/周;复方芪芎+TAK-242组给予复方芪芎颗粒液(0.03 g/mL)灌胃(1次/d),TAK-242 3 mg/(kg·d)腹腔注射(2次/周);空白组与模型组给予0.9%氯化钠溶液灌胃,1次/d。各组连续干预至第35天。分别记录造模后第14,17,20,23,26,29,32,35天大鼠足趾肿胀度、关节炎指数评分;干预至第35天取血检测血清白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、白细胞介素-8(IL-8)、肿瘤坏死因子-α(TNF-α)水平,HE染色和抗酒石酸酸性磷酸酶(TRAP)染色观察软骨组织形态和破骨细胞数量,免疫荧光染色观察软骨组织中TLR4、MCP-1、IL-6、TNF-α和滑膜组织中CD68阳性表达情况。结果实验第14~35天,模型组大鼠足趾肿胀度和关节炎指数均明显高于同期空白组(P均<0.05);第23~35天TAK-242组、复方芪芎+TAK-242组大鼠足趾肿胀度和第26~35天大鼠关节炎指数均明显低于同期模型组(P均<0.05);第32~35天TAK-242组、复方芪芎+TAK-242组大鼠足趾肿胀度和关节炎指数均明显低于同期复方芪芎组(P均<0.05);TAK-242组与复方芪芎+TAK-242组各时间点的足趾肿胀度和关节炎指数比较差异均无统计学意义(P均>0.05)。模型组大鼠血清IL-1β、IL-6、IL-8、TNF-α水平和软骨组织中TLR4、MCP-1、IL-6、TNF-α阳性细胞比例及滑膜组织中CD68+阳性细胞比例均明显高于空白组(P均<0.05),大鼠软骨损伤明显,破骨细胞数量增多;各药物组大鼠血清IL-1β、IL-6、IL-8、TNF-α水平和软骨组织中TLR4、MCP-1、IL-6、TNF-α阳性细胞比例及滑膜组织中CD68+阳性细胞比例均明显低于模型组(P均<0.05),软骨损伤明显减轻,破骨细胞数量减少,且TAK-242组和复方芪芎+TAK-242组上述各指标均明显低于中药组(P均<0.05),TAK-242组与复方芪芎+TAK-242组上述各指标比较差异均无统计学意义(P均>0.05)。结论 复方芪芎颗粒可以缓解类风湿关节炎大鼠关节炎症,保护软骨细胞,这可能与调控TLR4/MCP-1通路活性减少炎症因子浸润有关。

Objective It is to observe the effect of compound astragalus ligustici granule(CALG)on cartilage inflammation in rats with rheumatoid arthritis based on the Toll-like receptor 4(TLR4)/monocyte chemokine-1(MCP-1)pathway,and to explore its mechanism in the treatment of rheumatoid arthritis.Methods Fifty male SD rats were taken,in which 8 rats were randomly selected as the blank group,and the rest of the rats were modeled as collagen-induced arthritis(CIA).On the 14th day of modeling,30 successfully modeled rats were randomly divided into 8 rats in the model group,7 rats in the TAK-242 group,7 rats in the CALG group,and 8 rats in the CALG+TAK-242 group.Starting from day 15,the CALG group was given CALG solution 0.03 g/mL by gavage,once daily;the TAK-242 group was given TAK-2423 mg/(kg·d)by intraperitoneal injection,twice per week;the CALG+TAK-242 group was given CALG solution 0.3 g/kg by gavage once daily,and TAK-2423 mg/(kg·d)by intraperitoneal injection,twice per week;the blank group and model group were given 0.9%sodium chloride solution by gavage,once daily.Each group was continuously intervened until the 35th day.Toe swelling and arthritis index scores of the rats were recorded on days 14,17,20,23,26,29,32,and 35 after modeling,respectively;the serum levels of interleukin-1β(IL-1β),interleukin-6(IL-6),interleukin-8(IL-8),tumor necrosis factor-α(TNF-α)were determined after the rats blood was taken on the 35th day of treatment,the morphology of cartilage tissue and the number of osteoclasts were observed by HE staining and tartrate acid phosphatase(TRAP)staining,and the positive expressions of TLR4,MCP-1,IL-6,TNF-αin cartilage tissue,and CD68 in synovial tissue were detected by immunofluorescence staining.Results The toe swelling degree and arthritis index of the rats in the model group on the 14th to 35th day of experiment were significantly higher than those in the blank group at the same time period(all P<0.05);the toe swelling degree of the rats on the 23 rd to 35th day of experiment and arthritis index on the 26th to 35th day of experiment in the TAK-242 group and CALG+TAK-242 group were significantly lower than those in the model group at the same time period(all P<0.05);the toe swelling degree and arthritis index of the rats on the 32 nd to 35th day of experiment in the TAK-242 group and CALG+TAK-242 group were significantly lower than those in the CALG group at the same time period(all P<0.05);the differences in the toe swelling degree and arthritis index of the rats at each time points were not statistically significant between the TAK-242 group and CALG+TAK-242 group(all P>0.05).The serum levels of IL-1β,IL-6,IL-8,and TNF-α,the proportions of cells with positive TLR4,MCP-1,IL-6,and TNF-αin cartilage tissues and the proportion of cells with positive CD68 in synovial tissues of the rats in the model group were significantly higher than those in the blank group(all P<0.05),and the cartilage damage of the rats was obvious with increased osteoblasts;the serum levels of IL-1β,IL-6,IL-8,and TNF-α,the proportions of cells with positive TLR4,MCP-1,IL-6,and TNF-αin cartilage tissues and the proportion of cells with positive CD68 in synovial tissues of the rats in each treatment group were significantly lower than those in the model group(all P<0.05),the cartilage damage was significantly alleviated,the osteoclasts were decreased,and all of the above indicators in the TAK-242 group and the CALG+TAK-242 group were significantly lower than those in the CALG group(all P<0.05),the differences in all the indexes were not statistically significant between the TAK-242 group and CALG+TAK-242 group(all P>0.05).Conclusion Compound astragalus rhizome granules can alleviate joint inflammation and protect chondrocytes in rats with rheumatoid arthritis,which may be related to the modulation of TLR4/MCP-1 pathway activity to reduce the infiltration of inflammatory factors.