基于TLR4/MyD88/NF-kB信号通路探讨追风透骨胶囊减缓兔膝骨关节炎模型软骨退变的作用机制  

The mechanism of Zhuifeng Tougu Capsule for slowing down cartilage degeneration in rabbit model with knee osteoarthritis based on TLR4/MyD88/NF-κB signaling pathway

作　　者：曹寅生[1] 易强 邝高艳[1] 危建文 金久楚 罗振华[1] CAO Yinsheng;YI Qiang;KUANG Gaoyan;WEI Jianwen;JIN Jiuchu;LUO Zhenhua(The First Hospital of Hunan University of Chinese Medicine,Changsha,Hunan 410007,China;Hunan University of Chinese Medicine,Changsha,Hunan 410208,China)

机构地区：[1]湖南中医药大学第一附属医院,湖南长沙410007 [2]湖南中医药大学,湖南长沙410208

出　　处：《湖南中医药大学学报》2023年第2期240-248,共9页Journal of Hunan University of Chinese Medicine

基　　金：湖南省自然科学基金项目(2020JJ5422)。

摘　　要：目的 观察追风透骨胶囊对兔膝骨关节炎(knee osteoarthritis, KOA)模型关节软骨退变的干预作用,并基于Toll样受体4(Toll like receptor 4, TLR4)/髓细胞分化初级反应蛋白88(myeloid differentiation primary response protein 88, MyD88)/核因子kappa-B(nuclear factor kappa-B, NF-κB)信号通路探讨追风透骨胶囊的可能作用机制。方法 从50只6月龄雄性新西兰兔中随机选取10只作为正常组,其余40只为KOA造模组,用改良Videman造模法制备兔KOA模型。模型验证后,从正常组中随机选取6只作为空白组(A组),KOA造模组抽取30只随机分为模型组(B组)、硫酸氨基葡萄糖组(C组)、追风透骨胶囊低剂量组(D组)、追风透骨胶囊中剂量组(E组)、追风透骨胶囊高剂量组(F组),每组6只。A、B组予蒸馏水灌胃,C、D、E、F组予相应药物灌胃,疗程均为6周。给药结束后,取造模侧膝关节软骨,予大体及HE染色观察,并用Pelletier评分及Mankin评分评估;以Western blot、RT-PCR法检测软骨中TLR4、My D88、NF-κB蛋白及其mRNA的表达;免疫组化法检测白细胞介素-1β(interleukin-1β, IL-1β)、白细胞介素-6(interleukin-6, IL-6)、肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)的含量。结果 与A组比较,B组膝关节软骨破坏明显,Pelletier评分及Mankin评分均升高(P<0.01),软骨中TLR4、MyD88、NF-κB蛋白及其mRNA表达水平均上调(P<0.01),IL-1β、IL-6、TNF-α含量均升高(P<0.01)。与B组比较,C、D、E、F组的软骨损伤程度轻,软骨Mankin评分均降低(P<0.01),软骨中TLR4、MyD88、NF-κB蛋白及m RNA表达水平均下调(P<0.01),IL-1β、IL-6、TNF-α含量均降低(P<0.01),此外,F组软骨Pelletier评分较B组降低明显(P<0.05)。结论 追风透骨胶囊能延缓改良Videman造模法诱导的兔KOA模型关节软骨退变,其作用机制可能与下调软骨中TLR4、MyD88、NF-κB蛋白及m RNA的表达,抑制TLR4/MyD88/NF-κB信号通路的活化,降低IL-1β、IL-6、TNF-α在软骨中的含量,从�Objective To observe the intervention effects of Zhuifeng Tougu Capsule on articular cartilage degeneration in rabbit model with knee osteoarthritis(KOA), and to explore the possible mechanism of Zhuifeng Tougu Capsule based on Toll like receptor 4(TLR4)/Myeloid differentiation primary response protein 88(MyD88)/nuclear factor kappa-B(NF-κB) signaling pathway.Methods From 50 6-month-old male New Zealand rabbits, we randomly selected 10 rabbits as normal group, and the other 40 as KOA model group. The rabbit KOA model was prepared by the modified Videman modeling. After model verification, 6 rabbits from the normal group were randomly selected as the blank group(Group A), and 30 rabbits from the KOA model group were randomly divided into model group(Group B), glucosamine sulfate group(Group C), low-dose Zhuifeng Tougu Capsule group(Group D), medium-dose Zhuifeng Tougu Capsule group(Group E), and high-dose Zhuifeng Tougu Capsule group(Group F), with 6 rabbits in each group. Group A and Group B were given intragastric gavage of distilled water, and Groups C, D, E and F were given intragastric gavage of the corresponding drugs. All the above courses of treatment were 6 weeks. After administration, the cartilage of the knee joint in the model was taken for gross observation and HE staining observation, and was evaluated with Pelletier score and Mankin score;TLR4, MyD88, NF-κB protein and its mRNA expression in cartilage were detected by Western blot and RTPCR;the content of its interleukin-1β(IL-1β), interleukin-6(IL-6) and tumor necrosis factor-α(TNF-α) was detected by immunohistochemistry. Results Compared with Group A, in Group B, the cartilage of knee joint was damaged significantly, Pelletier score and Mankin score both increased(P<0.01), TLR4, MyD88, NF-κB protein and the expression levels of mRNA in cartilage were up-regulated(P<0.01), and the content of IL-1β, IL-6 and TNF-α increased(P<0.01). Compared with Group B, in Groups C, D, E and F, the degree of cartilage injury was mild, Mankin score of cart

关 键 词：膝骨关节炎 追风透骨胶囊 Toll样受体4 髓细胞分化初级反应蛋白88 核因子kappa-B 白细胞介素-1β 白细胞介素-6 肿瘤坏死因子-α 

分 类 号：R274.9[医药卫生—中西医结合]