甘草附子汤对膝关节骨关节炎大鼠NF-κB信号通路的影响

Effect of Gancao Fuzi decoction on NF-κB signaling pathway in knee osteoarthritis rats

目的 基于核因子-κB(NF-κB)信号通路探讨甘草附子汤(GCFZ)对膝关节骨关节炎(knee osteoarthritis,KOA)大鼠的作用及其机制。方法 将40只SD大鼠随机分为5组,每组8只,采用膝关节腔注射木瓜蛋白酶混合液的方法建立大鼠KOA模型,观察各组大鼠膝关节宽度、机械性痛阈和右下肢负重能力的变化;酶联免疫吸附测定法(Elisa)检测大鼠膝关节腔关节液肿瘤坏死因子-α(TNF-α)、白介素-6(IL-6)和白介素-1β(IL-1β)的水平;实时荧光定量聚合酶链反应(RT-PCR)方法检测膝关节滑膜组织TNF-α、IL-6、IL-1β mRNA表达水平;蛋白免疫印迹(Western blot)法检测膝关节滑膜组织NF-κB信号通路关键蛋白p65、抑制蛋白κBα(IκBα)、磷酸化抑制蛋白κBα(p-IκBα)、抑制蛋白激酶α/β(IKKα/β)、磷酸化抑制蛋白激酶α/β(p-IKKα/β)的表达情况。结果 第14、28天,与空白组比较,模型组膝关节宽度增加、机械性痛阈和右下肢负重百分比显著降低(P<0.01),关节液TNF-α、IL-6和IL-1β水平显著上升(P<0.01),膝关节滑膜组织TNF-α、IL-6和IL-1β mRNA表达提高(P<0.01),p65、p-IκBα和p-IKKα/β蛋白表达明显上调、IκBα蛋白降解明显。与模型组比较,第14、28天,3个给药组均能降低膝关节宽度(P<0.01)、提高大鼠机械性痛阈和右下肢负重能力(P<0.05);阳性药物组和GCFZ高剂量组能降低关节液TNF-α水平(P<0.05);3个给药组均能显著降低IL-6和IL-1β水平(P<0.05)、抑制滑膜组织TNF-α、IL-6和IL-1β mRNA表达(P<0.01);阳性药物和GCFZ能明显抑制KOA大鼠滑膜组织p65、IκBα蛋白的表达,抑制IκBα和IKKα/β蛋白的磷酸化。结论 甘草附子汤对KOA有一定的防治作用,作用机制可能与调控NF-κB信号通路有关。

Objective To investigate the effect and mechanism of Gancao Fuzi decoction(GCFZ) on knee osteoarthritis(KOA) rats based on NF-κB signaling pathway. Methods 40 SD rats were randomly divided into five groups, 8 rats per group. The rat KOA model was established by injecting papain mixture into the knee joint.The changes of knee joint width, mechanical pain threshold and weight-bearing capacity of right lower limb of rats in each group were observed. ELISA was used to measure the levels of TNF-α, IL-6 and IL-1β in the synovial fluid of rat knee joint. RT-PCR was used to determine the expression of TNF-α, IL-6 and IL-1β mRNA in knee joint synovial tissue. Western blot was used to detect the expression of NF-κB pathway proteins including p65,IκBα, p-IκBα, IKKα/β and p-IKKα/β in knee joint synovial tissues. Results On day 14 and 28, compared with the blank group, the knee joint width was obviously increased, but the mechanical pain threshold and the weightbearing percentage of the right lower limb were decreasedin the model group(P < 0.01). The levels of TNF-α, IL-6and IL-1β in the synovial fluid were elevated, and the expression of TNF-α, IL-6 and IL-1β mRNA in the synovial tissue of knee joint was significantly increased in the model group(P < 0.01). Meantime, the expression of p65,p-IκBα and p-IKKα/β proteins in synovial tissue was upregulated, and the degradation of IκBα protein was obvious. Compared with the model group, on days 14 and 28, all three treatment groups reduced the width of knee joint(P < 0.01), and elevated rat mechanical pain threshold and the weight-bearing capacity of the right lower limb(P < 0.05). The positive drug group and high dose GCFZ group reduced the level of TNF-α in the synovial fluid(P < 0.05). Three administration groups significantly suppressed the levels of IL-6 and IL-1β in the synovial fluid(P < 0.05), and inhibited the expression of TNF-α, IL-6 and IL-1β mRNA in synovial tissue(P <0.01). The positive drug and GCFZ inhibited the expression of p65 and IκBα proteins, and phosphorylation of IκBα and IKKα/β proteins in the synovial tissue of KOA rats. Conclusion GCFZ has a certain therapeutic effect on KOA, which is related to the regulation of NF-κB signaling.