Objective: To observe the effects of electroacupuncture (EA) penetration needling on Toll-like receptors 4/myeloid differentiation factor 88/nuclear factor-kappa B (TLR4/MyD88/NF-κB) signaling pathway in rat synovium and the serum-related inflammatory factors, so as to explore the mechanism of EA penetration needling on synovial inflammation in rats with knee osteoarthritis (KOA).
Methods: SD male rats were randomly divided into sham-operation group, model group, EA+penetration needling group, and conventional EA group, with 16 rats in each group. The rats model was prepared by anterior cruciate ligment transection and these rats were forced to exercise for 8 weeks after operation. After successful modeling, in the EA+penetration needling group, the needles were inserted at "Dubi" (ST35) "Neixiyan" (EX-LE4), and at "Xuehai"(SP10) "Liangqiu"(ST34) on the right hind limb, towards each other, 5-8 mm in depth, respectively. In the conventional EA group, the needles were inserted at ST35 and EX-LE4 on the right hind limb, obliquely, at 30° angle to the skin, 3-5 mm in depth; and were inserted at SP10 and ST34 on the right hind limb perpendicularly, 3-5 mm in depth. In these two groups, electric stimulation was operated with dense-disperse wave, 2 Hz/10 Hz in frequency and 0.5-1.5 mA in intensity, retained for 20 min in each treatment. The treatment was given once daily, 10 days as 1 course of treatment, and 2 courses were required at the interval of 2 days. After the intervention, the knee joint effusion was observed by musculoskeletal ultrasound; the contents of IL-1β, IL-6 and TNF-α in serum were determined by ELISA; the morphological changes in the synovium were observed after H.E. staining; the positive expression of NF-κB p65 in the synovial membrane was detected by immunohistochemical method; the expression levels of TLR4, MyD88, TRAF-6 and NF-κB p65 proteins in the synovial membrane were determined by Western blot.
Results: Compared with the sham-operation group, in the model group, the knee joint effusion was obviously increased, the synovial lining cells were distributed irregularly, the cells were disarranged, the pannus was formed largely, and a great number of the inflammatory cells were infiltrated; the contents of serum IL-1β, IL-6 and TNF-α, the positive expression of NF-κB p65, the protein expression levels of TLR4, MyD88, TRAF-6 and NF-κB p65 in the synovial tissue were increased (P<0.05). Compared with the model group, the knee joint effusion was reduced, the synovial lining cells were proliferated, a small number of the inflammatory cells were infiltrated, and the pannus was formed lightly; the contents of serum IL-1β, IL-6 and TNF-α, the positive expression of NF-κB p65, the protein expression levels of TLR4, MyD88, TRAF-6 and NF-κB p65 in the synovial tissue were lower (P<0.05) in the EA+penetration needling group and the conventional EA group. In the conventional EA group, the knee joint effusion was increased, the synovial lining cells were proliferated, the inflammatory cells were infiltrated largely, and the pannus was formed increasingly; the contents of serum IL-1β, IL-6 and TNF-α, and the protein expression levels of TLR4, MyD88 and NF-κB p65 in the synovial tissue were increased when compared with the EA+penetration needling group (P<0.05).
Conclusion: The EA+penetration needling can significantly relieve the synovial inflammatory reaction and the knee joint effusion in KOA rats. The mechanism is probably related to down-regulating the downstream inflammatory cascade through inhibiting the transduction of TLR4/MyD88/NF-κB signaling pathway.
目的：观察电针透刺法对大鼠滑膜Toll样受体4/髓样分化因子88/核转录因子-κB(TLR4/MyD88/NF-κB)信号通路及血清相关炎性因子的影响，探讨电针透刺法对膝关节骨关节炎(KOA)大鼠滑膜炎性反应的影响机制。方法：雄性SD大鼠随机分为假手术组、模型组、电针透刺组、普通电针组，每组16只。采用前交叉韧带离断术结合术后强迫运动制备KOA大鼠模型。电针透刺组于右侧“犊鼻”“内膝眼”予相互透刺5~8 mm，“血海”“梁丘”予相互透刺5~8 mm；普通电针组于右侧“犊鼻”“内膝眼”予向上与皮肤呈30°角斜刺3~5 mm，“血海”“梁丘”予直刺3~5 mm。两组均同样连接电针，疏密波，频率2 Hz/10 Hz，强度0.5~1.5 mA，1次/d，每次20 min，10 d为1个疗程，每疗程间休息2 d，共2个疗程。肌骨超声观察大鼠膝关节积液情况；ELISA法检测大鼠血清IL-1β、IL-6及TNF-α的含量；HE染色法观察大鼠滑膜形态学变化；免疫组织化学法检测大鼠滑膜组织中NF-κB p65的阳性表达；Western blot法检测大鼠滑膜组织中TLR4、MyD88、肿瘤坏死因子受体相关因子-6(TRAF-6)及NF-κB p65蛋白表达水平。结果：与假手术组比较，模型组膝关节积液明显增多，滑膜内衬层细胞分布不规则，细胞排列紊乱，生成大量血管翳，可见大量炎性细胞浸润，血清IL-1β、IL-6、TNF-α含量及滑膜组织中NF-κB p65阳性表达增加(P<0.05)，TLR4、MyD88、TRAF-6、NF-κB p65蛋白表达水平升高(P<0.05)；与模型组比较，电针透刺组和普通电针组膝关节积液明显减少，可见滑膜内衬层细胞增生、少量的炎性细胞浸润和血管翳生成，血清IL-1β、IL-6、TNF-α含量及滑膜组织NF-κB p65阳性表达减少(P<0.05)，TLR4、MyD88、TRAF-6、NF-κB p65蛋白表达水平降低(P<0.05)；与电针透刺组比较，普通电针组膝关节积液较多，血清IL-1β、IL-6、TNF-α含量及滑膜组织TLR4、MyD88、NF-κB p65蛋白表达水平均升高(P<0.05)。结论：电针透刺法可显著改善KOA大鼠膝关节滑膜炎性反应状态，减少膝关节积液，其作用机制可能是通过抑制TLR4/MyD88/NF-κB信号通路的转导而抑制其下游炎性级联反应。.
Keywords: Electroacupuncture; Knee osteoarthritis; Myeloid differentiation factor 88; Nuclear factor-kappa B; Penetration needling; Synovium; Toll-like receptors 4.