Objective: To observe the effect of acupuncture of "Yinlingquan"(SP9) and "Sanyinjiao"(SP6) on expression of phosphatidylinositol-3 kinase/protein kinase B/mammalian target protein of rapamycin (PI3K/Akt/mTOR) signaling in adjuvant arthritis (AA) rats, so as to explore its mechanism underlying improvement of AA.
Methods: Forty-eight male Wistar rats were randomly divided into normal control, AA model, acupuncture and medication (tripterygium wilfordii) groups, with 12 rats in each group. The AA model was established by putting the rats in a windy, cold and wet environment for 12 h, once every day for 21 days and injection of Freund's complete adjuvant (CFA) into the sole of the right hindlimb on the 21st day. Manual acupuncture stimulation was applied at SP9 and SP6 for 30 min/time, once a day for 21 days. Rats of the medication group received gavage of tripterygium wilfordii tablets solution (8 mg/kg), once a day for 21 days, and those of the normal control group and model group received gavage of the same amount of normal saline, once a day for 21 days. The degree of joint swelling and arthritis index (AI) were detected 1 day before modeling, 3 days after modeling, and 21 days after the treatment. Twenty-four hours after the last treatment, the contents of serum cytokines interleukin (IL)-17, IL-6 and tumor necrosis factor (TNF)-α were detected by enzyme-linked immunosorbent assay (ELISA); and changes of synovial ultrastructure were observed under electron microscope. Western blot was used to detect the expression levels of PI3K, Akt, phosphorylated protein kinase B (p-Akt), phosphorylated mammalian rapamycin target protein (p-mTOR), mTOR, microtubule associated protein 1 light chain 3B (LC3-Ⅱ) and mammalian atg6 homologous protein (Beclin-1) in the synovial membrane tissue.
Results: Compared with the normal control group, the joint swel-ling degree and AI, contents of serum TNF-α, IL-6 and IL-17, and the expression levels of PI3K, Akt, p-Akt, mTOR and p-mTOR in the synovium were increased in the model group (P<0.05), while the expression levels of LC3-Ⅱ and Beclin-1 proteins in the synovium were significantly decreased (P<0.05). Compared with the model group, the joint swelling degree and AI, contents of serum TNF-α, IL-6 and IL-17, and the expression levels of PI3K, Akt, p-Akt, mTOR and p-mTOR were significantly decreased in the acupuncture and medication groups (P<0.05), while the expresion levels of LC3-Ⅱ and Beclin-1 proteins were significantly increased （P<0.05）. Comparison between the two treatment groups showed that the therapeutic effects of acupuncture were obviously weaker than those of medication in down-regulating TNF-α and IL-6, Akt, p-Akt and mTOR levels (P<0.05) and in up-regulating Beclin-1 expression (P<0.05). Outcomes of electron microscope displayed widened nuclear membrane space, some fractured mitochondrial cristae with vacuoles, expanded rough endoplasmic reticulum, reduction of autophagosomes in the cytoplasm and ruptured synovial cell membrane in the model group, and increase of autophagosomes, deformed organelles in the acupuncture and medication groups.
Conclusion: Acupuncture can relieve the inflammatory reactions and joint synovial injury of the affected joint in AA rats which may be associated with its effects in inhibiting PI3K/Akt/mTOR signaling and increasing the auto-phagy level of synovial cells.
目的：基于磷脂酰肌醇-3激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/AKT/mTOR)信号通路，观察针刺“阴陵泉”“三阴交”对佐剂性关节炎(AA)大鼠膝关节滑膜细胞自噬水平的影响，探讨针刺治疗类风湿关节炎的可能机制。方法：Wistar大鼠随机分为正常组、模型组、针刺组、雷公藤组，每组12只。采用风、寒、湿环境因素+弗氏完全佐剂复合造模方法复制AA大鼠模型。针刺组针刺大鼠“阴陵泉”“三阴交”穴，30 min/次，1次/d，连续治疗21 d；雷公藤组予以雷公藤多苷片溶液灌胃给药（8 mg·kg-1·d-1），1次/d，连续治疗21 d。分别在致炎后第3 天、治疗第21天测量各组大鼠右后足跖容积并计算关节肿胀度(E)、评价关节炎指数(AI)；ELISA法检测血清肿瘤坏死因子 (TNF)-α、白细胞介素 (IL)-17、IL-6的含量；电镜下观察右膝关节滑膜超微结构的变化；Western blot法检测右膝关节滑膜组织中PI3K、AKT、磷酸化蛋白激酶B(p-AKT)、mTOR、磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)、微管相关蛋白1轻链3B (LC3-Ⅱ)、哺乳动物ATG6同源蛋白(Beclin-1)的表达。结果：与正常组比较，模型组大鼠E及AI显著升高(P<0.05)；治疗后针刺组、雷公藤组大鼠E及AI显著低于模型组(P<0.05)。与正常组比较，模型组血清TNF-α、IL-6、IL -17含量升高(P<0.05)，右膝关节滑膜组织PI3K、AKT、p-AKT、mTOR、p-mTOR表达升高(P<0.05)，Beclin-1、LC3-Ⅱ蛋白表达显著降低(P<0.05)；与模型组比较，针刺组和雷公藤组血清TNF-α、IL-6、IL-17含量降低(P<0.05)，右膝关节滑膜组织PI3K、AKT、p-AKT、mTOR、p-mTOR表达降低(P<0.05)，Beclin-1、LC3-Ⅱ蛋白表达显著升高 (P<0.05)；与雷公藤组比较，针刺组TNF-α、IL-6水平升高(P<0.05)，右膝关节滑膜组织AKT、p-AKT、mTOR蛋白表达升高(P<0.05)，Beclin-1表达降低(P<0.05)。模型组右膝关节滑膜细胞细胞器减少、损伤明显，自噬小体减少；针刺组自噬小体增多、清晰可见，可见不规则、变形细胞器；雷公藤组可见增多的自噬小体及变形、萎缩的细胞器。结论：针刺能够通过抑制PI3K/AKT/mTOR信号通路，提高AA大鼠滑膜细胞自噬水平，降低炎性因子及E、AI水平，从而减轻AA大鼠关节滑膜损伤，保护滑膜组织。.
Keywords: Acupuncture; Adjuvant arthritis; Autophagy; PI3K/AKT/mTOR signaling pathway; Synovial tissue.