[Study on the underlying mechanism of acupotomy in inhibiting bone destruction in collagen-induced arthritis rats based on OPG/RANKL/NF-κB signaling pathway].

OBJECTIVES: To explore the mechanism of acupotomy in inhibiting bone destruction in rats with collagen-induced arthritis(CIA).

METHODS: Twenty-four SD female rats were randomly allocated to blank control, model and acupotomy groups, with 8 rats in each group. The CIA model was established by subcutaneous injection of bovine type II collagen + Freund's incomplete adjuvant at the base of the tail (on day 0 and 7). The rats of the acupotomy group received alternative relaxing treatment of Tongbi Qiji technique (sternum-xiphoid) and Tongyang Qishu technique (the region around Dazhui[GV14]) with needle knife, once every 3 d, for a total of 9 sessions. The rats of the blank control and model groups received grabbing and fixing as those of the acupotomy group. After the intervention, the arthritis index (AI) score (0-4 points for each limb, 16 points in total) was evaluated. Micro-CT was used to observe the bone destruction of the ankle joint and to analyze the related bone parameters including bone mineral density (BMD), bone surface/bone volume (BS/BV), bone volume/ total volume (BV/TV), bone trabecular number (Tb.N), trabecular thickness (Tb.Th) and trabecular separation/spacing (Tb.Sp). The tartrate-resistant acid phosphatase (TRAP) staining was used to observe changes of the number of osteoclast (OC) in the ankle tissue, and the HE staining was used to observe histopathological changes of the ankle joint. The contents of serum tumor necrosis factor-α (TNF-α), interleukin (IL) -6 and IL-1β were detected by ELISA. The mRNA and protein expression levels of osteoprotegin (OPG), receptor activator of nuclear factor-κB ligand (RANKL), nuclear factor-κB (NF-κB) p65, and nuclear factor-κB inhibitory protein kinase β (IKKβ) in the ankle tissue were detected by real-time fluorescent quantitative PCR and Western blot, respectively.

RESULTS: Compared with the blank control group, the AI score, BS/BV, Tb.Sp, serum contents of TNF-α, IL-6 and IL-1β, and the expression levels of RANKL, NF-κB p65 and IKKβ mRNA and protein were significantly increased (P<0.01), while BMD, BV/TV, Tb.Th and Tb.N, and the expression levels of OPG mRNA and protein were all significantly decreased in the model group (P<0.01). After acupotomy intervention, both the increase of the AI score from day 14 to 28 after intervention, BS/BV, Tb.Sp, serum contents of TNF-α, IL-6 and IL-1β, and the expression levels of RANKL, NF-κB p65 and IKKβ mRNA and protein, and the decrease of BMD, BV/TV, Tb.Th and Tb.N, and the expression levels of OPG mRNA and protein were reversed in the acupotomy group (P<0.01, P<0.05). Micro-CT showed rough surface of the ankle joint, enlarged gap, significantly reduced bone mass and serious bone erosion in the model group, and improved surface density of the ankle joint, increased bone mass and decreased bone erosion in the acupotomy group. HE staining displayed an obvious proliferation of synovial cells in the ankle joint tissue, and infiltration of a large number of inflammatory cells in the model group;but no apparent proliferation of the synovial cells in the ankle joint tissue, and only a small amount of inflammatory cells in the acupotomy group.

CONCLUSIONS: Acupotomy can reduce inflammation, inhibit bone destruction in RA rats, which may be related to its function in regulating the OPG/RANKL/NF-κB signaling pathway, further inhibiting OC differentiation.