[The effect of heat-reinforcing needling on the level of TRAF6-K63 ubiquitination and NF-κB pathway in the synovial tissue of knee joint in rabbits with rheumatoid arthritis of cold syndrome].

Objectives: To observe the effect of heat-reinforcing needling on the level of tumor necrosis factor receptor associated factor 6 (TRAF6)-K63 ubiquitination and the nuclear factor-κB (NF-κB) pathway in the synovial tissue of the knee joints of a rabbit model with rheumatoid arthritis (RA) of cold syndrome, so as to explore the mechanism by which the heat-reinforcing needling inhibits inflammatory response in RA synovitis.

Methods: Eight out of 32 rabbits were randomly selected and assigned to the normal group. The remaining rabbits were induced to establish the RA cold syndrome model using ovalbumin combined with complete Freund's adjuvant and low-temperature cryogenic methods. After modeling evaluation, the rabbits were randomly divided into model group, inhibitor group, and heat-reinforcing needling group, with 6 rabbits in each group. Rabbits in the inhibitor group received intraperitoneal injection of C25-140 solution (2.5 mg/kg), twice a day for 14 days. Rabbits in the heat-reinforcing needling group received acupuncture at "Zusanli"(ST36) according to the operation standard of the heat-reinforcing needling and the needle was retained for 30 min, once a day, for 14 days. After the intervention, knee joint circumference and pain threshold of the rabbits were measured. Color Doppler ultrasound was used to observe changes in knee joint imaging. HE staining was performed to observe morphological changes in the synovial tissue. Immunohistochemical method was used to observe the expression of inflammatory factors tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 in the synovial tissue. Western blot was used to detect the protein expression of ubiquitin-conjugating enzyme E2 13 (Ubc13) and NF-κB p65 in the synovial tissue, and co-immunoprecipitation was used to detect the level of TRAF6-K63 ubiquitination in the synovial tissue.

Results: Compared with the normal group, the model group showed a decreased pain threshold (P<0.05), increased knee joint circumference (P<0.05), and increased average optical density of TNF-α, IL-1β, and IL-6 in the synovial tissue(P<0.05), as well as elevated protein expression of NF-κB p65 and TRAF6-K63 ubiquitination levels (P<0.05), while Ubc13 protein expression was decreased (P<0.05). Compared with the model group, the heat-reinforcing needling group and the inhibitor group had increased pain thresholds and reduced knee joint circumferences (P<0.05), and significantly reduced average optical density of TNF-α, IL-1β, and IL-6 in the synovial tissue (P<0.05), as well as decreased protein expression of NF-κB p65, TRAF6-K63 ubiquitination levels (P<0.05), while Ubc13 protein expression was increased (P<0.05). Compared to the heat-reinforcing needling group, the inhibitor group showed reduced pain threshold (P<0.05), and lower average optical density values of TNF-α, IL-1β, IL-6 in the synovial tissue (P<0.05), as well as decreased expression of NF-κB p65 protein, TRAF6-K63 ubiquitination levels (P<0.05), while Ubc13 protein expression was increased (P<0.05). The imaging changes of the knee joints and the pathological morphological changes of the synovial tissues in the two treatment groups are both less severe than those in the model group.

Conclusions: The heat-reinforcing needling may inhibit inflammatory response in synovitis by suppressing TRAF6-K63 ubiquitination levels, down-regulating the activity of the NF-κB pathway, and reducing the secretion of downstream inflammatory factors.