Effect of heat-reinforcing needling on the TLR4-MyD88-ERK1 / 2 signaling pathway in the synovial tissue of the knee joint of rabbits with rheumatoid arthritis with cold syndrome.

Objective We aimed to investigate the anti-inflammatory mechanism of heat-reinforcing needling on rheumatoid arthritis(RA) with cold syndrome. Methods The RA with cold syndrome model was established by injecting ovalbumin dry powder and Freund's complete adjuvant combined with cold freezing. Fifty rabbits(50% male and 50% female) were randomly divided into the normal group, the model group, the resatorvid(TAK-242) group, the heat-reinforcing needling group, and the lipopolysaccharide( LPS) combined with heat-reinforcing needling group, 10 rabbits in each group. Rabbits in the TAK-242 group were intraperitoneally injected with the TLR4 inhibitor TAK-242(0. 75 mg/ kg); rabbits in the heat-reinforcing needling group were treated with "Zusanli" (ST 36) with heat-reinforcing needling; and rabbits in the LPS combined with heat-reinforcing needling group were injected intraperitoneally with the TLR4 agonist LPS(0. 25 mg/ kg) on the basis of heat-reinforcing needling. Each group received continuous treatment for 14 days. The circumference and skin temperature of the knee joint of the rabbits were recorded; the histomorphology of the synovial membrane of the rabbit knee joints was observed by HE staining; the expression levels of TNF-α, IL-1β, and IL-6 in synovial tissues were detected by immunohistochemistry; the protein expression levels of TLR4, MyD88, and ERK1/2 in synovial tissues were detected by Western blotting; and the mRNA levels of mTOR and p70S6K were detected by real- time PCR. Results The circumference of the knee joint was lower and the skin temperature of the knee joint was higher(P<0. 01) in all intervention groups compared with the model group. The HE staining of the TAK-242 group and the heat-reinforcing needling group showed that the inner mucosa of the joint capsule was basically flat, the proliferation of pannus was reduced, and there was a small amount of inflammatory cell infiltration, while the LPS combined with the heat-reinforcing needling group showed that the inner mucosa of the joint capsule was rough, the pannus proliferated, and the inflammatory cell infiltration and aggregation were obvious. The expression of TNF-α, IL-1β, and IL-6(P <0. 01), the protein levels of TLR4, MyD88, and ERK1/2, and the mRNA levels of mTOR and p70S6K were lower (P<0. 01) in all intervention groups compared with the model group. The circumference of the knee joint was lower and the skin temperature of the knee joint was higher(P<0. 01) in the heat-reinforcing needling group and the TAK-242 group compared with the LPS combined with heat-reinforcing needling group; the levels of TNF-α, IL-1β, and IL-6(P <0. 01), the protein levels of TLR4, MyD88, and ERK1/ 2, and the mRNA levels of mTOR and p70S6K were lower(P<0. 01) in the heat-reinforcing needling group and the TAK-242 group compared with the LPS combined with the heat-reinforcing needling group. Conclusion Heat-reinforcing needling can effectively reduce knee swelling and inflammatory infiltration in RA with cold syndrome model rabbits. The possible mechanism is related to the inhibition of the TLR4-MyD88-ERK1/2 pathway, antagonism of cell proliferation activity, and a decrease of TNF-α, IL-1β, and IL-6 secretion by heat-reinforcing needling. [ABSTRACT FROM AUTHOR]