Effects of moxibustion on TRPV1 channel and TLR4 signaling pathway-mediated inflammatory network in articular synovial tissue of RA rats

To observe the effects of moxibustion at Shenshu (BL23) and Zusanli (ST36) point areas on the transient receptor potential vanilloid type 1 (TRPV1) channel and the inflammatory network mediated by the Toll-like receptor 4 (TLR4) signaling pathway in the articular synovial tissue of rheumatoid arthritis (RA) rat models, and to explore the target role of the TRPV1 channel in moxibustion intervention for synovial inflammatory pain in RA.

A total of 56 healthy male Sprague-Dawley rats were divided into 7 groups using the random number table method, namely a normal group, a model group, a moxibustion group, a TRPV1 agonist group (capsaicin group), a moxibustion + TRPV1 agonist group (moxibustion + capsaicin group), a TRPV1 antagonist group (capsazepine group), and a moxibustion + TRPV1 antagonist group (moxibustion + capsazepine group), with 8 rats in each group. Except for the normal group, the other 6 groups were established as RA models using a composite modeling method combining wind-cold-damp environmental factors with Freund’s complete adjuvant. After successful modeling, the moxibustion group received mild moxibustion with moxa sticks of 0.9 cm in diameter at bilateral Shenshu (BL23) and Zusanli (ST36) point areas, 30 min per time, once daily for 14 consecutive days. The TRPV1 agonist group and TRPV1 antagonist group were subcutaneously injected with capsaicin and capsazepine, respectively, at bilateral Shenshu (BL23) and Zusanli (ST36) point areas, once daily for 14 consecutive days. The moxibustion + TRPV1 agonist group and moxibustion + TRPV1 antagonist group received moxibustion intervention 30 min after subcutaneous injection of capsaicin or capsazepine at the above point areas, with the same moxibustion protocol as in the moxibustion group, for 14 consecutive days. After the intervention, hematoxylin-eosin staining and transmission electron microscopy were used to observe the pathological changes of synovial tissue and cells. Immunohistochemistry was applied to detect the contents of interleukin (IL)-1β, IL-6, IL-17, and tumor necrosis factor (TNF)-α in the synovial tissue. Western blotting and reverse transcription-polymerase chain reaction were performed to measure the relative expression levels of TLR4, myeloid differentiation factor 88 (Myd88), TRPV1 proteins and mRNAs in the synovial tissue. Enzyme-linked immunosorbent assay was used to determine the contents of IL-1β, IL-2, IL-6, IL-17, and TNF-α in the serum.

Compared with the normal group, the model group showed obvious pathological damage in rat synovial tissue, with significantly increased contents of IL-1β, IL-6, IL-17, and TNF-α (P<0.01 or P<0.05) in the synovial tissue, significantly upregulated protein expression of TLR4, Myd88, and TRPV1 (P<0.01), significantly increased relative mRNA expression levels of TLR4, Myd88, and TRPV1 (P<0.01), and significantly elevated serum contents of IL-1β, IL-2, IL-6, IL-17A, and TNF-α (P<0.01). Compared with the capsazepine group, the capsaicin group exhibited marked pathological damage in the synovial tissue, with significantly higher contents of IL-1β, IL-6, IL-17, and TNF-α in the synovial tissue (P<0.01), significantly increased protein expression of TLR4, Myd88, and TRPV1 (P<0.01), significantly elevated relative mRNA expression levels of TLR4, Myd88, and TRPV1 (P<0.01), and significantly higher serum contents of the above inflammatory cytokines (P<0.01). Compared with the model group, the moxibustion group had significantly alleviated pathological damage in the synovial tissue, with markedly decreased contents of IL-1β, IL-17, and TNF-α in the synovial tissue (P<0.01), no significant decrease in the IL-6 content (P>0.05), significantly downregulated protein expression of TLR4, Myd88, and TRPV1 (P<0.01), significantly reduced relative mRNA expression levels of TLR4, Myd88, and TRPV1 (P<0.01), and significantly lower serum contents of the above inflammatory cytokines (P<0.01); the capsaicin group showed aggravated pathological damage in the synovial tissue, with a significantly increased IL-6 content (P<0.01), but no significant elevation in the contents of IL-1β, IL-17, or TNF-α in the synovial tissue; meanwhile, the protein and mRNA expression levels of TLR4, Myd88, and TRPV1 in the synovial tissue and serum inflammatory cytokine contents were all significantly increased (P<0.01). Compared with the capsaicin group, the moxibustion + capsaicin group had significantly relieved pathological damage in the synovial tissue, with markedly decreased contents of IL-1β and IL-6 in the synovial tissue (P<0.01), but no significant reduction in the IL-17 or TNF-α content (P>0.05), significantly downregulated protein expression of TLR4, Myd88, and TRPV1 (P<0.01), significantly reduced relative mRNA expression levels of TLR4, Myd88, and TRPV1 (P<0.01), and significantly lower serum contents of the above inflammatory cytokines (P<0.01). Compared with the capsazepine group, the moxibustion + capsazepine group showed alleviated pathological damage in the synovial tissue, with insignificantly decreased contents of IL-1β, IL-6, IL-17, or TNF-α in the synovial tissue (P>0.05); the TLR4 protein expression was significantly decreased (P<0.01), while the protein expression of Myd88 and TRPV1 was reduced without statistical significance (P>0.05); the relative mRNA expression levels of TLR4 and Myd88 were significantly decreased (P<0.05, P<0.01), while that of TRPV1 was reduced without statistical significance (P>0.05); the serum contents of the above inflammatory cytokines were all significantly decreased (P<0.01).

Activation of the TRPV1 channel exacerbates the inflammatory response mediated by the TLR4 signaling pathway in RA model rats. Moxibustion can exert anti-inflammatory and analgesic effects and ameliorate RA synovial inflammatory injury by regulating the dual “point-synovium” TRPV1 channels and inhibiting the inflammatory response mediated by the TLR4 signaling pathway. The TRPV1 channel located on point receptors and the targeted synovium may be potential spatially specific therapeutic targets for moxibustion intervention for RA synovial inflammation.