Effects of moxibustion on Treg/Th17 cell and its signal pathway in mice with rheumatoid arthritis

Abstract

To observe the effects of moxibustion on Treg/Th17 imbalance and related signal pathway in mice with rheumatoid arthritis (RA), so as to explore the action mechanism of moxibustion on RA. Twenty-four DBA/1J male mice were randomly divided into a normal group, a model group, a sham moxibustion group and a moxibustion group, 6 mice in each one. RA model was induced by subcutaneous injection of typeⅡcollagen and adjuvant at tail in mice other than the normal group. The mice in the moxibustion group were treated with moxibustion at"Zusanli" (ST 36) and "Shenshu" (BL 23), 1 mg per cone, 6 cones per acupoint. The consecutive 6-day treatment was taken as one course, and totally 2 courses were given with an interval of 2 d between courses. The mice in the sham moxibustion group were treated with immobilization as the moxibustion group. The effects of moxibustion on joint swelling was evaluated by RA scale of collagen induced arthritis (CIA); the pathological changes of joint inflammation were observed by HE staining; the cell count of Th17 and Treg in spleen was analyzed by flow cytometry; the content of cytokine IL-1β, IL-6, IL-10, IL-17, IL-23, TGF-β and Galectin-9 were analyzed by ELISA; the mRNA and protein expression of Foxp3, Galectin-9, RORγt, CARMA1, NF-κB were analyzed by Real-time PCR and Western Blotting method. Ten to 12 d after the secondary immune, red and swelling of ankle joint, feet and toe joint were observed, indicating successful establishment of RA model. 15 d into moxibustion treatment, the joint swelling was improved in the moxibustion group and the sham moxibustion group, which was superior in the moxibustion group (P<0.05). As for pathological changes, compare with the normal group, the articular surface was rougher and synovial layer thinner in the model group, which was recovered to a certain extent in the sham moxibustion group; the articular surface was smooth and synovial layer was thicker in the moxibustion group, which was similar to the normal group. The results of flow cytometry test indicated the cell count of Treg in the model group was reduced but that of Th17 was increased than the normal group (both P<0.01); the moxibustion could increase significantly the cell count of Treg (P<0.05), but no effect was observed on Th17 (P>0.05). The results of ELISA test indicated the differences of increasing of IL-1β, IL-6, IL-17, IL-23, TGF-βas well as the reducing of IL-10 were not significant between the sham moxibustion group and the moxibustion group (all P>0.05); moxibustion treatment could increase the content of Galectin-9 which was reduced in RA mice (P<0.05). The results of RT-PCR and Western blotting test indicated the mRNA and protein expression of Foxp3, Galectin-9 were reduced in the model group (all P<0.01), which could be up-regulated by moxibustion treatment (P<0.05, P<0.01); the mRNA and protein expression of RORγt, CARMA1, NF-κB was increased (all P<0.01), which could be down-regulated by moxibustion treatment (P<0.05, P<0.01). Moxibustion could improve the swelling of joint and inflammatory reaction of joint synovial in RA mice; the mechanism may be related to the regulation of Treg cells number in spleen and the expression of Foxp3, Galectin-9, RORγt, CARMA1, NF-κB, mRNA and protein expression.