Artesunate Reverses Th17/Treg Imbalance Induced Inhibition Of IL-17 In Collagen-induced Arthritis Rats

Objective: Rheumatoid arthritis(RA) belongs to systemic autoimmune diseases, which with chronic inflammatory in small peripheral joint and tissue as the main performance.Chronic symmetric polyarthritis as the main feature of RA, small joints of hands and foot, wrist, knee were the most commonly affected joints.Systemic extra-articular manifestations symptoms such as fever, anemia, pulmonary fibrosis could be combined with RA.Chronic inflammation of synovial tissue, abnormal proliferation of synovial cell and systemic immune abnormalities in the involved joints were the mainly pathological changes of RA.Systemic immune system abnormalities caused chronic inflammation of synovium, resulte in the formation of pannus, secrete a variety of inflammatory cytokines, bone and cartilage damage, the destruction was similar to tumor destruction.Without proper treatment, RA could be repeatedly delayed healing, eventually leading to deformity and function of involved joints will loss.In clinical treatment mainly use corticosteroids, immunosuppressive agents, disease modifying anti-rheumatic drug(DMARDs) and biologics agents to treat RA.Corticosteroids and immunosuppressive agents had significant effect in the treatment of RA, but exist some serious adverse reactions, traditional DMARDs drugs effected slowly, which also exist some limitations.Biological agents could specifically antagonize the proinflammatory cytokines,but the costs was high, which exist some contraindications to use, lack of widely used in clinical application. Artemisinin was commonly used as anti-malarial drugs,artesunate was a kind of potential immune inhibitors with low toxicity and high efficiency as semi-synthetic artemisinin derivatives[1]. Studies found that artesunate could inhibit the secretion of TNF in synovial cells of RA by regulating the signaling pathway of NF-kappa B [2], play an important role to inhibit synovitis of RA[3], At the same time by inhibiting interoperability between proinflammatory cytokines reduce the formation of pannus, delay RA destruction of cartilage and bone tissue[4,5].Recent studies suggest that the imbalance of Th17 and Treg cells was an important mechanism in the pathogenesis of RA[6].Our previous experiments had proved that artesunate can upregulate the expression of Foxp3 of CIA rats synovial cells in vitro, and dose-dependent manner[7].Our subject successfully established model of collagen-induced arthritis(CIA) rats, observed the expression of Th17 / Treg in rats spleen lymphocytes in vivo after the intervention of different doses of artesunate. To investigate the regulation of Th17/Treg cell expression by artesunate, whether artesunate could increased the proportion of Treg cells in synovium,thus reducing the activity of IL – 17 in t synovium tissue.Methods : Established collagen induced arthritis. Arthritis index(AI) score as the accreditation standards.Successful model of rats were average divided into normal control group、CIA model group、different does of Artesunate’group, HCQ group、MTX group and MP group.Extract lymphocytes from spleen, The expression of Th17/Foxp3 of different groups in CIA rats were detected by Flow cytometry( FCM). Immunohistochemistry was used to detect IL-17 / Foxp3 expression in synovial tissue of rats in different groups.Results: 1.Induced CIA rats model successfully. 2.Evalution of clinical indicators:Symmetric arthritis happened at 9 days after the primary immunization in CIA model group, mainly performance as the swelling of small toe foot or ankle. Swollen of elbow and arm were appeared at14 days after immunization in some rats, foot or hand joint deformity began to appear after 10 weeks. More serious manifestations rats can not crawl or weight. 3.Artesunate had significant effect in improving arthritis index scores, ankle swelling, radiographic scores of CIA rats. 4. The change of Th17/Treg lymphocyte subsets: CIA model group compared with normal control group showed Treg lymphocyte decreased, Th17 lymphocyte increased. The lymphocytes expression of Foxp3 in drug intervention groups were higher than those in the CIA model group, the secretion of IL-17 declined, the difference between the groups was statistically significant(P<0.05). The Treg lymphocyte in 20mg/kg/day artesunate groups was higher, Th17 lymphocyte was lower than that of other Artesunate groups and Hydroxychloroquine groups,the difference was statistically significant(P<0.05). 5.The expression of IL-17/Foxp3 of involved joints synovium tissue of CIA rats: After the intervention of artesunate to CIA rats, all doses of artesunate could rise the percentage of Treg cells and decrease Th17 cells in the synovial tissue,of which the does of 20mg/kg/day was most significant.Conclusions: Artesunate could reverse Th17/Treg lymphocytes imbalance of CIA rats, while raising the expression of Foxp3,reducing the expression of IL-17 in synovial tissues of involved joints,which was positively correlated with the dose. The expression of Foxp3 in 20mg/kg/day artesunate group was higher than the other doses of artesunate group and hydroxychloroquine group, suggesting that it may be increase the expression of Foxp3 in lymphocytes to inhibit the secretion of inflammatory cytokines Th17, reversing Th17/Treg lymphocytes imbalance, thereby preventing the progression of RA.