Eleutheroside E Ameliorates Arthritis Severity In Collagen-induced Arthritis Mice Model By Suppressing Inflammatory Cytokine Release

Objectives: An effective and practical mice model of rheumatoid arthritis was established by injecting of bovine collagen type II. The changes of body weight, the general score of joint, joint radiological score, anti-II collagen antibody level and the level of inflammatory cytokines in serum were used to evaluated the severity of arthritis. The therapeutic effects of different doses of Eleutheroside E on the rheumatoid arthritis mice model were subsequently testified. Finally, the potential mechanism of Eleutheroside E was investigated.Methods: ①SPF male DBA/1 mice were feed until 7-8 weeks, weighting 18-25 g. The bovine collagen type II and complete Freund’s adjuvant emulsion was used to induce rheumatoid arthritis model by subcutaneous injection in the back as a primary immunization. After three weeks, the bovine collagen type II and incomplete Freund’s adjuvant emulsion was injected subcutaneously at the base of the tail as a boost immunization. ②The model mice were randomly divided into five groups including Tripterygium group(15mg/kg orally), low-dose EE group(15mg/kg orally), medium-dose EE group(30mg/kg orally) and high-dose EE group(60mg/kg orally), and the model group was injected with vehicle(orally) alone. ③The signs and symptoms of arthritis(including weight, joint swelling), radiographic changes and histopathological changes in different treatment groups were observed and recorded. The sera of CIA mice used in this experiment were collected and detected the concentration of cytokines and their anti-II collagen antibodies. ④THP-1 cells were co-cultured with different doses of EE for 48 h. The supernatants were collected and detected the level of TNF-α and IL-6.Results: ①The rheumatoid arthritis model was developed successfully by injecting bovine collagen type II. ②Compared to vehicle-treated CIA mice, 30 mg/kg and 60 mg/kg EE treatment obviously decreased the arthritis scores and body weight loss in CIA mice. The hind paw thickness in CIA mice was significantly decreased(P<0.01). Noticeably, the arthritis scores, body weight loss and the hind paw thickness of mice treated with 15 mg/kg EE were lower than those of vehicle-treated mice, but the statistical difference was not significant(P>0.05). ③Mammography and micro-CT examination found that the joint structure of the CIA model group mice was damaged, joint space disappeared and the bone surface is roughness. In 30 mg/kg and 60 mg/kg EE treatment groups, the extent of bone damage of mice were significantly reduced and the bone damage score were significantly decreased(P<0.01). But CIA mice treated with 15 mg/kg EE did not show significant difference compared with vehicle-treated mice(P>0.05). ④ The HE staining results showed that marked infiltration of inflammatory cells, pannus information, cartilage damage, and bone erosion in the joints of vehicle-treated CIA mice. However, the infiltration of inflammatory cells and formation of pannus in CIA mice were significantly reduced after treated with EE(30mg/kg and 60 mg/kg) and TG(15 mg/kg). Cartilage damage and bone erosion in the CIA mice were also significantly inhibited by EE(30 mg/kg and 60 mg/kg) treatment groups. But CIA mice treated with 15 mg/kg EE did not show significant difference compared to vehicle-treated mice. ⑤The level of anti-collagen II antibody showed that the antibody level in vehicle-treated CIA mice was very high. In the EE(30mg/kg and 60 mg/kg) treatment groups, the level of anti-collagen II antibody significantly decreased(P<0.01). Anti-collagen II antibody in mice treated with 15 mg/kg EE was significant lower than those in vehicle-treated mice, the difference was statistically significant(P<0.001). ⑥The level of cytokines showed that significant lower levels of TNF-α, IL-6, and IL-23 in EE treatment sera than those from vehicle-treated mice, the difference was statistically significant(P<0.05). Despite the level of IL-17 is lower than vehicle-treated mice, but no significant difference. ⑦THP-1 cells were cultured in vitro, the results showed that: Eleutheroside E is no toxic side effects on macrophages, and can inhibit the activition of macrophages, reduced secretion of TNF-α and IL-6, and showed time and dose dependence. Compared with the control group, the difference was statistically significant(P<0.05).Conclusion: ① The method of rheumatoid arthritis model was developed successfully. ②Eleutheroside E can significantly improve the symptoms of arthritis in CIA mice.③Eletheroside E can inhibit the production of inflammatory cytokines such as TNF-α, IL-6 and IL-23.