Protective Effect And Mechanism Of Alpha-melanocyte-stimulating Hormone On Collagen-induced Arthritis In Rats

PartⅠ Protective effect ofα-MSH on collagen-induced arthritis in rats Objective: To observe the suppressive effect of α-MSH on the development of collagen-induced arthritis in rats. Methods: Rat arthritis was setup by injecting bovine Ⅱ collagen(BⅡC). The rats were randomly divided into four groups of 6～8. group A: normal control; group B: CIA control; group C: low dose of α-MSH(0.25mg·kg-1·d-1); group D: high dose of α-MSH (1mg·kg-1·d-1). α-MSH or PBS was administered intraperitoneally twice everyday from the day of primary immunization for 7 days . The body weight, the hind paw volumes, the arthritic index, the pathological score and the radiographic changes of all rats were compared separately. Results: (1) Arthritis in rats took place in 2 weeks after the primary immunization. The symptoms included such as: redness and swelling of the joints, limited joints movement. Pathologys indicated synovial hyperplasia and inflammatory cell infiltrations, erosion of articular cartilage. Soft-tissue swelling , bone erosion, narrow of the joint cavity and periosteal new bone formation were demonstrated by the radiography. (2) The effects of α-MSH on the development of the arthritis: ① The state of arthritis: swelling of the paws were alleviated distinctively and the mean arthritic index was declined in the group D, the desease onset time delayed, but the arthritis was not completely prevented. ② Pathohistological results: the severity of intimal lining hyperplasia and mononuclear cell infiltrations was reduced, and no cartilage destruction was demonstrated in the group D. ③ Radiographical results: the radiography of the group D showed soft-tissue swelling, while osteoporosis,joint space narrow and articular bone erosion were indicated in group B and C. Conclusion:α-MSH may ameliorate the symptoms of CIA rats, decline the severity of synovial proliferation and inflammatory cell infiltrations, and inhibit the cartilage erosion in pathology. Part Ⅱ Immunological mechanism in the protective effect of α-MSH on collagen-induced arthritis in rats Objective:To explore the immunological mechanism in the protective effect of α-MSH on collagen-induced arthritis in rats. Methods: (1) Immunized cells from inguinal lymph nodes or spleen were cultured in the presence ofα-MSH(10-12mol/L～10-6mol/L)and/or 100(g/ml BⅡC. [3H] Thymidine was added and radioactivity incorporated into the cells for the effect of α-MSH on the proliferation of lymphocytes was determined by liquid scintillation counting. (2) Splenocytes and peritoneal macrophages from immunized rats were cultured withα-MSH(10-14mol/L～10-6mol/L)and/or 100 (g/ml BⅡC( or 10 (g/ml LPS). Culture supernatants were harvested and analyzed for IL-1(,IFN-( ,IL-10 by ELISA. TNF-α was detected by the L929 cytotoxicity using MTT colometeric assay. NO was measured by the method of nitrate reductase. (3) Flow cytometric analysis were performed to study the effects of α-MSH on the expressions of CD28 on the T-cells and CD86 on the macrophages. Results: (1)α-MSH(10-12mol/L～10-6mol/L)inhibited the BⅡC antigen-immunized T lymphocytes proliferation and maximal suppression was observed at a concentration of 10-8mol/L(P < 0.01). (2) Different concentrations of α-MSH(10-14mol/L～10-6 mol/L)could suppress the production of IL-1 (,IFN-( and TNF-α by BⅡC-stimulated spleen cells and IL-1 (,TNF-α and NO by peritoneal macrophages, enhanced the synthesis of IL-10 by peritoneal macrophages. Maximal effect was detected whenα-MSH was used at a concentrations of 10-10mol /L or 10-8mol/L(P < 0.01). (3) The expression of CD28 on T lymphocyte and CD86 on macrophage were reduced byα-MSH administration and maximal inhibitions of α-MSH on CD28 and CD86 were detected at a concentration of 10-10mol/L or 10-8mol/L separately(P < 0.05). Conclusion:α-MSH has a protective effect on CIA rats, with mechanisms of regulating the balance of Th1/Th2 cytokines, the production of NO a...