The Effect Of Metformin In Vivo On The Inhibition Of Inflammation And Joint Protection Of The Collagen-induced Arthritis (CIA) Rat Model

Background and objectiveRheumatoid arthritis (RA), with high morbidity, is a kind of systemic autoimmune disease which mainly involved peri articular, this disease is one of the causes of disability and loss of labor capacity in china. The pathology is characterized by erosion proliferative synovitis of the whole body joints, this progressive disease can recur repeatedly all life long and eventually lead to the deformity and non-function of joints. Although the etiology and pathogenesis of RA is not yet fully clear, studies have confirmed that Monocyte-macrophages and lymphocytes infiltrated in synovial tissues produce large amounts of cytokines, these cytokines by acting on multiple cells and mutual adjustment, to form a complex network, and eventually lead to the occurrence and development. Studies have confirmed, TNF-a is central to the pathogenesis of rheumatoid arthritis, which not only can mediate the inflammation and damage to joints of rheumatoid arthritis by NF-kB, JAK/STAT pathways, but also can promote the synoviocytes, fibroblasts, macrophages etc. produce a variety of inflammatory cytokines in the form of autocrine or paracrine, such as IL-1β, IL-6, etc., they all act on the inflammation and joint destruction of rheumatoid arthritis. So it is considered to be a revolutionary change in the treatment of RA that the appearance of TNF-a antagonists such the biological agents as etanercept, adalilmumab, etc. But biologics are expensive and its biological safety has not yet to be confirmed, so they are not widely used. Now the standard RA therapeutic schedule includes non-steroidal anti-inflammatory drugs (NSAID), hormones, disease-modifying antirheumatic drugs (DMARDs), but it is not able to improve the symptoms of patients in the long run and there are many severe side effects. Therefore, searching for new drugs to improve RA disease remains a challenging clinical problem we faced. In recent years, studies have shown that metformin is not only play the role of anti-inflammatory systematically, reducing the level of inflammatory factors and elevating anti-inflammatory cytokines; but also reduce the cartilage decomposition mediated by inflammatory cytokines, maintain the homeostasis of cartilage and protect it; and it can also adjust the production of cytokines of osteoblasts to inhibit osteoclast formation, reduce osteoclast differentiation and protect bone mass; then eventually achieves the aim of joints protection. In this study, we intend to demonstrate that metformin has the effects of anti-inflammatory and joint protection in the rat model of rheumatoid arthritis induced by the type Ⅱ collagen.Objective1. To verify the effect of metformin in vivo on the inhibition of inflammation of the collagen-induced arthritis (CIA) rat model;2. To verify the effect of metformin in vivo on the joint protection of the collagen-induced arthritis (CIA) rat model.Methods1. Under4℃, dissolved type Ⅱ collagen into0.01mol/L acetic acid, placed it through the night,till it fully dissolved.2. Under the ice bath,blended it with complete freund’s adjuvant to1:1then made the mixture into emulsion using high-speed homogenate machine,preserved it at4℃.3. Gave several spots intraperitoneal injection of0.3ml emulsion in the rat tail and the skin around anus.Repeat the experimental operation on the7th day to strengthen the induction.4. After the success of the induction, randomly divided the rats into groups and gave CIA rat models high doses of metformin (100mg/kg/d),low doses of metformin (50mg/kg/d) and methotrexate(2.7mg/kg/w) for treatment by gavage on the7th day.5. Evaluated the arthritis score of each group of rats and measured the width of talus every two days.Executed rats and drew materials on the28th day,then shot X-ray films and reconstruct MICRO-CT3D data of the ankle of rat and do the HE staining and detected the levels of inflammatory and anti-inflammatory factors in rat serum by Elisa.Results1. After continuous observation rat foot arthritis scoring and measurement from talus width:Arthritis scoring of high doses of metformin group was obviously lower than other induced groups (P<0.05),talus width is also much smaller (P<0.05);The rest of the treatment groups also has a certain effect on reducing arthritis score and the width of talus compared to the induced group (P<0.05)2. Elisa show:After the induction, inflammatory factors-TNF-a, IL-1β, IL-6has been significantly rised,to the contrary anti-inflammatory factor-IL-10has been significantly lowered in induced group. High doses of metformin can lower the level of inflammatory factors-TNF-α, IL-1β, IL-6(P<0.05), also can rise the level of anti-inflammatory factor-IL-10obviously (P<0.05). Low doses of metformin and MTX have the similar effect (P<0.05),but not more significant than high doses of metformin. Conclusion Metformin in vivo can significantly Inhibit the inflammation and protect the joint structure of the collagen-induced arthritis (CIA) rat model.3. X-ray radiography show:High doses of metformin group can significantly save joint structure and make joint clearance clear;The treatment effect of the rest treatment groups are not better than high dose group, also has severe soft tissue swelling,bone absorption,joint disintegration and severe damage. 4. MICRO-CT reconstruction results show:in induced group, bone absorption and dissolution are severe, it is very rough and voids at the bone surface, the joints had completely destroyed; high-dose metformin treatment groups:there is no severe bone absorption and dissolved phenomenon, it is just a bit rough at bone surface without voids, the structure of joints were completely preserved; methotrexate therapy group and low-dose metformin treatment group:there were also severe bone absorption and dissolution phenomena, the bone surface is very rough and a large numbers bone voids appeared there, the structure of joints were completely ruined,but not so severe as the induced group.5. HE staining show:HE staining results about induced group showed:the inflammatory cells, stained dark blue, emerged as flocculence, the bone mass was severely eroded by inflammatory cells, joint clearance was disappeared, and replaced by inflammatory cells completely. HE staining results about methotrexate therapy group and low-dose metformin treatment group:a large number of inflammatory cells located in the joint clearance, cartilage and bone mass, the joint clearance was messed up and flooded with inflammatory cells, the bone mass was also severely eroded by inflammatory cells; HE staining results about about high-dose metformin group showed:there was just a small amount of inflammatory cells and scattered in the joint space, the joint clearance was normal and clear, the bone mass was not severely eroded by inflammatory cells, the pink cartilage and dark red bone mass were visible clearly without little damage.Conclusions1. Metformin have a dose-dependent effect on the information of the collagen-induced arthritis (CIA) rat model in vivo;2. Metformin have a dose-dependent effect on the joint protection of the collagen-induced arthritis (CIA) rat model in vivo.