Combined Treatment With Anisodamine And Neostigmine In Collagen-induced Arthritis Mice

Backgrounds and Objectives:Rheumatoid arthritis (RA) is an auto-immune disease characterized by chronic inflammation of the synovium, which can cause tissue and organ damage resluting in disabiltiy. The traditional method for the treatment of RA, is the use of non-steroidal drugs, glucocorticoids, and disease-modifying antirheumatic drugs. The proinflammatory cytokines, which exist in synovial fluid and serum of RA patients, play a significant role in the initiation and progression of the chronic inflammatory process in RA synovial membrane.Recently, it has been demonstrated that the nervous system, via an inflammatory reflex of the vagus nerve, plays an important role in limiting inflammatory responses, termed as "the cholinergic antiinflammatory pathway". Acetylcholine, the principal neurotransmitter of the vagus nerve, is a key mediator of this cholinergic antiinflammatory pathway. This neurotransmitter can interact with members of the nicotinic acetylcholine receptor (nAChR) family, and in particular the α7subtype (a7nAChR) expressed by macrophages. Anisodamine produces more acetylcholine by blocking the M cholinergic receptors acting with a7nicotine receptor expressed by macrophages, and hence enhances the reaction of acetylcholine with a7nicotine receptor expressed by macrophages, thereby activating the cholinergic antiinflammatory pathway for the treatment of RA.However, to produce therapeutic effects, a very large dose of anisodamine must be administered, which is prone to result in atropine-like side effects; Cholinesterase inhibitors are likely to lead to the side effects of cardiovascular and digestive system caused by acetylcholine accumulation when taken alone, so we prescribe a course of treatment, which is a compound of an appropriate proportion of the muscarinic receptor blocking agents and cholinesterase inhibitors (ie, Compound Anisodamine) to activate the cholinergic anti-inflammatory pathway synergistically, and enhance its anti-inflammatory effect and reduce the side effects of the treatment program. Thus, it will provide a novel strategy for the treatment of RA. Thus, Our previous studies have demonstrated that the use of a combination of anisodamine and neostigmin has positive effects on a7nAChR, and a patent has been applied. Hence, this study aims to explore the combination for the treatment of RA. Methods:Collagen-induced arthritis (CIA) was induced in DBA/1J mice. Arthritic mice were treated with combination of anisodamine (25mg/kg, i.p) and neostigmine (50μg/kg, i.p) once a day, starting from the second immunization. Arthritic index and paw thickness were evaluated. In additions, radiological and histological scores were calculated. Serum inflammatory cytokines (TNFa, IL-6, IL-1β) and anti-type Ⅱ collagen-specific antibodies (IgG, IgG1, IgG2a) were measured using ELISA.Results:1. Our results showed that combination of anisodamine and neostigmine markedly reduced arthritic index and paw thickness.2. Radiological assessment revealed decreased cartilage damage and bone erosion in combined therapy group compared with CIA group.3. Histological staining results showed that the synovial joints and bone erosion of combined treatment group was significantly ligher than those in the control group.4. Combined therapy also significantly decreased the serum levels of TNFa, IL-6and IL-1β compared with CIA group.5. Anti-type Ⅱ collagen-specific antibodies (IgG, IgG2a) in the serum also decreased in treatment group compared with CIA group.Conclusion:These data provide the preliminary evidence that combined therapy of anisodamine and neostigmine inhibits joint inflammation and articular damage in the murine CIA model of rheumatoid arthritis.