Mechanisms And Active Substances Researches Of Cinnamomi Ramulus Against Rheumatoid Arthritis

Rheumatoid arthritis(RA)is a systemic inflammatory disease with progressive immune disorders.Perseverant synovitis and high levels of rheumatoid factor can lead to irreversible joint damage and severe disability,with great social implications in terms of treatment costs,disability and loss of work force.Cinnamomi ramulus is a significant Chinese traditional medicine.It has been used in combination with other traditional Chinese medicines to form “Guizhi Decoction”(such as a classical prescription Guizhi shaoyao zhimu decoction)to relieve inflammation and pain in patients with arthralgia syndrome.As the dominant medicine of Guizhi decoction,the mechanism of Cinnamomi ramulus in treating RA is still poorly understood.Objective:In this study,we constructed a collagen-induced arthritis(CIA)rat model and a TNF-α-induced synovial fibroblast(MH7A)model to explore the ameliorative effect of Cinnamomi ramulus extracts on arthritis symptoms in CIA rats,and the inhibitory effect on proliferation and migration of MH7 A cells.Finally,modern information technology was used to verify the active compounds and key target pathways of Cinnamomi ramulus.These results provide experimental basis for its traditional application in the treatment of arthralgia syndromes such as RA.Methods:1.After the establishment of type II collagen-induced arthritis(CIA)rat model,the rats were randomly assigned to receive methotrexate and Cinnamomi ramulus extracts.The arthritis index and paw volume of the rats were observed and recorded.The levels of inflammatory factors in serum of rats were detected by ELISA kit.The synovial tissue,joint and cartilage of CIA rats were observed by histopathological section.Micro CT was used to scan the joints of the rats and observe the degree of damage of the joints and cartilage.2.CCK-8 and cell clone assay were used to evaluate the effect of Cinnamomi ramulus extracts on the proliferation activity of synovial fibroblasts(MH7A)induced by TNF-α.Cell apoptosis was detected by flow cytometry and fluorescence staining.The distribution of cell cycle was analyzed by flow cytometry.The change of migration and invasion ability of MH7 A cells was studied by scratch and transwell assay.The effects of Cinnamomi ramulus extracts on the expression of apoptosis,cycle and invasion related genes and proteins in MH7 A cells were studied by q RT-PCR and western blot assay.3.GC-MS and UPLC-QE-MS/MS were used to qualitatively analyze the compounds from Cinnamomi ramulus.The differentially expressed genes(DEGs)of RA were obtained from GSE55457 and GSE55235 datasets of GEO database by R language.Combined with network pharmacology,the targets of Cinnamomi ramulus in RA treatment were enriched and analyzed.Finally,software such as Discovery Studio4.5 was used for molecular docking to evaluate the docking activity of active components and core targets.Results:1.After the treatment of Cinnamomi ramulus extracts,the symptoms of arthritis swelling and bone destruction of knee and ankle joints in CIA rats were significantly improved,and the proliferation of synovial cells and infiltration of inflammatory cells in knee joints were reduced.Serum inflammatory cytokines IL-17 A,TNF-α,IL-1β,IL-6 levels were down-regulated,showing a significant combined protective effect.2.At the cellular level,it was also proved that Cinnamomi ramulus extracts could inhibit the proliferation and migration of MH7 A cells induced by TNF-α,promote cell apoptosis and induce G2/M arrest.At the protein and gene levels,we also confirmed the regulatory effects of Cinnamomi ramulus extracts on apoptosis(C-Caspase 3,Bax and Bcl-2),cycle(CDC2,Cyclin B1,CDK4,P21 and P53 and Cyclin D)and invasion(MMP-1,MMP-2 and MMP-3).3.A total of 63 compounds were identified by GC-MS and UPLC-QE-MS/MS,and 559 related targets of 63 compounds were screened out.55 common genes were obtained after intersecting with DEGs of RA,which mainly involved TNF signaling pathway,osteoclast differentiation,IL-17 signaling pathway,apoptosis,Th17 cell differentiation,Th1 and Th2 cells differentiation,NF-kappa B signaling pathway,etc.The biological processes that may be affected include cytosolic calcium ion concentration,cell chemotaxis,regulation of inflammatory response and so on.The key targets of these biological processes and pathways include MAPK8、PTGS2、JUN、CCL5、STAT1,etc.Molecular docking validation also confirmed that compounds with high scores,such as cinnamaldehyde.Conclusion:Cinnamomi ramulus extracts could improve arthritis symptoms and bone injury in CIA rats by inhibiting synovial cell proliferation and inflammatory cell infiltration,inhibit proliferation,migration and invasion of MH7 A cells induced by TNF-α,promote cell apoptosis and G2/M cycle arrest.The results of network pharmacology and molecular docking confirmed that the mechanism of Cinnamomi ramulus in the treatment of RA was related to inflammatory response and apoptosis pathway,which is consistent with the experimental results of CIA rat and MH7 A cell model.