Therapeutic Effect And Potential Mechanism Of Fangchinoline On Experimental Rheumatoid Arthritis

Rheumatoid arthritis(RA)is a chronic,systemic autoimmune disease characterized by synovitis and erosive destruction of bone tissue.RA has a high incidence,high morbidity,and poor cure.The current treatment of RA mainly includes early and mid-term drug treatment and advanced joint replacement.Current RA treatment commonly used in clinic includes glucocorticoids(GC),nonsteroidal antiinflammatory drugs(NSAIDs),disease-modifying anti-rheumatic drugs(DMARDs)and biological agents.Although GC and NSAIDs can alleviate RA symptoms,they can't prevent the progression of the disease and prevent joint destruction.Although DMARDs and biological agents can prevent the process of bone destruction,long-term use has large toxic and side effects.and biological preparations are expensive and there are major infections risk.Therefore,finding new alternative or supplemental drugs has become a problem that needs urgent solution.Traditional Chinese medicines have the advantages of easy accessibility,multi-targeting,and low toxic and side effects,making them the first choice for alternative medicines for RA.Fanglinoline(Fan)is one of the main alkaloid active ingredients contained in dry roots.It has been proved to have antioxidant,anti-inflammatory,cardiovascular protection,neuroprotective,and hypoglycemic effects.It also have anti-cancer and other functions,but have not yet seen its research report on the treatment of RA.RA is an inflammatory disease,and its mainly damaged cells have been transformed into tumor cells by synovial fibroblasts.Therefore,we propose the hypothesis that Fan may have a therapeutic effect on RA.To this end,we used Collagen-induced rat model of rheumatoid arthritis(CIA)to detect the effect of Fan on RA.and analyzed the potential mechanism of Fan on RA treatment from the animal,cell and molecular level.The main experiment content and results:Part ?:In Vivo Experiment:Therapeutic Effect of Fan on CIA Rats160 g-180 g SPF grade female Wistar rats were selected and injected intradermally and subcutaneously with type II collagen to establish the CIA model.The negative control group was injected with the same amount of normal saline.When the CIA model was established(arthritis index score? 2),CIA rats were randomly divided into groups.Grouped status of this thesis:Control group without induction of CIA,intraperitoneal injection of normal saline(n=5);positive control group(ClA+Vehicle),daily intraperitoneal injection of normal saline(n=6);positive treatment group(CIA+MTX 3 mg·kg-1)was intraperitoneally injected with MTX(n=6)once every three days;lowdose treatment group(CIA+Fan 3 mg·kg-1)was intraperitoneally injected with Fan(n=6).The high-dose treatment group(CIA+Fan 6 mg·kg-1)was intraperitoneally injected with Fan(n=6)daily.Treatments were started with an arthritis index score?2 and continued for 14 days.After 14 days,the rats were evaluated for arthritis index,imaging,and scores to comprehensively evaluate Fan's treatment of CIA rats.Results:Fan treatment significantly inhibited the severity of CIA rats.The main manifestations of the Fan treatment group were significantly lower than those of the CIA control group in terms of joint index scores,paw swelling volume,and imaging scores.Moreover,the high-dose Fan treatment was superior to the MTX positive drug group.The results of pathological section analysis showed that compared with the CIA control group,the Fan treatment group significantly improved in the infiltration of inflammatory cells,synovial hyperplasia,vasospasm formation,destruction of bone and cartilage.The above results indicate that Fan has a significant therapeutic effect on type ? collagen-induced CIA rats.Part ?:In Vitro Experiment:Analysis of Possible Mechanism of Fan on RA TreatmentFibroblast-like synoviocytes(FLS)are the main damaged cells throughout the course of RA.FLS can release a variety of inflammatory factors that respond to and mediate inflammatory responses.Over-activation,hyperplasia,and invasiveness of FLS are thought to be the key to sustained inflammation of the RA synovium and erosive destruction of bone tissue.Therefore,in this paper,primary cultured CIA rat synovial fibroblast-like cells(CIA-FLS)were used as test cells to analyze whether Fan's inhibitory effect on RA was achieved by affecting the FLS over-activation phenotype.The main experiment contents include:(1)Detecting the toxicity of Fan by MTS assay;(2)Detecting the effect of Fan on the migration of CIA-FLS cells by cell scratch assay;(3)Detecting the effect of Fan on the proliferation of Fan CIA-FLS cells by EdU incorporation assay;(4)Flow cytometry was used to detect the effect of Fan on apoptosis of CIA-FLS cells;(5)Q-PCR was used to analyze the effect of Fan on the expression of inflammatory cytokines stimulated by TNFa in CIA-FLS cells.(6)Analysis of possible molecular mechanisms by Western-blot.Results:Through MTS assay,the median lethal concentration(IC50)of Fan to CIA-FLS was 4.114 ?M.The highest dose used in the paper was 2 ?M.Scratch test showed that Fan inhibited the migration of CIA-FLS cells in a concentration-dependent manner;EdU incorporation experiments showed that 1 ?M Fan can significantly inhibit the proliferation of TNF?-stimulated CIA-FLS cells;Q-PCR analysis found that Fan can Dose-dependent inhibition of the expression of the inflammatory factors IL-1?,IL6 and IL-8.Western blot analysis revealed that Fan inhibited the activation of MAPK(p38)and the degradation of I?B? in a dose-dependent manner.Immunofluorescence detection of p65 nuclear entry further confirmed the inhibitory effect of Fan on NF-?B signaling pathway.Conclusion:Animal experiments show that Fan can significantly reduce the incidence of CIA rats,and has obvious therapeutic effect on type ? collagen-induced RA.In vitro cell experiments show that Fan can significantly inhibit the overactive phenotype of CIA-FLS.Inhibition of FLS cell proliferation,migration and expression of inflammatory factors;this inhibition may be achieved by inhibiting p38 and NF-?B signaling pathways.In conclusion,our results suggest that Fan has the potential to develop alternative drugs for RA.providing the basis for the development of alternative clinical drugs for RA.