| Background : Rheumatoid arthritis(RA)is a chronic and symmetrical autoimmune disease with an unknown pathophysiological mechanism that affects about 1% to 2% of the world's population.If RA is not treated,it will cause disability and systemic complications,which may lead to early death and economic losses.With the increasing attention paid to RA,RA research has become an important topic in the field of rheumatology.Currently,the clinical therapeutic effect of drugs for RA is limited,and some drugs have high side effects and expensive.Therefore,new drugs still need to be developed for the treatment of RA.By inhibiting the activity of phosphodiesterase 5(PDE5),PDE5 blocks the hydrolysis of c GMP in cells to inactive 5 '-GMP,and increases the content of c GMP in vivo,thereby activating protein kinase G(PKG),further inhibiting the transcriptional activity of Wnt/ b-catenin and gene products.The PDE5 inhibitor(PDE5i)designed in this project is a compound designed and synthesized by the professor of school of pharmacy,sun yat-sen university on the basis of the marketed PDE5 inhibitor.Compared with other phosphodiesterase inhibitors,PDE5 i has better anti-inflammatory and pro-apoptotic effects through previous exploratory experiments.At the same time,PDE5 i has been observed to significantly improve the treatment of RA rats in previous experiments,and PDE5 i is expected to be one of the candidate drugs for the treatment of rheumatoid arthritis.Objective: In this study,the Freunds Adjuvant(10 mg/m L)prepared by BCG was used to induce the formation of rheumatoid arthritis model in rats,and PDE5 i was used for drug intervention for 4 weeks.The gait behavior,pain threshold,imaging observation,histopathological observation,immunological evaluation and enzyme-linked immunosorbent assay(ELISA)were used to evaluate the pharmacodynamic effect of PDE5 i on RA.Then the therapeutic mechanism ofRA-fibroblast synovial cells(MH7A)was investigated by western blot.Methods: The experiment was divided into three parts:(1)The RA model was established by subcutaneous injection of BCG freumann adjuvant(10mg/m L)into the left posterior foot of each rat.Rats with successful modeling were divided into normal group(normal saline i.p.),RA group(normal saline i.p.),high-dose PDE5 i group(3 mg/kg/d i.p.),low-dose PDE5 i group(1mg/kg/d i.p.),and methotrexate(MTX)group(1 mg/kg/d i.p.).Each group of 12 was administered for 28 days.(2)After treatment,gait analysis system and pain threshold were used to evaluate the behavioral changes in each group of RA.The pathological changes of the joints and immune organs of each group were compared by imaging observation,HE staining,saffron solid green staining and immunological evaluation.The content of c GMP in rat serum was detected by ELISA,which proved the targeting of PDE5 i.The contents of IL-1?,IL-6 and TNF-? in the serum of rats were detected by ELISA to explain the response of PDE5 i to the inflammatory condition and further evaluate the pharmacodynamic effect of PDE5 i on RA.(3)By culture of RA-fibroblast synovioid cells(MH7A)as the research object of mechanism,the RA group and the PDE5 i high,medium and low dose groups(concentrations of 32 n M,16 n M and 8 n M,respectively)were set.The protein expression of PKG,b-catenin,Cyclin D1 and DKK-1 in each group was detected by WB to explore the possible signaling pathway mediated by PDE5 i in RA.Results:(1)Compared with the normal group,all gait indexes of rats in RA group were significantly decreased,and there was a statistical difference(p<0.05),indicating that RA rats had difficulty walking.The pain threshold of rats in group R was also significantly lower than that in the normal group,with a significant statistical difference(p<0.01).Compared with the RA group,the PDE5 i group effectively improved the walking disorder and pain threshold(p<0.05).(2)The results of imaging,HE staining and saffron solid-green staining showed that the joints in the RA group were significantly eroded and damaged,with a large number of inflammatory cells infiltrating the joint cavity,synovial cell proliferation,and a large amount of fibrosis in the cartilage tissue.The bone and joint in the PDE5 i group improved the above conditions.The serum contents of Il-1?,Il-6 and TNF-? in RA rats were all increased by ELISA compared with the normal group(p<0.05).The contents of IL-1?,IL-6 and TNF-? decreased in the PDE5 i group,which wasstatistically significant compared with the RA group(p<0.05).Immunological evaluation results showed that the organ index of RA group was significantly higher than that of the normal group,and the difference was significant(p<0.05).The viscera index of the PDE5 i group decreased,which was statistically significant compared with that of the RA group(p<0.05).(3)WB experimental results showed that compared with RA group,the level of PKG protein expression in the PDE5 i group was increased in a concentration-dependent manner,and there was a statistical difference(p<0.05).Compared with the RA group,the level of ?-catenin protein in the PDE5 i group was decreased in a concentration-dependent manner,and there was a significant statistical difference(p<0.01).Conclusion:(1)PDE5i can effectively improve walking disorders and pain caused by rheumatoid arthritis.(2)PDE5i can effectively alleviate bone joint erosion,synovitis and other pathological conditions,and effectively regulate RA related functions by mediating the Wnt/?-catenin signaling pathway,thus improving and treating RA. |
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