| Objective: Rheumatoid arthritis(RA)is a chronic systemic inflammatory disease of symmetrical peripheral arthritis.The local persistent inflammatory reaction of the joint will lead to the irreversible destruction,deformity and loss of function of the joint in patients with rheumatoid arthritis.The key link of the occurrence and development of RA is fibroblast-like synoviocytes(FLS)in the synovium of the joint.The immune damage effect of FLS is mainly to release inflammatory factors through self release and recruitment of inflammatory cells.Further study of FLS related signaling pathways and influencing factors can provide a new way to prevent RA with FLS as the target.Mitogen activated protein kinase(MAPK)plays an important role in the development of RA.Three major subfamilies of MAPK are c-Jun N-terminal kinase(JNK),extracellular regulatory kinase(ERK)and p38 kinase.The JNK signaling pathway is involved in many biological processes.Many studies have shown the role of JNK activation in the development of RA.JNK activates the transcription factors involved in the expression of matrix metalloproteinase(MMP)gene,thus increasing the production of MMP,thus promoting the invasion of FLS and the destruction of joints.It is suggested that JNK activation is an important factor in the pathogenesis of rheumatoid arthritis.Caffeine is a central nervous system(CNS)stimulant of methylxanthine.It has been reported that caffeine has a potential antagonistic effect on methotrexate(MTX)used in the treatment of RA patients.A meta-analysis showed a significant association between coffee consumption and the risk of serum positive RA.These reports suggest that caffeine may play a role in the pathogenesis of RA.Exploring the mechanism of caffeine in the development of RA may open up new ideas for the treatment of RA.Therefore,it is of great significance to explore the mechanism of caffeine regulating the process of RA.In this dissertation,we intend to study the effects of caffeine on the proliferation and invasion of RA-FLS and its molecular mechanism,and explore the theoretical basis of caffeine promoting the development of RA.Methods: In this paper,human rheumatoid arthritis fibroblast like synovium cells were isolated and cultured from the synovium of patients with rheumatoid arthritis.The effect of caffeine on the proliferation of RA-FLS was detected by MTT assay.The effect of caffeine on the invasive ability of RA-FLS was detected by cell invasion test.To explore the possible mechanism of caffeine induced FLS growth and invasion,Western blot and real-time PCR were used to detect the effects of different concentrations of caffeine on the transcription and expression of cyclin D1,cyclin E,MMP2 and MMP9 in RA-FLS.In order to further study the molecular mechanism of caffeine promoting the proliferation of RA FLS,Western blot was used to detect the effects of different concentrations of caffeine on the expression of p-JNK,Atf2,c-jun and p-p65 proteins in RA-FLS.The effect of SP600125 on the expression of MMP2,MMP9,cyclin D1,cyclin E,c-jun and ATF2 protein in the downstream JNK protein of RA-FLS was detected by Western blot.MTT method was used to detect the effect of SP600125 on the proliferation of FLS induced by caffeine.The effect of SP600125 on caffeine induced invasion of RA-FLS was detected by cell invasion test.Results: The RA-FLS were treated with different concentrations of caffeine(0,250,500 and 750 ? g / ml)for 48 hours,and the growth rate was measured at 0,24,48,72 and 96 hours.In the caffeine treated group,the proliferation of FLS was significantly promoted in a dose-dependent manner.The cells were also treated with different concentrations of caffeine(0,250,500 and 750 g/ml)for 48 hours.Then the cells were inoculated in matrigel for about 20 hours to observe the invasive ability.The results showed that caffeine promoted the invasion of RA-FLS in a dose-dependent manner.Caffeine also increased the m RNA and protein expression of cyclin D1,cyclin E,MMP2 and MMP9 in a dose-dependent manner.In addition,caffeine can significantly up regulate the levels of phosphorylated JNK and JNK target protein,suggesting that caffeine can induce JNK activation.Caffeinated RA-FLS,downstream target ATF2 and c-Jun of JNK were up-regulated,suggesting that caffeine can activate JNK signaling pathway of RA-FLS.In addition,FLS was treated with caffeine for 48 hours,and SP600125,a JNK specific inhibitor,was added to the culture medium(concentration 5 ? m and 10 ? m)for 24 hours.Western blot,MTT and cell invasion test were carried out.The results showed that SP600125 inhibited the expression of p-JNK,MMP2,MMP9,cyclin D1,cyclin E,ATF2 and c-jun in a dose-dependent manner.In addition,SP600125 treatment also inhibited the cell growth rate.Cell invasion experiment showed that SP600125 significantly reduced the number of FLS cells compared with caffeine alone.These data suggest that JNK inhibition can reverse the biological role of caffeine in FLS.SP600125,a JNK inhibitor,can inhibit the effect of caffeine on the proliferation and invasion of FLS.SP600125 also blocked the expression of downstream target of JNK in a dose-dependent manner.Conclusion: Caffeine can promote the growth and invasion of RA-FLS.Caffeine also increased the m RNA and protein expression of cyclin D1,cyclin E,MMP2 and MMP9 in a dose-dependent manner.Caffeine can significantly up regulate the levels of phosphorylated JNK and JNK target protein,suggesting that caffeine can induce JNK activation.SP600125,a JNK inhibitor,can inhibit the effect of caffeine on the proliferation and invasion of FLS.SP600125 also blocked the expression of downstream target of JNK in a dose-dependent manner.The results show that caffeine promotes FLS proliferation and invasion by activating JNK signaling pathway,suggesting the role of caffeine in the development of rheumatoid arthritis. |
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