| BackgroundIn recent years, the researches indicate that Syk plays an important role in thepathogenesis of RA. We can block the chain reaction of inflammation and provide a newavenue for treatment of RA by suppressing activation of Syk, so Syk become a novel targetthat we study for the drug in the treatment of RA. The School of Pharmacy in East ChinaUniversity of Science and Technology found a small molecule compound (HL131012)which has high inhibitory effect on Syk, and then they composed some derivatives after aseries of structural modification study by the method of the traditional medicinal chemistrystructural optimization. Through the kinase inhibitory activity test, they preliminarilyscreened five kinds of small molecule compounds, and next in order to get the one whichhas greater biological activity by researching the inhibitory effect of small moleculecompounds on Syk in the cellular level, so they cooperate with us to study this subject.ObjectiveTo observe the effect of the small molecule compounds on proliferation and release ofinflammatory cytokines IL-6and MMP-3of FLS in rheumatoid arthritis and compare withthe positive drug R406, in order to screen the small molecule compound which has goodbiological activity and make further research about its effect on RA.We hope to provide acandidate drug to develop for the treatment of RA in the future.MethodsBy the method of MTT assay to test the half maximal inhibitory concentration (IC50)of small molecule compounds and positive drug R406on proliferation of FLS in24h,48h, 72h, then compare and screen the small molecule compounds that exhibit great inhibitoryeffect. By Enzyme-linked immunosorbent assay (ELISA) to detect the effect of the smallmolecule compounds and positive drug R406on release of IL-6and MMP-3of FLS withrheumatoid arthritis, then compare and select the small molecule compounds that exhibithigh inhibitoty effect.Statistics and calculation methods: All data were dealed with SPSS17.0statisticalsoftware. The measurement data was expressed as mean±S.D. Statistical analysis wasperformed using one-way ANOVA by SNK-q test for comparisons between means ofmultiple groups. The significance level P was set at0.01, P values less than0.01wereconsidered to be statistically significant.Results1. In the optical microscope, the growth of the third generation synoviocytes wasuniform, cell body presented long spindle and stretched out protuberance. By the methodof FCM, we got the results that the expression of CD90was positive and CD68wasnegative, in accordance with the typical morphological characteristics of fibroblast likesynoviocyte.2. The results of MTT showed that when when FLS was incubated at24h, the IC50oncell proliferation of HL131078was (16.30±1.739), compared with positive drug(28.02±1.611), the difference was statistically significant (P<0.01), at48h, the IC50on cellproliferation of HL131012, HL131074, HL131078and HL131087were respectively18.44±0.788,21.08±2.033,11.17±0.858,22.17±1.783, compared with positive drug(26.71±1.859), the differences were statistically significant (P<0.01), at72h, the IC50oncell proliferation of HL131012, HL131078and HL131087were respectively3.58±0.484,9.74±1.677,5.44±0.785, compared with positive drug (13.56±1.471), the differenceswere statistically significant (P<0.01). According to these data, we reached the conclusionthat when the FLS was incubated after24h,48h and72h, the comparisons of IC50betweenHL131078and positive drug, all differences were statistically significant(P<0.01).3. The results of ELISA indicated that the content of IL-6/MMP-3in high concentration of small molecule compounds groups, compared with that in lowconcentration of small molecule compounds groups, the differences were statisticallysignificant(P<0.01). In high concentration, the content of IL-6and MMP-3wererespectively in HL131012group (13.68±0.91,0.268±0.02), in HL131078group(17.69±0.42,0.313±0.014), compared with positive drug group (19.93±0.97,0.329±0.011),the differences were statistically significant (P<0.01).ConclusionsThe small molecule compound HL131078exhibited inhibitory effect on proliferationof FLS that induced by TNF-α. The small molecule compounds HL131012and HL131078can significantly inhibit the release of IL-6and MMP-3of FLS that induced by TNF-α.The small molecule compound HL131078exhibited good biological activity and can beused as the further researching object that probably provide the reference value fordevelopment of new drugs in the treatment of RA in the future. |
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