Study On The Mechanism Of The Genistein Inhibiting Angiogenesis In Rheumatoid Arthritis

Rheumatoid arthritis(rheumatoid arthritis,RA)is a systemic immune disease.In the pathological process of RA,angiogenesis plays a great role in promoting the proliferation of synovial cells by providing more oxygen and nutrients,and the spread of inflammatory factors by providing more channels.Therefore,inhibition of angiogenesis in the synovial membrane of the joint becomes an important target of the treatment of RA.Genistein(GEN),a natural compound extracted from leguminous plants,has been shown to inhibit the angiogenesis in tumor and inflammation in RA,but its effects on the angiogenesis of RA and the underlying molecular mechanisms remain to be clarified.In this work,the effects of genistein on angiogenesis and its mechanism were studied in vitro.To ensure the experimental concentration of genistein is in safe range,the effect of different concentrations of genistein on the survival rate of MH7 A and EA.hy926 cells in 24 h and 48 h was tested by CCK-8 method,and the results showed that the highest safety concentration of genistein in 48 h was 25 ?mol/L.Our preliminary study showed that genistein can inhibit the expression of IL-6,which is an important upstream factor of JAK/STAT pathway,we suppose IL-6 may have some influence on the expression of VEGF.For which,MH7 A cells were stimulated by TNF-?,and use fluorescence quantitative PCR and ELISA to verify whether the expression of IL-6 and VEGF could be inhibit by genistein.The results showed that the expression of IL-6 and VEGF in MH7 A cells was significantly increased by TNF-?,which could be effectively reversed when treated with genistein,suggested that genistein can effectively inhibit the inflammation,and had a potential inhibitory effect on the angiogenesis in inflammatory environment.IL-6 is an important inflammatory factor in the course of RA,and also has a certain role in promoting the angiogenesis.To test whether genistein can inhibit angiogenesis,we use IL-6 to simulate an inflammatory and angiogenesis promotive environment,the scratch experiment,transwell and the matrixgel tube formation experiment were cary on under this condition.The results showed that the migration and tube formation of vascular endothelial cells were enhanced by IL-6,and could be inhibited by the treatment of genistein,which indicate that genistein could inhibit angiogenesis in inflammatory environment.In order to simulate the microenvironment of RA,the effect of genistein on angiogenesis in RA was further investigated by the co-culture of MH7 A and EA.hy926 cells.The results show that treat with TNF-? only or co-culture with MH7 A only can both promote the migration of EA.hy926 cells.Treat MH7 A cells with TNF-? and then co-culture with EA.hy926 cells,the number of cell that go through the membrane increased.On this basis,we treat MH7 A with AG490,an inhibitor of JAK/STAT signaling pathway,and the number of EA.hy926 cell that go through the membrane decreased significantly,which indicated that blocking the JAK/STAT signaling pathway can decrease the contents that promote EA.hy926 cells migration.The effect of genistein on the inhibition of EA.hy926 cell migration was consistent with AG490,suggesting that genistein could inhibit the secretion of angiogenic factors by MH7 A cells through JAK/STAT pathway,and therefor inhibit the migration of EA.hy926.To further clarify the mechanism of genistein inhibiting the angiogenesis in RA,IL-6 were used to simulate inflammation and promote angiogenesis.In addition,to verify whether genistein could inhibit the expression of VEGF,JAK2,STAT3 in MH7 A cells in the condition of IL-6 stimulation,fluorescence quantitative PCR and ElISA were carried out.The cell immunofluorescence assay was used to verify whether genistein could inhibit the nucleus transfer of P-STAT3 in MH7 A cells under the condition of IL-6 stimulation.The results showed that the expression of VEGF decreased significantly and the protein secretion of VEGF declined significantly after treating MH7 A cells with genistein and AG490.After staining p-STAT3 by cell immunofluorescence,it was found that genistein inhibited the nucleus transfer of p-STAT3 in MH7 A cells stimulated by IL-6,which showed that genistein inhibited JAK2/STAT3,the downstream pathway of IL-6.To sum up,this study found that genistein can effectively inhibit the migration and formation of vascular endothelial cells in inflammatory environment,and act on the downstream pathway of IL-6(JAK2/STAT3 pathway)in MH7 A cells.Genistein can also inhibit the nucleus translocation of p-STAT3,and thus inhibiting the expression of VEGF and inhibiting angiogenesis in the inflammatory environment.These findings further verifies the important role of genistein in inhibiting angiogenesis in inflammatory environment,it also reveals the underlying molecular mechanism of the anti-angiogenesis role of genistein,which provides theoretical support for the prospect of genistein as a therapeutic drug for RA,and provides a theoretical reference for the role of Genistein in other angiogenesis-related diseases.