Icariin Regulates The EphA2-RhoA Signaling Pathway To Promote Osteogenic Differentiation Of MC3T3-E1 Cells In Porphyromonas Gingivalis Infection

Objective The purpose of this study was to investigate the effect of icariin(ICA)on osteogenic differentiation of MC3T3-E1 cells infected by Porphyromonas gingivalis(P.gingivalis)by regulating Eph A2-RhoA pathway,so as to provide a theoretical basis for the application of icariin in the treatment of periodontal diseases.Methods Porphyromonas gingivalis with a multiplicity of infection(MOI)of 50 was infected with MC3T3-E1 cells to establish a bacterial infection model,and apoptosis of MC3T3-E1 cells at 7 d,14 d and 21 d was detected by flow cytometry.The number of colonies in solid medium was observed by antibiotic protection method for 7 days,14 days and 21 days,and the effects of icariin at different concentrations(0.01,0.1,1,10 and 100 μM)on the proliferation of MC3T3-E1 cells were detected by CCK-8.The protein expressions of Eph A2,Runx2,ALP and OPN were detected by Western-blot,and the osteogenic differentiation function was determined by alkaline phosphatase staining and alizarin red staining.After Eph A2 was silenced in MC3T3-E1 cells,the expression of RhoA,Runx2,ALP and OPN protein was detected.Results1.Flow cytometry results confirmed that as the infection time of MC3T3-E1 cells by Porphyromonas gingivalis increased,the apoptosis rate of cells in the infected group did not increase significantly compared with the normal group,and the difference was not statistically significant(P >0.05).2.The results of the antibiotic protection method showed that Porphyromonas gingivalis invaded the cells at 7,14 and 21 days.3.CCK-8 assay showed that 0.01μM icariin could promote cell proliferation,but the difference was not statistically significant(P >0.05).0.1-10 μM icariin significantly promoted the proliferation of MC3T3-E1 cells in a concentration-and time-dependent manner.100μM of icariin inhibited cell proliferation and apoptosis occurred.4.Western-blot results showed that the drug group inhibited Eph A2 expression and enhanced ALP,OPN and Runx2 protein expression compared with the bacterial infection group(P < 0.05).5.The results of alkaline phosphatase assay showed that icariin at 0.1-10 μM could effectively improve the osteogenic differentiation of MC3T3-E1 cells under Porphyromonas gingivalis infection,with the most obvious effect of icariin at 10 μM.6.The results of alizarin red staining confirmed that intracellular mineral deposition was reduced in the Porphyromonas gingivalis infection state,but icariin at 0.1-10 μM promoted the mineralization ability of osteoblasts with a significant increase in mineral deposition,and the effect of 10 μM epimedium was most pronounced.7.After inhibition of Eph A2 in MC3T3-E1 cells,Western-blot results showed that the expression of Eph A2 and RhoA was significantly higher in the bacterial infection group compared with the control group(P <0.05),but the expression of Eph A2,RhoA was significantly decreased and the expression of ALP,OPN,Runx2 was increased under the effect of icariin(P <0.05).Conclusion Icariin can effectively improve osteogenic differentiation function in inflammatory states,which may be achieved through the regulation of Eph A2-RhoA.