The Effects Of Coronin-1 With Different Expression Levels On INOS And TLRs Expression And Apoptosis In Raw264.7 Cells

ObjectiveIn order to investigate the effects and molecular mechanisms of different expression levels of Coronin-1 on iNOS and TLRs expression and apoptosis in RAW264.7 cell line.Methods1.According to the difference of Coronin-1 expression level,the experiment was divided into three groups: RAW264.7-Cor.Plus,RAW264.7 and RAW264.7-Cor.Minus group.The level of iNOS mRNA was tested by Realtime PCR,and the levels of TLR2 and TLR4 and TLR9 were detected by Western-blot,the apoptosis percent was analyzed by Flow cytometry.2.Before and after treated with the CsA(a Calcineurin inhibitor)for 24 hours,the level of Calcineurin was determined by Western-blot.Three groups of cells and RAW264.7 cells treated with cytD(an actin polymerization inhibitor)for 48 hours were stained with TRITC-phalloidin,the percentage of F-actin rearrangement was calculated.3.Before and after treated with the CsA for 24 hours or cyt D for 48 hours respectively,the level of iNOS mRNA was tested by Realtime PCR,and the levels of TLR2 and TLR4 and TLR9 were detected by Western-blot,and the apoptosis percent was analyzed by Flow cytometry.Results1.There were no significant difference in the levels of iNOS mRNA and TLRs and apoptosis rate between RAW264.7 and RAW264.7-Cor.Minus group(P>0.05).The levels of iNOS mRNA and TLRs protein expression and apoptosis percent were significantly decreased in cells with Coronin-1 over-expression(RAW264.7-Cor.Plus)(P<0.05).The trend showed :RAW264.7 > RAW264.7-Cor.Plus.2.The level of CaN protein expression of macrophages was positive correlation to the difference of Coronin-1 expression level.The CaN expression level decreased along with the decline of Coronin-1 expression level in RAW264.7 cells.The level of CaN expression was significantly decreased in RAW264.7 and RAW264.7-Cor.Plus cells after treated with Cs A,showing the trend: RAW264.7-Cor.Plus > RAW264.7 > RAW264.7-Cor.Minus;RAW264.7 > RAW264.7 + Cs A;RAW264.7-Cor.Plus > RAW264.7-Cor.Plus + Cs A(P<0.05).By TRITC-phalloidin staining,the F-actin rearrangement rate of RAW264.7-Cor.plus cells was significantly higher than that of RAW264.7 cells(P<0.05).The F-actin rearrangement rate of RAW264.7 treated-in cyt D was significantly lower than that of the corresponding control group(P<0.05).3.After treated with Cs A,the levels of iNOS mRNA and TLRs protein expression and apoptosis percent were significantly increased in cells with Coronin-1 over-expression(RAW264.7-Cor.Plus)(P<0.05).After treated with cytD,there were no significant difference in the levels of iNOS mRNA and apoptosis rate between the treated cells and untreated control cells(P>0.05),but the level of TLRs protein expression was significantly decreased(P<0.05),which was independent of the level of Coronin-1 expression.Conclusion1.The Coronin-1 over-expression inhibited the levels of iNOS mRNA and TLRs protein expression and apoptosis percent.2.The Coronin-1 over-expression promoted the the expression of CaN and the F-actin rearrangement.The level of CaN was suppressed in macrophages treated-in CsA.The F-actin rearrangement was inhibited when treated with cytD which was independent of the different levels of Coronin-1 expression.3.The Coronin-1 over-expression not only promoted the F-actin rearrangement,but also inhibited the expression of TLRs and iNOS mRNA and cell apoptosis(P<0.05),which molecular mechanism is associated with that Coronin-1 promoted Ca2+ signaling pathway regulated by calcineurin and the process was independent of F-actin-mediated processes.