The Impact Of Cyclophilin A On Expression Of NF-κB、M-CSF And ZFP36in RAW264.7Cells Induced By Ox-LDL

1. The expression of Cyclophilin A, NF-κB, M-CSF and ZFP36inRAW264.7cells induced by oxidized low density lipoprotein.Objectives To detect the expression of Cyclophilin A, Nuclear Factor-κB(NF-κB),Macrophage Colony Stimulating Factor(M-CSF) and Zinc Finger Protein-36(ZFP36)expression level in RAW264.7cells treated by oxidized low density lipoprotein(ox-LDL) in different time.Methods RAW264.7cells were incubated with ox-LDL for different time (0,4,8,12h), Reverse Transcription Polymerase Chain Reaction (RT-PCR) and Western Blot(WB) were applied to test the Cyclophilin A expression in mRNA and protein.Western Blot (WB) was applied to test the NF-κB, M-CSF and ZFP36expressions inprotein.ELISA detect the content change of M-CSF.Results Incubation of RAW264.7cells and ox-LDL for4hours,8hours and2hoursthe mRNA and protein expression of Cyclophilin A is time up-regulated Comparingwith the regular RAW264.7cells by both RT-PCR and Western-blot method (P<0.05),8h treatment group appears to be the most obviously change one in both mRNA andprotein aspects.So, in follow-up experiments, we choose incubation of RAW264.7cells and ox-LDL for8hours as our first choice. Synchronous detection of the proteinexpression of NF-κB p65results show the same tendency of Cyclophilin A, comparedwith control group, the protein expressions of groups processed by ox-LDL for4h,8h and12h were significantly enhanced(P<0.05),8h group shows obviously effects. onthe other hand, I-κBα protein expression level present the time dependencedowntrend(P<0.05),8h also shows obviously effects.Western-blot test also foundZFP36protein expression in ox-LDL processed cells for4,8, and12h decreasedsignificantly, the expression quantity of8h group changes obviously, there aresignificant differences between these4groups(P<0.05). ELISA tests showed that theexpression of M-CSF was significantly enhanced, the effect of up-regulation is mostsignificant in8h group (P<0.05).Summaries Ox-LDL time up-regulates the expression of cyclophilin A, NF-κB andM-CSF and down-regulates the expression of ZFP36.2. The effect of Cyclophilin A siRNA on NF-κB, M-CSF andZFP36in RAW264.7cells induced by ox-LDLObjectives To detect the expression level of protein NF-κB, M-CSF and ZFP36inRAW264.7cells treated by Cyclophilin A siRNAMethods With the help of Invitrogen company LipofectamineTM2000transfectinstruction manual,we transfected RAW264.7cells with Cyclophilin A siRNA，RT-PCR and Western-blot were respectively used to detect the expression of mRNAand protein changes of Cyclophilin A to make sure the Cyclophilin A siRNARAW264.7cells model was builded.Based on the results of our first part, we choose50mg/Land8h as our treatment conditions in our following experiments. NF-κB andZFP36proteins expression were detected by Western-blot. ELISA method was used todetect the expression of M-CSF. Results RT-PCR and Western-blot methods were separately used to detected theexpressions of Cyclophilin A at mRNA and protein level (P<0.05) in RAW264.7cellsand found the expression of Cyclophilin A in the group which was treated byCyclophilin A siRNA transfect reagent is largely down-regulated comparing with theregular RAW264.7cells group and blank control group.These results mentionedabove proved the Cyclophilin A siRNA RAW264.7cells model established and can beused in subsequent experiments. Cyclophilin A siRNA treated RAW264.7cellsinduced by ox-LDL significantly reduced the NF-κB p65protein expression and theI-κBα protein expression significantly increased, differences between these groupswere significant (P<0.05). Western blot detected the protein expression of transfectiongroup also increased significantly with significant difference (P<0.05), ELISA testshows the M-CSF expression is reduced significantly (P<0.05).Summaries Cyclophilin A siRNA treatment can down-regulate the expression ofNF-κB and M-CSF in RAW264.7cell and increase the expression of ZFP36.Conclusions1. ox-LDL time up-regulated the expression of cyclophilin A, NF-κB and M-CSFand down-regulated the expression of ZFP36.2. Cyclophilin A siRNA treatment can down-regulate the expression of NF-κBand M-CSF in RAW264.7cell and increase the expression of ZFP36.