Effects Of Targeted Silencing Notch Genes On NF-?B Pathway In Macrophages Of Patients With Atherosclerosis

ObjectiveTo observe the expression of DLL4,JAG1,P65,P52 and I?B? within macrophages of patients with atherosclerosis which is silenced by Notchl-siRNA,Notch2-siRNA,Notch3-siRNA.To investigate the mechanism and crosstalk between Notch and NF-?B signaling pathway in patients with atherosclerosis and try to find new methods to prevent atherosclerosis.Methods1.The mononuclear cells from peripheral blood of patients with AS were induced to macrophages,which were then silenced by Notch1-siRNA,Notch2-siRNA,Notch3-siRNA and NC-siRNA.2.RT-PCR technologies and Western blot analysis were applied to assess the expression levels of P65,P52,DLL4,JAG1 and I?B? in the signaling pathway.3.Electrophoretic mobility shift assay(EMSA)was used to observe the NF-?B DNA binding activity.4.Subcellular distributions of P65 and P52 were detected through immunofluorescence.Results1.RT-PCR results showed that,after Notch-siRNA being transfected into macrophages,the expression of NF-?B P65,NF-?B P52 gradually declined in the Notch3-siRNA group(0.709±0.007,0.853±0.061),the Notch2-siRNA group(0.557±0.031,0.822±0.077)and the Notchl-siRNA group(01114±0.014,0.519±0.035)(P<0.01),all of which significantly decreased compared with the control group(1.004±0.105,1.001±0.056)and the negative control group(1.043±0.096,1.010±0.054)(P<0.05 or 0.01).The expression of I?Ba(1.811 ±0.172,3.253±0.169,5.295±0.433)gradually increased(P<0.01),all of which significantly increased compared with the control(1.003±0.087)and the negative control group(1.027±0.052)(P<0.05 or 0.01),especially in the Notchl-siRNA group.The expression levels of DLL4 and JAG1 in the Notchl-siRNA group(0.248±0.027,0.296±0.026),the Notch2-siRNA group(0.563±0.0178,0.495±0.016)and the Notch3-siRNA group(0.782±0.058,0.655±0.048)were significantly lower than those in the control group and the negative control group(P<0.01),and the difference was statistically significant(P<0.01).The Notchl-siRNA group decreased most significantly.2.Western Blot results showed that,after Notch-siRNA being transfected into macrophages,the protein expression of NF-?B P65,NF-?B P52,DLL4 and JAG1 gradually declined in the Notch3-siRNA group,the Notch2-siRNA group and the Notchl-siRNA group(P<0.01),all of which significantly decreased compared with the control group and the negative control group(P<0.05 'or 0.01).The protein expression of I?B? gradually increased(P<0.01),all of which significantly increased compared with the control group and the negative control group(P<0.05 or 0.01),especially in the Notch1-siRNA group.3.The NF-?B DNA binding activity were gradually inhibited in the Notch3-siRNA group,the Notch2-siRNA group and the Notchl-siRNA group,all of which also significantly decreased compared to the control group and the negative control group.4.The fluorescence intensity of P65 and P52 decreased both in the nucleus and cytoplasm compared to the control group and the negative control group(P<0.01),which declined more obviously in the nucleus.Conclusions1.After the Notch1,Notch2 and Notch3 gene being silenced in the macrophages,the expression of P65 and P52 can be down-regulated at the transcriptional level and the expression of I?B? is up-regulated,all of which prevent the P65 and P52 proteins transferring to the nucleus.Meanwhile it also reduce the nuclear protein NF-?B DNA binding activity,thus affect the downstream gene transcription activity.All of these eventually lead NF-?B signaling pathway to be inhibited.A positive relationship may exist between Notch signaling pathway and NF-?B pathway in patients with AS.2.Notchl pathway may play more important role in the regulation on NF-?B signaling pathway compared with Notch2 pathway and Notch 3 pathway.3.The expression of DLL4,JAG1 was inhibited at the transcription level after the Notchl,Notch2 and Notch3 gene being silenced in the macrophages,especially in the Notchl-siRNA group.