Effect And Mechanism Of Sirtl On Inflammatory Response Of Human Coronary Endothelial Cells Via NF-?B Signaling Pathway

OBJECTIVE: In this study,oxidized low density lipoprotein(ox-LDL)was used to induce the injury of human coronary artery endothelial cells(HCAEC),and to establish the atherosclerosis model,so as to investigate the anti-inflammatory effects and mechanisms of silencing information regulator 1(SIRT1)exerted in the treatment of atherosclerosis(As).Methods: 1.Verification of Sirt1 expression.HCAEC in logarithmic growth phase was divided into normal control group,empty plasmid transfection group,empty plasmid transfection + ox-LDL group,Sirt1 overexpression + ox-LDL group,Sirt1 interference + ox-LDL group,and NF-?B block(PDTC)+Sirt1 interference + ox-LDL group.Lipofectamine 2000 lipofection was used to transfect the endothelial cells line of the human coronary artery for 24 h,then it was added with 100?g / ml of ox-LDL to conduct the continue training for 24 h.The transfection efficiency was observed under fluorescence microscope.RT-PCR was used to detect the mRNA level of cytokine Sirt1,and Western blot was used to detect the protein expression level of SIRT1 in cells.2.Research on the role of SIRT1 in the inflammatory response of HCEAC.HCAEC in logarithmic growth phase was divided into normal control group,empty plasmid transfection group,empty plasmid transfection + ox-LDL group,Sirt1 overexpression + ox-LDL group and Sirt1 interference + ox-LDL group.RT-PCR was used to detect the mRNA level of ICAM-1;Western blot was used to detect the expression level of ICAM-1 protein;ELISA was used to detect the expressions of IL-1 and hs-CRP in each group.3.Exploration on the mechanism of SIRT1 exerts anti-inflammatory effects.(1)HCAEC in logarithmic growth phase was divided into normal control group and ox-LDL group(100?g / ml).Immunofluorescence was used to observe the positioning situation of NF-?B.(2)HCAEC in logarithmic growth phase was divided into empty plasmid transfection + ox-LDL group,PDTC+Sirt1 interference + ox-LDL.RT-PCR was used to detect the mRNA level of ICAM-1;Western blot was used to detect the expression level of ICAM-1 protein;ELISA was used to detect the expressions of IL-1 and hs-CRP in each group.(3)HCAEC in logarithmic growth phase was divided into normal control group,empty plasmid transfection + ox-LDL group,Sirt1 overexpression + ox-LDL group,Sirt1 interference + ox-LDL group.Western blot was used to detect the expression level of ICAM-1 protein;ELISA was used to detect the protein expression level of acetylated NF-?B and NF-?B proteins.Result: 1.The solution was changed after 24 hours of transfection of liposome Lipofectamine 2000 to HCAEC cells,and then 100?g / ml of ox-LDL was added in it to conduct the continue training for 24 h.The transfection group was successfully transfected under fluorescence microscope.The mRNA and protein levels verified that SIRT1 could be able to express successfully.2.Sirt1 overexpression could inhibite the RNA level expression and the protein expression,inflammatory factor hs-CRP and IL-1 protein expression of ox-LDL-induced adhesion molecule ICAM-1(P<0.05);Sirt1 interference could promote RNA level expression and protein expression,inflammatory factor hs-CRP,IL-1 protein expression of ox-LDL-induced adhesion molecule ICAM-1(P<0.05).3.Normal cells NF-?B diffuse throughout the cell,100?g / ml of ox-LDL promotes NF-?B into the nucleus;Sirt1 interference+ox-LDL+NF-?B block group(PDTC)was compared with Sirt1 interference +ox-LDL group,the mRNA expression of ICAM-1 was decreased(P<0.05),and the protein expression of ICAM-1,IL-1 and hs-CRP was decreased(P<0.05).The overexpression of SIRT1 inhibited the protein expression of ox-LDL-induced transcription factor NF-?B and acetylated NF-?B(P<0.05),SIRT1 interference promoted the protein expression of ox-LDL-induced transcription factor NF-?B and acetylated NF-?B(P<0.05).Conclusion: SIRT1 can inhibit the inflammatory response in HCEAC induced by ox-LDL and produce anti-inflammatory effects by inhibiting the activation of NF-?B pathway.Therefore,SIRT1 may play a role in the treatment of atherosclerosis through anti-inflammatory effects.