Study Of Resveratrol Intervention And SIRT1/NF-?B/CD40 Pathway Effect On Immune-inflammation In Atherosclerosis

Objective:Research on the effects of resveratrol has proved considerably in prolonging life-span,anti-oxidant,anti-aging,anti-inflammation,etc.However,the anti-inflammatory mechanism is still unclear.As CD40-CD40 L interaction is critical in the pathogenesis of atherosclerosis,we tend to find the relationship between resveratrol and CD40 signaling pathway.Here,by creating in vitro and in vivo atherosclerosis model,we study the possible molecular mechanism of resveratrol against CD40 signaling pathway via SIRT1 activation to explore the biological sense and clinical significance of resveratrol.Methods:1.Effect of resveratrol on cell viability,inflammatory factors,chemokine factors,and reactive oxygen species levels in TNF-? induced HUCECs: Cell viability was evaluated.IL-6,MCP-1 levels were examined by using ELISA.ROS level was detected by using flow cytometry.2.Cellular location of SIRT1 influenced by resveratrol: Effects of resveratrol and SIRT1 si RNA on subcellular location of SIRT1 in TNF-? induced HUVECs location was observed by using immunofluorescence staining method.3.Differently expressed genes detection especially CD40 and SIRT1 in resveratrol treated HUVECs: Differently expressed genes were screened by using whole genome oligo microarray.SIRT1,CD40,NF-?B and p38 MAPK mRNA levels were detected by real-time PCR and protein levels were detected by WB in HUVECs.4.Endogenous interaction between SIRT1/p38 MAPK/NF-?B and CD40: Endogenous interaction between SIRT1 and NF-?B was observed by using co-immunoprecipitation assay.Regulation of NF-?B/CD40 transcriptional activity was examined by using luciferase reporter gene assay.Effect of p38 MAPK on NF-?B activity was also examined by using NF-?B reporter gene assay.5.Effect of resveratrol on atherosclerotic mice lipid metabolism and plaque size: LDLR-/-mice were separated in four groups: normal food group,high fat food group,high fat food with solvent group and high fat food with resveratrol group.TCH,TG,HDL-C and LDL-C were detected and CD40 expression was calculated.6.Statistical analysis: Data were presented as means ±SD,Student's t-test and ANOVA test were processed with SPSS software.A p value < 0.05 was regarded as statistically significant.Results:1.Effect of resveratrol on cell viability,inflammatory factors,chemokine factors,and reactive oxygen species(ROS)levels in TNF-? induced HUCECs: Cell viability assay CCK-8 showed that 10 ?g/L of TNF-? alone inhibited HUVECs growth while concurred with 10-20 ?mol/L resveratrol protected the cell viability in a dose dependent manners.IL-6 and MCP-1 levels in cell supernatant were increased after treated with TNF-? while combined with resveratrol significantly decreased the levels in HUVECs.Flow cytometry analysis showed the ROS of HUVECs treated with TNF-? was obviously higher than resveratrol treated cells.2.Cellular location of SIRT1 influenced by resveratrol: Immunofluorescence observation showed that SIRT1 protein subcellular localization both expressed in the cytoplasm and nuclear,resveratrol prompts SIRT1 transfer from cytoplasm to nuclear.3.Differently expressed genes detection especially CD40 and SIRT1 in resveratrol treated HUVECs: Results of whole genome oligo microarray combined with WB and PCR showed that resveratrol activated SIRT1 and repressed CD40,NF-?B,phosphor-p38 MAPK expression in TNF-? induced HUVECs.4.Endogenous interaction between SIRT1/p38 MAPK/NF-?B and CD40: NF-?B reporter gene assay showed that resveratrol inhibited the NF-?B activity through SIRT1 activation.WB results showed that resveratrol inhibited p38 MAPK phosphorylation by SIRT1 activation.The luciferase reporter gene assay showed that NF-?B prompted CD40 transcriptional activity.5.Effect of resveratrol on lipid metabolism and plaque size in atherosclerotic mice: Results showed that resveratrol reduced TCH,TG and LDL-C but increased the level of HDL-C.HE staining and immunohistochemistry results showed that resveratrol reduced the plaque size and CD40 expression in atherosclerotic mice.Conclusions:1.Resveratrol inhibited TNF-?esveratd HUVECs inflammation may through SIRT1 activation,which subsequently repressed p38 MAPK and NF-?B activity,and finally reduced CD40 transcriptional and protein levels.2.With high fat intervention,the LDLR-/-mouse model of atherosclerosis is successfully established.Resveratrol helps to alleviate the atherosclerosis inflammation and reduce atherosclerotic plaque size,of which the mechanism may involve CD40 pathway inhibition.