Chrysin Alleviates Vascular Endothelial Inflammation Via Inhibiting The NF-?B Signaling Pathway

Part ?:Chrysin alleviates expression of adhesion molecules on endothelial cell in inflammatory stateObjective:Early reports indicate that the occurrence of vascular endothelial inflammation is closely related to various vascular diseases such as atherosclerosis,thromboangiitis obliterans,diabetic vascular disease,etc.Flavonoids have been shown to be effective in preventing arteriosclerosis,lowering blood lipids and cholesterol,and reducing the incidence of coronary heart disease.However,studies on flavonoids in endothelial inflammation have been rarely mentioned.The aim of this experiment was to investigate the effects of a flavonoid,chrysin,on endothelial cell inflammation and expression of adhesion molecules.Methods:Primary human umbilical vein endothelial cells(HUVEC)were incubated with four concentrations of extreme low,low,medium and high concentrations of chrysin for 6 hours,then stimulated endothelial cells with interleukin 1?(IL-1?)to mimic the model of endothelial cell inflammation.Cell adhesion assays was used to detect the effect of different drug concentrations on the adhesion rate of human monocytes(THP-1)to HUVECs.Western-blot and real-time PCR were used to detect the effects of chrysin on messenger RNA and protein expression levels of intercellular adhesion molecule 1(ICAM-1),vascular adhesion molecule 1(VCAM-1)and E-selectin.Results:Chrysin reduces the adhesion rate of THP-1 to HUVECs which are in inflammatory state effectively,and inhibits IL-1?-induced the increase of mRNA levels and protein levels of ICAM-1,VCAM-1 and E-selectin concentration-dependently.Conclusions:In inflammatory state of endothelial cells,chrysin can effectively inhibit the adhesion between primary human umbilical vein endothelial cells and monocytes,and suppress the transcription and translation of adhesion molecules to alleviate the occurrence of endothelial cell inflammation.Part ?:Chrysin affects the expression of adhesion molecules via NF-?B signaling pathwayObjective:The previous part of the experiment demonstrated that chrysin has a significant concentration-dependent inhibition of endothelial cell inflammation.According to previous studies,nuclear factor ?B(NF-?B)is important in the inflammatory response of endothelial cells,especially in the role of regulating adhesion molecules,we have further studied the molecular mechanism of inhibition of endothelial cell inflammation by chrysin.Methods:After human umbilical vein endothelial cells were incubated with different concentrations of chrysin,they were stimulated by interleukin-1?.The nuclear factor ?B p65 subunit and nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor,alpha(I?B?)in NF-?B pathway were detected by western-blot,and it is aimed to investigate how the expression of phosphorylated protein and total protein was influenced by different concentrations of chrysin.The translocation of p65 in endothelial cells was detected by p65 immunofluorescence staining.After treatment of the drug,proteins in HUVECs were Isolated From cytoplasm and nucleus and the expression levels of p65 and IKBa in both cytoplasm and nucleus were then detected by western-blot.After incubation of Human embryonic kidney 293 cells(HEK-293T)with different concentrations of chrysin,the effects of promoter activities of ICAM-1,VCAM-1 and E-selectin were detected by a dual luciferase reporter assay system.Results:Under the treatment of HUVEC with different concentrations of chrysin,p-65 and p-I?B? decreased in a concentration-dependent manner,and the total p65 expression remained unchanged,while the total IKBa expression was firstly decreased by IL-1?,and then increased concentration-dependently.After incubation with medium and high concentrations of chrysin,the increase of p65 in cytoplasm of HUVEC was detected by immunofluorescence,while the p65 in nucleus was decreased.After the proteins wereextracted from cytoplasm and nucleus,at medium chrysin concentration,p65 in the cytoplasm was slightly increased,and at high concentrations,p65 in the cytoplasm was reduced;p65 in the nucleus was decreased concentration-dependently.The promoter activities of ICAM-1 and VCAM-1 in 293T cells after incubation with chrysin were significantly inhibited in a concentration-dependent manner,while E-selectin only showed a downward trend.Conclusions:In endothelial cells,chrysin inhibits IL-1?-induced activation of the NF-?B signaling pathway and simultaneously inhibits the nuclear translocation of NF-?B,increasing I?B? degradation.Chrysin can significantly reduce the transcriptional activity of the promoters of ICAM-1 and VCAM-1 and attenuate the transcriptional activity of the E-selectin promoter.Part ?:Chrysin reduces vascular inflammation in murine model of acute inflammationObjective:To investigate the effect of chrysin on vascular inflammation in mice.Methods:The mice were intraperitoneally injected with lipopolysaccharide(LPS)after 7 days of continuous administration of low and high dose of chrysin and positive control drug.After 18 hours,the mice were sacrificed and Lung,liver and kidney were harvest.Then Tissue sectioning and HE staining were used to observe endovascular and perivascular leukocyte infiltration,erythrocyte aggregation and interstitial edema.Results:Vascular injury,such as leukocyte infiltration,erythrocyte aggregation and interstitial edema in mice pretreated with chrysin decreased in a dose-dependent manner.Conclusions:Chrysin attenuates leukocyte-endothelial cell adhesion and inflammation,and also inhibits the permeability when endothelium was damaged,and further studies should be done to investigate the potential use of chrysin in diseases such as diabetic vascular disease or atherosclerosis.