To Investigate The Anti-inflammatory Effects Of Two Active Components In Aina Balsam Oil On Vascular Endothelial Cells

Vascular endothelial cells(VECs),as a layer of specialized cells in the vascular cavity,are the key regulatory interface between blood and tissues.Inflammatory mediators can damage the barrier function of endothelial cells,leading to increased permeability,increased expression of surface adhesion factors,monocyte adhesion and foam cell formation,resulting in vascular dysfunction.Therefore,regulating the inflammatory pathway may be an effective way to repair damaged vascular endothelium and regulate its dysfunction in the future.Previous studies found that Blumea balsamifera oil had good anti-inflammatory effect.The chemical components of Blumea balsamifera oil were analyzed by GC/MS,and then the anti-inflammatory activities of its main components were screened.It was found that(-)-Linalool and?-Caryophyllene(BCP)had better anti-inflammatory activities.However,their anti-inflammatory effect on VECs and the possible mechanism are still unknown.Therefore,this paper aims to explore the anti-inflammatory effects of(-)-Linalool and BCP on VECs and their possible mechanisms.In this study,the MTT method was used to detect the cell vitality of(-)-Linalool and BCP on VECs.A cell inflammation model of VECs was builded by lipopolysaccharide(LPS).The effects of(-)-Linalool and BCP on the expression of PGE₂,COX-2,iNOS,IL-6,TNF-?,CD14,TLR4,My D88,VCAM-1,ICAM-1,E-selectin and MCP-1 were detected by ELISA and qRT-PCR.The effects of(-)-Linalool and BCP on the expression of E-selectin,MCP-1,NF-?Bp65 and I?B?in LPS stimulated VECs were detected by Western blot.Then TMT quantitative proteomics was used to identify the proteins in blank control group(K)and LPS group(M)and BCP+LPS group(Y).Through GO function and KEGG signal pathway enrichment analysis,candidate proteins related to inflammation were screened and verified by Western blot and qRT-PCR.Next,the correlation between candidate proteins and NF-?B signaling pathway related proteins was analyzed by using STRING database,and the target genes were screened.si RNA interference was used to silence the target gene.The expression of SOS1,NF-?B related protein,downstream inflammatory factors and adhesion factors were detected by immunofluorescence,qRT-PCR,ELISA and Western blot.MTT results showed that(-)-Linalool and BCP within 300?g/m L had no significant effect on the viability of chicken pulmonary artery vascular endothelial cells(chPAEC),but also had no significant effect on the viability of human vascular endothelial cells(EA.hy926)(concentration?200?g/m L).The results of ELISA and qRT-PCR showed that:LPS concentration of 4?g/m L can significantly increase the secretion of PGE₂,COX-2,iNOS of chPAEC(P<0.01),1?g/m L can significantly increase the expression of TNF-?and IL-6 mRNA of EA.hy926(P<0.001).However,the concentration of(-)-Linalool and BCP within(40-120?g/m L)can significantly inhibit the secretion of PGE₂,COX-2,iNOS,VCAM-1,and ICAM-1 of chPAEC stimulated by LPS(P<0.05),and significantly reduced the mRNA expression of IL-6,TNF-?,CD14,TLR4 and My D88.(-)-Linalool and BCP concentrations within(30-90?g/m L)can significantly inhibit LPS stimulation of IL-6,TNF-?,VCAM-1,ICAM-1,E-selectin,MCP-1 mRNA and protein expression(P<0.05)in EA.hy926cells.Western blot results showed that(-)-Linalool and BCP significantly reduced the expression of NF-?Bp65 protein in LPS stimulated chPAEC(P<0.01),it can also significantly reduced the protein expression of E-selectin,MCP-1,NF-?Bp65,and significantly increased the protein expression of I?B-?in LPS stimulated EA.hy926cells(P<0.01).TMT quantitative proteomics technology identified a total of 4800proteins in the M and K groups and M and Y groups in EA.hy926 cells.According to the criteria of FC>1.2 and FC<1/1.2 and P<0.05,168 differences were screened out.There were 25 up-regulations and 3 down-regulations in the M vs K group,while 63up-regulations and 77 down-regulations in the M vs Y group.After GO function and KEGG pathway analysis,candidate genes SOS1,GEM,Akrin2,Scribble related to inflammation were screened out,and the verification results were consistent with the proteomics data.Meanwhile,it has been verified that BCP can inhibit the expression of SOS1,GEM,Akirin2,and Scribble.Akirin2,SOS1,and NF-?B signaling pathway related proteins are analyzed through STRING database,and the correlation between SOS1 is stronger.After silencing the SOS1 gene by si RNA,the results of immunofluorescence,qRT-PCR,ELISA and Western blot showed that the expression of SOS1 mRNA and protein in EA.hy926 cells decreased significantly(P<0.05),and NF-?B signaling pathway NF-?Bp65 protein was significantly down-regulated(P<0.05),I?B-?protein was significantly up-regulated(P<0.05),downstream inflammatory factors and adhesion factors:IL-6,TNF-?,VCAM-1,ICAM-1,E-selectin,MCP-1 mRNA and protein were significantly down-regulated(P<0.05).The results suggest that LPS can increase the expression of VECs inflammatory factors and adhesion factors,(-)-Linalool and BCP could reduce the secretion of adhesion factors and inflammatory factors by inhibiting the activation of NF-?B signaling pathway,thereby alleviating the inflammatory damage of VECs caused by LPS.Further studies have found that BCP can regulate the activation of NF-?Bp65 by inhibiting the expression of SOS1,reduce the secretion of inflammatory factors and adhesion factors to protect VECs from damage,which lays the foundation for further research on the anti-inflammatory mechanism of(-)-Linalool and BCP.