Effects Of ApoM On Expression Of Adhesion Molecules In Endothelial Cells Induced By TNF-α

Objective To investigate the effect of the apoliprotein M(Apo M)bound Sphingosine 1-phosphate(S1P)and PKCδ on the induction of inflammatory cytokine ICAM-1in EA.hy926 cells.Methods(1)The EA.hy926 cells were transfected with human Apo M gene using lentiviral vectors.And then,two groups were divided,the group of Apo M overexpression(Apo M-OE)and the group of normal expression of Apo M for negative control(NC).The NC cells were divided into blank treatment group and TNF-α treatment group.The Apo M-OE cells were divided into blank treatment group and TNF-α treatment group.The expression of ICAM-1 m RNA was detected by PCR.(2)The NC cells were divided into blank treatment group and S1 P treatment group.The Apo M-OE cells were divided into blank treatment group and S1 P treatment group.The expression of ICAM-1 m RNA was detected by PCR.(3)NC cells were divided into TNF-α treatment group and S1P+TNF-α-treated group.The Apo M-OE cells were divided into TNF-α group and S1P+TNF-α-treated group.The expression of ICAM-1 m RNA was detected by PCR.(4)The NC cells were divided into TNF-α treatment group and Rotterin+TNF-α treatment group.The Apo M-OE cells were divided into TNF-α group and Rotterin+TNF-α group.The expression of ICAM-1 m RNA was detected by PCR.Results(1)The results of PCR showed that the expression of Apo M m RNA in Apo M-OE group was significantly higher than that in NC group.(2)The expression of ICAM-1 m RNA in the TNF-α treated group was significantly higher than that in the control group.After overexpression of Apo M,the level of ICAM-1 m RNA in the TNF-α treated group was significantly higher than that in the control group.The TNF-α treated group in the NC cell was significantly increased.The expression of ICAM-1 m RNA was significantly higher than that of Apo M-OE cells treated with TNF-α,indicating that overexpression of Apo M inhibited TNF-α-mediated ICAM-1 elevation in EAhy926 cells;The Two-way ANOVA results showed that TNF-α and Apo M have an interactive effect on the expression of ICAM-1.(3)There was no significant difference in the expression of ICAM-1 between the in NC blank control group and the Apo M-OE blank control group;the expression level of ICAM-1 m RNA in the Apo M-OE S1P-treated group was significantly lower than that in the NC control group,indicating that,under the condition of no TNF-α-mediated inflammation,Apo M overexpression and S1 P can significantly down-regulate ICAM-1 expression.The results of the two-way ANOVA suggest that there is no interaction between Apo M and S1 P in influencing ICAM-1 expression.(4)All the groups were pretreated with TNF-α.There was no significant difference in ICAM-1 m RNA between the Blank treatment group and the S1 P treatment group in NC cells.There was no significant difference in ICAM-1 m RNA between the Blank treatment group and the S1 P group in the Apo M-OE cells,which indicated that,compared with apo M-OEgroup,apo M bound S1 P can not significantly inhibit the expression of ICAM-1.(5)All the groups were pretreated with TNF-α.In NC cells,the expression of ICAM-1 m RNA of Blank treatment group was significantly lower than that of Rottlerin treatment group,which showed that the effect of TNF-α induced ICAM-1 expression was significantly augmented after inhibition of PKCδ activity.The expression of ICAM-1 m RNA of Rottlerin treatment group in Apo M-OE cells was significantly lower than that of Rottlerin group in NC cells,which indicated that Apo M could significantly inhibit the expression of Rotterin mediated the epression of ICAM-1.Conclusion TNF-α induced the increase of ICAM-1 m RNA level.In the absence of TNF-α,S1 P could inhibit the expression of ICAM-1in the Apo M-OE cells.Under TNF-α-mediated inflammatory conditions,overexpression of Apo M could inhibit the expression of ICAM-1.Besides,the expression of ICAM-1 in EA.hy926 cells was increased after blocking PKCδ by Rotterin,suggesting that PKCδ might play an anti-inflammatory role in endothelial cells.Overexpression of Apo M could inhibit the inceased ICAM-1 m RNA level induced by inactivation of PKCδ.In conclusion,Apo M may play anti-inflammatory effects through inhibiting the expression of ICAM-1mediated by Rottlerin in the endothelial cells.