The Effect Of Apolipoprotein M On The Expression And Phosphorylation Of Endothelial Nitric Oxide Synthase

Objective:More and more studies have shown that apolipoprotein M（apoM）is involved in many pathophysiological processes in human,as a component of high density lipoprotein（HDL）and a physiological carrier of 1-phosphate sphingosine（S1P）.Apo M is closely related to endothelial protection and inflammation.Endothelial nitric oxide synthase（eNOS）is enriched in endothelial cells,which is closely related to endothelial cell inflammation,vascular relaxation and platelet adhesion and aggregation.It is the aim of this study to explore whether apoM involved in the pathophysiological process of the body by mediating the changes in the total amount of eNOS and its activity.Approach:（1）Human umbilical vein-derived EA.hy926 cells is a permanent human hybrid endothelial cell line,which were infected with negative control lentivirus and lentivirus carrying apoM gene,obtaining stable expressed EA.hy926 cells transfected by negative control lentivirus(apoM^{T gN})and EA.hy926 cells transfected by lentivirus carrying apoM gene(apoM^{T g}),when the cells grew to 70%⁸0%confluency,replaced with 1%BSA and continue to culture cells 12 hours.Tumor necrosis factor-α（TNF-α）（10ng/ml）was used for the treatment of cells for 12 hours respectively,collected cells and extracted total RNA from cells,detect the expression of eNOS mRNA,assessment of the effect of apoM on the expression level of eNOS.（2）Sixteen apoM knock-out(apoM^-/-)C57BL/6male mice aged 8 to10-weeks-old（approximately 25 g body weight）and age and sixteen gender matched wild-type(apoM^+/+)C57BL/6 mice were applied in the present study.ApoM^-/-mice were randomly divided into control group and 12 hours group,with 8 mice in each group.Apo M^+/+mice were randomly divided into the same groups as the apoM^-/-mice.The mice of control group were not accepted any treatment.The mice in 12 hrs group were assigned to receive intraperitoneal（i.p.）injection of lipopolysaccharides（LPS）（10mg/kg）and mice were sacrificed at 12 hrs.The kidney tissues were collected and detected mRNAexpression of eNOS mRN A.（3）Subculture of apoM^{T gN}and apoM^{T g},when they reach a certain amount inoculate into 6-wells plates.Replaced with 1%BSA and continue to culture cells 12 hours when the cells grew to 70%⁸0%confluency,collecting cell and extraction of total protein,analysis and comparison of the effects of cell over expressed apoM on eNOS and eNOS Ser¹¹⁷⁷phosphorylation site.Results:（1）ApoM^T g g can enhance the expression of eNOS mRNA compared with apoM^{T gN}.After 12 hours of TNF-αtreatment,the expression of eNOS mRNA in apoM^Tgg cells was significantly increased compared with that in the blank control group and the apoM^{T gN}cells treated with TNF-α12 hours.（2）Under physiological conditions,compared with apo M^+/+mice,the expression level of eNOS has no obvious change in apo M^-/-mice.After treated with LPS for 12 hours,eNOS mRN A expression of apoM^+/+mice was significantly increased compared with apo M^-/-mice.ENOS mRNA expression of apoM^+/+mice also increased significantly after treated with LPS for 12 hours.（3）Compared with the corresponding control group apoM^{T gN},the apoM^{T g}group has no obvious effect on the total amount of eNOS protein,but could significantly increase the level of phosphorylation of eNOS Ser¹177177 in the endothelial cells.Conclusions:ApoM can up-regulate expression of eNOS mRNA under inflammatory conditions.ApoM can enhance the phosphorylation of eNOS Ser¹¹⁷⁷to play an anti-inflammatory effect.