Apolipoprotein M Inhabit The Expression Of IL-1? And MCP-1 In Inflammatory Response In Vivo And In Vitro

Objective:Apolipoprotein M?apoM?is one of the high density lipoprotein?HDL?apolipoproteins and it could function as physiological carrier of sphingosine-1-phosphate?S1P?in vivo.It is well known that HDL could decrease inflammatory responses,which recently being suggested via the apoM-S1P pathway.In the present study,we further investigated importance of apoM on the inhibitory effects of HDL on inflammatory responses.Approach:?1?Forty-eight apoM knock-out(apoM^-/-)C57BL/6 male mice aged 8 to10-weeks-old?approximately 25g body weight?and forty-eight age and gender matched wild-type(apoM^+/+)C57BL/6 mice were applied in the present study.ApoM^-/-mice were randomly divided into control group and other 5 groups?1 h,3 hrs,6 hrs,12 hrs and 24 hrs resp.?with 8 mice in each group.ApoM^+/+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 other 5 groupswere randomly assigned to receive intraperitoneal?i.p.?injection of lipopolysaccharides?LPS??10mg/kg?and mice were sacrificed at 1 h,3 hrs,6hrs,12 hrs and 24 hrs respectively.The liver tissues were collected and detected mRNA expression of Interleukine-1 beta?IL-1??and monocyte chemotactic protein 1?MCP-1?.Moreover,the cell culture experiments were performed,the cells,a permanent human hybrid endothelial cell line?EA.hy926?,were cultured in the presence of recombinant human apoM?rec-apoM?and with over-expressed apoM(apoM^Tg)and were then administered with tumor necrosis factor-??TNF-??for investigating effects of apoM on the production of inflammation cytokines,such as IL-1?and MCP-1.?2?The EA.hy926cells were infected with negative control lentivirus and lentivirus carrying apoM gene,the total RNA and total cellular protein were obtained from stable cell lines,detect the expression of Sphingosine-1-phosphate receptor 1?S1P1?mRNA and protein in the cells were infected with negative control lentivirus(apoM^TgN cell)and lentivirus carrying apo M gene(apoM^Tg cell).Selecte for 8¹0 weeks,weighting about 25g healthy male apoM^+/+and apoM^-/-C57BL/6 mice each 8 animals were sacrificed,The expression level of S1P1mRNA was detected in the aorta of mice,and the expression level of S1P1 was detected by immunohistochemistry.?3?16 male C57BL/6 of apoM^+/+mice and 16 apoM^-/-mice were selected randomly and divided respectively into two group with 8 mice in each group?Normal Saline group and LPS group?,All animals were received intraperitoneal injection of corresponding reagent,sacrificed at one hours and extracted total RNA from their livers,and then detect the mRNA expression of 3?-hydroxysterol?-24-reductase?DHCR24?and IL-1?.Moreover,the apoM^TgN cell and apoM^Tg cell were infected or not be infected with TNF-?and then were extracted total RNA from cell to detect the mRNA expression of DHCR24 and IL-1?.Results:?1?The mRNA levels of IL-1?and MCP-1 in liver tissues were significantly higher after LPS administration in apoM^-/-mice,compared to those in apoM^+/+mice.In cell culture experiments,when cells were pre-cultured with rec-apoM or cells overexpressed apoM(apoM^Tg)showed decreased expressions of IL-1?and MCP-1 after TNF-?treatment compared to the normal apoM expressed cells(apoM^TgN).?2?The expression of S1P1mRNA and protein in cells of apoM over expressioned was higher than that in control group.The expression level of S1P1 mRNA and protein in the aorta of apoM^+/+mice was higher than that of apoM^-/-mice.?3?Under physiological conditions or no inflammatory stimulation,the expression of DHCR24 mRNA in cells of apoM over expressioned was higher than that in control group.The expression level of DHCR24 mRNA in the liver of apoM^+/+mice was higher than that of apoM^-/-mice.Conclusions:ApoM can increase the expression of S1P1 and DHCR24,it have inhibitory effects against the mRNA expression of inflammatory factor such as IL-1?and MCP-1 induced by LPS or TNF-?in vivo and in vitro,which might be mediated via the S1P1 and DHCR24 pathways.