Role Of VGSCs In Phenotypic Switching In Mouse RAW264.7Cells And In Circulating And Intraplaque Monocyte/Macrophage Subsets Of Hyperlipidemic Mouse Model

Objective:Monocytes/macrophages represent an important part of the innate immune system providing organism with first line defense and at the same time responsible for maintenance of homeostasis. Inflammatory stimulation leads to the founctional change of monocytes. Monocytes/macrophages are heterogeneous groups, and can be divided into distinct subtypes. It is believed that these subpopulations differ in immune functions, moreover, abnormal proportion of the subsets can be linked to pathogenesis of virous disease. Therefore, the modulation of mononuclear phagocytes could be a potential therapeutic target for limiting the initiation and progression of certain disease. Phenytoin (PHT) is a non-selective antagonist of voltage-gated sodium channels (VGSCs). Previous studies havn showed that epilepsy patients receiving PHT therapy have a reduced incidence of ischemic heart disease compared with control group. It is well known that VGSCs are responsible for electrogenesis in excitable cells such as neurons, skeletal and cardiac muscles. However, VGSCs are also expressed in non-excitable cells including macrophages. Thereby, we hypothesize that VGSCs antagonist may confer its cardioprotective effect through modulating the phenotypes of monocyte/macrophage.Methods:1. Peripheral blood monocytes (Ly6G-CDllb+) were isolated from C57BL/6J mice using magnetic activated cell sorting. Then a PCR-based approach was used to detect mRNA expression of VGSCs (a subunits:NaV1.1-NaV1.9and NaVX;(3subunits:Scnlb-Scn4b) in purified mouse peripheral blood monocytes and RAW264.7macrophages;2. Determined the co-expression and abundance of VGSCs subunits in mouse RAW264.7macrophages and peripheral blood monocytes, then specific RNA interference (RNAi) plasmid was transfected into RAW264.7macrophages. Stable cell lines of low expressing specific VGSCs subtype were obtained in the presence of hygromycin B. Experiments were performed in stable cell lines:1) Efficiency of RNAi was quantified by real-time PCR;2) RAW264.7cell proliferation was measured by CCK-8;3) Flow cytometry was used to analyze cell cycle and phagocytosis;4) Migratory ability was detected by transwell chamber migration assay;5) Macrophage polarization of stable cell line induced by lipopolysaccharide or interleukin-4was measured by real-time PCR, the phenotypic markers are as followed, M1:iNOS; M2:Arg-1. The cell cycle of RAW264.7cells induced by PHT was analyzed by flow cytometry.3. C57BL/6J and ApoE-/-mice (6-8weeks) were used for in vivo study. Animal were grouped into high-fat and standard chow diet groups.10weeks later, mice consuming high fat were given PHT or normal saline for another4weeks, while the mice consuming clow diet were given normal saline at the same time. The proportions of monocyte/macrophage subsets were analyzed by flow cytometry at10and14weeks. Plasma triglycerides and total cholesterol concentration were measured by ELISA.Results:1) In purified mouse peripheral blood monocytes, the major expressed VGSCs isoforms were NaV1.1, NaV1.3-NaV1.9, NaVX and Scnlb-Scn4b, while NaV1.1, NaV1.3-NaV1.7, NaV1.9and NaVX were expressed in RAW264.7cells. NaV1.4and NaV1.9were the highest abundances in RAW264.7cells.2) The expression of NaV1.4was reduced by75%in NaV1.4knockdown cells. Silencing of NaV1.4could decrease RAW264.7cells’phagocytic and migratory ability. Moreover, PHT could inhibit RAW264.7cell growth, though NaV1.4knockdown had no effect on cell proliferation. The mRNA expression level of iNOS was down-regulated, and Argl was up-regulated in NaV1.4knockdown stable cell line.3) High fat diet could increase the proportion of Ly6Chi monocyte and Ly6ChiF4/80+macrophage. Compared with the saline group, Ly6Chi monocytes and Ly6ChiF4/80+macrophages decreased, while the Ly6Cl0monocyte and Ly6CloF4/80+macrophage had a opposite trend.4) High fat diet could increase the level of plasma triglyceride and cholesterol, and PHT had no effect on the lipid level.Conclusion:Blocking VGSCs in macrophages could inhibit macrophage proliferation and M1phenotype transition. VGSCs antagonist could inhibit the increase of pro-inflammatory monocyte (Ly6Chi) and macrophage (Ly6ChiF4/80+) induced by hyperlipidemia. The present study demonstrates that the VGSCs in monocyte/macrophage maybe a novel therapeutic target for astherosclerosis.