Effects Of Triggering Receptor Expressed On Myeloid Cells-2on The Proliferation And Apoptosis In Mouse Fibroblasts And The Regulation Of VIP

Triggering receptor expressed on myeloid cells-2(TREM-2), firstly detected on immature dendritic cell (DC) surface in2000, is one of the receptor family of triggering receptor expressed on myeloid cells (TREM), mainly expressed in macrophages, fibroblasts and other cells. It could activate some T cells through extracellular regulated protein kinase (ERK) and tyrosine protein kinase (TPK). The activation of ERK under the stimulation of lipopolysaccharide (LPS) could induce apoptosis in DC. Vasoactive intestinal peptide (VIP) is one of the important neuropeptides in lung. However, it is uncertain that whether VIP could impact apoptosis, proliferation and inflammation through regulating the expression of TREM-2.Objective:to explore the relationship of TREM-2and mouse fibroblast apoptosis and proliferation, and to investigate the effect of VIP on the expression of TREM-2in LPS-stimulated mouse fibroblast and its mechanism.Method:(1) Effect of TREM-2on mouse fibroblast cell apoptosis and proliferation:Construct TREM-2over-expression plasmid and grouped according to the following transient transfection to mouse fibroblasts:①control group, empty plasmid group, TREM-2over-expression plasmid group. estimate the transfection efficiency at12h and24h after transfection by fluorescence microscopy; using RT-PCR to detect TREM-2mRNA expression; flow cytometry was employed to detect cell cycle;②LPS group, LPS+transfection reagent group, LPS+empty plasmid group and LPS+TREM-2over-expression plasmid group. PI staining detection of apoptosis in mouse fibroblast cell apoptosis.(2) Effect of VIP on LPS-stimulated mouse fibroblast cells TREM-2expression and its mechanism:RT-PCR and flow cytometry were used to detect the TREM-2expression under stimulation of LPS present or absent of VIP respectively on mRNA and protein levels. And the PKC signaling pathway inhibitor (H-7), PKA signaling pathway inhibitor (H-89), MAPK signaling pathway inhibitor (PD98059) and CaM signaling pathway inhibitor (W-7) were used to investigate the mechanism of VIP.Results:1. Effect of TREM-2over-expression on mouse fibroblast cell proliferation and apoptosis:(1) Fluorescence microscopy results showed that:there was green fluorescence under fluorescence microscopy at12h after transfection; and the ratio of green fluorescence (+) cell was about30%after24h.(2) RT-PCR results showed that:the TREM-2mRNA expression in TREM-2over-expression plasmid group (1.02±0.14) was significantly increased compared with the control group (0.22±0.03) and the empty plasmid group (0.25±0.03),(p<0.01).(3) Flow cytometry showed that:compared with the empty plasmid group (16.11±1.52)%, the ratio of G2/M phase in TREM-2over-expression plasmid group (21.98±2.23)%was significantly increased (p<0.05).(4) Apoptosis detection results showed that: LPS could induce apoptosis significantly in fibroblast. There was no difference between LPS group and LPS+empty plasmid group, while TREM-2over-expression could significantly reduce the LPS-induced apoptosis.2. Effect of VIP on LPS-stimulated TREM-2expression in mouse fibroblast and its mechanism:(1) RT-PCR results showed that:LPS reduced mouse fibroblast TREM-2mRNA expression (p<0.05); VIP could increased TREM-2mRNA expression in time-dependent manner (0h,3h,6h,12h,24h), which reached the peak at6h (p<0.01), and in dose-dependent (10-10mol/L,10-9mol/L,10-8mol/L,10-7mol/L) manner, which showed the most obvious effect at10-8mol/L (p<0.01).The effect of VIP (10-8mol/L) on the LPS-stimulated TREM-2mRNA expression at6h could be blocked by H-7, H-89, PD98059and W-7(p<0.05). (2) Flow cytometry results showed that:LPS reduced mouse fibroblast TREM-2expression (6848±1723.15; p<0.05), and VIP could upregulate TREM-2expression (23618±2888.44; p<0.01).The effect of VIP (10-8mol/L) on the LPS-stimulated TREM-2expression at6h could be blocked by H-7, H-89, PD98059and W-7(p<0.05).Conclusions:1. TREM-2inhibits apoptosis of mouse fibroblast.2. TREM-2promotes proliferation of mouse fibroblast.3. LPS reduced mouse fibroblast TREM-2expression.4. VIP could up-regulate TREM-2expression in LPS stimulated mouse fibroblast through PKC, PKA, MAPK and CaM intracellular signal transduction pathway.