Effect Of Vasoactive Intestinal Peptide On The Expression Of TREM-1in Acute Lung Injury And Its Mechanism

Acute lung injury (ALI) and its severe form acute respiratory distress syndrome (ARDS) are breath failure, caused by a variety of factors. Excessive inflammation plays an important role in ALI/ARDS, and effectively inhibition of the "waterfall" inflammation in early phase of ALI/ARDS can improve the outcome of ALI/ARDS. Triggering receptor expressed on myeloid cells-1(TREM-1) is an immunoglobulin superfamily receptor, expressed on macrophage and granulocyte myeloid cell. It is reported that TREM-1could amplify the inflammation involving in TLR4. Vasoactive intestinal peptide (VIP) is one of the most abundant neuropeptides in lung. Our preliminary studies confirmed that VIP could inhibit the expression of pro-inflammatory cytokines like TNF-α, presenting anti-inflammatory characteristics. However, the effect of VIP on the expression of TREM-1in ALI mice is unclear.Objective:To investigate the TREM-1expression of mouse lungs in ALI and the effect of VIP on the expression of TREM-1.Methods: 1. A mouse model of acute lung injury (ALI) by intraperitoneal injection of lipopolysaccharide (LPS) was established to observe the expression pattern of TREM-1in mouse with ALI. VIP lentiviral vector was tracheal injected into mouse to observe the effect of high expression of VIP to TREM-1expression in mouse with ALI;2. Observe the effect of VIP (10-10,10-9,10-8and10-7M) on the TREM-1mRNA expression in LPS-stimulated murine macrophage. RT-PCR and flow cytometry was used to detect the mRNA and protein of TREM-1, resepectively. PDTC, NF-κB inhibitor, was used to investigate the expression of TREM-1of LPS-stimulated murine macrophage. Western Blot was used to detect the I-κB expression.Results:1. The effect of VIP on TREM-1mRNA expression in lung of ALI miceALI animal model was established, RT-PCR results showed that TREM-1mRNA expression in mice lungs with ALI was increased significantly after intraperitoneal injection of LPS for6h. Compared with GFP+LPS group (0.630±0.039), and VIP could reduce ALI in mice lungs of TREM-1mRNA expression (0.531±0.0271)(P<0.05).2. Effect of VIP on TREM-1mRNA expression in LPS-stressed murine macrophage 2.1Effect of VIP on TREM-1mRNA expression in murine macrophageRT-PCR results showed that:After treatment with different concentrations of VIP (10-10,10-9,10-8and10-7M)(P>0.05) or treatment with different times (2,4,6,12and24h)(P>0.05), TREM-1mRNA expression in murine macrophages did not change significantly.2.2Effect of VIP on TREM-1mRNA expression in LPS-stressed murine macrophageRT-PCR results showed that LPS could increase the expression of TREM-1mRNA significantly3h after stimulation (0.062±0.005), and the expression reached the peak after6h (0.107±0.015, P<0.05). LPS could increase the TREM-1mRNA expression in dose-dependent manner: the TREM-1mRNA level was increased as the concentration of LPS increased gradually. TREM-1mRNA expression reached the peak when the concentration of LPS was1μg/ml (0.165±0.015, P<0.05).Compared with the expression of TREM-1mRNA after2h (0.364±0.018) with VIP pretreatment, the expression was significantly decreased at6h point (0.251±0.008), and reached the lowest at12h point (0.178±0.006)(P<0.05). The expression of TREM-1mRNA was decreased gradually as the concentration of VIP increased (10-10,10-9,10-8and10-7M), and it reached the lowest when the concentration of VIP was10-7M (0.249±0.010, P<0.05). The results of flow cytometry showed that:LPS (1μg/ml) could increase TREM-1expression (117.46±6.225, P<0.05), and VIP (10-8M) could reduce the expression of TREM-1significantly (90.89±3.635, P<0.05).3. Effect of VIP on I-κB expression in LPS-stressed murine macrophageRT-PCR results showed that:PDTC statistically inhibited LPS-induced murine macrophage TREM-1mRNA expression (0.135±0.009, P<0.05).The results of Western Blot showed that:VIP didn’t impact the the expression of I-κB in mouse macrophages with the resting state (P>0.05); compared with the LPS group (0.186±0.022), the expression of I-κB was significantly increased in VIP±LPS group (0.317±0.034, P<0.05).Conclusion:1. The expression of TREM-1mRNA was increased in ALI, and VIP could reduce the expression of TREM-1mRNA;2. LPS induce TREM-1expression in murine macrophage. VIP can inhibit that effect and its mechanism may be related to NF-κB activation.