Effect of neuregulin receptor degradation protein 1 regulation of macrophage polarization on function of fibroblasts in vitro

Abstract

Objective To explore the effect of E3 ligase neuregulin receptor degradation protein 1 (Nrdp1) regulating macrophage polarization on the function of fibroblasts. Methods Lipopolysaccharide (LPS) and interferon-γ (IFN-γ) were combined to induce mouse primary peritoneal macrophages to M1 macrophages (M1), and IL-13 was used to induce macrophages to M2 macrophages (M2). Then flow cytometry and Western blotting were used to detect M1 surface marker protein CD11c and its specific secretion inducible nitric oxide synthase (iNOS), and M2 surface marker protein CD206 and the secretion arginase 1 (Arg1). After the mouse macrophages was transfected with siRNA-Nrdp1 transiently for 24 h, the expression of Nrdp1 was detected by Western blotting. A Transwell co-culture system was established for macrophages and fibroblasts, and was induced under different conditions. The co-cultured cells were divided into LPS group (co-induction of LPS and IFN-γ), LPS-NC group (co-induction and siRNA of irrelevant sequence), LPS-siRNA-Nrdp1 group (co-induction of LPS and siRNA-Nrdp1), IL-13 group, IL-13-NC group and IL-13-siRNA-Nrdp1 group. After co-culture for 8, 12 and 24 h, CCK-8 assay was employed to detect the proliferation of fibroblasts, and ELISA to measure the contents of transforming growth factor β1 (TGF-β1) and type Ⅰ procollagen. Analysis of variance of factorial designs and Bonferroni test were used for statistical analysis for the data. Results The proportion of CD11c positive cells was significantly higher in the LPS-induced macrophages than the non-induced cells (P < 0.05), and similar result was seen in the IL-13-induced macrophages for CD206 expression (P < 0.05). Western blot analysis indicated the levels of iNOS and Arg1 were obviously higher in the LPS- and IL-13-induced macrophages than the non-induced control cells, respectively (P < 0.01). Transient transfection of siRNA-Nrdp1 resulted in notably lower expression of Nrdp1 when compared with the blank control and negative control transfection (both P < 0.01). In the co-culture system for 24 h, CCK8 assay showed the count of fibroblasts was significantly lower in the IL-13-siRNA-Nrdp1 group than the IL-13 and IL-13-NC groups (P < 0.01), and ELISA indicated that the content of TGF-β1 was lower in the IL-13-siRNA-Nrdp1 group than the IL-13 and IL-13-NC groups (P < 0.01), and that of type Ⅰ procollagen was lower in the IL-13-siRNA-Nrdp1 group than the IL-13 groups (P < 0.01). Conclusion Interference of Nrdp1 synthesis by siRNA can affect the polarization of macrophages to M2 type, inhibit the proliferation of fibroblasts in the co-culture system of M2 type macrophages and fibroblasts, and reduce the TGF-β1 and type Ⅰ procollagen in the co-culture system.

Keywords

• macrophage
• polarization
• neuregulin receptor degradation protein 1
• transforming growth factor β1
• procollagen type ⅰ