神经调节素受体降解蛋白1调控巨噬细胞极化对成纤维细胞功能的影响

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

目的探讨E3连接酶神经调节素受体降解蛋白1(neuregulin receptor degradation protein 1,Nrdp1)调控巨噬细胞极化对成纤维细胞功能的影响。方法分别以内毒素(lipopolysaccharide, LPS)和γ干扰素(interferon-γ,IFN-γ)混合剂、白细胞介素13(interleukin-13,IL-13)诱导小鼠原代腹腔巨噬细胞为M1型巨噬细胞(简称M1)和M2型巨噬细胞(简称M2);流式细胞仪、Western blot分别检测M1表面标记蛋白CD11c、特异性分泌物——诱导型一氧化氮合酶(induciblenitricoxide synthase, iNOS)及M2表面标记蛋白CD206、特异性分泌物——精氨酸酶1(arginine1,Arg1)的表达。转染siRNA-Nrdp1 24 h, Western blot检测Nrdp1表达;建立巨噬细胞与成纤维细胞的Transwell共培养体系,按添加的诱导剂不同,分为LPS组(LPS及IFN-γ共同诱导)、LPS-NC组(siRNA阴性Nrdp1无关序列对照组)、LPS-siRNA-Nrdp1组(转染siRNA-Nrdp1质粒)及对应的IL-13组、IL-13-NC组、IL-13-siRNA-Nrdp1组;培养8、12、24 h, CCK-8检测成纤维细胞增殖情况,ELISA检测转化生长因子β1(transforming growth factor-beta1, TGF-β1)、Ⅰ型前胶原含量。对数据行析因设计方差分析、Bonferroni法检验。结果 LPS诱导的巨噬细胞中,M1表面标记蛋白CD11c阳性细胞比例显著高于对照组(P<0.05);IL-13诱导的巨噬细胞中,M2表面标记蛋白CD206阳性细胞比例显著高于未诱导组(P<0.05);LPS组M1特异性分泌物iNOS表达量显著高于对照组(P<0.01)及IL-13组(P<0.01);IL-13组M2型特异性分泌物Arg1显著高于对照组及LPS组(P<0.01)。siRNA-Nrdp1干扰巨噬细胞24 h后,Western blot检测结果显示,干扰组(siRNA-Nrdp1组)Nrdp1的蛋白表达量明显低于空白对照组(P<0.01)及阴性对照组(P<0.01)。共培养24 h后,CCK-8检测结果显示,IL-13-siRNA-Nrdp1组成纤维细胞增殖数量明显低于IL-13组及IL-13-NC组(P<0.01);ELISA检测结果显示,IL-13-siRNA-Nrdp1组TGF-β1含量明显低于IL-13组及IL-13-NC组(P<0.01),IL-13-siRNA-Nrdp1组Ⅰ型前胶原含量明显低于IL-13组(P<0.01)。结论通过siRNA干扰Nrdp1合成,影响巨噬细胞向M2型极化,可抑制M2型巨噬细胞与成纤维细胞共培养体系中成纤维细胞的增殖,减少共培养体系中成纤维细胞TGF-β1及Ⅰ型前胶原的分泌。

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 CD11 c 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 CD11 c 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.