Gao Yunxing, Fu Yu, Chen Xiao, Li Zepeng, He Xiaowei, Li Xianwei
Departments of Medical Microbiology and Immunology of School of Basic Medicine, Wannan Medical College, Wuhu 241002, China.
Department of Clinical Medicine of School of Clinical Medicine, Wannan Medical College, Wuhu 241002, China.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi. 2023 Aug;39(8):693-700.
Objective To investigate the effects and mechanism of Interleukin-33 (IL-33) mediated proliferation and differentiation of pulmonary myofibroblasts (MFbs) in pulmonary fibrosis (PF). Methods C57BL/6 mice were randomly divided into four groups: a control group, a bleomycin (BLM) group, a BLM combined with IL-33 group and a BLM combined with anti-IL-33 antibody group, 12 mice in each group. The PF model was induced by intratracheal injection of BLM (5000 U/kg). The degrees of fibrosis were examined using HE and Masson staining. ELISA was used to measure the plasma levels of IL-33. Immunohistochemical staining was used to measure the expression of alpha smooth muscle actin (α-SMA) in lung tissue. Primary pulmonary fibroblasts were isolated and cultured from lung tissues of mice. The cells were divided into four groups: a control group, an IL-33 group, an IL-33 combined with dimethyl sulfoxide (DMSO) group and an IL-33 combined with pyrrolidine dithiocarbamate (PDTC) group. The cells were treated with DMSO or PDTC for 1 hour and then with IL-33 for 48 hours. Cell proliferation was measured by 5-ethynyl-2'-deoxyuridine (EdU) assay and cell cycle was measured by flow cytometry. Transwell assay was used to analyze cell migration. Real-time quantitative PCR was used to measure the expression of collagen type I (Col1), Col3 and α-SMA mRNA. The protein levels of IL-33, Col1, Col3, α-SMA, eukaryotic initiation factor 3a (eIF3a), phosphorylated IκBα (p-IκBα) (total lysate), p-NF-κB p65(total lysate) and NF-κB p65 (nucleus) were measured by Western blot analysis. Results In vivo, compared with the control group, the expressions of IL-33, p-IκBα (total lysate), p-NF-κB p65 (total lysate), NF-κB p65(nucleus), eIF3a, α-SMA, Col1 and Col3 in the BLM group significantly increased. Compared with the BLM group, the expressions of p-IκBα (total lysate), p-NF-κB p65 (total lysate), NF-κB p65 (nucleus), eIF3a, α-SMA, Col1 and Col3 in the IL-33 group increased further and the PF was further aggravated. But the effect of anti-IL-33 antibody was just opposite to that of IL-33. In vitro, IL-33 markedly induced the proliferation and migration of pulmonary fibroblasts, and significantly up-regulated the expression of p-IκBα (total lysate), p-NF-κB p65(total lysate), NF-κB p65 (nucleus), eIF3a, α-SMA, Col1 and Col3. But all these effects of IL-33 were reversed by pyrrolidine dithiocarbamate. Conclusion The results suggest that IL-33 may promote the expression of eIF3a by activating NF-κB signaling pathway, thus inducing the proliferation and differentiation of MFbs and promoting the occurrence and development of PF.
目的 探讨白细胞介素-33（IL-33）介导的肺成纤维细胞（MFbs）增殖和分化在肺纤维化（PF）中的作用及机制。方法 将C57BL/6小鼠随机分为四组：对照组、博来霉素（BLM）组、BLM联合IL-33组和BLM联合抗IL-33抗体组，每组12只小鼠。通过气管内注射BLM（5000 U/kg）诱导PF模型。采用HE和Masson染色检测纤维化程度。ELISA法检测血浆IL-33水平。免疫组化染色检测肺组织中α平滑肌肌动蛋白（α-SMA）的表达。从小鼠肺组织中分离培养原代肺成纤维细胞。将细胞分为四组：对照组、IL-33组、IL-33联合二甲基亚砜（DMSO）组和IL-33联合吡咯烷二硫代氨基甲酸盐（PDTC）组。细胞先用DMSO或PDTC处理1小时，然后用IL-33处理48小时。采用5-乙炔基-2'-脱氧尿苷（EdU）检测法检测细胞增殖，流式细胞术检测细胞周期。Transwell检测法分析细胞迁移。实时定量PCR检测Ⅰ型胶原（Col1）、Ⅲ型胶原（Col3）和α-SMA mRNA的表达。采用蛋白质免疫印迹分析检测IL-33、Col1、Col3、α-SMA、真核起始因子3a（eIF3a）、磷酸化IκBα（p-IκBα）（总裂解物）、磷酸化NF-κB p65（总裂解物）和NF-κB p65（细胞核）的蛋白水平。结果 在体内，与对照组相比，BLM组中IL-33、p-IκBα（总裂解物）、p-NF-κB p
