METHODS: Transfection of HEK293T cells and AMs with plasmids carrying TNFAIP3 and C/EBPβ was performed, followed by co-culturing AMs with pulmonary fibroblasts. Immunoblotting analysis was then utilized to assess the expression of TNFAIP3, C/EBPβ, and collagen type 1 (Col1). Quantitative PCR analysis was conducted to quantify the mRNA levels of C/EBPβ, IL-10, and TGF-β1. STRING database analysis, and immunoprecipitation assays were employed to investigate the interactions between TNFAIP3 and C/EBPβ.
RESULTS: TNFAIP3 expression was significantly reduced in SSc-ILD AMs, correlating with increased Col1 production in fibroblasts. Overexpression of TNFAIP3 inhibited this pro-fibrotic activity. Conversely, C/EBPβ expression was elevated in SSc-ILD AMs, and its reduction through TNFAIP3 restoration decreased pro-fibrotic cytokines IL-10 and TGFβ1 levels. Protein-protein interaction studies confirmed the regulatory relationship between TNFAIP3 and C/EBPβ.
CONCLUSIONS: This study highlights the important role of TNFAIP3 in regulating pulmonary fibrosis in SSc-ILD by modulating C/EBPβ expression in AMs. These findings suggest that targeting TNFAIP3 could be a potential therapeutic strategy for managing SSc-ILD patients.
方法:用携带TNFAIP3和C/EBPβ的质粒转染HEK293T细胞和AMs,然后将AMs与肺成纤维细胞共培养。然后使用免疫印迹分析来评估TNFAIP3,C/EBPβ的表达,和胶原蛋白1型(Col1)。进行定量PCR分析以定量C/EBPβ的mRNA水平,IL-10和TGF-β1。STRING数据库分析,和免疫沉淀试验用于研究TNFAIP3和C/EBPβ之间的相互作用。
结果:TNFAIP3在SSc-ILDAMs中的表达显著降低,与成纤维细胞中Col1产生增加相关。TNFAIP3的过表达抑制了这种促纤维化活性。相反,SSc-ILDAMs中C/EBPβ表达升高,其通过TNFAIP3恢复的减少降低了促纤维化细胞因子IL-10和TGFβ1的水平。蛋白质-蛋白质相互作用研究证实了TNFAIP3和C/EBPβ之间的调节关系。
结论:本研究强调了TNFAIP3通过调节AM中C/EBPβ的表达在调节SSc-ILD肺纤维化中的重要作用。这些发现表明靶向TNFAIP3可能是治疗SSc-ILD患者的潜在治疗策略。