Targeting USP11 may alleviate radiation-induced pulmonary fibrosis by regulating endothelium tight junction

Radiation-induced pulmonary fibrosis (RIPF) is a major side effect after radiotherapy for thoracic malignancies. However, rare anti-RIPF therapeutics show definitive effects for treating this disease. Ubiquitin-specific peptidase 11 (USP11) has been reported to promote transforming growth factor β (TGFβ) signaling which plays an essential role underlying RIPF. Herein, we explored the role of USP11 on RIPF. In the present study, USP11-knockout (Usp11-/-) mice were used to explore the effects of USP11 on RIPF. The lung tissue was obtained after receiving 30 Gy X-ray irradiation. The expression of USP11, TGF-β1, and a-SMA was determined by immunohistochemical and Western Blot, respectively. γ-H2AX foci and TUNEL positive cells were detected by fluorescent technique to assess DNA damage and apoptosis. High-throughput proteomic analysis was applied to further explore the related mechanisms. The transwell co-culture method was used to investigate bystander effects in HELF cells induced by irradiated HMEC-1 cells in vitro. Here we found that radiation activated USP11 in vivo and in vitro. Our results showed that USP11 deficiency effectively decreased serum TGF-β1 level, suppressed α-SMA expression, and mitigated pulmonary fibrosis. In addition, fewer γ-H2AX foci and decreased apoptotic cells were identified after irradiation in the primary cells isolated from the lungs of Usp11-/- mice. High-throughput proteomics analysis results showed that 22-upregulated and 158-downregulated proteins were identified in the lung tissues of Usp11-/- mice after irradiation. Furthermore, gene set enrichment analysis (GSEA) revealed that USP11 deficiency affects the tight junction signaling pathway. We verified that USP11 deficiency remarkably reinforced tight junction in the endothelial cells and alleviated TGF-β1 to inhibit fibrosis of fibroblast cells. The present study preliminarily showed that USP11-knockout mitigated RIPF via reinforcement endothelial barrier function.放射性肺纤维化（Radiation-induced pulmonary fibrosis, RIPF）是胸部恶性肿瘤放疗后常见的主要不良反应，但目前尚无针对RIPF的治疗药物具备确凿疗效。已有研究表明，泛素特异性蛋白酶11（Ubiquitin-specific peptidase 11, USP11）可调控转化生长因子β（Transforming growth factor β, TGFβ）信号通路，而该通路在RIPF的发生发展中发挥关键作用。本研究旨在探究USP11在RIPF中的作用。本研究采用USP11基因敲除（Usp11⁻/⁻）小鼠，探究USP11对RIPF的影响：实验小鼠接受30 Gy X射线照射后采集肺组织，分别采用免疫组化法与蛋白质免疫印迹（Western Blot）检测USP11、TGF-β1及α平滑肌肌动蛋白（α-SMA）的表达水平；通过荧光技术检测γ-H2AX焦点与TUNEL阳性细胞，以评估DNA损伤与细胞凋亡情况；采用高通量蛋白质组学分析进一步探究潜在分子机制；此外，体外采用Transwell共培养法，探究照射后人微血管内皮细胞（HMEC-1 cells）诱导的人胚肺成纤维细胞（HELF cells）的旁观者效应。研究发现，辐射可在体内及体外激活USP11。结果显示，USP11基因敲除可有效降低血清TGF-β1水平、抑制α-SMA表达，并减轻肺纤维化程度。此外，从Usp11⁻/⁻小鼠肺组织分离的原代细胞经照射后，其γ-H2AX焦点数量与凋亡细胞比例均显著降低。高通量蛋白质组学分析结果表明，照射后Usp11⁻/⁻小鼠肺组织中共鉴定出22个上调蛋白与158个下调蛋白。进一步基因集富集分析（GSEA）结果显示，USP11缺失可调控紧密连接信号通路。本研究验证发现，USP11基因敲除可显著增强内皮细胞的紧密连接功能，并缓解TGF-β1介导的成纤维细胞纤维化过程。本研究初步证实，USP11基因敲除可通过增强内皮屏障功能，减轻放射性肺纤维化。