Copper oxide nanoparticles (Nano-CuO) are one of the most produced and used nanomaterials. Previous studies have shown that exposure to Nano-CuO caused acute lung injury, inflammation, and fibrosis. However, the mechanisms underlying Nano-CuO-induced lung fibrosis are still unclear. Here, we hypothesized that exposure of human lung epithelial cells and macrophages to Nano-CuO would upregulate MMP-3, which cleaved osteopontin (OPN), resulting in fibroblast activation and lung fibrosis. A triple co-culture model was established to explore the mechanisms underlying Nano-CuO-induced fibroblast activation. Cytotoxicity of Nano-CuO on BEAS-2B, U937* macrophages, and MRC-5 fibroblasts were determined by alamarBlue and MTS assays. The expression or activity of MMP-3, OPN, and fibrosis-associated proteins was determined by Western blot or zymography assay. Migration of MRC-5 fibroblasts was evaluated by wound healing assay. MMP-3 siRNA and an RGD-containing peptide, GRGDSP, were used to explore the role of MMP-3 and cleaved OPN in fibroblast activation. Exposure to non-cytotoxic doses of Nano-CuO (0.5 and 1 µg/mL) caused increased expression and activity of MMP-3 in the conditioned media of BEAS-2B and U937* cells, but not MRC-5 fibroblasts. Nano-CuO exposure also caused increased production of cleaved OPN fragments, which was abolished by MMP-3 siRNA transfection. Conditioned media from Nano-CuO-exposed BEAS-2B, U937*, or the co-culture of BEAS-2B and U937* caused activation of unexposed MRC-5 fibroblasts. However, direct exposure of MRC-5 fibroblasts to Nano-CuO did not induce their activation. In a triple co-culture system, exposure of BEAS-2B and U937* cells to Nano-CuO caused activation of unexposed MRC-5 fibroblasts, while transfection of MMP-3 siRNA in BEAS-2B and U937* cells significantly inhibited the activation and migration of MRC-5 fibroblasts. In addition, pretreatment with GRGDSP peptide inhibited Nano-CuO-induced activation and migration of MRC-5 fibroblasts in the triple co-culture system. Our results demonstrated that Nano-CuO exposure caused increased production of MMP-3 from lung epithelial BEAS-2B cells and U937* macrophages, which cleaved OPN, resulting in the activation of lung fibroblasts MRC-5. These results suggest that MMP-3-cleaved OPN may play a key role in Nano-CuO-induced activation of lung fibroblasts. More investigations are needed to confirm whether these effects are due to the nanoparticles themselves and/or Cu ions.

中文翻译：

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MMP-3 介导的 OPN 裂解参与氧化铜纳米颗粒诱导的成纤维细胞激活

氧化铜纳米粒子（Nano-CuO）是生产和使用最多的纳米材料之一。先前的研究表明，接触纳米氧化铜会导致急性肺损伤、炎症和纤维化。然而，纳米CuO诱导肺纤维化的机制仍不清楚。在这里，我们假设人肺上皮细胞和巨噬细胞暴露于纳米 CuO 会上调 MMP-3，从而裂解骨桥蛋白 (OPN)，导致成纤维细胞活化和肺纤维化。建立三重共培养模型来探索纳米 CuO 诱导成纤维细胞活化的机制。通过 alamarBlue 和 MTS 测定确定纳米 CuO 对 BEAS-2B、U937* 巨噬细胞和 MRC-5 成纤维细胞的细胞毒性。通过蛋白质印迹或酶谱分析测定 MMP-3、OPN 和纤维化相关蛋白的表达或活性。通过伤口愈合测定评估 MRC-5 成纤维细胞的迁移。使用 MMP-3 siRNA 和含有 RGD 的肽 GRGDSP 来探索 MMP-3 和切割的 OPN 在成纤维细胞活化中的作用。暴露于非细胞毒性剂量的 Nano-CuO（0.5 和 1 µg/mL）会导致 BEAS-2B 和 U937* 细胞的条件培养基中 MMP-3 的表达和活性增加，但 MRC-5 成纤维细胞不会增加。纳米 CuO 暴露还导致裂解 OPN 片段的产生增加，但通过 MMP-3 siRNA 转染消除了这种现象。来自暴露于纳米 CuO 的 BEAS-2B、U937* 或 BEAS-2B 和 U937* 共培养物的条件培养基引起未暴露的 MRC-5 成纤维细胞的激活。然而，将 MRC-5 成纤维细胞直接暴露于纳米 CuO 并不会诱导其活化。在三重共培养系统中，将 BEAS-2B 和 U937* 细胞暴露于 Nano-CuO 会导致未暴露的 MRC-5 成纤维细胞活化，而在 BEAS-2B 和 U937* 细胞中转染 MMP-3 siRNA 显着抑制 MRC-5 成纤维细胞的活化和迁移。此外，GRGDSP 肽预处理抑制了三重共培养系统中纳米 CuO 诱导的 MRC-5 成纤维细胞的活化和迁移。我们的结果表明，纳米 CuO 暴露导致肺上皮 BEAS-2B 细胞和 U937* 巨噬细胞产生 MMP-3 增加，从而裂解 OPN，从而激活肺成纤维细胞 MRC-5。这些结果表明，MMP-3 裂解的 OPN 可能在纳米 CuO 诱导的肺成纤维细胞活化中发挥关键作用。需要更多的研究来确认这些影响是否是由纳米颗粒本身和/或铜离子造成的。