Date on Master's Thesis/Doctoral Dissertation

12-2022

Document Type

Doctoral Dissertation

Degree Name

Ph. D.

Department

Environmental and Occupational Health Sciences

Degree Program

Public Health Sciences with a specialization in Environmental Health, PhD

Committee Chair

Zhang, Qunwei

Committee Co-Chair (if applicable)

Chen, Shao-yu

Committee Member

Chen, Shao-yu

Committee Member

Hoyle, Gary

Committee Member

Neal, Achel

Committee Member

Yu, Jerry

Author's Keywords

Nano-CuO; MMP-3; cell junction-associated proteins; fibroblast activation; pulmonary inflammation and fibrosis

Abstract

Copper oxide nanoparticles (Nano-CuO) are wildly used in medical and industrial fields and our daily necessities. However, the biosafety assessment of Nano-CuO is far behind their rapid development. This study was to investigate the potential mechanisms underlying Nano-CuO-induced pulmonary inflammation and fibrosis, especially to determine whether Nano-CuO exposure could dysregulate MMP-3, an important mediator in pulmonary fibrosis, and its roles in Nano-CuO-induced pulmonary inflammation and fibrosis. Aim 1 was to investigate whether exposure to Nano-CuO caused MMP-3 dysregulation in lung epithelial cells and the role of MMP-3 in Nano-CuO-induced alteration of cell junction-associated proteins. The potential mechanisms that might be involved in these effects were also explored. The results demonstrated that exposure to Nano-CuO, but not Nano-TiO2, caused increased ROS generation, MAPKs activation, and MMP-3 expression. Nano-CuO-induced ROS generation was not observed in mitochondrial DNA-depleted BEAS-2B ρ0 cells, indicating that mitochondria may be the main source of Nano-CuO-induced ROS generation, which was also confirmed by pretreatment of cells with Mito-TEMPO, a specific mitochondrial ROS inhibitor. In addition, pretreatment of cells with ROS scavengers or inhibitors or depleting mitochondrial DNA significantly attenuated Nano-CuO-induced MAPKs activation and MMP-3 upregulation, and pretreatment of cells with MAPKs inhibitors abolished Nano-CuO-induced MMP-3 upregulation, suggesting Nano-CuO-induced MMP-3 upregulation is through Nano-CuO-induced ROS generation and MAPKs activation. Moreover, exposure of BEAS-2B cells to Nano-CuO for 48 h resulted in decreased expression of tight junction-associated proteins such as zonula occludens protein-1 (ZO-1), occludin, and claudin-1, and adherens junction-associated protein E-cadherin, which were inhibited by MMP-3 siRNA transfection, suggesting an important role of MMP-3 in Nano-CuO-induced alterations of cell junction-associated proteins. Aim 2 was to examine whether Nano-CuO exposure could activate fibroblasts and the role of MMP-3 and OPN in this process. The results showed that exposure to non-cytotoxic doses of Nano-CuO caused a dose-dependent increase in MMP-3 expression and activity and OPN expression in both BEAS-2B cells and PMA-differentiated U937 macrophages (U937*), but not in MRC-5 fibroblasts. Nano-CuO exposure also increased the production of MMP-3-cleaved OPN fragment, and the cleavage of OPN was abolished by MMP-3 siRNA transfection in BEAS-2B and U937* cells. 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, which was reflected by increased expression of α-smooth muscle actin (α-SMA), Col1A1, and fibronectin. However, direct exposure to Nano-CuO did not induce the activation of MRC-5 fibroblasts. 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, suggesting that MMP-3 released from Nano-CuO-exposed BEAS-2B and U937* cells may play a key role in Nano-CuO-induced activation of MRC-5 fibroblasts. Furthermore, pretreated MRC-5 fibroblasts with GRGDSP, an RGD-containing peptide that interrupts the binding of MMP-3-cleaved OPN to its cell surface receptors, attenuated Nano-CuO-induced activation and migration of fibroblasts in the triple co-culture system, indicating that MMP-3-cleaved OPN is engaged in the activation of MRC-5 fibroblasts caused by Nano-CuO. Aim 3 was to investigate the role of MMP-3 in Nano-CuO-induced lung injury and fibrosis in vivo. Our results demonstrated that mice intratracheally exposed to Nano-CuO (0, 25, 50, 100 µg per mouse) resulted in a dose-dependent increase in acute lung inflammation at day 3 after exposure, which was evidenced by elevated neutrophil count, and the levels of total protein and LDH in BALF. Nano-CuO-induced acute lung inflammation was further confirmed histologically by the infiltration of excessive neutrophils into lung alveolar space and interstitial tissues. In the time-course study, Nano-CuO exposure caused increases in the neutrophil count, and the levels of total protein and LDH in BALF, which appeared as early as day 1 after exposure, peaked at day 3 and then declined. Although the total cell count, macrophage count, and LDH level in BALF decreased from day 14 after exposure, their levels were still higher than those in the control group at days 28 and 42 post-exposure, suggesting that Nano-CuO exposure caused chronic inflammation in mouse lungs. Nano-CuO-caused fibrosis were observed in the lung sections evidenced by trichrome staining at days 14, 28, and 42 post-exposure. Nano-CuO-induced fibrosis was further confirmed by increased hydroxyproline content and fibrosis-related proteins, such as α-SMA, Col1A1, and fibronectin. Nano-CuO exposure also caused persistent expression of MMP-3 in mouse lungs from day 1 to day 42 post-exposure. Elevated MMP-3 protein levels in BALF were confirmed by Western blot. To explore the role of MMP-3 in Nano-CuO-induced lung inflammation and fibrosis, mouse Ambion® In vivo Pre-designed MMP-3 siRNA was chosen. MMP-3 siRNA treatment significantly ameliorated the acute lung inflammation and injury caused by Nano-CuO. MMP-3 siRNA treatment also restored the downregulated cell junction-associated proteins such as ZO-1, occludin, and E-cadherin in mouse lungs caused by Nano-CuO. Furthermore, MMP-3 siRNA treatment alleviated Nano-CuO-induced chronic inflammation and fibrosis in mouse lungs. These results suggest that MMP-3 plays important roles in Nano-CuO-induced pulmonary inflammation, injury, and fibrosis. In summary, this study showed that exposure to Nano-CuO could cause ROS generation which further leads to MMP-3 production in lung epithelial cells. Increased MMP-3 production caused disruption of cell junction-associated proteins, initiating and promoting Nano-CuO-induced lung inflammation, injury, and fibrosis. Findings in this study could provide insights into the interventions that prevent metal nanoparticle-induced lung injury and fibrosis. Findings in this study also provide the scientific basis to establish the exposure limits of metal oxide nanoparticles.

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