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Cited by 24Interstitial Lung Diseases and Idiopathic Pulmonary FibrosisOccupational and environmental lung diseasesNanoparticles: synthesis and applications

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MMP-3-mediated cleavage of OPN is involved in copper oxide nanoparticle-induced activation of fibroblasts

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Zhang, Yuanbao, Mo, Yiqun, Zhang, Yue, Yuan, Jiali, Zhang, Qunwei·2023·Particle and Fibre Toxicology
DOI 10.1186/s12989-023-00532-yPMC PMC10201731
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Abstract

Background: 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. Methods: 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. Results: 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. Conclusions: 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.

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Figures

Fig. 1 Cytotoxicity of Nano-CuO on BEAS-2B cells, U937-derived macrophages (U937*), and MRC-5 fibroblasts. BEAS-2B (a), U937* macrophages (b), and MRC-5 fibroblasts (c) were seeded into 96-well plates and treated with 0.5, 1, 2, 5, and 10 µg/mL of Nano-CuO for 24 h (a and b) and 48 h (c), respectively. Cells without treatment were used as control. The cytotoxicity was determined by alamarBlue assay. Data represent mean ± SE (n = 6). * Significant difference as compared to the control group, p < 0.05
Fig. 1 Cytotoxicity of Nano-CuO on BEAS-2B cells, U937-derived macrophages (U937*), and MRC-5 fibroblasts. BEAS-2B (a), U937* macrophages (b), and MRC-5 fibroblasts (c) were seeded into 96-well plates and treated with 0.5, 1, 2, 5, and 10 µg/mL of Nano-CuO for 24 h (a and b) and 48 h (c), respectively. Cells without treatment were used as control. The cytotoxicity was determined by alamarBlue assay. Data represent mean ± SE (n = 6). * Significant difference as compared to the control group, p < 0.05CC BY · PubMed Central
Fig. 2 Increased expression and activity of MMP-3 in the conditioned media of BEAS-2B cells and U937* macrophages exposed to Nano-CuO. BEAS-2B cells (a and c) and U937* macrophages (b and d) were exposed to 0.5 and 1 µg/mL of Nano-CuO for 12 h. After treatment, the conditioned media were collected. Cells without treatment were used as control. MMP-3 protein level in the conditioned media was detected by Western blot, while its activity was detected by β-casein zymography. Equal protein loading was verified by Coomassie Brilliant Blue staining. The activity of MMP-3 was also determined in the conditioned media from co-culture system (e). a, b, and e were the results of a single experiment, while c and d were normalized band densitometry readings averaged from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05
Fig. 2 Increased expression and activity of MMP-3 in the conditioned media of BEAS-2B cells and U937* macrophages exposed to Nano-CuO. BEAS-2B cells (a and c) and U937* macrophages (b and d) were exposed to 0.5 and 1 µg/mL of Nano-CuO for 12 h. After treatment, the conditioned media were collected. Cells without treatment were used as control. MMP-3 protein level in the conditioned media was detected by Western blot, while its activity was detected by β-casein zymography. Equal protein loading was verified by Coomassie Brilliant Blue staining. The activity of MMP-3 was also determined in the conditioned media from co-culture system (e). a, b, and e were the results of a single experiment, while c and d were normalized band densitometry readings averaged from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05CC BY · PubMed Central
Fig. 3 Increased expression of OPN in BEAS-2B cells and U937* macrophages exposed to Nano-CuO. BEAS-2B cells, U937* macrophages, or co-culture of BEAS-2B and U937* macrophages at a ratio of 1:1 or 9:1 (BEAS-2B:U937*) were exposed to 0.5 and 1 µg/mL of Nano-CuO for 12 h. After treatment, the cells and conditioned media were collected. Cells without treatment were used as control. Full-length OPN (66 kDa) in the cells and cleaved OPN (40 kDa N-terminal fragment) in the conditioned media were detected by Western blot. Equal protein loading was verified by β-actin expression (a and b) or Coomassie Brilliant Blue staining (c and d). a and c were the results of a single experiment. b and d were results of three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05
Fig. 3 Increased expression of OPN in BEAS-2B cells and U937* macrophages exposed to Nano-CuO. BEAS-2B cells, U937* macrophages, or co-culture of BEAS-2B and U937* macrophages at a ratio of 1:1 or 9:1 (BEAS-2B:U937*) were exposed to 0.5 and 1 µg/mL of Nano-CuO for 12 h. After treatment, the cells and conditioned media were collected. Cells without treatment were used as control. Full-length OPN (66 kDa) in the cells and cleaved OPN (40 kDa N-terminal fragment) in the conditioned media were detected by Western blot. Equal protein loading was verified by β-actin expression (a and b) or Coomassie Brilliant Blue staining (c and d). a and c were the results of a single experiment. b and d were results of three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05CC BY · PubMed Central
Fig. 4 The role of MMP-3 on Nano-CuO-induced production of cleaved OPN in BEAS-2B cells and U937* macrophages. BEAS-2B cells (a and b) and U937* macrophages (c and d) were transfected with 30 nM of MMP-3 siRNA or Negative Control No. 2 siRNA. After transfection, the cells were exposed to 1 µg/mL of Nano-CuO for 12 h. The conditioned media were collected to detect the level of cleaved OPN (40 kDa N-terminal fragment) by Western blot. Equal protein loading was verified by Coomassie Brilliant Blue staining. a and c were the results of a single experiment. b and d were normalized band densitometry readings averaged from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05; # Significant difference as compared to the Nano-CuO-treated group transfected with Negative Control No. 2 siRNA, p < 0.05
Fig. 4 The role of MMP-3 on Nano-CuO-induced production of cleaved OPN in BEAS-2B cells and U937* macrophages. BEAS-2B cells (a and b) and U937* macrophages (c and d) were transfected with 30 nM of MMP-3 siRNA or Negative Control No. 2 siRNA. After transfection, the cells were exposed to 1 µg/mL of Nano-CuO for 12 h. The conditioned media were collected to detect the level of cleaved OPN (40 kDa N-terminal fragment) by Western blot. Equal protein loading was verified by Coomassie Brilliant Blue staining. a and c were the results of a single experiment. b and d were normalized band densitometry readings averaged from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05; # Significant difference as compared to the Nano-CuO-treated group transfected with Negative Control No. 2 siRNA, p < 0.05CC BY · PubMed Central
Fig. 5 Direct Nano-CuO exposure had no effects on activation of MRC-5 fibroblasts. MRC-5 cells were treated with 0.5 and 1 µg/mL of Nano-CuO for 48 h (a and b) or with 1 µg/mL of Nano-CuO for 12, 24, 48, and 72 h (c and d). Cells without treatment were used as control. Proteins were isolated from the cells to detect the expression of α-SMA, Col1A1, and fibronectin. a and c were the results of a single Western blot experiment. b and d were the average expression levels of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3)
Fig. 5 Direct Nano-CuO exposure had no effects on activation of MRC-5 fibroblasts. MRC-5 cells were treated with 0.5 and 1 µg/mL of Nano-CuO for 48 h (a and b) or with 1 µg/mL of Nano-CuO for 12, 24, 48, and 72 h (c and d). Cells without treatment were used as control. Proteins were isolated from the cells to detect the expression of α-SMA, Col1A1, and fibronectin. a and c were the results of a single Western blot experiment. b and d were the average expression levels of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3)CC BY · PubMed Central
Fig. 6 Activation of MRC-5 fibroblasts cultured in the conditioned media from Nano-CuO-exposed BEAS-2B or U937* cells. a is the experimental protocol. BEAS-2B or U937* cells were treated with 1 µg/mL of Nano-CuO for 12 h and the conditioned media (CM) were collected to culture MRC-5 fibroblasts. After 0, 12, 24, 48, and 72 h culture, the MRC-5 fibroblasts were collected for protein isolation and Western blot. b and d were the results of a single Western blot experiment. c and e were the average expression levels of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05. Graphics in a were created with BioRender (https://BioRender.com)
Fig. 6 Activation of MRC-5 fibroblasts cultured in the conditioned media from Nano-CuO-exposed BEAS-2B or U937* cells. a is the experimental protocol. BEAS-2B or U937* cells were treated with 1 µg/mL of Nano-CuO for 12 h and the conditioned media (CM) were collected to culture MRC-5 fibroblasts. After 0, 12, 24, 48, and 72 h culture, the MRC-5 fibroblasts were collected for protein isolation and Western blot. b and d were the results of a single Western blot experiment. c and e were the average expression levels of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05. Graphics in a were created with BioRender (https://BioRender.com)CC BY · PubMed Central
Fig. 7 Conditioned media from Nano-CuO-exposed BEAS-2B or U937* cells enhanced migration of unexposed MRC-5 fibroblasts. BEAS-2B or U937* cells were treated with 0 or 1 µg/mL of Nano-CuO for 12 h and the conditioned media (CM) were collected to culture MRC-5 fibroblasts for 48 h. Migration of MRC-5 cells was detected by wound healing assay. a and b were representative images. c and d were the average closure area from three independent experiments. Scale bars in a and b represent 200 µm. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05
Fig. 7 Conditioned media from Nano-CuO-exposed BEAS-2B or U937* cells enhanced migration of unexposed MRC-5 fibroblasts. BEAS-2B or U937* cells were treated with 0 or 1 µg/mL of Nano-CuO for 12 h and the conditioned media (CM) were collected to culture MRC-5 fibroblasts for 48 h. Migration of MRC-5 cells was detected by wound healing assay. a and b were representative images. c and d were the average closure area from three independent experiments. Scale bars in a and b represent 200 µm. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05CC BY · PubMed Central
Fig. 8 Cultured in the conditioned media from Nano-CuO-exposed co-culture of BEAS-2B and U937* cells caused activation of unexposed MRC-5 fibroblasts. a is the experimental protocol of the co-culture experiments. Co-culture of BEAS-2B and U937* cells with ratio of 1:1 or 9:1 (BEAS-2B:U937*) was treated with 1 µg/mL of Nano-CuO for 12 h and the conditioned media (CM) were collected. MRC-5 fibroblasts were then cultured in the conditioned media for 0, 12, 24, 48, and 72 h. Proteins from MRC-5 cells were isolated to detect the expression of α-SMA, Col1A1, and fibronectin. b and d were the results of a single Western blot experiment. c and e were the average expression levels of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05. Graphics in a were created with BioRender (https://BioRender.com)
Fig. 8 Cultured in the conditioned media from Nano-CuO-exposed co-culture of BEAS-2B and U937* cells caused activation of unexposed MRC-5 fibroblasts. a is the experimental protocol of the co-culture experiments. Co-culture of BEAS-2B and U937* cells with ratio of 1:1 or 9:1 (BEAS-2B:U937*) was treated with 1 µg/mL of Nano-CuO for 12 h and the conditioned media (CM) were collected. MRC-5 fibroblasts were then cultured in the conditioned media for 0, 12, 24, 48, and 72 h. Proteins from MRC-5 cells were isolated to detect the expression of α-SMA, Col1A1, and fibronectin. b and d were the results of a single Western blot experiment. c and e were the average expression levels of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05. Graphics in a were created with BioRender (https://BioRender.com)CC BY · PubMed Central
Fig. 9 Indirect exposure to Nano-CuO caused activation of MRC-5 fibroblasts in a triple co-culture system. a is the experimental protocol for the triple co-culture experiments. BEAS-2B cells and U937* cells were seeded at the ratio of 1:1 (b and c) or 9:1 (d and e) (BEAS-2B:U937*) in the inserts of Transwell® units and MRC-5 cells were seeded in the lower chambers of the units. After establishment of the triple co-culture system, BEAS-2B and U937* cells were exposed to 0, 0.5 and 1 µg/mL of Nano-CuO for 12 h, then the inserts were taken out. MRC-5 cells in the lower chamber were incubated for another 36 h and proteins were isolated to detect the expression of α-SMA, Col1A1, and fibronectin. b and d were the results of a single Western blot experiment. c and e were the average expression levels of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05. Graphics in a were created with BioRender (https://BioRender.com)
Fig. 9 Indirect exposure to Nano-CuO caused activation of MRC-5 fibroblasts in a triple co-culture system. a is the experimental protocol for the triple co-culture experiments. BEAS-2B cells and U937* cells were seeded at the ratio of 1:1 (b and c) or 9:1 (d and e) (BEAS-2B:U937*) in the inserts of Transwell® units and MRC-5 cells were seeded in the lower chambers of the units. After establishment of the triple co-culture system, BEAS-2B and U937* cells were exposed to 0, 0.5 and 1 µg/mL of Nano-CuO for 12 h, then the inserts were taken out. MRC-5 cells in the lower chamber were incubated for another 36 h and proteins were isolated to detect the expression of α-SMA, Col1A1, and fibronectin. b and d were the results of a single Western blot experiment. c and e were the average expression levels of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05. Graphics in a were created with BioRender (https://BioRender.com)CC BY · PubMed Central
Fig. 10 The role of MMP-3 on Nano-CuO-induced activation of MRC-5 fibroblasts in the triple co-culture system. BEAS-2B and U937* cells were seeded at the ratio of 1:1 (a and b) or 9:1 (c and d) (BEAS-2B:U937*) in the inserts of Transwell® units and transfected with 30 nM of MMP-3 siRNA or Negative Control No. 2 siRNA. After transfection, the cells were exposed to 1 µg/mL of Nano-CuO for 12 h. After exposure, the inserts were taken out and the MRC-5 fibroblasts seeded in the lower chamber were incubated for another 36 h. The proteins isolated from MRC-5 fibroblasts were used to detect the expression levels of α-SMA, Col1A1, and fibronectin by Western blot. a and c were the results of a single Western blot experiment. b and d were the average expression level of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05; # Significant difference as compared to the Nano-CuO-treated group transfected with Negative Control No. 2 siRNA, p < 0.05
Fig. 10 The role of MMP-3 on Nano-CuO-induced activation of MRC-5 fibroblasts in the triple co-culture system. BEAS-2B and U937* cells were seeded at the ratio of 1:1 (a and b) or 9:1 (c and d) (BEAS-2B:U937*) in the inserts of Transwell® units and transfected with 30 nM of MMP-3 siRNA or Negative Control No. 2 siRNA. After transfection, the cells were exposed to 1 µg/mL of Nano-CuO for 12 h. After exposure, the inserts were taken out and the MRC-5 fibroblasts seeded in the lower chamber were incubated for another 36 h. The proteins isolated from MRC-5 fibroblasts were used to detect the expression levels of α-SMA, Col1A1, and fibronectin by Western blot. a and c were the results of a single Western blot experiment. b and d were the average expression level of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05; # Significant difference as compared to the Nano-CuO-treated group transfected with Negative Control No. 2 siRNA, p < 0.05CC BY · PubMed Central
Fig. 11 MMP-3 siRNA transfection attenuated Nano-CuO-induced migration of MRC-5 fibroblasts in the triple co-culture system. BEAS-2B and U937* cells were seeded at a ratio of 1:1 (a and c) or 9:1 (b and d) (BEAS-2B:U937*) in the inserts of Transwell® units and transfected with 30 nM of MMP-3 siRNA or Negative Control No. 2 siRNA. After transfection, the cells were exposed to 1 µg/mL of Nano-CuO for 12 h. After exposure, the inserts were taken out and the MRC-5 fibroblasts seeded in the lower chamber were incubated for another 36 h. Migration of MRC-5 cells was detected by wound healing assay. Cells without any treatments were used as the control. a and b were representative images. c and d were the average closure area from three independent experiments. Scale bars in a and b represent 200 µm. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05; # Significant difference as compared to the Nano-CuO-treated group with control siRNA transfection, p < 0.05
Fig. 11 MMP-3 siRNA transfection attenuated Nano-CuO-induced migration of MRC-5 fibroblasts in the triple co-culture system. BEAS-2B and U937* cells were seeded at a ratio of 1:1 (a and c) or 9:1 (b and d) (BEAS-2B:U937*) in the inserts of Transwell® units and transfected with 30 nM of MMP-3 siRNA or Negative Control No. 2 siRNA. After transfection, the cells were exposed to 1 µg/mL of Nano-CuO for 12 h. After exposure, the inserts were taken out and the MRC-5 fibroblasts seeded in the lower chamber were incubated for another 36 h. Migration of MRC-5 cells was detected by wound healing assay. Cells without any treatments were used as the control. a and b were representative images. c and d were the average closure area from three independent experiments. Scale bars in a and b represent 200 µm. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05; # Significant difference as compared to the Nano-CuO-treated group with control siRNA transfection, p < 0.05CC BY · PubMed Central
Fig. 12 The role of MMP-3-cleaved OPN in Nano-CuO-induced activation of MRC-5 fibroblasts in the triple co-culture system. BEAS-2B and U937* cells were seeded at the ratio of 1:1 (a and b) or 9:1 (c and d) (BEAS-2B:U937*) in the inserts, and MRC-5 fibroblasts were seeded in the lower chambers of Transwell® units. MRC-5 fibroblasts were pretreated with 200 µg/mL of GRGDSP peptides, then BEAS-2B and U937* cells were exposed to 1 µg/mL of Nano-CuO for 12 h. After exposure, the inserts were taken out and the MRC-5 fibroblasts were incubated for another 36 h. The proteins from MRC-5 fibroblasts were isolated to determine the activation of MRC-5 fibroblasts by Western blot. a and c were the results of a single Western blot experiment. b and d were the average expression levels of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05; # Significant difference as compared to the Nano-CuO-treated alone group, p < 0.05
Fig. 12 The role of MMP-3-cleaved OPN in Nano-CuO-induced activation of MRC-5 fibroblasts in the triple co-culture system. BEAS-2B and U937* cells were seeded at the ratio of 1:1 (a and b) or 9:1 (c and d) (BEAS-2B:U937*) in the inserts, and MRC-5 fibroblasts were seeded in the lower chambers of Transwell® units. MRC-5 fibroblasts were pretreated with 200 µg/mL of GRGDSP peptides, then BEAS-2B and U937* cells were exposed to 1 µg/mL of Nano-CuO for 12 h. After exposure, the inserts were taken out and the MRC-5 fibroblasts were incubated for another 36 h. The proteins from MRC-5 fibroblasts were isolated to determine the activation of MRC-5 fibroblasts by Western blot. a and c were the results of a single Western blot experiment. b and d were the average expression levels of α-SMA, Col1A1, and fibronectin normalized to β-actin from three independent experiments. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05; # Significant difference as compared to the Nano-CuO-treated alone group, p < 0.05CC BY · PubMed Central
Fig. 13 Pretreatment of GRGDSP peptides inhibited Nano-CuO-induced migration of MRC-5 fibroblasts in the triple co-culture system. BEAS-2B and U937* cells were seeded at the ratio of 1:1 (a and c) or 9:1 (b and d) (BEAS-2B:U937*) in the inserts and MRC-5 fibroblasts were seeded in the lower chambers of Transwell® units. MRC-5 fibroblasts were pretreated with 200 µg/mL of GRGDSP peptides, then BEAS-2B and U937* cells were exposed to 1 µg/mL of Nano-CuO for 12 h. After exposure, the inserts were taken out and the MRC-5 fibroblasts were incubated for another 36 h. Migration of MRC-5 cells was detected by wound healing assay. a and b were representative images. c and d were the average closure area from three independent experiments. Scale bars in a and b represent 200 µm. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05; # Significant difference as compared to the Nano-CuO-treated alone group, p < 0.05
Fig. 13 Pretreatment of GRGDSP peptides inhibited Nano-CuO-induced migration of MRC-5 fibroblasts in the triple co-culture system. BEAS-2B and U937* cells were seeded at the ratio of 1:1 (a and c) or 9:1 (b and d) (BEAS-2B:U937*) in the inserts and MRC-5 fibroblasts were seeded in the lower chambers of Transwell® units. MRC-5 fibroblasts were pretreated with 200 µg/mL of GRGDSP peptides, then BEAS-2B and U937* cells were exposed to 1 µg/mL of Nano-CuO for 12 h. After exposure, the inserts were taken out and the MRC-5 fibroblasts were incubated for another 36 h. Migration of MRC-5 cells was detected by wound healing assay. a and b were representative images. c and d were the average closure area from three independent experiments. Scale bars in a and b represent 200 µm. Data represent mean ± SE (n = 3). * Significant difference as compared to the control group, p < 0.05; # Significant difference as compared to the Nano-CuO-treated alone group, p < 0.05CC BY · PubMed Central
Fig. 14 Schematic diagram of potential mechanisms of Nano-CuO-induced activation of fibroblasts. Exposure of lung epithelial BEAS-2B cells and U937* macrophages to Nano-CuO caused increased expression and activity of MMP-3 and increased production of MMP-3-cleaved OPN, leading to the activation of fibroblasts. Graphics were created with BioRender (https://BioRender.com)
Fig. 14 Schematic diagram of potential mechanisms of Nano-CuO-induced activation of fibroblasts. Exposure of lung epithelial BEAS-2B cells and U937* macrophages to Nano-CuO caused increased expression and activity of MMP-3 and increased production of MMP-3-cleaved OPN, leading to the activation of fibroblasts. Graphics were created with BioRender (https://BioRender.com)CC BY · PubMed Central

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Sorted by citations

Metadata

Authors
Zhang, Y., Mo, Y., Zhang, Y., Yuan, J., & Zhang, Q.
Affiliations
  • Northwestern University (US)
  • University of Louisville (US)
Corresponding author
Zhang, Qunwei
ORCID
Venue
Particle and Fibre Toxicology
Publisher
Springer Science and Business Media LLC
Published
2023-05-22
Volume
20
Issue
1
Pages
22-22
ISSN
1743-8977 (ISSN-L)
DOI
10.1186/s12989-023-00532-y
PMC
PMC10201731
OpenAlex
W4377291875
Cited by
24
References
2
Citations by year
2026: 5 · 2025: 10 · 2024: 9
FWCI
4.42
Type
journal article
Language
en
Open access
Licence
Locations
  • Particle and Fibre Toxicology (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
National Institutes of Health (ES023696)
Topics
  • Health Sciences › Medicine › Pulmonary and Respiratory Medicine › Interstitial Lung Diseases and Idiopathic Pulmonary Fibrosis
  • Health Sciences › Medicine › Pulmonary and Respiratory Medicine › Occupational and environmental lung diseases
  • Physical Sciences › Materials Science › Materials Chemistry › Nanoparticles: synthesis and applications
Keywords
copper oxide nanoparticles, MMP-3, fibroblast activation, pulmonary fibrosis, nanoparticle toxicity, macrophages, lung epithelial cells, fibroblast migration, RGD peptide, lung injury, GRGDSP peptide, GRGDS peptide
MeSH terms
Cell Line, Copper, Epithelial Cells, Fibroblasts, Humans, Lung, Macrophages, Coculture Techniques, Matrix Metalloproteinase 3, Osteopontin, Metal Nanoparticles

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