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Silica promotes the transdifferentiation of rat circulating fibrocytes in vitro.

Yao W, Liu S, Li J, Hao C - Mol Med Rep (2015)

Bottom Line: The mRNA expression levels of collagen I, collagen III and α‑smooth muscle actin (SMA) in cFbs were analyzed by reverse transcription‑quantitative polymerase chain reaction.The results indicated that in the cell model, the free silica effectively increased the protein and mRNA expression levels of collagen‑I, collagen‑III and α‑SMA.The free silica significantly promoted the transdifferentiation of cFbs into myofibroblasts in a dose‑and time-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Public Health, Zhengzhou University, Zhengzhou, Henan 450001, P.R. China.

ABSTRACT
To investigate the effects of silica on circulating fibrocytes (cFbs), the present study established a primary culture model of rat alveolar macrophages and cFbs in vitro. Macrophages were treated with free silica, and their supernatant was used to stimulate cFbs. The mRNA expression levels of collagen I, collagen III and α‑smooth muscle actin (SMA) in cFbs were analyzed by reverse transcription‑quantitative polymerase chain reaction. The intracellular and extracellular protein expression levels of collagen I, collagen III and α‑SMA were detected by ELISA and immunofluorescence staining. The results indicated that in the cell model, the free silica effectively increased the protein and mRNA expression levels of collagen‑I, collagen‑III and α‑SMA. The free silica significantly promoted the transdifferentiation of cFbs into myofibroblasts in a dose‑and time-dependent manner.

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Protein expression levels of collagen I, collagen III and α-SMA in cFbs, as determined by ELISA. (A) The cFbs were treated with the supernatant of AM treated with SiO2 (0, 20, 40, 60 and 80 µg/ml) for 24 h, and untreated cells served as the control group. The protein expression levels of collagen I, collagen III and α-SMA were quantified and normalized relative to the control group. The bands were quantified by densitometric analysis. (B-D) Expression levels of collagen I, collagen III and α-SMA following treatment with AM supernatant (0 and 80 µg/ml SiO2) for 3, 6, 9, 12, 15 and 18 days for collagen I and collagen III, and for 13, 16, 19, 22, 25 and 28 days for α-SMA. The cFb supernatant was collected and protein expression was determined by ELISA. The expression levels of collagen I and collagen III were quantified and normalized relative to the control group. The bands were quantified by densitometric analysis. Values are expressed as the mean ± standard deviation from three independent experiments (n=6). *P<0.01, vs. the control. cFbs, circulating fibrocytes; α-SMA, α smooth muscle actin; SiO2, crystalline silica; AM, alveolar macrophages.
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f4-mmr-12-04-5828: Protein expression levels of collagen I, collagen III and α-SMA in cFbs, as determined by ELISA. (A) The cFbs were treated with the supernatant of AM treated with SiO2 (0, 20, 40, 60 and 80 µg/ml) for 24 h, and untreated cells served as the control group. The protein expression levels of collagen I, collagen III and α-SMA were quantified and normalized relative to the control group. The bands were quantified by densitometric analysis. (B-D) Expression levels of collagen I, collagen III and α-SMA following treatment with AM supernatant (0 and 80 µg/ml SiO2) for 3, 6, 9, 12, 15 and 18 days for collagen I and collagen III, and for 13, 16, 19, 22, 25 and 28 days for α-SMA. The cFb supernatant was collected and protein expression was determined by ELISA. The expression levels of collagen I and collagen III were quantified and normalized relative to the control group. The bands were quantified by densitometric analysis. Values are expressed as the mean ± standard deviation from three independent experiments (n=6). *P<0.01, vs. the control. cFbs, circulating fibrocytes; α-SMA, α smooth muscle actin; SiO2, crystalline silica; AM, alveolar macrophages.

Mentions: In a further experiment, an ELISA was used to investigate the effects of SiO2 on the protein expression levels of collagen I, collagen III and α-SMA in the cFbs cultured with the supernatant of AM treated with various concentrations of SiO2 for various durations. The cFbs were treated with AM supernatant treated with SiO2 at concentrations of 0, 20, 40, 60 and 80 µg/ml for 24 h, and untreated cFbs served as a control group. The protein expression levels of collagen I, collagen III and α-SMA were quantified and normalized relative to the control group. As shown in Fig. 4A, a marked increase was observed in collagen I, collagen III and α-SMA expression in the cFbs treated with SiO2 at concentrations of 20, 40, 60 and 80 µg/ml for 24 h, as compared with that in the control group. No statistically significant difference was observed between the 60 µg/ml and 80 µg/ml treatment groups (P>0.05; n=6). Following cFbs treatment with 80 µg/ml SiO2 for various durations, the protein expression levels of collagen I and collagen III began to increase on day 3, and those of α-SMA on day 13, whereas the expression levels of collagen I and collagen III in the 0 µg/ml SiO2 and the control groups began to increase on day 6, and those of α-SMA on day 19. In the treatment group, the expression levels of collagen I and collagen III were significantly upregulated on days 3, 6, 9, 12, 15 and 18, as compared with those in the control group (P<0.05; Fig. 4B and C), whereas the expression of α-SMA was significantly upregulated on days 13, 16, 19, 22, 25 and 28 (P<0.05; Fig. 4D). These results suggested that SiO2 stimulated collagen I, collagen III and α-SMA expression at the protein level and accelerated the processes of transdifferentiation of cFbs into myofibroblasts in a dose-dependent manner.


Silica promotes the transdifferentiation of rat circulating fibrocytes in vitro.

Yao W, Liu S, Li J, Hao C - Mol Med Rep (2015)

Protein expression levels of collagen I, collagen III and α-SMA in cFbs, as determined by ELISA. (A) The cFbs were treated with the supernatant of AM treated with SiO2 (0, 20, 40, 60 and 80 µg/ml) for 24 h, and untreated cells served as the control group. The protein expression levels of collagen I, collagen III and α-SMA were quantified and normalized relative to the control group. The bands were quantified by densitometric analysis. (B-D) Expression levels of collagen I, collagen III and α-SMA following treatment with AM supernatant (0 and 80 µg/ml SiO2) for 3, 6, 9, 12, 15 and 18 days for collagen I and collagen III, and for 13, 16, 19, 22, 25 and 28 days for α-SMA. The cFb supernatant was collected and protein expression was determined by ELISA. The expression levels of collagen I and collagen III were quantified and normalized relative to the control group. The bands were quantified by densitometric analysis. Values are expressed as the mean ± standard deviation from three independent experiments (n=6). *P<0.01, vs. the control. cFbs, circulating fibrocytes; α-SMA, α smooth muscle actin; SiO2, crystalline silica; AM, alveolar macrophages.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4581811&req=5

f4-mmr-12-04-5828: Protein expression levels of collagen I, collagen III and α-SMA in cFbs, as determined by ELISA. (A) The cFbs were treated with the supernatant of AM treated with SiO2 (0, 20, 40, 60 and 80 µg/ml) for 24 h, and untreated cells served as the control group. The protein expression levels of collagen I, collagen III and α-SMA were quantified and normalized relative to the control group. The bands were quantified by densitometric analysis. (B-D) Expression levels of collagen I, collagen III and α-SMA following treatment with AM supernatant (0 and 80 µg/ml SiO2) for 3, 6, 9, 12, 15 and 18 days for collagen I and collagen III, and for 13, 16, 19, 22, 25 and 28 days for α-SMA. The cFb supernatant was collected and protein expression was determined by ELISA. The expression levels of collagen I and collagen III were quantified and normalized relative to the control group. The bands were quantified by densitometric analysis. Values are expressed as the mean ± standard deviation from three independent experiments (n=6). *P<0.01, vs. the control. cFbs, circulating fibrocytes; α-SMA, α smooth muscle actin; SiO2, crystalline silica; AM, alveolar macrophages.
Mentions: In a further experiment, an ELISA was used to investigate the effects of SiO2 on the protein expression levels of collagen I, collagen III and α-SMA in the cFbs cultured with the supernatant of AM treated with various concentrations of SiO2 for various durations. The cFbs were treated with AM supernatant treated with SiO2 at concentrations of 0, 20, 40, 60 and 80 µg/ml for 24 h, and untreated cFbs served as a control group. The protein expression levels of collagen I, collagen III and α-SMA were quantified and normalized relative to the control group. As shown in Fig. 4A, a marked increase was observed in collagen I, collagen III and α-SMA expression in the cFbs treated with SiO2 at concentrations of 20, 40, 60 and 80 µg/ml for 24 h, as compared with that in the control group. No statistically significant difference was observed between the 60 µg/ml and 80 µg/ml treatment groups (P>0.05; n=6). Following cFbs treatment with 80 µg/ml SiO2 for various durations, the protein expression levels of collagen I and collagen III began to increase on day 3, and those of α-SMA on day 13, whereas the expression levels of collagen I and collagen III in the 0 µg/ml SiO2 and the control groups began to increase on day 6, and those of α-SMA on day 19. In the treatment group, the expression levels of collagen I and collagen III were significantly upregulated on days 3, 6, 9, 12, 15 and 18, as compared with those in the control group (P<0.05; Fig. 4B and C), whereas the expression of α-SMA was significantly upregulated on days 13, 16, 19, 22, 25 and 28 (P<0.05; Fig. 4D). These results suggested that SiO2 stimulated collagen I, collagen III and α-SMA expression at the protein level and accelerated the processes of transdifferentiation of cFbs into myofibroblasts in a dose-dependent manner.

Bottom Line: The mRNA expression levels of collagen I, collagen III and α‑smooth muscle actin (SMA) in cFbs were analyzed by reverse transcription‑quantitative polymerase chain reaction.The results indicated that in the cell model, the free silica effectively increased the protein and mRNA expression levels of collagen‑I, collagen‑III and α‑SMA.The free silica significantly promoted the transdifferentiation of cFbs into myofibroblasts in a dose‑and time-dependent manner.

View Article: PubMed Central - PubMed

Affiliation: Department of Public Health, Zhengzhou University, Zhengzhou, Henan 450001, P.R. China.

ABSTRACT
To investigate the effects of silica on circulating fibrocytes (cFbs), the present study established a primary culture model of rat alveolar macrophages and cFbs in vitro. Macrophages were treated with free silica, and their supernatant was used to stimulate cFbs. The mRNA expression levels of collagen I, collagen III and α‑smooth muscle actin (SMA) in cFbs were analyzed by reverse transcription‑quantitative polymerase chain reaction. The intracellular and extracellular protein expression levels of collagen I, collagen III and α‑SMA were detected by ELISA and immunofluorescence staining. The results indicated that in the cell model, the free silica effectively increased the protein and mRNA expression levels of collagen‑I, collagen‑III and α‑SMA. The free silica significantly promoted the transdifferentiation of cFbs into myofibroblasts in a dose‑and time-dependent manner.

Show MeSH
Related in: MedlinePlus