Impaired autophagy-accelerated senescence of alveolar type II epithelial cells drives pulmonary fibrosis induced by single-walled carbon nanotubes

Abstract

Background: The rapid increase in production and application of carbon nanotubes (CNTs) has led to wide public concerns in their potential risks to human health. Single-walled CNTs (SWCNTs), as an extensively applied type of CNTs, have shown strong capacity to induce pulmonary fibrosis in animal models, however, the intrinsic mechanisms remain uncertain.

Results: In vivo experiments, we showed that accelerated senescence of alveolar type II epithelial cells (AECIIs) was associated with pulmonary fibrosis in SWCNTs-exposed mice, as well as SWCNTs-induced fibrotic lungs exhibited impaired autophagic flux in AECIIs in a time dependent manner. In vitro, SWCNTs exposure resulted in profound dysfunctions of MLE-12 cells, characterized by impaired autophagic flux and accelerated cellular senescence. Furthermore, the conditioned medium from SWCNTs-exposed MLE-12 cells promoted fibroblast-myofibroblast transdifferentiation (FMT). Additionally, restoration of autophagy flux with rapamycin significantly alleviated SWCNTs-triggered senescence and subsequent FMT whereas inhibiting autophagy using 3-MA aggravated SWCNTs-triggered senescence in MLE-12 cells and FMT.

Conclusion: SWCNTs trigger senescence of AECIIs by impairing autophagic flux mediated pulmonary fibrosis. The findings raise the possibility of senescence-related cytokines as potential biomarkers for the hazard of CNTs exposure and regulating autophagy as an appealing target to halt CNTs-induced development of pulmonary fibrosis.

Keywords: Alveolar type II epithelial cells; Autophagy; Carbon nanotube; Pulmonary fibrosis; Senescence.

Fig. 1

Single-walled carbon nanotubes (SWCNTs) induced pulmonary fibrosis and senescence of AECIIs in mouse lung tissues. Mice were exposed to 40 μg SWCNT by intratracheal instillation, then lung tissues were collected on days 3, 7 and 28. A Scheme of workflow for evaluation of cellular senescence and pulmonary fibrosis induced by SWCNTs in vivo. B–D Western Blotting analysis of p21 and p16 protein expressions (n = 3) in lung tissues. Contents of TGF-β (E) and PAI-1 (F) in bronchoalveolar lavage fluid (BALF) quantified by ELISA (n = 5). HE staining (G) and Masson’s trichrome staining (H) of mouse lung tissues (200×). I The semiquantitative Ashcroft scores for the severity of pulmonary fibrosis (n = 4). J The hydroxyproline (HYP) level (n = 4) in lung tissues of mice. K Immunohistochemistry (IHC) of COL I expression (n = 5) in lung tissues of mice. L, M The correlation of hydroxyproline contents in mice lung tissues and senescence-associated secretory phenotype (SASP) factors (TGF-β and PAI-1) in BALF. N Immunostaining for p16 (a senescence-related marker) and SP-C (an AECIIs marker) from SWCNTs-exposed lung tissues of mice on day 28 was detected by immunofluorescence (IF) (400×). *P < 0.05 vs CTRL group

Fig. 2

SWCNTs impaired autophagic flux in AECIIs of mouse lung tissue. Mice were exposed to 40 μg SWCNTs by intratracheal instillation, then autophagy of the lung tissue was evaluated. A–D Western Blotting analysis of Atg5, LC3BI, LC3BII and p62 protein expressions (n = 3) in lung tissues of mice. E The LC3B expressions (n = 5) in lung tissues were evaluated by IHC on days 3, 7 and 28 upon SWCNTs administration (200×). F Immunostaining for LC3B and SP-C from SWCNTs-exposed lung tissues of mice on day 28 was detected by IF (200×). *P < 0.05 vs CTRL group

Fig. 3

SWCNTs impaired autophagic flux and triggered senescence of MLE-12 cells in vitro. A The cytotoxicity of SWCNTs (0, 5, 10, 25 and 50 μg/ml) at different time points (0, 6, 12, 24 and 48 h) was determined by the modified MTT assay. MLE-12 cells were treated with 10 μg/ml SWCNTs for 6, 12 and 24 h, and then the effects of SWCNTs on autophagy and senescence in MLE-12 cells were evaluated. B Scheme of workflow for time effects of SWCNTs on autophagy and senescence in MLE-12 cells and fibroblast-to-myofibroblast transdifferentiation (FMT) in lung fibroblasts in vitro. C–F Western Blotting analysis of Atg5, LC3BI, LC3BII and p62 protein expressions in MLE-12 cells (n = 3). G LC3B puncta in MLE-12 cells was observed by IF (200×). H–J Western Blotting analysis of p21 and p16 protein expressions in MLE-12 cells (n = 3). K SA-β-gal activity was determined by X-gal staining in MLE-12 cells (400×). L The number of SA-β-gal positive MLE-12 cells per field (n = 8). Contents of TGF-β (M) and PAI-1 (N) in the supernatants of culture medium quantified by ELISA (n = 5). *P < 0.05 vs CTRL group

Fig. 4

SWCNTs-exposed MLE-12 cells caused FMT. MLE-12 cells were treated with 10 μg/ml SWCNTs for 6, 12 and 24 h, and then cell-free culture medium, as conditioned medium (CM), was used to culture primary lung fibroblasts for 48 h. A, B mRNA expressions of α-SMA and COLI in lung fibroblasts were measured by qRT-PCR (n = 4). C, D Western Blotting analysis of α-SMA protein expression in lung fibroblasts (n = 3). E COLI positive expression in lung fibroblasts was evaluated by IHC (n = 5) (200×). *P < 0.05 vs CTRL group

Fig. 5

Restoration of autophagic flux using rapamycin alleviated FMT in lung fibroblasts by suppressing SWCNTs-triggered senescence in MLE-12 cells. MLE-12 cells were exposed to 10 μg/ml SWCNTs for 24 h with or without rapamycin pretreatment, and then the cell-free culture medium, as CM, was used to culture primary lung fibroblasts for 48 h. A Scheme of workflow for evaluating the effects of rapamycin on autophagy and senescence in MLE-12 cells and FMT in lung fibroblasts after SWCNTs exposure in vitro. B, C Western Blotting analysis of Atg5, LC3BI, LC3BII and p62 protein expressions in MLE-12 cells (n = 3). D LC3B puncta in MLE-12 cells was observed by IF (200×). E, F Western Blotting analysis of p21 and p16 protein expressions in MLE-12 cells (n = 3). G SA-β-gal activity was determined by X-gal staining in MLE-12 cells (400×). H The number of SA-β-gal positive MLE-12 cells per field (n = 8). Contents of TGF-β (I) and PAI-1 (J) in the supernatants of culture medium quantified by ELISA (n = 5). K mRNA expressions of α-SMA and COLI in lung fibroblasts were measured by qRT-PCR (n = 4). L, M Western Blotting analysis of α-SMA protein expression in lung fibroblasts (n = 3). N COLI positive expression in lung fibroblasts was evaluated by IHC (n = 5) (200×). *P < 0.05 vs CTRL group. ^#P < 0.05 vs SWCNT group

Fig. 6

Inhibition of autophagy using 3-MA aggravated FMT in lung fibroblasts by promoting SWCNTs-triggered senescence in MLE-12 cells. MLE-12 cells were exposed to 10 μg/ml SWCNTs for 24 h with or without 3-MA pretreatment,, and then the cell-free culture medium, as CM, was used to culture primary lung fibroblasts for 48 h. A Scheme of workflow for evaluating the effects of 3-MA on autophagy and senescence in MLE-12 cells and FMT in lung fibroblasts after SWCNTs exposure in vitro. B, C Western Blotting analysis of Atg5, LC3BI, LC3BII and p62 protein expressions in MLE-12 cells (n = 3). D LC3B puncta in MLE-12 cells was observed by IF (200×). E, F Western Blotting analysis of p21 and p16 protein expressions in MLE-12 cells (n = 3). G SA-β-gal activity was determined by X-gal staining in MLE-12 cells (400×). H The number of SA-β-gal positive MLE-12 cells per field (n = 8). Contents of TGF-β (I) and PAI-1 (J) in the supernatants of culture medium quantified by ELISA (n = 5). K mRNA expressions of α-SMA and COLI in lung fibroblasts were measured by qRT-PCR (n = 4). L, M Western Blotting analysis of α-SMA protein expression in lung fibroblasts (n = 3). N COLI positive expression in lung fibroblasts was evaluated by IHC (n = 5) (200×). *P < 0.05 vs CTRL group. ^#P < 0.05 vs SWCNT group