Early Pulmonary Fibrosis-like Changes in the Setting of Heat Exposure: DNA Damage and Cell Senescence

Abstract

It is well known that extreme heat events happen frequently due to climate change. However, studies examining the direct health impacts of increased temperature and heat waves are lacking. Previous reports revealed that heatstroke induced acute lung injury and pulmonary dysfunction. This study aimed to investigate whether heat exposure induced lung fibrosis and to explore the underlying mechanisms. Male C57BL/6 mice were exposed to an ambient temperature of 39.5 ± 0.5 °C until their core temperature reached the maximum or heat exhaustion state. Lung fibrosis was observed in the lungs of heat-exposed mice, with extensive collagen deposition and the elevated expression of fibrosis molecules, including transforming growth factor-β1 (TGF-β1) and Fibronectin (Fn1) (p < 0.05). Moreover, epithelial-mesenchymal transition (EMT) occurred in response to heat exposure, evidenced by E-cadherin, an epithelial marker, which was downregulated, whereas markers of EMT, such as connective tissue growth factor (CTGF) and the zinc finger transcriptional repressor protein Slug, were upregulated in the heat-exposed lung tissues of mice (p < 0.05). Subsequently, cell senescence examination revealed that the levels of both senescence-associated β-galactosidase (SA-β-gal) staining and the cell cycle protein kinase inhibitor p21 were significantly elevated (p < 0.05). Mechanistically, the cGAS-STING signaling pathway evoked by DNA damage was activated in response to heat exposure (p < 0.05). In summary, we reported a new finding that heat exposure contributed to the development of early pulmonary fibrosis-like changes through the DNA damage-activated cGAS-STING pathway followed by cellular senescence.

Keywords: DNA damage; cGAS–STING pathway; heat exposure; lung fibrosis; senescence.

Figure 1

Heat exposure-induced early pulmonary fibrosis-like changes in mice. (A,B) Representative images and quantitative analysis of Masson’s staining of the lung tissue. Scale bar = 100 μm. Locally magnified images of lung tissue sections (scale bar = 50 µm). (C) Gene expression of TGF-β1, Fn1, and Col1a1 in lung tissues was quantitated using qRT-PCR. (D–F) Western blot for lung TGF-β1 and Fn1 protein and quantification analysis. (n = 7) (* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. Control).

Figure 2

Heat exposure-induced EMT in the lung tissue of mice. (A) Gene expressions of E-cadherin, Vimentin, α-SMA, CTGF, Snail1 and Slug in the lung tissues were quantitated using qRT-PCR. (B–D) Western blot for lung E-cadherin and CTGF protein and quantification analysis. (E,F) Lung E-cadherin was measured using IHC and quantification analysis. Scale bar = 100 μm. Locally enlarged images of lung tissue sections (scale bar = 50 µm). (n = 7) (* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. Control).

Figure 3

Heat exposure-induced lung cellular senescence in mice. (A,B) Lung β-gal was measured using IHC and quantification analysis. Scale bar = 100 μm. Locally enlarged images of lung tissue sections (scale bar =50 µm). (C) Gene expressions of P21, P16, and Ki67 in the lung tissues were quantitated using qRT-PCR. (D,E) Western blot for the lung P21 protein and quantification analysis. (n = 7) (* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. Control).

Figure 4

Heat exposure-induced DNA damage and cGAS–STING pathway activation. (A,B) Lung γ-H2AX was measured using IHC and quantification analysis. Scale bar = 100 μm. Locally enlarged images of lung tissue sections (scale bar = 50 µm). (C,D) Western blot for lung γ-H2AX protein and quantification analysis. (E–G) Western blot for lung cGAS and STING protein and quantification analysis. (n = 7) (* p < 0.05, ** p < 0.01 and *** p < 0.001 vs. Control).

Figure 5

Schematic diagram of the mechanisms underlying heat exposure-induced early pulmonary fibrosis-like changes. Heat exposure induced DNA damage in mice lung tissues, which subsequently led to the activation of the cGAS–STING signaling pathway, further inducing lung cell senescence and ultimately leading to the development of early pulmonary fibrosis-like changes.