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. 2023 Oct 24;14(10):698.
doi: 10.1038/s41419-023-06179-z.

Sohlh2 promotes pulmonary fibrosis via repression of p62/Keap1/Nrf2 mediated anti-oxidative signaling pathway

Lanlan Liu  1 , Xiaoli Zhang  1 , Ruihong Zhang  1 , Liyan Wang  2 , Sujuan Zhi  1 , Xiaoning Feng  1 , Xuyue Liu  1 , Ying Shen  1 , Jing Hao  3
Affiliations

Sohlh2 promotes pulmonary fibrosis via repression of p62/Keap1/Nrf2 mediated anti-oxidative signaling pathway

Lanlan Liu et al. Cell Death Dis. .

Abstract

Disturbance in the redox balance of alveolar epithelial cells (AECs) was considered as a causal factor for pulmonary fibrosis. The regulatory mechanisms of redox hemostasis in the development of pulmonary fibrosis remain largely unknown. Using a type II AEC-specific Sohlh2 conditional knock-in (CKI) mouse model, we found that Sohlh2, a basic HLH transcription factor, accelerated age-related pulmonary fibrosis. High-fat diet (HFD) resulted in a tremendous increase in lung inflammation and fibrotic changes in the lung tissues of Sohlh2 CKI mice. Sohlh2 overexpression led to a significant rise of intracellular ROS and apoptosis in the lung, mouse primary AECIIs, and human A549 cells, which was attenuated by ROS inhibitor (NAC). Sohlh2 enhanced oxidative stress via repressing p62/Keap1/Nrf2 mediated anti-oxidative signaling pathway. p62, a direct target of Sohlh2, mediated Sohlh2 effects on ROS generation and apoptosis in A549 cells. Hence, our findings elucidate a pivotal mechanism underlying oxidative stress-induced pulmonary fibrosis, providing a framework for aging-related disorder interventions.

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Conflict of interest statement

The authors declare that there is no conflict of interest regarding the publication of this paper.

Figures

Fig. 1
Fig. 1. The generation of inducible AECII-specific Sohlh2 knock-in mice.
a Schema demonstrating the process of AECII-specific mice (Sohlh2 KI) mice generation using tamoxifen-inducible Sftpc-promoter driven CreERT2. Sohlh2loxP/loxPSftpcCreERT2- male mice were used as controls. b Sohlh2loxP/loxP mice were crossed to SftpcCreERT2+ mice, and genotypes of offspring mice were identified by PCR (n = 3 mice per group). c Representative images and quantification analysis of Western blot assay showing Sohlh2 protein levels in the Control and Sohlh2 KI mouse lungs (n = 4 mice per group). Data are presented as the mean ± SD. *P < 0.05.
Fig. 2
Fig. 2. Sohlh2 overexpression in AECIIs causes spontaneous age-related pulmonary fibrosis.
a Representative images of reconstructed anatomical X-ray MicroCT obtained from 2 M and 8 M mice showed parenchymal opacity. b Representative images showing gross lung morphology of 2-, 4-, and 8-month-old mice. c Representative images and quantification analysis of HE and Masson staining in sections from the indicated 2 M, 4 M, and 8 M lungs of the Control and Sohlh2 KI mice. Scale bars: 50 μm. d Representative images of immunofluorescence staining in the lung tissue samples of 8 M mice showing Sohlh2 (red), α-SMA (red), FN (red), and nuclei (blue). Scale bars: 50 μm. e, f qPCR and Western blot analysis of the expression of fibrosis-related genes in the indicated Control- and Sohlh2 KI-lungs. g qPCR analysis of proinflammatory cytokines in the Control- and Sohlh2 KI-lungs. h The total cells, macrophages, and neutrophils in the BALF of 8 M Control and Sohlh2 KI mice. i ELISA analysis of proinflammatory cytokines in the indicated lungs and BALF. j Representative images and quantification analysis of TUNEL staining in sections from the indicated 8 M Control- and Sohlh2 KI-lungs. Scale bars: 50 μm. Data are presented as the mean ± SD. ns. P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 3
Fig. 3. Sohlh2 aggravates the occurrence and progression of pulmonary fibrosis induced by HFD.
a Representative images of reconstructed anatomical X-ray MicroCT showing parenchymal opacity of lungs obtained from HFD-fed mice. b Representative images showing gross lung morphology of HFD-fed mice (left), n = 5 mice per group; representative photomicrographs of HE-stained lung sections (middle). Scale bars: 100 μm; representative images of Masson’s trichrome-stained lung sections at 8 weeks after HFD treatment (right). Scale bars: 100 μm. The histogram on the right is the statistical analysis of Masson’s trichome staining. c, d qPCR and Western blot analysis of profibrotic gene expression at mRNA and protein levels from the indicated lung tissues of HFD mice. e Immunofluorescence staining analysis of representative mice lung tissue samples of HFD mice showing Sohlh2 (red), α-SMA (red), FN (red), and nuclei (blue). Scale bars: 50 μm. f qPCR analysis of proinflammatory cytokines mRNA levels from the indicated lung tissues after 8-week HFD treatment. (n = 3). g The total cells, macrophages, and neutrophils in the BALF of the Control and Sohlh2 KI mice after 8-week HFD treatment. h ELISA analysis of proinflammatory cytokines in the Control- and Sohlh2 KI-lungs and the indicated BALF after 8-week HFD treatment. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 4
Fig. 4. Sohlh2 enhances oxidative stress in the lungs and cultured AECIIs.
a Representative photographs of DHE fluorescent imaging of lung tissue sections. Scale bars: 100 μm. b The levels of MDA in the lung tissues of 8 M and HFD mice were detected. c Representative TEM images of AECIIs from the Control and Sohlh2 KI mice after 8-week HFD treatment. Mitochondrial profiles showed enlarged swollen mitochondria in Sohlh2 KI AECIIs. Boxed regions are enlarged at the right. Scale bars: 1 μm. d Under an inverted phase-contrast microscope, the morphology of murine primary AECIIs (left). Scale bars: 50 μm; the expression of cell surfactant protein SP-C was measured by immunofluorescence staining (right). Scale bars: 50 μm. e ROS levels were measured by DHE fluorescent intensity in murine primary AECIIs and A549 cells treated with or without 300 μM PA. Scale bars: 50 μm. f Detection of ROS level and average immunofluorescence intensity of mouse primary AECIIs and human A549 cells treated with or without PA by FACS. g The levels of MDA were shown in Sohlh2 overexpression A549 cells treated by PA. h qPCR analysis of proinflammatory cytokines mRNA levels from mouse primary AECIIs and human A549 cells treated by PA. (n = 3). i ELISA analysis of IL-6, TNF-α, and TGF-β1 in the culture medium of Sohlh2 overexpression A549 cells treated by PA. j Representative images and quantification analysis of TUNEL staining in mouse primary AECIIs and human A549 cells treated with or without PA. (n = 3). Scale bars: 20 μm. k Percentages of Annexin V-positive cells upon PA treatment were examined by FACS. Cells treated with PBS served as the control. (n = 3). Data are presented as the mean ± SD. ns. P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 5
Fig. 5. NAC attenuated Sohlh2-mediated oxidative stress in AECIIs and pulmonary fibrosis.
Sohlh2 overexpression and the control A549 cells were pretreated with or without NAC (5 mM), then treated with 300 μM PA for 48 h. a Representative photographs of DHE fluorescent imaging of A549 cells. Scale bars: 50 μm. b The ROS levels in the indicated A549 cells were detected by FACS. c Representative images and quantification analysis of TUNEL staining in A549 cells. (n = 3). Scale bars: 20 μm. d qPCR analysis of proinflammatory cytokines mRNA levels in the indicated A549 cells. (n = 3). e ELISA analysis of IL-6, TNF-α, and TGF-β1 in the supernatant of A549 cells obtained from different groups. f Representative images showing gross lung morphology of mice obtained from the Control/HFD, Sohlh2 KI/HFD, Control/HFD + NAC, and Sohlh2 KI/HFD + NAC groups. (n = 5). g Representative images and quantification analysis of HE and Masson staining in sections from the Control and Sohlh2 KI lungs after 8-week HFD and NAC treatment. Scale bars: 100 μm. h Representative images of DHE fluorescent imaging of lung tissue sections in four groups. Scale bars: 100 μm; Representative images showing α-SMA (red) and nuclei (blue) in lung sections. Scale bars: 50 μm. i, j qPCR and Western blot analysis of profibrotic gene expression at mRNA and protein levels from indicated lung tissues. k qPCR analysis of proinflammatory cytokines mRNA levels from indicated lung tissues. Data are presented as the mean ± SD. ns. P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 6
Fig. 6. Sohlh2 inhibits the activation of the p62/Keap1/Nrf2 signaling pathway by repressing p62 transcription in the lungs and AECIIs.
a qPCR analysis of Sohlh2, p62, Keap1, Nrf2 and its target genes in murine primary AECIIs and human A549 cells treated by 300 μM PA; qPCR analysis of Sohlh2, p62, Nrf2, and its downstream target genes expression in the indicated lungs of 8 M and HFD-fed mice. b Representative Western blot and quantification analysis showing the expression levels of Sohlh2, total Nrf2, and nuclear Nrf2 in Sohlh2 overexpressing A549 cells treated by PA. n = 4. GAPDH or β-Tubulin was used as a loading control for total or cytosolic proteins, and Histone H3 was used as a loading control for nuclear proteins. c Representative Western blot and quantification analysis showing the protein expression levels of Sohlh2, p62, Keap1, and total Nrf2 in mouse primary AECIIs and A549 cells treated by PA. d, e Representative Western blot and quantification analysis showing the expression levels of Sohlh2, p62, Keap1, and total Nrf2 in the Control and Sohlh2 KI lungs of 4 M, 8 M, and HFD-fed mice. f Predictive binding sites of Sohlh2 to p62 promoter region. g ChIP analysis of forced Sohlh2 expression and A549 cells using an anti-Sohlh2 antibody for p62 promoter. h p62 promoter-luciferase reporter activity in Sohlh2 overexpression and Sohlh2 shRNA A549 cells. Data are presented as the mean ± SD. ns. P > 0.05, *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 7
Fig. 7. p62 overexpression prevents AECIIs from Sohlh2-induced oxidative stress damage and blocks the effect of Sohlh2 on the Keap1/Nrf2 signaling pathway.
p62 overexpression or control plasmid was transfected into the control and Sohlh2 overexpressing A549 cells. a ROS levels were measured by DHE fluorescent intensity in four group A549 cells treated by PA. Scale bars: 50 μm. b Detection of ROS levels in four group A549 cells treated by PA by FACS. c Representative images and quantification analysis of TUNEL staining in four group A549 cells treated by PA. (n = 3). Scale bars: 20 μm. d Percentages of Annexin V-positive cells upon PA treatment were examined by FACS. e, f qPCR and ELISA analysis of the expression of proinflammatory cytokines from four group A549 cells treated by PA. g Representative Western blot images and quantification analysis showing the expression levels of Sohlh2, total Nrf2, and nuclear Nrf2 in the indicated A549 cells. h Representative Western blot images and quantification analysis showing the expression levels of Sohlh2, p62, Keap1, and total Nrf2 in the indicated A549 cells. i qPCR analysis of the expression of Nrf2 target genes (HO1, NQO1, Gsta1) in the indicated A549 cells. Data are presented as the mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.
Fig. 8
Fig. 8. Sohlh2 promotes pulmonary fibrosis via suppressing the activation of the p62/Keap1/Nrf2 signaling pathway and aggravating oxidative stress of AECIIs.
In the occurrence and progression of pulmonary fibrosis, Sohlh2 can downregulate the transcriptional activity of p62 by directly binding to the promoter region of p62, activating the Keap1/Nrf2 signaling pathway to result in the production of ROS in AECIIs, leading to inflammation, cells apoptosis, and fibrosis of lung tissues under different conditions. Therefore, targeted Sohlh2 may prevent age-related and stress-induced pulmonary fibrosis and provide a new way for clinical treatment of IPF with anti-oxidation.
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