| Background:Idiopathic pulmonary fibrosis(IPF)is an age-related interstitial lung disease characterized by progressive scarring of lung tissue and reduced gas exchange,leading to respiratory failure.IPF commonly occurs in the middle-aged and elderly,and the median survival time is 3～5 years.The causes of death were respiratory failure and secondary pulmonary infection.At present,there is a lack of specific drugs for the treatment of IPF.Therefore,revealing the pathogenesis of IPF is expected to provide new therapeutic targets for patients with IPF.The molecular mechanism of the occurrence and progression of IPF is not completely clear.Many experimental evidences show that oxidative stress damage of type Ⅱ alveolar epithelial cells(AECⅡs)is critical in IPF.AECⅡs are considered "stem cells" of the alveolar epithelium.They proliferate and differentiate into type Ⅰ alveolar epithelial cells(AECⅠs)during lung injury,ensure the integrity of the alveolar structure,and play an important role in repairing lung injury.In patients with IPF and murine models of pulmonary fibrosis,the injury and apoptosis of alveolar epithelial cells significantly increase,and there are apparent mitochondrial dysfunction and accumulation of reactive oxygen species(ROS)in AECⅡs.However,in the process of occurrence and progression of IPF,the exact molecular mechanism of regulating AECⅡs oxidative stress remains unclear.Oxidative stress is the imbalance between oxidation and anti-oxidation in the body,which leads to excessive production of highly active molecules,such as ROS,which is closely related to aging and disease.Excessive ROS in the lung could stimulate fibroblasts to produce collagen I,induce apoptosis of alveolar epithelial cells and imbalance of interstitial MMPs/TIMPs,and enhance the secretion of IL-1β,TNF-α,PDGF,TGF-β1,and other cytokines,which result in pulmonary fibrosis and loss of respiratory function.p62/Keap1/Nrf2 signaling pathway is an essential intracellular defense mechanism against oxidative stress.SQSTM1/p62 is an essential selective autophagy junction protein,which contains the ubiquitin-related domain to bind to Kelch-like ECH-associatedproteinl(Keap1).Keap1 degradation is induced by p62-mediated autophagy,resulting in the release and translocation of Keapl-binding nuclear factor erythroid 2-related factor 2(Nrf2)to the nucleus,inducing the expression of antioxidant genes,such as Heme oxygenase 1(HO1),NAD(P)H:quinone oxidoreductase-1(NQO1),and Glutathione Stransferase(GST).The expression of p62 in lung homogenate of patients with IPF was significantly lower than that of the normal controls,suggesting the inhibition of the p62/Keap1/Nrf2 signaling pathway in lung tissues,may be involved in the enhancement of oxidative stress and promoting the occurrence and progression of IPF.Therefore,exploring the new regulatory factors of the p62/Keap1/Nrf2 signaling pathway in the occurrence and progression of IPF may provide new targets for the clinical treatment of IPF.Sohlh2 is a member of the basic helix-loop-helix(bhlh)transcription factor family,which can bind to the conserved E-box sequence of the target gene promoters and regulate the expression of downstream target genes.Our previous data have confirmed that Sohlh2 is a novel tumor suppressor that can inhibit breast and ovarian cancer progression.Our recent experimental results showed that Sohlh2 regulated oxidative stress caused by chemotherapeutic drugs in cancer cells.This study used a murine model of pulmonary fibrosis with AECII-specific Sohlh2 expression,overexpressing Sohlh2 murine primary AECⅡs and human A549 cells to explore the molecular mechanism in pulmonary fibrosis.Methods:(1)The generation of inducible AECⅡs-specific Sohlh2 knock-in miceTo generate the mice with tamoxifen-inducible Sohlh2 knock-in specifically in AECⅡs,Sohlh2lox/loxP mice were crossed to SftpcCreERT2+ mice.To induce recombination by CreERT2,5 consecutive intraperitoneal tamoxifen(100 mg/kg/dose)injections,prepared with sunflower seed oil,were given from six weeks of age.Sohlh2loxP/loxPSftpcCreERT2-and Sohlh2loxP/loxPSftpcCreERT2+mice were used as the Control or the Sohlh2 KI mice for experiments,and a Western blot assay was performed to detect the expression of Sohlh2 in the mouse lung tissues.(2)To investigate the effects of Sohlh2 in the occurrence and progression of pulmonary fibrosis in miceThe lung tissues from two months old(2M),four months old(4M),and eight months old(8M)Control and Sohlh2 KI mice were collected.MicroCT,Hematoxylin-eosin staining(HE),Masson staining,Western blot,and immunofluorescent staining(IF)assays were performed to investigate the effects of Sohlh2 on the occurrence and progression of pulmonary fibrosis in mice.The regulation of Sohlh2 on lung inflammation and apoptosis in the development of pulmonary fibrosis in mice was detected by qPCR,ELISA,and TUNEL assays.(3)To investigate the effects of Sohlh2 in the progression of pulmonary fibrosis induced by a high-fat diet in mice6-week-old Control and Sohlh2 KI mice were treated with a high-fat diet(HFD)for eight weeks.In the seventh week,MicroCT recorded the degree of pulmonary fibrosis.After one week,the mice were euthanized,and the lung tissues of the Control mice and the Sohlh2 KI mice were collected.The effects of Sohlh2 in pulmonary fibrosis induced by a high-fat diet in mice were analyzed by HE staining,Masson staining,Western blot,and IF assays.qPCR,ELISA,and TUNEL assays were performed to investigate the effects of Sohlh2 in lung tissue inflammatory response and apoptosis induced by a high-fat diet in mice.(4)To investigate the effects of Sohlh2 on the oxidative stress in lung tissues of IPF and PA-induced AECⅡsThe levels of ROS and malondialdehyde(MDA)in lung tissues of 2M,4M,8M,and HFDinduced Control and Sohlh2 KI mice were detected by DHE staining and TBA assays to explore the regulation of Sohlh2 on oxidative stress in the lung tissues of IPF.Primary AECⅡs overexpressing Sohlh2,Sohlh2 overexpression and Sohlh2 knockdown human A549 cells were cultured in vitro and treated with 300uM palmitic acid(PA).The effects of Sohlh2 on oxidative stress,inflammation,and apoptosis of AECⅡs were investigated by the methods of DHE staining,TBA,qPCR,ELISA,TUNEL,and flow cytometry.(5)To investigate whether N-Acetyl-L-cysteine could reduce the oxidative stress of AECⅡs and pulmonary fibrosis induced by Sohlh2High-fat diet mice were intragastrically ingested N-Acetyl-L-cysteine(NAC).HE staining,Masson staining,qPCR,ELISA,Western blot,and IF assays were utilized to determine whether Sohlh2 promoted the occurrence and development of pulmonary fibrosis through oxidative stress in vivo.A549 cells overexpressing Sohlh2 and knockdown Sohlh2 were cultured in vitro and added PA after pretreatment with NAC.The effects of NAC on oxidative stress,inflammation,and apoptosis of AECⅡs induced by Sohlh2 were examined by DHE staining,qPCR,ELISA,TUNEL,and FACS assays.(6)To investigate the regulation of Sohlh2 in the p62/Keap1/Nrf2 signaling pathwayThe effects of Sohlh2 on the expression of p62,Keap1,Nrf2,and their downstream target genes in mouse lung tissues and AECⅡs were detected by qPCR and Western blot assays.Whether Sohlh2 could directly regulate the transcription of p62 was studied by ChIP and luciferase reporter assay.p62 overexpression plasmid was transfected into Sohlh2 overexpression A549 cells to investigate whether p62 mediated the effects of Sohlh2 on promoting oxidative stress,inflammation,apoptosis,and activation of Keap1/Nrf2 signaling pathway in the AECⅡs by qPCR,Western blot,DHE staining,ELISA,TUNEL,and FACS assays.Results:(1)The generation of inducible AECⅡs-specific Sohlh2 knock-in miceTo obtain Sohlh2loxP/loxPSftpcCreERT2+male mice,Sohlh2loxP/loxP mice were crossed to SftpcCreERT2+ mice.In the sixth week,100 mg/kg of tamoxifen was injected intraperitoneally for 5 consecutive days to induce the expression of Sohlh2 in AECⅡs.Western blot results showed that the expression of Sohlh2 in lung tissues of the Sohlh2 KI mice was significantly higher than that of the Control mice.(2)Sohlh2 overexpression promotes the occurrence and progression of pulmonary fibrosisThe degree of pulmonary fibrosis in the Control mice and the Sohlh2 KI mice at the age of 2M,4M,and 8M were detected by MicroCT.The results showed that the Sohlh2 KI mice obtained more severe pulmonary fibrosis compared to the Control mice in an age-dependent manner.The HE and Masson staining results showed that the lung tissue structure of the Control mice and the Sohlh2 KI mice at two months old has no significant difference.Pulmonary fibrosis appeared in the Sohlh2 KI mice at four months old.The lung tissue structure of the Sohlh2 KI mice at eight months old was more severe than that of the Control mice:with more infiltration of inflammatory cells,the thickened alveolar septum,and an increase in collagen fiber deposition.qPCR and ELISA assays were performed to detect the level of inflammation in the lung tissues of eight months old mice.The results showed that the mRNA levels of IL-6,IL-1β,TNF-α,and NLRP3 in lung tissues of the Sohlh2 KI mice were significantly elevated compared with the Control mice.The construction of IL-6,TNF-α,and TGF-β1 in lung tissue homogenate and bronchoalveolar lavage fluid(BALF)of the Sohlh2 KI mice were significantly higher than those of the Control mice.The results of qPCR and Western blot assays showed that the expression of ColI,α-SMA,FN,and TGF-β1 in lung tissues of the Sohlh2 KI mice was significantly upregulated compared with the Control mice,which were consistent with the results of immunofluorescent staining.TUNEL results showed that the number of apoptotic cells in lung tissues of the Sohlh2 KI mice was higher than that of the Control mice.Taken together,overexpression of Sohlh2 promotes the occurrence and progression of pulmonary fibrosis.(3)Sohlh2 aggravates the progression of pulmonary fibrosis induced by a high-fat diet in mice6-week-old Control mice and Sohlh2 KI mice were treated with HFD for eight weeks,and there was no significant difference in weight gain between the two groups.MicroCT results showed that prominent fibrosis was observed in lung tissues of the Sohlh2 KI mice compared with the Control mice.HE and Masson staining results showed that the Sohlh2 KI mice had more severe lung tissue injury,inflammatory cell infiltration,alveolar septum thickening,and collagen fiber deposition compared with the control group.The results of qPCR showed that the mRNA levels of IL-6,IL-1β,TNF-α,and NLRP3 in lung tissues of the Sohlh2 KI mice were upregulated compared to the Control mice.Meanwhile,ELISA results showed that the construction of IL-6,TNF-α,and TGF-β1 in lung homogenate and BALF of the Sohlh2 KI mice were significantly higher than those of the Control mice.The results of qPCR and Western blot showed that the expression of ColI,α-SMA,FN,and TGF-β1 in lung tissues of the Sohlh2 KI mice was upregulated.The results of immunofluorescent staining showed that the expression of Sohlh2,α-SMA,and FN in lung tissues of the Sohlh2 KI mice was increased.The results of TUNEL showed that apoptotic cells increased in lung tissues of the Sohlh2 KI mice.Collectively,these results suggest that Sohlh2 aggravates HFD-induced pulmonary fibrosis in mice.(4)Sohlh2 enhances oxidative stress in the lung tissues of IPF and PA-induced AECⅡsThe results of DHE staining showed that compared with the Control mice,ROS levels in lung tissues of the Sohlh2 KI mice gradually increased with age,and ROS levels in the lung tissues of HFD-induced Sohlh2 KI mice were significantly enhanced.DHE staining and FACS analysis showed that overexpression of Sohlh2 elevated ROS production in AECⅡs,while knockdown of Sohlh2 resulted in the opposite effects.The results of the TBA assay showed that overexpression of Sohlh2 could increase the level of MDA in the lung tissues of IPF and AECⅡs,while knockdown of Sohlh2 decreased the level of MDA.The inflammatory reaction of AECⅡs treated with PA was examined by qPCR and ELISA assays.The results showed that overexpressing Sohlh2 augmented the expression of IL-6,TNF-α,and TGF-β1 in AECⅡs,while knockdown of Sohlh2 significantly repressed the expression of IL-6,TNF-α,and TGF-β1.Excessive ROS could lead to the apoptosis of alveolar epithelial cells.Therefore,we further detected the cell apoptosis of PA-induced AECⅡs by TUNEL and FACS analysis.The results showed that the overexpression of Sohlh2 promoted the apoptosis of AECⅡs,while the apoptosis of AECⅡs was significantly lower in the Sohlh2 knockdown group than that of the control group.Collectively,the data suggest that Sohlh2 enhances oxidative stress in the lung tissues of IPF and PA-induced AECⅡs.We next examined mitochondrial ultrastructural changes in AECⅡs in the murine model of HFD-induced lung fibrosis through transmission electron microscopy(TEM).AECⅡs of the Sohlh2 KI mice exposed to HFD showed swollen mitochondria with disrupted cristae,compared to the controls.(5)NAC reduces the oxidative stress of AECⅡs and pulmonary fibrosis caused by Sohlh2To explore whether the oxidative stress of AECⅡs mediated the effects of Sohlh2 in pulmonary fibrosis,NAC,an inhibitor of oxidative stress,was utilized to treat A549 cells before the addition of PA.The results of DHE staining,FACS,TUNEL,qPCR,and ELISA assays showed that NAC could partially block the effects of Sohlh2 on oxidative stress,apoptosis,and inflammation response of AECⅡs.HFD-induced Control and Sohlh2 KI mice were fed with NAC;the results of HE staining,Masson staining,and DHE staining showed that NAC reduced the infiltration of inflammatory cells,collagen fiber deposition,and oxidative stress in lung tissues of the Sohlh2 KI mice.The results of qPCR and Western blot assays showed that NAC partially blocked the upregulation of ColⅠ,α-SMA,and FN in lung tissues of the Sohlh2 KI mice.qPCR results showed that NAC attenuated the secretion of pro-inflammatory cytokines such as IL-6,IL-1β,and TNF-α in lung tissues of the Sohlh2 KI mice.The results of ELISA showed that NAC decreased the upregulation of IL-6,TNF-α,and TGF-β1 in lung homogenate and BALF of the Sohlh2 KI mice.Taken together,these results suggest that NAC reduces the oxidative stress of AECⅡs and pulmonary fibrosis caused by Sohlh2.(6)Sohlh2 activates the Keapl/Nrf2 signaling pathway by downregulating p62 transcriptionqPCR results showed that Sohlh2 downregulated the mRNA levels of p62 and Nrf2 downstream target genes such as HO1,NQO1,and GST in mouse lung tissues and AECⅡs;knockdown of Sohlh2 obtained the opposite results.The results of the Western blot assay showed that Sohlh2 increased the expression of Keap1 in mouse lung tissues and AECⅡs,decreased the expression of p62 and total Nrf2,and reduced the entry of Nrf2 into the nucleus.In contrast,the knockdown of Sohlh2 resulted in the opposite effects.The results of ChIP and luciferase reporter assays showed that Sohlh2 could directly bind to the promoter region of p62 and inhibit its transcription activity.In comparison,the knockdown of Sohlh2 promoted the transcription activity of p62.To investigate whether p62 mediated the effects of sohlh2 in ACEIIs,the p62 overexpression plasmid was transfected into Sohlh2 overexpressing A549 cells.The results of qPCR and Western blot assays showed that p62 could partially block the effect of Sohlh2 on the activation of the Keap1/Nrf2 signaling pathway in AECⅡs.The results of DHE staining and FACS confirmed that p62 could partially inhibit the effects of Sohlh2 on the oxidative stress and apoptosis of AECⅡs.The results of qPCR and ELISA assays showed that p62 could partially inhibit the expression of inflammatory factors in Sohlh2 overexpression AECⅡs.Conclusion:1.Sohlh2 promotes the occurrence and progression of idiopathic pulmonary fibrosis;2.Sohlh2 suppresses the activation of the p62/Keap1/Nrf2 signaling pathway and aggravates oxidative stress of AECⅡs during pulmonary fibrosis. |
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