Abstract Oxidative damage exacerbates pulmonary fibrosis by impairing alveolar type II epithelial (AT2) cell function. This study demonstrates that the SUMO-specific protease 1 (SENP1)-Sirtuin 3 (Sirt3) axis, critical for mitochondrial redox regulation, is suppressed in AT2 cells during lung injury. In bleomycin-induced pulmonary fibrosis models, activating the SENP1-Sirt3 axis via Sirt3 SUMOylation site mutation (Sirt3 K223R) reduced Superoxide Dismutase 2 (SOD2) acetylation, thereby lowering mitochondrial reactive oxygen species (mtROS) accumulation and apoptosis. This intervention increased AT2 cell proliferation and differentiation into alveolar type I cells while reducing Keratin 8 (KRT8)^+ transitional cell number, a profibrotic population. Additionally, SENP1-Sirt3 activation attenuated inflammation and fibrosis in lung tissue. Transcriptomic analysis linked the axis to enhanced Wnt signaling and lipid metabolism pathways, promoting AT2 stemness. Antioxidant N-acetylcysteine (NAC) supplementation mirrored these benefits, reinforcing ROS clearance as a therapeutic mechanism. These findings highlight SENP1-Sirt3 as a pivotal regulator of AT2 resilience, offering a potential strategy to mitigate fibrosis by targeting mitochondrial oxidative stress and cellular plasticity. Keywords: SENP1-Sirt3 axis, AT2 cell, Oxidative damage, Mitochondria, Lung fibrosis 1. Introduction Alveolar type II epithelial cells (AT2 cells), play a pivotal role in maintaining lung homeostasis and facilitating injury repair [[41]1]. However, their dysfunction is a central driver in the pathogenesis of pulmonary fibrosis [[42]2]. Under physiological conditions, AT2 cells proliferate and differentiate into alveolar type I epithelial (AT1) cells to restore alveolar integrity after injury [[43]3]. In fibrotic lungs, this differentiation capacity is impaired, leading to abnormal proliferation and the emergence of pathological transitional cell states, Keratin 8^+ (KRT8^+) cells [[44]4]. These dysfunctional cells lose regenerative potential and instead secrete pro-fibrotic mediators such as Transforming Growth Factor Beta (TGF-β) that activate fibroblasts, driving excessive collagen deposition and extracellular matrix remodeling [[45]5]. Mitochondria play a pivotal role in regulating the proliferation and differentiation of AT2 cells, which are critical for maintaining lung homeostasis and repair [[46]6]. As the primary energy producers, mitochondria supply adenosine triphosphate (ATP) necessary for AT2 cell division and differentiation into AT1 cells [[47]7]. Chronic stress or injury triggers mitochondrial hyperplasia, an adaptive response to sustain energy production and promote AT2 cell survival. Conversely, acute damage leads to mitochondrial degradation via autolysis, impairing ATP synthesis and stalling proliferation. Mitochondrial cristae remodeling and matrix swelling disrupt oxidative phosphorylation (OXPHOS), reducing ATP output and hindering differentiation of AT2 cells [[48]8,[49]9]. Mitochondria also regulate redox balance [[50]7,[51]8]. Dysfunctional mitochondria generate excess ROS, activating stress pathways that promote fibrotic transitions in AT2 cells. Also, the redox balance within mitochondria influences signaling cascades like Wingless-related integration site (Wnt)/β-catenin, which governs AT2 cell fate decisions [[52]10]. However, targeting mitochondria to regulate the function of AT2 cells and ameliorate the progression of pulmonary fibrosis remains a significant challenge. Emerging evidence highlights the SUMO-specific protease 1 (SENP1)-Sirtuin 3 (Sirt3) axis as a critical regulator of mitochondrial redox homeostasis across multiple tissues [[53][11], [54][12], [55][13], [56][14]]. Calorie restriction enhances mitochondrial translocation of SENP1, which activates mitochondrial deacetylase Sirt3 via deSUMOylation, thereby amplifying antioxidant defenses [[57]12]. This mechanism extends to T cells, where SENP1-Sirt3 signaling promotes T cells differentiation by modulating mitochondrial redox balance [[58]15]. Compelling studies demonstrate its protective roles in ischemia/reperfusion injuries, SENP1-mediated Sirt3 deSUMOylation underpins intra-arterial cooling-induced neuroprotection and ameliorates acute kidney injury by enhancing mitochondrial stress responses, reducing inflammation and fibrosis [[59]11,[60]14]. Similarly, hepatic ischemia/reperfusion-induced oxidative damage is attenuated through Sirt3 deSUMOylation, preserving mitochondrial integrity [[61]13]. While these tissue-specific benefits suggest systemic effects through oxidative stress reduction, comprehensive validation of its pan-tissue efficacy remains imperative. Herein, we found that SENP1-Sirt3 signaling was suppressed in bleomycin (BLM)-treated AT2 cells. We further constructed Sirt3 SUMOylation site mutation (Sirt3 K223R) mice to mimic the activation of SENP1-Sirt3 axis, and investigated the impact of SENP1-Sirt3 axis on AT2 cell activity and its role in pulmonary fibrosis progression. Our findings demonstrate that Sirt3 K223R mice effectively attenuates BLM-induced acute mortality, alveolar epithelial cell apoptosis, inflammatory responses, and fibrotic development. Mechanistically, SENP1-Sirt3 axis activation reduces mitochondrial reactive oxygen species (mtROS) production by deacetylating Superoxide Dismutase 2 (SOD2) in AT2 cells, thereby enhancing SOD2 activity and decreasing the population of KRT8^+ transitional cells. These results suggest that ameliorating mitochondrial oxidative stress in AT2 cells through SENP1-Sirt3 axis activation represents a promising therapeutic strategy for pulmonary fibrosis. 2. Results 2.1. SENP1-Sirt3 signaling within AT2 cells is downregulated during lung injury As mitochondrial dysfunction drives oxidative stress, metabolic reprogramming, and fibroblast activation in fibrotic lung tissue, investigating mitochondrial function regulation is essential to understanding the pathogenesis and progression of pulmonary fibrosis [[62]16,[63]17]. BLM is a cytostatic drug used in cancer therapy that binds to oxygen and divalent iron ions to form an active form, which produces a large amount of ROS and causes DNA breakage and cell apoptosis [[64]18]. The model of lung injury induced by BLM has been widely used to study lung tissue damage repair and pulmonary fibrosis [[65][19], [66][20], [67][21], [68][22]]. Based on this, we established a BLM-induced lung injury model ([69]Fig. 1A). We compared the body weight of mice before and after modeling and found that BLM-induced lung injury caused a sharp decline in body weight ([70]Fig. 1B). Meanwhile, we also examined apoptosis in lung cells using Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) staining and observed significant cell death ([71]Fig. 1C and D). To investigate the changes in AT2 cells during lung injury, we stained AT2 cells with the specific protein marker surfactant protein C (proSPC) on day 7 post-injury and observed a significant reduction in their numbers ([72]Fig. 1E and F). The SENP1-Sirt3 signaling pathway is closely associated with mitochondrial function [[73]12]. To further determine the changes of mitochondria in AT2 cells during oxidative injury in lung tissues, we treated A549 (which exhibits AT2 cell characteristics) with BLM for 48 h in vitro. We found that mitochondrial SENP1 protein levels decreased, accompanied by increased SUMOylation of Sirt3 and markedly elevated acetylation and SUMOylation levels in mitochondria after BLM challenge ([74]Fig. 1G). Also, primary AT2 cells isolated from bleomycin-treated mice also showed the consistent results ([75]Supplementary Fig. 1A). Subsequently, we examined mitochondrial morphological alterations in injured AT2 cells using electron microscopy and observed a significant decrease in both mitochondrial number and cristae quantity, accompanied by swelling of the mitochondrial matrix ([76]Fig. 1H–J). Meanwhile, we also detected mitochondrial ROS and ATP levels in AT2 cells, and found that bleomycin treatment significantly increased mitochondrial ROS levels while markedly reducing ATP production capacity in AT2 cells ([77]Fig. 1K and L). These results indicate that bleomycin-induced lung injury is accompanied by downregulation of the SENP1-Sirt3 signaling and a decrease in mitochondrial quantity and function in AT2 cells. Fig. 1. [78]Fig. 1 [79]Open in a new tab BLM-induced pulmonary injury leads to downregulation of the SENP1-Sirt3 axis and mitochondrial morphological disruption. (A) Schematic of BLM challenge. (B) Analysis of WT-PBS and WT-BLM mice body weight change, n = 5. (C) Representative images of lung tissues in mice treated with or without BLM with TUNEL staining. Scale bars, 100 μm. (D) Statistical analysis of TUNEL staining positive cells, each data point represents in an individual mouse (n = 5/group). (E) Representative images of lung tissues in mice treated with or without BLM with immunofluorescence (IF) staining. The AT2 cells were stained with Surfactant protein C (proSPC); the nuclei were stained with DAPI. Scale bars, 100 μm. (F) Statistical analysis of proSPC positive cells, each data point represents in an individual mouse (n = 5/group). (G) Western blot was used to detect the changes in SENP1-Sirt3 axis in mitochondrial lysates of A549 cells treated with BLM (10 μM) for 48 h. The Sirt3 SUMOylation was detected with Sirt3 antibody after immunoprecipitation by SUMO1 antibody. Mitochondrial pan-SUMOylation and pan-acetylation was detected by SUMO1 antibody and acetyl-Lysine (AcK) antibody, respectively. (H) Transmission electron microscopy observation of AT2 cell mitochondria with and without BLM challenge, red arrows indicate mitochondria, yellow stars indicate lamellar bodies. Scale bars, 500 nm. (I) Statistical analysis of mitochondrion number of AT2 cells, each data point represents in an individual AT2 cell (n = 15/group). (J) Statistical analysis of the cristae number per mitochondria in AT2 cells, each data point represents in an individual mitochondrion (n = 30/group). (K) Mitochondrial ROS level detected by flow cytometry and labeled with MitoSOX in AT2 cells in mice treated with or without BLM, each data point represents in an individual mouse (n = 4/group). (L) ATP level detected in AT2 cells in mice treated with or without BLM, each data point represents in an individual mouse (n = 4/group). Data are presented as the means ± SEM. ∗∗p < 0.01; ∗∗∗∗p < 0.0001. 2.2. Sirt3 K223R protects against BLM-induced pulmonary inflammation in mice To evaluate whether improving mitochondrial function alleviates BLM-induced lung injury, we constructed Sirt3 K223R (abbreviated as Sirt3 KR) mice to mimic the activation of the SENP1-Sirt3 signaling, thereby enhancing mitochondrial function and assessing its role in lung injury pathogenesis. In this study, we treated wild type (WT) and Sirt3 KR mice with high and low doses of BLM intratracheally. While high-dose bleomycin caused mortality in wild-type mice within 14 days, Sirt3 KR mice resisted this injury ([80]Supplementary Fig. 1B and 1C). Through observation, we found that activation of SENP1-Sirt3 axis protected mice against acute death, weight loss and lung injury hemorrhage induced by high dose of BLM ([81]Supplementary Fig. 1C–E). To better assess this protective capacity of Sirt3 KR mice, we chose low-dose bleomycin to induce lung injury and observed alterations in lung tissue. When the dose of BLM was reduced to 0.67 μg/g, we found that neither WT nor KR mice exhibited death ([82]Fig. 2A and B). Through HE staining of BLM-induced lung tissues at 7- and 14-day, we observed thickened alveolar septa due to collagen deposition and inflammatory exudate, fragmented alveolar walls, and enlarged alveolar spaces, while Sirt3 KR mice exhibited markedly attenuated histological damage severity ([83]Fig. 2C). Due to the damage of alveolar epithelium, followed by the appearance of infiltrated immune cells in the alveoli (day 4–7), the lung tissue will undergoes inflammatory response and remodeling [[84]5,[85]19]. To further explore whether SENP1-Sirt3 axis activation can reduce inflammatory response after BLM challenge, WT and Sirt3 KR mice were analyzed on day 7. Sirt3 KR mice lost less body weight compared with WT littermates after BLM challenge ([86]Supplementary Fig. 2A). Bronchoalveolar lavage fluid (BALF) of Sirt3 KR mice had few cells and low protein content ([87]Fig. 2D and E). Giemsa staining of alveolar lavage fluid revealed that Sirt3 KR significantly reduced inflammatory cell infiltration into the lungs during injury ([88]Fig. 2F). In addition, lower levels of the proinflammatory cytokine Tumor Necrosis Factor-alpha (TNF-α) and Interleukin-6 (IL-6) were detected in the lung tissue of Sirt3 KR mice ([89]Supplementary Fig. 2B). Therefore, these results suggest that activating the SENP1-Sirt3 axis alleviates BLM-induced lung injury and reduces pulmonary inflammation occurrence during the early injury phase. Fig. 2. [90]Fig. 2 [91]Open in a new tab Sirt3 KR mice reduce BLM-induced pulmonary inflammation and fibrosis. (A) Schematic of BLM challenge. (B) Survival curve (n = 5/group). (C) HE staining of mouse lung tissue sections. Scale bars, 100 μm. (D) Statistical analysis of cell number in BALF, each data point represents in an individual mouse (n = 3/group). (E) Statistical analysis of protein concentration in BALF, each data point represents in an individual mouse (n = 3/group). (F) Giemsa staining of BALF. Scale bars, 100 μm. (G) Masson staining of mouse lung tissue sections. Scale bars, 100 μm. (H) Ashcroft score analysis, each data point represents in an individual mouse (n = 3/group). (I) Western blot was used to detect the protein expression of collagen1 (Colla1) and α-SMA in the lung tissue, each lane represents in an individual mouse (n = 3/group). Data are presented as the means ± SEM. ∗∗p < 0.01. 2.3. Sirt3 K223R reduces BLM-induced pulmonary fibrosis in mice Pulmonary inflammation, driven by persistent injury or infection, initiates fibrosis via immune cell activation and pro-fibrotic cytokines. Inflammation disrupts repair mechanisms, promoting fibroblast proliferation, excessive collagen deposition, and alveolar destruction. Severe pulmonary fibrosis develops in the lungs of mice by day 14 following low-dose BLM treatment ([92]Fig. 2G and H). Masson staining revealed that WT mice exhibited severely distorted alveolar architecture with extensive collagen deposition, while Sirt3 KR mice showed only partially disrupted alveolar structure and larger fibrotic areas. The Ashcroft score of Sirt3 KR mice was significantly lower compared to wild-type controls ([93]Fig. 2G and H). Detection of type I collagen and α-smooth muscle actin (α-SMA) further confirmed these results ([94]Fig. 2I). These findings suggest that activation of the SENP1-Sirt3 axis mitigates the progression of fibrosis by alleviating inflammation during the early phase of injury. 2.4. Sirt3 K223R enhances the activity of AT2 cells to alleviate lung injury To investigate the mechanisms by which Sirt3 KR AT2 cells alleviates pulmonary inflammation and fibrosis in a BLM-induced lung injury model, we sorted out AT2 cells from WT and Sirt3 KR mice on day 7 after BLM challenge and performed the transcriptomic analysis. Principal Component Analysis and the gene expression heatmap showed gene expression was significantly different between WT and KR after BLM challenge ([95]Fig. 3A and B). Enrichment analysis of down-regulated differential gene pathways (KR-BLM vs WT-BLM) showed that chemokine signaling pathway, lung fibrosis, Interleukin-5 (IL-5) signaling pathway, matrix metalloproteinase, apoptosis and oxidative damage response were markedly enriched ([96]Fig. 3C and [97]Supplementary Fig. 3A), among which IL-5 is closely related to fibrosis, and inhibition of IL-5 production can significantly reduce lung fibrosis [[98][23], [99][24], [100][25]]. Matrix metalloproteinases (MMPs) play an important role in the process of pulmonary fibrosis, and some MMPs are detected to be increased in the process of pulmonary fibrosis, including MMP8, MMP9, MMP10 and MMP14 etc [[101]26]. Likewise, inhibition of Mitogen-Activated Protein Kinase (MAPK) signaling also alleviates pulmonary fibrosis [[102][27], [103][28], [104][29]]. Gene set enrichment analysis (GSEA) showed overview of proinflammatory and profibrotic mediators, lung fibrosis and apoptosis were markedly enriched ([105]Fig. 3D). Enrichment analysis of up-regulated differential gene pathways (KR-BLM vs WT-BLM) showed that cholesterol metabolism with Bloch and Kandutsch Russell pathways, fatty acid biosynthesis, Peroxisome Proliferator-Activated Receptor (PPAR) signaling pathway, elongation of very long chain fatty acids, Wnt signaling pathway and pluripotency, glutathione and one carbon metabolism, Sterol Regulatory Element Binding Transcription Factor (SREBF) and MicroRNA-33 (miR33) in cholesterol and lipid homeostasis and mechanisms associated with pluripotency were markedly enriched ([106]Fig. 3E and [107]Supplementary Fig. 3B). GSEA showed overview of sterol regulatory element binding proteins SREBP signaling, PPAR signaling pathway and peroxisome were markedly enriched ([108]Fig. 3F). AT2 cell activity relies on lipid metabolism to synthesize pulmonary surfactant phospholipids and cholesterol [[109]30]. Key regulators like SREBP drive lipid biosynthesis, sustaining alveolar stability [[110]31]. Dysregulated lipid metabolism impairs surfactant production, exacerbating lung injury and fibrosis by compromising AT2-mediated repair [[111]32]. These data highlight Sirt3-KR enhances AT2 cell activity by promoting lipid metabolism, thereby alleviating pulmonary inflammation and fibrosis. Fig. 3. [112]Fig. 3 [113]Open in a new tab Transcriptome analysis of WT and Sirt3 KR AT2 cells after BLM challenge. (A) Principal component analysis. (B) Heat map of gene expression in each group. (C) Pathways enrichment analysis results of down-regulated differential gene in Sirt3 K223R AT2 cells compared with WT AT2 cells after BLM challenge. (D) Gene Set Enrichment Analysis of down-regulated differential gene in Sirt3 K223R mice compared with Sirt3 WT mice after BLM challenge. (E) Pathways enrichment analysis results of up-regulated differential gene in Sirt3 K223R mice compared with Sirt3 WT mice after BLM challenge. (F) Gene Set Enrichment Analysis of up-regulated differential gene in Sirt3 K223R mice compared with Sirt3 WT mice after BLM challenge. 2.5. Sirt3 K223R promotes the proliferation and differentiation of AT2 cell Having identified that AT2 cell activity is regulated by the SENP1-Sirt3 signaling through transcriptomic analysis, we will next investigate whether the proliferation and differentiation capacities of AT2 cells are altered in Sirt3 KR mice. After BLM challenge, alveolar epithelial cells will undergo apoptosis and reduce on day 2–4 [[114]5]. Therefore, we detect the number of alveolar epithelial cells in the lung tissue on day 3 were detected, Sirt3 KR mice retained larger population of both AT1 ([115]Supplementary Fig. 4A and 4B) and AT2 ([116]Fig. 4A and B) cells compared with WT littermates after BLM challenge. TUNEL staining and Bax protein expression of lung tissues also confirmed that Sirt3 KR mice could attenuate apoptosis after BLM challenge ([117]Supplementary Fig. 4C–E). In normal lung tissue, the turnover rate of AT2 cells is very low, however, when the alveolar structure is destroyed, AT2 cells can proliferate and differentiate into AT1 cells to repair the damaged alveolar structure [[118]33]. To further analyze the role of SENP1-Sirt3 axis in AT2 cells during the lung injury, lung tissue was analyzed on day 14 after BLM challenge and we found that AT2 cells from Sirt3 KR mice showed increased proliferation and differentiation activity ([119]Fig. 4C–F). This result suggests that Sirt3 KR regulates the proliferation and differentiation of AT2 cells to stabilize alveolar cell populations. Fig. 4. [120]Fig. 4 [121]Open in a new tab Analysis of the number and function of WT and Sirt3 KR AT2 cells in vivo and in vitro. (A) Representative IF images of detecting the expression of proSPC of mouse lung on day 3 after BLM challenge. Scale bars, 100 μm. (B) Statistical analysis of AT2 cells number, each data point represents in an individual mouse (n = 3/group). (C) Representative IF images of detecting the expression of Ki67 in AT2 cells of mouse lung on day 14 after BLM challenge. Scale bars, 100 μm. Yellow arrows point to AT2 cells that express the proliferation marker Ki67. (D) Statistical analysis of Ki67^+ AT2 cells in AT2 cells of mouse lung on day 14 after BLM challenge, each data point represents in an individual mouse (n = 3/group). (E) Representative IF images of detecting the expression of HOPX^+ in AT2 cells of mouse lung on day 14 after BLM challenge, yellow arrows point to AT2 cells that express the AT1 cells marker HOPX. Scale bars, 100 μm. (F) Statistical analysis of HOPX^+ AT2 cells in AT2 cells of mouse lung on day 14 after BLM challenge, each data point represents in an individual mouse (n = 3/group). (G) Schematic of AT2 cell co-culture with lung fibroblast organoid. (H) Representative results of AT2 cell co-culture with lung fibroblast organoid. Scale bars, 100 μm. (I) Statistical analysis of AT2 cell co-culture with lung fibroblast organoid number, each data point represents in an individual mouse (n = 3/group). (J) qPCR detecting the expression of Sftpc (K) Axin2 (L) Hopx, each data point represents in an individual mouse (n = 3/group). Data are presented as the means ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. To isolate AT2 cells conveniently to study the effect of SENP1-Sirt3 axis, we generated KR; Sftpc-creER; Rosa26-EYFP mice by crossing WT; Sftpc-creER; Rosa26-EYFP and KR mice ([122]Supplementary Fig. 5A–C). AT2 cells are able to form alveolar organoids when cultured in Matrigel-embedded 3D conditions, with cellular composition varying depending on culture methods. We then utilized two culture systems: feeder free culture ([123]Supplementary Fig. 5D) and co-culture ([124]Fig. 4G). Under feeder free culture conditions, supplementing media with specific inhibitors or agonists maintains AT2 cells in their undifferentiated state, resulting in organoids predominantly composed of homogeneous AT2 populations. We found that activation of SENP1-Sirt3 axis could promote AT2 cells to form more alveolar organoids and proliferate more AT2 cells under feeder free culture conditions ([125]Supplementary Fig. 5E–G). Also, it could promote AT2 cells to form more alveolar organoids and express higher stemness-related genes (Axin2) and AT1 cell marker genes (Hopx) under co-culture conditions ([126]Fig. 4H–L). The results in two culture systems demonstrate that the SENP1-Sirt3 signaling promotes the proliferation and stemness of AT2 cells. To assess the effect of SENP1-Sirt3 axis on AT2 cells in vitro after BLM challenge, feeder free alveolar organoids were challenge with BLM for 48 h and we found that alveolar organoids formed by Sirt3 KR AT2 cells were plumper and more intact with better light transmission ([127]Supplementary Fig. 6A–B). We further digested BLM-challenged alveolar organoids into single-cell suspensions for re-culture, we found AT2 cell alveolar organoids from Sirt3 KR mice still form more alveolar organoids under feeder free culture conditions ([128]Supplementary Fig. 6C–D). Moreover, AT2 cells were isolated from WT and Sirt3 KR mice on day 7 after BLM challenge to perform feeder free alveolar organoids culture, likewise, alveolar organoids formed by Sirt3 KR AT2 cells still form a larger number of alveolar organoids under feeder free culture conditions ([129]Supplementary Fig. 6E–G). These findings indicate that Sirt3 KR mice resist BLM-induced injury by modulating AT2 cell functions. 2.6. Sirt3 K223R drives Wnt signaling to enhance the activity of AT2 cells Recent studies have shown that there is a distinct population of KRT8 transitional cells during alveolar regeneration after BLM challenge, which are an intermediate cell population from the differentiation of AT2 to AT1 cells, characterized with cell cycle arrest, pro-fibrosis and senescence-associated secretory phenotype [[130]4,[131]5,[132][34], [133][35], [134][36]]. Our immunofluorescence staining results confirmed that this cell population was abundant after BLM challenge and significantly less in the lung tissue of KR mice compare to WT mice ([135]Fig. 5A and B). A decrease in KRT8^+ cells of Sirt3 KR mice suggests a reduced number of AT2 cells entering the KRT8^+ intermediate state, which is associated with increased differentiation toward terminally differentiated AT1 cells. Fig. 5. [136]Fig. 5 [137]Open in a new tab Sirt3 KR reduces KRT8^+ cells and promotes Wnt signaling. (A) Representative IF images of detecting the expression of KRT8 in AT2 cells of mouse lung on day 7 after BLM challenge. Yellow arrows point to AT2 cells that express KRT8. Scale bars, 100 μm. (B) Statistical analysis of KRT8^+ cells in AT2 cells of mouse lung on day 7 after BLM challenge, each data point represents in an individual mouse (n = 4/group). (C) Representative IF images of detecting the expression of Axin2 in AT2 cells of mouse lung on day 7 after BLM challenge, yellow circles indicate AT2 cells that express Axin2. Scale bars, 100 μm. (D) Statistical analysis of KRT8^+ cells in AT2 cells of mouse lung on day 7 after BLM challenge, each data point represents in an individual mouse (n = 3/group). (E) Schematic diagram of the experiment. (F) Western blot was used to detect the protein expression of β-catenin and Axin2 in AT2 cells after BLM challenge. Data are presented as the means ± SEM. ∗∗p < 0.01. The canonical Wnt signaling is initiated through paracrine or autocrine Wnt ligands binding to frizzled family receptors, leading to cytoplasmic accumulation of β-catenin protein and subsequent nuclear translocation to regulate downstream target gene expression, including Axin2 [[138]33,[139]37]. In murine lung tissue, Wnt ligands activating AT2 cells primarily originate from fibroblast-secreted Wnt5a or autocrine Wnt7b protein [[140]33]. Notably, Axin2^+ AT2 cells represent a stem-like subpopulation constituting approximately 1 % of total AT2 cells, which undergo substantial proliferation following lung injury [[141]33]. To confirm the effect of SENP1-Sirt3 axis activation on Wnt signaling activity in AT2 cells, immunofluorescence staining of lung tissue sections were performed to observe the expression of Axin2 protein in AT2 cells on day 7 after BLM challenge and we found more AT2 cells expressed Axin2 protein in Sirt3 KR mice compare to WT mice ([142]Fig. 5C and D). Finally, AT2 cells, isolated from Sirt3 KR mice compare to WT mice after BLM challenge with 24 h, also expressed more β-catenin and Axin2 protein ([143]Fig. 5E and F). These results align with transcriptomic data, demonstrating that Sirt3 KR mice exhibited a significant reduction in KRT8^+ transitional AT2 cells compared to WT controls. 2.7. Sirt3 K223R alleviates mitochondrial oxidative damage in AT2 cells As the major deacetylase in mitochondria, Sirt3 enhances mitochondrial function by deacetylating substrates [[144]38]. Transcriptome data analysis revealed that Sirt3 KR significantly inhibits the BLM-induced elevation of oxidative damage ([145]Fig. 3A and [146]Supplementary Fig. 3C). Based on previous studies, antioxidant enzyme SOD2 scavenges ROS in mitochondria, and it is considered to be a substrate of Sirt3 [[147]14,[148]39,[149]40]. We hypothesized that Sirt3 KR may eliminate ROS produced in mitochondria by regulating the acetylation level of SOD2 in AT2 cells, resulting in reduced apoptosis. To verify our hypothesis, we detected the expression of Bax, SOD2 and the acetylation level of SOD2 in alveolar organoids after BLM challenge, the results showed that alveolar organoids formed by Sirt3 KR AT2 cells had lower expression level of Bax and acetylation level of SOD2 ([150]Fig. 6A and B). Moreover, propidium iodide (PI) staining and mtROS staining also further indicated that alveolar organoids formed by Sirt3 KR AT2 cell produced less ROS and less apoptosis after BLM challenge ([151]Fig. 6C–F). These results suggest that Sirt3-mediated SOD2 deacetylation critically mitigates mtROS-driven apoptosis, highlighting its therapeutic potential in lung injury models where oxidative mitochondrial damage is pathogenic. Fig. 6. [152]Fig. 6 [153]Open in a new tab Sirt3 KR alleviates mitochondrial ROS level in AT2 cells. (A) Schematic of feeder free organoids treated with BLM. (B) Western blot was used to detect the protein expression of Bax, SOD2 and ac-SOD2 (K68) in AT2 cells feeder free organoids after BLM challenge. (C) PI staining of AT2 cells feeder free organoids after BLM challenge. Scale bars, 200 μm. (D) Statistical analysis of PI staining fluorescence intensity, each data point represents in an individual mouse (n = 4/group). (E) mtROS staining of AT2 cells feeder free organoids after BLM challenge. Dashed circles indicate mtROS stained by mitoSOX Red. Scale bars, 200 μm. (F) Statistical analysis of mtROS staining fluorescence intensity, each data point represents in an individual mouse (n = 4/group). Data are presented as the means ± SEM. ∗∗∗p < 0.001. 2.8. NAC promotes AT2 cell activity and mitigates pulmonary fibrosis In order to understand the effect of ROS-scavenging on the activity of AT2 cells, we employed N-acetylcysteine (NAC), a well-characterized antioxidant, in a feeder-free alveolar organoid culture system derived from AT2 cells [[154]35] ([155]Supplementary Fig. 7A). NAC was selected for its ability to directly neutralize ROS and replenish glutathione, thereby mitigating oxidative stress. Concurrently, NAC supplementation markedly improved organoid formation efficiency and AT2 cell proliferation ([156]Supplementary Fig. 7B–D), suggesting that ROS clearance fosters a pro-regenerative microenvironment. Organoids treated with NAC exhibited a significant reduction in mtROS production, as quantified by MitoSOX fluorescence assays ([157]Supplementary Fig. 7E–F). consistent with enhanced redox homeostasis. These findings align with our earlier observations in Sirt3 KR models, where deacetylation of SOD2 amplified mitochondrial antioxidant capacity [[158]14]. However, unlike Sirt3 KR which targets enzymatic activity of SOD2 via post-translational modification NAC operates through direct ROS scavenging, highlighting distinct yet complementary mechanisms. The data imply that SENP1-Sirt3 signaling may synergize with antioxidant therapies to amplify AT2 cell resilience during alveolar repair. Specifically, SENP1-mediated deSUMOylation of Sirt3 could stabilize its activity, while NAC alleviates ROS overload, collectively preserving mitochondrial integrity. This dual approach-combining SENP1-Sirt3 axis and broad-spectrum ROS neutralization may optimize AT2 cell survival and proliferation in oxidative injury models, such as BLM-induced pulmonary fibrosis. Further studies are warranted to dissect crosstalk between these mechanisms and their therapeutic potential. To further investigate the therapeutic potential of NAC in promoting AT2 cell activity and alleviating pulmonary fibrosis, we conducted in vivo experiments using a BLM-induced lung injury model ([159]Fig. 7A). Mice receiving intraperitoneal NAC supplementation post-BLM challenge exhibited attenuated weight loss ([160]Fig. 7B), a hallmark of systemic inflammation and metabolic stress in fibrotic progression. Histopathological and biochemical analyses revealed that NAC significantly reduced collagen deposition and fibrotic lesion area ([161]Fig. 7C and D), as quantified by hydroxyproline assays and Masson staining, indicating suppression of extracellular matrix remodeling. Notably, immunofluorescence and flow cytometry demonstrated enhanced AT2 cell proliferative activity ([162]Fig. 7E and F) and reduced generation of KRT8^+ transitional AT2 cells ([163]Fig. 7G and H), a subpopulation associated with maladaptive differentiation and pro-fibrotic signaling. These findings align with in vitro organoid data, suggesting NAC mitigates oxidative stress-driven AT2 cell dysfunction and preserves alveolar epithelial homeostasis. However, unlike genetic Sirt3 activation, NAC transient ROS scavenging may lack sustained metabolic reprogramming effects. Combined with prior results, this supports a dual therapeutic strategy: targeting both acute ROS overload and mitochondrial resilience pathways to optimize AT2 cell repair capacity in fibrotic lungs ([164]Fig. 8). Further studies should explore timing, dosage, and synergy between these approaches for clinical translation. Fig. 7. [165]Fig. 7 [166]Open in a new tab NAC treatment promotes AT2 cells proliferation and differentiation. (A) Schematic of experiment, each mouse received 100 μL of NAC/PBS solution at a concentration of 60 mg/μL via intraperitoneal injection. (B) Analysis of body weight change in mice, each data point represents in an individual mouse (n = 4/group). (C) HE & Masson staining of mouse lung tissue sections after BLM challenge. Scale bars, 100 μm. (D) Ashcroft score, each data point represents in an individual mouse (n = 4/group). (E) Representative IF images showing that NAC supplementation promoted the proliferation and differentiation of AT2 cells (day 14 lung tissue sections after BLM challenge) and reduced the production of KRT8^+AT2 cells (day 7 lung tissue sections after BLM challenge). White arrows point to AT2 cells that express the proliferation marker Ki67. Yellow arrowheads point to AT2 cells that express the AT1 cells marker HOPX. Yellow arrows point to AT2 cells that express KRT8. Scale bars, 100 μm. (F) The proportion of Ki67-expressing AT2 cells in each group was statistically analyzed, each data point represents in an individual mouse (n = 4/group). (G) The proportion of HOPX^+AT2 cells in each group was statistically analyzed, each data point represents in an individual mouse (n = 4/group). (H) The proportion of KRT8^+AT2 cells in each group was statistically analyzed, each data point represents in an individual mouse (n = 4/group). Data are presented as the means ± SEM. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001. Fig. 8. [167]Fig. 8 [168]Open in a new tab Activation of the SENP1-Sirt3 axis protects against pulmonary oxidative damage by regulating the activity of AT2 cells. External injury stimuli trigger apoptosis in alveolar epithelial cells, compromising alveolar structural integrity. Within AT2 cell mitochondria, reduced activity of the SENP1-Sirt3 axis leads to increased acetylation of SOD2. This promotes the accumulation of mtROS, impairs AT2 cell function, and reduces Wnt signaling and metabolic activity. Consequently, more differentiation-arrested KRT8^+ transitional AT2 cells are generated. These cells release pro-fibrotic cytokines, ultimately inducing the development of pulmonary fibrosis. 3. Discussion SENP1 and Sirt3, key enzymes mediating protein post-translational modifications, are critical for maintaining lung tissue homeostasis [[169][41], [170][42], [171][43], [172][44], [173][45]]. Notably, Sirt3 expression is consistently reduced in AT2 cells across multiple experimental lung disease models [[174]43,[175]46,[176]47]. Our study demonstrated that the SENP1-Sirt3 regulatory axis becomes functionally compromised in AT2 cells following pulmonary injury. Compared to WT controls, Sirt3 KR mice exhibited reduced mortality, attenuated alveolar epithelial injury, diminished inflammatory responses, and milder pulmonary fibrosis. These findings highlight the essential role of SENP1-Sirt3 axis in mitigating pathological progression during lung injury. AT2 cells are central drivers of pulmonary fibrosis progression, where injury triggers inflammatory factor release and immune cell recruitment to initiate alveolar remodeling-a pathological cascade consistent with our murine model phenotypes [[177]48], Emerging single-cell evidence reveals that fibrotic AT2 cells undergo dual pathological reprogramming: cell cycle arrest prevents regenerative proliferation, while differentiation blockade promotes accumulation of KRT8^+ transitional AT2 cells [[178]4,[179]5,[180]34,[181]49]. These maladaptive subpopulations secrete pro-fibrotic mediators and sustain inflammatory microenvironments. Our transcriptomic profiling demonstrated that SENP1-Sirt3 axis activation suppresses three critical drivers of AT2 cell dysfunction: (1) proinflammatory signaling, (2) fibrogenic pathways, and (3) apoptosis networks (Bax/Bcl-2) following bleomycin injury. Immunofluorescence analyses corroborated these findings, showing reduced KRT8^+ transitional AT2 cell generation in Sirt3 KR models compared to WT controls. Collectively, these data establish that SENP1-Sirt3 axis activation preserves AT2 cell homeostasis by simultaneously resolving inflammatory-fibrotic signaling and restoring differentiation competence, thereby counteracting BLM-induced epithelial injury. Wnt signaling plays an important role in AT2 cell stemness. the residual AT2 cells after lung injury proliferate and differentiate into AT1 cells under the effect of Wnt signaling to repair damaged alveoli [[182]33]. Our in vivo mouse experiments and in vitro organoid culture experiments demonstrated that SENP1-Sirt3 axis activation promoted the proliferation and differentiation of AT2 cells, further confirmed by RNA-seq analysis showing that SENP1-Sirt3 axis activation up-regulate Wnt signaling after BLM challenge. In addition, we also demonstrated that reducing mtROS production by NAC supplementation promoted AT2 cell activity to form more alveolar organoids. There is an interplay between ROS signaling and Wnt signaling, loss of the Wnt target gene Myc preventing ROS accumulation [[183]50], overproduction of ROS inhibiting the Wnt/β-catenin signaling [[184]51], and NAC supplementation reactivated the Wnt/β-catenin pathway by scavenging ROS [[185]52]. Mitochondria have pivotal effect on the fate of AT2 cells and pulmonary fibrosis [[186]53]. Excessive ROS production leads to AT2 cell apoptosis and pulmonary fibrosis [[187]54,[188]55]. The Sirt3-SOD2-mtROS production nexus has been reported in some studies, however, our study made it clear that SENP1-Sirt3 axis activation removed SOD2 acetylation modification, alleviate mtROS overproduction induced apoptosis and promoted the activity in AT2 cells. The absence of the mitochondrial fusion proteins mitofusin1 (MFN1) and mitofusin2 (MFN2) in mice AT2 cells leads to morbidity and lung fibrosis and loss of MFN1, MFN2 or inhibiting lipid synthesis via fatty acid synthase deficiency in AT2 cells exacerbates BLM-induced lung fibrosis [[189]56]. Lipid deficiency also contributes to impaired progenitor renewal capacity of AT2 cells in aging and idiopathic lung fibrosis [[190]57]. Additionally, overexpression of fatty acid synthase in AT2 cells attenuates BLM-induced lung fibrosis by restoring mitochondrial dysfunction in mice [[191]58]. Our RNA-seq analysis also revealed that metabolic pathways, especially lipid-related metabolic pathways were upregulated, including cholesterol metabolism, fatty acid biosynthesis, PPAR signaling pathway, elongation of very long chain fatty acids in KR AT2 cell after BLM challenge. AT2 cells show decreased mitochondrial number and size, swollen mitochondria with disrupted cristae and decreased Optic Atrophy 1 (OPA1) protein expression after BLM challenge, our previous work has proved that SENP1-Sirt3 axis activation could remove the deacetylation of ATP-dependent zinc metalloprotease (YME1L1), regulating mechanism in controlling OPA1 cleavage and subsequent OPA1-mediated mitochondrial fusion in T cell memory development [[192]15]. Further studies are needed to determine whether SENP1-Sirt3 axis activation affects lipid metabolism pathways through YME1L1-OPA1 pathway by regulating mitochondrial fusion in AT2 cells. Given the technical challenges associated with isolating and maintaining human primary AT2 cells, we initially utilized A549 cells and primary mouse cells to explore the alteration of SENP-Sirt3 signaling under oxidative stress. However, this limitation should be considered when extrapolating our in vitro results to human pulmonary physiology. Future studies should aim to validate these findings using primary human AT2 cells, as their use would enhance the translational relevance of our observations and provide a more accurate reflection of alveolar epithelial behavior in pathological contexts [[193]1]. Also, immune cells, especially alveolar macrophages, play important roles in pulmonary fibrosis [[194][59], [195][60], [196][61], [197][62], [198][63]]. Our previous study showed that SENP1-Sirt3 axis regulates the function of T cells and macrophage [[199]15,[200]64,[201]65]. Considering KR mice are systemic mutant mice, the role of immune cells in pulmonary fibrosis needs to be further measured. An important limitation is the use of the BLM mouse model, which induces reversible fibrosis, distinct from the irreversible progression in human patients [[202]19]. In mice, BLM triggers acute injury with transient fibrosis that partially resolves over time, whereas human pulmonary fibrosis involves chronic, unresolved inflammation and persistent extracellular matrix accumulation [[203]49,[204]53]. While our findings on the SENP1-Sirt3 axis highlight its role in mitigating oxidative stress and promoting AT2 cell resilience, the reversible nature of this model limits direct translation to human progressive disease. Future studies using models mimicking chronic, irreversible fibrosis are needed to validate therapeutic relevance. In summary, we have revealed the alteration of SENP1-Sirt3 axis in AT2 cells during pulmonary fibrosis, and highlight that activation of SENP1-Sirt3 axis regulate SOD2 acetylation level, alleviate ROS overproduction induced apoptosis, enhance AT2 cell activity, and reduce pulmonary fibrosis. Consequently, targeting SENP1-Sirt3 axis of AT2 cells might offer a potential therapeutic approach for pulmonary fibrosis. 4. Materials and methods 4.1. Mouse lines Sirt3 wild-type and K223R mice were described in our previous studies [[205]12]. Sftpc-creER; Rosa26-EYFP mice were kindly gifted by Prof. Pengfei Sui (Shanghai Institute of Biochemistry and Cell Biology, Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences). EYFP-lineage labeled AT2 cell was inducted by intraperitoneal injecting 50 mg/kg tamoxifen (T6906; TargetMol) in corn oil (MB1148; Meilunbio) for 4 consecutive days. Subsequent studies were initiated at least 2 weeks after the completion of tamoxifen treatment. Age matched male adult (8–12 week old) mice were used in each independent experiment. All mice were bred on the C57BL/6 background and maintained under specific pathogen–free conditions with a 12-h light/12-h dark cycle and a temperature of 22 ± 2 °C and a humidity of 40–70 %. Animal experiments were performed in compliance with the “Guide for the Care and Use of Laboratory Animals”, which were approved by the Experimental Animal Ethical Committee at Shanghai Jiao Tong University School of Medicine. The ethical approval number for animal experiments is JUMC2023-126-A. 4.2. Cell culture Human pulmonary epithelial cell line A549 and primary mouse lung fibroblasts were cultured in DMEM/F12 supplemented with 10 % FBS and antibiotics in 5 % CO2 at 37 °C in a humidified atmosphere. 4.3. Alveolar organoids culture For the feeder free organoids culture, sorted AT2 cells were mixed in culture medium and the equal volume of growth factor-reduced Matrigel (356231, Corning) and 5000 cells per 20 μL gel-containing drops were placed on the bottom of 24 well plate or 2500 cells per 10 μL gel-containing drops were placed on the bottom of 48 well plate. The drops were incubated for 20 min at 37 °C to solidify the gels, and 200 μL or 400 μL of medium was added to each well in 48-well or 24-well plates, respectively. Medium was changed every 3 days. Y27632 was included in the medium for the first 3 days of the sorting or passaging to increase cell viability. Organoids were imaged using Leica M205 FCA. The composition of culture media is described in [206]Supplementary Table 3. For the co-culture with lung fibroblast organoids, in brief, 5000 freshly isolated EYFP^+ AT2 cells and 50000 primary fibroblasts were resuspended in DMEM/F12 and mixed in a 1:1 ratio with growth-factor-reduced matrigel (356231, Corning). Then, 90 μL mixture was pipetted into a 24-well transwell insert (3470, Corning). 500 μL culture media was added in the lower chamber and changed every other day. Organoids were imaged using Leica M205 FCA. The composition of culture media is described in [207]Supplementary Table 4. 4.4. Mouse model of bleomycin-induced lung injury For animal studies, the BLM treatment was administered as a single dose via intratracheal injection at concentrations of 0.67 μg/g or 1.33 μg/g body weight (BLM, catalog no. S1214, Sellect) dissolved in 50 μL PBS, following anesthesia with isoflurane. The duration of treatment corresponded to the experimental time points, with mice sacrificed at specific intervals (e.g., day 3, 7, or 14 post-BLM challenge) based on the experimental objectives (e.g., analyzing acute injury, inflammation, or fibrosis progression). For histological analysis, upon sacrifice, lungs were inflated with 4 % paraformaldehyde (PFA) and fixed overnight at 4 °C, then washed with PBS and processed into either paraffin sections (6 μm thickness) or cryosections (10 μm thickness). PFA fixation was used for both paraffin and cryosection preparation; paraffin sections were utilized for Hematoxylin-Eosin (HE), Masson's trichrome, and TUNEL staining, while cryosections were employed for immunofluorescence to preserve antigenicity. Immunofluorescence staining involved primary antibodies (diluted 1:100–1:500) incubated overnight at 4 °C, followed by fluorochrome-conjugated secondary antibodies (1:200 dilution, ThermoFisher), with specific antibody details provided in [208]Supplementary Table 1. 4.5. Mouse lung tissue dissociation Lung dissociation was performed as described previously [[209]35]. Briefly, lungs were intratracheally inflated with 1 mL of enzyme solution containing dispase (5 U/mL), DNase I (0.33 U/mL) and collagenase type I (450 U/mL) in DMEM/F12. Lungs were then inflated with 0.5 mL of 2 % low melting agarose and lung lobes were separated and incubated with 5 mL enzyme solution for 30 min at 37 °C with rotation. The reaction was quenched with an equal amount of DMEM/F12 + 10 % FBS medium and filtered through a 70 μm strainer. The cell pellet was resuspended in red blood cell lysis buffer (100 μM EDTA, 10 mM KHCO[3], 155 mM NH[4]Cl) for 3 min, washed with DMEM/F12 containing 10 % FBS and filtered through a 40 μm strainer. Total cells were centrifuged at 400g for 5min at 4 °C and the cell pellet was processed for AT2 cells and lung fibroblast isolation by FACS. EYFP^+AT2 cells and EYFP^− cells were sorted out and EYFP^− cells were cultured in a type I collagen (40136ES10, Yeasen) coated cell culture dish at 37 °C and 5 % CO[2] for 2 days. The primary lung fibroblasts were cultured no more than 3 passages. 4.6. Histology After mice were sacrificed, lungs were inflated with 4 % paraformaldehyde (PFA) and fixed at 4 °C overnight, followed by 3 washes with PBS. Lungs tissues and were then prepared for paraffin (6 μm) or cryo (10 μm) sectioning. HE (C0105S, Beyotime), Masson's trichrome (G1346, Solarbio) and TUNEL staining (C1090, Beyotime) were conducted with paraffin sections according to the manufacturer's instructions. The degree severity of lung fibrosis was quantified by Hubner modified Ashcroft scoring method [[210]66]. 4.7. BLM treatment of organoid cultures In feeder-free alveolar organoid cultures, BLM (catalog no. S1214, Sellect) treatment for 24 h or 48 h was initiated at 10 or 14 days after seeding as demonstrated in schematic of [211]Fig. 6A, [212]Supplementary Fig. 6A or [213]Supplementary Fig. 6C. Specifically, sorted AT2 cells were mixed with Matrigel and seeded, with Y27632 included in the medium for the first 3 days post-seeding to enhance cell viability. BLM was dissolved in PBS prior to use. The final concentration applied to organoid cultures was 10 μM, prepared by diluting the BLM stock solution into the culture medium to achieve this working concentration. PBS was used as vehicle-treated controls at the same volume as the BLM treatment group to account for potential solvent effects. 4.8. Detection of cell death and mtROS For detection in organoids: After removing the culture medium, alveolar organoids were washed three times with sterile PBS. They were then stained with PI (G1707, Servicebio) or MitoSOX Red ([214]M36008, Thermo Fisher Scientific) following the manufacturer's instructions to detect cell death and mtROS production, respectively. All images were acquired using an OLYMPUS IX73 microscope, underwent threshold processing, and the integrated fluorescence intensities of PI and MitoSOX Red were quantified using the integrated density measurement function in ImageJ software. For detection in animal tissues: Immediately after mouse sacrifice, fresh lung tissues were digested to prepare single-cell suspensions. The suspensions were stained with MitoSOX Red and analyzed via flow cytometry as previously described [[215]14]. 4.9. Immunofluorescence Mouse lungs paraffin sections were deparaffinized, rehydrated and antigen retrieved by boiling in sodium citrate solution for 15 min. Lung cryosections were washed with PBS to remove OCT (Sakura, 4583) and blocked in TBS containing 5 % donkey serum (Servicebio, G1217) and 0.1 % Triton X-100 (Sangon Biotech) for 1 h. Sections were then incubated with primary antibodies (1:100–1:500 dilution) 4 °C for overnight and fluorochrome conjugated secondary antibodies (1:200 dilution, ThermoFisher) for 2 h at room temperature. After antibody staining, nuclei were stained with DAPI (1:1000, Beyotime) and sections were mounted with 50 % glycerol. Fluorescence images were acquired using Leica DMi8. Six to ten random views of each lung section at 20 × were analyzed using Leixa LAS X software. The antibodies used were as [216]Supplementary Table 1. 4.10. Fluorescence intensity quantification The integrated fluorescent density (IFD) was quantified using ImageJ/Fiji software (Version 2.1.0/1.53c). Raw 16-bit TIFF images (acquired with an OLYMPUS IX73×microscope, 10× objective) were imported into ImageJ without any post-processing (e.g., brightness/contrast adjustments) to preserve original signal intensity. For each sample, 5 non-overlapping, randomly selected fields of view (FOVs) were analyzed. Regions of interest (ROIs) were manually drawn around the entire area of interest (e.g., bronchiolar epithelium or alveolar regions) to ensure consistency across samples. A cell-free area adjacent to the ROI was selected as the background region. The mean fluorescence intensity (MFI) of the background was subtracted from the MFI of the ROI to eliminate non-specific signal. IFD was calculated as: IFD= (MFI of ROI−MFI of background) × Area of ROI. This formula accounts for both signal intensity and the size of the fluorescent region, providing a robust measure of total signal. 4.11. Bronchoalveolar lavage fluid (BALF) processing Mice were sacrificed by carbon dioxide with blood removal and perfused with 5 mL of saline solution thorough the right ventricle, after which a catheter was introduced into the trachea. A 1 mL syringe was loaded with 1 mL of sterile PBS with protease inhibitors, and then BALF was collected. This was repeated 3 times. Lavage fluid was centrifuged for 10 min at 1500g at 4 °C. Cell counts of the pooled lung lavage fluid were made with a hemocytometer. The protein concentration of the cell-free BALF was determined by the method of BCA (P0010, Beyotime). Giemsa staining of BALF was conducted according to the manufacturer's instructions (G1079, Servicebio). 4.12. Mitochondria isolation In brief, approximate 10^7 cell lysates from tissues or cultured cell were incubated in ice-cold Tissue Dissociation (TD) buffer (135 mM NaCl, 5 mM KCl, 25 mM Tris-HCl pH 7.5) containing EDTA-free protease inhibitor mixture (1 tablet/50 mL TD buffer) (Roche). After centrifuging at 600g for 10 min, the pellet was washed with 10 mL ice-cold TD buffer and centrifuged again at 600g for 10 min. The pellet was then re-suspended in ice-cold Mannitol-Sucrose (MS) buffer containing 210 mM mannitol, 70 mM sucrose, 5 mM Tris-HCl (pH 7.5), 1 mM EGTA, 0.5 mg/mL BSA and protease inhibitor mixture. The lysate was further homogenized in this buffer with a glass–Teflon motorized homogenizer for 70 times. The mitochondrial fraction was isolated by differential centrifugation 1,300g for 5 min to remove pellet and 17,000g for 15 min to remove supernatant. Subsequently, the pellet was stored in 50 μL MS buffer at −80 °C and for further analysis. 4.13. ATP level measurement ATP levels were measured immediately from freshly isolated AT2 cells without prior storage or culture. The AT2 cell was isolated from mice lung on day 7 after BLM challenge. The amount of ATP level per milligram of protein was measured using an ATP assay kit (Meilunbio, MA0440) following the manufacturer's instructions. 4.14. Quantitative RT-PCR Total alveolar organoids RNA was isolated by using TRIzol reagent according to the manufacturer's instructions (Tiangen). Organoids (∼10^6 cells) were lysed in 1 mL TRIzol reagent, vortexed for 15 s, and incubated at room temperature for 5 min. After adding 200 μL chloroform, samples were shaken vigorously for 15 s, incubated for 2 min, then centrifuged at 12,000g for 15 min at 4 °C. The aqueous phase was transferred to a new tube, mixed with 500 μL isopropanol, and incubated for 10 min. Following centrifugation (12,000g, 10 min, 4 °C), the RNA pellet was washed with 1 mL 75 % ethanol (DEPC-treated), centrifuged at 7,500g for 5 min, air-dried, and the pellet RNA was eluted with 30 μL of RNase-free water, and its concentration and purity were assessed using a NanoDrop One spectrophotometer (Thermo Fisher; A260/A280 ratio >1.8, A260/A230 ratio >2.0). RNA integrity was verified by 1 % agarose gel electrophoresis (distinct 28S and 18S rRNA bands with a 2:1 intensity ratio). cDNA was synthesized from 1 μg purified RNA by using HiScript III All-in-one RT SuperMix Perfect for qPCR (Vazyme). Reactions were prepared in a 20 μL volume containing 5 μM random hexamers, 10 mM dNTP mix, 1 × RT buffer, 5 mM DTT, 20 U RNase recombinant ribonuclease inhibitor, and 10 U reverse transcriptase. Samples were incubated at 23 °C for 10 min (primer annealing), 55 °C for 10 min (reverse transcription), and 80 °C for 10 min (enzyme inactivation). Negative controls (no-RT reactions) were included by omitting the reverse transcriptase to monitor genomic DNA contamination. The resulting cDNA was diluted 1:5 with RNase-free water before use in qPCR. qPCR was performed using a SYBR Green Supermix PCR kit (Roche). Real-time qRT-PCR was performed using gene-specific primer sets ([217]Supplementary Table 2). Gene expression was assessed in triplicate and normalized to a reference gene, Actb. 4.15. Western blot analysis All samples used for western blot were either freshly excised lung tissues from sacrificed mice or freshly harvested organoids. Tissues or organoids were lysed in RIPA buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1 % NP-40; 0.5 % sodium deoxycholate; 0.1 % SDS) supplemented with protease inhibitor cocktail (cOmplete, Roche) and phosphatase inhibitors (PhosSTOP, Roche) to prevent protein degradation and dephosphorylation. The protein suspensions were centrifuged (12000g, 10 min, 4 °C), and the supernatants were then concentrated prior to separation using denaturing SDS-PAGE gels. The electrophoretically separated proteins were transferred from the SDS-PAGE gel to a PVDF membrane. After blocking with 5 % BSA for 1 h at room temperature, the membrane was sequentially incubated with the primary antibody at 4 °C overnight and the secondary antibody at room temperature for 1 h. Finally, proteins were visualized using ECL western blotting substrate. The primary antibodies used were as follows: [218]Supplementary Table 1. 4.16. Immunoprecipitation Extracted proteins were quantified and transferred to a new tube, and 50 % protein A/G-agarose was added. The antigen-antibody complex was incubated on a rotary shaker overnight at 4 °C, followed by centrifugation at 13,000g for 5 s. The precipitate was washed three times with RIPA lysis buffer, while the supernatant was collected for analysis using SDS-PAGE and western blotting methods. 4.17. RNA sequencing of AT2 cells Total RNAs were extracted from AT2 cells using TRIzol reagent according to the manufacturer's instructions (Tiangen). RNA quality was determined by 260:280 ratio and the RNA integrity number (RIN) determined by an Agilent Technologies 2100 Bioanalyzer. RNA samples with a 260:280 ratio >1.8 and a RIN >7 were used for the library construction using the TruSeq Stranded mRNA Library Preparation kit (Illumina), according to manufacturer's instructions. Then libraries with different indexs were multiplexed and loaded on an Illumina HiSeq/Illumina Novaseq/MGI2000 instrument for sequencing using a 2 × 150 paired-end (PE) configuration according to manufacturer's instructions. After the data preprocessing, gene level fragments per kilobase of exon per million fragments mapped and significant changes in gene and transcript expression (thresholds: linear fold change >1.5 or < -1.5, Benjamini Hochberg FDR (Padj) < 0.05) were then calculated using a variety of approaches. We used the clusterProfiler program [[219]67] and GSEA [[220]68] to identify the enriched biological processes and pathways among the differentially expressed genes. 4.18. Transmission electron microscopy Lung tissues were gently taken from the mice. The tissues were cut into smaller pieces (3 × 3 mm) while keeping it immersed in the fixative (a mixture of 2 % paraformaldehyde and 2.5 % glutaraldehyde). The tissue fixation was continued overnight at 4 °C using a shaker/rotator and washed, dehydrated, and embedded in resin according to standard procedures. Ultrathin sections (70–90 nm thickness) were cut using a Leica UC7 ultramicrotome with a diamond knife and collected onto 200-mesh copper grids coated with a Formvar/carbon film. Images were acquired using a HITACHI H-7650 or PHILIPS CM-120 Transmission Electron Microscope. AT2 cells were identified by their characteristic lamellar bodies. Mitochondria were evaluated based on the number in each AT2 cell. Cristae were assessed for density (number per one mitochondrion) using high-magnification (×30,000 - × 50,000) micrographs. 4.19. Statistical analysis All results were expressed as means ± SEM, for statistical analysis, which was performed using GraphPad Prism 9. For evaluation of group differences, the unpaired 2-tailed Student's t-test was used assuming equal variance. Differences for which p < 0.05 were considered statistically significant. Non-quantitative results were representative of at least three independent experiments. CRediT authorship contribution statement Mingming Zhang: Writing – original draft, Validation, Methodology, Investigation, Data curation. Xin Lin: Methodology, Formal analysis, Data curation. Jianli He: Methodology. Yong Zuo: Methodology. Qiuju Fan: Methodology. Innocent Agida: Methodology. Hongsheng Tan: Methodology. Caiying Zhu: Methodology. Jinke Cheng: Writing – review & editing, Supervision, Resources, Funding acquisition, Conceptualization. Tianshi Wang: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization. Ethics approval and consent to participate The animal studies were reviewed and approved by the Animal Care Committee of Shanghai Jiao Tong University School of Medicine (Shanghai, China). Funding This study was funded by in part by the National Key Research and Development Program of China 2020YFA0803600 (J.C.), by National Natural Science Foundation of China 82230049 (J.C.), 82030075 (J.C.), 92049113 (T.W.), 32070759 (T.W.). We thank for all members of the Core Facility of Basic Medical Sciences, Shanghai Jiao Tong University School of Medicine for technical assistance. Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Jinke Cheng reports financial support was provided by the National Key Research and Development Program of China. Jinke Cheng reports financial support was provided by National Natural Science Foundation of China. Tianshi Wang reports financial support was provided by National Natural Science Foundation of China. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Footnotes ^Appendix A Supplementary data to this article can be found online at [221]https://doi.org/10.1016/j.redox.2025.103752. Contributor Information Jinke Cheng, Email: jkcheng@shsmu.edu.cn. Tianshi Wang, Email: tianshi777@shsmu.edu.cn. Appendix A. Supplementary data The following is the Supplementary data to this article: Multimedia component 1 [222]mmc1.pdf^ (302.4MB, pdf) Data availability The data that has been used is confidential. References