Male C57BL/6 wild-type mice (8 weeks old) were purchased from GemPharmatech (Nanjing, China). All mice were housed under controlled environmental conditions with a 12-hour light/dark cycle and provided with water and food prior to experiments. The study protocol was approved by the Animal Review Committee of Zhongshan Hospital, Fudan University (2022/02/01).

SENP5^flox/flox mice and Sftpc-Cre mice were generated by GemPharmatech (Nanjing, China) via the CRISPR-Cas9 system. To generate alveolar epithelial cell-specific SENP5-deficient mice, Sftpc-Cre mice were crossed with SENP5^flox/flox mice to obtain Sftpc-Cre-SENP5^flox/flox mice (SENP5 conditional knockout (cKO)). Littermate SENP5^flox/flox mice were used as controls.

The LPS-induced ALI model was established using 8-week-old male C57BL/6 mice. The mice were randomly divided into control and ALI groups. Briefly, mice were anesthetized with isoflurane, and a midline neck incision was made to expose the trachea. The isoflurane concentration was titrated between 3% and 5% to maintain an appropriate depth of anesthesia. A total of 100 µL lipopolysaccharide (LPS) (0111: B4, L2630, Sigma, St. Louis, MO, USA) was slowly administered intratracheally at a dose of 1 mg/kg. This dosage was selected based on well-established protocols, which have been demonstrated to induce robust and reproducible acute lung injury characterized by significant inflammatory cell infiltration, pulmonary edema, and alveolar damage within 24–48 hours [12, 13, 14]. The control group received an equal volume (100 µL) of sterile PBS. 24 h or 48 h after LPS inhalation, the mice were euthanized via intraperitoneal injection 1% pentobarbital (150 mg/kg), followed by cervical dislocation, and the mice’s lung tissues were collected for further analysis.

Total RNA was extracted from control (NC) and SENP5 knockdown BEAS-2B cells using Trizol reagent (Thermo Fisher Scientific, Waltham, MA, USA). RNA quality was verified on an Agilent 2100 bioanalyzer (Agilent Technologies, Santa Clara, CA, USA), and sequencing libraries were prepared with the NEBNext® Ultra™ RNA Library Prep Kit (NEB, Ipswich, MA, USA). The libraries were sequenced on an Illumina NovaSeq 6000 platform (Illumina, San Diego, CA, USA) by Sangon Biotech (Shanghai, China). Subsequent bioinformatic analysis was performed as follows: Raw reads were quality-controlled using FastQC and processed with fastp to remove adapters and low-quality bases. The clean reads were then aligned to the reference genome using HISAT2, and gene expression was quantified using featureCounts. Differential expression analysis was conducted using the DESeq2 version 1.40.2 (Bioconductor, Boston, MA, USA) in R Studio 2024.12.0+467 (Posit Software, PBC, Boston, MA, USA) with normalization based on the median of ratios method. Genes with $|$log₂ fold change$|$ $>$2 and adjusted p value 0.05 were defined as significantly differentially expressed.

BEAS-2B human bronchial epithelial cells were purchased from American Type Culture Collection (ATCC) and cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (Gibco, Grand Island, NY, USA), 100 U/mL penicillin, and 100 µg/mL streptomycin (Gibco, USA) at 37 °C with 5% CO₂. To induce an inflammatory response, BEAS-2B cells were treated with LPS (10 µg/mL) (0111:B4, L4391, Sigma, USA). The cell line used in this study was routinely tested for mycoplasma contamination and was confirmed to be negative. Besides, the cell line was validated by STR profiling.To knockdown SENP5 expression, lentivirus targeting SENP5 (Genepharma, Shanghai, China) and empty vectors (NC) were used to infect the BEAS-2B cells. Six hours after infection, the medium was replaced. Cells were then cultured for an additional 48 h before harvesting for subsequent experiments. All cell experiments were conducted with three biological replicates per group.

Cell viability was assessed using the CCK-8 assay. For CCK-8, BEAS-2B cells were seeded in 96-well plates at a density of 1 $\times$ 10⁴ cells in each well for 24 h. A total of 10 µL of the CCK-8 solution was added to each well, followed by 2 h of incubation at 37 ℃. The optical density (OD) value at 450 nm was measured using a microplate reader (Thermo Fisher Scientific).

The apoptosis rate in BEAS-2B cells was determined by flow cytometry (CytoFLEX LX, Beckman Coulter, Brea, CA, USA) using an Annexin V-APC/PI kit (Vazyme, Nanjing, China). Data analysis was conducted with FlowJo software 10.8.1 (TreeStar, Ashland, OR, USA).

Mice’s left lung tissues were fixed in 4% paraformaldehyde at room temperature overnight and then embedded in paraffin. Sections were cut at 4 µm thickness for staining. Then sections were deparaffinized in xylene twice and rehydrated through a graded ethanol series (100%, 90%, and 70%). The sections were washed in distilled water before hematoxylin and eosin staining. Subsequently, sections were sequentially dehydrated through 95% and 100% ethanol solutions and cleared in xylene. Finally, lung sections were sealed with neutral gum for microscopic examination of the pathological changes of lung injury.

The mouse lung tissues were embedded in paraffin and then deparaffinized and rehydrated before antigen retrieval. Later, these sections were subjected to 3% H₂O₂ and 10% goat serum at room temperature for 30 min. After cleaning, the SENP5 primary antibody was added and incubated overnight at 4 °C. The following day, a horseradish peroxidase (HRP)-conjugated secondary antibody (1:1000 dilution) was applied for 15 minutes at room temperature and a diaminobenzidine (DAB) substrate was used for chromogenic development. The immunohistochemical detection of lung samples was conducted through the GeneTech kit (Shanghai, China). All experiments were conducted independently three times.

Total RNA was extracted from BEAS-2B cells and lung tissue using Trizol reagent (Thermo Fisher Scientific, USA). cDNA was synthesized using SuperRT III All-in-one RT Mix for qPCR (with gDNA Remover) (Yeasen, Shanghai, China). Quantitative PCR was performed using the Hieff® qPCR SYBR Green Master Mix (Yeasen, Shanghai, China) using the CFX-96 real-time PCR detection machine (Bio-Rad, Hercules, CA, USA) according to the manufacturer’s instructions: 95 °C for 5 min, 45 cycles of 95 °C for 10 s, 60 °C for 30 s, and 72 °C for 20 s. The primers were synthesized by Sangon Biotech (Shanghai, China). Relative mRNA expression levels were analyzed through the 2^{-Δ⁢Δ⁢Ct} method. The primer sequences are listed in Table 1. All experiments are representative of three independent experiments.

Total protein was extracted from BEAS-2B cells and lung tissues by RIPA buffer (Yeasen, Shanghai, China) supplemented with 1 mM PMSF and protease inhibitor cocktail at 4 ℃. Protein concentrations were determined using a BCA protein assay kit (Epizyme, Shanghai, China). Samples (40 µg) were separated by 8–12% SDS-PAGE gels and transferred to PVDF membranes (Millipore, Burlington, MA, USA). Membranes were then incubated with the primary antibodies at 4 ℃ overnight, followed by incubation with HRP-conjugated anti-mouse and anti-rabbit IgG antibodies at room temperature for 1 h. The following antibodies were used in experiments: SENP5-Specific Polyclonal antibody (1:1000, 19529-1-AP, Proteintech, Wuhan, China); $\beta$-actin Monoclonal antibody (1:1000, 30101ES10, Yeasen); mTOR Monoclonal antibody (1:1000, 66888-1-Ig, Proteintech); Phospho-mTOR (1:1000, ab109268, Abcam, Cambridge, MA, USA); p70 Polyclonal antibody (1:1000, 14485-1-AP, Proteintech); Phospho-p70 Polyclonal antibody (1:1000, 28735-1-AP, Proteintech). Target bands were detected using a digital image system (Tanon, Shanghai, China).

In this study, all data were presented as mean $\pm$ standard deviation (SD). Statistical analysis was performed using GraphPad Prism Software 10.0 (San Diego, CA, USA). Comparisons between two groups were conducted using Student’s t-test, while one-way ANOVA was applied for comparisons across multiple groups. Survival analysis was carried out using the Kaplan–Meier method, with between-group differences evaluated by the log-rank test. A p-value 0.05 was considered statistically significant relative to the control group.

To validate the role of SENP5 in lung injury, we first established an LPS-induced BEAS-2B human epithelial cell injury model. SENP5 expression level was upregulated at both mRNA and protein levels in BEAS-2B cells at 24 h and 48 h after LPS stimulation (Fig. 1A,B), accompanied by elevated pro-inflammatory cytokine expression (Fig. 1C). In addition, we further established an LPS-stimulated lung injury mouse model. For this, C57BL/6 mice were intratracheally administered with PBS or LPS (1 mg/kg). Western blot analysis revealed that a marked upregulation of SENP5 in mouse lung tissues following LPS challenge (Fig. 1D). Compared with PBS-treated mice, LPS-treated lung injury mice exhibited significant lung damage, including pulmonary edema, inflammatory cell infiltration, and alveolar wall thickening in a time-dependent manner (Fig. 1E). Consistently, the immunohistochemistry staining results from pulmonary tissues further confirmed that SENP5 significantly increased at 24 h and 48 h after LPS treatment (Fig. 1F). These findings suggest that SENP5 was highly expressed during LPS-induced lung injury.

Fig. 1.

SENP5 was upregulated in LPS-stimulated BEAS-2B cells and inflammatory mouse lung tissues. (A) Relative mRNA expression levels of SENP5 in LPS-stimulated BEAS-2B cells at the indicated time points (n = 3). (B) Representative Western blot images of SENP5 protein levels in LPS-stimulated BEAS-2B cells at the indicated time points (n = 3). (C) Relative mRNA expression levels of pro-inflammatory cytokines in LPS-stimulated BEAS-2B cells at the indicated time points (n = 3). (D) Representative Western blot images of SENP5 protein levels in mice lung tissues undergoing LPS-induced ALI at the indicated time points (n = 3). (E) Representative H&E staining images of mouse lung tissues undergoing LPS-induced ALI at the indicated time points (n = 3). Upper scale bar, 100 µm; lower scale bar, 625 µm. (F) Representative images of immunohistochemical staining of SENP5 in mice lung tissues undergoing LPS-induced ALI at the indicated time points (n = 3). Upper scale bar, 100 µm; lower scale bar, 625 µm. All the data are expressed as the mean $\pm$ SD; *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001; determined by one-way ANOVA with Tukey’s post hoc test. SENP5, SUMO-specific peptidase 5; LPS, lipopolysaccharide; BEAS-2B, human normal lung epithelial cells; ALI, acute lung injury; H&E, hematoxylin-eosin; SD, standard deviation; ANOVA, analysis of variance.

To explore the underlying biological function of SENP5, we also analyzed LPS-induced inflammatory response, cell proliferation, and apoptosis in BEAS-2B cells transfected with SENP5 lentivirus shRNAs. Compared to the control (NC) group, the #1 and #2 SENP5 shRNA group exhibited a significant decrease in expression of up to 70% to 80% at mRNA and protein levels (Fig. 2A,B). Thus, the #1 and #2 SENP5 shRNAs were utilized for further experiments. To explore the effect of SENP5 knockdown on apoptosis and inflammatory cytokines, BEAS-2B cells were treated with LPS for 24 h and 48 h after transfecting SENP5 shRNAs. As illustrated in Fig. 2C, the knockdown of SENP5 distinctly caused a decrease in cell viability when cotreated with LPS. Besides, the inflammatory response was markedly exacerbated, with significantly elevated mRNA levels of IL-6, TNF-$\alpha$, and IL-1$\beta$ in SENP5-knockdown cells after LPS exposure (Fig. 2D,E). In parallel, flow cytometry analysis revealed that SENP5 knockdown increased the apoptotic rate following LPS stimulation (Fig. 2F).

Fig. 2.

SENP5 knockdown exacerbated LPS-induced inflammatory response and apoptosis in BEAS-2B epithelial cells. (A) Relative mRNA expression levels of SENP5 in BEAS-2B cells transfected with control or SENP5-targeting shRNAs (n = 3). (B) Representative Western blot images of SENP5 protein levels in BEAS-2B cells (n = 3). (C) Cell viability assessed by CCK-8 assay in LPS-stimulated BEAS-2B cells following SENP5 knockdown (n = 3). (D,E) Relative mRNA expression levels of pro-inflammatory cytokines in BEAS-2B cells at 24 h and 48 h (n = 3). (F) Flow cytometry analysis and quantification of apoptosis rate in BEAS-2B cells following SENP5 knockdown. All the data are expressed as the mean $\pm$ SD; *p 0.05, **p 0.01, ***p 0.001, ****p 0.0001; determined by one-way ANOVA with Tukey’s post hoc test.

To validate the biological significance of SENP5, we established conditional SENP5 cKO mice by crossing floxed SENP5 mice (SENP5^flox/flox) with Sftpc-cre mice and then validated the mice genotype (Supplementary Fig. 1). H&E staining results from mouse lung tissues indicated that SENP5 cKO mice exhibited disseminated thickening of alveolar septa, interstitial exudation, together with significant inflammatory cell infiltration after LPS administration (Fig. 3A). Immunohistochemistry staining results showed SENP5 deficiency further increased cell apoptosis and inhibited proliferation, as reflected by the expressions of caspase3 and ki67 (Fig. 3B,C). Survival analysis showed that SENP5 cKO mice had a limited probability of survival compared to SENP5^flox/flox controls after LPS challenge (Fig. 3D). Taken together, these results revealed that SENP5 deficiency facilitated apoptosis and inhibited proliferation in vivo.

Fig. 3.

SENP5 deficiency facilitated apoptosis and inhibited proliferation in lung tissues. (A) Representative images of H&E staining of lung tissues from SENP5^flox/flox and SENP5 cKO mice (n = 3). Left scale bar, 625 µm; Right scale bar, 100 µm. (B) Representative images of immunohistochemical staining and quantification of caspase-3 in SENP5^flox/flox and SENP5 cKO mice lung tissues. Left scale bar, 625 µm; Right scale bar, 100 µm. (C) Representative images of immunohistochemical staining and quantification of Ki-67 in SENP5^flox/flox and SENP5 cKO mice lung tissues. Left scale bar, 625 µm; Right scale bar, 100 µm. (D) The survival curve of SENP5^flox/flox and SENP5 cKO mice undergoing LPS challenge (n = 10). All the data are expressed as the mean $\pm$ SD; *p 0.05, **p 0.01; determined by Student’s t-test (B,C) or log-rank test (D). cKO, conditional knockout.

To gain better insight into the molecular mechanism of SENP5, RNA sequencing was performed in NC and SENP5-knockdown BEAS-2B cells. In total, 667 differentially expressed genes ($|$log₂ FC$|$ $>$2 and p value 0.05) were created as an overall Volcano plot. Differentially expressed genes with the most significant differences were then selected in SENP5 knockdown epithelial cells (Fig. 4A). We identified SLC7A5, a neutral amino acid transporter, as one of the most significantly downregulated genes. Thus, we proposed that SENP5 deficiency interfered with SLC7A5 expression in BEAS-2B cells. To investigate whether the SENP5-mediated apoptosis was linked to SLC7A5, we next examined the expression of SLC7A5 in LPS-stimulated BEAS-2B cells. LPS stimulation induced a time-dependent increase in SLC7A5 expression at both mRNA and protein levels (Fig. 4B,C). Consistent with the RNA sequencing results, the upregulation of SLC7A5 was abolished upon SENP5 knockdown (Fig. 4D,F).

Fig. 4.

SENP5 deficiency facilitated apoptosis through inhibiting SLC7A5/mTOR signaling pathway in BEAS-2B epithelial cells. (A) Volcano plot of differentially expressed genes between NC and SENP5 knockdown BEAS-2B cells. DEGs were identified based on $|$log₂ FC$|$ $>$2 and p value 0.05. (B) Relative mRNA expression levels of SLC7A5 in LPS-stimulated BEAS-2B cells at the indicated time points (n = 3). (C) Representative Western blot images of LAT1 protein levels in LPS-stimulated BEAS-2B cells at the indicated time points (n = 3). (D,F) Relative mRNA expression of SLC7A5 in SENP5 knockdown BEAS-2B cells treated with LPS for 24 h (D) or 48 h (F) (n = 3). (E,G) Representative Western blot images of LAT1, mTOR, p-mTOR, p70, and p-p70 protein levels in SENP5 knockdown BEAS-2B cells treated with LPS for 24 h (E) or 48 h (G) (n = 3). All the data are expressed as the mean $\pm$ SD; **p 0.01. ***p 0.001, ****p 0.0001; determined by one-way ANOVA with Tukey’s post hoc test. SLC7A5/mTOR, Solute carrier family 7 member 5/Mechanistic target of rapamycin; NC, Negative control; DEGs, Differential expressed genes; LAT1, L-type amino acid transporter 1.

SLC7A5 (LAT1), as an amino acid transporter, was reported to be linked with the activation of the mTOR pathway in cancers. Given the established role of SLC7A5 in amino acid transport and mTOR activation, we next investigated the downstream pathway. Western blot analysis showed that SENP5 deficiency could reduce LPS-induced phosphorylation of mTOR and its crucial downstream effector p70, indicating suppression of mTORC1 signaling (Fig. 4E,G). To summarize, SENP5 deficiency was suggested to induce apoptosis to aggravate LPS-induced ALI via inhibiting the SLC7A5/mTOR pathway in vitro.