All animal operations were followed by the Guide for the Care and Use of Laboratory Animals and approved by Tianjin Third Central Hospital.

Mice model

A total of 80 male C57BL/6 mice (8-week-old, Vital River, China) were used in this study. All mice were received adaptive feeding for 1 week. All the mice were divided into eight groups, including sham, LPS, LPS+adeno-associated virus (AAV)-sh-NC, LPS+AAV-shTRIM27#1, LPS+AAV-shTRIM27#2, LPS+AAV-sh-NC+AAV-empty, LPS+AAV-sh-TRIM27#1+AAV-empty, and LPS+AAV-sh-TRIM27#1+AAV-NOX4 (n = 10 per group) at random. The mice were received intratracheal injection with 5 mg/kg LPS. Correspondingly, sham mice were received intratracheal injection with equal dose of phosphate buffer saline (PBS, Sigma). After injection for 24 h, the mice were sacrificed, and lungs were removed for further study. Three weeks before LPS injection, mice were received intratracheal administration with 6 × 1010 vector genomes (vg) of AAV-sh-TRIM27#1 (dissolved in 50 μl of PBS), AAV-sh-TRIM27#2, or AVV-NOX4 after anesthesia. Meanwhile, AAV-sh-NC or AVV-empty was served as negative control, respectively. The AAV vectors were purchased from Hanbio Company (China).

Lung wet/dry (W/D) ratio

The wet weight of lung tissues was measured, the dry weight of lung tissues was measured after desiccated at 80 °C for 48 h, and the wet/dry (W/D) weight ratio was calculated.

Histological examination and lung injury score

The lung tissues were washed with PBS, fixed, paraffin-embedded, and cut into 4-μm sections. Thereafter, the sections were deparaffinized with xylene, rehydrated in ethanol, boiled in retrieval buffer for 5 min, and stained with hematoxylin–eosin (H&E). To assess the lung injury of mice, the system comprises contained four categories (alveolar edema, alveolar hemorrhage, interstitial thickening, and neutrophil infiltration), and each category was graded from 0 (normal) to 4 (severe): 0—no injury, 1—injury to 25% of the field, 2—injury to 25–50% of the field, 3—injury to 50–75% of the field, and 4—diffuse injury.

Biological detection

Inflammatory cytokines including IL-1β, TNF-α, IL-6, and MPO, were measured by the ELISA using commercial ELISA kits (R&D Systems), respectively.

TUNEL staining assay

The cell apoptosis assay was performed by the TUNEL Kit (Roche, USA). The paraffin sections were de-waxed, dehydration, and incubated with protease K for 15 min at 37 ℃. Thereafter, the sections were blocked with TUNEL reaction solution in the dark for 15 min at 37 ℃, followed by POD incubation for 30 min at 37 ℃. Then, the sections were added with DAB for 10 min and then re-stained with hematoxylin. By captured under a fluorescence microscope (Leica, Germany), the cell apoptosis rate was calculated.

Immunofluorescence

The sections were stained with PPARγ antibody (1/400; Abcam, UK), p-p65 antibody (1/400; Abcam, UK), NOX4 antibody (1/400; Abcam, UK), DHE antibody (1/400; Abcam, UK), or TRIM27 antibody (1/400; Abcam, UK) at 4 °C, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 555) (1/1000; Abcam, UK) or Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (1/1000; Abcam, UK) for 2 h at room temperature. Subsequently, the sections were stained with a DAPI solution for 8 min at room temperature, and the images were acquired under a fluorescent microscope (Leica, Germany).

Measurement of superoxide dismutase (SOD) and malondialdehyde (MDA) and dihydroethidium (DHE)

The SOD and MDA contents in lung tissues were measured by tauperoxide dismutase (T-SOD) assay kit (Nanjing Jiancheng, China) and cell malondialdehyde assay kit (Nanjing Jiancheng, China), according to the manufacturer’s instructions, respectively.

For DHE intensity measurement, the lung sections were incubated with 2 μmol/L DHE (Sigma-Aldrich, USA) in the dark for 30 min. After washing three times with PBS, the fluorescent images were captured under a fluorescent microscope (Leica, Germany) and the relative DHE intensity was calculated.

Ubiquitination analysis

The TC-1 cells (mouse alveolar epithelial cells) were purchased from the Science Cell Laboratory and cultured in DMEM (Thermo Fisher Scientific, USA) supplemented with 10% FBS (Gibco, USA) at 37 ℃ in 5% CO2. The TC-1 cells transfected with sh-TRIM27#1 or not were pre-treated by 100 ng/mL LPS for 6 h, lysed with RIPA buffer and then reacted with PPARγ antibody. Subsequently, the immunoprecipitated complexes were subjected western blot analysis using Ubiquitin (Ub) antibody (Abcam; ab7780).

Quantitative real time-PCR (qRT-PCR)

The total RNA was isolated from lung tissues with TRIzol reagent (Invitrogen, USA) and then were converted to cDNA with SuperScript IV (Invitrogen, USA) and amplified using SYBR Green Master Mix (Takara, Japan). The relative mRNA levels were normalized to GAPDH and calculated using 2−△△Ct formula. The primers were as follows: GAPDH-forward: 5′-TCATTGACCTCAACTACAGGT-3′ and GAPDH-reverse: 5′-CTAAGCAGTTGGTGGTGCAG-3′; IL-6-forward: 5′-CCGGAGAGGAGACTTCACAG-3′ and IL-6-reverse: 5′-TGGTCTTGGTCCTTAGCCAC-3′; Il-1β-forward: 5′-GGAGAAGCTGTGGCAGCTA-3′ and Il-1β-reverse: 5′-GCTGATGTACCAGTTGGGGA-3′; TNF-α-forward: 5′-GACCCTCACACTCAGATCAT-3′ and TNF-α-reverse: 5′-TTGAAGAGAACCTGGGAGTA-3′.

Western blot

Proteins were extracted from tissues with RIPA lysis and separated by SDS-PAGE as previously described [16]. The primary antibodies were listed as below: TRIM27 (1/800; Abcam, UK), GAPDH (1/800; Abcam, UK), Bax (1/800; Abcam, UK), Bcl-2 (1/800; Abcam, UK), cleaved-caspase3 (1/800; Abcam, UK), caspase3 (1/800; Abcam, UK), cleaved-caspase9 (1/800; Abcam, UK), caspase9 (1/800; Abcam, UK), PPARγ (1/800; Abcam, UK), p-p65 (1/800; Abcam, UK), p65 (1/800; Abcam, UK), and NOX4 (1/800; Abcam, UK).

Statistical analysis

The data in this study were shown as mean ± SD, repeated three times and analyzed by GraphPad Prism7.0 (USA). For survival analysis of mice, the Kaplan–Meier method with log-rank tests was performed to plot survival curves. The comparison among three groups was performed by ANOVA with Tukey’s post hoc tests. The p < 0.05 was defined as statistically significant.