Animal model of chronic Asthma

All animal experiments were performed according to the Guide for the Care and Use of Laboratory Animals, and approved by the ethical committee of Shengjing Hospital of China Medical University. Female C57BL/6 mice at the age of 6 weeks were purchased from Changsheng (China). Chronic asthmatic model was induced as the previous described [36]. Briefly, the mice were sensitized with 20 µg ovalbumin (OVA; Aladdin, China) and 2 mg Al(OH)3 by two intraperitoneal injections on days 0 and 14. From days 21, mice were challenged with 1.5% aerosolized OVA for 45 min. The OVA aerosol challenge was conducted 3 times/week (every other day, a total of 8 weeks). Twenty-four hours following the last challenge, mice were sacrificed. The controls (Sham) were sensitized and challenged with saline. Adenovirus expressing SYVN1 (AdSYVN1) or negative control (AdNC) (2.5 × 106 PFU) was prepared to intratracheal injection for 3 days before the first aerosol. Four weeks later, the adenovirus delivery was repeated. The schematic timeline for animal protocols is shown in Fig. 2A.

Construction of recombinant adenovirus

To construct the recombinant adenovirus expressing SYVN1, the SYVN1 gene sequences were synthesized by General Bio (China) and inserted into the NotI and HindIII sites of pShuttle-CMV vectors (Fenghui, China). The plasmids were transfected into HEK-293 A cells (iCell, China) using Lipofectamine 3000 (Invitrogen, USA) to produce the recombinant adenovirus.

Lung histopathology

Upon sacrifice, the lung tissues were fixed in 4% paraformaldehyde and embedded in paraffin. Then, the paraffin-embedded lung Sect. (5-µm thick) were subjected to Hematoxylin and eosin (H&E), periodic acid-Schiff (PAS) and Masson staining as standard protocols to determine inflammation, mucus metaplasia and collagen deposition. The morphological photographs were observed under the BX53 microscope (Olympus, Japan) equipped with a DP73 Olympus camera (Olympus, Japan) via 10×, 20×, or 40× objective lenses. Images were obtained using CellSens software (Olympus, Japan) and analyzed using Image-Pro Plus 6.0 software (Media Cybernetics, USA).

The inflammatory score was assessed using the method reported by Cho et al. [37]. In brief, inflammation was scored as following: score 0 (no inflammatory cells were detectable), score 1 (occasional observation of inflammatory cells), score 2 (bronchi or vessels were surrounded by 1–3 layers of inflammatory cells), score 3 (bronchi or vessels were surrounded by 4–5 layers of inflammatory cells), and score 4 (bronchi or vessels were surrounded by more than 5 layers of inflammatory cells).

The goblet cell hyperplasia was scored according to the previous study based on the ratio of goblet cells in the epithelium [38]: score 0 (no goblet cells), score 1 (< 25% goblet cells), score 2 (25–50% goblet cells), score 3 (51–75% goblet cells), and score 4 (> 75% goblet cells).

To assess collagen deposition, the staining area (blue) and total area were quantified and the percentage of collagen fibers were calculated as the collagen area (blue)/total area as previously reported [39].

Immunohistochemical staining

For immunohistochemical staining, the paraffin-embedded sections of lung tissues were incubated with the α-smooth muscle actin (α-SMA) antibody (Affinity, China) or SYVN1 antibody (Proteintech, China) overnight at 4 °C. Then, the HRP-conjugated goat anti-rabbit antibody (ThermoFisher, USA) was used to incubate for 60 min at 37 °C. The results were visualized by the BX53 microscope (Olympus, Japan) equipped with a DP73 Olympus camera (Olympus, Japan) via 20× objective lenses and obtained using CellSens software (Olympus, Japan).

Cell culture and treatment

BEAS-2B cells (iCell, China) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal bovine serum (FBS) in an incubator with 5% CO2 condition at 37 °C. To induce epithelial-mesenchymal transition, the cells were treated with TGF-β1 (SinoBiological, China) at the dose of 2.5, 5 or 10 ng/ml for 24 h.

To overexpress SYVN1 or SIRT2 expression, the plasmids for targeting SYVN1 (SYVN-oe), SIRT2 (SIRT2-oe) or negative control (Vector) were transfected into BEAS-2B cells for 48 h using Lipofectamine 3000 (Invitrogen, USA), according to the manufacturer’s protocol. In addition, the ER stress inhibitor 4-PBA (5 mM; MACKLIN, China) was administrated to the cells for 2 h prior to TGF-β1 treatment.

Co-immunoprecipitation (Co-IP)

Co-IP assay was carried out using a Pierce Co-IP kit (ThermoFisher, USA) according to the manufacture’s protocol. Cells were pretreated with 10 µM MG132 (Aladdin, China) for 6 h, and then were collected and lysed with an immunoprecipitation buffer (Beyotime, China) to extract proteins. The extracts were incubated with the AminoLink Plus Coupling Resin along with the immobilized SIRT2 antibody (Santa Cruz, USA). After incubation, the immune complex was washed and determined by Western blot.

Immunofluorescence staining

The paraffin-embedded lung sections and fixed cells were conducted to immunofluorescence staining. In brief, the tissue slides or cells were incubated with primary antibodies against E-cadherin (Affinity, China), Vimentin (Affinity, China), GRP78 (Proteintech, China), CHOP (Affinity, China) overnight at 4 °C, followed by the incubation of Cy3-conjugated anti-rabbit (Invitrogen, USA) or Cy3-conjugated anti-mouse (Invitrogen, USA) for 60 min at room temperature. Photographs were captured under the BX53 microscope (Olympus, Japan) equipped with a DP73 Olympus camera (Olympus, Japan) via 40× objective lenses (blue filter for DAPI, red filter for GRP78, CHOP, E-cadherin and Vimentin) using CellSens software (Olympus, Japan). In addition, primary antibodies against SYVN1 (Proteintech, China) and SIRT2 (Santa Cruz, USA) were used to incubate with cells overnight at 4 °C to identify the colocalization of SYVN1 and SIRT2 in cells. Then, the FITC-conjugated anti-rabbit (Abcam, UK) and Cy3-conjugated anti-mouse (Invitrogen, USA) secondary antibodies were added and incubated for 60 min at room temperature. Images were taken with a laser scanning confocal microscope (C2, Nikon, Japan) via 60× objective lenses (blue filter for DAPI, red filter for SIRT2, green filter for SYVN1).

Real-time PCR

Total RNA was extracted using the TRIpure reagent (Bioteke, China) and was reverse transcribed to cDNA using a BeyoRT II M-MLV Reverse Transcriptase (Beyotime, China). Real-time PCR was carried out using the specific primers (Table  1) and SYBR Green probes (Solarbio, China) with the Exicycler96 Real-time PCR system (Bioneer, Korea). The GAPDH expression was used as an internal control. The 2−ΔΔCt method was used to obtain mRNA levels.

Western blot

The lung tissues and cells were lysed with a RIPA buffer (Solarbio, China) supplemented a protease inhibitor. After quantification, protein extracts were subjected to SDS-PAGE and transferred to PVDF membranes. The membranes were blocked with 5% non-fat milk for 1 h and incubated with primary antibodies overnight at 4 °C, including SYVN1 antibody (Proteintech, China), SIRT2 antibody (Affinity, China), α-SMA antibody (Affinity, China), TGF-β antibody (Affinity, China), collagen I antibody ((Affinity, China), E-cadherin antibody (Affinity, China), Vimentin antibody (Affinity, China), GRP94 antibody (Affinity, China), GRP78 antibody (Proteintech, China), CHOP antibody (Affinity, China), p-PERK antibody (Affinity, China), PERK (Affinity, China), p-IRE antibody (Affinity, China), IRE antibody (Affinity, China), ATF6 antibody (Proteintech, China), Ubiquitin (linkage specific K48) antibody (Abcam, UK), Histone antibody (Proteintech, China) and GAPDH antibody (Proteintech, China). Subsequently, the HRP-conjugated anti-rabbit (Solarbio, China) and HRP-conjugated anti-mouse (Solarbio, China) secondary antibodies were used for incubation for 1 h at 37 °C. The protein bands were visualized using ECL chemiluminescence solution (Solarbio, China). The gray values were analyzed using Gel-Pro-Analyzer software (Media Cybernetics, USA). The target bands were standardized to the internal control and then normalized against the mean of protein level in the control group.

Statistical analysis

Data were shown as mean ± SD, and data difference analysis was performed using GraphPad Prism 8.0. Comparisons between two groups were analyzed by t test. One-way ANOVA following Bonferroni’s test was conducted for multiple comparisons. The statistical significance was identified when p < 0.05.