Experimental mice and asthma models

Female C57 mice and HMGB1f/f, sftpc-Cre; HMGB1f/f mice were provided by the Animal Experimental Center of Yanbian University and Sai Ye Biotechnology Co., Ltd. (Suzhou, China). Mice weighed 20 ± 2 g and were housed SPF grade. Ethical guidelines were adhered to in the approval of all animal experiments. ([JI] 2020-00093).

Female C57 mice were divided into four groups (n = 12/group) (A) Control; (B) asthma; (C) asthma + agomir negative control (agomir NC); (D) asthma + miR-130b-3p agomir group. On Day 0, 10 μg of HDM (XPB46D3A4, Greer Laboratories, Lenoir, NC, USA) was added to 40 μl of saline [19]. Mice in groups C and D were sensitized by intranasal instillation (i.n.), followed by challenged i.n. the same dosage and volume of HDM on days 7–11. Control group mice were instilled with 40 μl of saline on days 0 and 7–11. Group C and D mice, from sensitization to treatment, were injected twice into the tail vein (i.v.) every seven days with Agomir NC or miR-130b-3p agomir (RiboBio synthesis, Guangdong, China) (20 nmol/mouse) after sensitization, and mice in groups A and B were given the same amount of saline. Conditional knockout mouse asthma models were established as described above. On day 14, the mice were humanely sacrificed by intraperitoneal injection of pentobarbital (200 mg/kg), and BALF was randomly taken from three mice. Mediastinal lymph nodes and lung tissues were collected for further detection.

Histological staining

Lung sections embedded in paraffin were subjected to staining with hematoxylin–eosin (HE), Masson, and periodic acid-Schiff (PAS) staining kits (G1120; G1340; G1280 Solibao, Beijing, China) to assess infiltration of inflammatory cells, deposition of collagen, and production of airway mucus. Immunohistochemical (IHC) staining for HMGB1/TLR4/DRP1 was conducted following the manufacturer's instructions (Beijing Zhongsui, PV-9000, China). Image acquisition was performed using a slide scanning image system (Shenzhen QiangSheng Technology).

Flow cytometry

In order to determine the percentage of eosinophils in BALF, the cells were centrifuged, suspended, and then incubated with leukocyte antibody APC-Cy7-CD45.2 (25–0454-U025, MBL, JAPAN) eosinophil antibodies PE-siglec-F (155506, Biolegend, USA), Percp-Cy5.5 CD11c (45–0114-82, Invitrogen, Carlsbad, CA, USA) at 4 °C for 30 min, and then measured after washing. For detecting the ratios of IL-4 to IFN-γ in CD4 positive cells of mediastinal lymph nodes of asthmatic mice, cells were treated with cell-surface antibody FITC-labeled CD4 (D341-4, MBL, JAPAN), followed by incubation with perforation and membrane rupture buffer (00–8333-56, 00–8222-49, eBioscience, USA) and APC-conjugated IFN-γ antibody (554413, BD, USA) and intracellular staining with PE-Cy7-conjugated IL-4 antibody (25–7042-42, eBioscience). The cell samples were then analyzed using Cytoflex (Beckman Coulter, Inc.) and the data analysis was conducted with CytoExpert2.4 software.

Extraction and detection of lung single-cell suspension standard operation

Under completely aseptic condition, the 30 mg of lung tissues were cut into small pieces measuring 1–2 mm3 with ophthalmic scissors and homogenized with a homogenizer for 1 min. The tissues pieces were added to fresh collagenase V digestive juice preheated to 37 °C and the sample was placed in a water bath and agitated gently at a temperature of 37 °C every 3 to 5 min. Almost completely digested lung tissues were observed at approximately 60 min, and then, the digestion was terminated by ice bath, followed by gently resuspending of the dispersed cells. After 200 mesh stainless steel mesh filtration was followed by centrifugation (300G, 5 min) and hemocytes were lysed for precipitation; 2 ml of 4 °C PBS containing 1% BSA was added to obtain mouse lung single cell suspension [20].

To label ATII cells, we incubated single cell suspensions with surface antibodies including APC-A750-CD45.2 (leukocytes, 25 −0454-U025, MBL), PC7-CD31 (endothelial cell, 561410, BD), Epcam (epithelial cell, 563477, BD, USA), PC5.5-MHC-II (ATII cell surface marker, 50–5321-U025, MBL), after processed with perforation and membrane rupture buffer, cells were incubated with FITC-proSP-C (surfactant protein C, ATII cell-specific surfactant protein, ab90713, abcam) [21]. CD45.2−CD31−EpCAM+ cells (referred to as EpCAM+ cells) were analyzed for pro-c expression. In EpCAM+ cells, pro-c+ cells also expressed MHCII+, the CD45.2− /CD31−/Epcam−+/proSP-C+/MHC-II+ cells were defined as ATII cells. Afterwards, ATII cells were stained for TLR4 (ab22048, abcam) and mucin domain-containing protein 3 (TIM-3) (20–5870-U025, TONBO). For intracellular labeling, cells were incubated with HMGB1 (ab79823, abcam), IL-1β (47–7114-82, eBioscience), IFN-γ (554413, BD Biosciences), IL-4 (554436, BD), DRP1 (8570S, CST) antibodies and stained with IgG (H + L) Fluor647 (1:200, S0013, affinity).

Cell culture and treatment

The MLE-12 mouse alveolar epithelial cell line was sourced from Shanghai Fuheng Biological Co., Ltd. Cells were cultured in DMEM/F-12 (VivaCell) culture medium containing 10% fetal bovine serum (Gibco BRL) and 1% penicillin/streptomycin (Gibco BRL) at 37 °C in a 5% CO2 constant temperature cell incubator to observe their growth status. The human bronchial epithelial cell line BEAS-2B was obtained from the American Type Culture Collection in Rockville, MD. The cells were grown in DMEM (VivaCell) supplemented with 10% fetal bovine serum and 1% penicillin /streptomycin at 37 °C in an atmosphere containing 5% CO2. In order to transfect miRNA-130b-3p mimics (synthesized by RiboBio, Guangdong, China) or NC (100 nM) into the MLE-12 cells, Lipofectamine 3000 (Invitrogen) was used for 24 h. After the transfection, the cells were treated with recombinant protein HMGB1 (10 μg/ml, Stemcell, USA) for 24 h. In the case of siRNA transfection, the cells received co-transfection of HMGB1 siRNA (1.5 μM) or NC siRNA (RiboBio) for 48 h.

Quantitative PCR

Total RNA and miRNAs were extracted from both MLE-12 cells and mouse lung tissues using specific extraction kits. Real-time PCR was conducted on an Azure cielo 6 system using either a three-step SYBR green RT-PCR kit or a miRcute RT-PCR kit, following instructions provided with the reagents. The data obtained from the PCR analysis were normalized to either Glyceraldehyde‑3‑phosphate dehydrogenase (GAPDH) or U6.

Dual-luciferase reporter analysis

By utilizing the miRwalk website (https://www.mirwalk.org/), we detected a possible interaction site between miR-130b-3p and HMGB1 3'UTR. To investigate this issue, we generated wild-type (WT) and mutant plasmids using RiboBio's pmiR-RB-ReportTM vector. Luciferase activity was measured using a GLOMAX96 spectrophotometer after transfection of HEK293 cells. Renilla (480 nm) and Firefly (560 nm) specificity values were determined for predictive analysis.

Immunofluorescence staining

MLE-12 cells treated with 0.1% Triton X-100 (Sigmal-Aldrich) were permeabilized for 15 min. Subsequently, the cells were blocked with 10% Bovine Serum Albumin (BSA) from Sigmal-Aldrich for one hour. The cells were then co-labeled with Rabbit-HMGB1 monoclonal antibody (ab79823, Abcam) and Mouse-SFTPC (H00006440-M01, Abnova, Taiwan, China), then MIE12 was incubated with anti-rabbit 488 (4412S, CST) and anti-mouse cy3 (ab97035, abcam) for 2 h. Lung paraffin sections were processed as described above. The mouse anti-HMGB1 (190377, Abcam), TLR4 (ab22048, Abcam), and DRP1 (611738, BD) were double-stained with rabbit anti-SFTPC (A1835, Abclonal), respectively. After overnight incubation at 4 °C, lung tissues were incubated with anti-rabbit 488 (IgG, 4412S, CST) and anti-mouse cy3 (ab97035, abcam) for 2 h. ROS were detected in lung sections after incubation with dihydroethidium (DHE) (Apexbio, USA). The photographs obtained using the Cytation™5 (BioTek Instruments) instrument provided clear images for analysis. The quantification of the images using Image J software (National Institutes of Health, Bethesda, MD).

Mitochondrial mass assessment

mtROS was measured using MitoSOX dye (M36008, Thermo Fisher) with a final concentration of 5 μM for 10 min at 37 °C. Assessment of mitochondrial membrane potential (MMP) was carried out using JC-1 dye (C2006, Beyotime), with changes in MMP indicated by alterations in the red/green fluorescence ratio. MitoTracker-Red dye (M7512, Thermo Fisher) was applied to the cells at a concentration of 100 μM for 30 min at 37 °C, followed by permeabilization and overnight incubation at 4 °C with anti- rabbit DRP1 (8570S, CST) and anti-rabbit MFN1 (14739S, CST) antibodies, respectively. Finally incubated for 2 h with donkey anti-sheep IgG-488 (abcam, ab150129). Mitochondrial morphology was photographed using the Cytation™5 imaging system.

TUNEL

Apoptosis was identified by the TUNEL kit (C1090, Beyotime). Permeabilized MLE-12 cells were fixed and incubated in a dark chamber at 37 °C for 60 min. In the case of lung paraffin sections, the paraffin was removed and incubated with proteinase K for 20 min and incubated under the same conditions.

Western blot

To extract proteins, RIPA lysate (Solaibao, R0020) was used for lysis of cells and lung tissues. The concentration of the total protein was then measured using a BCA kit (Solaibao, PC0020). Following this, 20 μg of proteins were separated through 10%−12% SDS-PAGE, then transferred onto PVDF membranes. After being blocked with TBST supplemented with 5% skim milk for 1 h, the bands overnight at 4 °C with primary antibodies and subsequently incubated with secondary antibodies for 2 h. The primary antibodies used in this process were HMGB1 (ab79823), Fis1 (ab229969), Bcl-2 (ab194583) which were acquired from Abcam; Cleaved-caspase-1(AF4005) was purchased from Affinity, Drp1 (8570), MFN1 (14739), Bax (41162), nod-like receptor family pyrin domain containing-3 (NLRP3) (13158), IL-1β (12242S), LaminB1 (13435), p-Drp1 (637) (4867), Cleaved-caspase-9 (20750), Cleaved-caspase-3 (9664), β-actin (3700), were purchased from CST. Secondary antibodies were IgG anti-rabbit and anti-mouse (97080; 6789, abcam). Protein expressions were analyzed with Quantity One software (BioRad, Hercules, CA).

Statistical analysis

SPSS 22.0 (IBM Co., Armonk, NY, USA) and GraphPad Prism 7.0 software were utilized for data analysis. The data were presented as mean values ± standard deviation (S.D). Group comparisons were carried out using analysis of variance and t-test, with statistical significance defined as a p-value less than 0.05.