Mechanism of miR-130b-3p in relieving airway inflammation in asthma through HMGB1-TLR4-DRP1 axis

doi:10.21203/rs.3.rs-4826024/v1

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Mechanism of miR-130b-3p in relieving airway inflammation in asthma through HMGB1-TLR4-DRP1 axis

https://doi.org/10.21203/rs.3.rs-4826024/v1

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Asthma is a chronic inflammatory respiratory disease characterized by recurrent breathing difficulties caused by airway obstruction and hypersensitivity. Although there is diversity in their specific mechanisms, microRNAs (miRNAs) have a significant impact on the development of asthma. Currently, the contribution of miR-130b-3p to asthma remains elusive. The goal of this study was to examine whether miR-130b-3p attenuates house dust mite (HDM)-induced asthma through High-mobility group box protein 1 (HMGB1)/Toll-like receptor 4 (TLR4)/mitochondrial fission protein (DRP1) signaling pathway. We elucidate that miR-130b-3p can bind to the HMGB1 3'UTR, attenuating HMGB1 mRNA and protein levels, and nucleo-cytoplasmic translocation of HMGB1. We observed that miR-130b-3p agomir or HMGB1 CKO attenuated HDM-induced airway inflammation and hyperresponsiveness, and decreased Th2-type cytokines in bronchoalveolar lavage fluid (BALF) and mediastinal lymph nodes. Further, HMGB1 CKO contributes to alleviating Th2 inflammation in AT-II cells (CD45.2⁻/CD31⁻/Epcam⁻/proSP-C⁺/MHC-II⁺) from lung single cell suspensions of asthmatic mice by flow cytometry. Our findings identified miR-130b-3p as a potent regulator in asthma that exerts its anti-inflammatory effects by targeting HMGB1 and the subsequent HMGB1/TLR4/Drp1 axis, presenting a prospective novel therapeutic avenue for asthma management.

miR-130b-3p

HMGB1

asthma.

Bronchial asthma is a well-known chronic disease closely linked to the immune system, resulting from an imbalance in immune cells. It is principally characterized by persistent airway inflammation, which culminates in airflow obstruction and compromised pulmonary function^[1]. Currently, asthma poses a great threat to public health, highlighting the necessity for identifying effective therapeutic targets for this condition^[2].

The pro-inflammatory factor HMGB1 is one of the common markers of damage in the nucleus of mammalian cells. Upon activation by specific stimuli, HMGB1 migrates from the nucleus to the cytoplasm and is subsequently released into the extracellular milieu where it acts as a potent pro-inflammatory mediator^[3]. This process plays a crucial role in the inflammatory response within the body. HMGB1 is observed at increased levels in the airways of asthmatics and acts as an extracellular signaling molecule that interacts with pattern-recognition receptors (e.g., TLR4), leading to an inflammatory cascade response that enhances airway smooth muscle contraction^[4]. Studies have shown that therapeutic interventions targeting HMGB1 using HMGB1 antibodies can suppress airway inflammation and potentially modulate lung fibroblast activation, thereby reversing airway remodeling^[5]. In addition, the promotion of the HMGB1/TLR4/Myeloid differentiation primary response 88 (MyD88)/Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling pathway could potentially explain how the reduction of Heat shock factor 1 (HSF1) exacerbates OVA-induced airway inflammation and hyperreactivity in mice^[6].

Highly conserved small-molecule non-coding RNA, also known as micro RNA (miRNAs), as important regulators of post-transcriptional expression of target genes, play a crucial role in the development of inflammatory and autoimmune diseases^[7]. Previous articles have confirmed that miR-192-5p attenuates airway remodeling and autophagy in asthma through the regulation of matrix metalloproteinase 16 (MMP-16) and autophagy gene 7 (ATG7)^[8]. In addition, miR-370, delivered by M2 macrophage -derived exosomes, has been shown to retard asthma progression by blocking the fibroblast growth factor 1 (FGF1)/p38 mitogen-activated protein kinase (MAPK)/signal transduction and transcription activator 1 (STAT1) pathway^[9]. Relevant to our preliminary Gene Chip analysis in lung tissues of asthmatic mice, among the miRNAs which have possible binding site to HMGB1 (predicted by miRwalk website), we focused on the miRNA with anti-inflammatory effect and down-regulated in asthmatic lungs. MiR-130b-3p exhibited down-regulation in lung tissues of ovalbumin (OVA)-induced asthma model mouse^[10]. MiR-130b-3p mimics markedly diminish acute lung injury and inflammatory factors in septic mice^[11]. Hence, miR-130b-3p may be an important regulator in the pathologic process of asthma, although, studies of specific molecular mechanisms have been lacking.

Mitochondrial homeostasis is instrumental in cellular signal transduction, with excessive production of ROS, dysregulation of Nox activity, and mitochondrial dysfunction leading to impaired antioxidant reactions and the occurrence of asthma^{[12, 13]}. Alveolar type II (ATII) cells have the unique ability to produce and release pulmonary surfactant. In addition, these cells function as progenitor cells in the context of lung diseases^[14]. Mitochondrial function is critical to the metabolic capacity of ATII cells, and mitochondrial impairment is strongly associated with the propagation of inflammation and pulmonary fibrosis^{[13, 15, 16]}. Notably, aberrant production of mitochondrial ROS (mtROS) in ATII cells is implicated in the pathophysiology of pulmonary fibrosis.^[17]. Among these, Drp1 and mitochondrial fusion protein Mitofusion 1 (Mfn1) proteins, which are associated with mitochondrial dynamics, have an essential impact in boosting mitochondrial function^[18]. Nevertheless, these mechanisms and functions underlying ATII cells in allergic airway inflammation remain elusive.

In this paper, miR-130b-3p agomir/mimic was applied as a therapeutic intervention, using WT mice and HMGB1 CKO (Sftpc-Cre; HMGB1^f/f) mice to elucidate their impact the asthma pathogenesis. These findings may offer a potential alternative strategy for managing asthma.

2.1 Experimental mice and asthma models

Female C57 mice and HMGB1^f/f, sftpc-Cre; HMGB1^f/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.

2.2 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).

2.3 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 ℃ 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.

2.4 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 1min. The tissues pieces were added to fresh collagenase V digestive juice preheated to 37 ℃ and the sample was placed in a water bath and agitated gently at a temperature of 37 ℃ 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).

2.5 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 hours. After the transfection, the cells were treated with recombinant protein HMGB1 (10 µg/ml, Stemcell, USA) for 24 hours. In the case of siRNA transfection, the cells received co-transfection of HMGB1 siRNA (1.5 µM ) or NC siRNA (RiboBio) for 48 hours.

2.6 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.

2.7 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.

2.8 Immunofluorescence staining

MLE-12 cells treated with 0.1% Triton X-100 (Sigmal-Aldrich) were permeabilized for 15 minutes. 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 ℃, 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).

2.9 Mitochondrial mass assessment

mtROS was measured using MitoSOX dye (M36008, Thermo Fisher) with a final concentration of 5 µM for 10 minutes at 37 ℃. 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 minutes at 37 ℃, followed by permeabilization and overnight incubation at 4 ℃ 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.

2.10 TUNEL

Apoptosis was identified by the TUNEL kit (C1090, Beyotime). Permeabilized MLE-12 cells were fixed and incubated in a dark chamber at 37℃ for 60 minutes. 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.

2.11 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 hour, the bands overnight at 4 ℃ 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).

2.12 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.

3.1 HMGB1 is a direct target of miR-30b-3p

The miRwalk database used predictive analysis to identify a potential binding site for miR-130b-3p in the 3'UTR of the HMGB1 mRNA (Fig. 1A). Subsequent confirmation was achieved through dual luciferase reporter assays, demonstrating a significant decrease in luciferase activity when miR-130b-3p mimics were co-transfected with the wild-type 3'UTR of HMGB1, as opposed to the mutated 3'UTR of HMGB1 (Fig. 1B). Following stimulation of MLE-12 with r-HMGB1, a notable reduction in miR-130b-3p expression was observed, accompanied by an up-regulation of HMGB1 mRNA levels compared to the control group (Fig. 1C). Concurrently, a decrease in nuclear HMGB1 and an increase in cytoplasmic HMGB1 were observed (Fig. 1D-E). Fluorescence assay revealed that cytoplasmic green fluorescence was enhanced in the r-HMGB1 group, indicating nucleo-cytoplasmic translocation, whereas miR-130b-3p mimics reversed this translocation (Fig. 1F). Additionly, in the human bronchial epithelial cell line BEAS-2B, miR-130b-3p also could inhibit the tanslocation of the HMGB1 (Figure S1). Collectively, these findings validate the targeted regulation of HMGB1 by miR-130b-3p and suggested that augmenting miR-130b-3p levels could potentially suppress HMGB1-mediated pro-inflammatory responses.

3.2 MiR-130b-3p agomir reduces airway inflammation in asthmatic mice

In order to explore the impact of miR-130b-3p on airway inflammation in asthmatic mice, we established an asthma model induced by HDM in WT mice (Fig. 2A). We observed the levels of HMGB1 protein were enhanced in both the HDM and miR-130b-3p NC groups compared with the control group, while was attenuated after miR-130b-3p agomir intervention (Fig. 2B-C). Detection of mRNA levels presented high expression of HMGB1, while the level of miR-130b-3p was decreased in the HDM group (Fig. 2D). Subsequently, HE staining showed that the airways were infiltrated by a large number of inflammatory cells, the pulmonary interstitium and airway smooth muscle were thickened, the alveoli were atrophied and collapsed, and a large area of alveoli fused and disorganized resulting in narrowing of the lumen. However, the miR-130b-3p agomir-treated group showed significantly improved airway inflammation and preserved alveolar structural integrity than HDM and NC groups. Furthermore, Masson staining showed increased collagen fiber deposition and destruction of the alveolar space structure in the bronchi, pulmonary interstitium, alveolar walls, and alveolar septa in the HDM group, while miR-130b-3p agomir significantly inhibited the pathological changes. PAS staining showed excessive mucus production in the airways of the HDM group, which was markedly decreased in the miR-130b-3p agomir group. Taken together, the aforementioned findings suggest that miR-130b-3p has a notable role in improving airway inflammation in mice with asthma.

3.3 The inflammation of the airways in HMGB1 CKO mice with asthma is blocked.

Firstly, HDM-induced asthma models were established in Sftpc-Cre; HMGB1^f/f mice (Fig. 3A). Analysis revealed that HMGB1 mRNA levels were significantly decreased and miR-130b-3p expression was increased in lung tissues of HMGB1 CKO mice when compared with HMGB1^f/f mice (Fig. 3B). Western blot results confirmed the reduced expression of HMGB1 protein, consistent with the genetic findings (Fig. 3C-3D). Histological analysis revealed that Sftpc-Cre; HMGB1^f/f mice had attenuated peribronchial inflammatory cell infiltration, significantly diminished alveolar structure destruction, and reduced collagen fiber deposition area and mucus production (Fig. 3E). In parallel, HDM -administrated Sftpc-Cre; HMGB1^f/f mice indicated a decreased percentage of eosinophils in BLAF, a significant downregulation of CD4⁺IL-4⁺ cells in lymph nodes, while the quantity of CD4⁺IFN-γ⁺ cells was increased (Fig. 3F). Collectively, HMGB1 CKO ameliorates airway inflammation and adjusts Th1/Th2 imbalance in asthmatic mice.

3.4 HMGB1 CKO improves Th1/Th2 inflammation in AT-II cells of asthmatic mice

AT-II cells were separated from single-cell suspensions of mouse lung tissues and labeled using flow cytometry by CD45.2⁻/CD31⁻/EpCAM⁺/proSP-C⁺/MHCII⁺ as described^[21]. Then, the levels of HMGB1, TLR4, IL-4, IFN-γ, and TIM-3 (T-cell immunoglobulin and mucin domain, HMGB1 as a ligand can bind to TIM3 on CD4 + T cells) were assessed. Results showed attenuation of HMGB1, TLR4, IL-4, and TIM3 in Sftpc-Cre; HMGB1^f/f mice, while IFN-γ displayed an opposite trend. Additionally, levels of DRP1 and IL-1β (Pro-IL-1β converts NLRP3 inflammasomes into mature and active forms of IL-1β) were examined in AT-II cells, with a noticeable decrease in IL-1β and DRP1 expression observed in the HMGB1 CKO group. As previously noted, the HMGB1 CKO contributes to the mitigation of Th1/Th2 inflammation in the AT-II cells of mice with asthma (Fig. 4A-4B).

3.5 HMGB1 CKO mitigates HMGB1, TLR4, and DRP1 expression in AT-II cells of asthmatic mice

The study assessed the co-localization of alveolar epithelial cell-specific markers (SFTPC) with HMGB1, TLR4, and DRP1 in lung tissues through immunofluorescence assay. As indicated in Fig. 5A-5C, the fluorescence intensity of HMGB1, TLR4, and DRP1 was markedly reduced in Sftpc-Cre; HMGB1^f/f mice following administration of HDM compared to the HDM-treated HMGB1^f/f group. Similarly, IHC staining showed that expression of HMGB1, TLR4, and DRP1 decreased in lung tissues of Sftpc-Cre; HMGB1^f/f mice (Fig. 5D). Thus, these findings underscore the inhibitory impact of HMGB1 CKO on the HMGB1, TLR4, and DRP1 expression in AT-II cells in asthmatic mice.

3.6 HMGB1 CKO relieves mitochondrial damage, apoptosis and NLRP3 inflammasome

The expression profiles of mitochondria dynamic-related proteins were measured. We observed a distinct decline in the expression of mitochondrial fission protein DRP1, p-DRP1(S616) and Fis1 after HMGB1 CKO, while in contrast, fusion protein MFN1 was elevated (Fig. 6A, 6B). Concurrently, the levels of apoptosis-related proteins were further assessed. These results supported that pro-apoptotic executioner protein BAX and Cleaved-caspase-3/9 expression was down-regulated, whereas anti-apoptotic protein (Bcl-2) expression was up-regulated in HDM-Sftpc-Cre;HMGB1^f/f mice compared to HDM-HMGB1^f/f mice (Fig. 6C, 6D). Furthermore, NLRP3-inflammasome, Cleaved- caspase-1 and IL-1β protein expression were detected (Fig. 6E, 6F). TUNEL assay revealed a reduction of apoptosis within alveolar cells of HDM administrated Sftpc-Cre; HMGB1^f/f mice, as indicated by diminished red fluorescence (Fig. 6G). In addition, a significant decrease in ROS levels was observed using DHE staining in the HMGB1 CKO mice (Fig. 6H). These findings suggest that HMGB1 CKO in AT-II epithelial cells effectively mitigates mitochondrial damage, apoptosis, and NLRP3 inflammasome events in asthmatic mice.

3.7 Interfering HMGB1 inhibits r-HMGB1-stimulated mitochondrial damage

Initial fluorescence assays were conducted to assess mtROS generation in MLE-12 cells using MitoSOX Dye. r-HMGB1 treatment augmented mtROS production, an effect that was mitigated following si-HMGB1 and miR-130b-3p mimic interference (Fig. 7A,7E). Correspondingly, analysis using JC-1 staining to observe MMP further confirmed the reduction in the ratio of red to green fluorescence in the r-HMGB1 group and its rescue after si-HMGB1 and miR-130b-3p inhibition (Fig. 7B,7F). Moreover, the co-localization of DRP1 and MFN1 with Mitochondrial Red staining indicated enhanced recruitment of DRP1 to the outer membrane of mitochondrial and a decrease in MFN1 in the r-HMGB1 group. However, si-HMGB1 or transfection with miR mimics attenuated this phenomenon (Fig. 7C-7D). Similary, reduced DRP1 recruitment observed also in the BEAS-2B cells (Figure S2). These alterations were reversed upon administration of si-HMGB1 and miR-130b-3p, as demonstrated by Western blot (Fig. 7G-7H). In summary, these experiments validated the protective role of HMGB1 interference against r-HMGB1-mediated mitochondrial dysfunction.

3.8 Interfering HMGB1 inhibit r-HMGB1-stimulated apoptosis and NLRP3 inflammasome

The detection of apoptosis-related proteins, such as Bcl2, BAX, Cleaved-caspase-3, Cleaved-caspase-9, and NLRP3 inflammasome, in MLE-12 cells following interference was conducted through Western blot analysis. In the group challenged with r-HMGB1, there was increased expression of apoptotic-related proteins (expect Bcl2) and NLRP3 inflammasome, which was reversed upon treatment with si-HMGB1 or miR-130b-3p mimic (Fig. 8A-8D). These findings were further supported by TUNEL staining (Fig. 8E-8F), confirming that HMGB1 interference inhibited r-HMGB1-induced apoptosis and NLRP3 inflammasome activation.

New evidence in the literature confirms that miRNA has been implicated in key roles in asthma-associated signaling pathways^[22]. This study identified the regulatory effect of miR-130b-3p on airway inflammation by specifically targeting HMGB1. Additionally, we found that both in vivo HMGB1 CKO and in vitro, si-HMGB1 administration curtail the release of Th2 cytokines, attenuate asthma-associated inflammation, and mitigate mitochondrial damage. The symptomatic relief is manifested by subdued eosinophil count in BALF, reduced IL-4 levels in lymph nodes in Sftpc-Cre; HMGB1^f/f asthmatic mice, and diminished HMGB1/TLR4/DRP1 expressions in alveoli by immunohistochemistry and immunofluorescence analyses. Upon this, our study focuses on the effects of miR-130b-3p and HMGB1 CKO on asthma disease.

MiR-130b-3p is closely associated with inflammatory diseases. It has been documented that miR-130b-3p attenuates infectious cardiomyopathy by modulating the AMPK/mTOR signaling pathway and directly targeting ACSL4 against iron ptosis^[23]. Synovial mesenchymal stem cell-derived extracellular vesicles attenuate chondrocyte injury during osteoarthritis by miR-130b-3p-mediated inhibition of the LRP12/AKT/β-catenin axis^[24]. Besides, Several investigations have supported the regulatory role of miR-130b-3p in lung inflammatory diseases^{[11, 25, 26]}. Notably, miR-130b-3p is documented to inhibits M1 polarization of lung macrophages by attenuating Irf1 release, thereby reducing inflammation in lung tissue of LPS-treated mice^[25]. Meanwhile, delivery vectors such as mesenchymal stem cell-derived exosomes (MSCs-Exo) facilitate the delivery of miR-130b-3p to the site of acute lung injury induced by LPS damage and play a protective role^[26]. However, the nexus between miR-130b-3p and mitochondrial dynamics remains sparsely studied. Here, we demonstrate that miR-130b-3p inhibits HMGB1-TLR4-DRP1 signaling axis-mediated airway inflammation, and decreases alveolar epithelial cell apoptosis and related inflammatory factor production, while concurrently safeguarding against mitochondrial insult.

The therapeutic potential of HMGB1 antagonists has been extensively validated across models of inflammatory disease^[27]. There is increasing evidence that HMGB1 regulates inflammation and immune responses through different receptors^[28]. Relevant studies have reported that Clara cell 16KDa protein (CC16) attenuates HDM-mediated airway inflammation and injury by inhibiting airway epithelial cell apoptosis in an HMGB1-dependent manner^[29]. In addition, fibroblast growth factor 10 has demonstrated anti-inflammatory and cytoprotective feats through curtailment of the HMGB1/TLR4 axis, leading to attenuate lung injury^[30]. Some data suggest that microsatellite DNA-derived oligodeoxynucleotide (MS19) may have a therapeutic effects on ALI by inhibiting the HMGB1-TLR4-NF-κB pathway^[31]. Activation of the NLPR3 inflammasome in human bronchial epithelial cells is associated with HMGB1^[32]. N-exo-miRNA182-5p alleviates sepsis-induced acute lung injury by targeted NOX4/Drp-1/NLRP3 signaling pathway^[33]. Together, these findings emphasize that HMGB1/TLR4/Drp1 /NLRP3 is an important node in asthma regulation, underpinning we used Sftpc-Cre; HMGB1^f/f mice to characterize the role of HMGB1 CKO in mitigating hyperresponsive airway inflammation. Our explorations were extended to validate changes in the TLR4/Drp-1/NLRP3 signaling axis after conditional knock-out of HMGB1 and si-HMGB1 and concluded that inhibition of HMGB1 significantly attenuates TLR4/Drp1/NLRP3 signaling pathway, further alleviate HDM-induced inflammatory lesions in asthma.

The development of asthma might be influenced by the mitochondria^[34]. For instance, miR-30a negates Drp1-induced mitochondrial fission and subsequent AECs-II apoptosis^[35]. Conversely, miR-181b-5p effectively downregulates DRP1 expression levels^[36]. Studies have shown that miR-20b can directly regulate MFN1 and MFN2 to inhibit hyperoxia-induced apoptosis and acute lung injury^[37]. Furthermore, it was found by querying the literature that the up-regulation of miR-146a-5p could improve pulmonary inflammation, damage to the cellular barrier, and cell death caused by PAF in human small airway epithelial cells^[38]. Disruption of mitochondrial function is a known prelude to programmed cell death^[39]. Here, Our inquiry hereby confirms that miR-130b-3p could attenuate mitochondrial insult and apoptosis by targeting HMGB1 to down-regulate Drp1 and augment MFN1 expression. The literature denotes that HMGB1 intensifies asthma-associated airway inflammation via escalated ER-mitochondrial Ca²⁺ trafficking and ROS production^[40]. HMGB1 is also involved in ERK/Drp1-dependent mitochondrial fission in pulmonary hypertension^[41]. We posit that HMGB1 CKO and si-HMGB1 can attenuate the disruption of mitochondrial function, reinstating cellular energy homeostasis.

In summary, we suggested that miR-130b-3p ameliorates airway inflammation, mitochondrial damage and apoptosis in asthmatic mice by targeting the HMGB1/TLR4/DRP1 axis, and may provide new methods for treating asthma.

HDM: House dust mite; HMGB1: High-mobility group box protein 1; TLR4: Toll-like receptor 4; DRP1:Mitochondrial fission protein; CKO: Conditional knockout; Sftpc-cre; HMGB1^f/f : HMGB1 gene specifically in alveolar type II epithelial cells; MLE-12 cells: Mouse lung epithelial cells; r-HMGB1: Recombinant HMGB1; BALF: Bronchoalveolar lavage fuid; ROS: Reactive oxygen species; miRNAs: Highly conserved small non-coding RNAs; ATII: Type II alveolar epithelial cells; mtROS: Mitochondrial ROS; Mfn1: Mitochondrial fusion protein Mitofusion1; HE: Hematoxylin-eosin; PAS: Period-Schiff; IHC: Immunohistochemistry; IFC: Immunofluorescence; GAPDH: Glyceraldehyde‑3‑phosphate dehydrogenase; BSA: Bovine Serum Albumin; DHE: Dihydroethidium; FIS1: Mitochondrial fission 1 protein; BCL2: Antiapoptotic protein; p-DRP1(S616): phosphorylated at Serine 616; NLRP3: Nod-like receptor family pyrin domain containing-3; IL-1β: Interleukin-1β; BAX/Cleaved-caspase-9/Cleaved-caspase-3: Apoptotic protein; IL-4 : Type 2 cytokine; IFN-γ :Type 1 cytokine; TIM-3: T-cell immunoglobulin and mucin domain.

Acknowledgments

Not applicable.

Author Contributions

Xue Han: Conceptualization, Data curation, Investigation, Software, Validation, Writing – original draft. Yilan Song: Conceptualization, Methodology, Software, Writing – review & editing.

Yihua Piao: Data curation, Formal Analysis, Writing – review & editing. Zhiguang Wang: Investigation, Methodology, Writing – review & editing. Yan Li: Investigation, Methodology, Writing – review & editing. Qingsong Cui: Investigation, Methodology, Writing – review & editing. Guanghai Yan : Conceptualization, Funding acquisition, Software, Writing – review & editing. Hongmei Piao: Conceptualization, Funding acquisition, Project administration, Software, Writing – review & editing. All authors read and approved the final manuscript.

Funding

This work was supported by a grant from National Natural Science Foundation of China (No. 82060004, 82160004). Higher Education Discipline Innovation Project (111 Project, No. D18012). Jilin Provincial Department of Education Project (JJKH20240690KJ). Jilin Provincial Science and Technology Department Project (20240404025ZP). Jilin Provincial Science and Technology Department Project (YDZJ202401083ZYTS). The funder has no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.

Data availability

The datasets analyzed during the current study are available from the corresponding author on reasonable request.

Ethics approval

This study did not involve human subjects. The animal experimental were approved by the Animal Experimental Center of Yanbian University ([JI] 2020-00093).

Competing interests

The authors declare that they have no competing interests.

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FigureS120240703151250.pdf
MiR-130b-3p agomir reduces airway inflammation in mice with asthma. (A) Schematic representation of the HDM-mediated asthma model in WT mice. (B-C) HMGB1 levels were measured by Western blot and their relative density. (D) The mRNA levels of miR-130b-3p and HMGB1 in lung tissues. (E) Histological analysis of lung sections. Scale bar: 100 µm. p < 0.05,p < 0.01, versus control, p < 0.05 versus miR-130b-3p agomir group.
FigureS220240703151250.pdf
HMGB1 CKO improves Th1/Th2 inflammation in AT-II cells of asthmatic mice. Isolated AT-II cells from lung single cell suspensions were labeled, and then using flow cytometry analyzed them. (A-B) Schematic diagram of the gating strategy of AT-II cells (CD45.2/CD31/EpCAM/proSP-C/MHCII), and further analysis of HMGB1, TLR4, DRP1, IL-1β, IL-4, IFN-γ, and TIM3, and cell proportion. p < 0.05,p < 0.01, p < 0.001.

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Mechanism of miR-130b-3p in relieving airway inflammation in asthma through HMGB1-TLR4-DRP1 axis

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Abstract

Figures

1. Introduction

2. Materials and Methods

2.1 Experimental mice and asthma models

2.2 Histological staining

2.3 Flow cytometry

2.4 Extraction and detection of lung single-cell suspension Standard operation

2.5 Cell culture and treatment

2.6 Quantitative PCR

2.7 Dual-luciferase reporter analysis

2.8 Immunofluorescence staining

2.9 Mitochondrial mass assessment

2.10 TUNEL

2.11 Western blot

2.12 Statistical analysis

3. Result

3.1 HMGB1 is a direct target of miR-30b-3p

3.2 MiR-130b-3p agomir reduces airway inflammation in asthmatic mice

3.4 HMGB1 CKO improves Th1/Th2 inflammation in AT-II cells of asthmatic mice

3.5 HMGB1 CKO mitigates HMGB1, TLR4, and DRP1 expression in AT-II cells of asthmatic mice

3.6 HMGB1 CKO relieves mitochondrial damage, apoptosis and NLRP3 inflammasome

3.7 Interfering HMGB1 inhibits r-HMGB1-stimulated mitochondrial damage

3.8 Interfering HMGB1 inhibit r-HMGB1-stimulated apoptosis and NLRP3 inflammasome

Discussion

Conclusion

Abbreviations

Declarations

References

Supplementary Files

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