[miR-16-5p alleviates airway inflammation in asthmatic rats by regulating TXNIP and suppressing the NLRP3 signaling axis]

Asthma is a chronic inflammatory airway disease. Thioredoxin-interacting protein (TXNIP), a thioredoxin (TRX)-binding protein, is involved in the regulation of inflammatory responses and apoptosis. The NOD-like receptor thermal protein domain associated protein 3 (NLRP3) inflammasome is a key mediator of inflammatory responses. MicroRNAs (miRNAs) are small non-coding RNAs. Previous studies have demonstrated that miR-16-5p expression is significantly downregulated in bronchial epithelial cells of asthmatic rats. However, whether miR-16-5p regulates the NLRP3 signaling pathway by targeting TXNIP and thereby influences airway inflammation in asthma remains unclear. This study aims to investigate the mechanism by which miR-16-5p alleviates ovalbumin (OVA)-induced airway inflammation in asthmatic rats through regulation of TXNIP and suppression of the NLRP3 signaling axis.

After one week of acclimatization, male Sprague-Dawley (SD) rats were randomly assigned to a control group, model group, miR-16-5p mimic group, NC mimic group, miR-16-5p inhibitor group, and NC inhibitor group (n=6 per group). Rats were sensitized on days 1, 8, and 15 with a mixture of 10% OVA and 10% aluminum hydroxide. From day 21, asthma was induced by inhalation of 4% OVA aerosol for one consecutive week. Beginning on day 21, rats received intratracheal administration of 20 μg/0.1 mL miR-16-5p mimic, NC mimic, miR-16-5p inhibitor, or NC inhibitor, respectively, 1 hour before aerosol challenge once daily for 7 days. At the end of the experiment, bronchoalveolar lavage fluid (BALF) and lung tissues were collected. miR-16-5p expression was determined by real-time reverse transcription polymerase chain reaction (real-time RT-PCR). Dual-luciferase reporter assays were performed to verify the targeting relationship between miR-16-5p and TXNIP. Inflammatory cells in BALF were counted microscopically. Levels of interleukin (IL)-1β, IL-6, tumor necrosis factor-alpha (TNF-α), and monocyte chemoattractant protein-1 (MCP-1) were measured by enzyme-linked immunosorbent assay (ELISA). Histopathological changes in lung tissues were evaluated by hematoxylin and eosin (HE) staining. Goblet cells were assessed using periodic acid-Schiff (PAS) staining, and bronchial collagen deposition was examined by Masson's trichrome staining. Immunohistochemistry was used to detect TXNIP and NLRP3 protein expression. Protein expression levels of TXNIP, NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC), cleaved-cysteine-dependent aspartate-directed protease-1 (cleaved-caspase-1), and IL-1β were determined by Western blotting.

miR-16-5p expression was significantly downregulated in asthmatic rats. Dual-luciferase reporter assays confirmed that TXNIP was a direct target of miR-16-5p. Compared with the control group, rats in the model group exhibited lung tissue injury, airway mucus hypersecretion, goblet cell hyperplasia, and extensive collagen deposition. The number of inflammatory cells in BALF was increased. Levels of IL-1β, IL-6, TNF-α, and MCP-1 were significantly elevated (P<0.05). In addition, protein expression levels of TXNIP, NLRP3, ASC, cleaved-caspase-1, and IL-1β in lung tissues were significantly increased (all P<0.05). Compared with the model group, overexpression of miR-16-5p improved the pathological score of lung tissues in asthmatic rats, although the difference was not statistically significant (P>0.05). However, goblet cell hyperplasia, collagen deposition, and inflammatory cell infiltration were significantly reduced. Levels of IL-1β, IL-6, TNF-α, and MCP-1 were significantly decreased (all P<0.05). Protein expression levels of TXNIP, NLRP3, cleaved-caspase-1, and IL-1β were also significantly reduced (all P<0.05). In contrast, airway inflammatory responses were further aggravated in rats treated with the miR-16-5p inhibitor.

miR-16-5p may alleviate airway inflammation in asthmatic rats by negatively regulating TXNIP and subsequently suppressing activation of the NLRP3 inflammasome.