miR-125b-5p Alleviates Allergic Airway Inflammation By Targeting TNFR2 To Regulate M1 Macrophage Polarization

Objective In this study, we explored the mechanism of action of miR-125b-5p in asthma-related airway inflammation (airway inflammation) by targeting TNFR2 to regulate M1 macrophage polarization. Methods In in vitro experiments, RT–qPCR was initially employed to quantify the expression levels of miR-125b-5p in lipopolysaccharide (LPS)-induced RAW264.7 cells. Following the transfection or inhibition of miR-125b-5p, flow cytometry was used to assess the proportion of M1 macrophages and changes in related cytokines. Moreover, RNA sequencing was performed to identify the target genes of miR-125b-5p, which were validated by flow cytometry and RT–qPCR. Western blot analysis was performed under both transfection and inhibition conditions of miR-125b-5p to examine the alterations in the NF-κB signalling pathway. In in vivo experiments, an asthma model was established through ovalbumin (OVA) induction, and histopathological scoring was conducted along with the detection of specific IgE in nasal lavage fluid and serum. Histopathological changes were observed using haematoxylin and eosin (H&E), periodic acid–Schiff (PAS), and toluidine blue staining, whereas flow cytometry was used to evaluate the changes in eosinophils in the lungs. Results In in vitro LPS-induced RAW264.7 cells, miR-125b-5p expression was significantly downregulated. LPS stimulation significantly increased the proportion of M1 macrophages and upregulated the expression of iNOS, IL-1β, IL-6, and TNF-α. Transfecting miR-125b-5p significantly reduced the proportion of M1 macrophages and suppressed the expression of iNOS, IL-1β, IL-6, and TNF-α. Conversely, the inhibition of miR-125b-5p led to a significant increase in M1 macrophage polarization and elevated levels of pro-inflammatory cytokines. TNFR2 was identified as a direct target of miR-125b-5p. Transfection with a miR-125b-5p mimic decreased TNFR2 expression, whereas transfection with an miR-125b-5p inhibitor increased TNFR2 expression. Knocking down TNFR2 significantly reduced the proportion of M1 macrophages and cytokines (iNOS, IL-1β, IL-6, TNF-α). In contrast, TNFR2 overexpression exacerbated M1 polarization and cytokine production. Additionally, transfecting miR-125b-5p or knocking down TNFR2 attenuated NF-κB activation, while inhibiting miR-125b-5p or overexpressing TNFR2 enhanced NF-κB signalling pathway activation. In vivo , OVA-induced mice showed reduced miR-125b-5p expression. Treatment with an miR-125b-5p agomir restored its levels and improved outcomes, significantly lowering histopathological scores, eosinophil infiltration in bronchoalveolar lavage fluid and lung tissue, serum specific IgE levels, and glycogen accumulation compared with those in untreated OVA-induced mice. Conclusion miR-125b-5p targets TNFR2 to modulate macrophage polarization and plays a crucial role in asthma. Our findings suggest new potential targets for therapy and diagnosis.