Effect of pirfenidone on pulmonary fibrosis in acute lung injury via the regulation of the miR-34a-5p/TGF-β1/SMAD pathway.

BACKGROUND: Pulmonary fibrosis (PF) is a common phenomenon in acute lung injury (ALI), and miR-34a-5p plays a significant role in injury-like illness. This study aims to investigate the molecular regulatory mechanisms of miR-34a-5p and pirfenidone (PFD) in the pathological process of PF, providing novel experimental data to elucidate the molecular mechanisms underlying the therapeutic effects of miR-34a-5p and PFD in the treatment of PF. METHODS: In mouse alveolar macrophage (MH-S) cells, quantitative polymerase chain reaction (qPCR) was employed to assess miR-34a-5p expression. Western blotting was used to examine the protein levels of TGF-β1, p-SMAD3, p-SMAD4, CD68, CD206, Arg-1, and YM-1. Cell Counting Kit-8 (CCK8) assays were conducted to asses cell viability. Animal models were established in male C57BL/6 mice, and lung tissues were obtained for histological analyses. Immunohistochemistry (IHC) was performed to measure the expression of CD206 and YM-1. RESULTS: In IL4-treated MH-S cells, the expression of miR-34a-5p was significantly upregulated according to qPCR, and miR-34a-5p inhibition could improve the cell viability. Western blot data showed that when induced by IL4, the levels of CD68, CD206, Arg-1, and YM-1 were significantly decreased, and miR-34a-5p was also suppressed. M2 macrophage polarization was mediated by miR-34a-5p. Furthermore, after treatment with PFD in IL4-treated MH-S cells, cell viability was increased, which was reversed when miR-34a-5p expression was elevated. Protein expression of CD68, CD206, Arg-1, and YM-1 was significantly reduced. With IL4 treatment, the protein levels of TGF-β1, p-SMAD2, and p-SMAD3 were significantly increased but then decreased when PFD was added. In vivo, miR-34a-5p expression was reduced after the addition of PFD, and the protein expression of CD206 and YM-1 was significantly decreased according to IHC. CONCLUSIONS: PFD could influence ALI by regulating miR-34a-5p through the TGF-β1/SMAD pathway, which suggests miR-34a-5p as a therapeutic target for ALI treatment.