Study on the anti-pulmonary fibrosis mechanism of Renshen Pingfei formula by inhibiting M2 macrophage polarization and regulating TGF - β/SMAD signaling pathway

Ethnopharmacological relevance: Renshen Pingfei formula (RSPF), a traditional Chinese medicine (TCM) formulation,has been historically prescribed in China for treating "Fei Bi" (pulmonary stagnation syndrome), which symptoms and pathogenesis aligns with those of idiopathic pulmonary fibrosis (IPF).

Aim of study: This study aimed to investigate the effects of RSPF on bleomycin-induced pulmonary fibrosis in mice and explore its regulatory effects on the TGF-β/SMAD signaling pathway and M2 macrophages polarization.

Materials and methods: LC-MS/MS was used to detect and identify the main active components of RSPF. In vivo studies were conducted using forty-eight C57BL/6 mice divided into six groups (n = 8): control, BLM, low, medium, or high RSPF dose (8.8, 17.5, or 35 g/kg, respectively), and Nintedanib (60 mg/kg/d). A pulmonary fibrosis mouse model was established by intratracheal instillation of bleomycin at 5 mg kg^-1. Histopathological changes in the lung tissue were assessed using hematoxylin-eosin (HE), Masson, and Sirius red staining. Enzyme-linked immunosorbent assay (ELISA) was employed to measure IL-1β and TNF-α levels in bronchoalveolar lavage fluid (BALF). Immunofluorescence analysis was performed to determine the expression levels of Arg-1⁺/F4/80⁺ and CD206⁺/F4/80⁺ cells. Flow cytometry was used to detect the expression of CD206 in interstitial macrophages (IMs) and alveolar macrophages (AMs). Furthermore, in vitro studies were employed using RSPF applied to IL-4-stimulated bone marrow-derived macrophages (BMDMs), co-cultured with alveolar epithelial cells. Flow cytometry was used to quantify M1 (CD86) and M2 (CD206) polarization. RT-qPCR, Western blot, and immunofluorescence were used to evaluate the fibrosis/EMT markers (E-cadherin, Vimentin, α-smooth muscle actin [α-SMA], fibronectin [FN], type I Collagen [Collagen I], TGF-β1, and SMAD2/3 phosphorylation).

Results: Seventy-eight active components were identified in RSPF. In vivo studies, RSPF increased the body weight of mice, improved Collagen deposition and fibrosis in IPF, upregulated E-cadherin protein levels, and downregulated the expression of fibrosis-related markers such as Vimentin, α-SMA, FN, and Collagen I. Additionally, RSPF inhibited IL-1β and TNF-α levels in BALF and suppressed the protein expression levels of TGF-β, p-Smad2/SMAD2, and p-Smad3/SMAD3. Further mechanistic studies indicated that RSPF downregulated the expression of Arg-1⁺/F4/80⁺ and CD206⁺/F4/80⁺ and decreased CD206 expression in IMs. In vitro studies, RSPF suppressed M2 polarization (CD206, Arg1), downregulated fibrotic markers (Collagen 1α1, α-SMA), and inhibited TGF-β1/Smad signaling.

Conclusion: RSPF alleviated pathological damage and fibrosis in the lung tissue of IPF mice, thereby delaying the progression of IPF. Its anti-fibrotic mechanism involved dual suppression of TGF-β1/Smad signaling and M2 macrophage polarization.

Macrophage polarization; Pulmonary fibrosis; Renshen pingfei formula; SMAD; TGF-β.