OBJECTIVE To investigate the effects and underlying mechanisms of Renshen Pingfei San (RSPFS) on bleomycin (BLM)-induced pulmonary fibrosis in aged mice.

METHODS Fifty 48-week-old male C57BL/6J mice were randomly assigned to a blank control group, BLM group, pirfenidone (PFD) group, and RSPFS high-, medium-, and low-dose groups (27.14, 13.57, and 6.79 g· kg⁻¹· d⁻¹). A pulmonary fibrosis model was induced by BLM. Drug intervention started on d7 after modeling and continued for 14 days. Body weight was recorded throughout the experiment. On d21 after modeling, lung function was evaluated using a whole-body plethysmography (WBP) system, and pulmonary imaging was assessed by Micro-CT. The lung coefficient and hydroxyproline (HYP) content in lung tissue were determined. Masson staining and hematoxylin and eosin (HE) staining were performed to evaluate histopathological changes. Western blot was used to detect the expression of PI3K/AKT/P21 pathway-related proteins and EMT-associated markers (E-cadherin, Vimentin, and α-SMA) in lung tissue. In vitro, RLE-6TN cells were treated with BLM and then intervened with RSPFS high/medium/low dose-containing serum and LY294002. Cellular ultrastructure was observed by transmission electron microscopy (TEM). β-galactosidase (SA-β-gal) staining was used to observe cell senescence, and DNA-binding fluorescent dye (Hoechst33342) staining was performed to observe senescence-related changes such as chromatin aggregation. Western blot was conducted to assess the expression of the PI3K/AKT/P21 pathway and E-cadherin and Vimentin proteins in RLE-6TN.

RESULTS Compared with the blank control group, mice in the BLM group showed a significant decrease in body weight on d14 and d21 (P<0.01), accompanied by reduced tidal volume (TV) and minute ventilation (MV) (P<0.01), markedly increased lung density on Micro-CT (P<0.01), and significantly elevated pathological scores and HYP content (P<0.05, P<0.01). Compared with the BLM group, the RSPFS high-dose group exhibited increased body weight on d14 (P<0.05) and a more significant improvement on d21 (P<0.01), along with improved TV and MV (P<0.01), reduced Micro-CT lung density, decreased pathological scores (P<0.05, P<0.01), and reduced lung tissue HYP content (P<0.05). In lung tissue, compared with the blank control group, the BLM group showed significantly increased expression of p-PI3K, p-AKT, P21, Vimentin, and α-SMA (P<0.01), with decreased E-cadherin expression (P<0.01). Compared with the BLM group, RSPFS high- and medium-dose groups showed significantly reduced levels of p-PI3K, p-AKT, P21, Vimentin, and α-SMA (P<0.01) and increased E-cadherin expression (P<0.01). In vitro, compared with the blank control group, BLM-treated RLE-6TN cells exhibited impaired morphology and ultrastructure, enhanced SA-β-gal staining, and increased Hoechst 33342 nuclear fluorescence intensity. Compared with the BLM group, RSPFS-containing serum group showed improved cellular morphology, attenuated SA-β-gal staining, and reduced Hoechst fluorescence intensity. Moreover, compared with the blank control group, BLM increased the expression of p-PI3K, p-AKT, P21, and Vimentin (P<0.05, P<0.01) and decreased E-cadherin (P<0.01). Compared with the BLM group, all BLM+RSPFS serum groups and the BLM+LY294002 group showed decreased expression of p-PI3K, p-AKT, P21, and Vimentin (P<0.05, P<0.01) and increased E-cadherin (P<0.01).

CONCLUSION RSPFS has an anti-BLM-induced pulmonary fibrosis effect in aging mice. In vitro and in vivo experiments suggest that it can slow down aging and inhibit epithelial-mesenchymal transition (EMT) by regulating the PI3K/AKT/P21 signaling pathway, thereby improving idiopathic pulmonary fibrosis.