# WuYeLuGen Granule Attenuates Bleomycin‐Induced Pulmonary Fibrosis in Rats by Inhibiting the TGF‐β1/Smad Signaling Pathway and Epithelial–Mesenchymal Transition

## TL;DR

WuYeLuGen Granule reduces lung scarring in rats by blocking a key signaling pathway and preventing cell transformation linked to fibrosis.

## Contribution

The study reveals that WuYeLuGen Granule inhibits TGF-β1/Smad signaling and epithelial-mesenchymal transition to treat pulmonary fibrosis.

## Key findings

- WYLG granules reduced fibrosis, inflammation, and collagen in BLM-induced rat lungs.
- WYLG inhibited TGF-β1/Smad signaling and epithelial-mesenchymal transition in fibroblasts.
- The TGF-β1/Smad pathway agonist reversed WYLG's antifibrotic effects, confirming the mechanism.

## Abstract

Pulmonary fibrosis is a chronic disease characterized by progressive interstitial lung changes affecting alveolar epithelial cells and pulmonary vessels. Following COVID‐19, it has emerged as a significant sequela in severe cases, often with a poor prognosis. WuYeLuGen (WYLG) Granule, derived by Xue’s Wuye Lugen Granule, exerts effects of replenishing qi, nourishing yin, clearing heat, and resolving dampness. While clinical and experimental studies provide evidence to support WYLG’s efficacy against early‐stage pulmonary fibrosis, its underlying mechanisms remain incompletely understood.

Active components of WYLG were identified using LC‐MS/MS. CCK8 assays were performed to determine the optimal concentrations of WYLG‐containing serum and TGF‐β1. WYLG granules were administered to bleomycin (BLM)‐induced rats and WYLG‐containing serum was applied to TGF‐β1‐stimulated rat pulmonary fibroblasts (RPFs). Hematoxylin‐Eosin (HE) and Masson staining were used to assess the protective effects of WYLG on rat lung tissues, while enzyme‐linked immunosorbent assay (ELISA) was employed to evaluate lung inflammation. Flow cytometry analyzed RPF cell proliferation, scratch assays examined cell migration, and Western blot detected the expression of fibrotic and pathway‐related proteins. Immunofluorescence was used to confirm the efficacy of WYLG in reducing RPF cell fibrosis.

LC‐MS/MS identified 18 active components in WYLG, primarily derived from Salvia miltiorrhiza and Astragalus membranaceus. The optimal concentration for TGF‐β1‐induced RPF stimulation was 10 ng/mL, and the optimal concentration of WYLG‐containing serum was 10%. In BLM‐induced rats, WYLG granules significantly alleviated pulmonary fibrosis, reduced inflammatory cell infiltration and collagen deposition, downregulated IL‐6 and α‐SMA levels, and upregulated E‐cadherin expression. Mechanistically, WYLG treatment decreased the levels of TGF‐β1 and p‐Smad2/Smad2, while increasing Smad7 levels in rat lung tissue. In TGF‐β1‐stimulated RPF, WYLG‐containing serum normalized cell proliferation, inhibited cell migration, reduced collagen I and α‐SMA expression, and increased E‐cadherin expression. Consistent with animal experiments, WYLG‐containing serum also downregulated TGF‐β1 and p‐Smad2/Smad2 levels in RPFs. Additionally, the TGF‐β1/Smad pathway agonist SRI‐011381 reversed the inhibitory effects of WYLG on RPF fibrosis, further confirming that WYLG exerts its antifibrotic effect through the TGF‐β1/Smad pathway.

WYLG markedly alleviates pulmonary fibrosis both in vivo and in vitro by inhibiting the TGF‐β1/Smad signaling pathway and regulating epithelial‐to‐mesenchymal transition, highlighting its potential as a therapeutic agent for progressive pulmonary fibrosis.