Salvia miltiorrhiza Restrains Reactive Oxygen Species-Associated Pulmonary Fibrosis via Targeting Nrf2-Nox4 Redox Balance

Pulmonary fibrosis (PF) is characterized by myofibroblast activation, which can be triggered by oxidative stress. In this study, we investigated the antifibrotic effect of the ethyl acetate extract of Salvia miltiorrhiza (EASM) on PF and examined the underlying molecular mechanism. EASM suppressed myofibroblast activation with reduced extracellular matrix deposition in the lungs of mice subjected to bleomycin (BLM) challenge, demonstrating the inhibitory effects on PF. EASM positively alleviated oxidative stress by upregulating nuclear factor-erythroid 2-related factor 2 (Nrf2) and concomitantly downregulating NADPH Oxidase 4 (NOX4) in the lungs of BLM-treated mice. This effect was also observed in an in vitro model of transforming growth factor beta 1 (TGF-$\beta$1)-stimulated fibroblast activation. EASM reduced Reactive Oxygen Species (ROS) generation in fibroblasts by stabilizing Nrf2 protein with promoting kelch-like ECH-associated protein 1 (Keap1) degradation. Nrf2 knockdown in the lungs of BLM-treated mice diminished the inhibitory effects of EASM on fibrosis, providing evidence in vivo to address the unique role of Nrf2. Additionally, EASM inhibited TGF-$\beta$1/SMAD3 signaling by downregulating protein kinase C delta (PKC-$\delta )$ and SMAD3 phosphorylation (p-Smad3), which led to suppression of the TGF-$\beta$1-induced fibrogenic response. These results indicate that EASM exhibits potent antifibrotic activity in vitro and in vivo, which might be associated with activation of Nrf2 pathway and inhibition of TGF-$\beta$1/SMAD3 pathway. Our findings support that EASM may act as an effective antifibrotic remedy for PF.