Tanshinone IIA Inhibits NADPH Oxidase 4 Expression by Regulating Sestrin2-Mediated AMPK/mTOR Signaling Pathway to Alleviate Myofibroblast Activation in Pulmonary Fibrosis

Oxidative stress serves as a driving force for myofibroblast activation in pulmonary fibrosis (PF). As a main enzymatic source of reactive oxygen species (ROS), NADPH oxidase 4 (Nox4) plays a critical role in modulating myofibroblast activation, and has thus emerged as a potential therapeutic target for PF. Tanshinone IIA (Tan-IIA), the most abundant fat-soluble component found in the root and rhizome of Salvia miltiorrhiza Bge., has been demonstrated to suppress ROS-mediated myofibroblast activation by inhibiting Nox4, and thereby ameliorating PF. However, the mechanism through which Tan-IIA regulates Nox4 to prevent myofibroblast activation remains unclear. This study aimed to investigate the protective effects of Tan-IIA against myofibroblast activation in PF, and to elucidate the upstream molecular mechanisms involved in Nox4 regulation. Tan-IIA inhibited myofibroblast activation by reducing extracellular matrix deposition in a mouse model of bleomycin-induced PF. Furthermore, Tan-IIA enhanced the expression of Sestrin2 (Sesn2), while concurrently suppressing Nox4 expression. This effect was verified using an in vitro model of transforming growth factor beta 1 (TGF-β1)-stimulated myofibroblast activation. We further demonstrated that Sesn2 was required for Tan-IIA to act against TGF-β1-induced myofibroblast activation by inhibiting Nox4-mediated oxidative stress. Additionally, both in vitro and in vivo studies revealed that Tan-IIA activates AMP-activated protein kinase (AMPK) and inhibits mammalian target of rapamycin (mTOR) via the upregulation of Sesn2. The findings indicate that Tan-IIA suppresses Nox4 by regulating the Sesn2/AMPK/mTOR signaling pathway, which highlights the crucial effect Sesn2 has in modulating Nox4 expression to prevent myofibroblast activation during PF.