Eupatilin inhibits pulmonary fibrosis by activating Sestrin2/PI3K/Akt/mTOR dependent autophagy pathway

Idiopathic pulmonary fibrosis (IPF) is a progressive chronic inflammatory disease with poor clinical outcomes and ineffective drug treatment options. Eupatilin is a major component extracted from the traditional herbal medicine Artemisia asiatica Nakai. Notably, it was demonstrated to have an anti-fibrosis effect in endometrial fibrosis, vocal fold, and hepatic fibrosis. Its role and mechanism in IPF remain unclear.This study used the TGF-β1-induced human embryonic lung fibroblasts (MRC-5) activation, IPF lung fibroblasts, and bleomycin-induced lung fibrosis mice model. Western blot, immunofluorescence staining, quantitative real time-PCR, hematoxylin and eosin staining, Masson's trichrome staining, and immunohistochemistry were used to evaluate the effects of eupatilin on fibroblast activation, pulmonary fibrosis, and autophagy. The autophagosomes were observed with a transmission electron microscope (TEM). RNA sequencing was used to determine the signaling pathway and key regulator related to autophagy.Eupatilin significantly decreased the expression of Col1A1, fibronectin, α-SMA, and SQSTM1/p62. In contrast, it increased the expression of LC3B II/I and the number of autophagosomes in TGF-β1 treated MRC-5, IPF lung fibroblasts, and bleomycin-induced lung fibrosis mice model; it also alleviated bleomycin-induced lung fibrosis. The KEGG pathway mapping displayed that PI3K/Akt and Sestrin2 were associated with the enhanced fibrogenic process. Eupatilin suppressed the phosphorylation of PI3K/Akt/mTOR. Autophagy inhibitor 3-methyladenine (3-MA) and Akt activator SC-79 abrogated the anti-fibrotic effect of eupatilin. Sestrin2 expression was also downregulated in TGF-β1 treated lung fibroblasts and lung tissues of the bleomycin-induced pulmonary fibrosis mice model. Furthermore, eupatilin promoted Sestrin2 expression, and the knockdown of Sestrin2 significantly aggravated the degree of fibrosis, increased the phosphorylation of PI3K/Akt/mTOR, and decreased autophagy.These findings indicate that eupatilin ameliorates pulmonary fibrosis through Sestrin2/PI3K/Akt/mTOR-dependent autophagy pathway.