Yangqing Chenfei formula attenuates silica-induced pulmonary fibrosis by suppressing activation of fibroblast via regulating PI3K/AKT, JAK/STAT, and Wnt signaling pathway

Abstract

BackgroundYangqing Chenfei formula (YCF) has been demonstrated its clinical efficiency on silicosis patients. However, the effect of YCF against silicotic fibrosis and its mechanism remain unclear. PurposeThis study is aimed to investigate active compounds and molecular mechanism of YCF in treating silicosis. MethodYCF was orally administrated to silicosis rats induced by crystalline silica. The effective fraction of YCF and the compounds was isolated and identified by using macroporous resin and HPLC-MS, respectively. The targets and potential molecular mechanism of YCF against silicotic fibrosis were investigated through pharmacological network and RNA-sequencing analysis and in vitro-experimental validation. ResultsYCF could remarkably improve the lung function and pathological changes of silicotic rats, reduce the aggregation of fibrocytes and deposition of ECM, such as collagen I, III, FN, and α-SMA, and suppress the TGF-β/Smad3 signaling. Furthermore, YCF6, the effective fraction derived from YCF, could significantly inhibit fibroblast activation induced by TGF-β. Then, 135 compounds were identified from YCF6 by using HPLC-MS, and Network pharmacology analysis predicted total 941 targets for these compounds. Moreover, 409 differentially expressed genes of fibroblast activation induced by TGF-β were identified. Then, integrated analysis of the 941 targets with 409 differentially expressed genes showed that YCF6 contains multiple compounds, such as tangeretin, L-Malic acid, 2-Monolinolein etc., which inhibits fibroblast activation probably by targeting different proteins, such as PIK3CA, AKT1, JAK2, STAT3, GSK3β, leading to regulate the signal network, such as PI3K/AKT signaling pathway, JAK/STAT signaling pathway, and Wnt signaling pathway. Finally, in vitro experiment indicated that tangeretin, the active compound contained in YCF6, could significantly inhibit TGF-β induced fibroblast activation. Moreover, YCF6 and tangeretin could markedly inhibit the activation of PI3K/AKT, JAK/STAT, and Wnt pathway. ConclusionYCF contained multiple compounds and targeted various proteins that regulated the fibroblast activation, which might be the molecular mechanisms of it in treating silicosis.