Abstract
BackgroundReactive oxygen species (ROS) levels largely determine pulmonary fibrosis. Antioxidants have been found to ameliorate lung fibrosis after long-term paraquat (PQ) exposure. The effects of antioxidants, however, on the signalling pathways involved in PQ-induced lung fibrosis have not yet been investigated sufficiently. Here, we examined the impacts of ligustrazin on lung fibrosis, in particular ROS-related autophagy and pro-fibrotic signalling pathways, using a murine model of PQ-induced lung fibrosis.MethodsWe explored the effects of microRNA-193 (miR-193a) on Hedgehog (Hh) and PI3K/Akt/mTOR signalling and oxidative stress in lung tissues. Levels of miR-193a, protein kinase B (Akt), phosphoinositide 3-Kinase (PI3K), ceclin1, mammalian target of rapamycin (mTOR), sonic hedgehog (SHH), myosin-like Bcl2 interacting protein (LC3), smoothened (Smo), and glioma-associated oncogene-1 (Gli-1) mRNAs were determined with quantitative real-time PCR. Protein levels of PI3K, p-mTOR, p-Akt, SHH, beclin1, gGli-1, LC3, smo, transforming growth factor-β1 (TGF-β1), mothers against DPP homologue-2 (Smad2), connective tissue growth factor (CTGF), collagen I, collagen III, α-smooth muscle actin (α-SMA) nuclear factor erythroid 2p45-related factor-2 (Nrf2), and p-Smad2 were detected by western blotting. In addition, α-SMA, malondialdehyde, ROS, superoxide dismutase (SOD), oxidised and reduced glutathione, hydroxyproline, and overall collagen levels were identified in lung tissues using immunohistochemistry.ResultsLong-term PQ exposure blocked miR-193a expression, reduced PI3K/Akt/mTOR signalling, increased oxidative stress, inhibited autophagy, increased Hh signalling, and facilitated the formation of pulmonary fibrosis. Ligustrazin blocked PI3K/Akt/mTOR and Hh signalling as well as reduced oxidative stress via increasing miR-193a expression and autophagy, all of which reduced pulmonary fibrosis. These effects of ligustrazin were accompanied by reduced TGF-β1, CTGF, and Collagen I and III expression.ConclusionsLigustrazin blocked PQ-induced PI3K/Akt/mTOR and Hh signalling by increasing miR-193a expression, thereby attenuating PQ-induced lung fibrosis.
Highlights
Reactive oxygen species (ROS) levels largely determine pulmonary fibrosis
Results miR-193a decreased p-Akt, Beclin1, LC3-II, and autophagy levels in A549 cells To investigate the impacts of miR-193a on p-Akt, Beclin1 and LC3-II in A549 cells, miR-193a mimic, miR-193a mimic-negative control (NC), miR-193a inhibitor, and miR-193a inhibitor-NC were transfected into the cells
Increased miR-193a levels significantly increased p-Akt, Beclin1 and LC3-II levels, thereby increasing autophagy in A549 cells (Fig. 1a-f). These findings suggested that miR-193a controlled autophagy by regulating the p-Akt, Beclin1 and LC3-II pro-autophagy pathways
Summary
Reactive oxygen species (ROS) levels largely determine pulmonary fibrosis. Antioxidants have been found to ameliorate lung fibrosis after long-term paraquat (PQ) exposure. Oxidative stress is an important molecular mechanism of PQ-induced pulmonary fibrosis [4] that often occurs in the lung after PQ exposure, disturbing the balance between oxides and peroxides and increasing reactive oxygen species (ROS) levels [5]. Previous studies have made the PQ model one of the best characterized models of fibrosis, as this method invokes a highly reproducible oxidative stress response that leads to fibroblast proliferation, collagen deposition, and incurable pulmonary fibrosis [11]. In this evaluation, we utilized a PQ-induced pulmonary fibrosis model to analyze the mechanism of ligustrazin against pulmonary fibrosis
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