Arctigenin alleviates TGF-β1-induced epithelial-mesenchymal transition and PAI-1 expression via AMPK/NF-κB pathway in peritoneal mesothelial cells

https://doi.org/10.1016/j.bbrc.2019.09.130Get rights and content

Highlights

  • Arctigenin dose-dependently alleviatesTGF-β1-induced EMT and PAI-1 in HPMCs.

  • Arctigenin activates AMPK but inhibits NF-κB signaling in TGF-β1-induced HPMCs.

  • Arctigenin attenuates TGF-β1-induced NF-κB binding capacity to PAI-1 promoter.

  • Compound C abrogates the inhibitory effect of Arctigenin on TGF-β1-induced EMT.

Abstract

Peritoneal fibrosis (PF) caused by long-term peritoneal dialysis is closely associated with the epithelial-mesenchymal transition (EMT) of human peritoneal mesothelial cells (HPMCs). Moreover, the anti-fibrotic role of Arctigenin (Arc) has been reported in several fibrosis disorders. Therefore, the preventive effect of Arc on transforming growth factor-β1 (TGF-β1)-induced EMT and the underlying mechanisms in HPMCs was investigated in this study. Firstly, the PD model was established by TGF-β1 stimulation in cultured HPMCs in vitro, we found that TGF-β1 significantly increased the EMT markers (α-SMA, vimentin, and fibronectin) and plasminogen activator inhibitor type 1 (PAI-1) expressions, but decreased epithelial marker (E-cadherin). Co-treatment with Arc (10, 20, 40 μM) ameliorated TGF-β1-induced EMT in a dose-dependent manner, and the expression of PAI-1 was also inhibited by Arc, which was abrogated by restoration of PAI-1. Moreover, Arc enhanced the phosphorylated AMP-activated protein kinase (AMPK), but inhibited the phosphorylated IκBα level and nuclear translocation of nuclear factor κB (NF-κB) p65 in TGF-β1-induced HPMCs. ChIP and Luciferase reporter assays verified that the increased binding capacity of NF-κB to the promoter of PAI-1 induced by TGF-β1 was reversely attenuated by Arc in HPMCs. However, the effect of Arc on TGF-β1-induced NF-κB activation, PAI-1 expression and EMT in HPMCs was attenuated by AMPK agonist Compound C. In conclusion, these data demonstrated that Arc suppressed TGF-β1-induced EMT by activating the AMPK/NF-κB pathway to inhibit PAI-1 expression in HPMCs. Therefore, Arc might act as a potential therapeutic agent for PD treatment.

Introduction

Peritoneal dialysis (PD) as one of the established renal replacement therapies is widely applied in patients with end-stage renal disease (ESRD) [1]. However, chronic exposure to the peritoneal dialysis fluid (PDF) during the long-term PD treatment often causes the peritoneal dysfunction and ultrafiltration failure, and subsequently leads to peritoneal fibrosis (PF) [2]. In response to PD fluids stimulation, epithelial-mesenchymal transition (EMT) of peritoneal mesothelial cells (PMCs) facilitates the peritoneal epithelial phenotype transdifferentiate into mesenchymal characteristics, leads to the loss of intercellular tight junctions, allowance of PMCs invading and migrating into the submesothelial compact zone and synthesis of excessive extracellular matrix (ECM) components, which mainly contributes to the initiation and progression of PF [3]. It has been suggested that various stimulators including transforming growth factor-β1 (TGF-β1) that involved in PF triggers the PMC phenotypic conversion and mesenchymal transition, ultimately leads to EMT of PMCs, however, the underlying molecular mechanisms remains undefined [4].

Plasminogen activator inhibitor-1 (PAI-1), also known as SERPIEN1, is a single-chain glycoprotein that belongs to the serpin family of protease inhibitors [5]. It has been demonstrated that PAI-1 prevents the activation of urokinase-type plasminogen activator (uPA)/tissue-type plasminogen activator (tPA), which results in the deterred conversion of plasminogen to plasmin and the inhibition of plasmin-dependent matrix metalloproteinases (MMP) activation [6]. However, sustained PAI-1 activity contributes to the abundant collagen deposition through the suppression of proteolytic-mediated ECM degradation in various tissue types, for instance, previous studies have suggested that PAI-1 expression is elevated in liver, cardiac, renal, and pulmonary fibrosis in response to persistent stimulation of fibrotic effectors, such as TGF-β1 [7]. Thus, inhibition or knockdown of PAI-1 expression has been confirmed to exert protective effect against fibrogenesis in various in vitro and in vivo models [8]. Recently, the upregulation of PAI-1 has been considered to be a biomarker of peritoneal membrane alterations in clinical PD patients, suggesting the potential therapeutic target to prevent peritoneal modifications, especially fibrosis [9].

Arctigenin (Arc) is a type of dibenzylbutyrolactone lignin derived from Arctium lappa, whose seed has been widely applied to treat infections, inflammations, and fibrogensis in many Asian countries [10]. For instance, Arc has been proved to suppress the EMT of renal tubules by decreasing the secretion of TGF-β1 in unilateral ureteral obstruction-induced damage and fibrogenesis [11]. In oral submucous fibrosis, Arc abolished the arecoline-induced migration, invasion, and myofibroblast characteristics of bibrotic buccal mucosal fibroblasts (fBMFs) in a dose-dependent manner [12]. Moreover, Arc repressed TGF-β1-induced EMT and monocyte chemoattractant protein-1 expression via the reactive oxygen species -mediated ERK/NF-κB signaling pathway in renal tubular epithelial cells [13]. Nevertheless, it is not yet elucidate whether the Arc exerts anti-fibrotic effects in peritoneal fibrosis.

In this study, the effects of Arc on the EMT-related events of TGF-β1-stimulated PMCs were investigated and the potential underlying mechanisms was explored. Intriguingly, we verified that Arc alleviated TGF-β1-induced EMT in PMCs, which was partly through PAI-1-mediated signaling pathway.

Section snippets

Cell culture and treatment

Human PMC cell line (HPMCs) was obtained from ATCC (NY, USA) and cultured in DMEM/F12 medium (Gibco, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) in a humidified atmosphere with 5% CO2 at 37 °C. The cells were incubated serum free for 12 h prior to stimulation with recombinant human TGF-β1 (2 ng/ml, MN, USA). Arc (Fig. 1A, Sigma-Aldrich, MO, USA) was dissolved in DMSO (Sigma-Aldrich), and then treated HPMCs with Arc at the concentration of 1, 2.5, 5, 10, 20, 40 μM in the

Effect of Arc on TGF-β1-induced EMT in HPMCs

Firstly, the cytotoxicity of Arc on HPMCs was examined by using CCK-8 assay. Arc at the concentrations of 1–20 μM showed minimal inhibitory effect on cell viability, but significantly decreased after 40 μM Arc treatment (Fig. 1B). Then, the proper concentrations of Arc on TGF-β1-induced proliferation of HPMCs was determined, we found that treating HPMCs with TGF-β1 for 24 h exhibited a significantly increased viability of HPMCs (P < 0.05), however, pretreatment with Arc (5, 10, 20 μM)

Discussion

In the past decades, studies have found that PMCs are the main cell population in the peritoneum and EMT of PMCs directly leads to the onset and progression of PF [16]. Currently, the application of active natural materials extracted from traditional herbal medicine as the alternative treatment agents have been convincingly demonstrated to have suppressive effects on the fibrotic development in peritoneum. For instance, Curcumin has been suggested to inhibit the PD solutions-induced EMT of PMCs

Declaration of competing interest

The authors declared that no conflicts of interest were disclosed.

Acknowledgement

This study was supported by “Science & Technology Research and Development Program of Shaanxi Province (No. S2016YFSF0644)”.

References (29)

  • H.B. Lee et al.

    Mechanisms of epithelial-mesenchymal transition of peritoneal mesothelial cells during peritoneal dialysis

    J. Korean Med. Sci.

    (2007)
  • D.E. Vaughan et al.

    Plasminogen activator inhibitor-1 is a marker and a mediator of senescence

    Arterioscler. Thromb. Vasc. Biol.

    (2017)
  • P. Flevaris et al.

    The role of plasminogen activator inhibitor type-1 in fibrosis

    Semin. Thromb. Hemost.

    (2017)
  • R. Rabieian et al.

    Plasminogen activator inhibitor type-1 as a regulator of fibrosis

    J. Cell. Biochem.

    (2018)
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