Interaction Mechanisms Between the NOX4/ROS and RhoA/ROCK1 Signaling Pathways as New Anti- fibrosis Targets of Ursolic Acid in Hepatic Stellate Cells.

Chenkai Huang,Anjiang Wang,Dakai Gan,Xuan Zhu,Jiang Chen,Fangyun Luo,Bimin Li,Sizhe Wan

doi:10.3389/fphar.2019.00431

Abstract

BackgroundStudies have shown that both NOX4 and RhoA play essential roles in fibrosis and that they regulate each other. In lung fibrosis, NOX4/ROS is located upstream of the RhoA/ROCK1 signaling pathway, and the two molecules are oppositely located in renal fibrosis. Currently, no reports have indicated whether the above mechanisms or other regulatory mechanisms exist in liver fibrosis.ObjectivesTo investigate the effects of the NOX4/ROS and RhoA/ROCK1 signaling pathways on hepatic stellate cell (HSC)-T6 cells, the interaction mechanisms of the two pathways, and the impact of UA on the two pathways to elucidate the role of UA in the reduction of hepatic fibrosis and potential mechanisms of HSC-T6 cell proliferation, migration, and activation.MethodsStable cell lines were constructed using the lentiviral transduction technique. Cell proliferation, apoptosis, migration, and invasion were examined using the MTS, TdT-mediated dUTP nick-end labeling, cell scratch, and Transwell invasion assays, respectively. The DCFH-DA method was used to investigate the ROS levels in each group. RT-qPCR and western blotting techniques were utilized to assess the mRNA and protein expression in each group. CoIP and the Biacore protein interaction analysis systems were used to evaluate protein interactions.ResultsThe NOX4/ROS and RhoA/ROCK1 signaling pathways promoted the proliferation, migration, and activation of HSCs. UA inhibited cell proliferation, migration, and activation by inhibiting the activation of the two signaling pathways, but the mechanism of apoptosis was independent of these two pathways. The NOX4/ROS pathway was upstream of and positively regulated the RhoA/ROCK1 pathway in HSCs. No direct interaction between the NOX4 and RhoA proteins was detected.ConclusionThe NOX4/ROS and RhoA/ROCK1 signaling pathways are two critical signaling pathways in a series of behavioral processes in HSCs, and NOX4/ROS regulates RhoA/ROCK1 through an indirect pathway to control the activation of HSCs. Additionally, NOX4/ROS and RhoA/ROCK1 constitute a new target for UA antifibrosis treatment.

Highlights

Hepatic fibrosis is the process by which a variety of etiologies cause the proliferation and transformation of hepatic stellate cell (HSC) into MFBs, leading to extracellular matrix (ECM) deposition or scar formation (Schuppan et al, 2018)
NOX4 and ras homolog family member A (RhoA) Are Required for the Proliferation of HSCs, and ursolic acid (UA) Suppresses Their Expression via NOX4
Transforming growth factor-β1 (10 ng/mL) was added to the culture medium for 48 h, and the Nox4 mRNA and protein expression levels were compared between the scramble lentivirus control HSCs (CON group) and NOX4 shRNA lentivirus transduction HSCs (NOX4i group)

Summary

Introduction

Hepatic fibrosis is the process by which a variety of etiologies cause the proliferation and transformation of HSCs into MFBs, leading to extracellular matrix (ECM) deposition or scar formation (Schuppan et al, 2018). The NOX family participates in the regulation of signal transduction in HSCs by generating ROS and plays a vital role in the activation of HSCs and the pathogenesis of hepatic fibrosis (Paik et al, 2011). Aoyama et al (2012) showed that both TGF-β1 and Ang II upregulate NOX4 expression and that a dual inhibitor of NOX1/4, GKT137831, inhibits ROS production and hepatic fibrosis. These findings indicate that NOX4 mediates the signal transduction of TGF-β1 and other major hepatic fibrogenic factors in HSCs, leading to their activation. No reports have indicated whether the above mechanisms or other regulatory mechanisms exist in liver fibrosis

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