ASK1 Enhances Angiotensin II-Induced Liver Fibrosis In Vitro by Mediating Endoplasmic Reticulum Stress-Dependent Exosomes

Abstract EN

Background.

Apoptosis signal-regulating kinase 1 (ASK1) has been reported to induce fibrotic signaling in the setting of oxidative stress.

However, the role of ASK1 and its mechanism of action in angiotensin II- (Ang II-) induced liver fibrosis remain largely unknown.

Methods.

Human hepatic LX-2 stellate cells were treated with Ang II alone or cotreated with Ang II plus an ASK1 inhibitor (GS-4997) or siRNA-targeting ASK1.

Immunofluorescent staining, real-time PCR, and western blotting were used to determine the expressionof α-SMA, Col I, and Col III expression.

Cell viability was assessed by the CCK-8 assay.

The concentrations of IL-1β, IL-18, and TNF-α in conditioned medium were determined by ELISA.

The levels of intracellular ROS in LX-2 cells were analyzed using a ROS assay kit.

Exosome size was determined by electron microscopy.

Results.

Ang II markedly increased the expression of extracellular matrix (ECM) proteins (α-SMA, Col I, and Col III) and proinflammatory cytokines (IL-1β, IL-18, and TNF-α).

Ang II also increased the expression of endoplasmic reticulum stress (ERS) markers (GRP78, p-PERK, and CHOP) and p-ASK1.

Results also showed that pretreatment with GS-4997 or siRNA could abolish all the abovementioned effects on LX-2 cells.

Furthermore, we found that exosome release caused by ASK1-mediated ERS was involved in the activation of LX-2 cells by Ang II.

The activation of LX-2 cells could be blocked by treating the exosomes with annexin.

Conclusions.

In summary, we found that ASK1 mediates Ang II-activated ERS in HSCs and the subsequent activation of HSCs, suggesting a promising strategy for treating liver fibrosis.