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.

中文翻译：

---

ASK1 通过介导内质网应激依赖性外泌体增强体外血管紧张素 II 诱导的肝纤维化

背景。据报道，细胞凋亡信号调节激酶 1 (ASK1) 在氧化应激环境中诱导纤维化信号传导。然而，ASK1 的作用及其在血管紧张素 II- (Ang II-) 诱导的肝纤维化中的作用机制仍然很大程度上未知。方法。人肝 LX-2 星状细胞用 Ang II 单独处理或用 Ang II 加 ASK1 抑制剂 (GS-4997) 或靶向 siRNA 的 ASK1 共同处理。免疫荧光染色、实时荧光定量PCR和蛋白质印迹用于确定α- SMA、Col I和Col III表达的表达。通过CCK-8测定评估细胞活力。IL-1 β、IL-18 和 TNF- α的浓度在条件培养基中通过ELISA确定。使用 ROS 测定试剂盒分析 LX-2 细胞中的细胞内 ROS 水平。通过电子显微镜确定外泌体大小。结果。Ang II 显着增加细胞外基质 (ECM) 蛋白（α- SMA、Col I 和 Col III）和促炎细胞因子（IL-1 β、IL-18 和 TNF- α ）的表达）。Ang II 还增加了内质网应激 (ERS) 标志物（GRP78、p-PERK 和 CHOP）和 p-ASK1 的表达。结果还表明，用 GS-4997 或 siRNA 预处理可以消除上述对 LX-2 细胞的所有影响。此外，我们发现由 ASK1 介导的 ERS ​​引起的外泌体释放与 Ang II 激活 LX-2 细胞有关。可以通过用膜联蛋白处理外泌体来阻断 LX-2 细胞的活化。结论。总之，我们发现 ASK1 介导 HSC 中的 Ang II 激活的 ERS ​​和随后的 HSC 激活，这表明治疗肝纤维化的有希望的策略。