ASK1 Enhances Angiotensin II-Induced Liver Fibrosis In Vitro by Mediating Endoplasmic Reticulum Stress-Dependent Exosomes.
ASK1 Enhances Angiotensin II-Induced Liver Fibrosis In Vitro by Mediating Endoplasmic Reticulum Stress-Dependent Exosomes.
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DOI：
10.1155/2020/8183713
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发表时间：
2020
影响因子：
4.6
通讯作者：
Li Q
中科院分区：
文献类型：
--
作者：
Fang PP;Pan CW;Lin W;Li J;Huang SS;Zhou GY;Du WJ;Li Q
据报道，细胞凋亡信号调节蛋白1(ASK1)在氧化应激过程中诱导纤维化信号转导。然而，ASK1在血管紧张素II-(Ang II-)诱导的肝纤维化中的作用及其作用机制尚不清楚。人肝LX-2星状细胞用Ang II单独处理，或与Ang II加ASK1抑制剂(GS-4997)或靶向ASK1的siRNA共同处理。免疫荧光染色、实时定量聚合酶链式反应和免疫印迹法检测α-SMA、Col I、Col III的表达。CCK-8比色法测定细胞存活率。用双抗体夹心法测定条件培养液中IL-1β、IL-18和肿瘤坏死因子-α的浓度。用ROS试剂盒检测LX-2细胞内ROS水平。用电子显微镜测定外切体的大小。Ang II显著增加细胞外基质蛋白(α-SMA、Col I和Col III)和促炎细胞因子(IL-1β、IL-18和Tf F-α)的表达。Ang II还增加内质网应激(ERS)标志物(GRP78、p-PERK和CHOP)和p-ASK1的表达。GS-4997或siRNA可阻断上述对LX-2细胞的作用。此外，我们还发现，ASK1介导的ERS引起的外切体释放参与了Ang II对LX-2细胞的激活。用Annexin处理外切体可以阻断LX-2细胞的激活。综上所述，我们发现ASK1介导了血管紧张素转换酶II激活的肝星状细胞内皮细胞以及随后激活的肝干细胞，这提示了一种治疗肝纤维化的有前景的策略。
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. 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. 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. 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.
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