Department of Hepatopancreatobiliary Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, China.
Department of Surgery, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang Province, China.
J Surg Res. 2023 Jan;281:245-255. doi: 10.1016/j.jss.2022.08.010. Epub 2022 Oct 6.
Heme oxygenase-1 (HO-1) is a protective protein in oxidative stress response. LXA4 is an "inflammatory braking signal" that is widely studied at present. The purpose of this study was to elucidate that LXA4 can protect cells by inducing HO-1 in human pulmonary microvascular endothelial cells (HPMECs) as in vitro model to explain acute lung injury after severe acute pancreatitis.
This study was performed in two parts: (1) To investigate the mechanisms of lipoxin A4-induced HO-1 expression in vitro, the study subjects were divided into four groups: a control group, LXA4 group (50 ng/mL LXA4), inhibitor group (50 ng/mL LXA4 + 20 μM LY294002 or 50 ng/mL LXA4 + 2 nmol/mL Bis II), and agonist group (50 ng/mL insulin-like growth factor 1, PMA). Western blotting was used to detect the expression of p-Akt, Akt, protein kinase C (PKC), p-Nrf2, Nrf2, and Keap1, and the location of Nrf2 was detected using immunofluorescence. The activation of antioxidant responsive element induced by Nrf2 was detected using Electrophoretic Mobility Shift Assay and (2) to investigate the cytoprotection of HO-1 induced by LXA4 in vitro, the subjects were divided into four groups: a control group, tumor necrosis factor α (TNF-α) group (50 ng/mL), LXA4 group (50 ng/mL TNF-α + 50 ng/mL LXA4), and Zinc protoporphyrin IX group (pretreated with 0.5 μM Zinc protoporphyrin IXfor 12 h, followed by 50 ng/mL TNF-α + 50 ng/mL LXA4). BCECF/AM-labeled THP-1 cells were used to analyze the adhesion of HPMECs, and a mitochondrial membrane potential assay kit with JC-1 was used to analyze the apoptosis of HPMECs.
In part one, (1) LXA4 upregulated the expression of HO-1 in a dose-dependent manner and (2) LXA4 activated the PI3K/Akt and PKC pathways and modulated the phosphorylation and subsequent depolymerization of Nrf2 from Keap1, promoting the translocation of Nrf2 to the nucleus. In part two, (1) LXA4 reversed the changes in mitochondrial membrane potential to alleviate apoptosis in HPMECs and (2) LXA4 attenuated the adhesion of HPMECs induced by TNF-α.
LXA4 can activate the PI3K/Akt and PKC pathways and induce the phosphorylation of Nrf2, resulting in the upregulation of HO-1. In addition, LXA4 alleviates adhesion and protects mitochondrial function by upregulating the expression of HO-1, which exerts cytoprotection in severe acute pancreatitis-induced lung injury.
血红素加氧酶-1（HO-1）是氧化应激反应中的一种保护性蛋白。脂氧素 A4 是目前广泛研究的“炎症制动信号”。本研究旨在阐明脂氧素 A4 可以通过诱导人肺微血管内皮细胞（HPMEC）中的 HO-1 来保护细胞，作为急性肺损伤后重症急性胰腺炎的体外模型。
本研究分为两部分：（1）为了研究脂氧素 A4 诱导 HO-1 表达的体外机制，将研究对象分为四组：对照组、脂氧素 A4 组（50ng/mL 脂氧素 A4）、抑制剂组（50ng/mL 脂氧素 A4+20μM LY294002 或 50ng/mL 脂氧素 A4+2nmol/mL Bis II）和激动剂组（50ng/mL 胰岛素样生长因子 1，PMA）。Western blot 用于检测 p-Akt、Akt、蛋白激酶 C（PKC）、p-Nrf2、Nrf2 和 Keap1 的表达，并通过免疫荧光检测 Nrf2 的位置。使用电泳迁移率变动分析检测 Nrf2 诱导的抗氧化反应元件的激活。（2）为了研究脂氧素 A4 在体外诱导的 HO-1 细胞保护作用，将研究对象分为四组：对照组、肿瘤坏死因子 α（TNF-α）组（50ng/mL）、脂氧素 A4 组（50ng/mL TNF-α+50ng/mL 脂氧素 A4）和锌原卟啉 IX 组（用 0.5μM 锌原卟啉 IX 预处理 12h，然后用 50ng/mL TNF-α+50ng/mL 脂氧素 A4）。使用 BCECF/AM 标记的 THP-1 细胞分析 HPMEC 的粘附，使用带有 JC-1 的线粒体膜电位测定试剂盒分析 HPMEC 的凋亡。
在第一部分中，（1）脂氧素 A4 呈剂量依赖性地上调 HO-1 的表达，（2）脂氧素 A4 激活了 PI3K/Akt 和 PKC 途径，并调节了 Nrf2 从 Keap1 上的磷酸化和随后的解聚，促进 Nrf2 向核内易位。在第二部分中，（1）脂氧素 A4 逆转了线粒体膜电位的变化，减轻了 HPMEC 的凋亡，（2）脂氧素 A4 减轻了 TNF-α诱导的 HPMEC 粘附。
脂氧素 A4 可以激活 PI3K/Akt 和 PKC 途径，并诱导 Nrf2 的磷酸化，从而上调 HO-1 的表达。此外，脂氧素 A4 通过上调 HO-1 的表达减轻粘附并保护线粒体功能，从而在重症急性胰腺炎诱导的肺损伤中发挥细胞保护作用。
