Department of Emergency Medicine, Shengjing Hospital of China Medical University, 36 Sanhao Street, Shenyang, 110004, People's Republic of China.
Department of Endocrinology, Shengjing Hospital of China Medical University, Shenyang, 110004, People's Republic of China.
Inflammation. 2017 Aug;40(4):1319-1330. doi: 10.1007/s10753-017-0575-8.
Acute lung injury (ALI) is a major complication soon after paraquat poisoning and rapidly progresses with high mortality. However, the specific mechanism underlying paraquat-induced ALI is still unclear. In this study, the mechanism underlying the protective effects of SP600125 on paraquat-induced ALI was investigated according to oxidative stress, inflammation, and apoptosis. The rats were randomly assigned into the control group (CON), the paraquat poisoning group (PQ), and the PQ + SP600125 group (SP). A549 cells were divided into the Con group, Pq group, and Sp group. H&E staining and detection of lung wet/dry ratio were employed to evaluate lung injury. Annexin V-PI staining was done to evaluate A549 cell apoptosis. Dihydroethidium fluorescence was used to measure reactive oxygen species (ROS) in the lungs and A549 cells. ELISA was performed to detect TNF-α and IL-6 in the supernatant of bronchoalveolar lavage fluid (BALF) and A549 cells. RT-qPCR was done to measure the messenger RNA (mRNA) expression of TNF-α and IL-6 in the lungs and A549 cells. Western blotting assay was performed to detect the protein expression of phospho-JNK, total JNK, and cleaved caspase-3. Electrophoretic mobility shift assay was employed to detect the DNA binding activities of AP-1 and P-p65. JNK inhibitor SP600125 reduced JNK phosphorylation, downregulated cleaved caspase-3 protein level, decreased AP-1 transcriptional activity and ROS level, and reduced the transcription and expression of TNF-α and IL-6, which improved ALI and cell apoptosis after paraquat poisoning. Our results indicate that JNK/AP-1 mediates ALI as well as oxidative stress and inflammation deterioration secondary to paraquat poisoning.
急性肺损伤（ALI）是百草枯中毒后不久的主要并发症，死亡率很高，且迅速进展。然而，百草枯诱导的 ALI 的具体机制仍不清楚。在这项研究中，根据氧化应激、炎症和细胞凋亡，研究了 SP600125 对百草枯诱导的 ALI 的保护作用机制。大鼠被随机分为对照组（CON）、百草枯中毒组（PQ）和 PQ+SP600125 组（SP）。A549 细胞分为 Con 组、Pq 组和 Sp 组。采用 H&E 染色和肺湿/干比检测评估肺损伤。用 Annexin V-PI 染色评估 A549 细胞凋亡。用二氢乙啶荧光法检测肺和 A549 细胞中的活性氧（ROS）。通过 ELISA 检测支气管肺泡灌洗液（BALF）和 A549 细胞上清液中 TNF-α和 IL-6 的含量。通过 RT-qPCR 检测肺和 A549 细胞中 TNF-α和 IL-6 的 mRNA 表达。通过 Western blot 检测磷酸化 JNK、总 JNK 和 cleaved caspase-3 的蛋白表达。通过电泳迁移率变动分析检测 AP-1 和 P-p65 的 DNA 结合活性。JNK 抑制剂 SP600125 降低了 JNK 磷酸化，下调了 cleaved caspase-3 蛋白水平，降低了 AP-1 转录活性和 ROS 水平，降低了 TNF-α和 IL-6 的转录和表达，改善了百草枯中毒后的 ALI 和细胞凋亡。我们的结果表明，JNK/AP-1 介导了 ALI 以及百草枯中毒后氧化应激和炎症恶化。
