Su Chia-Cheng, Wang Shu-Chi, Chen I-Chen, Chiu Fang-Yen, Liu Po-Len, Huang Chi-Han, Huang Kuan-Hua, Fang Shih-Hua, Cheng Wei-Chung, Huang Shu-Pin, Yeh Hsin-Chih, Liu Ching-Chih, Lee Po-Yen, Huang Ming-Yii, Li Chia-Yang
Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Division of Urology, Department of Surgery, Chi-Mei Medical Center, Tainan, Taiwan.
Front Pharmacol. 2021 May 11;12:652860. doi: 10.3389/fphar.2021.652860. eCollection 2021.
Zerumbone is a natural product isolated from the pinecone or shampoo ginger, (L.) Smith, which has a wide range of pharmacological activities, including anti-inflammatory effects. However, the effects of zerumbone on activation of the NLRP3 inflammasome in macrophages have not been examined. This study aimed to examine the effects of zerumbone on LPS-induced inflammatory responses and NLRP3 inflammasome activation using murine J774A.1 cells, murine peritoneal macrophages, and murine bone marrow-derived macrophages. Cells were treated with zerumbone following LPS or LPS/ATP treatment. Production of nitric oxide (NO) was measured by Griess reagent assay. The levels of IL-6, TNF-α, and IL-1β secretion were analyzed by ELISA. Western blotting analysis was performed to determine the expression of inducible NO synthase (iNOS), COX-2, MAPKs, and NLRP3 inflammasome-associated proteins. The activity of NF-κB was determined by a promoter reporter assay. The assembly of NLRP3 was examined by immunofluorescence staining and observed by confocal laser microscopy. Our experimental results indicated that zerumbone inhibited the production of NO, PGE and IL-6, suppressed the expression of iNOS and COX-2, repressed the phosphorylation of ERK, and decreased the activity of NF-κB in LPS-activated J774A.1 cells. In addition, zerumbone suppressed the production of IL-1β and inhibited the activity of NLRP3 inflammasome in LPS/ATP- and LPS/nigericin-activated J774A.1 cells. On the other hand, we also found that zerumbone repressed the production of NO and proinflammatory cytokines in LPS-activated murine peritoneal macrophages and bone marrow-derived macrophages. In conclusion, our experimental results demonstrate that zerumbone effectively attenuates the LPS-induced inflammatory response in macrophages both and by suppressing the activation of the ERK-MAPK and NF-κB signaling pathways as well as blocking the activation of the NLRP3 inflammasome. These results imply that zerumbone may be beneficial for treating sepsis and inflammasome-related diseases.
姜酮是从松果或红球姜（Zingiber zerumbet (L.) Smith）中分离出的一种天然产物，具有广泛的药理活性，包括抗炎作用。然而，姜酮对巨噬细胞中NLRP3炎性小体激活的影响尚未得到研究。本研究旨在使用小鼠J774A.1细胞、小鼠腹腔巨噬细胞和小鼠骨髓来源的巨噬细胞，研究姜酮对脂多糖（LPS）诱导的炎症反应和NLRP3炎性小体激活的影响。细胞在LPS或LPS/ATP处理后用姜酮处理。通过格里斯试剂法测定一氧化氮（NO）的产生。通过酶联免疫吸附测定（ELISA）分析白细胞介素-6（IL-6）、肿瘤坏死因子-α（TNF-α）和白细胞介素-1β（IL-1β）的分泌水平。进行蛋白质免疫印迹分析以确定诱导型一氧化氮合酶（iNOS）、环氧化酶-2（COX-2）、丝裂原活化蛋白激酶（MAPKs）和NLRP3炎性小体相关蛋白的表达。通过启动子报告基因测定法确定核因子-κB（NF-κB）的活性。通过免疫荧光染色检测NLRP3的组装，并通过共聚焦激光显微镜观察。我们的实验结果表明，姜酮在LPS激活的J774A.1细胞中抑制了NO、前列腺素E（PGE）和IL-6的产生，抑制了iNOS和COX-2的表达，抑制了细胞外信号调节激酶（ERK）的磷酸化，并降低了NF-κB的活性。此外，姜酮在LPS/ATP和LPS/尼日利亚菌素激活的J774A.1细胞中抑制了IL-1β的产生并抑制了NLRP3炎性小体的活性。另一方面，我们还发现姜酮在LPS激活的小鼠腹腔巨噬细胞和骨髓来源的巨噬细胞中抑制了NO和促炎细胞因子的产生。总之，我们的实验结果表明，姜酮通过抑制ERK-MAPK和NF-κB信号通路的激活以及阻断NLRP3炎性小体的激活，有效地减轻了LPS诱导的巨噬细胞炎症反应。这些结果表明姜酮可能对治疗败血症和炎性小体相关疾病有益。
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