Department of Respiratory Medicine, the Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450014, China.
Department of Thoracic and Cardiovascular Surgery, the Second Affiliated Hospital of Zhengzhou University, Zhengzhou 450014, China.
Life Sci. 2019 Apr 15;223:137-145. doi: 10.1016/j.lfs.2019.03.017. Epub 2019 Mar 8.
Excessive inflammatory response and oxidative stress are considered as important pathogenic factors in the development of acute lung injury. Isorhynchophylline (IRN), a tetracyclic oxindole alkaloid isolated from Uncaria rhynchophylla, possesses anti-inflammatory and anti-oxidant activities. Our study aimed to investigate the effects and potential mechanisms of IRN on lipopolysaccharide (LPS)-stimulated murine alveolar macrophage cell lines MH-S and NR8383.
CCK-8 assay was used to evaluate the cytotoxicity of IRN and LPS. Inflammatory response was assessed by detecting the mRNA expressions and release of tumor necrosis factor α (TNF-α), interleukin (IL)-1β, IL-6, and plasminogen activator inhibitor-1 (PAI-1) using qRT-PCR and ELISA. The expressions of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 were examined by qRT-PCR and western blot. Oxidative stress was evaluated by detecting malondialdehyde (MDA) level and the activities of superoxide dismutase (SOD), glutathione peroxidase (GSH-Px) and catalase (CAT). The changes of the toll like receptor (TLR4)/nuclear factor-kappa B (NF-κB)/nod-like receptor protein 3 (NLRP3) inflammasome pathway was detected by western blot.
Treatment with LPS or IRN for 24 h showed no cytotoxicity on MH-S and NR8383 cells. IRN pretreatment inhibited LPS-induced production of inflammatory cytokines, expressions of iNOS and COX-2, and oxidative stress in murine alveolar macrophages. Additionally, IRN inhibited LPS-induced activation of TLR4/NF-κB/NLRP3 inflammasome pathway in MH-S cells. Mechanistically, inhibition of TLR4/NF-κB/NLRP3 inflammasome pathway by si-TLR4 suppressed LPS-induced inflammation and oxidative stress in murine alveolar macrophages.
IRN exerted anti-inflammatory and anti-oxidant effects on LPS-stimulated murine alveolar macrophages via inhibition of the TLR4/NF-κB/NLRP3 inflammasome pathway.
过度的炎症反应和氧化应激被认为是急性肺损伤发展的重要致病因素。异钩藤碱（IRN）是从钩藤中分离出的一种四环吲哚生物碱，具有抗炎和抗氧化作用。本研究旨在探讨 IRN 对脂多糖（LPS）刺激的鼠肺泡巨噬细胞系 MH-S 和 NR8383 的作用及潜在机制。
用 CCK-8 法检测 IRN 和 LPS 的细胞毒性。采用 qRT-PCR 和 ELISA 检测肿瘤坏死因子-α（TNF-α）、白细胞介素（IL）-1β、IL-6 和纤溶酶原激活物抑制剂-1（PAI-1）的 mRNA 表达和释放，评估炎症反应。用 qRT-PCR 和 Western blot 检测诱导型一氧化氮合酶（iNOS）和环氧化酶（COX）-2 的表达。通过检测丙二醛（MDA）水平和超氧化物歧化酶（SOD）、谷胱甘肽过氧化物酶（GSH-Px）和过氧化氢酶（CAT）的活性来评估氧化应激。用 Western blot 检测 toll 样受体（TLR4）/核因子-κB（NF-κB）/核苷酸结合寡聚化结构域样受体蛋白 3（NLRP3）炎症小体通路的变化。
用 LPS 或 IRN 处理 24 小时对 MH-S 和 NR8383 细胞无细胞毒性。IRN 预处理抑制 LPS 诱导的鼠肺泡巨噬细胞炎症细胞因子产生、iNOS 和 COX-2 表达和氧化应激。此外，IRN 抑制 LPS 诱导的 MH-S 细胞 TLR4/NF-κB/NLRP3 炎症小体通路的激活。机制上，si-TLR4 抑制 TLR4/NF-κB/NLRP3 炎症小体通路抑制 LPS 诱导的鼠肺泡巨噬细胞炎症和氧化应激。
IRN 通过抑制 TLR4/NF-κB/NLRP3 炎症小体通路，对 LPS 刺激的鼠肺泡巨噬细胞发挥抗炎和抗氧化作用。
