Abstract：
BACKGROUND: Uncontrolled inflammation causes health problems. Extracellular signal-regulated kinase (ERK) phosphorylates signal transducer and activator of transcription 3 (STAT3) at Ser727, resulting in inflammation. The leaf of Vernonia amygdalina (VA) is a medicinal herb for managing inflammation-associated diseases. Oral administration or topical application of VA leaf extract exerts anti-inflammatory effects in rat models. However, the anti-inflammatory mechanisms of the herb are not fully understood.
OBJECTIVE: In this study, we aimed to investigate the involvement of ERK/STAT3 (Ser727) signaling in the anti-inflammatory effects of an ethanolic extract of VA leaves.
METHODS: Extracts of VA leaves were prepared with different concentrations of ethanol. A LPS-stimulated RAW264.7 cell model was used for in vitro assays, and a TPA (12-O-tetradecanoylphorbol-13-acetate)-induced ear edema mouse model was employed for in vivo assays. The 95% ethanol extract of VA leaves (VAE) exerted the strongest inhibitory effect on nitric oxide (NO) production in LPS-stimulated macrophages; thus it was selected for use in this study. Hematoxylin and eosin (H&E) staining was used to examine pathological conditions of mouse ear tissues. Griess reagent was employed to examine NO generation in cell cultures. Immunoblotting and ELISA were used to examine protein levels, and RT-qPCR was employed to examine mRNA levels.
RESULTS: Topical application of VAE ameliorated mouse ear edema induced by TPA. VAE suppressed the phosphorylation of ERK (Thr202/Tyr204) and STAT3 (Ser727); and decreased protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-6, IL-1β and tumor necrosis factor-α (TNF-α) in the mouse ear tissues and in LPS-stimulated RAW 264.7 cells. VAE also inhibited NO production, and lowered mRNA levels of IL-6, IL-1β and TNF-α in the macrophages.
CONCLUSIONS: VAE ameliorates TPA-induced mouse ear edema. Suppression of ERK/STAT3 (Ser727) signaling is involved in VAE\'s anti-inflammatory effects. These novel data provide further pharmacological justifications for the medicinal use of VA in treating inflammation-associated diseases, and lay the groundwork for developing VAE into a new anti-inflammatory agent.
OBJECTIVE: In this study, we aimed to investigate the involvement of ERK/STAT3 (Ser727) signaling in the anti-inflammatory effects of an ethanolic extract of VA leaves.
METHODS: Extracts of VA leaves were prepared with different concentrations of ethanol. A LPS-stimulated RAW264.7 cell model was used for in vitro assays, and a TPA (12-O-tetradecanoylphorbol-13-acetate)-induced ear edema mouse model was employed for in vivo assays. The 95% ethanol extract of VA leaves (VAE) exerted the strongest inhibitory effect on nitric oxide (NO) production in LPS-stimulated macrophages; thus it was selected for use in this study. Hematoxylin and eosin (H&E) staining was used to examine pathological conditions of mouse ear tissues. Griess reagent was employed to examine NO generation in cell cultures. Immunoblotting and ELISA were used to examine protein levels, and RT-qPCR was employed to examine mRNA levels.
RESULTS: Topical application of VAE ameliorated mouse ear edema induced by TPA. VAE suppressed the phosphorylation of ERK (Thr202/Tyr204) and STAT3 (Ser727); and decreased protein levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), interleukin (IL)-6, IL-1β and tumor necrosis factor-α (TNF-α) in the mouse ear tissues and in LPS-stimulated RAW 264.7 cells. VAE also inhibited NO production, and lowered mRNA levels of IL-6, IL-1β and TNF-α in the macrophages.
CONCLUSIONS: VAE ameliorates TPA-induced mouse ear edema. Suppression of ERK/STAT3 (Ser727) signaling is involved in VAE\'s anti-inflammatory effects. These novel data provide further pharmacological justifications for the medicinal use of VA in treating inflammation-associated diseases, and lay the groundwork for developing VAE into a new anti-inflammatory agent.
摘要：
背景：未控制的炎症会导致健康问题。细胞外信号调节激酶（ERK）在Ser727处磷酸化信号转导子和转录激活因子3（STAT3），导致炎症。Vernoniaamgdalina（VA）的叶子是一种用于治疗炎症相关疾病的草药。口服或局部施用VA叶提取物在大鼠模型中发挥抗炎作用。然而,该草药的抗炎机制尚未完全了解。
目的：在本研究中，我们旨在研究ERK/STAT3(Ser727)信号在VA叶乙醇提取物抗炎作用中的作用.
方法：用不同浓度的乙醇制备VA叶提取物。LPS刺激的RAW264.7细胞模型用于体外测定，和TPA（12-O-十四烷酰基佛波醇-13-乙酸酯）诱导的耳水肿小鼠模型用于体内测定。VA叶（VAE）的95％乙醇提取物对LPS刺激的巨噬细胞中一氧化氮（NO）的产生具有最强的抑制作用；因此，将其选择用于本研究。苏木精和伊红(H&E)染色用于检查小鼠耳组织的病理状况。使用Griess试剂检查细胞培养物中的NO生成。免疫印迹和ELISA用于检测蛋白质水平，采用RT-qPCR检测mRNA水平。
结果：局部应用VAE可改善TPA诱导的小鼠耳部水肿。VAE抑制了ERK（Thr202/Tyr204）和STAT3（Ser727）的磷酸化；并降低了诱导型一氧化氮合酶（iNOS）的蛋白质水平，环氧合酶-2（COX-2），小鼠耳组织和LPS刺激的RAW264.7细胞中的白介素（IL）-6，IL-1β和肿瘤坏死因子-α（TNF-α）。VAE还抑制NO的产生，并降低巨噬细胞中IL-6，IL-1β和TNF-α的mRNA水平。
结论：VAE可改善TPA诱导的小鼠耳部水肿。ERK/STAT3(Ser727)信号传导的抑制涉及VAE的抗炎作用。这些新数据为VA在治疗炎症相关疾病中的药用用途提供了进一步的药理学理由。并为将VAE开发成新型抗炎药奠定基础。
目的：在本研究中，我们旨在研究ERK/STAT3(Ser727)信号在VA叶乙醇提取物抗炎作用中的作用.
方法：用不同浓度的乙醇制备VA叶提取物。LPS刺激的RAW264.7细胞模型用于体外测定，和TPA（12-O-十四烷酰基佛波醇-13-乙酸酯）诱导的耳水肿小鼠模型用于体内测定。VA叶（VAE）的95％乙醇提取物对LPS刺激的巨噬细胞中一氧化氮（NO）的产生具有最强的抑制作用；因此，将其选择用于本研究。苏木精和伊红(H&E)染色用于检查小鼠耳组织的病理状况。使用Griess试剂检查细胞培养物中的NO生成。免疫印迹和ELISA用于检测蛋白质水平，采用RT-qPCR检测mRNA水平。
结果：局部应用VAE可改善TPA诱导的小鼠耳部水肿。VAE抑制了ERK（Thr202/Tyr204）和STAT3（Ser727）的磷酸化；并降低了诱导型一氧化氮合酶（iNOS）的蛋白质水平，环氧合酶-2（COX-2），小鼠耳组织和LPS刺激的RAW264.7细胞中的白介素（IL）-6，IL-1β和肿瘤坏死因子-α（TNF-α）。VAE还抑制NO的产生，并降低巨噬细胞中IL-6，IL-1β和TNF-α的mRNA水平。
结论：VAE可改善TPA诱导的小鼠耳部水肿。ERK/STAT3(Ser727)信号传导的抑制涉及VAE的抗炎作用。这些新数据为VA在治疗炎症相关疾病中的药用用途提供了进一步的药理学理由。并为将VAE开发成新型抗炎药奠定基础。
