Karunarathne Wisurumuni Arachchilage Hasitha Maduranga, Lee Kyoung Tae, Choi Yung Hyun, Jin Cheng-Yun, Kim Gi-Young
Department of Marine Life Sciences, Jeju National University, Jeju 63243, Republic of Korea.
Forest Biomaterials Research Center, National Institute of Forest Science, Jinju 52817, Republic of Korea.
Phytomedicine. 2020 May 17;76:153237. doi: 10.1016/j.phymed.2020.153237.
Hibiscus syriacus L. has been used as a medicinal plant in many Asian countries. However, anti-inflammatory activity of H. syriacus L. remains unknown.
This study was aimed to investigating the anti-inflammatory effect of anthocyanin fractions from the H. syriacus L. variety Pulsae (PS) on the lipopolysaccharide (LPS)-induced inflammation and endotoxic shock.
MTT assay and flow cytometry analysis were performed to determine cytotoxicity of PS. RT-PCR, western blotting, and ELISA were conducted to evaluate the expression of proinflammatory mediators and cytokines. Molecular docking study predicted the binding scores and sites of PS to TLR4/MD2 complex. Immunohistochemical assay was conducted to evaluate the binding capability of PS to TLR4/MD2 and nuclear translocation of NF-κB p65. A zebrafish endotoxic shock model was used to evaluate anti-inflammatory activity of PS in vivo.
PS suppressed LPS-induced nitric oxide and prostaglandin E secretion concomitant with the downregulation of inducible nitric oxide synthase and cyclooxygenase-2 expression. Furthermore, PS inhibited the production of proinflammatory cytokines such as TNF-α, IL-6, and IL-12 in LPS-stimulated RAW 264.7 macrophages. Additionally, molecular docking data showed that PS mostly fit into the hydrophobic pocket of MD2 and bound to TLR4. In particular, apigenin-7-O-glucoside powerfully bound to MD2 and TLR4 via hydrogen bonding. Additionally, immunohistochemistry assay revealed that PS inhibited LPS-induced TLR4 dimerization or expression on the cell surface, which consequently decreased MyD88 recruitment and IRAK4 phosphorylation, resulting in the inhibition of NF-κB activity. PS also attenuated LPS-mediated mortality and abnormality in zebrafish larvae and diminished the recruitment of neutrophils and macrophages at the inflammatory site accompanied by the low levels of proinflammatory mediators and cytokines.
PS might be a novel immunomodulator for the effective treatment of LPS-mediated inflammatory diseases.
木槿在许多亚洲国家一直被用作药用植物。然而，木槿的抗炎活性尚不清楚。
本研究旨在探讨木槿品种“普赛”（PS）中的花青素组分对脂多糖（LPS）诱导的炎症和内毒素休克的抗炎作用。
进行MTT法和流式细胞术分析以确定PS的细胞毒性。采用RT-PCR、蛋白质印迹法和酶联免疫吸附测定法评估促炎介质和细胞因子的表达。分子对接研究预测了PS与TLR4/MD2复合物的结合分数和位点。进行免疫组织化学测定以评估PS与TLR4/MD2的结合能力以及NF-κB p65的核转位。使用斑马鱼内毒素休克模型在体内评估PS的抗炎活性。
PS抑制LPS诱导的一氧化氮和前列腺素E分泌，同时下调诱导型一氧化氮合酶和环氧化酶-2的表达。此外，PS抑制LPS刺激的RAW 264.7巨噬细胞中促炎细胞因子如TNF-α、IL-6和IL-12的产生。此外，分子对接数据显示PS大多适合MD2的疏水口袋并与TLR4结合。特别是，芹菜素-7-O-葡萄糖苷通过氢键与MD2和TLR4强力结合。此外，免疫组织化学测定显示PS抑制LPS诱导的TLR4二聚化或细胞表面表达，从而减少MyD88募集和IRAK4磷酸化，导致NF-κB活性受到抑制。PS还减轻了LPS介导的斑马鱼幼虫死亡率和异常情况，并减少了炎症部位中性粒细胞和巨噬细胞的募集，同时伴有促炎介质和细胞因子水平降低。
PS可能是一种新型免疫调节剂，可有效治疗LPS介导的炎症性疾病。
