Centre for Drug and Herbal Development, Faculty of Pharmacy, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.
Department of Biochemistry, Faculty of Medicine, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia.
Int Immunopharmacol. 2024 Jun 15;134:112148. doi: 10.1016/j.intimp.2024.112148. Epub 2024 May 7.
Vascular inflammation is the key event in early atherogenesis. Pro-inflammatory endothelial cells induce monocyte recruitment into the sub-endothelial layer of the artery. This requires endothelial expression of adhesion molecules namely intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), alongside chemokines production. Christia vespertilionis (L.f.) Bakh.f. (CV) possesses anti-inflammatory property. However, its potential anti-atherogenic effect in the context of vascular inflammation has yet to be explored.
To evaluate the anti-atherogenic mechanism of 80% ethanol extract of CV leaves on tumor necrosis factor-α (TNF-α)-activated human umbilical vein endothelial cells (HUVECs).
Qualitative analysis of the CV extract was carried out by using liquid chromatography with tandem mass spectrometry (LC-MS/MS). The cell viability of HUVECs treated with CV extract was determined by MTT assay. The effect of CV extract on monocyte adhesion was determined by monocyte-endothelial adhesion assay. Protein expressions of ICAM-1, VCAM-1 and nuclear factor-kappa B (NF-κB) signaling pathway were determined by western blot while production of monocyte chemoattractant protein-1 (MCP-1) was determined by ELISA.
LC-MS/MS analysis showed that CV extract composed of five main compounds, including schaftoside, orientin, isovitexin, 6-caffeoyl-D-glucose, and 3,3'-di-O-methyl ellagic acid. Treatment of CV extract at a concentration range from 5 to 60 µg/mL for 24 h maintained HUVECs viability above 90 %, therefore concentrations of 20, 40 and 60 μg/mL were selected for the subsequent experiments. All concentrations of CV extract showed a significant inhibitory effect on monocyte adhesion to TNF-α-activated HUVECs (p < 0.05). In addition, the protein expressions of ICAM-1 and VCAM-1 were significantly attenuated by CV in a concentration dependent manner (p < 0.001). At all tested concentrations, CV extract also exhibited significant inhibition on the production of MCP-1 (p < 0.05). Moreover, CV extract significantly inhibited TNF-α-induced phosphorylation of inhibitor of nuclear factor-κB kinase alpha/beta (IKKα/β), inhibitor kappa B-alpha (IκBα), NF-κB and nuclear translocation of NF-κB (p < 0.05).
CV extract inhibited monocyte adhesion to endothelial cells by suppressing protein expressions of cell adhesion molecules and production of chemokines through downregulation of NF-κB signaling pathway. Thus, CV has the potential to be developed as an anti-atherogenic agent for early treatment of atherosclerosis.
血管炎症是动脉粥样硬化早期发生的关键事件。促炎内皮细胞诱导单核细胞募集到动脉的亚内皮层。这需要内皮细胞表达细胞间黏附分子-1（ICAM-1）和血管细胞黏附分子-1（VCAM-1）等黏附分子，以及产生趋化因子。蝙蝠葛（L.f.）Bakh.f.（CV）具有抗炎作用。然而，其在血管炎症背景下的抗动脉粥样硬化作用尚未得到探索。
评估 80%乙醇 CV 叶提取物对肿瘤坏死因子-α（TNF-α）激活的人脐静脉内皮细胞（HUVEC）的抗动脉粥样硬化机制。
采用液相色谱-串联质谱（LC-MS/MS）对 CV 提取物进行定性分析。MTT 法测定 CV 提取物处理的 HUVEC 细胞活力。单核细胞-内皮细胞黏附试验测定 CV 提取物对单核细胞黏附的影响。Western blot 测定 ICAM-1、VCAM-1 和核因子-κB（NF-κB）信号通路蛋白表达，ELISA 测定单核细胞趋化蛋白-1（MCP-1）的产生。
LC-MS/MS 分析显示，CV 提取物由 5 种主要化合物组成，包括梓醇、荭草苷、异荭草苷、6-咖啡酰-D-葡萄糖和 3,3'-二-O-甲基鞣花酸。浓度范围为 5 至 60μg/mL 的 CV 提取物处理 24 小时，维持 HUVEC 活力高于 90%，因此选择 20、40 和 60μg/mL 浓度进行后续实验。所有浓度的 CV 提取物均显著抑制 TNF-α 激活的 HUVEC 上单核细胞的黏附（p<0.05）。此外，CV 以浓度依赖的方式显著减弱 ICAM-1 和 VCAM-1 的蛋白表达（p<0.001）。在所有测试浓度下，CV 提取物对 MCP-1 的产生也表现出显著抑制作用（p<0.05）。此外，CV 提取物显著抑制 TNF-α 诱导的核因子-κB 激酶α/β（IKKα/β）、κB 抑制物α（IκBα）、NF-κB 的磷酸化和 NF-κB 的核转位（p<0.05）。
CV 提取物通过下调 NF-κB 信号通路抑制细胞黏附分子的蛋白表达和趋化因子的产生，抑制单核细胞与内皮细胞的黏附。因此，CV 具有作为抗动脉粥样硬化药物用于早期治疗动脉粥样硬化的潜力。
