粗壮女贞水提物对氧化三甲胺诱导的血管内皮细胞炎症保护作用及机制

Protective effects and mechanism of Ligustrum robustum water extract on trimethylamine N-oxide induced inflammation in vascular endothelial cells

中文关键词:  粗壮女贞水提取物  香蜂草苷  芦丁  氧化三甲胺  血管内皮细胞  炎症因子

英文关键词:Ligustrum robustum water extract  didymin  rutin  trimethylamine N-oxide  vascular endothelial cells  inflammatory factors

中文摘要:

      目的 探究粗壮女贞(Ligustrum robustum, LR)水提物对氧化三甲胺(trimethylamine N-oxide, TMAO)诱导的血管内皮细胞炎症反应的抑制作用及机制。方法 采用TMAO诱导EA.hy926细胞炎症反应, 设置对照组、TMAO组(800 μmol/L TMAO)、LR低、中、高剂量组(0.5、1.0、2.0 mg/mL)、香蜂草苷低、中、高剂量组(20、40、60 μmol/L)和芦丁低、中、高剂量组(20、40、60 μmol/L)。酶联免疫法(enzyme-linked immunosorbent sorbent assay, ELISA)测定各组细胞培养上清液中白细胞介素6 (interleukin-6, IL-6)、白细胞介素8 (interleukin-8, IL-8)、肿瘤坏死因子-α (tumor necrosis factor-α, TNF-α)、细胞间黏附分子(intercellular adhesion molecule-1, ICAM-1)的分泌量。实时荧光定量聚合酶链式反应(realtime fluorescence quantitative polymerase chain reaction, RT-qPCR)法和western blot法检测炎症通路相关基因和蛋白质表达水平; 间接免疫荧光法观察核转录因子-κB (nuclear factor-κB, NF-κB)的核定位。结果 TMAO诱导后EA.hy926细胞IL-6、IL-8、TNF-α、ICAM-1分泌量增加(P<0.05、P<0.01), 相较于TMAO组, 除LR水提物低剂量对IL-6、TNF-α及LR水提物高剂量对ICAM-1分泌量无影响外(P>0.05), LR水提物预处理可下调IL-6、IL-8、TNF-α、ICAM-1分泌量(P<0.05、P<0.01), 香蜂草苷和芦丁预处理可下调TNF-α、ICAM-1分泌量(P<0.05)。LR水提物、香蜂草苷和芦丁预处理可使p38、Nf-κb mRNA水平降低(P<0.05), p38、NF-κB p65蛋白磷酸化比例降低(P<0.05), NF-κB核定位减少。结论 TMAO可诱导血管内皮细胞炎症反应, LR水提物、香蜂草苷和芦丁可以通过抑制EA.hy926细胞p38/NF-κB通路的激活达到抗炎作用。

英文摘要:

      Objective To investigate the anti-inflammatory effects and mechanism of Ligustrum robustum (LR) water extract on the inflammatory response of vascular endothelial cells induced by trimethylamine N-oxide (TMAO). Methods TMAO was used to induce EA.hy926 cells to establish a vascular endothelial cell inflammation model, control group, TMAO group (800 μmol/L TMAO), LR water extract low, medium and high dose group (0.5, 1.0, 2.0 mg/mL), didymin low, medium, high dose group (20, 40, 60 μmol/L), rutin low, medium, high dose group (20, 40, 60 μmol/L) were set up. Enzyme-linked immunosorbent sorbent assays (ELISA) was performed to determine the secretion of interleukin-6 (IL-6), interleukin-8 (IL-8), tumor necrosis factor-α (TNF-α), intercellular adhesion molecule-1 (ICAM-1). The expression of genes and proteins associated to inflammatory pathway were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR) and western blot assay, respectively. Nuclear factor-κB (NF-κB) localization was observed by indirect immunofluorescence. Results The secretion of IL-6, IL-8, TNF-α and ICAM-1 increased in EA.hy926 cells after TMAO treatment (P<0.05), but decreased after pre-treatment with LR water extract (P<0.05, P<0.01), except that the low dose of LR water extract had no effect on the IL-6 and TNF-α secretion and the high dose of LR water extract had no effect on ICAM-1 secretion (P>0.05), didymin or rutin compared with TMAO group (P<0.05). With pre-treatment with LR water extract, the secretion levels of TNF-α and ICAM-1 were down-regulated by didymin and rutin pre-treatment (P<0.05), Under the pre-treatment with LR water extract, didymin and rutin, p38 and Nf-κb mRNA levels and phosphorylation ratios of p38 (P<0.05), NF-κB were downregulated and the NF-κB nuclear localization was reduced. Conclusion TMAO can induce inflammatory response in vascular endothelial cells, while LR water extract, didymin and rutin can inhibit the activation of p38/NF-κB pathway in EA.hy926 cells to achieve anti-inflammatory effect.