摘要
目的:研究艳山姜挥发油(essential oil of Alpinia zerumber fructus,EOAZF)对脂多糖(lipopolysaccharide,LPS)诱导人主动脉内皮细胞(human aortic endothelial cells,HAECs)炎性损伤的保护作用。方法:体外传代培养HAECs,先用不同剂量EOAZF(0.05,0.1 mg·L-1)和阿司匹林(0.5 mmol·L-1)预处理1 h,另设空白组,再与LPS(5 mg·L-1)共同作用12 h。采用噻唑蓝(MTT)比色法检测细胞活性;苏木素-伊红染色进行细胞形态学观察;细胞划痕修复实验分析HAECs划痕修复能力;蛋白免疫印迹法(Western blot)检测血管细胞黏附分子-1(vascular cell adhesion molecule-1,VCAM-1),半胱氨酸天冬氨酸蛋白酶-3(Caspase-3),Toll样受体4(Toll-like receptor 4,TLR4),核转录因子-κB抑制蛋白激酶(inhibitor of nuclear factor kappa-B kinase,IKK),磷酸化NF-κB抑制蛋白激酶(phospho-inhibitor of nuclear factor kappa-B kinase,p-IKK)蛋白的表达;实时荧光定量聚合酶链式反应(Real-time PCR)检测细胞中TLR4和VCAM-1 mRNA的表达;免疫荧光法观察NF-κB p65的核转位情况。结果:与空白组比较,LPS能诱导HAECs形态学损伤,降低细胞存活率及划痕修复能力,同时上调VCAM-1,Caspase-3,TLR4,IKK及PIKK的表达。免疫荧光实验结果分析表明LPS能够诱导NF-κB p65的核转位。与模型组比较,EOAZF预处理后,能够改善LPS诱导的HAECs形态学损伤,提高细胞存活率及划痕修复能力,抑制VCAM-1,Caspase-3,TLR4,IKK及P-IKK的表达及NF-κB p65的核转位。结论:EOAZF对LPS诱导的HAECs炎性损伤具有保护作用,其机制与调控TLR4/IKK/NF-κB-p65信号有关。
Objective: To study the protective effect of essential oil of Alpinia zerumber fructus( EOAZF) on the inflammatory injury of human aortic endothelial cells( HAECs) induced by lipopolysaccharide( LPS). Method: HAECs were cultured in vitro. HAECs were pre-treated with different doses of EOAZF( 0. 05,0. 1 mg·L-1) and aspirin( 0. 5 mmol·L-1) for 1 h,then incubated with lipopolysaccharide( LPS)( 5 mg·L-1)for 12 h. The effect of EOAZF on the cell viability of HAECs induced by LPS was detected by methyl thiazolyl tetrazolium( MTT) assay,hematoxylin eosin( HE) staining was used to observe the cell morphology,and wound healing assay was used to analyze the ability of HAECs wound healing ability. Western blot was used to detect the protein expressions of vascular cell adhesion molecule-1( VCAM-1),cysteine aspartase-3( Caspase-3),Toll like receptor 4( TLR4),nuclear factor kappa B inhibitory protein kinase( IKK) and phosphorylated nuclear factor kappa inhibitory protein kinase( p-IKK),and real-time quantitative polymerase chain reaction( Real-time PCR)was used to detect the mRNA expressions of TLR4 and VCAM-1. The nuclear translocation of nuclear factor kappaB p65( NF-κB p65) was observed by immunofluorescence. Result: Compared with control group,LPS induced HAECs morphological damage,decreased cell viability and wound healing ability,and increased the expressions of VCAM-1,Caspase-3,TLR4,IKK and p-IKK. Immunofluorescence results showed that LPS induced nuclear translocation of NF-κB p65. Compared with model group,the pre-treatment of EOAZF improved the morphological damage of HAECs induced by LPS,increased cell viability and wound healing ability,inhibited the expressions of VCAM-1,Caspase-3,TLR4,IKK and p-IKK,as well as significantly inhibited the nuclear transposition of NF-κB p65. Conclusion: EOAZF has the protective effect on the inflammatory injury of HAECs induced by LPS,and its mechanism may be related to the regulation of TLR4/IKK/NF-κB p65 signaling.
作者
林丹
石廷雨
杨红
吴国萍
付凌云
陶玲
沈祥春
LIN Dan;SHI Ting-yu;YANG Hong;WU Guo-ping;FU Ling-yun;TAO Ling;SHEN Xiang-chun(Key Laboratory of Optimal Utilization of Natural Medicine Resources,Guizhou Medical University,Guiyang 550025,China)
出处
《中国实验方剂学杂志》
CAS
CSCD
北大核心
2018年第19期139-144,共6页
Chinese Journal of Experimental Traditional Medical Formulae
基金
国家自然科学基金项目(81360650)
贵州省留学人员科技活动经费项目(黔人项目资助合同[2013]02号)
贵州省高等教育科技创新团队项目(黔教合人才团队字[2014]31)
贵州省科技创新团队项目(黔科合人才团队[2015]4025号)
贵州省高层次创新型人才百层次人才项目(贵州省科技厅黔科合人才[2015]4029号)
贵阳市科技基金项目(筑科合同[20151001]药07号)
关键词
艳山姜挥发油
脂多糖
阿司匹林
内皮细胞损伤
TOLL样受体4
核转录因子-ΚB
essential oil of Alpinia zerumber fructus
lipopolysaccharide
aspirin
endothelial cell injury
Toll like receptor 4
nuclear factor-kappa B