摘要
为研究槲皮素对脂多糖(Lipopolysaccharides,LPS)诱导人脑微血管内皮细胞(Human brain microvascular endothelial cell,HBMEC)的作用机制,体外培养HBMEC,用LPS诱导HBMEC造模,将细胞分为正常组、模型组、槲皮素组及依达拉奉组,采用噻唑蓝法检测LPS诱导炎症因子变化的时间和槲皮素对HBMEC增殖能力的影响,用实时定量PCR法检测肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)mRNA、趋化因子C—C基元配体20(chemokine C—C motif ligand 20,CCL20)mRNA和白细胞介素-8(interleukin-8,IL-8)mRNA的表达,用酶联免疫吸附法检测TNF-α,CCL20和IL-8的含量,用蛋白免疫印迹法检测p-NF-ΚB p65,NF-ΚB p65,p-IKKα和IKKα的蛋白表达。结果发现,与正常组比,1 mg·L^-1的LPS组细胞活力明显下降(P<0.01);模型8 h组TNF-αmRNA,CCL20 mRNA,IL-8 mRNA表达显著上升(P<0.01);槲皮素各给药组(12.5,25,50,100,200,400μmol·L^-1)细胞活力变化不明显。与模型组相比,200μmol·L^-1槲皮素组的模型细胞增殖明显(P<0.01);槲皮素组和依达拉奉组TNF-αmRNA,CCL20 mRNA,IL-8 mRNA表达和TNF-α,CCL20,IL-8的含量显著下降(P<0.05);槲皮素组蛋白数量比n(p-NF-ΚBp65)/n(NF-ΚBp65),n(p-IKKα)/n(IKKα)显著减小(P<0.01),而依达拉奉组n(p-NF-ΚBp65)/n(NF-ΚBp65),n(p-IKKα)/n(IKKα)无明显变化。说明槲皮素具有抑制LPS诱导HBMEC的损伤作用,可能是通过下调IKKα/NF-ΚB p65信号通路减少炎症相关因子TNF-α,CCL20和IL-8的表达。这为槲皮素应用于脑血管病变引起的帕金森病临床防治提供了实验依据。
In order to study the mechanism of quercetin on human brain microvascular endothelial cells(HBMEC)induced by lipopolysaccharide(LPS),HBMEC was cultured in vitro,and induced by LPS.The cells were divided into four groups:the normal group,model group,quercetin group,and edaravone group.The time of LPS-induced inflammatory factors and the impact of quercetin on the proliferation of HBMEC were detected by the thiazolyl blue method.Tumor necrosis factor-α(TNF-α)mRNA and chemokine were detected by real-time quantitative PCR.The expression of chemokine C—C motif ligand 20(CCL20)mRNA and interleukin-8(IL-8)mRNA,and the detection of TNF-α,CCL20 and IL-8 were conducted by the enzyme-linked immunosorbent assay.The protein content of p-NF-ΚB p65,NF-ΚB p65,p-IKKαand IKKαwas detected by Western blot.Compared with that of the normal group,the cell viability of the LPS group at 1 mg/L was significantly decreased(P<0.01);the release of TNF-αmRNA,CCL20 mRNA and IL-8 mRNA in the model group was significantly increased(P<0.01);the cell viability of the drug-administered group(12.5,25,50,100,200,400μmol/L)was not significant.Compared to the model group,model cells in the 200μmol/L quercetin group proliferated significantly(P<0.01);TNF-αmRNA,CCL20 mRNA,IL-8 mRNA release and the content of TNF-α,CCL20 and IL-8 in quercetin and edaravone groups were significantly decreased(P<0.05);the protein expression ratio of n(p-NF-ΚBp65)/n(NF-ΚBp65)and n(p-IKKα)/n(IKKα)in the quercetin group was significantly decreased(P<0.01).In the edaravone group,the protein expression ratio of n(p-NF-ΚBp65)/n(NF-ΚBp65)and n(p-IKKα)/n(IKKα)did not change significantly.Quercetin can inhibit the injury induced by LPS-induced HBMEC,possibly by the mechanism of down-regulating the IKKα/NF-ΚB p65 signaling pathway to reduce the expression of TNF-α,CCL20 and IL-8.It should be beneficial to use quercetin in clinical prevention and treatment on cerebrovascular diseases such as Parkinson's disease.
作者
秦劭晨
王爱梅
张晋岳
李若瑜
李万婷
QIN Shao-chen;WANG Ai-mei;ZHANG Jin-yue;LI Ruo-yu;LI Wan-ting(The Affiliated Hospital,Shanxi University of Chinese Medicine,Taiyuan 030024,China)
出处
《湖南师范大学自然科学学报》
CAS
北大核心
2019年第6期38-45,共8页
Journal of Natural Science of Hunan Normal University
基金
山西省卫生和计划生育委员会科研课题(201601119)
山西中医药大学科技创新能力培育计划项目(2019PY-051)
