黑米花色苷矢车菊素-3-O-β-葡萄糖苷对脂多糖诱导的人THP-1单核细胞炎症损伤的保护作用

Protective Effect of Black Rice-Derived Cyanidin-3-O-β-Glucoside on Lipopolysaccharide-Induced Inflammatory Injury in Human Monocyte Cell Line THP-1

单核细胞介导的炎症反应在动脉粥样硬化发生和发展过程中起关键作用。花色苷是一种具有多种生物学活性的多酚类黄酮化合物,富含于各种深色的蔬菜、水果及谷类中,其中矢车菊素-3-O-β-葡萄糖苷(cyanidin-3-O-β-glucoside,Cy-3-g)是花色苷中重要的单体,本研究旨在探讨黑米来源的Cy-3-g对脂多糖(lipopolysaccharide,LPS)诱导的人单核白血病细胞(Tohoku hospital pediatrics-1,THP-1)炎症损伤的作用及可能的分子机制。采用乙醇-盐酸溶液浸提、大孔树脂吸附洗脱等步骤得到黑米花色苷粗提物,然后用中压液相层析联合紫外检测提取得到纯化的Cy-3-g(纯度>96.5%)。用不同质量浓度(0、0.10、0.25μg/mL和0.50μg/mL)Cy-3-g与THP-1细胞共孵育4 h,然后加入LPS(质量浓度50 ng/mL)与细胞继续孵育48 h,用酶联免疫吸附试验法测定培养液中白细胞介素-1β(interleukin-1β,IL-1β)、IL-6、IL-8、IL-10和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平,用定量聚合酶链式反应(polymerase chain reaction,PCR)检测细胞中IL-1β、IL-6、IL-8、IL-10、TNF-α、Toll-样受体4(Toll-like receptor 4,TLR4)和核因子κB(nuclear factor kappa B,NF-κB)p65的mRNA表达水平,用Western blot蛋白免疫印迹法检测TLR4、NF-κB p65、核因子κB抑制因子(inhibitor of NF-κB,IκBα)蛋白表达水平。结果表明,与溶剂对照组相比,LPS诱导的THP-1细胞IL-1β、IL-6和TNF-α mRNA相对表达量和释放水平可被不同质量浓度Cy-3-g显著抑制(P<0.05、P<0.01、P<0.001),在LPS作用下,质量浓度0.25、0.50μg/mL Cy-3-g能够显著促进IL-10 mRNA表达(P<0.01、P<0.001),质量浓度0.10、0.25、0.50μg/mL Cy-3-g能够显著促进IL-10释放(P<0.05、P<0.01、P<0.001),但是不同质量浓度Cy-3-g对LPS诱导的细胞IL-8 m RNA表达水平和释放水平的促进作用无显著差异(P>0.05)。质量浓度0.25、0.50μg/m L Cy-3-g能够显著抑制TLR4和NF-κB p65的m RNA和蛋白表达(P<0.05、P<0.001、P<0.01),同时能够抑制IκBα和NF-κB p65的磷酸化,并抑制LPS对IκBα的降解作用。与溶剂对照组相比,NF-κB的特异性抑制剂Bay11-7082可高度显著下调LPS诱导的THP-1细胞IL-1β、IL-6和TNF-α释放水平(P<0.001),但是与质量浓度0.50μg/mL Cy-3-g联合使用时无协同抑制效果(P>0.05)。综上,黑米花色苷Cy-3-g能够对LPS诱导的THP-1单核细胞炎症损伤起到保护作用,其机制可能是下调TLR4/NF-κB介导的信号通路。

Monocyte-mediated inflammatory responses play a key role in the progression of atherosclerosis.Anthocyanins are polyphenolic flavonoids that are abundant in various dark-colored vegetables,fruits and cereals,which possess a wide range of biological activities.Cyanidin-3-O-β-glucoside(Cy-3-g)is an important anthocyanin monomer.The current study aimed to explore the effects of black rice-derived Cy-3-g on lipopolysaccharide(LPS)-induced inflammatory responses in Tohoku hospital pediatrics-1(THP-1)as well as its possible mechanism of action.Crude anthocyanins from black rice were obtained by extraction with ethanol-hydrochloric acid solution and macroporous resin adsorption and purified by medium-pressure liquid chromatography with UV detection.Cy-3-g with a purity greater than 96.5%was obtained.THP-1 monocytes were sequentially incubated with different concentrations(0,0.1,0.25 and 0.5μg/mL)Cy-3-g for 4 h,and LPS(50 ng/mL)for additional 48 h.The levels of interleukin(IL)-1β,IL-6,IL-8,IL-10 and tumor necrosis factor(TNF)-αin the culture were determined by enzyme-linked immunosorbent assay(ELISA),and the mRNA expression levels of the genes encoding IL-1β,IL-6,IL-8,IL-10,TNF-α,Toll-like receptor 4(TLR4)and nuclear factor kappa B(NF-κB)p65 were measured by quantitative polymerase chain reaction(PCR).Moreover,the protein expression levels of TLR4,NF-κB p65 and inhibitor of NF-κB(IκBα)were detected by Western blot.The LPS-induced increase in the relative mRNA expression levels and release levels of IL-1β,IL-6 and TNF-αin THP-1 cells was significantly inhibited by all concentrations of Cy-3-g tested(P<0.05,P<0.01 and P<0.001,respectively).Cy-3-g at 0.25 and 0.50μg/mL significantly blunted the inhibitory effect of LPS on IL-10 mRNA expression(P<0.01 and P<0.001,respectively).Cy-3-g at 0.10,0.25 and 0.50μg/mL significantly mitigated the inhibitory effect of LPS on IL-10 release(P<0.05,P<0.01 and P<0.001,respectively),but had no significant influence on LPS-induced promotion of IL-8 mRNA expression and release levels(P>0.05).Cy-3-g at 0.25 and 0.50μg/mL significantly inhibited the mRNA and protein expression of TLR4 and NF-κB p65(P<0.05,P<0.01 and P<0.001,respectively),suppressed the expression of phosphorylated IκBαand NF-κB,and inhibited LPS-induced IκBαdegradation.Bay11-7082,a specific inhibitor of NF-κB,significantly decreased the release of IL-1β,IL-6 and TNF-αin LPS-stimulated THP-1 cells(P<0.001),but had no synergistic effects with 0.50μg/mL Cy-3-g(P>0.05).Black rice-derived Cy-3-g exerts a protective effect on LPS-induced inflammatory injury of THP-1 monocytes possibly through down-regulating the TLR4/NF-κB signaling pathway.