摘要
目的:研究绿原酸对巨噬细胞源性泡沫细胞凋亡的影响及其作用机制。方法:以氧化低密度脂蛋白(OxLDL)诱导小鼠腹腔巨噬细胞为巨噬细胞源性泡沫细胞,MTT法检测不同浓度绿原酸对泡沫细胞活性的影响,并用流式细胞术检测绿原酸对细胞凋亡率的影响;用Western Blot法检测绿原酸对凋亡相关蛋白表达的影响,用荧光定量PCR法检测绿原酸对Toll样受体4(TLR4)信号通路相关分子的m RNA表达水平的影响;用双荧光素酶报告基因法检测核因子κB(NF-κB)的转录活性,初步探讨其作用机制。结果:绿原酸浓度依赖性降低巨噬细胞源性泡沫细胞的活力并增加细胞凋亡率,增加凋亡相关蛋白Bax/Bcl-2的比例,抑制TLR4/NF-κB信号通路。结论:绿原酸可抑制巨噬细胞源性泡沫细胞生长并诱导其凋亡,提示绿原酸具有抗动脉粥样硬化的作用,其机制可能与TLR4/NF-κB信号通路有关。
Objective:To investigate the apoptotic effects of chlorogenicacidin macrophage-derived foam cells. Method:Oxidized low-density lipoprotein(OxLDL)was used to induce mouse peritoneal macrophages transfer into foam cells. The effects of chlorogenic acid on the cell viability and apoptosis in foam cell were examined by the MTT assay and the flow cytometry,respectively. Western blotting assay was used to test the expression level of the apoptotic proteins. Real-time quantitative PCR(q-PCR) was used to determine the effects of chlorogenic acid on the mRNA expression of TLR4 signaling molecules. Dual-luciferase reporter gene assay was used to evaluate the effects of chlorogenic acid on the transcriptional activity of NF-κB. Result:Chlorogenic acid dose-dependently inhibited the cell viability and induced apoptosis in macrophage-derived foam cells. The Western blot results showed thatchlorogenic acid enhanced the protein expression ratio of Bax/Bcl-2. Results of Q-PCRand dual-luciferase reporter gene assay showed that chlorogenic acidsuppressed TLR4/NF-κB signaling pathway. Conclusion:Chlorogenic acidinhibited the cell viability and induced apoptosis in macrophage-derived foam cells,which associated with inhibiting the TLR4/NF-κB signaling pathway. These results suggested the antiatherosclerosis activity of chlorogenic acid.
引文
[1]许颖智,张军平,李明,等.四妙勇安汤抑制动脉粥样硬化易损斑块炎症反应机制[J].科技导报,2010,28(23):95-98.
[2]Go AS,Mozaffarian D,Roger VL,et al.Heart disease and stroke statistics-2013 update:a report from the American Heart Association.Circulation,2013,127(1):143-152.
[3]高媛,康林.巨噬细胞源性泡沫细胞的功能变化研究进展[J].中国实用医药,2008,18(3):184-186.
[4]汪玮琳,谢云.泡沫细胞形成与动脉粥样硬化发生机制的研究进展[J].继续医学教育,2015,4(29):68-72.
[5]Babaev VR,Chew JD,Ding L,et al.Macrophage EP4 deficiency increases apoptosis and suppresses early atherosclerosis,Cell Metab,2008,8(6):492-501.
[6]Hamada M,Nakamura M,Tran MT,et al.,MafB promotes atherosclerosis by inhibiting foam-cell apoptosis,Nat Commun,2014,5:3147.
[7]Hundal RS,Salh BS,Schrader JW,et al.Oxidized low density lipoprotein inhibits macrophage apoptosis through activation of the PI 3-kinase/PKB pathway,Lipid Res,2001,42(9):1483-1491.
[8]Arai S,Shelton JM,Chen M,et al.A role for the apoptosis inhibitory factor AIM/Spalpha/Api6 in atherosclerosis development,Cell Metab,2005,1(3):201-213.
[9]成蓓,余其振,柯丽,等.泡沫细胞形成过程中TOLL样受体4表达的变化[J].心血管康复医学杂志,2005,2(15):114-116.
[10]刘祥兰,刘重芳.金银花中氯原酸提取工艺的比较和优化研究[J].中成药,2000,22(6):402-404.
[11]霍晓芳,唐彦萍,张庆军,等.绿原酸对脂多糖诱导的巨噬细胞的影响[J].遵义医学院学报,2003,6(26):507-510.
[12]Zeng P,Liu B,Wang Q,Fan Q,Diao JX,Tang J,Fu XQ,Sun XG.Apigenin Attenuates Atherogenesis through Inducing Macrophage Apoptosis via Inhibition of AKT Ser473 Phosphorylation and Downregulation of Plasminogen Activator Inhibitor-2.Oxid Med Cell Longev,2015,2015:379538.
[13]Ross R.Atherosclerosis-an inflammatory disease.N Engl JMed,1999,340(2):115-126.
[14]Galkina E,Ley K.Immune and inflammatory mechanisms of atherosclerosis.Annu Rev Immunol,2009,27:165-197.
[15]Matsuura E,Kobayashi K,Tabuchi M,Lopez LR.Oxidative modification of low-density lipoprotein and immune regulation of atherosclerosis.Prog Lipid Res,2006,45(6):466-486.
[16]褚现明,李冰,安毅,等.炎症与动脉粥样硬化关系研究进展[J].中国分子心脏病学杂志,2010,11(3):184-188.
[17]Lan Wu.Effect of chlorogenic acid on antioxidant activity of FlosLonicerae extracts.J Zhejiang UnivSci B,2007,8(9):673-679.
[18]Ong KW,Hsu A,Tan BK.Anti-diabetic and anti-lipidemic effects of chlorogenic acid are mediated by ampk activation.BiochemPharmacol,2013,85(9):1341-1351.
[19]Gaestel M,Kotlyarov A,Kracht M.Targeting innate immunity protein kinase signalling in inflammation.Nat Rev Drug Discov,2009,8:480-499.
[20]王彤,刘存志,刘玉珍,等.bcl-2/bax基因调控机体细胞凋亡的机制研究进展[J].中国老年学杂志,2008,28(16):1658-1660.
[21]Autret A,Martin S J.Bcl-2 family proteins and mitochondrial fission/fusion dynamics[J].Cell Mol Life Sci,2010,67(10):1599-1606.
[22]Mohan S,Abdelwahab SI,Kamalidehghan B,Syam S,May KS,Harmal NS,Shafifiyaz N,Hadi AH,Hashim NM,Rahmani M,Taha MM,Cheah SC,Zajmi A.Involvement of NF-κB and Bcl2/Bax signaling pathways in the apoptosis of MCF7 cells induced by a xanthone compound Pyranocycloartobiloxanthone A.Phytomedicine,2012,19(11):1007-1015.
