摘要
目的观察白藜芦醇苷对氧化低密度脂蛋白(ox-LDL)诱导THP-1巨噬细胞(MDM)炎性增殖与细胞因子表达的影响及机制。方法培养THP-1巨噬细胞,分为对照组(仅加入普通培养基)、ox-LDL组(80μmol/L ox-LDL处理细胞24 h)、白藜芦醇苷处理组(100μmol/L白藜芦醇苷预处理2 h后,再加入80μmol/L ox-LDL处理24 h)、EX-527组(10μmol/L EX-527预处理细胞2 h)。CCK-8法检测各组细胞增殖率,分光光度计检测细胞内超氧化物歧化酶(SOD)以及丙二醛(MDA)含量,2',7'-二氯二氢荧光素二乙酸酯(DCFH-DA)负载法检测活性氧(ROS)的含量;实时定量PCR法检测白藜芦醇苷对组蛋白去乙酰化酶沉默信息调节因子1(SIRT1)、单核细胞趋化因子1(MCP-1)、肿瘤坏死因子α(TNF-α)、白细胞介素6(IL-6)mRNA水平的影响,Western blot法检测SIRT1、MCP-1、TNF-α、IL-6的蛋白水平。结果 ox-LDL促进THP-1巨噬细胞增殖。100μmol/L白藜芦醇苷组明显抑制ox-LDL诱导的细胞增殖,同时明显增加SOD含量,降低胞内MDA与ROS含量。ox-LDL抑制THP-1巨噬细胞中SIRT1 mRNA与蛋白表达,促进MCP-1、TNF-α、IL-6 mRNA与蛋白表达。白藜芦醇苷显著增加ox-LDL诱导后SIRT1 mRNA与蛋白表达,抑制MCP-1、TNF-α、IL-6 mRNA与蛋白表达,EX-527能抑制白藜芦醇苷上述保护作用。结论白藜芦醇苷可通过上调SIRT1表达提高抗巨噬细胞增殖的能力,减少ox-LDL诱导细胞内活性氧生成、抑制炎性因子表达。
Objective To investigate the effects of polydatin on cell proliferation and cytokine expression in THP-1 monocyte-derived macrophages( MDMs) induced by oxidized low-density lipoprotein( ox-LDL),and the possible mechanisms.Methods MDMs were divided into the control group( only treated with ordinary culture medium),ox-LDL group( treated wtih80 μmol/L ox-LDL for 24 hours),polydatin treatment group( treated with 100 μmol/L polydatin for 2 hours prior to the treatment with 80 μmol/L ox-LDL for 24 hours) and EX-527 treatment group( treated with 10 μmol/L SIRT1 inhibitor EX-527 for 2 hours prior to the treatment with ox-LDL and polydatin). The effects of polydatin on ox-LDL-induced oxidative proliferation and cytokine expression in MDMs were evaluated by CCK-8 assay. Spectrofluorometry was used to determine the intracellular level of superoxide dismutase( SOD) and malondialdehyde( MDA). DCFH-DA loading was used to detect the content of reactive oxidative species( ROS). The levels of silent mating type information regulator 1( SIRT1),monocyte chemoattractant protein-1( MCP-1),tumor necrosis factor-α( TNF-α) and interleukin-6( IL-6) were examined by real-time quantitative PCR and Western blotting. Results Polydatin( 100 μmol/L) inhibited the proliferation of MDMs induced by ox-LDL,decreased the levels of MDA and ROS,whereas the level of SOD increased. The mRNA and protein levels ofSIRT1 in MDMs were inhibited by ox-LDL,whereas the expressions of MCP-1,TNF-α and IL-6 were promoted. Pre-treatment with EX-527 attenuated the inhibitory effects of polydatin on the proliferation of MDMs,inhibited the expressions of SIRT1,promoted the expressions of MCP-1,TNF-α and IL-6. Conclusion Polydatin up-regulates the expression of SIRT1 to increase the ability of anti-macrophage proliferation,reduce the level of the intracellular ROS induced by ox-LDL,and inhibit the expression of inflammatory cytokines.
引文
[1]Lv J X,Kong Q,Ma X.Current advances in circulating inflammator biomarkers in atherosclerosis and related cardio-cerebrovascular diseases[J].Chronic Dis Transl Med,2017,3(4):207-212.
[2]Wang X Q,Liu Z H,Xue L,et al.C1q/TNF-related protein 1 links macrophage lipid metabolism to inflammation and atherosclerosis[J].Atherosclerosis,2016,250:38-45.
[3]Tajfard M,Latiff L A,Rahimi H R,et al.Serum concentrations of MCP-1 and IL-6 in combination predict the presence of coronary artery disease and mortality in subjects undergoing coronary angiography[J].Mol Cell Biochem,2017,435(1/2):37-45.
[4]Yang Q,Yuan H,Chen M,et al.Metformin ameliorates the progression of atherosclerosis via suppressing macrophage infiltration and inflammatory responses in rabbits[J].Life Sci,2018,198:56-64.
[5]Fran9a C N,Izar M C O,Hortêncio M N S,et al.Monocyte subtypes and the CCR2 chemokine receptor in cardiovascular disease[J].Clin Sci(Lond),2017,131(12):1215-1224.
[6]Weiskirchen S,Weiskirchen R.Resveratrol:How much wine do you have to drink to stay healthy?[J].Adv Nutr,2016,7(4):706-718.
[7]Pang N,Chen T,Deng X,et al.Polydatin prevents methylglyoxalinduced apoptosis through reducing oxidative stress and improving mitochondrial function in human umbilical vein endothelial cells[J/OL].Oxid Med Cell Longev,2017,2017:7180943.DOI:10.1155/2017/7180943.Epub 2017 Sep 13.
[8]Deng X,Zheng H,Li D,et al.microRNA-34a regulates proliferation and apoptosis of gastric cancer cells by targeting silent information regulator 1[J].Exp Ther Med,2018,15(4):3705-3714.
[9]Kitada M,Ogura Y,Koya D.The protective role of Sirt1 in vascular tissue:its relationship to vascular aging and atherosclerosis[J].Aging(Albany NY),2016,8(10):2290-2307.
[10]Shimada T,Furuta H,Doi A,et al.Des-acyl ghrelin protects microvascular endothelial cells from oxidative stress-induced apoptosis through sirtuin 1 signaling pathway[J].Metabolism,2014,63(4):469-474.
[11]Hirano T,Mori Y.Anti-atherogenic and anti-inflammatory properties of glucagon-like peptide-1,glucose-dependent insulinotropic polypepide,and dipeptidyl peptidase-4 inhibitors in experimental animals[J].J Diabetes Investig,2016,7(Suppl 1):80-86.
[12]Ooi B K,Goh B H,Yap W H.Oxidative stress in cardiovascular diseases:Involvement of Nrf2 antioxidant redox signaling in macrophage foam cells formation[J/OL].Int J Mol Sci,2017,5:18(11):E2336.DOI:10.3390/ijms18112336.
[13]Yamagata K,Tusruta C,Ohtuski A,et al.Docosahexaenoic acid decreases TNF-α-induced lectin-like oxidized low-density lipoprotein receptor-1 expression in THP-1 cells[J].Prostaglandins Leukot Essent Fatty Acids,2014,90(4):125-132.
[14]Chen L,Yang W,Guo Y,et al.Exosomal lncRNA GAS5 regulates the apoptosis of macrophages and vascular endothelial cells in atherosclerosis[J/OL].PLo S One,2017,12(9):e0185406.DOI:10.1371/journal.pone.0185406.e Collection 2017.
[15]Qin Z.The use of THP-1 cells as a model for mimicking the function and regulation of monocytes and macrophages in the vasculature[J].Atherosclerosis,2012,221(1):2-11.
[16]Bryk D,Olejarz W,Zapolska-Downar D.The role of oxidative stress and NADPH oxidase in the pathogenesis of atherosclerosis[J/OL].Postepy Hig Med Dosw(Online),2017,71(0):57-68.DOI:10.5604/17322693.1229823.
[17]Engin A.The pathogenesis of obesity-associated adipose tissue inflammation[J].Adv Exp Med Biol,2017,960:221-245.
[18]Bian T,Li H,Zhou Q,et al.Humanβ-defensin 3 reduces TNF-α-induced inflammation and monocyte adhesion in human umbilical vein endothelial cells[J/OL].Mediators Inflamm,2017,2017:8529542.DOI:10.1155/2017/8529542.Epub 2017 Feb 28.
[19]Liu R,Shen H,Ma J,et al.Extracellular vesicles derived from adipose mesenchymal stem cells regulate the phenotype of smooth muscle cells to limit intimal hyperplasia[J].Cardiovasc Drugs Ther,2016,30(2):111-118.
[20]Ling Y,Chen G,Deng Y,et al.Polydatin post-treatment alleviates myocardial ischaemia/reperfusion injury by promoting autophagic flux[J].Clin Sci(Lond),2016,130(18):1641-1653.
[21]Chen L,Lan Z.Polydatin attenuates potassium oxonate-induced hyperuricemia and inflammation by inhibiting NF-κB/NLRP3activation via the AMPK/SIRT1 pathway[J].Food Funct,2017,8(5):1785-1792.
[22]Han J,Chen D,Liu D,et al.Modafinil attenuates inflammation via inhibiting Akt/NF-κB pathway in apo E-deficient mouse model of atherosclerosis[J].Inflammopharmacology,2018,26(2):385-393.
[23]Liu W,Chen P,Deng J,et al.Resveratrol and polydatin as modulators of Ca2+mobilization in the cardiovascular system[J].Ann N Y Acad Sci,2017,1403(1):82-91.
[24]Huang Q H,Xu L Q,Liu Y H,et al.Polydatin protects rat liver against ethanol-induced injury:Involvement of CYP2E1/ROS/Nrf2and TLR4/NF-κB p65 pathway[J/OL].Evid Based Complement Alternat Med,2017,2017:7953850.DOI:10.1155/2017/7953850.Epub 2017 Nov 8.
[25]Ma Y,Gong X,Mo Y,et al.Polydatin inhibits the oxidative stressinduced proliferation of vascular smooth muscle cells by activating the e NOS/SIRT1 pathway[J].Int J Mol Med,2016,37(6):1652-1660.