小檗碱抑制氧化型低密度脂蛋白诱导的人脐静脉内皮细胞增殖及其分子机制
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摘要
目的探讨小檗碱对氧化型低密度脂蛋白(ox-LDL)诱导人脐静脉内皮细胞(HUVEC)增殖的抑制作用及其相关信号通路改变。方法体外培养HUVEC,以ox-LDL刺激增殖并以不同浓度小檗碱干预,采用细胞计数试剂盒8与Ed U掺入实验检测小檗碱对HUVEC增殖的抑制作用,通过实时荧光定量PCR及Western blot分析相关基因和蛋白表达及信号通路变化。结果小檗碱(1~50 mg/L)呈浓度依赖性显著抑制ox-LDL诱导的HUVEC增殖,并降低增殖细胞核抗原(PCNA)、核因子кB(NF-κB)、凝集素样氧化型低密度脂蛋白受体1(LOX-1)的表达;该作用与PI3K/Akt、ERK1/2及p38MAPK信号通路蛋白的磷酸化水平降低相关。结论小檗碱通过下调PCNA、NF-κB与LOX-1表达从而抑制ox-LDL诱导的HUVEC增殖,PI3K/Akt、ERK1/2及p38MAPK信号通路参与其中。
Aim To investigate the inhibitory effect of berberine on the proliferation of human umbilical vein endothelial cell(HUVEC) induced by oxidized low density lipoprotein(ox-LDL) and the changes of related signaling pathways.Methods HUVEC was cultured in vitro,and the proliferation of HUVEC was stimulated by ox-LDL and intervened with berberine at different concentrations.The inhibitory effect of berberine on proliferation of HUVEC was detected by cell counting kit 8 and Ed U incorporation experiment.The expressions of related genes and proteins and the changes of signaling pathway were analyzed by real-time quantitative PCR and Western blot.Results In a concentration dependent manner,berberine(1~50 mg/L) significantly inhibited the HUVEC proliferation induced by ox-LDL,and reduced the expressions of proliferating cell nuclear antigen(PCNA),nuclear factor κB(NF-κB) and lectin-like oxidized low density lipoprotein receptor-1(LOX-1).These effects were associated with decreased phosphorylation levels of PI3K/Akt,ERK1/2 and p38 MAPK signaling pathways.Conclusion Berberine inhibits the proliferation of HUVEC induced by ox-LDL through the down-regulation of PCNA,NF-κB and LOX-1 expressions.PI3 K/Akt,ERK1/2 and p38 MAPK signaling pathways are involved in this process.
Aim To investigate the inhibitory effect of berberine on the proliferation of human umbilical vein endothelial cell(HUVEC) induced by oxidized low density lipoprotein(ox-LDL) and the changes of related signaling pathways.Methods HUVEC was cultured in vitro,and the proliferation of HUVEC was stimulated by ox-LDL and intervened with berberine at different concentrations.The inhibitory effect of berberine on proliferation of HUVEC was detected by cell counting kit 8 and Ed U incorporation experiment.The expressions of related genes and proteins and the changes of signaling pathway were analyzed by real-time quantitative PCR and Western blot.Results In a concentration dependent manner,berberine(1~50 mg/L) significantly inhibited the HUVEC proliferation induced by ox-LDL,and reduced the expressions of proliferating cell nuclear antigen(PCNA),nuclear factor κB(NF-κB) and lectin-like oxidized low density lipoprotein receptor-1(LOX-1).These effects were associated with decreased phosphorylation levels of PI3K/Akt,ERK1/2 and p38 MAPK signaling pathways.Conclusion Berberine inhibits the proliferation of HUVEC induced by ox-LDL through the down-regulation of PCNA,NF-κB and LOX-1 expressions.PI3 K/Akt,ERK1/2 and p38 MAPK signaling pathways are involved in this process.
引文
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[8]Li W,Liu Y,Wang B,et al.Protective effect of berberine against oxidative stress-induced apoptosis in rat bone marrow-derived mesenchymal stem cells[J].Exp Ther Med,2016,12(6):4 041-048.
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[11]王银,徐瑞霞,马春艳.TNNI3K基因过表达诱导小鼠胚胎干细胞向心肌细胞分化[J].中国动脉硬化杂志,2016,24(4):325-333.
[12]Zhang Z,Zhang M,Li Y,et al.Simvastatin inhibits the additive activation of ERK1/2 and proliferation of rat vascular smooth muscle cells induced by combined mechanical stress and ox-LDL through LOX-1 pathway[J].Cell Signal,2013,25(1):332-340.
[13]Zhang L,Jia YH,Zhao XS,et al.Trichosanatine alleviates oxidized low-density lipoprotein induced endothelial cells injury via inhibiting the LOX-1/p38MAPK pathway[J].Am J Transl Res,2016,8(12):5 455-464.
[14]Dandapat A,Hu C,Sun L,et al.Small concentrations of ox-LDL induce capillary tube formation from endothelial cells via LOX-1-dependent redox-sensitive pathway[J].Arterioscler Thromb Vasc Biol,2007,27(11):2 435-442.
[15]Chen C,Yu Z,Li Y,et al.Effects of berberine in the gastrointestinal tract--a review of actions and therapeutic implications[J].Am J Chin Med,2014,42(5):1 053-070.
[16]Huang LH,Pan XP,Gong KR,et al.Anti-inflammatory effects of three kinds of traditional Mongolian medicine monomer and its combination on LPS-stimulated RAW264.7 macrophages[J].Eur Rev Med Pharmacol Sci,2016,20(5):950-958.
[17]Park KS,Kim JB,Lee SJ,et al.Berberine-induced growth inhibition of epithelial ovarian carcinoma cell lines[J].J Obstet Gynaecol Res,2012,38(3):535-540.
[18]Park KS,Kim JB,Bae J,et al.Berberine inhibited the growth of thyroid cancer cell lines 8505C and TPC1[J].Yonsei Med J,2012,53(2):346-351.
[19]Chen J,Wu FX,Luo HL,et al.Berberine upregulates miR-22-3p to suppress hepatocellular carcinoma cell proliferation by targeting Sp1[J].Am J Transl Res,2016,8(11):4 932-941.
[20]Li DX,Zhang J,Zhang Y,et al.Inhibitory effect of berberine on human skin squamous cell carcinoma A431 cells[J].Genet Mol Res,2015,14(3):10 553-568.
[21]Kang MR,Chung IK.Down-regulation of DNA topoisomerase II alpha in human colorectal carcinoma cells resistant to a protoberberine alkaloid,berberrubine[J].Mol Pharmacol,2002,61(4):879-884.
[22]Hsieh YS,Kuo WH,Lin TW,et al.Protective effects of berberine against low-density lipoprotein(LDL)oxidation and oxidized LDLinduced cytotoxicity on endothelial cells[J].J Agric Food Chem,2007,55(25):10 437-445.
[23]Huang Z,Wang L,Meng S,et al.Berberine reduces both MMP-9and EMMPRIN expression through prevention of p38 pathway activation in PMA-induced macrophages[J].Int J Cardiol,2011,146(2):153-158.
[24]Bae YA,Cheon HG.Activating transcription factor-3 induction is involved in the anti-inflammatory action of berberine in RAW264.7murine macrophages[J].Korean J Physiol Pharmacol,2016,20(4):415-424.
[25]Liang KW,Ting CT,Yin SC,et al.Berberine suppresses MEK/ERK-dependent Egr-1 signaling pathway and inhibits vascular smooth muscle cell regrowth after in vitro mechanical injury[J].Biochem Pharmacol,2006,71(6):806-817.
[26]Li JJ.Inflammation in coronary artery diseases[J].Chin Med J(Engl),2011,124(21):3 568-575.
[27]Gu L,Bai W,Li S,et al.Celastrol prevents atherosclerosis via inhibiting LOX-1 and oxidative stress[J].PLo S One,2013,8(6):e65 477.
[28]Liu J,Jia YJ,Li XL,et al.RANTES gene G-403A polymorphism and coronary artery disease:a Meta analysis of observational studies[J].PLo S One,2012,7(10):e47 211.
[2]Seibold S,Schurle D,Heinloth A,et al.Oxidized LDL induces proliferation and hypertrophy in human umbilical vein endothelial cells via regulation of p27Kip1 expression:role of Rho A[J].J Am Soc Nephrol,2004,15(12):3 026-034.
[3]Kuhlmann CR,Schafer M,Li F,et al.Modulation of endothelial Ca2+activated K+channels by oxidized LDL and its contribution to endothelial proliferation[J].Cardiovasc Res,2003,60(3):626-634.
[4]Kuo HP,Chuang TC,Yeh MH,et al.Growth suppression of HER2-overexpressing breast cancer cells by berberine via modulation of the HER2/PI3K/Akt signaling pathway[J].J Agric Food Chem,2011,59(15):8 216-224.
[5]Cecil CE,Davis JM,Cech NB,et al.Inhibition of H1N1 influenza A virus growth and induction of inflammatory mediators by the isoquinoline alkaloid berberine and extracts of goldenseal(Hydrastis canadensis)[J].Int Immunopharmacol,2011,11(11):1706-714.
[6]Meng S,Wang LS,Huang ZQ,et al.Berberine ameliorates inflammation in patients with acute coronary syndrome following percutaneous coronary intervention[J].Clin Exp Pharmacol Physiol,2012,39(5):406-411.
[7]Sefidabi R,Mortazavi P,Hosseini S.Antiproliferative effect of berberine on canine mammary gland cancer cell culture[J].Biomed Rep,2017,6(1):95-98.
[8]Li W,Liu Y,Wang B,et al.Protective effect of berberine against oxidative stress-induced apoptosis in rat bone marrow-derived mesenchymal stem cells[J].Exp Ther Med,2016,12(6):4 041-048.
[9]Deng YX,Zhang XJ,Shi QZ,et al.Anti-hyperglycemic effects and mechanism of traditional Chinese medicine Huanglian Wan in streptozocin-induced diabetic rats[J].J Ethnopharmacol,2012,144(2):425-432.
[10]Heinloth A,Heermeier K,Raff U,et al.Stimulation of NADPH oxidase by oxidized low-density lipoprotein induces proliferation of human vascular endothelial cells[J].J Am Soc Nephrol,2000,11(10):1 819-825.
[11]王银,徐瑞霞,马春艳.TNNI3K基因过表达诱导小鼠胚胎干细胞向心肌细胞分化[J].中国动脉硬化杂志,2016,24(4):325-333.
[12]Zhang Z,Zhang M,Li Y,et al.Simvastatin inhibits the additive activation of ERK1/2 and proliferation of rat vascular smooth muscle cells induced by combined mechanical stress and ox-LDL through LOX-1 pathway[J].Cell Signal,2013,25(1):332-340.
[13]Zhang L,Jia YH,Zhao XS,et al.Trichosanatine alleviates oxidized low-density lipoprotein induced endothelial cells injury via inhibiting the LOX-1/p38MAPK pathway[J].Am J Transl Res,2016,8(12):5 455-464.
[14]Dandapat A,Hu C,Sun L,et al.Small concentrations of ox-LDL induce capillary tube formation from endothelial cells via LOX-1-dependent redox-sensitive pathway[J].Arterioscler Thromb Vasc Biol,2007,27(11):2 435-442.
[15]Chen C,Yu Z,Li Y,et al.Effects of berberine in the gastrointestinal tract--a review of actions and therapeutic implications[J].Am J Chin Med,2014,42(5):1 053-070.
[16]Huang LH,Pan XP,Gong KR,et al.Anti-inflammatory effects of three kinds of traditional Mongolian medicine monomer and its combination on LPS-stimulated RAW264.7 macrophages[J].Eur Rev Med Pharmacol Sci,2016,20(5):950-958.
[17]Park KS,Kim JB,Lee SJ,et al.Berberine-induced growth inhibition of epithelial ovarian carcinoma cell lines[J].J Obstet Gynaecol Res,2012,38(3):535-540.
[18]Park KS,Kim JB,Bae J,et al.Berberine inhibited the growth of thyroid cancer cell lines 8505C and TPC1[J].Yonsei Med J,2012,53(2):346-351.
[19]Chen J,Wu FX,Luo HL,et al.Berberine upregulates miR-22-3p to suppress hepatocellular carcinoma cell proliferation by targeting Sp1[J].Am J Transl Res,2016,8(11):4 932-941.
[20]Li DX,Zhang J,Zhang Y,et al.Inhibitory effect of berberine on human skin squamous cell carcinoma A431 cells[J].Genet Mol Res,2015,14(3):10 553-568.
[21]Kang MR,Chung IK.Down-regulation of DNA topoisomerase II alpha in human colorectal carcinoma cells resistant to a protoberberine alkaloid,berberrubine[J].Mol Pharmacol,2002,61(4):879-884.
[22]Hsieh YS,Kuo WH,Lin TW,et al.Protective effects of berberine against low-density lipoprotein(LDL)oxidation and oxidized LDLinduced cytotoxicity on endothelial cells[J].J Agric Food Chem,2007,55(25):10 437-445.
[23]Huang Z,Wang L,Meng S,et al.Berberine reduces both MMP-9and EMMPRIN expression through prevention of p38 pathway activation in PMA-induced macrophages[J].Int J Cardiol,2011,146(2):153-158.
[24]Bae YA,Cheon HG.Activating transcription factor-3 induction is involved in the anti-inflammatory action of berberine in RAW264.7murine macrophages[J].Korean J Physiol Pharmacol,2016,20(4):415-424.
[25]Liang KW,Ting CT,Yin SC,et al.Berberine suppresses MEK/ERK-dependent Egr-1 signaling pathway and inhibits vascular smooth muscle cell regrowth after in vitro mechanical injury[J].Biochem Pharmacol,2006,71(6):806-817.
[26]Li JJ.Inflammation in coronary artery diseases[J].Chin Med J(Engl),2011,124(21):3 568-575.
[27]Gu L,Bai W,Li S,et al.Celastrol prevents atherosclerosis via inhibiting LOX-1 and oxidative stress[J].PLo S One,2013,8(6):e65 477.
[28]Liu J,Jia YJ,Li XL,et al.RANTES gene G-403A polymorphism and coronary artery disease:a Meta analysis of observational studies[J].PLo S One,2012,7(10):e47 211.