PI3K介导的Nrf2磷酸化激活在槲皮素抑制山岗橐吾碱肝细胞毒性中的作用
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摘要
目的:观察磷脂酰肌醇-3-激酶(phosphoinositide-3-kinase,PI3K)介导的核转录因子E2相关因子2(nuclear factor E2-related factor 2,Nrf2)激活在槲皮素(quercetin,Quer)抑制山岗橐吾碱(clivorine,Cliv)诱导的人正常肝L-02细胞毒性中的作用。方法:L-02细胞与Quer(1,10,25,50μmol·L-1)预孵15 min后,再与Cliv(50μmol·L-1)孵育48 h,采用噻唑蓝(thiazolam blue,MTT)比色法检测细胞存活率。检测细胞内的活性氧(reactive oxygen species,ROS)和还原型谷胱甘肽(reduced glutathione,GSH),并进一步通过双荧光素酶报告基因法检测Nrf2的核转位激活。蛋白免疫印迹法(Western blot)检测Nrf2和PI3K的磷酸化。实时荧光定量聚合酶链式反应(Real-time PCR)分析Nrf2下游基因血红素加氧酶-1 (heme oxygenase-1,Hmox1),NADPH醌氧化还原酶-1 (NADPH:quinone oxidoreductase-1,Nqo1),谷氨酰-半胱氨酸连接酶催化亚基(catalytic subunit of glutamate-cysteine ligase,Gclc)和谷氨酰-半胱氨酸连接酶调节亚基(modifier subunit of glutamate-cysteine ligase,Gclm) mRNA的表达。结果:与空白组比较,Quer(10,25,50μmol·L-1)能明显逆转Cliv降低的L-02细胞存活率(P <0. 05),且能逆转Cliv降低的L-02细胞内GSH含量(P <0. 05),升高ROS水平(P <0. 01)。Quer能诱导PI3K的磷酸化(P <0. 05),Quer可以诱导Nrf2的磷酸化和转录激活(P <0. 05)。Quer能使Nrf2下游mRNA表达水平提高(P <0. 05),PI3K抑制剂LY294002(LY)能阻断Quer对Hmox1 mRNA表达的升高作用(P <0. 01)。结论:Quer通过诱导Nrf2磷酸化激活逆转了Cliv诱导的肝细胞毒性,PI3K在Quer诱导的Nrf2磷酸化激活中发挥了重要作用。
Objective: To observe the effect of phosphoinositide-3-kinase(PI3 K)-mediated nuclear factor E2-related factor 2(Nrf2) phosphorylated activation in quercetin(Quer) in prohibiting clivorine(Cliv)-induced cytotoxicity in L-02 cells. Method: Human normal liver L-02 cells were pre-incubated with Quer(1,10,25,50 μmol·L-1) for 15 min,and then incubated with Cliv(50 μmol·L-1) for 48 h. The cell viability was evaluated by thiazolam blue(MTT) assay. Cellular reactive oxygen species(ROS) and reduced glutathione(GSH) were detected. The transcriptional activation of Nrf2 was measured by dual-luciferase reporter assay. The phosphorylation of Nrf2 and PI3 K was measured by Western blot, and downstream genes, including heme oxygenase 1(Hmox1),NADPH: quinone oxidoreductase 1(Nqo1),catalytic subunit of glutamate-cysteine ligase(Gclc),modifier subunit of glutamate-cysteine ligase(Gclm),were measured by Real-time PCR. Result: Quer(10,25,50 μmol·L-1) reversed Cliv-induced decreased cell viability(P < 0. 05),GSH(P < 0. 05) and ROS levels(P < 0. 01). Quer induced phosphorylation of PI3 K(P < 0. 05). Quer induced phosphorylation and subsequent transcriptional activation of Nrf2(P < 0. 05),and enhanced mRNA expressions of Nrf2 downstream genes(P < 0. 05). The increased Hmox1 mRNA expression was reduced by PI3 K inhibitor LY294002(LY)(P <0. 01). Conclusion: Quer alleviates Cliv-induced hepatotoxicity by inducing Nrf2 phosphorylated activation,and PI3 K plays an important role in regulating Quer-induced Nrf2 phosphorylated activation.
Objective: To observe the effect of phosphoinositide-3-kinase(PI3 K)-mediated nuclear factor E2-related factor 2(Nrf2) phosphorylated activation in quercetin(Quer) in prohibiting clivorine(Cliv)-induced cytotoxicity in L-02 cells. Method: Human normal liver L-02 cells were pre-incubated with Quer(1,10,25,50 μmol·L-1) for 15 min,and then incubated with Cliv(50 μmol·L-1) for 48 h. The cell viability was evaluated by thiazolam blue(MTT) assay. Cellular reactive oxygen species(ROS) and reduced glutathione(GSH) were detected. The transcriptional activation of Nrf2 was measured by dual-luciferase reporter assay. The phosphorylation of Nrf2 and PI3 K was measured by Western blot, and downstream genes, including heme oxygenase 1(Hmox1),NADPH: quinone oxidoreductase 1(Nqo1),catalytic subunit of glutamate-cysteine ligase(Gclc),modifier subunit of glutamate-cysteine ligase(Gclm),were measured by Real-time PCR. Result: Quer(10,25,50 μmol·L-1) reversed Cliv-induced decreased cell viability(P < 0. 05),GSH(P < 0. 05) and ROS levels(P < 0. 01). Quer induced phosphorylation of PI3 K(P < 0. 05). Quer induced phosphorylation and subsequent transcriptional activation of Nrf2(P < 0. 05),and enhanced mRNA expressions of Nrf2 downstream genes(P < 0. 05). The increased Hmox1 mRNA expression was reduced by PI3 K inhibitor LY294002(LY)(P <0. 01). Conclusion: Quer alleviates Cliv-induced hepatotoxicity by inducing Nrf2 phosphorylated activation,and PI3 K plays an important role in regulating Quer-induced Nrf2 phosphorylated activation.
引文
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[20]方静,陈江,彭君伟,等.基于Nrf2通路探讨薏苡附子败酱散治疗溃疡性结肠炎的作用机制[J].中国实验方剂学杂志,2018,24(13):85-92.
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[22] LIN M,ZHAI X,WANG G,et al. Salvianolic acid B protects against acetaminophen hepatotoxicity by inducing Nrf2 and phaseⅡdetoxification gene expression via activation of the PI3K and PKC signaling pathways[J]. J Pharmacol Sci, 2015, 127(2):203-210.
[23] LI L,DONG H,SONG E,et al. Nrf2/ARE pathway activation, HO-1 and NQO1 induction by polychlorinated biphenyl quinone is associated with reactive oxygen species and PI3K/Akt signaling[J].Chem Biol Interact,2014,209(1):56-67.
[24] PANG C,ZHENG Z Y,SHI L,et al. Caffeic acid prevents acetaminophen-induced liver injury by activating the Keap1-Nrf2 antioxidative defense system[J]. Free Radic Biol Med,2016,91:236-246.
[25] YANG X,LI W,SUN Y,et al. Comparative study of hepatotoxicity of pyrrolizidine alkaloids retrorsine and monocrotaline[J]. Chem Res Toxicol,2017,30(2):532-539.
[26] JI L L,LIU T Y,WANG Z T. Pyrrolizidine alkaloid clivorine induced oxidative injury on primary cultured rat hepatocytes[J]. Hum Exp Toxicol,2010,29(4):303-309.
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[31] SHI L,HAO Z, ZHANG S, et al. Baicalein and baicalin alleviate acetaminophen-induced liver injury by activating Nrf2 antioxidative pathway:the involvement of ERK1/2 and PKC[J]. Biochem Pharmacol,2018,150:9-23.
[32] Jaiswal A K. Nrf2 signaling in coordinated activation of antioxidant gene expression[J]. Free Radic Biol Med,2004,36(10):1199-1207.
[33] Toroser D,Yarian C S,Orr W C,et al. Mechanisms of gamma-glutamylcysteine ligase regulation[J]. Biochim Biophys Acta,2005,1760(2):233-244.
[34] Choi A M, Alam J. Heme oxygenase-1:function,regulation,and implication of a novel stress-inducible protein in oxidant-induced lung injury[J]. Am J Respir Cell Mol Biol,1996,15(1):9-19.
[35]夏小俊,金中初. NQO1酶及其被氧环境诱导表达的研究进展[J].生理科学进展,2002,33(3):225-229.
[2] Kuhara K,Takanashi H,Hirono I,et al. Carcinogenic activity of clivorine, a pyrrolizidine alkaloid isolated from Ligularia Dentata[J]. Cancer Lett,1980,10(2):117-122.
[3] JI L L,ZHAO X G,CHEN L,et al. Pyrrolizidine alkaloid clivorine inhibits human normal liver L-02 cells growth and activates p38 mitogen-activated protein kinase in L-02 cells[J]. Toxicon,2002,40(12):1685-1690.
[4] JI L L, SHENG Y C, ZHENG Z Y, et al. The involvement of p62-Keap1-Nrf2 antioxidant signaling pathway and JNK in the protection of natural flavonoid quercetin against hepatotoxicity[J]. Free Radic Biol Med,2015,85:12-23.
[5]蒋萍,杨阳,季莉莉,等.黄芩素对4种肝损伤物质造成肝细胞毒性的拮抗作用[J].中国实验方剂学杂志,2012,18(6):145-148.
[6] Kawabata K,Mukai R,Ishisaka A. Quercetin and related polyphenols:new insights and implications for their bioactivity and bioavailability[J]. Food Funct,2015,6(5):1399-1417.
[7]李韶静,廖应芬,杜青.槲皮素纳米载药系统的研究与应用[J].中国中药杂志,2018,43(10):1978-1984.
[8]朱玲玲,张洋,窦勤玲,等.槲皮素对U937细胞迁移和侵袭能力及MMP-2,MMP-9表达的影响[J].中国实验方剂学杂志,2018,24(16):146-151.
[9] Amaral S,Mira L,Nogueira J M F,et al. Plant extracts with anti-inflammatory properties—a new approach for characterization of their bioactive compounds and establishment of structure-antioxidant activity relationships[J]. Bioorg Med Chem,2009,17(5):1876-1883.
[10]骆明旭,罗丹,赵万红.槲皮素药理作用研究进展[J].中国民族民间医药,2014,23(17):12-14.
[11] Kelly G S. Quercetin. Monograph[J]. Altern Med Rev,2011,16(2):172-194.
[12] Boots A W,Haenen G R,Bast A. Health effects of quercetin:from antioxidant to nutraceutical[J]. Eur J Pharmacol,2008,585(2/3):325-337.
[13] Hertog M G,Feskens E J,Hollman P C,et al. Dietary antioxidant flavonoids and risk of coronary heart disease:the zutphen elderly study[J]. Lancet, 1993, 342(8878):1007-1011.
[14] Manach C,Morand C,Texier O,et al. Quercetin metabolites in plasma of rats fed diets containing rutin or quercetin[J]. J Nutr,1995,125(7):1911-1922.
[15] Hollman P C,de Vires J H,van Leeuwen S D,et al.Absorption of dietary quercetin glycosides and quercetin in healthy ileostomy volunteers[J]. Am J Clin Nutr,1995,62(6):1276-1282.
[16]李航,段惠军. Nrf2/ARE信号通路及其调控的抗氧化蛋白[J].中国药理学通报,2011,27(3):300-303.
[17] YU X, Kensler T. Nrf2 as a target for cancer chemoprevention[J]. Mutat Res,2005,591(1/2):93-102.
[18]高之心,李元海,鲁显福.转录因子NF-E2相关因子2-抗氧化反应元件信号路径及其肝脏保护作用进展[J].国际麻醉学与复苏杂志,2013,34(2):182-185.
[19]谢晨,陈韩英,钟晶,等.紫草素通过上调Nrf2途径及干预胞内氧化还原平衡稳态诱导A549细胞凋亡[J].中国药理学通报,2014,30(10):1445-1451.
[20]方静,陈江,彭君伟,等.基于Nrf2通路探讨薏苡附子败酱散治疗溃疡性结肠炎的作用机制[J].中国实验方剂学杂志,2018,24(13):85-92.
[21] ZHANG J Q,SHI L,XU X N,et al. Therapeutic detoxification of quercetin against carbon tetrachlorideinduced acute liver injury in mice and its mechanism[J]. J Zhejiang Univ Sci B, 2014, 15(12):1039-1047.
[22] LIN M,ZHAI X,WANG G,et al. Salvianolic acid B protects against acetaminophen hepatotoxicity by inducing Nrf2 and phaseⅡdetoxification gene expression via activation of the PI3K and PKC signaling pathways[J]. J Pharmacol Sci, 2015, 127(2):203-210.
[23] LI L,DONG H,SONG E,et al. Nrf2/ARE pathway activation, HO-1 and NQO1 induction by polychlorinated biphenyl quinone is associated with reactive oxygen species and PI3K/Akt signaling[J].Chem Biol Interact,2014,209(1):56-67.
[24] PANG C,ZHENG Z Y,SHI L,et al. Caffeic acid prevents acetaminophen-induced liver injury by activating the Keap1-Nrf2 antioxidative defense system[J]. Free Radic Biol Med,2016,91:236-246.
[25] YANG X,LI W,SUN Y,et al. Comparative study of hepatotoxicity of pyrrolizidine alkaloids retrorsine and monocrotaline[J]. Chem Res Toxicol,2017,30(2):532-539.
[26] JI L L,LIU T Y,WANG Z T. Pyrrolizidine alkaloid clivorine induced oxidative injury on primary cultured rat hepatocytes[J]. Hum Exp Toxicol,2010,29(4):303-309.
[27] LIU J,WANG X,YONG H, et al. Preparation,characterization,digestibility and antioxidant activity of quercetin grafted cynanchum auriculatum starch[J]. Int J Biol Macromol,2018,114:130-136.
[28] Kaspar J W,Niture S K,Jaiswal A K. Nrf2:INrf2(Keap1)signaling in oxidative stress[J]. Free Radic Biol Med,2009,47(9):1304-1309.
[29] ZHANG D D. Mechanistic studies of the Nrf2-Keap1signaling pathway[J]. Drug Metab Rev,2006,38(4):769-789.
[30] Jain A K,Jaiswal A K. GSK-3 acts upstream of fyn kinase in regulation of nuclear export and degradation of NF-E2related factor 2[J]. J Biol Chem,2007,282(22):16502-16510.
[31] SHI L,HAO Z, ZHANG S, et al. Baicalein and baicalin alleviate acetaminophen-induced liver injury by activating Nrf2 antioxidative pathway:the involvement of ERK1/2 and PKC[J]. Biochem Pharmacol,2018,150:9-23.
[32] Jaiswal A K. Nrf2 signaling in coordinated activation of antioxidant gene expression[J]. Free Radic Biol Med,2004,36(10):1199-1207.
[33] Toroser D,Yarian C S,Orr W C,et al. Mechanisms of gamma-glutamylcysteine ligase regulation[J]. Biochim Biophys Acta,2005,1760(2):233-244.
[34] Choi A M, Alam J. Heme oxygenase-1:function,regulation,and implication of a novel stress-inducible protein in oxidant-induced lung injury[J]. Am J Respir Cell Mol Biol,1996,15(1):9-19.
[35]夏小俊,金中初. NQO1酶及其被氧环境诱导表达的研究进展[J].生理科学进展,2002,33(3):225-229.