生姜提取物对苯并芘染毒小鼠Ⅱ相酶的诱导作用及对Nrf2-Keap1通路的影响
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摘要
研究生姜提取物对苯并芘(benzo[α]pyrene,BαP)染毒小鼠抗氧化酶及Ⅱ相酶的影响及调控机制。用100、200、400 mg/(kg·d)的生姜提取物(分别标记为LG、MG和HG)对昆明小鼠连续灌胃给药14 d,然后腹腔注射50 mg/(kg·d) BαP,测定小鼠血清及肝肾中谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)、过氧化氢酶(catalase,CAT)和超氧化物歧化酶(superoxide dismutase,SOD)以及醌氧化还原酶(quinone reductase,QR)、血氧合酶1(hemooxygenase 1,HO-1)和谷胱甘肽-S-转移酶(glutathione-S-transferase,GST)的活力及相应蛋白和mRNA的表达水平,分析了核转录因子E2相关因子2(nuclear factor (erythroid-derived 2)-like 2,Nrf2)和Kelch样环氧氯丙烷相关蛋白1(Kelch like ECH associated protein 1,Keap1)的表达情况。结果表明:与BαP损伤组相比,BαP+MG和BαP+HG能显著诱导小鼠血清中CAT和GSH-Px活力及肝肾中CAT、SOD和GSH-Px的活力(P<0.05);BαP+MG和BαP+HG处理导致动物肝脏中QR、HO-1和GST的活力分别提高了21.7%和29.05%、23.99%和56.39%、32.40%和15.08%。与BαP处理组相比,BαP+MG极显著提高了Nrf2的蛋白表达水平(21.9%),抑制了Keap1蛋白表达(34.7%)(P<0.01)。高效液相色谱-轨道离子阱质谱联用方法从生姜提取物中共鉴定出19种成分。因此,生姜提取物能显著诱导BαP染毒小鼠中抗氧化酶和Ⅱ相酶,并且该过程与Nrf2-Keap1信号通路相关。
The effect of ginger extract on antioxidant and phase II detoxifying enzymes in mice exposed to benzo(α)pyrene(BαP) was investigated and the underlying regulatory mechanism was elucidated. Kunming mice were administered by gavage with ginger extract at 100(low-dose group, LG), 200(medium-dose group, MG) and 400 mg/(kg·d)(high-dose group, HG) for 14 consecutive days before intraperitoneal injection with 50 mg/(kg·d) of BαP. Glutathione peroxidase(GSHPx), catalase(CAT), superoxide dismutase(SOD), quinone reductase(QR), hemooxygenase 1(HO-1) and glutathione-Stransferase(GST) activity and their protein and mRNA expression were analyzed. The expression levels of nuclear factor(erythroid-derived 2)-like 2(Nrf2) and Kelch like ECH-associated protein 1(Keap1) were examined as well. Results showed that BαP + MG and BαP + HG significantly(P < 0.05) elevated CAT and GSH-Px activities in the serum and CAT, SOD and GSH-Px activities in the liver and kidney in mice compared to BαP alone. Meanwhile, the administration of BαP + MG and BαP + HG led to a significant increase of QR, HO-1 and GST activity by 21.7%, 23.99% and 32.40% in liver, and by 29.05%, 56.39% and 15.08% in kidney, respectively. In contrast to BαP, BαP + MG treatment led to a 21.9% increase of Nrf2 protein expression and a 34.7% reduction of Keap1 protein expression. Totally, 19 compounds were identified in ginger extract by high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(HPLC/LTQOrbitrap-MS). Our results suggest that ginger extract was able to induce antioxidant and phase II enzymes in mice exposed to BαP by a mechanism related to Nrf2-Keap1 signaling pathway.
The effect of ginger extract on antioxidant and phase II detoxifying enzymes in mice exposed to benzo(α)pyrene(BαP) was investigated and the underlying regulatory mechanism was elucidated. Kunming mice were administered by gavage with ginger extract at 100(low-dose group, LG), 200(medium-dose group, MG) and 400 mg/(kg·d)(high-dose group, HG) for 14 consecutive days before intraperitoneal injection with 50 mg/(kg·d) of BαP. Glutathione peroxidase(GSHPx), catalase(CAT), superoxide dismutase(SOD), quinone reductase(QR), hemooxygenase 1(HO-1) and glutathione-Stransferase(GST) activity and their protein and mRNA expression were analyzed. The expression levels of nuclear factor(erythroid-derived 2)-like 2(Nrf2) and Kelch like ECH-associated protein 1(Keap1) were examined as well. Results showed that BαP + MG and BαP + HG significantly(P < 0.05) elevated CAT and GSH-Px activities in the serum and CAT, SOD and GSH-Px activities in the liver and kidney in mice compared to BαP alone. Meanwhile, the administration of BαP + MG and BαP + HG led to a significant increase of QR, HO-1 and GST activity by 21.7%, 23.99% and 32.40% in liver, and by 29.05%, 56.39% and 15.08% in kidney, respectively. In contrast to BαP, BαP + MG treatment led to a 21.9% increase of Nrf2 protein expression and a 34.7% reduction of Keap1 protein expression. Totally, 19 compounds were identified in ginger extract by high performance liquid chromatography coupled with linear ion trap-Orbitrap mass spectrometry(HPLC/LTQOrbitrap-MS). Our results suggest that ginger extract was able to induce antioxidant and phase II enzymes in mice exposed to BαP by a mechanism related to Nrf2-Keap1 signaling pathway.
引文
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[27]PANDA A K,CHAKRABORTY D,SARKAR I,et al.New insights into therapeutic activity and anticancer properties of curcumin[J].Journal of Experimental Pharmacology,2017,9:31-45.DOI:10.2147/JEP.S70568.
[28]DAVATGARAN-TAGHIPOUR Y,MASOOMZADEH S,FARZAEI M H,et al.Polyphenol nanoformulations for cancer therapy:experimental evidence and clinical perspective[J].International Journal of Nanomedicine,2017,12:2689-2702.DOI:10.2147/IJN.S131973.
[29]ZHU W,CROMIE M M,CAI Q,et al.Curcumin and vitamin E protect against adverse effects of benzo[a]pyrene in lung epithelial cells[J].PLoS ONE,2014,9(3):1-11.DOI:10.1371/journal.pone.0092992.
[30]BODDUPALLI S,MEIN J R,JAMES D R,et al.Induction of phase 2 antioxidant enzymes by broccoli sulforaphane:perspectives in maintaining the antioxidant activity of vitamins A,C,and E[J].Frontiers in Nutrigenomics,2012,3(7):1-15.DOI:10.3389/fgene.2012.00007.
[2]MARX W,RIED K,MCCARTHY A L,et al.Ginger-Mechanism of action in chemotherapy-induced nausea and vomiting:a review[J].Critical Reviews in Food Science and Nutrition,2017,57(1):141-146.DOI:10.1080/10408398.2013.865590.
[3]VIPIN A V,RAKSHA RAO K,KURREY N K,et al.Protective effects of phenolics rich extract of ginger against aflatoxin B,-induced oxidative stress and hepatotoxicity[J].Biomedicine&Pharmacotherapy,2017,91:415-424.DOI:10.1016/j.biopha.2017.04.107.
[4]ABOLAJI A O,OJO M,AFOLABI T T,et al.Protective properties of 6-gingerol-rich fraction from Zingiber officinale(Ginger)on chlorpyrifos-induced oxidative damage and inflammation in the brain,ovary and uterus of rats[J].Chemico-Biological Interactions,2017,270:15-23.DOI:10.1016/j.cbi.2017.03.017.
[5]CHEN H,FU J,CHEN H,et al.Ginger compound[6]-shogaol and its cysteine-conjugated metabolite(M2)activate Nrf2 in colon epithelial cells in vitro and in vivo[J].Chemical Research in Toxicology,2014,27(9):1575-1585.DOI:10.1021/tx50021 lx.
[6]WEINSTEIN I B,JEFFREY A M,JENNETTE K W,et al.Benzo(a)pyrene diol epoxides as intermediates in nucleic acid binding in vitro and in vivo[J].Science,1976,193:592-595.DOI:10.1126/science.959820.
[7]KIM K B,LEE B M.Oxidative stress to DNA,protein,and antioxidant enzymes(superoxide dismutase and catalase)in rats treated with benzo(a)pyrene[J].Cancer Letters,1997,113(1/2):205-212.DOI:10.1016/S0304-3835(97)04610-7.
[8]KENSLER T W,WAKABAYASHI N,BISWAL S.Cell survival responses to environmental stresses via the Keapl-Nrf2-ARE pathway[J].Annual Reviews Pharmacology and Toxicology,2007,47(1):89-116.DOI:10.1146/annurev.pharmtox.46.120604.141046.
[9]SINGH R P,PADMAVATHI B,RAO A R.Modulatory influence of Adhatoda vesica(Justicia adhatoda)leaf extract on the enzymes of xenobiotic metabolism,antioxidant status and lipid peroxidation in mice[J].Molecular and Cellular Biochemistry,2000,213(1):99-109.DOI:10.1023/A:1007182913931.
[10]LI Feng,NITTERANON V,TANG X Z,et al.In vitro antioxidant and anti-inflammatory activities of 1-dehydro-[6]-gingerdione,6-shogaol,6-dehydroshogaol and hexahydrocurcumin[J].Food Chemistry,2012,135(2):332-337.DOI:10.1016/j.foodchem.2012.04.145.
[11]刘步云,王永丽,张健,等.不同品种生姜的氧化及抗炎症活性[J].食品与发酵工业,2015,41(11):81-86.DOI:10.13995/j.cnki.l 1-1802/ts.201511015.
[12]YU S,KHOR T O,CHEUNG K L,et al.Nrf2 expression is regulated by epigenetic mechanisms in prostate cancer of TRAMP mice[J].PLoS ONE,2010,5(1):e8579.DOI:10.1371/journal.pone.0008579.
[13]史巧巧,席俊,陆启玉.食品中苯并芘的研究进展[J].食品工业科技,2014,35(5):379-381.DOI:10.13386/j.issn1002-0306.2014.05.066.
[14]MOHAMED O I,EL-NAHAS A F,EL-SAYED Y S,et al.Ginger extract modulates Pb-induced hepatic oxidative stress and expression of antioxidant gene transcripts in rat liver[J].Pharmaceutical Biology,2015,54(7):1164-1172.DOI:10.3109/13880209.2015.1057651.
[15]姜卫星.生姜醇提取物对大鼠肝脏缺血再灌注损伤后细胞凋亡和炎症反应的影响[J].现代中西医结合杂志,2016,25(9):932-936.DOI:10.3969/j.issn.1008-8849.2016.09.007.
[16]KOTA N,KRISHNA P,POLASA K.Alterations in antioxidant status of rats following intake of ginger through diet[J].Food Chemistry,2008,106(3):991-996.DOI:10.1016/j.foodchem.2007.07.073.
[17]EL-SHARAKY A S,NEW AIRY A A,KAMEL M A,et al.Protective effect of ginger extract against bromobenzene-induced hepatotoxicity in male rats[J].Food and Chemical Toxicology,2009,47(7):1584-1590.DOI:10.1016/j.fct.2009.04.005.
[18]JANA S,MANDLEKAR S.Role of phaseⅡdrug metabolizing enzymes in cancer chemoprevention[J].Current Drug Metabolism,2009,10(6):595-616.DOI:10.2174/138920009789375379.
[19]NIRMALA K,KRISHNA T P,POLASA K.Modulation of xenobiotic metabolism in ginger(Zingiber officinale Roscoe)fed rats[J].International Journal of Nutrition and Metabolism,2010,2(3):56-62.
[20]LI Feng,WANG Yongli,PARKIN K L,et al.Isolation of quinone reductase(QR)inducing agents from ginger rhizome and their in vitro anti-inflammatory activity[J].Food Research International,2011,44(6):1597-1603.DOI:10.1016/j.foodres.2011.04.010.
[21]胡流芳,王迎,任汝静,等.Keap1-Nrf2/ARE信号通路的抗氧化应激作用及其调控机制[J].国际药学研究杂志,2016,43(1):146-152.DOI:10.13220/j.cnki.j ipr.2016.01.022.
[22]TAGUCHI K,MOTOHASHI H,YAMAMOTO M.Molecular mechanisms of the Keapl-Nrf2 pathway in stress response and cancer evolution[J].Genes Cells,2011,16(2):123-140.DOI:10.1111/j.1365-2443.2010.01473.x.
[23]RUSHMORE T H,KING R G,PAULSON K E,et al.Regulation of glutathione S-transferase Ya subunitgene expression:identification of a unique xenobiotic-responsive element controlling inducible expression by planar aromatic compounds[J].Proceedings of the National Academy of Sciences,1990,87(10):3826-3830.DOI:10.1073/pnas.87.10.3826.
[24]BAK M J,OK S,JUN M,et al.6-Shogaol-rich extract from ginger up-regulates the antioxidant defense systems in cells and mice[J].Molecules,2012,17(7):8037-8055.DOI:10.3390/molecules17078037.
[25]AGGARWAL B B,SHISHODIA S.Molecular targets of dietary agents for prevention and therapy of cancer[J].Biochemical Pharmacology,2006,71(10):1397-1421.DOI:10.1016/j.bcp.2006.02.009.
[26]SINHA D,BISWAS J,SUNG B,et al.Chemopreventive and chemotherapeutic potential of curcumin in breast cancer[J].Current Drug Targets,2012,13(14):1799-1819.DOI:10.2174/138945012804545632.
[27]PANDA A K,CHAKRABORTY D,SARKAR I,et al.New insights into therapeutic activity and anticancer properties of curcumin[J].Journal of Experimental Pharmacology,2017,9:31-45.DOI:10.2147/JEP.S70568.
[28]DAVATGARAN-TAGHIPOUR Y,MASOOMZADEH S,FARZAEI M H,et al.Polyphenol nanoformulations for cancer therapy:experimental evidence and clinical perspective[J].International Journal of Nanomedicine,2017,12:2689-2702.DOI:10.2147/IJN.S131973.
[29]ZHU W,CROMIE M M,CAI Q,et al.Curcumin and vitamin E protect against adverse effects of benzo[a]pyrene in lung epithelial cells[J].PLoS ONE,2014,9(3):1-11.DOI:10.1371/journal.pone.0092992.
[30]BODDUPALLI S,MEIN J R,JAMES D R,et al.Induction of phase 2 antioxidant enzymes by broccoli sulforaphane:perspectives in maintaining the antioxidant activity of vitamins A,C,and E[J].Frontiers in Nutrigenomics,2012,3(7):1-15.DOI:10.3389/fgene.2012.00007.