Effects of phytochemicals sulforaphane on uridine diphosphate-glucuronosyltransferase expression as well as cell-cycle arrest and apoptosis in human colon cancer Caco-2 cells

Our study investigated the effects of the chemopreventive agent sulforaphane (SFN) on human colon cancer Caco-2 cells and potential underlying mechanisms of the effects. When treated with SFN at hypotoxic levels, UDP-glucuronosyltransferase 1A (UGT1A) was induced in a dose-dependent manner. SFN at 25 μM showed the highest UGT1A-induction activity, inducing UGT1A1, UGT1A8, and UGT1A10 mRNA expression, and increasing UGT-mediated N-hydroxy-PhIP glucuronidation. SFN- induced UGT1A expression may have resulted from Nrf2 nuclear translocation or activation. At higher concentrations, e.g. 75 μM, SFN caused G1/G2 cell cycle arrest and induced apoptosis possibly through reducing anti-apoptotic bcl-2 expression and increasing apoptosis-inducing bax expression in Caco-2 cells. Taken together, these results demonstrated the chemopreventive effects of SFN on human colon cancer Caco-2 cells may have been partly attributed to Nrf2-mediated UGT1A induction and apoptosis induction, and our studies provided theoretic and experimental basis for clinical application of SFN to human colon cancer prevention.     

慢性阻塞性肺疾病大鼠转录因子KLF2对Nrf2调控γ-GCS表达的影响

目的:研究肺Krüppel样转录因子(KLF2/LKLF)在慢性阻塞性肺疾病(COPD)大鼠肺组织中的表达,探讨KLF2与红系衍生因子2(Nrf2)之间的关系以及对抗氧化酶γ-谷氨酰半胱氨酸合酶(γ-GCS)表达的影响。方法:复制大鼠COPD模型,应用免疫组化、Western blot、原位杂交和RT-PCR方法检测KLF2、Nrf2、γ-GCS蛋白及mRNA的表达,并行相关分析。同时利用免疫共沉淀技术(CO-IP)研究KLF2与Nrf2蛋白之间的相互关系。结果:免疫组化及Western blot结果显示COPD组KLF2、Nrf2、γ-GCS蛋白水平较对照组明显升高(P<0.05);原位杂交及RT-PCR显示KLF2、γ-GCS mRNA水平在COPD组显著高于对照组(P<0.01),而Nrf2 mRNA在两组表达无明显差异(P>0.05);CO-IP结果显示在Nrf2抗体捕获的免疫沉淀中,KLF2抗体可杂交出明显的蛋白条带(P<0.01);直线相关分析发现KLF2蛋白与Nrf2蛋白呈正相关(P<0.05),KLF2、Nrf2蛋白与γ-GCS蛋白及mRNA均呈正相关(均P<0.05)。结论:KLF2在COPD肺组织中表达上调,可能通过活化Nrf2,促进Nrf2核积聚上调γ-GCS的基因表达,在氧化失衡中发挥作用。     

(−)-Epigallocatechin Gallate Protects Against Cerebral Ischemia-Induced Oxidative Stress via Nrf2/ARE Signaling

(?)-Epigallocatechin gallate (EGCG) has recently been shown to exert neuroprotection in a variety of neurological diseases; however, its role and the underlying mechanisms in cerebral ischemic injury are not fully understood. This study was conducted to investigate the potential neuroprotective effects of EGCG and the possible role of the nuclear factor erythroid 2-related factor 2 (Nrf2)/antioxidant response element (ARE) pathway in the putative neuroprotection against experimental stroke in rats. The results revealed that EGCG exhibit significant neuroprotection, as evidenced by reduced infarction size and the decrease in transferase dUTP nick end labeling-positive neurons. Furthermore, EGCG also enhanced levels of Nrf2 and its downstream ARE pathway genes such as heme oxygenase-1, glutamate-cysteine ligase modulatory subunit and glutamate-cysteine ligase regulatory subunit, as compared to control groups. In accordance with its induction of Nrf2 activation, EGCG exerted a robust attenuation of reactive oxygen species generation and an increase in glutathione content in ischemic cortex. Taken together, these results demonstrated that EGCG exerted significant antioxidant and neuroprotective effects following focal cerebral ischemia, possibly through the activation of the Nrf2/ARE signaling pathway.     

Suppressive effect of an inducible nitric oxide inhibitor, ONO-1714, on AOM-induced rat colon carcinogenesis.

The expression of inducible nitric oxide synthase (iNOS) is markedly elevated in rat colon cancers induced by azoxymethane (AOM). In addition, iNOS can be detected in most adenomas and dysplastic aberrant crypt foci (ACF), suggesting that iNOS plays an important role in colon carcinogenesis. In the present study, the effect of an iNOS inhibitor, ONO-1714 ((1S,5S,6R,7R)-7-chloro-3-imino-5-methyl-2-azabicyclo[4.1.0] heptane hydrochloride), on AOM-induced rat colon carcinogenesis was investigated. Male F344 rats were treated with 15 mg/kg body weight of AOM once a week, for 2 weeks. ONO-1714 was given to the rats at doses of 10, 20, 50, and 100 ppm in diet for 4 weeks from the day before the first carcinogen treatment. The number of AOM-induced ACF in the rats receiving 10, 20, 50 and 100 ppm ONO-1714 were 94, 73 (P < 0.05), 71 (P < 0.005), and 53% (P < 0.0005), respectively, of the control value. Moreover, the mean number of aberrant crypts per focus was significantly lowered in 100 ppm ONO-1714 group (P < 0.05). Then, the effects of long-term treatment (32 weeks) with 50 and 100 ppm ONO-1714 on AOM-induced colorectal tumor development were examined. Although incidences and multiplicities of colon tumors did not significantly differ among the groups, number of tumors developing in the middle part of colon were reduced with both 50 and 100 ppm doses (P < 0.05). Furthermore, colon tumor volume tended to be decreased by ONO-1714 treatment, and the number of colon tumors more than 3mm in diameter was significantly lowered in the 100 ppm ONO-1714 group (P < 0.01). These results suggest that iNOS plays roles in both early and late stages of colon carcinogenesis.     

银杏叶提取物对对乙酰氨基酚诱导的小鼠急性肝损伤的保护作用

目的：探究银杏叶提取物（GBE）对对乙酰氨基酚（APAP）诱导的小鼠急性肝损伤的保护作用及其机制。方法：30只小鼠随机分为对照组、模型组、GBE低、中、高剂量组（50,100,and 200 mg·kg^-1）,每组6只。除对照组外,剩余小鼠腹腔注射APAP （300 mg/kg）一次,随后GBE低、中、高剂量组按照相应剂量灌胃给药,治疗2 d后取材。观察各组肝脏大体情况和肝组织的病理组织学变化;取血测定各组小鼠血清中ALT、AST的活性和TNF-α、IL-6的水平;取肝检测各组肝组织中SOD、MPO的活性和GSH、MDA的含量;通过Western blot检测各组肝组织中Nrf2、HO-1蛋白的表达量。结果：与对照组相比,模型组肝脏明显肿大,病理表现差,血清中ALT、AST、TNF-α、IL-6的水平显著升高（P<0.01）,肝组织中GSH的含量和SOD的活性显著降低（P<0.01）,MDA的含量和MPO的活性显著升高（P<0.01）,Nrf2、HO-1蛋白表达明显下调（P<0.01）。与模型组相比,GBE组肝脏肿大减轻,病理表现有所改善,血清中ALT、AST、TNF-α、IL-6的水平显著降低（P<0.01）,肝组织中GSH的含量和SOD的活性显著提高（P<0.01）,MDA的含量和MPO的活性显著降低（P<0.01）,Nrf2、HO-1蛋白表达上调（P<0.05）,其中高剂量GBE组治疗效果最明显。结论：GBE可对APAP诱导的小鼠急性肝损伤具有保护作用,其作用机制可能是通过Nrf2/HO-1抗氧化途径发挥作用。     

Green Tea Polyphenol (?)-Epigallocatechin-3-Gallate Restores Nrf2 Activity and Ameliorates Crescentic Glomerulonephritis

Crescentic glomerulonephritis (GN) is the most severe form of GN and is associated with significant morbidity and mortality despite aggressive immunotherapy with steroids, cytotoxic drugs, and plasmapheresis. We examined the therapeutic efficacy of the green tea polyphenol (−)-epigallocatechin-3-gallate (EGCG, 50 mg/kg BW/day x3weeks), a potent anti-inflammatory and anti-oxidant agent, on experimental crescentic GN induced in 129/svJ mice by administration of rabbit anti-mouse glomerular basement membrane sera. Routine histology and key molecules involved in inflammatory and redox signaling were studied. EGCG treatment significantly reduced mortality, decreased proteinuria and serum creatinine, and markedly improved renal histology when compared with vehicle-treated mice. The improvements in renal function and histology were accompanied by the restoration of Nrf2 signaling (which was impaired in vehicle-treated mice) as shown by increased nuclear translocation of Nrf2 and cytoplasmic glutamate cysteine ligase catalytic subunit, glutamate cysteine ligase modifier subunit, and glutathione peroxidase. EGCG-treated mice also showed reduction in p-Akt, p-JNK, p-ERK1/2 and p-P38 as well as restoration of PPARγ and SIRT1 levels. Lower dose of EGCG (25 mg/kg BW/day x2 weeks) treatment also significantly decreased proteinuria and serum creatinine, and markedly improved renal histology when compared with vehicle-treated mice. Thus, our data illustrate the efficacy of EGCG in reversing the progression of crescentic GN in mice by targeting multiple signaling and inflammatory pathways as well as countering oxidative stress.     

Nrf2激动剂CDDO-Im对高脂饮食诱导的肥胖小鼠肝脏脂肪变性的影响

摘要 目的：探究Nrf2激动剂CDDO-Im对高脂饮食诱导的肥胖小鼠肝脏脂肪变性的作用。方法：33只雄性C57BL/6J小鼠随机分为两组：一组16只饲喂普通饲料,另一组17只饲喂高脂饲料建立肥胖模型。造模成功后将小鼠随机分成四组：普通饲料溶剂对照组（Control ND组）、普通饲料Nrf2激动剂组（Nrf2(+) ND组）、高脂饲料溶剂对照组（Control HFD组）和高脂饲料Nrf2激动剂组（Nrf2(+) HFD组）。分别给予Nrf2激动剂CDDO-Im和等体积溶剂灌胃干预6周后,检测各组小鼠血清甘油三酯（TG）、总胆固醇（T-CHO）和低密度脂蛋白-胆固醇（LDL-C）。苏木素-伊红（HE）染色观察肝脏组织形态学变化。RT-qPCR检测肝脏Nrf2下游抗氧化基因Nqo1、Ho1和Gclc的mRNA表达水平,Western Blot检测肝脏NQO1、HO-1和GCLC的蛋白表达水平。结果：与正常小鼠相比,肥胖小鼠的体重、TG和LDL-C升高（P＜0.05）,肝脏脂肪变性增加,GCLC的蛋白表达水平降低（P＜0.05）。在肥胖小鼠中,与溶剂对照组相比,Nrf2激动剂组小鼠的体重、血清TG降低（P＜0.05）,肝脏脂肪变性减轻,Nqo1和Gclc的mRNA表达水平升高（P＜0.05）,NQO1和GCLC的蛋白表达水平升高（P＜0.05）。结论：Nrf2激动剂CDDO-Im可改善高脂饮食诱导的肥胖小鼠肝脏脂肪变性,可能与Nrf2激动剂CDDO-Im激活抗氧化基因的表达来减轻肝细胞氧化应激有关。     

Vanadium inhibits placental glutathione S-transferase (GST-P) positive foci in 1,2-dimethyl hydrazine induced rat colon carcinogenesis

Vanadium (V) has recently been found to possess potent anti-neoplastic activity in rat colon carcinogenesis. In the present study attempts have been made to investigate the expression of the number and area of aberrant crypt foci positive for placental glutathione S-transferase (GST-P) during 1,2-dimethyl hydrazine (DMH)-induced rat colon carcinogenesis. Male Sprague Dawley rats were randomly divided into four groups. Rats in group A were designed as normal controls. Group B animals received DMH once a week (20 mg/kg body wt.) intraperitoneally for 16 weeks. Group C rats received the same treatment of DMH as in group B, along with 0.5-ppm vanadium as ammonium monovanadate ad libitum in drinking water throughout the experiment. Vanadium alone was given to Group D rats without any DMH injection. The expression of the number and the area of aberrant crypt foci (ACF) positive for GST-P was maximum in DMH-treated group. Vanadium-treated rats significantly reduced (P < 0.001) the expression of GST-P positive ACF cells (by 71.13%) for the entire period of the study. Moreover the histopathological examination also showed that vanadium action could minimize the aberrant crypt foci (P < 0.001). Furthermore, vanadium supplementation also elevated SOD activities in both liver and colon (P < 0.01, P < 0.02 and P < 0.01, P < 0.02 respectively) when compared to their carcinogen counterparts. Our results confirm that vanadium is particularly effective in limiting the action of the carcinogen, thereby establishing its anticarcinogenicity in chemically induced rat colon carcinogenesis.     

依达拉奉通过Nrf2信号分子调节氧化应激减轻脑缺血再灌注诱导的神经损伤

本研究旨在探讨依达拉奉(edaravone,ED)在脑缺血再灌注损伤中发挥神经元保护作用与Nrf2信号分子间的关系。体内实验利用脑内脑中动脉闭塞(middle cerebral artery occlusion model,MCAO)建立SD大鼠脑缺血再灌注损伤模型,体外实验采用过氧化氢(H2O2)损伤PC12细胞建立氧化应激模型。通过TTC染色、HE染色、Nissl染色来检测大脑的病理状态。测定活性氧(reactive oxygen species,ROS)、丙二醛(malondialdehyde,MDA)含量、超氧化物歧化酶(superoxide dismutase,SOD)活性,来反映氧化应激水平。此外,通过Hoechst 33342染色和线粒体膜电位(mitochondrial membrane potential,MTP)测定,探究细胞水平的损伤。采用免疫组织化学和蛋白质印记测定Nrf2的表达。构建Nrf2敲除的PC12细胞系,证实Nrf2信号分子抑制氧化应激损伤的作用。结果提示,经依达拉奉给药后,在动物体内水平,TTC染色证实,脑缺血再灌注损伤(cerebral ischemia reperfusion injury,CIRI)大鼠的脑组织梗死体积减小(P<0.001),ROS和MDA水平下降(P<0.01),SOD活性上升(P<0.01);在细胞水平,凋亡细胞减少(P<0.05),MTP上升(P<0.01),ROS和MDA水平下降,SOD活性上升(P<0.01);在分子水平,免疫组化和Western印迹结果均提示,Nrf2蛋白质含量较正常组增加。H2O2诱导Nrf2基因敲除的PC12细胞损伤加重,且依达拉奉的治疗效果明显削弱。综上所述,Nrf2在依达拉奉减轻脑缺血再灌注诱导的氧化应激损伤中发挥关键作用。     

Nrf2 attenuates inflammatory response in COPD/emphysema: Crosstalk with Wnt3a/β‐catenin and AMPK pathways

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow limitation and abnormal inflammatory response. Wnt/β‐catenin and AMP‐activated protein kinase (AMPK) have been shown to modulate lung inflammatory responses and injury. However, it remains elusive whether Wnt/β‐catenin and AMPK modulate nuclear factor erythroid‐2 related factor‐2 (Nrf2)‐mediated protective responses during the development of emphysema. Here we showed that treatment with a Wnt pathway activator (LiCl) reduced elastase‐induced airspace enlargement and cigarette smoke extract (CSE)‐induced lung inflammatory responses in WT mice, which was associated with increased activation of Nrf2 pathway. Interestingly, these effects of LiCl were not observed in Nrf2^?/? mice exposed to elastase. In normal human bronchial epithelial (NHBE) cells, Wnt3a overexpression up‐regulated, whereas Wnt3a knockdown further down‐regulated the levels of Nrf2 and its target proteins heme oxygenase‐1 (HO‐1) and NAD(P)H: quinone oxidoreductase 1 (NQO1) by CSE treatment. In contrast, Nrf2 deficiency did not have any effects on Wnt/β‐catenin pathway in mouse lungs and NHBE cells. Both elastase and CSE exposures reduced AMPK phosphorylation. A specific AMPK activator metformin increased Wnt3a, β‐catenin, Nrf2 phosphorylation and activation but reduced the levels of IL‐6 and IL‐8 in NHBE cells and mouse lungs exposed to CSE. Furthermore, Nrf2 deficiency abolished the protection of metformin against CSE‐induced increase in IL‐6 and IL‐8 in NHBE cells. In conclusion, Nrf2 mediates the protective effects of both Wnt3a/β‐catenin and AMPK on lung inflammatory responses during the development of COPD/emphysema. These findings provide potential therapeutic targets for the intervention of COPD/emphysema.     

白藜芦醇抑制肠癌细胞焦亡的作用*

目的: 研究白藜芦醇(Res)对肠癌细胞焦亡的影响。方法: ①葡聚糖硫酸钠(DSS)诱发小鼠结肠癌(CRC)实验:30只C57BL/6小鼠随机分为正常对照组(Contro组),氧化偶氮甲烷(AOM)组,AOM/DSS组,AOM/DSS+Res组和Res组,每组6只,造模周期共70 d。第1周第1日对AOM组、AOM/DSS组和AOM/DSS+Res组小鼠AOM(10 mg/kg)腹腔注射一次,无菌水饮水,1% DSS水供AOM/DSS组和AOM/DSS+Res组饮用,对AOM/DSS+Res组和Res组小鼠灌胃给予Res(50 mg/kg),造模结束后,取小鼠结肠组织固定、包埋、切片; IHC和Western blot检测小鼠结肠组织NLRP3、Caspase-1、IL-18蛋白表达情况。②离体实验:HCT 116细胞给予Res(2.4 μg/L)以及转染miR-31,加Res实验分为4组,分别标记0 h、12 h、24 h、48 h组;细胞转染分组为5组,即control组、miR-31 mimic组、miR-31 mimic+Res组、miR-31inhibitor组、miR-31inhibitor+Res组,48 h后收集细胞,每组设置三个复孔,并通过Western blot检测细胞NLRP3、Caspase-1、GSDMD-N、IL-18和IL-1β蛋白表达情况。结果: 动物实验:与control组相比较,AOM/DSS组NLRP3、Caspase-1、IL-18蛋白表达显著升高(P<0.01),AOM/DSS+Res组NLRP3、Caspase-1、IL-18蛋白表达水平相较于AOM/DSS组有显著下降(P<0.01);细胞实验:与control组相比,miR-31 mimic组NLRP3(P<0.01)、GSDMD-N(P<0.05)、IL-18(P<0.01)蛋白表达显著升高, miR-31 inhibitor组NLRP3、GSDMD-N、IL-18蛋白表达显著降低(P<0.05)。结论: Res可通过细胞焦亡抑制结肠癌。     

Nrf2-Keap1 signaling pathway regulates human UGT1A1 expression in vitro and in transgenic UGT1 mice

The formation of beta-D-glucopyranosides (glucuronides) by the UDP-glucuronosyltransferases (UGTs) is a significant metabolic pathway that facilitates the elimination of small hydrophobic molecules such as drugs, dietary constituents, steroids, and bile acids. We elucidate here that an anti-oxidative response leads to induction of UGT1A1 through the Nrf2-Keap1 pathway. When human HepG2 cells were treated with the prooxidants tert-butylhydroquinone and beta-naphthoflavone, cellular UGT1A1 glucuronidation activities were increased. The induction of UGT1A1 proceeded following the overexpression of Nrf2 and was blocked following overexpression of Keap1, demonstrating that Keap1 suppresses Nrf2 activation of the UGT1A1 gene. Loss of function analysis for Nrf2 conducted by small interfering RNA revealed that induction of UGT1A1 was not seen in Nrf2 knock-out cells. To examine the contribution of oxidants toward the regulation of human UGT1A1 in vivo, transgenic mice bearing the human UGT1 locus (Tg-UGT1) were treated with tert-butylhydroquinone. Human UGT1A1 was markedly increased in small and large intestines as well as in liver. Gene mapping experiments including transfections of UGT1A1 reporter gene constructs into HepG2 cells coupled with functional analysis of Nrf2 expression and binding to anti-oxidant-response elements (ARE) resulted in identification of an ARE in the phenobarbital-response enhancer module region of the UGT1A1 gene. The ARE flanks the recently identified Ah receptor xenobiotic-responsive element. The results suggest that Nrf2-Keap1-dependent UGT1A1 induction by prooxidants might represent a key adaptive response to cellular oxidative stress that defends against a variety of environmental insults, including electrophile attacks and chemical carcinogenesis.     

Nrf2 expression modifies influenza A entry and replication in nasal epithelial cells

Influenza infection is a major cause of morbidity and mortality worldwide, especially during pandemics outbreaks. Emerging data indicate that phase II antioxidant enzyme pathways could play a role in virus-associated inflammation and immune clearance. While Nrf2-dependent gene expression is known to modify inflammation, a mechanistic role in viral susceptibility and clearance has yet to be elucidated. Therefore, we utilized differentiated human nasal epithelial cells (NEC) and an enzymatic virus-like particle entry assay, to examine the role Nrf2-dependent gene expression has on viral entry and replication. Herein, lentiviral vectors that express Nrf2-specific short hairpin (sh)-RNA effectively decreased both Nrf2 mRNA and Nrf2 protein expression in transduced human NEC from healthy volunteers. Nrf2 knockdown correlated with a significant increase in influenza virus entry and replication. Conversely, supplementation with the potent Nrf2 activators sulforaphane (SFN) and epigallocatechin gallate (EGCG) significantly decreased viral entry and replication. The suppressive effects of EGCG on viral replication were abolished in cells with knocked-down Nrf2 expression, suggesting a causal relationship between the EGCG-induced activation of Nrf2 and the ability to protect against viral infection. Interestingly, the induction of Nrf2 via nutritional supplements SFN and EGCG increased antiviral mediators/responses: RIG-I, IFN-β, and MxA at baseline in the absence of infection. Our data indicate that there is an inverse relationship between the levels of Nrf2 expression and the viral entry/replication. We also demonstrate that supplementation with Nrf2-activating antioxidants inhibits viral replication in human NEC, which may prove to be an attractive therapeutic intervention. Taken together, these data indicate potential mechanisms by which Nrf2-dependent gene expression regulates susceptibility to influenza in human epithelial cells.     

Specific induction of hepatic cytochrome p4501a-2 in C57BL/6 and DBA/2 mice treated with 2-amino-3-methylimidazo[4,5-f]quinoline (IQ)

The food mutagen/carcinogen amino-3-methylimidazo[4,5-f]quinoline (IQ) is activated by cytochrome p4501a-2 via N-hydrox-ylation; various P450s may contribute to detoxification via ring hydroxylation. Alterations in P450 levels by IQ treatment might therefore influence its toxicity. To examine the role of Ah locus genotype on the biochemical effects of IQ, C57BL/6 (Ah^bAh^b; p450Ia-½ inducible) and DBA/2 (Ah_dAh_d, noninducible) mice of both sexes were given IQ at varying doses, with different vehicles and routes of administration. Livers taken after 24 hours were assessed for total cytochrome p450 and activities of ethoxyresorufin-O-deethylase (EROD, a p4501a-l activity, inducible in Ah^b mice), meth-oxyresorufin-O-demethylase (MROD, a p4501a-2 activity), and benzyloxyresorufin-O-dealkylase (BzROD, an activity of p4502b). There was little effect on total cytochrome p450, but all three enzyme activities were often induced, a maximum of 2.5-fold, in both sexes and in DBA/2 as well as C57BL/6 mice. However, Western immunoblot analysis with monoclonal antibodies demonstrated an increase only in p4501a-2 protein. p4501a-l remained undetectable. A monoclonal antibody to p4502-b recognized one protein band in liver mi-crosomes from males and two bands in female mice of both strains. Amounts of these proteins were not altered by IQ treatment. Thus, IQ specifically, if moderately, induces its activating enzyme, p4501a-2, in a process that was not clearly related to Ah responsiveness.