Carvacrol modulates instability of xenobiotic metabolizing enzymes and downregulates the expressions of PCNA,MMP-2, and MMP-9 during diethylnitrosamine-induced hepatocarcinogenesis in rats

Vascular calcification (VC) is highly associated with increased morbidity and mortality in patients with advanced chronic kidney disease. Paracrine/autocrine factors such as vasoactive peptides are involved in VC development. Here, we investigated the expression of the novel peptide C-type natriuretic peptide (CNP) in the vasculature, tested its ability to prevent VC in vivo and in vitro, and examined the mechanism involved. Rat aortic VC was induced by vitamin D3 plus nicotine (VDN). CNP (500 ng/kg/h) was administered by mini-osmotic pump. Calcification was examined by von Kossa staining; CNP and cyclic guanosine monophosphate (cGMP) contents were detected by radioimmunoassay, and mRNA and protein levels were examined by real-time PCR and Western blot analysis in aortas and calcified vascular smooth muscle cells (VSMCs). VDN-treated rat aortas showed higher CNP content and decreased expression of its receptor natriuretic peptide receptor B, along with increased vascular calcium deposition and alkaline phosphatase (ALP) activity. Low CNP levels were accompanied by increased vascular calcium deposition and ALP activity in VDN-treated rats when compared to vehicle treatment, which was further confirmed in cultured VSMCs. Administration of CNP greatly reduced VC in VDN-treated aortas compared with controls, which was confirmed in calcified VSMCs. The decrease in alpha-actin expression was ameliorated by CNP in vitro. Moreover, protein expression levels of osteopontin (OPN) were significantly up-regulated in calcified aortas, and CNP increased OPN expression in calcified aortas. Furthermore, CNP downregulated OPN and bone morphogenic protein 2 (BMP-2) expression in calcified aortas and VSMCs. Modulation of OPN and BMP-2 expression by CNP and the beneficial effects of CNP on calcified VSMCs were blocked significantly by protein kinase G inhibitor H7. Impaired local endogenous CNP and its receptor system may be associated with increased mineralization in vivo in rat aortas with VC, and administration of CNP inhibits VC development in vivo and in vitro, at least in part, via a cGMP/PKG pathway.     

Induction of NAD(P)H:quinone oxidoreductase 1 expression by cysteine via Nrf2 activation in human intestinal epithelial LS180 cells

In this study, we examined the effects of 20 amino acids on the expression level of NAD(P)H:quinone oxidoreductase 1 (NQO1) in human intestinal LS180 cells. Five amino acids were associated with significant increases in NQO1 mRNA expression; the most substantial increase was induced by cysteine, which markedly increased the NQO1 mRNA level in a time- and dose-dependent manner. Cysteine also increased the protein level of NQO1 and its enzymatic activity in LS180 cells. Furthermore, cysteine significantly up-regulated NQO1 promoter activity, and this induction was completely abolished by mutation of the antioxidant response element, a binding site of the nuclear factor erythroid 2-related factor 2 (Nrf2). Knockdown experiment using siRNA against Nrf2 showed the involvement of Nrf2 on cysteine-induced increase in NQO1 mRNA expression. Further, cysteine treatment increased the amount of Nrf2 protein in the nucleus and decreased the amount of Kelch-like ECH-associated protein 1 (a suppressor protein of Nrf2) in the cytosol, suggesting that Nrf2 was activated by cysteine. Oral administration of cysteine to mice significantly increased NQO1 mRNA levels in the mouse intestinal mucosa. These findings show that cysteine induces NQO1 expression in both in vitro and in vivo systems and also suggest that Nrf2 activation is involved in this induction.     

Bach1与Nrf2调控γ-谷氨酰半胱氨酸合成酶对大鼠慢性阻塞性肺疾病的作用

为了探讨Bach1 (BTB and CNC homology 1)与红系衍生的核因子相关因子2（Nrf2）和γ-谷氨酰半胱氨酸合成酶（γ-GCS）的表达变化在慢性阻塞性肺疾病（COPD）中的作用和意义,用气管内注入脂多糖及熏香烟的复合刺激法建立大鼠COPD 模型．观察两组大鼠的肺组织病理学改变,测两组大鼠的肺功能指标;应用免疫组化、Western印迹、原位杂交和逆转录 聚合酶链反应（RT-PCR）等方法检测Bach1与Nrf2及γ GCS 在两组大鼠的肺组织中的表达．结果显示,COPD组的肺功能指标（FEV_0.3、FEV_0.3/FVC%、PEF）明显恶化;光镜下肺组织病理改变符合COPD的特征性改变;γ GCS与Nrf2 的mRNA及蛋白质表达在COPD组大鼠肺组织中明显增强;而Bach1 mRNA及蛋白质在COPD组和对照组的表达无明显差别．且核/胞浆分离技术表明,Nrf2蛋白在对照组主要表达于胞浆,胞核中表达水平较弱,在COPD组胞浆、胞核均有表达,但是在胞核中的水平明显升高;Bach1蛋白在对照组主要表达于胞核中,胞浆中无明显表达,在COPD组主要表达于胞浆,胞核中无明显表达．相关性分析表明,γ-GCS mRNA与FEV_0.3、FEV_0.3/FVC%、PEF均呈负相关;Nrf2蛋白表达与γ GCS mRNA呈正相关;Bach1蛋白与γ GCS mRNA呈负相关．上述结果提示,Bach1与Nrf2和γ-GCS可能均在大鼠COPD的发病中发挥作用,且Bach1和Nrf2可能通过竞争性机制调控γ-GCS的表达,影响COPD的发生和发展．     

Scutellarin ameliorates nonalcoholic fatty liver disease through the PPARγ/PGC-1α-Nrf2 pathway

Nonalcoholic fatty liver disease (NAFLD) is characterised by excessive accumulation of hepatic lipids and oxidative injury of hepatocytes. Scutellarin is a flavonoid glycoside having antioxidative stress activity. Our current study aims to investigate the molecular mechanism of scutellarin ameliorating NAFLD. Scutellarin treatment was applied to male C57BL/6 mice maintained on a high-fat diet (HFD) and HepG2 cells challenged with oleic acid. The antioxidation biochemical indicators and lipid levels in the liver and cells were detected by kits. Liver pathology was observed by light microscope, Oil Red O staining, and transmission electron microscope (TEM). In addition, quantitative real-time polymerase chain reactions (qRT-PCR) and western blot assays were employed to detect the mRNA and protein levels of various antioxidative-related genes in the presence or absence of peroxisome proliferator-activated receptor gamma (PPARγ); inhibitor GW9662. Our results showed that scutellarin could significantly reduce blood lipid levels and enhance antioxidative capacities in both the models. In addition, scutellarin treatment conspicuously activated PPARγ, peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), nuclear factor erythroid-2-related factor (Nrf2), haem oxygenase-1 (HO-1), glutathione S-transferase (GST), and NAD(P)H quinone dehydrogenase one (NQO1), while it significantly inhibited nuclear factor kappa B (NF-κB), Kelch-like ECH-associated protein 1 (Keap1) at both the mRNA and protein levels. However, after interfered by GW9662, scutellarin effect was significantly decreased. The experimental data demonstrated that scutellarin showed strong hypolipidaemic, antioxidative, and liver protective activity which could be attributed to its regulating activity in the PPARγ/PGC-1α-Nrf2 signaling pathway.     

核因子相关因子2调节大鼠气道上皮细胞γ-谷氨酰半胱氨酸合成酶表达

红系衍生的核因子相关因子2(Nrf2)是调节抗氧化基因表达的关键转录因子.γ-谷氨酰半胱氨酸合成酶(γ-GCS)是肺内主要的抗氧化基因,但Nrf2如何调节γ-GCS表达目前仍不清楚.该文采用香烟烟雾提取物(CSE)诱导大鼠气道上皮细胞为研究对象,探索Nrf2调节γ-GCS基因表达机制.细胞免疫荧光显示,Nrf2蛋白质在CSE处理1、3和6h组,主要在胞核中表达.细胞免疫化学与Western印迹显示,γ-GCS蛋白质在CSE1、3、6h组表达明显增强,其中Western印迹结果高于对照组(P0.05);Nrf2胞核蛋白质表达增强.p-aPKCι/ζ蛋白质在CSE1、3h组表达增强,与对照组相比差异显著(P0.05).RT-PCR结果表明,γ-GCSmRNA在CSE1、3、6h组表达明显高于对照组(P0.05).γ-GCS活性在CSE1、3、6h组增高并高于对照组(P0.05).GSH含量在CSE3、6h组明显高于对照组(P0.05).预先加入aPKCι/ζ抑制剂RO813220,Nrf2胞浆蛋白质表达增强,GSH含量、p-aPKCι/ζ蛋白质、γ-GCS蛋白质与其mRNA和活性均明显低于CSE3h组(P0.05).相关性分析显示Nrf2与γ-GCS、γ-GCS活性、p-aPKCι/ζ呈正相关,p-aPKCι/ζ与Nrf2、γ-GCS、γ-GCS活性呈正相关(P0.05).以上结果表明,CSE可能通过aPKCι/ζ-Nrf2信号通路调节γ-GCS的表达水平和活性.     

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。     

Nrf2/HO-1/HMGB1信号通路在白血病细胞K562/A02化疗耐药中的机制研究

目的:通过观察高迁移率族蛋白1(HMGB1)、转录因子NF-E2相关因子2(Nrf2)及血红素加氧酶1(HO-1)基因沉默对白血病化疗耐药细胞(K562/A02细胞株)的影响,探讨该信号通路在白血病化疗耐药中的作用及其可能机制。方法:将HMGB1基因、Nrf2基因及HO-1基因的特异性干扰RNA分别转染阿霉素耐药细胞株K562/A02,荧光实时定量(RT-PCR)方法检测HMGB1、Nrf2及HO-1的mRNA表达水平,Western blot方法检测HMGB1、Nrf2及HO-1的蛋白表达水平,免疫荧光方法检测Nrf2的蛋白表达,并使用CCK-8方法检测转染前后K562/A02细胞株的细胞活性。结果:HMGB1基因、Nrf2基因或HO-1基因沉默的K562/A02细胞活性皆显著低于对照组及空白组(P0.05),化疗敏感性恢复。结论:HMGB1高表达导致了白血病细胞株K562/A02对阿霉素的化疗耐药,Nrf2/HO-1信号通路参与了HMGB1诱导的K562/A02细胞的化疗耐药,其表达上调可恢复K562/A02细胞对阿霉素的敏感性。     

Peroxynitrite activates NF-E2-related factor 2/antioxidant response element through the pathway of phosphatidylinositol 3-kinase: the role of nitric oxide synthase in rat glutathione S-transferase A2 induction.

The protective adaptive response to electrophiles and reactive oxygen species is mediated by the induction of phase II detoxifying genes through antioxidant response elements (AREs). Our previous study showed that sulfur amino acid deprivation (SAAD) produces peroxides and induces rat glutathione S-transferase A2 (rGSTA2) through NF-E2-related factor 2 (Nrf2)/ARE activation via the pathway of phosphatidylinositol 3-kinase (PI3-kinase). The current study was designed to investigate the role of peroxynitrite in Nrf2/ARE activation and rGSTA2 induction. L-Arginine deficiency or N(G)-nitro-L-arginine methyl ester (L-NAME) reduced peroxide production induced by SAAD in H4IIE cells. Northern and Western blot analyses revealed that the levels of rGSTA2 mRNA and protein were significantly increased 24h after incubation of the cells in SAAD medium, which was inhibited by L-arginine deficiency or L-NAME. Subcellular fractionation and gel shift analyses revealed that SAAD increased the level of nuclear Nrf2 and activated ARE, which were also blocked by L-arginine deficiency or L-NAME. Whereas the exogenous NO donor S-nitroso-N-acetyl-penicillamine (SNAP) alone failed to significantly induce rGSTA2, SNAP enhanced SAAD-inducible rGSTA2 expression, verifying the notion that peroxynitrite derived from NO contributes to rGSTA2 induction. 3-Morpholinosydnonimine (SIN-1), which decomposes and yields peroxynitrite, increased the rGSTA2 mRNA and protein levels in a dose-dependent manner. SIN-1 increased the level of nuclear Nrf2 and activated Nrf2/ARE, which was supershifted by anti-Nrf2 and anti-Maf antibodies. SIN-1 increased the activity of PI3-kinase, as monitored by phosphorylation of Akt. SIN-1-inducible rGSTA2 expression was inhibited by PI3-kinase inhibitors. These results provide evidence that peroxynitrite plays an essential role in nuclear translocation of Nrf2 and ARE activation through the pathway of PI3-kinase and that nitric oxide synthase is involved in the induction of rGSTA2.     

Constitutive activation of nuclear factor-E2-related factor 2 induces biotransformation enzyme and transporter expression in livers of mice with hepatocyte-specific deletion of Kelch-like ECH-associated protein 1

Chemicals that activate nuclear factor-E2-related factor 2 (Nrf2) often increase multidrug-resistance-associated protein (Mrp) expression in liver. Hepatocyte-specific deletion of Kelch-like ECH-associated protein 1 (Keap1) activates Nrf2. Use of hepatocyte-specific Keap1 deletion represents a nonpharmacological method to determine whether constitutive Nrf2 activation upregulates liver transporter expression in vivo. The mRNA, protein expression, and localization of several biotransformation and transporters were determined in livers of wild-type and hepatocyte-specific Keap1-null mice. Sulfotransferase 2a1/2, NADP(H):quinone oxidoreductase 1, cytochrome P450 2b10, 3a11, and glutamate-cysteine ligase catalytic subunit expression were increased in livers of Keap1-null mice. Organic anion-transporting polypeptide 1a1 expression was nearly abolished, as compared to that detected in livers of wild-type mice. By contrast, Mrp 1-5 mRNA and protein levels were increased in Keap1-null mouse livers, with Mrp4 expression being more than 15-fold higher than wild types. In summary, Nrf2 has a significant role in affecting Oatp and Mrp expressions.     

Berry anthocyanins suppress the expression and secretion of proinflammatory mediators in macrophages by inhibiting nuclear translocation of NF-κB independent of NRF2-mediated mechanism

The objectives of this study were to compare the anti-inflammatory effects of anthocyanins from blueberry (BBA), blackberry (BKA), and blackcurrant (BCA) and to determine the relationship between their antioxidant capacity and anti-inflammatory effect in macrophages. Major anthocyanins in BBA, BKA and BCA were malvidin-3-glucoside (16%), cyanidin-3-glucoside (98%) and delphinidin-3-rutinoside (44%), respectively. BKA showed higher total antioxidant capacity than BBA and BCA. RAW 264.7 macrophages were incubated with 0–20 μg/ml of BBA, BKA and BCA, and subsequently activated by lipopolysaccharide (LPS) to measure proinflammatory cytokine production. Interleukin 1β (IL-1β) messenger RNA (mRNA) levels were significantly decreased by all berry anthocyanins at 10 μg/ml or higher. Tumor necrosis factor α (TNFα) mRNA levels and secretion were also significantly decreased in LPS-treated macrophages. The levels of the repression were comparable for all berry anthocyanins. LPS-induced nuclear factor κB (NF-κB) p65 translocation to the nucleus was markedly attenuated by all of the berry anthocyanins. In bone marrow-derived macrophages (BMMs) from nuclear factor E2-related factor 2 wild-type (Nrf2^+/+) mice, BBA, BKA and BCA significantly decreased cellular reactive oxygen species (ROS) levels with a concomitant decrease in IL-1β mRNA levels upon LPS stimulation. However, in the BMM from Nrf2^−/− mice, the anthocyanin fractions were able to significantly decrease IL-1β mRNA despite the fact that ROS levels were not significantly affected. In conclusion, BBA, BKA and BCA exert their anti-inflammatory effects in macrophages, at least in part, by inhibiting nuclear translocation of NF-κB independent of the NRF2-mediated pathways.     

基于PERK/Nrf2/HO-1信号通路研究瑞马唑仑对心肌缺血再灌注损伤大鼠铁死亡的影响

摘要 目的：基于蛋白激酶R样内质网激酶（PERK）/核因子E2相关因子2（Nrf2）/血红素氧合酶-1（HO-1）信号通路探究瑞马唑仑对心肌缺血再灌注损伤（MIRI）大鼠铁死亡的影响。方法：将90只SD大鼠随机分为假手术（Sham）组、MIRI组、低剂量-瑞马唑仑组（L-瑞马唑仑组，5 mg/kg）、高剂量-瑞马唑仑组（H-瑞马唑仑组，20 mg/kg）、H-瑞马唑仑+PERK抑制剂组（瑞马唑仑20 mg/kg+GSK2606414 1 mg/kg），每组18只。采用结扎冠状动脉左前降支（LAD）0.5 h、再灌注2 h制备MIRI大鼠模型，于再灌注2 h后即刻尾静脉注射给药，再灌注24 h后进行组织取材。酶联免疫吸附（ELISA）法检测血清心肌损伤标志物[肌酸激酶同工酶（CK-MB）、心肌肌钙蛋白I（cTnI）]水平；HE染色观察心肌组织病理改变；Tunel染色检测心肌细胞凋亡；透射电镜观察心肌细胞超微结构变化；检测心肌组织中铁死亡相关标志物[铁、活性氧（ROS）、谷胱甘肽（GSH）、丙二醛（MDA）]水平；蛋白质印迹法（Western Blot）检测心肌组织中PERK/Nrf2/HO-1信号通路相关蛋白表达。结果：与Sham组相比，MIRI组心肌结构受损，纤维排列紊乱，线粒体呈现显著的铁死亡特征（膜固缩，膜密度增加，嵴减少），血清中CK-MB、cTnI水平，心肌细胞凋亡率及心肌组织中铁、ROS、MDA水平升高（P<0.05），心肌组织中GSH水平及p-PERK/PERK、核Nrf2/Nrf2、HO-1蛋白表达降低（P<0.05）；与MIRI组相比，L-瑞马唑仑组和H-瑞马唑仑组心肌组织上述病理改变明显减轻，血清CK-MB、cTnI水平，心肌细胞凋亡率及心肌组织中铁、ROS、MDA水平降低（P<0.05），心肌组织中GSH水平及p-PERK/PERK、核Nrf2/Nrf2、HO-1蛋白表达升高（P<0.05）；与H-瑞马唑仑组相比，H-瑞马唑仑+PERK抑制剂组心肌组织上述病理改变加重，血清CK-MB、cTnI水平，心肌细胞凋亡率及心肌组织中铁、ROS、MDA水平升高（P<0.05），心肌组织中GSH水平及p-PERK/PERK、核Nrf2/Nrf2、HO-1蛋白表达降低（P<0.05）。结论：瑞马唑仑可通过抑制铁死亡减轻大鼠MIRI，可能通过激活PERK/Nrf2/HO-1信号通路而实现。