Nur77 modulates hepatic lipid metabolism through suppression of SREBP1c activity

NR4A nuclear receptors are induced in the liver upon fasting and regulate hepatic gluconeogenesis. Here, we studied the role of nuclear receptor Nur77 (NR4A1) in hepatic lipid metabolism. We generated mice expressing hepatic Nur77 using adenoviral vectors, and demonstrate that these mice exhibit a modulation of the plasma lipid profile and a reduction in hepatic triglyceride. Expression analysis of >25 key genes involved in lipid metabolism revealed that Nur77 inhibits SREBP1c expression. This results in decreased SREBP1c activity as is illustrated by reduced expression of its target genes stearoyl-coA desaturase-1, mitochondrial glycerol-3-phosphate acyltransferase, fatty acid synthase and the LDL receptor, and provides a mechanism for the physiological changes observed in response to Nur77. Expression of LXR target genes Abcg5 and Abcg8 is reduced by Nur77, and may suggest involvement of LXR in the inhibitory action of Nur77 on SREBP1c expression. Taken together, our study demonstrates that Nur77 modulates hepatic lipid metabolism through suppression of SREBP1c activity.     

CYP2E1 potentiation of LPS and TNFα-induced hepatotoxicity by mechanisms involving enhanced oxidative and nitrosative stress, activation of MAP kinases, and mitochondrial dysfunction

The mechanisms by which alcohol causes cell injury are not clear. A major mechanism that is the focus of considerable research is the role of lipid peroxidation and oxidative stress in alcohol toxicity. Many pathways have been suggested to play a role in how alcohol induces oxidative stress. Considerable attention has been given to alcohol-elevated production of lipopolysaccharide (LPS) and TNFα and to alcohol induction of CYP2E1. These two pathways are not exclusive of each other, however, associations and interactions between them, especially in vivo, have not been extensively evaluated. We have shown that increased oxidative stress from induction of CYP2E1 in vivo sensitizes hepatocytes to LPS and TNF toxicity and that oxidants, such as peroxynitrite, activation of p38 and JNK MAP kinases, inactivation of NF-kB protective pathways and mitochondrial dysfunction are downstream mediators of this CYP2E1-LPS/TNF potentiated hepatotoxicity. This review will summarize studies showing potentiated interactions between these two risk factors in promoting liver injury and the mechanisms involved.     

Structures of the N-linked carbohydrate of ascorbic acid oxidase from zucchini

N-glycan moiety of ascorbic acid oxidase from zucchini (Cucurbita pepo) has been described to be a core-pentasaccharide with a xylose [D'Andrea et al. (1988) Glycoconjugate J 5:151-7]. Ascorbic acid oxidase is sometimes used to characterize antibodies directed against carbohydrate determinants on plant glycoproteins. To prevent misinterpretations of immunological data, the structure of the N-glycan of ascorbic acid oxidase has been reinvestigated. The oligosaccharides were released by almond N-glycosidase and analysed as their pyridylamino derivatives by 2D-HPLC and exoglycosidase digestions. The main structure resembled the typical complex plant N-glycan consisting of a core-pentasaccharide decorated with xylose and 3-linked fucose. The other abundant species lacked the fucose residue. Small amounts of these glycans carried a GlcNAc residue on the 6-arm. Therefore, ascorbic acid oxidase will not only react with antibodies directed against the xylosylated region but also with those binding to N-glycans with 3-linked fucose.     

Cellular Caspase-3 Contributes to EV-A71 2Apro-Mediated Down-Regulation of IFNAR1 at the Translation Level

Enterovirus A71(EV-A71) is the major pathogen responsible for the severe hand, foot and mouth disease worldwide, for which few effective antiviral drugs are presently available. Interferon-a(IFN-a) has been used in antiviral therapy for decades; it has been reported that EV-A71 antagonizes the antiviral activity of IFN-a based on viral 2 Apro-mediated reduction of the interferon-alpha receptor 1(IFNAR1); however, the mechanism remains unknown. Here, we showed a significant increase in IFNAR1 protein induced by IFN-a in RD cells, whereas EV-A71 infection caused obvious downregulation of the IFNAR1 protein and blockage of IFN-a signaling. Subsequently, we observed that EV-A71 2 Apro inhibited IFNAR1 translation by cleavage of the eukaryotic initiation factor 4 GI(eIF4GI), without affecting IFNAR1 m RNA levels induced by IFN-a. The inhibition of IFNAR1 translation also occurred in puromycin-induced apoptotic cells when caspase-3 cleaved e IF4 GI. Importantly, we verified that 2 Aprocould activate cellular caspase-3, which was subsequently involved in e IF4 GI cleavage mediated by 2 Apro. Furthermore, inhibition of caspase-3 activation resulted in the partial restoration of IFNAR1 in cells transfected with 2 A or infected with EV-A71, suggesting the pivotal role of both viral 2 Aproand caspase-3 activation in the disturbance of IFN-a signaling. Collectively, we elucidate a novel mechanism by which cellular caspase-3 contributes to viral 2 Apro-mediated down-regulation of IFNAR1 at the translation level during EV-A71 infection, indicating that caspase-3 inhibition could be a potential complementary strategy to improve clinical anti-EV-A71 therapy with IFN-a.     

Increase of hepatic fat accumulation by liver specific expression of Hepatitis B virus X protein in zebrafish

The pathogenesis of fatty liver disease remains largely unknown. Here, we assessed the importance of hepatic fat accumulation on the progression of hepatitis in zebrafish by liver specific expression of Hepatitis B virus X protein (HBx). Transgenic zebrafish lines, GBXs, which selectively express the GBx transgene (GFP-fused HBx gene) in liver, were established. GBX Liver phenotypes were evaluated by histopathology and molecular analysis of fatty acid (FA) metabolism-related genes expression. Most GBXs (66–81%) displayed obvious emaciation starting at 4 months old. Over 99% of the emaciated GBXs developed hepatic steatosis or steatohepatitis, which in turn led to liver hypoplasia. The liver histology of GBXs displayed steatosis, lobular inflammation, and balloon degeneration, similar to non-alcoholic steatohepatitis (NASH). Oil red O stain detected the accumulation of fatty droplets in GBXs. RT-PCR and Q-rt-PCR analysis revealed that GBx induced hepatic steatosis had significant increases in the expression of lipogenic genes, C/EBP-α, SREBP1, ChREBP and PPAR-γ, which then activate key enzymes of the de novo FA synthesis, ACC1, FAS, SCD1, AGAPT, PAP and DGAT2. In addition, the steatohepatitic GBX liver progressed to liver degeneration and exhibited significant differential gene expression in apoptosis and stress. The GBX models exhibited both the genetic and functional factors involved in lipid accumulation and steatosis-associated liver injury. In addition, GBXs with transmissible NASH-like phenotypes provide a promising model for studying liver disease.     

<Emphasis Type="Italic">p</Emphasis>-hydroxybenzyl alcohol prevents brain injury and behavioral impairment by activating Nrf2, PDI,and neurotrophic factor genes in a rat model of brain ischemia

The therapeutic goal in treating cerebral ischemia is to reduce the extent of brain injury and thus minimize neurological impairment. We examined the effects of p-hydroxybenzyl alcohol (HBA), an active component of Gastrodia elata Blume, on transient focal cerebral ischemia-induced brain injury with respect to the involvement of protein disulphide isomerase (PDI), nuclear factor-E2-related factor 2 (Nrf2), and neurotrophic factors. All animals were ovariectomized 14 days before ischemic injury. Ischemic injury was induced for 1 h by middle cerebral artery occlusion (MCAO) followed by 24-h reperfusion. Three days before MCAO, the vehicle-treated and the HBA-treated groups received intramuscular sesame oil and HBA (25 mg/kg BW), respectively. 2,3,5-Triphenyltetrazolium chloride (TTC) staining showed decreased infarct volume in the ischemic lesion of HBA-treated animals. HBA pretreatment also promoted functional recovery, as measured by the modified neurological severity score (mNSS; p < 0.05). Moreover, expression of PDI, Nrf2, BDNF, GDNF, and MBP genes increased by HBA treatment. In vitro, H₂O₂-induced PC12 cell death was prevented by 24 h HBA treatment, but bacitracin, a PDI inhibitor, attenuated this cytoprotective effect in a dose-dependent manner. HBA treatment for 2 h also induced nuclear translocation of Nrf2, possibly activating the intracellular antioxidative system. These results suggest that HBA protects against brain damage by modulating cytoprotective genes, such as Nrf2 and PDI, and neurotrophic factors.     

Methylseleninic acid activates Keap1/Nrf2 pathway via up-regulating miR-200a in human oesophageal squamous cell carcinoma cells

Oesophageal squamous cell carcinoma (ESCC) occurs at a very high rates in certain regions of China. There are increasing evidences demonstrating that selenium could act as a potential anti-oesophageal cancer agent, but the precise mechanisms involved are still not completely understood. Methylseleninic acid (MSA), as a potent second-generation selenium compound, is a promising chemopreventive agent. Previous studies demonstrated that the kelch-like ECH-associated protein 1 (Keap1)/nuclear factor E2-related factor 2 (Nrf2) system plays a critical role in cancer prevention, but little is known about its association with MSA in ESCC cells. In the present study, we observed that MSA treatment significantly down-regulated Keap1, induced nuclear accumulation of Nrf2 and enhance the antioxidant response element (ARE) promoter activity in ESCC cells. MSA could also significantly induce miR-200a expression and inhibit Keap1 directly. Antagomir-200a could attenuate MSA treatment-induced Keap1 down-regulation in ESCC cells. Moreover, MSA-induced miR-200a expression was dependent on the mediation of Krüpple-like factor 4 (KLF4). These results reaffirm the potential role of MSA as a chemopreventive agent via the regulation of KLF4/miR-200a/Keap1/Nrf2 axis in ESCC cells.     

miR-380调控羊驼黑色素细胞MAPK/ERK信号通路

miR-380是不同羊驼毛色中差异表达的基因之一,但是否与黑色素生成有关未见报道。为了丰富调控黑色素生成的机制,挖掘黑色素生成路径中所涉及到的更多新的基因并揭示miR 380在黑色素细胞中的功能,本实验通过生物信息学方法预测出MAPK信号通路的成员MAP3K6是miR-380的靶基因之一。在293T细胞中共转染miR-380和MAP3K6后,与对照组相比双荧光报告酶活性下降（28.92 ± 25.63）%(P<0.01) ,下降趋势明显,说明MAP3K6可能是miR-380的靶基因之一;在羊驼黑色素细胞中转染miR-380后,MAP3K6、MEK1、ERK1/2、CREB和MITF在转录水平的表达量与NC组相比具有显著下降趋势,其中CREB下降趋势尤为显著（64.20 ± 54.30）%（P<0.01）,Western印迹检测MAP3K6、p-MEK1、p-ERK1/2、CREB和MITF在蛋白质水平的表达与NC组相比下降趋势明显且p-MEK1和CREB基因下降极为显著,分别为（29.09 ± 10.68）%（P<0.001）和（47.12 ± 6.70）%（P<0.001）,抑制组则反之。通过 Masson-Fontana黑色素颗粒染色法检测miR-380抑制黑色素细胞产生黑色素颗粒,用紫外分光度法检测真黑素（eumelanin,EM）和褐黑素（pheomelanin,PM）,含量结果提示EM与PM含量分别下降为（38.63 ± 2.00）%（P<0.01）,（54.10 ± 5.73）%（P<0.001）且PM含量下降极为显著。综上所述miR-380通过靶向抑制MAP3K6等基因的表达,从而对MAPK/ERK信号通路起调控作用,最终影响黑色素生成生物学功能,此研究对哺乳动物毛色形成机制和防止皮肤受紫外辐射有重要意义。     

组蛋白赖氨酸甲基转移酶2D调控H9c2细胞中HIF-1α/VEGF通路

组蛋白赖氨酸甲基转移酶2D (histone-lysine N-methyltransferase 2D, KMT2D) 作为主要的组蛋白3第4位赖氨酸 (H3K4) 甲基转移酶,在调控胚胎发育、组织分化、代谢和肿瘤抑制方面发挥重要作用。在小鼠体内,敲除Kmt2d会导致严重的心脏发育缺陷最终造成胚胎期死亡。低氧诱导因子-1α (hypoxia-inducible factor 1α, HIF-1α) 作为调节细胞应对低氧的关键转录因子,能够调控多种下游基因转录。有相关研究揭示,表观遗传调控者能够调节HIF-1α的稳定性和活性。同样,作为表观遗传调控者的组蛋白甲基转移酶KMT2D是否参与低氧条件下HIF-1α对下游基因的调控,目前仍未知。在本研究中,观察在Kmt2d正常或缺乏的情况下,心肌细胞H9c2对低氧环境的应答反应。结果显示,与常氧条件相比,低氧状态下HIF-1α、组蛋白乙酰化酶P300、KMT2D及其介导的H3K4一甲基化 (H3K4 mono-methylation, H3K4me1)的蛋白质水平增加 (P<0.05);HIF-1α下游基因血管内皮生长因子 (vascular endothelial growth factor, Vegf) 的mRNA表达水平明显上调 (P<0.01)。染色质免疫共沉淀实验 (chromatin immunoprecipitation assay, ChIP-qPCR) 检测结果显示,H3K4me1和组蛋白3第27位赖氨酸乙酰化 (histone 3 lysine 27 acetylation, H3K27ac) 在Vegf基因启动子区域的结合丰度明显增加 (P<0.05)。低氧条件下沉默Kmt2d之后,H3K4me1蛋白水平和Vegf的mRNA表达下降 (P<0.05)。本研究表明,低氧条件下KMT2D参与调控HIF-1α和下游基因Vegf的表达。     

组蛋白赖氨酸甲基转移酶2D调控H9c2细胞中HIF-1α/VEGF通路

组蛋白赖氨酸甲基转移酶2D (histone-lysine N-methyltransferase 2D, KMT2D) 作为主要的组蛋白3第4位赖氨酸 (H3K4) 甲基转移酶,在调控胚胎发育、组织分化、代谢和肿瘤抑制方面发挥重要作用。在小鼠体内,敲除Kmt2d会导致严重的心脏发育缺陷最终造成胚胎期死亡。低氧诱导因子-1α (hypoxia-inducible factor 1α, HIF-1α) 作为调节细胞应对低氧的关键转录因子,能够调控多种下游基因转录。有相关研究揭示,表观遗传调控者能够调节HIF-1α的稳定性和活性。同样,作为表观遗传调控者的组蛋白甲基转移酶KMT2D是否参与低氧条件下HIF-1α对下游基因的调控,目前仍未知。在本研究中,观察在Kmt2d正常或缺乏的情况下,心肌细胞H9c2对低氧环境的应答反应。结果显示,与常氧条件相比,低氧状态下HIF-1α、组蛋白乙酰化酶P300、KMT2D及其介导的H3K4一甲基化 (H3K4 mono-methylation, H3K4me1)的蛋白质水平增加 (P<0.05);HIF-1α下游基因血管内皮生长因子 (vascular endothelial growth factor, Vegf) 的mRNA表达水平明显上调 (P<0.01)。染色质免疫共沉淀实验 (chromatin immunoprecipitation assay, ChIP-qPCR) 检测结果显示,H3K4me1和组蛋白3第27位赖氨酸乙酰化 (histone 3 lysine 27 acetylation, H3K27ac) 在Vegf基因启动子区域的结合丰度明显增加 (P<0.05)。低氧条件下沉默Kmt2d之后,H3K4me1蛋白水平和Vegf的mRNA表达下降 (P<0.05)。本研究表明,低氧条件下KMT2D参与调控HIF-1α和下游基因Vegf的表达。     

Salvianic acid A alleviates chronic alcoholic liver disease by inhibiting HMGB1 translocation via down‐regulating BRD4

Alcoholic liver disease (ALD) is the major cause of chronic liver disease and a global health concern. ALD pathogenesis is initiated with liver steatosis, and ALD can progress to steatohepatitis, fibrosis, cirrhosis and even hepatocellular carcinoma. Salvianic acid A (SAA) is a phenolic acid component of Danshen, a Chinese herbal medicine with possible hepatoprotective properties. The purpose of this study was to investigate the effect of SAA on chronic alcoholic liver injury and its molecular mechanism. We found that SAA significantly inhibited alcohol‐induced liver injury and ameliorated ethanol‐induced hepatic inflammation. These protective effects of SAA were likely carried out through its suppression of the BRD4/HMGB1 signalling pathway, because SAA treatment largely diminished alcohol‐induced BRD4 expression and HMGB1 nuclear translocation and release. Importantly, BRD4 knockdown prevented ethanol‐induced HMGB1 release and inflammatory cytokine production in AML‐12 cells. Similarly, alcohol‐induced pro‐inflammatory cytokines were blocked by HMGB1 siRNA. Collectively, our results reveal that activation of the BRD4/HMGB1 pathway is involved in ALD pathogenesis. Therefore, manipulation of the BRD4/HMGB1 pathway through strategies such as SAA treatment holds great therapeutic potential for chronic alcoholic liver disease therapy.     

Structural similarity between binding sites in influenza sialidase and isocitrate dehydrogenase: implications for an alternative approach to rational drug design.

Using searching techniques based on algorithms derived from graph theory, we have established a similarity between a 3-dimensional cluster of side chains implicated in drug binding in influenza sialidase and side chains involved in isocitrate binding in Escherichia coli isocitrate dehydrogenase. The possible implications of the use of such comparative methods in drug design are discussed.     

Sex differences in hepatic gene expression in a rat model of ethanol-induced liver injury.

Sex differences in susceptibility to alcohol-induced liver injury have been observed in both humans and experimental animal models. Using a standard model of alcohol-induced fatty liver injury and microarray analysis, we have identified differential expression of hepatic genes in both sexes. The genes that exhibit differential expression are of three types: those that are changed only in male rats fed alcohol, those that change in only female rats fed alcohol, and those that change in both sexes, although not always in the same manner. Certain of the differentially expressed genes have previously been identified as participants in the induction of alcohol-induced liver injury. However, this analysis has identified a number of genes that heretofore have not been implicated in alcoholic liver injury; such genes may provide new areas of investigation into the pathogenesis of this disease.     

中国人群中抗结核药物引发肝损伤的易感基因标记研究

为系统性研究中国人群中抗结核药物引发肝损伤的易感基因标记,本研究以41例抗结核药物引发肝损伤的病人和39例健康对照为研究对象,采用Haloplex捕获测序的方法对其基因组中药物代谢、转运和免疫相关通路的109个基因进行靶向测序。用Plink软件对DNA突变位点与肝损伤的发生进行关联分析,以千人基因组计划东亚人群作为对照组,对显著性位点进行验证,并用SIFT和Polyphen2软件对预测显著关联的位点进行功能预测。结果发现UGT1A4 rs2011404(X^2=4.6809,P=0.0305)是抗结核药物引发的肝损伤的易感基因标记,且rs2011404突变可能引起UGT1A4蛋白的功能障碍。本研究为临床上对抗结核药物的合理用药提供了有益的参考。     

Genetic ablation and short-duration inhibition of lipoxygenase results in increased macroautophagy

12/15-lipoxygenase (12/15-LOX) is involved in organelle homeostasis by degrading mitochondria in maturing red blood cells and by eliminating excess peroxisomes in liver. Furthermore, 12/15-LOX contributes to diseases by exacerbating oxidative stress-related injury, notably in stroke. Nonetheless, it is unclear what the consequences are of abolishing 12/15-LOX activity. Mice in which the alox15 gene has been ablated do not show an obvious phenotype, and LOX enzyme inhibition is not overtly detrimental. We show here that liver histology is also unremarkable. However, electron microscopy demonstrated that 12/15-LOX knockout surprisingly leads to increased macroautophagy in the liver. Not only macroautophagy but also mitophagy and pexophagy were increased in hepatocytes, which otherwise showed unaltered fine structure and organelle morphology. These findings were substantiated by immunofluorescence showing significantly increased number of LC3 puncta and by Western blotting demonstrating a significant increase for LC3-II protein in both liver and brain homogenates of 12/15-LOX knockout mice. Inhibition of 12/15-LOX activity by treatment with four structurally different inhibitors had similar effects in cultured HepG2 hepatoma cells and SH-SY5Y neuroblastoma cells with significantly increased autophagy discernable already after 2 hours. Hence, our study reveals a link between ablation or inhibition of 12/15-LOX and stimulation of macroautophagy. The enhanced macroautophagy may be related to the known tissue-protective effects of LOX ablation or inhibition under various diseased conditions caused by oxidative stress and ischemia. This could provide an important cleaning mechanism of cells and tissues to prevent accumulation of damaged mitochondria and other cellular components.     

The unconventional secretory machinery of fibroblast growth factor 2

Unconventional secretory proteins represent a subpopulation of extracellular factors that are exported from eukaryotic cells by mechanisms that do not depend on the endoplasmic reticulum and the Golgi complex. Various pathways have been implicated in unconventional secretion including those involving intracellular membrane-bound intermediates and others that are based on direct protein translocation across plasma membranes. Interleukin 1β (IL1β) and fibroblast growth factor 2 (FGF2) are classical examples of unconventional secretory proteins with IL1β believed to be present in intracellular vesicles prior to secretion. By contrast, FGF2 represents an example of a non-vesicular mechanism of unconventional secretion. Here, the author discusses the current knowledge about the molecular machinery being involved in FGF2 secretion. To reveal both differential and common requirements, this review further aims at a comprehensive comparison of this mechanism with other unconventional secretory processes. In particular, a potentially general role of tyrosine phosphorylation as a regulatory signal in unconventional protein secretion will be discussed.