Autophagy: a cyto-protective mechanism which prevents primary human hepatocyte apoptosis during oxidative stress

The role of autophagy in the response of human hepatocytes to oxidative stress remains unknown. Understanding this process may have important implications for the understanding of basic liver epithelial cell biology and the responses of hepatocytes during liver disease. To address this we isolated primary hepatocytes from human liver tissue and exposed them ex vivo to hypoxia and hypoxia-reoxygenation (H-R). We showed that oxidative stress increased hepatocyte autophagy in a reactive oxygen species (ROS) and class III PtdIns3K-dependent manner. Specifically, mitochondrial ROS and NADPH oxidase were found to be key regulators of autophagy. Autophagy involved the upregulation of BECN1, LC3A, Atg7, Atg5 and Atg 12 during hypoxia and H-R. Autophagy was seen to occur within the mitochondria of the hepatocyte and inhibition of autophagy resulted in the lowering a mitochondrial membrane potential and onset of cell death. Autophagic responses were primarily observed in the large peri-venular (PV) hepatocyte subpopulation. Inhibition of autophagy, using 3-methyladenine, increased apoptosis during H-R. Specifically, PV human hepatocytes were more susceptible to apoptosis after inhibition of autophagy. These findings show for the first time that during oxidative stress autophagy serves as a cell survival mechanism for primary human hepatocytes.     

厄贝沙坦对人脐静脉内皮细胞株EA.hy926增殖、凋亡、VEGF mRNA表达的影响

目的:应用不同浓度厄贝沙坦对人脐静脉内皮细胞株EA.hy 926的增殖、凋亡生物学效应及血管发生主要基因VEGFmRNA的表达进行体外研究,探讨厄贝沙坦对内皮细胞的血管生成效应。方法:各种浓度厄贝沙坦对人脐静脉内皮细胞株EA.hy926共同孵育24 h。细胞增殖采用CCK8法分析,Annexin V/PI双染法检测细胞凋亡。RT-PCR验证VEGFmRNA的表达。结果:厄贝沙坦各浓度干预组细胞形态无明显变化,CCK8结果提示厄贝沙坦各干预组相比对照组细胞增殖活力增高(P<0.05),呈浓度非依赖性。流式细胞仪分析厄贝沙坦各浓度干预组细胞无明显凋亡。RT-PCR发现厄贝沙坦1×10-4,1×10-5,1×10-6mol/L浓度组VEGFmRNA表达增高(P<0.05)。结论:厄贝沙坦促进EA.hy926细胞株细胞增殖,上调VEGFmRNA的表达。这提示除了降压效应,血管紧张素受体拮抗剂在缺血性心脏病如慢性心力衰竭治疗中具有一定作用。     

Beneficial effects of edaravone, a novel antioxidant, in rats with dilated cardiomyopathy

Edaravone, a novel antioxidant, acts by trapping hydroxyl radicals, quenching active oxygen and so on. Its cardioprotective activity against experimental autoimmune myocarditis (EAM) was reported. Nevertheless, it remains to be determined whether edaravone protects against cardiac remodelling in dilated cardiomyopathy (DCM). The present study was undertaken to assess whether edaravone attenuates myocardial fibrosis, and examine the effect of edaravone on cardiac function in rats with DCM after EAM. Rat model of EAM was prepared by injection with porcine cardiac myosin 28 days after immunization, we administered edaravone intraperitoneally at 3 and 10 mg/kg/day to rats for 28 days. The results were compared with vehicle-treated rats with DCM. Cardiac function, by haemodynamic and echocardiographic study and histopathology were performed. Left ventricular (LV) expression of NADPH oxidase subunits (p47(phox), p67(phox), gp91(phox) and Nox4), fibrosis markers (TGF-β(1) and OPN), endoplasmic reticulum (ER) stress markers (GRP78 and GADD 153) and apoptosis markers (cytochrome C and caspase-3) were measured by Western blotting. Edaravone-treated DCM rats showed better cardiac function compared with those of the vehicle-treated rats. In addition, LV expressions of NADPH oxidase subunits levels were significantly down-regulated in edaravone-treated rats. Furthermore, the number of collagen-III positive cells in the myocardium of edaravone-treated rats was lower compared with those of the vehicle-treated rats. Our results suggest that edaravone ameliorated the progression of DCM by modulating oxidative and ER stress-mediated myocardial apoptosis and fibrosis.     

Notch1 control of oligodendrocyte differentiation in the spinal cord

We have selectively inhibited Notch1 signaling in oligodendrocyte precursors (OPCs) using the Cre/loxP system in transgenic mice to investigate the role of Notch1 in oligodendrocyte (OL) development and differentiation. Early development of OPCs appeared normal in the spinal cord. However, at embryonic day 17.5, premature OL differentiation was observed and ectopic immature OLs were present in the gray matter. At birth, OL apoptosis was strongly increased in Notch1 mutant animals. Premature OL differentiation was also observed in the cerebrum, indicating that Notch1 is required for the correct spatial and temporal regulation of OL differentiation in various regions of the central nervous system. These findings establish a widespread function of Notch1 in the late steps of mammalian OPC development in vivo.     

High Boiling Compounds of Neutral Essential Oil from Tea

Glucoamylase, acid protease, and acid carboxypeptidase in namazake were inactivated with microbubble super critical carbon dioxide (SC CO₂) at 25 MPa, 35°C for 30 min. After the treatment, their activities decreased from 330, 20.1, and 115 U/ml sake to 51.8, 1.8, and 0 U/ml sake, respectively. During the storage at 20°C for 60 days, no increase in amino acid value was observed. On the other hand, glucose concentration in the sake treated with SC CO₂ increased as well as namazake. The formation of lactic acid, acetic acid, and ethanol were depressed, and acidity was constant during storage. On the contrary, the decrease of pyruvic and malic acid after 20 days of storage could be depressed. Analysis of volatile compounds suggests that the treatment with SC CO₂ did not cause the formation of volatile compounds in contrast to heat-treatment. As a result of sensory evaluation, the sake treated with SC CO₂ at 20 MPa, 35°C for 30 min was given sensory scores near to those of namazake.     

Endothelin-1 Up-Regulates p115RhoGEF in Embryonic Rat Cardiomyocytes During the Hypertrophic Response

In cardiomyocytes, certain extracellular stimuli that activate heterotrimeric G protein-coupled receptors (GPCRs) can induce hypertrophy by regulating gene expression and increasing protein synthesis. We investigated if rat embryonic cardiomyocytes (H9c2) underwent variations in the expression levels and subcellular distribution of key components of GPCR-activated signaling pathways during endothelin-1 (ET-1)-induced hypertrophic response. A significant increase of p115RhoGEF protein level was evident in ET-1-treated cells. Real-time quantitative PCR showed RhoGEF mRNA levels were significantly increased. Inhibition of the Rho-associated kinase (ROCK) caused a significant decrease of p115RhoGEF protein in the nuclear fraction, whereas an inhibitor of PKC induced a redistribution of the protein between membrane/organelle and nuclear fractions. The ROCK inhibitor also decreased H9c2 cell hypertrophic response. These results indicate that ROCK and its downstream target molecules, which are involved in inducing the hypertrophic response, are also implicated in signaling the up-regulation of the p115RhoGEF protein.     

HBx参与肝细胞癌发生发展调控的分子机制

肝细胞癌 (hepatocellular carcinoma, HCC)是我国最常见的恶性肿瘤之一,而HBV慢性感染是肝癌发生的主要原因.乙型肝炎病毒(HBV)中X基因编码的一种多功能蛋白(HBx),参与众多重要生物学过程的调控,并促进肝细胞癌的发生. 早期研究表明,HBx在HCC发生过程中发挥重要的调控功能,但其确切分子机制尚未完全明确. 近几年,HBx参与生物学过程的分子机制研究有了较快的进展. 有趣的是,研究发现,HBx在不同的细胞系以及HBV感染的不同阶段发挥促抑凋亡的双重作用,HBx还参与细胞自噬的调控. 此外,在HBx参与细胞增殖及肿瘤侵袭和转移等方面,也产生了一些新的认识. 本文将从HBx对肝细胞凋亡、自噬和增殖的调控及其对肝癌细胞转移和侵袭的调控等方面,对HBx参与肝细胞癌发生发展调控机制做一综述.     

Protracted low-dose radiation priming and response of liver to acute gamma and proton radiation

Abstract

This study evaluated liver from C57BL/6 mice irradiated with low-dose/low-dose-rate (LDR) γ-rays (0.01 Gy, 0.03 cGy/h), with and without subsequent exposure to acute 2 Gy gamma or proton radiation. Analyses were performed on day 56 post-exposure. Expression patterns of apoptosis-related genes were strikingly different among irradiated groups compared with 0 Gy (p < 0.05). Two genes were affected in the Gamma group, whereas 10 were modified in the LDR + Gamma group. In Proton and LDR + Proton groups, there were six and 12 affected genes, respectively. Expression of genes in the Gamma (Traf3) and Proton (Bak1, Birc2, Birc3, Mcl1) groups was no longer different from 0 Gy control group when mice were pre-exposed to LDR γ-rays. When each combined regimen was compared with the corresponding group that received acute radiation alone, two genes in the LDR + Gamma group and 17 genes in the LDR + Proton group were modified; greatest effect was on Birc2 and Nol3 (> 5-fold up-regulated by LDR + Protons). Oxygen radical production in livers from the LDR + Proton group was higher in LDR, Gamma, and LDR + Gamma groups (p < 0.05 vs. 0 Gy), but there were no differences in phagocytosis of E. coli. Sections stained with hematoxylin and eosin (H&E) suggested more inflammation, with and without necrosis, in some irradiated groups. The data demonstrate that response to acute radiation is dependent on radiation quality and regimen and that some LDR γ-ray-induced modifications in liver response were still evident nearly 2 months after exposure.     

Dual functions of autophagy in the response of breast tumor cells to radiation

In MCF-7 breast tumor cells, ionizing radiation promoted autophagy that was cytoprotective; pharmacological or genetic interference with autophagy induced by radiation resulted in growth suppression and/or cell killing (primarily by apoptosis). The hormonally active form of vitamin D, 1,25D₃, also promoted autophagy in irradiated MCF-7 cells, sensitized the cells to radiation and suppressed the proliferative recovery that occurs after radiation alone. 1,25D₃ enhanced radiosensitivity and promoted autophagy in MCF-7 cells that overexpress Her-2/neu as well as in p53 mutant Hs578t breast tumor cells. In contrast, 1,25D₃ failed to alter radiosensitivity or promote autophagy in the BT474 breast tumor cell line with low-level expression of the vitamin D receptor. Enhancement of MCF-7 cell sensitivity to radiation by 1,25D₃ was not attenuated by a genetic block to autophagy due largely to the promotion of apoptosis via the collateral suppression of protective autophagy. However, MCF-7 cells were protected from the combination of 1,25D₃ with radiation using a concentration of chloroquine that produced minimal sensitization to radiation alone. The current studies are consistent with the premise that while autophagy mediates a cytoprotective function in irradiated breast tumor cells, promotion of autophagy can also confer radiosensitivity by vitamin D (1,25D₃). As both cytoprotective and cytotoxic autophagy can apparently be expressed in the same experimental system in response to radiation, this type of model could be utilized to distinguish biochemical, molecular and/or functional differences in these dual functions of autophagy.