脂联素抑制内质网应激干预慢性间歇性缺氧所致肾损伤

目的探讨脂联素(Ad)对慢性间歇性缺氧(CIH)所致肾损伤的干预作用及相关机制。方法 60只成年Wistar大鼠随机分成4组:正常对照(NC)组、NC+Ad组、CIH组和CIH+Ad组。其中CIH组和CIH+Ad组大鼠接受CIH处理4个月。其余2组接受正常空气处理,同时NC+Ad组和CIH+Ad组大鼠接受Ad(10l人開)治疗,每周2次,持续4个月。荧光显微镜下观察活性氧(ROS)的水平。TUNEL染色检测肾脏细胞凋亡情况。Western blotting检测各组大鼠肾脏组织中GRP78、CHOP、IRE1、PERK、pro-ATF6蛋白的表达,以反映各组大鼠内质网应激情况。采用SPSS 17.0软件进行统计学分析。计量资料用均数±标准差(±s)表示,组间比较用t检验。结果实验满4个月时,组间比较示各参数在NC与NC+Ad组间差异均无统计学意义(P均0.05)。与NC组和NC+Ad组比较:(1)ROS水平在CIH组显著增高,而CIH+Ad组低于CIH组,但仍高于NC组和NC+Ad组(尸均0.05);(2)肾细胞凋亡率和反映凋亡的caspase-12和caspase-3蛋白水平在CIH组明显增加,CIH+Ad组较CIH组明显减少,但仍然高于NC组和NC+Ad组(P均0.05);(3)CIH组肾脏组织的GRP78、CHOP、IRE1、PERK蛋白水平明显增加,在CIH+Ad组明显减少,但仍高于NC组和NC+Ad组(P均0.05);pro-ATF6蛋白水平在CIH组明显降低,在CIH+Ad组有所增加,但仍然低于NC组和NC+Ad组(P均0.05)。结论 CIH可以通过激活ROS和ERS相关的细胞凋亡途径导致肾脏损伤,而补充外源性Ad后,可能通过抑制ROS,进而抑制ERS,保护肾脏细胞。     

Expression pattern of mda-7/IL-24 receptors in liver cancer cell lines

BACKGROUND:The mda-7/IL-24 receptor belongs to the typeⅡcytokine receptor family,and its two heterodimeric receptors are IL-22R1/IL-20R2 and IL-20R1/IL-20R2. Mda-7/IL-24 receptor expression in liver cancer cell lines has not yet been described.This information may be helpful for further clinical gene therapy. METHODS:With normal skin total RNA as template,the cDNA sequences of IL-20R1,IL-20R2 and IL-22R were amplified by RT-PCR.Total RNA was extracted from cultured liver cancer cell lines and a normal liver...     

Inhibition of tumor necrosis factor-α-dependent cardiomyocyte apoptosis by metallothionein

Myocardial cell death is an important cellular event of heart failure. Tumor necrosis factor-α (TNF) accumulates in the failing heart and causes myocyte apoptosis, but the mechanism of this action is unclear. This study was undertaken to examine the relationship between TNF-induced cardiomyocyte apoptosis and activation of p38 mitogen-activated protein kinase (MAPK) through oxidative stress. Primary cultures of neonatal cardiomyocytes isolated from transgenic mouse hearts that overexpress metallothionein (MT) as well as cardiomyocytes isolated from wild-type mice were used. The treatment of wild-type cardiomyocytes with TNF at 10 ng/mL induced apoptosis, as detected by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling and confirmed by Annexin V-fluorescein isothiocyanate binding. The apoptotic effect of TNF was significantly inhibited in the MT-overexpressing cardiomyocytes. Corresponding to the apoptotic effect, TNF at 10 ng/mL caused rapid phosphorylation of p38 MAPK in wild-type cardiomyocytes. The activation of p38 MAPK was further confirmed by an in vivo experiment treating the mice with TNF and measuring p38 MAPK activity using an immune complex kinase assay. The activation of p38 MAPK was not observed in the MT-overexpressing cardiomyocytes either in vitro or in vivo. Importantly, TNF-induced accumulation of reactive oxygen species was dramatically reduced in the MT-overex-pressing cardiomyocytes as determined by a carboxy-H₂-DCFDA staining method. This study thus suggests that p38 MAPK activation is likely involved in TNF-induced cardiomyocyte apoptosis, which is also related to reactive oxygen species accumulation.     

软脂酸通过上调还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶表达促进血管内皮细胞凋亡

目的探讨软脂酸(PA)诱导的血管内皮细胞凋亡中还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶的作用。方法人脐静脉内皮细胞(HUVEC)贴壁培养后分为对照组;PA(200、400、800μmol/L)浓度组(分别为PA200组、PA400组、PA800组);NADPH氧化酶抑制剂apocynin(50、100、200 mol/L)干预组(分别为apo50+PA400组、apo100+PA400组、apo200+PA400组)。采用流式细胞仪检测细胞凋亡率;提取细胞蛋白,采用Westernblot技术检测NADPH氧化酶亚基p47phox蛋白表达的水平。倒置共聚焦荧光显微镜检测活性氧的产生。结果 PA呈浓度依赖性诱导HUVEC凋亡;与对照组比较,PA400、PA800组HUVEC凋亡率明显升高,P47phox蛋白表达明显增强(P<0.05),PA400组活性氧产生明显升高(P<0.05);与PA400组比较,apo100+PA400组、apo200+PA400组HUVEC凋亡率明显降低,p47phox蛋白表达明显减弱,活性氧产生明显降低(P<0.05)。结论 PA呈浓度依赖性诱导HUVEC凋亡,apocynin能部分抑制PA的上述作用,其机制与下调NADPH氧化酶亚基p47phox蛋白表达及活性氧产生有关。     

Attenuation of cold-induced apoptosis by exogenous melatonin in carrot suspension cells: the possible involvement of polyamines

Pretreatment with 43 nM (10 ng/mL) to 86 nM melatonin for 5 days significantly attenuated cold-induced apoptosis in carrot suspension cells (Daucus carota L.) as evidenced by the TUNEL procedure, DNA fragmentation and the morphological changes revealed by electronic microscopy observations. The antiapoptotic effect of melatonin was initially thought to be a result of its antioxidant actions. In our study, however, reactive oxygen species (ROS) generation remained unaffected by melatonin treatment, suggesting that melatonin plays its protective role not related to its direct ROS scavenger. At the same time, notable increases in putrescine and spermidine levels were observed in melatonin-treated cells, which may be responsible for the alleviation of the cold-induced apoptosis. The possible involvement of polyamines in the antiapoptotic effect of melatonin was further confirmed by the inhibitory effect of exogenous polyamines on apoptosis as displayed by the DNA laddering assay.     

Mpv17l protects against mitochondrial oxidative stress and apoptosis by activation of Omi/HtrA2 protease

Cellular localization determines whether the serine protease HtrA2 exerts pro- or antiapoptotic functions. In contrast to the well-characterized proapoptotic function of cytosolic HtrA2, mechanisms underlying the mitochondrial protective role are poorly understood. Mpv17l is a transmembrane protein previously implicated in peroxisomal reactive oxygen species metabolism and a close homolog of the inner mitochondrial membrane protein Mpv17. Here we demonstrate a previously undescribed direct interaction between Mpv17l and HtrA2 in mitochondria. The interaction is mediated by a PDZ domain and induces protease activation of HtrA2. HtrA2 inhibits mitochondrial superoxide generation, stabilizes mitochondrial membrane potential, and prevents apoptosis at baseline and in response to extracellular inducers of mitochondrial stress. The physiological role of Mpv17l is underscored by the finding that oxidative stress-induced downregulation of Mpv17l is a consistent feature in renal injury models. Our findings identify Mpv17l as a unique interacting protein and regulator of HtrA2 protease mediating antioxidant and antiapoptotic function in mitochondria.     

Adverse Effects of Cigarette and Noncigarette Smoke Exposure on the Autonomic Nervous System : Mechanisms and Implications for Cardiovascular Risk

This review summarizes the detrimental effects of cigarette and noncigarette emission exposure on autonomic function, with particular emphasis on the mechanisms of acute and chronic modulation of the sympathetic nervous system. We propose that the nicotine and fine particulate matter in tobacco smoke lead to increased sympathetic nerve activity, which becomes persistent via a positive feedback loop between sympathetic nerve activity and reactive oxidative species. Furthermore, we propose that baroreflex suppression of sympathetic activation is attenuated in habitual smokers; that is, the baroreflex plays a permissive role, allowing sympathoexcitation to occur without restraint in the setting of increased pressor response. This model is also applicable to other nontobacco cigarette emission exposures (e.g., marijuana, waterpipes [hookahs], electronic cigarettes, and even air pollution). Fortunately, emerging data suggest that baroreflex sensitivity and autonomic function may be restored after smoking cessation, providing further evidence in support of the health benefits of smoking cessation.     

腺病毒介导MDA-7/IL-24选择性促进肝癌细胞的凋亡和增殖阻滞

目的 观察MDA-7／IL-24基因对不同p53状态人肝癌细胞HepG2、MHCC97L以及Hep3B和正常的肝细胞L02的选择性杀伤作用，为肝癌的基因治疗提供理论基础。方法 将携带人MDA-7／IL-24基因的腺病毒Ad．mda-7感染人正常肝细胞L02和不同p53状态的肝癌细胞HepG2，MHCC97L、Hep3B。通过逆转录聚合酶链反应方法观察MDA7／IL24基因的表达，酶联免疫吸附法检测细胞培养上清液中MDA-7／IL-24蛋白的浓度，通过四甲基偶氮唑盐染色法及Hoechst染色观察MDA-7／IL-24对肝癌细胞的生长抑制和杀伤作用，Annexin-V和碘化丙啶双染后流式细胞仪检测细胞的凋亡，应用流式细胞仪检测细胞周期。结果 复制缺陷型腺病毒能介导外源基因MDA-7／IL-24在肝癌细胞株HepG2，MHCC97L和Hep3B以及正常细胞L02中高效表达。细胞培养上清液中有MDA-7／IL-24蛋白表达。MDA-7／IL-24能明显抑制各种肝癌细胞的生长，Hoechst染色提示MDA-7／IL-24促进肝癌细胞的凋亡，流式细胞仪提示MDA-7能选择性杀伤肝癌细胞而对正常的肝细胞无影响，细胞周期分析提示MDA-7／IL-24阻滞肝癌细胞在G2／M期，同时对正常的肝细胞没有促凋亡作用和增殖阻滞作用。结论 复制缺陷型重组腺病毒载体Ad．mda-7能介导MDA-7／IL-24基因在人肝癌细胞中高效表达，选择性地杀伤肝癌细胞HepG2、MHCC97L和Hep3B，促进细胞增殖阻滞及诱导肿瘤细胞凋亡而与肿瘤细胞的P53基因的状态无关，同时对正常的肝细胞L02无任何毒性作用。     

Potential role of the ubiquitin-proteasome system in atherosclerosis: aspects of a protein quality disease

Misfolded or damaged proteins are recognized intracellularly by protein quality mechanisms. These include chaperones and the ubiquitin-proteasome system, which aim at restoration of protein function and protein removal, respectively. A number of studies have outlined the functional significance of the ubiquitin-proteasome system for the heart and, as of recently, for the vascular system. This review summarizes these recent findings with a focus on atherosclerosis. In particular, this paper reflects on the viewpoint of atherosclerosis as a protein quality disease.     

cGMP/PKG pathway mediates myocardial postconditioning protection in rat hearts by delaying normalization of intracellular acidosis during reperfusion

Ischemic postconditioning has been demonstrated to limit infarct size in patients, but its molecular mechanisms remain incompletely understood. Low intracellular pH (pHi) inhibits mitochondrial permeability transition, calpain activation and hypercontracture. Recently, delayed normalization of pHi during reperfusion has been shown to play an important role in postconditioning protection, but its relation with intracellular protective signaling cascades is unknown. The present study investigates the relation between the rate of pHi normalization and the cGMP/PKG pathway in postconditioned myocardium. In isolated Sprague-Dawley rat hearts submitted to transient ischemia both, postconditioning and acidic reperfusion protocols resulted in a similar delay in pHi recovery measured by ³¹P-NMR spectroscopy (3.6 ± 0.2 min and 3.5 ± 0.2 min respectively vs. 1.4 ± 0.2 min in control group, P < 0.01) and caused equivalent cardioprotection (48% and 41% of infarct reduction respectively, P < 0.01), but only postconditioning increased myocardial cGMP levels (P = 0.02) and activated PKG. Blockade of cGMP/PKG pathway by the addition of the guanylyl cyclase inhibitor ODQ or the PKG inhibitor KT5823 during reperfusion accelerated pHi recovery and abolished cardioprotection in postconditioned hearts, but had no effect in hearts subjected to acidic reperfusion suggesting that PKG signaling was upstream of delayed pHi normalization in postconditioned hearts. In isolated cardiomyocytes the cGMP analog 8-pCPT-cGMP delayed Na⁺/H⁺-exchange mediated pHi normalization after acidification induced by a NH₄Cl pulse. These results demonstrate that the cGMP/PKG pathway contributes to postconditioning protection at least in part by delaying normalization of pHi during reperfusion, probably via PKG-dependent inhibition of Na⁺/H⁺-exchanger.     

Inhibition of bile salt-induced apoptosis by cyclic AMP involves serine/threonine phosphorylation of CD95

BACKGROUND AND AIMS: Cyclic AMP (cAMP) inhibits bile salt-induced hepatocyte apoptosis; the underlying mechanisms are unclear. METHODS: The effects of cAMP on taurolithocholate-3-sulfate-(TLCS)- or glycochenodesoxycholate (GCDC)-induced CD95 (Fas/APO-1) activation and apoptosis were studied in 24-hour cultured rat hepatocytes and in perfused rat liver. RESULTS: TLCS induced a rapid oxidative stress response, c-Jun-N-terminal kinase (JNK) and epidermal growth factor (EGF) receptor (EGF-R) activation, subsequent EGF-R/CD95 association and CD95 tyrosine phosphorylation, CD95 membrane targeting, death-inducing signal complex (DISC) formation and hepatocyte apoptosis. None of these responses was triggered by cAMP; however, cAMP induced H89-sensitive serine/threonine phosphorylation of CD95. Similar data were obtained with GCDC, another proapoptotic bile acid. cAMP did not prevent the TLCS-induced oxidative stress response, JNK activation and EGF-R/CD95 association, however abolished EGF-R activation and subsequent CD95 tyrosine phosphorylation, CD95 membrane trafficking, and DISC formation in a H89-sensitive way. Also in presence of TLCS, cAMP induced rapid Ser/Thr phosphorylation of CD95 within 10 min. The effects of cAMP on the various steps of CD95 activation were also found in the intact perfused rat liver. Evidence is given that a cAMP-induced Ser/Thr phosphorylation favors internalization of CD95. CONCLUSIONS: Inhibition of bile salt-induced apoptosis by cAMP involves both PKA-dependent Ser/Thr phosphorylation of the CD95 and inhibition of EGF-R activation, which results in an inhibition of CD95 tyrosine phosphorylation, CD95 membrane targeting, and DISC formation. CD95 regulation involves complex phosphorylations with CD95-tyrosine phosphorylation favoring CD95 membrane trafficking and DISC formation, whereas CD95 Ser/Thr phosphorylation inhibits these processes.     

IL-24: A new player in the pathogenesis of pro-inflammatory and allergic skin diseases

Interleukin (IL)-24 is a member of the IL-20 family of cytokines and is produced by various types of cells, such as CD4⁺ T cells, NK cells, mast cells, keratinocytes, bronchial epithelial cells, and myofibroblasts. Previous studies suggest that IL-24 plays an essential role in the pathogenesis of pro-inflammatory autoimmune disorders such as psoriasis, arthritis, and inflammatory bowel diseases. However, the role of IL-24 in the pathogenesis of allergic diseases has been elusive. It has already been reported that IL-24 is involved in the pathogenesis of allergic lung and skin diseases. Moreover, we have recently revealed for the first time the pivotal functions of IL-24 in IL-13–mediated skin barrier dysfunction in atopic dermatitis (AD), which is known to be a characteristic of AD caused by Th2 cytokines such as IL-4 or IL-13. In this review, we show recent advances in the basic characteristics of IL-24 and its novel functions in the pathogenesis of allergic skin inflammation, focusing on AD. A better understanding of the role of IL-24 in allergic diseases can lead to the development of new therapeutic options.     

Effects of mesenchymal stromal cells play a role the oxidant/antioxidant balance in a murine model of asthma

Asthma is a heterogeneous disease characterised by chronic airway inflammation. One of the most devastating consequences of this inflammatory process is the generation of reactive oxygen and nitrogen species responsible for oxidative stress. The aim of this study is to analyse the efficiency of treatment with human bone marrow-derived mesenchymal stromal cells (hMSC) in maintaining the oxidative balance in a murine model of allergic asthma by quantifying nitrotyrosine in lung tissues. After confirmation of asthma in the experimental model, samples of lung parenchyma were submitted to immunohistochemical assessment. Intravenous administration of hMSC reduced the levels of nitrotyrosine in the ASTHMA-hMSC group compared to those in the ASTHMA-SAL group. In conclusion, therapeutic administration of hMSC had a beneficial effect on oxidative stress, reducing the levels of nitrotyrosine in lung tissues in a model of allergic asthma.     

Proteomic analysis of age dependent nitration of rat cardiac proteins by solution isoelectric focusing coupled to nanoHPLC tandem mass spectrometry

Protein nitration occurs as a result of oxidative stress induced by reactive oxygen (ROS) and reactive nitrogen species (RNS). Therefore, protein nitration serves as a hallmark for protein oxidation in vivo. We have previously reported on age dependent protein nitration in cardiac tissue of Fisher 344 BN-F1 rats analyzed by two-dimensional gel electrophoresis; however, only one specific nitration site was identified [Kanski, J., Behring, A., Pelling, J., Schöneich, C., 2005a. Proteomic identification of 3-nitrotyrosine-containing rat cardiac proteins: effects of biological aging. Am. J. Physiol. Heart Circ. Physiol. 288, H371–381]. In the present report, we used solution phase isoelectric focusing (IEF) followed by nanoHPLC–ESI-MS/MS that allowed us to obtain good MS/MS data to identify specific sites of protein nitration in cardiac tissue. As expected, more nitrated proteins were detected in cardiac tissue of old rats, including myosin heavy chain, neurofibromin, tropomyosin and nebulin-related anchoring protein. The post-translational modification of these cytoskeletal proteins may provide some rationale for the age-dependent functional decline of the heart.     

Intercepting the Lipid-Induced Integrated Stress Response Reduces Atherosclerosis

Background

Eukaryotic cells can respond to diverse stimuli by converging at serine-51 phosphorylation on eukaryotic initiation factor 2 alpha (eIF2α) and activate the integrated stress response (ISR). This is a key step in translational control and must be tightly regulated; however, persistent eIF2α phosphorylation is observed in mouse and human atheroma.