A Linear Fragment of Unacylated Ghrelin (UAG6−13) Protects Against Myocardial Ischemia/Reperfusion Injury in Mice in a Growth Hormone Secretagogue Receptor-Independent Manner

Unacylated ghrelin (UAG), the most abundant form of ghrelin in circulation, has been shown to exert cardioprotective effect in experimental cardiopathies. The present study aimed to investigate the cardioprotective effect of a linear bioactive fragment of UAG against myocardial ischemia-induced injury and dysfunction in C57BL/6 wild type mice and the mechanisms involved. Treatments were administered at doses of 100 (UAG), 1,000 and 3,000 (UAG6−13) nmol/kg at 12 h interval during 14 days prior to 30 min left coronary artery ligation and reperfusion for a period of 6 or 48 h. The infarct area was decreased in a dose-dependent manner at 48 h of reperfusion, with a reduction of 54% at the highest dose of UAG6−13 tested. Myocardial hemodynamics were improved as demonstrated by an increase in cardiac output, maximum first derivative of left ventricular pressure, and preload recruitable stroke work, a load-independent contractility index. Six hours after reperfusion, circulating levels of IL-6 and TNF-α pro-inflammatory cytokines were reduced, and the effect was maintained at 48 h for TNF-α. 5′ AMP-activated protein kinase (AMPK) was activated, while acetyl-CoA carboxylase (ACC) activity was inhibited, along with a decrease in apoptotic protein levels. In isolated hearts, the effect of UAG6−13 was unaffected by the presence of D-Lys3-GHRP-6, a ghrelin receptor (GHSR1a) antagonist, suggesting that the peptide acted through a GHSR1a-independent pathway. The results support the therapeutic application of UAG bioactive peptide fragments against myocardial ischemia/reperfusion injury

Genetic ablation of calcium-independent phospholipase A2γ induces glomerular injury in mice

Glomerular visceral epithelial cells (podocytes) play a critical role in the maintenance of glomerular permselectivity. Podocyte injury, manifesting as proteinuria, is the cause of many glomerular diseases. We reported previously that calcium-independent phospholipase A(2)γ (iPLA(2)γ) is cytoprotective against complement-mediated glomerular epithelial cell injury. Studies in iPLA(2)γ KO mice have demonstrated an important role for iPLA(2)γ in mitochondrial lipid turnover, membrane structure, and metabolism. The aim of the present study was to employ iPLA(2)γ KO mice to better understand the role of iPLA(2)γ in normal glomerular and podocyte function as well as in glomerular injury. We show that deletion of iPLA(2)γ did not cause detectable albuminuria; however, it resulted in mitochondrial structural abnormalities and enhanced autophagy in podocytes as well as loss of podocytes in aging KO mice. Moreover, after induction of anti-glomerular basement membrane nephritis in young mice, iPLA(2)γ KO mice exhibited significantly increased levels of albuminuria, podocyte injury, and loss of podocytes compared with wild type. Thus, iPLA(2)γ has a protective functional role in the normal glomerulus and in glomerulonephritis. Understanding the role of iPLA(2)γ in glomerular pathophysiology provides opportunities for the development of novel therapeutic approaches to glomerular injury and proteinuria

Role of calcium-independent phospholipase A2 gamma (iPLA2y) in glomerular epithelial cell injury

In experimental membranous nephropathy, complement C5b-9-induces glomerular epithelial cell (GEC)/podocyte injury and proteinuria. The effects of C5b-9 are mediated via signaling pathways, including calcium-independent phospholipase A2γ (iPLA2γ), and mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38. The iPLA2γ pathway is cytoprotective. First, we studied the mechanisms of iPLA2γ activation and cytoprotection. iPLA2γ activitation was monitored by quantifying prostaglandin E2 (PGE2) production. In GECs, iPLA2γ localized at the endoplasmic reticulum and mitochondria, which was dependent on the N-terminal region of iPLA2γ. Complement-mediated production of PGE2 was amplified in GECs that overexpress iPLA2γ, compared with control cells, and was blocked by the iPLA2γ inhibitor bromoenol lactone in both iPLA2γ-overexpressing and control GECs. Complement-induced activation of iPLA2γ was mediated via ERK and p38 pathways, but not JNK pathway. In COS-1 cells that overexpress iPLA2γ and cyclooxygenase-1, PGE2 production was induced by co-expression of constitutively active MEK1 or MAPK interacting kinase 1 (MNK1) as well as by stimulation with epidermal growth factor (EGF) + ionomycin. Complement- and EGF + ionomycin-stimulated iPLA2γ activity was attenuated by the S511A/S515A double mutation. Moreover, complement and EGF + ionomycin enhanced phosphorylation of Ser-511. Thus, stimulation of iPLA2γ was dependent on an increase in cytosolic Ca2+ concentration and phosphorylation of Ser-511 and/or Ser-515 via MNK1. Phosphorylation of Ser-511 and/or Ser-515 plays a key role in the catalytic activity and signaling of iPLA2γ. Next, we addressed the cytoprotective function of iPLA2γ by examining if iPLA2γ is involved in the adaptive unfolded protein response (UPR). In GECs, iPLA2γ amplified tunicamycin-induced activating transcription factor-6 (ATF6) activation and upregulated the ER chaperones, grp94 and grp78, which are downstream of ATF6 and enhance protein-folding capacity of the ER. These effects were dependent on iPLA2γ catalytic activity, but not on prostanoids. Furthermore, ATF6 amplification occurred only when the full length iPLA2γ was expressed, but not an N-terminally truncated mutant, which does not associate with the membrane of the ER. Induction of the ATF6 pathway of the UPR and its amplification by iPLA2γ limited tunicamycin-induced GEC injury. Finally, to better understand the role of iPLA2γ in normal podocyte function and in podocyte injury in vivo, we employed iPLA2γ knockout (KO) mice. Deletion of iPLA2γ caused neither albuminuria nor morphological changes in the glomerulus. However, after induction of anti-glomerular basement membrane nephritis, iPLA2γ KO mice exhibited significantly increased levels of albuminuria, compared to wild type (WT) mice. Furthermore, in contrast to WT mice, iPLA2γ KO mice exhibited a marked loss of podocytes, implying that iPLA2γ has a protective role in glomerulonephritis. Collectively, this work characterizes the mechanism of iPLA2γ activation in complement-mediated GEC injury, shows that the cytoprotective effect of iPLA2γ involves the unfolded protein response, ATF6, and that iPLA2γ has a protective role in glomerulonephritis. Defining the role of iPLA2γ provides opportunities for development of novel therapeutic approaches to GEC injury and proteinuria.Dans la néphropathie membraneuse expérimentale, le complément C5b-9 induit des dommages aux cellules épithéliales glomérulaires (CGE)/podocytes et cause la protéinurie. Les effets du C5b-9 sont médiés par des voies de signalisation, y compris la phospholipase A2γ indépendante du calcium (iPLA2γ), et des protéines kinases activées par les mitogènes (MAPK) telles que ERK, JNK et p38. La voie iPLA2γ est cytoprotectrice. Tout d'abord, nous avons étudié les mécanismes d'activation de et de cytoprotection de iPLA2γ. L'activation de iPLA2γ a été suivie par la quantification de la production de prostaglandine E2 (PGE2). Dans les CGE, iPLA2γ est localisée au réticulum endoplasmique (RE) et aux mitochondries. Cette localisation était dépendante de la région N-terminale de iPLA2γ. La production de la PGE2 induite par le complément était amplifiée dans les CGE qui surexpriment iPLA2γ, par rapport aux cellules de contrôle. Cette amplification était bloquée par un inhibiteur de iPLA2γ (bromoenol lactone) à la fois dans les CGE surexprimant iPLA2γ et les CGE de contrôle. L'activation du iPLA2γ induite par le complément était médiée par des voies de signalisation ERK et p38, mais pas par JNK. Dans les cellules COS-1 qui surexpriment iPLA2γ et la cyclo-oxygénase-1, la production de PGE2 était induite par la co-expression de la kinase constitutivement active MEK1 ou de MNK1 ainsi que par la stimulation par le facteur de croissance épidermique (EGF) + ionomycine. L'activité iPLA2γ stimulé par le complément, EGF et ionomycine, était atténuée par la double mutation, S511A/S515A. En outre, la stimulation par le complément et par la combinaison EGF + ionomycine augmentait la phosphorylation de Ser-511. Ainsi, la stimulation de iPLA2γ était dépendant d'une augmentation de la concentration de calcium cytosolique et de la phosphorylation de Ser-511 et/ou Ser-515 par l'intermédiaire de MNK1. La phosphorylation de Ser-511 et/ou Ser-515 joue un rôle clé dans l'activité catalytique et la signalisation de iPLA2γ.Ensuite, nous avons examiné la fonction cytoprotectrice de iPLA2γ en examinant si iPLA2γ est impliqué dans la ‘‘unfolded protein response'' ou UPR. Dans les CGE, iPLA2γ amplifiait l'activation transcription factor 6 (ATF6) induite par la tunicamycine et a augmentait l'expression des chaperons du RE grp94 et grp78, qui sont en aval du ATF6 et améliorent la capacité de pliage des protéines dans ce compartiment. Ces effets étaient dépendants de l'activité catalytique de iPLA2γ, mais pas des prostanoïdes. En plus, l'amplification de ATF6 se produisait seulement en présence d'une forme intacte de iPLA2γ, mais pas en présence d'un mutant portant une troncation N-terminale et incapable de s'associer avec la membrane du RE. L'induction de la voie de ATF6 de l'UPR et son amplification par iPLA2γ limitait les dommages induits par la tunicamycine dans les CGE.Enfin, pour mieux comprendre le rôle de iPLA2γ dans la fonction normale des podocytes et dans les dommages aux podocytes in vivo, nous avons utilisé des souris knock-out iPLA2γ (KO). La déplétion de iPLA2γ ne cause pas d'albuminurie, ni de changements morphologiques dans le glomérule. Cependant, après l'induction de néphrite par anticorps anti-membrane glomérulaire basale, les souris KO iPLA2γ démontraient une albuminurie augmentée par rapport aux souris de type sauvage (WT). De plus, contrairement aux souris WT, les souris KO iPLA2γ présentaient une diminution marquée des podocytes, ce qui implique que iPLA2γ a un rôle protecteur dans la glomérulonéphrite.Mis ensemble, ces travaux caractérisent le mécanisme d'activation des iPLA2γ dans le dommage aux CGE induit par le complément. Ils montrent que l'effet cytoprotecteur de iPLA2γ implique l'UPR, ATF6, et que iPLA2γ a un rôle protecteur dans la glomérulonéphrite. Définir le rôle de iPLA2γ offre des possibilités pour le développement de nouvelles approches thérapeutiques pour les dommages aux CGE et pour la protéinurie

New Adenosine Derivatives from Aizoon canariense L.: In Vitro Anticholinesterase, Antimicrobial, and Cytotoxic Evaluation of Its Extracts

Aizoaceae is a large succulent family characterized by many psychoactive species. Aizoon canariense L., a wild neglected plant traditionally used in gastrointestinal ailments, has been the subject of a limited number of phytochemical and biological studies. Therefore, herein, we investigated the in vitro cytotoxic, antimicrobial, and anticholinesteraseactivity of the aerial parts of A. canariense L. and analyzed the phytochemical compositions of the lipoidal and alkaloidal fractions. Petroleum ether extract showed the presence of behenic and tricosylic acid, while an in-depth investigation of the alkaloidal fraction revealed the identification of new adenine based alkaloids (1–5), which were isolated and identified for the first time from Aizoon canariense L. Their structures were elucidated based on extensive spectroscopic analyses. The alkaloidal extract showed a powerful cytotoxic effect (IC50 14–28 μg/mL), with the best effect against colon carcinoma, followed by liver and breast carcinomas. The alkaloidal extract also had a potent effect against Candida albicans and Escherichia coli, with minimum inhibitory concentrations (MIC) values of 312.5 and 625 µg/mL. The in vitro anticholinesterase activity was potent, with IC50 < 200 ng/mL for the tested extracts compared with 27.29 ± 0.49 ng/mL for tacrine

Telomerase RNA component lncRNA as potential diagnostic biomarker promotes CRC cellular migration and apoptosis evasion via modulation of β-catenin protein level

Aim: Long non-coding RNA (LncRNA) telomerase RNA component (TERC) has telomerase-dependent and independent activity in numerous cancer types. The present study purposes to demonstrate the role of lncRNA TERC as a diagnostic serum biomarker in colorectal cancer (CRC) patients and the molecular mechanism of lncRNA TERC in inducing tumor in CRC cell lines. Materials and methods: PCR array was performed to examine lncRNAs dysregulated in CRC. LncRNA TERC expression level was evaluated in 70 CRC patients and 35 control subjects using RT-qPCR. Then transfection was performed to build down-expression models of lncRNA TERC. ROC curve analysis was applied to assess the diagnostic value of serum LncRNA CRC. In addition, RT-qPCR was used to detect expression level of lncRNA TERC and β-catenin mRNA. Moreover, ELISA and Western blot were used to detect the level of β-catenin protein in sera of CRC patients and cell lines. The biological functions such as cell growth and migration of CRC cells were assessed using a wound healing assay. Cell cycle analysis and apoptosis analysis were performed using flow cytometry. Results: The lncRNA TERC is overexpressed in the sera of CRC patients with high diagnostic and stage discrimination accuracy. Furthermore, lncRNA TERC expression was upregulated in CRC cell lines and lncRNA TERC silencing induced cell arrest and apoptosis and inhibited cell migration. Furthermore, inhibition of lncRNA TERC reduces β-catenin protein levels. Conclusion: The lncRNA TERC could be considered as an early stages CRC diagnostic biomarker with a good ability to discriminate between CRC stages. lncRNA TERC induces CRC by promoting cell migration and evading apoptosis by elevating the level of β-catenin protein

Serum Neutrophil Gelatinase-Associated Lipocalin (NGAL) in HCV-Positive Egyptian Patients Treated with Sofosbuvir

Background. Direct-acting antivirals (DAAs) made a drastic change in the management of HCV infection. Sofosbuvir is one of the highly potent DAAs, eliminated mainly through the kidney. But concerns about renal safety during treatment may limit its use. Neutrophil gelatinase-associated lipocalin (NGAL) has been proven as a predictor of renal tubular injury. Hence, the aim of this work was to assess serum neutrophil gelatinase-associated lipocalin (NGAL) in HCV-positive patients before and after treatment with the sofosbuvir-based antiviral regimen. Methods. This prospective study included 87 Egyptian patients with chronic HCV infection treated with sofosbuvir plus daclatasvir with or without ribavirin for 12 weeks. Serum NGAL was measured before and at the end of treatment (EOT). Analysis of NGAL and estimated glomerular filtration rate (eGFR) evolution was done. Results. Our results showed a statistically significant decrease in serum NGAL (P=0.02) with a nonsignificant reduction in eGFR (P=0.06). Moreover, changes in serum NGAL levels (baseline compared to EOT) in patients ranked by KDIGO-CKD classification showed a significant decrease in stages 1 and 2 (P=0.14 and 0.034, respectively) and a nonsignificant decrease in stage 3 (P=0.25). Also, eGFR changes after treatment in patients ranked by the same classifications showed a nonsignificant reduction in all stages (P>0.05). Conclusions. Sofosbuvir appears to have no nephrotoxic effects and is safe to treat patients with chronic HCV infection