Cardioprotective Effect of Licochalcone D against Myocardial Ischemia/Reperfusion Injury in Langendorff-Perfused Rat Hearts.

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RESEARCH ARTICLE Cardioprotective Effect of Licochalcone D against Myocardial Ischemia/Reperfusion Injury in Langendorff-Perfused Rat Hearts Xuan Yuan1☯, Hai-tao Niu1☯, Peng-long Wang3, Jie Lu1, Hong Zhao3, Shi-han Liu1, Qiusheng Zheng2,3*, Chang-gui Li1* 1 The Affiliated Hospital of Qingdao University, Qingdao, 266003, Shandong, China, 2 Binzhou Medical College, Yantai, 264000, Shandong, China, 3 Key Laboratory of Xinjiang Endemic Phytomedicine Resources, Ministry of Education, School of Pharmacy, Shihezi University, Shihezi, 832002, Xinjiang, China ☯ These authors contributed equally to this work. * (QSZ); (CGL) Abstract OPEN ACCESS Citation: Yuan X, Niu H-t, Wang P-l, Lu J, Zhao H, Liu S-h, et al. (2015) Cardioprotective Effect of Licochalcone D against Myocardial Ischemia/ Reperfusion Injury in Langendorff-Perfused Rat Hearts. PLoS ONE 10(6): e0128375. doi:10.1371/ journal.pone.0128375 Academic Editor: Xin-Liang Ma, Thomas Jefferson University, UNITED STATES Received: November 27, 2014 Accepted: April 25, 2015 Published: June 9, 2015 Copyright: © 2015 Yuan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This study was supported by the National Natural Science Foundation of China (No. 81260338), the Xinjiang Production and Construction Corps Funds for Distinguished Young Scientists (No. 2011CD006), and International Cooperation Projects (No. 2012BC001) to QZ. Postdoctoral Innovation Funds of Shandong Province to XY (No. 2014007). Competing Interests: The authors have declared that no competing interests exist. Flavonoids are important components of ‘functional foods’, with beneficial effects on cardiovascular function. The present study was designed to investigate whether licochalcone D (LD) could be a cardioprotective agent in ischemia/reperfusion (I/R) injury and to shed light on its possible mechanism. Compared with the I/R group, LD treatment enhanced myocardial function (increased LVDP, dp/dtmax, dp/dtmin, HR and CR) and suppressed cardiac injury (decreased LDH, CK and myocardial infarct size). Moreover, LD treatment reversed the I/ R-induced cleavage of caspase-3 and PARP, resulting in a significant decrease in proinflammatory factors and an increase in antioxidant capacity in I/R myocardial tissue. The mechanisms underlying the antiapoptosis, antiinflammation and antioxidant effects were related to the activation of the AKT pathway and to the blockage of the NF-κB/p65 and p38 MAPK pathways in the I/R-injured heart. Additionally, LD treatment markedly activated endothelial nitric oxide synthase (eNOS) and reduced nitric oxide (NO) production. The findings indicated that LD had real cardioprotective potential and provided support for the use of LD in myocardial I/R injury. Introduction Cardiovascular diseases are the main causes of human disability and mortality worldwide [1]. Coronary artery ischemia/reperfusion (I/R) injury is known to occur during the restoration of coronary blood flow after a period of myocardial ischemia and triggers myocardial cell injury and necrosis [2]. I/R injury is an intricate process that implicates many mechanisms. In addition, apoptosis, inflammation and oxidative damage play important roles in I/R injury progression [3–5]. Additionally, antiapoptosis, antiinflammation, and antioxidation have been reported to protect the heart from I/R injury, offering additional evidence that apoptosis, inflammation and oxidative injury are involved in I/R injury [6, 7]. Hence, the modulation of PLOS ONE | DOI:10.1371/journal.pone.0128375 June 9, 2015 1 / 14 Cardioprotective Effect of Licochalcone D against Myocardial I/R Fig 1. Chemical structure of licochalcone D. doi:10.1371/journal.pone.0128375.g001 apoptosis, inflammation and oxidative damage and of related cascade responses is considered a crucial therapeutic strategy for treating cardiovascular disease. Flavonoids are the most potent and versatile biologically active compounds in edible plants and have beneficial influences on the cardiovascular system, including anti-hypertensive [8– 11], anti-atherosclerotic [12–14], and anti-platelet effects [15]. Indeed, experimental and epidemiological studies have suggested that this phytochemical class affects circulatory functions [16, 17]. Additionally, the antioxidant property of flavonoids is responsible for preventing some cardiovascular diseases [18–20]. More importantly, flavonoids produced cardioprotective effects in experimental models of myocardial ischemia/reperfusion (I/R) injury [21–27]. Licochalcone D (LD, Fig 1), which is flavonoid compound primarily existing in the root of Glycyrrhiza inflata, possesses antioxidant and antiinflammatory properties [28–30]. Furthermore, LD exerts its antiinflammatory effect by suppressing mast cell degranulation and LPS-induced phosphorylation of NF-κB [31, 32]. However, the mechanism by which this component exerts effects on I/R injury remains unclear. Therefore, the purpose of the present study was to investigate whether LD might prevent myocardial I/R injury and to determine the potential mechanisms involved. Materials and Methods Male Sprague-Dawley (SD) rats (250–280 g) were obtained from Xinjiang Medicine University Medical Laboratory Animal Center (SDXK (Xin) 2011–004), Xinjiang Medical University, Xinjiang, China. The rats were maintained under standard animal care conditions (22 ± 3°C and 60% humidity), with a commercial standard mouse diet (Shihezi University Laboratory Animal Center, Xinjiang, China) and water ad libitum. And all experimental procedures were approved by the Ethics Committee of Shihezi University. Isolated rat heart preparation The male SD rats (250–280 g) were anesthetized by an intraperitoneal injection of 60 mmol/L chloral hydrate (0.35 g/kg). To anticoagulate, 250 U/kg of heparin were administered intraperitoneally to the rats. Thoracic surgery was performed to remove the heart. Hearts were excised quickly and immersed in ice-cold Krebs-Henseleit buffer [33] (118 mM NaCl, 1.2 mM KH2PO4, 4.7 mM KCl, 1.2 mM CaCl2, 1.2 mM MgSO4, 24.9 mM NaHCO3 and 11.1 mM glucose, pH 7.4) comprised of 95% O2 5% CO2 to suppress contraction and reduce oxygen PLOS ONE | DOI:10.1371/journal.pone.0128375 June 9, 2015 2 / 14 Cardioprotective Effect of Licochalcone D against Myocardial I/R consumption. Immediately, the heart was mounted on Langendorff's apparatus and equilibrated with a gas mixture bubbled with 95% O2, 5% CO2 at 37°C. The above course was finished within 2 min. A water-filled latex balloon, coupled to a pressure transducer (Statham), was inserted into the left ventricular cavity through the left auricle, and the volume of the balloon was adjusted to maintain a stable left ventricular end-diastolic pressure of 5–12 mmHg during initial equilibration [16]. Measurement of heart hemodynamic parameters The following functional parameters were continuously monitored with a computer-based data acquisition system (PowerLab/4S with Chart 5 software, AD Instruments, Gladstone, Australia): left ventricular systolic pressure (LVSP), left ventricular end-diastolic pressure (LVEDP), left ventricular developed pressure (LVDP, LVDP = LVSP-LVEDP), maximum rise/ down velocity of left intraventricular pressure (dp/dtmax and dp/dtmin), coronary flow (CF) and heart rate (HR). The heart effluents were collected at 1 min intervals to determine the coronary flow (CF). The recovery of LVDP, dp/dtmax, dp/dtmin, CF and HR were expressed as the percent of 1 minute before ischemia. Experimental groups The isolated rat hearts were randomly divided into three groups (n = 8): control group (Sham), I/R group and I/R with LD. Control group hearts were perfused for the 90 min stabilization period. I/R group hearts were stabilized for 30 min, and then global ischemia (no flow) and reperfusion were subjected to 15 min and 45 min, respectively. Hearts in LD group hearts were stabilized for 20 min, instead K-H buffer with indicated concentration of LD, global ischemia and reperfusion were established for 10 min, 15 min and 45 min, respectively (Fig 2). Left ventricular (LV) function was measured at baseline, just prior to ischemia and 45 min after reperfusion, because the recovery of LV function after reperfusion reached a maximum within 45 min. Enzyme activity assays Lactate dehydrogenase (LDH) and creatine kinase (CK) activity was measured to assess the extent of myocardial cell injury [34]. Briefly, samples were collected from the coronary effluent at the end of the experiment, and the activities of LDH and CK were assayed using LDH and CK kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Determination of myocardial infarct size The myocardial infarct size (INF) was determined by means of a double-staining technique and was evaluated by a digital imaging system described previously [35]. At the end of the Fig 2. Experimental protocol doi:10.1371/journal.pone.0128375.g002 PLOS ONE | DOI:10.1371/journal.pone.0128375 June 9, 2015 3 / 14 Cardioprotective Effect of Licochalcone D against Myocardial I/R reperfusion, the heart was removed, washed in phosphate buffered saline, frozen (−20°C, 30min), and then sliced into 1mm sections perpendicularly along the long axis from apex to base. The slices were incubated in 1% triphenyltetrazolium chloride (TTC, Biodee, Beijing, China) in pH 7.4 buffer at 37°C for 10–15 min, fixed in 10% formaldehyde solution, and imaged using a digital camera. Then the infarct (white), non-infarct (red) areas were measured an Image-Pro Plus 7.0 (Media Cybernetics, Wyoming, USA). The myocardial infarct size was represented as a percentage of infarct area (INF) over total area of sections (LV, left ventricle area) × 100%. Nitrate plus nitrite (NOx) measurement NOx, the stable end product of NO, has been tested as an index of bioavailability of NO. The level of myocardial NOx was assessed using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). NOx was designated as μmol/g protein. Its concentration was determined at an optical density of 550 nm in a spectrophotometric method. Protein determination was carried out according to the Bradford method [36]. Oxidative stress assay After 45 min of the perfusion, hearts were harvested and frozened at -70°C for following experiments. The hearts were grinded by a liquid nitrogen-chilled tissue pulverizer, weighed the cardiac tissues, and then homogenized in bufer and centrifuged using homogenizer. The superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione/glutathione disulide (GSG/GSSH) concentrations were measured with or without the inhibitors (MK0026, PDTC, or SB203580) according to the instruction of the kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). The levels of MDA were expressed as nanomole MDA per milligram protein. Inflammatory cytokines measurement To investigate the LD’s role in I/R-induced inflammation response, the levels of proinflammatory cytokines (IL-6, TNF-α and CRP) were detected with or without the inhibitors (MK0026, PDTC, or SB203580) according to the instruction of the Rat Interleukin 6 ELISA Kit, Rat Tumor Necrosis Factor α ELISA Kit, and Rat CRP/C-Reactive Protein ELISA Kit (Sigma-Aldrich, St. Louis, MO, USA) Determination of caspase-3, PARP, eNOS, iNOS expression, iNOS, total or phosphorylaed AKT, NF-κB/p65 and p38 MAPK by Western blot assay Frozen myocardial tissue samples were ground with a mortar and pestle, and subsequently placed into a tissue grinder with lysis buffer. The protein concentration was determined with a Bio-Rad protein assay (Bio-Rad Laboratories, Hercules, CA, USA). The lysate samples were separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), nitrocellulose membranes (Amersham Biosciences, New Jersey, USA), and blocked with 5% nonfat milk in Tris-buffered saline with Tween (TBST) for 2 h at room temperature. Membranes were incubated with an antibody against cleaved caspase-3, cleavedPARP, and phosphorylated eNOS (Cell Signaling Technology, MA, USA), or iNOS (BD Bioscience Laboratories, CA, USA), total or phosphorylaed AKT, NF-κB/p65 and p38 MAPK (Sigma-Aldrich, MO, USA) in 5% milk/TBST at 4°C overnight. The membrane was then washed with TBST and incubated with horseradish peroxidase-conjugated antibody (Cell Signaling Technology) for 1 h at room temperature. The blots were developed with an enhanced PLOS ONE | DOI:10.1371/journal.pone.0128375 June 9, 2015 4 / 14 Cardioprotective Effect of Licochalcone D against Myocardial I/R chemiluminescence detection kit (Thermo, NY, USA) and were exposed on Kodak radiographic film. The immunoblotting was visualized with EC3 Imaging System, and analyzed with Vision Works LS software (UVP, CA, USA). Terminal DNA breakpoints in situ 3-hydroxy end labeling (TUNEL) assay The TUNEL staining kit was purchased from Roche Group (Basel, Switzerland). According to the manufacturer’s instruction, the tissue sections were deparaffinized by immersing slides in xylene, and rehydrated by sequentially immersing the slides through graded ethanol washes. The sections were treated with protease K (20 ug/mL) for 15 min, then were immersed in TUNEL reaction mixture for 60 min at 37°C in a humid chamber, and incubated with converter-POD was used to incubate the slides for 30 min at 37°C. The slides were observed by optical microscopy. Statistical analysis Data were presented as mean±SD from at least six independent experiments and statistical comparisons were evaluated with One-way ANOVA and followed by Student's t-test. Differences were considered significant at p < 0.05. Analyses were performed using the SPSS 17 software (SPSS Inc., Chicago, IL, USA, 2008). Results LD improved the recovery of I/R-altered cardiac function The effects of LD (0.1, 0.5, 1, 2 and 4 μg/mL) on hemodynamic parameters (LVDP, ±dp/dtmax, CF and HR) in the control group, I/R group and LD-treated group hearts were shown in Fig 3. The hearts subjected to 15 min of ischemia, followed by 45 min of reperfusion, showed a significant decrease in the values of LVDP (49.38%), dp/dtmax (47.30%) dp/dtmin (44.46%), CF (71.40%) and HR (69.47%) after 45 min of reperfusion. Compared with unprotected I/R hearts, LD pre-treatment increased the cardiac functional index, and 1 μg/mL of LD significantly improved the values of LVDP, dp/dtmax, dp/dtmin, CF and HR but had little effect on cardiac function (S1 Table). LD decreased myocardial injury in I/R rats Considering the significant cardioprotective effects of LD at 1 μg/mL, this concentration was chosen for the subsequent assays. Myocardial infarct size (INF), lactate dehydrogenase (LDH) and creatine kinase (CK) levels were measured to examine whether LD might reduce myocardial injury. As shown in Fig 4, the myocardial INF (39.94±8.89%), LDH leakage (58.5±8.43) and CK (126.36±14.13) markedly increased in the hearts of rats in the I/R group after 15 min of ischemia, followed by 45 min of reperfusion, compared to controls. In contrast, LD pretreatment significantly reduced the I/R-induced increase in myocardial INF (11.30±7.78%), LDH (27.5±7.26) and CK (72.00±17.24) release in rat heart. LD suppressed myocardial apoptosis Apoptosis is the major form of cell death after a short period of ischemia that is followed by reperfusion; therefore, we investigated the effects of LD treatment on myocardial apoptosis-related proteins (caspase-3 and PARP) in I/R cardiac tissue. As shown in Fig 5, I/R induced increased activation of apoptosis, as illustrated by the cleaved caspase-3 protein expression, which is associated with a 1.47-fold increase in cleaved PARP, compared with the control PLOS ONE | DOI:10.1371/journal.pone.0128375 June 9, 2015 5 / 14 Cardioprotective Effect of Licochalcone D against Myocardial I/R Fig 3. Effect of LD on cardiac function in rats subjected to I/R (n = 8). The three time points (15 min, 30 min and 45 min) represente the the time points after the start of reperfusion. *P<0.05, **P<0.01 compared with control group; #P<0.05, ##P<0.01 compared with I/R group. doi:10.1371/journal.pone.0128375.g003 group. LD treatment substantially reduced this I/R-induced cleavage of both caspase-3 and PARP. LD treatment alleviated inflammation in I/R cardiac tissue Previous studies have indicated that LD has antiinflammation effect and that the inflammatory response participates in I/R-induced cardiac injury; thus the effects of LD on inflammatory cytokines (IL-6, TNF-α and CRP) were also detected in the ischemic heart. I/R injury significantly increased the IL-6 expression level and activated TNF-α and CRP expression. The IL-6 expression level in the LD treatment group (62.12±5.05 pg/mL) was markedly lower than that in the I/R group (109.83±13.62 pg/mL). Compared with the I/R group, the activities of TNF-α Fig 4. LD reduced I/R-induced myocardial injury. (A) The myocardial infarct size. (B) The level of LDH. (C) The level of CK. **P<0.01 compared with control group; ##P<0.01 compared with I/R group. doi:10.1371/journal.pone.0128375.g004 PLOS ONE | DOI:10.1371/journal.pone.0128375 June 9, 2015 6 / 14 Cardioprotective Effect of Licochalcone D against Myocardial I/R Fig 5. LD suppressed myocardial apoptosis. (A) Cleaved caspase-3 and PARP expressions in cardiac tissue were analyzed via Western blot. (B) Quantitative analysis of cleaved caspase-3 and PARP protein levels. *P<0.05, **P<0.01 compared with control group; #P<0.05, ##P<0.01 compared with I/ R group. doi:10.1371/journal.pone.0128375.g005 and CRP significantly enfocar la importancia de la producción mediática de los niños en su descubrimiento del mundo, sobre todo utilizando el periódico escolar y la imprenta. Asimismo las asociaciones de profesores trabajaron en esta línea e incluso la enseñanza católica se comprometió desde los años sesenta realizando trabajos originales en el marco de la corriente del Lenguaje Total. Páginas 43-48 45 Comunicar, 28, 2007 En el ámbito de los medios, también desde el principio del siglo XX hay ciertas corrientes de conexión. Pero es a lo largo de los años sesenta cuando se constituyeron asociaciones de periodistas apasionados por sus funciones de mediadores, que fomentaron la importancia ciudadana de los medios como algo cercano a los jóvenes, a los profesores y a las familias. Así se crearon la APIJ (Asociación de Prensa Información para la Juventud), la ARPEJ (Asociación Regional de Prensa y Enseñanza para la Juventud), el CIPE (Comité Interprofesional para la Prensa en la Escuela) o la APE (Asociación de Prensa y Enseñanza), todas ellas para la prensa escrita Estas asociaciones fueron precedidas por movimientos surgidos en mayo de 1968, como el CREPAC que, utilizando películas realizadas por periodistas conocidos, aclaraba temas que habían sido manipulados por una televisión demasiado próxima al poder político y realizaba encuentros con grupos de telespectadores. cipio del siglo XX, y nos han legado textos fundadores muy preciados, importantes trabajos de campo y muchos logros educativos y pedagógicos. La educación en medios ha tenido carácter de oficialidad de múltiples maneras, aunque nunca como una enseñanza global. Así la campaña «Operación Joven Telespectador Activo» (JTA), lanzada al final de los años setenta y financiada de manera interministerial para hacer reflexionar sobre las prácticas televisuales de los jóvenes, la creación del CLEMI (Centro de Educación y Medios de Comunicación) en el seno del Ministerio de Educación Nacional en 1983, la creación de la optativa «Cine-audiovisual» en los bachilleratos de humanidades de los institutos en 1984 (primer bachillerato en 1989) y múltiples referencias a la educación de la imagen, de la prensa, de Internet. La forma más visible y rápida de evaluar el lugar de la educación en medios es valorar el lugar que se le ha reservado en los libros de texto del sistema educa- 2. Construir la educación en los medios sin nombrarla El lugar que ocupa la edu- La denominación «educación en medios», que debería cación en los medios es muy ambiguo, aunque las cosas están cambiando recientemente. entenderse como un concepto integrador que reagrupase todos los medios presentes y futuros, es a menudo percibida En principio, en Francia, co- por los «tradicionalistas de la cultura» como una tendencia mo en muchos otros países, la educación en los medios no es hacia la masificación y la pérdida de la calidad. una disciplina escolar a tiempo completo, sino que se ha ido conformado progresivamente a través de experiencias y reflexiones teóricas que han tivo en Francia. Una inmersión sistemática nos permi- permitido implantar interesantes actividades de carác- te constatar que los textos oficiales acogen numerosos ter puntual. Se ha ganado poco a poco el reconoci- ejemplos, citas, sin delimitarla con precisión. miento de la institución educativa y la comunidad es- colar. Podemos decir que ha conquistado un «lugar», 3. ¿Por qué la escuela ha necesitado casi un siglo en el ámbito de la enseñanza transversal entre las dis- para oficilializar lo que cotidianamente se hacía en ciplinas existentes. ella? Sin embargo, la escuela no está sola en esta aspi- Primero, porque las prácticas de educación en me- ración, porque el trabajo en medios es valorado igual- dios han existido antes de ser nombradas así. Recor- mente por el Ministerio de Cultura (campañas de foto- demos que no fue hasta 1973 cuando aparece este grafía, la llamada «Operación Escuelas», presencia de término y que su definición se debe a los expertos del colegios e institutos en el cine ), así como el Minis- Consejo Internacional del Cine y de la Televisión, que terio de la Juventud y Deportes que ha emprendido en el seno de la UNESCO, definen de esta forma: numerosas iniciativas. «Por educación en medios conviene entender el estu- Así, esta presencia de la educación en los medios dio, la enseñanza, el aprendizaje de los medios moder- no ha sido oficial. ¡La educación de los medios no apa- nos de comunicación y de expresión que forman parte rece oficialmente como tal en los textos de la escuela de un dominio específico y autónomo de conocimien- francesa hasta 2006! tos en la teoría y la práctica pedagógicas, a diferencia Este hecho no nos puede dejar de sorprender ya de su utilización como auxiliar para la enseñanza y el que las experiencias se han multiplicado desde el prin- aprendizaje en otros dominios de conocimientos tales Páginas 43-48 46 Comunicar, 28, 2007 como los de matemáticas, ciencias y geografía». A pe- mente en todas las asignaturas. Incluso los nuevos cu- sar de que esta definición ha servido para otorgarle un rrículos de materias científicas en 2006 para los alum- reconocimiento real, los debates sobre lo que abarca y nos de 11 a 18 años hacen referencia a la necesidad no, no están totalmente extinguidos. de trabajar sobre la información científica y técnica y En segundo lugar, porque si bien a la escuela fran- el uso de las imágenes que nacen de ella. cesa le gusta la innovación, después duda mucho en Desde junio de 2006, aparece oficialmente el tér- reflejar y sancionar estas prácticas innovadoras en sus mino «educación en medios» al publicar el Ministerio textos oficiales. Nos encontramos con una tradición de Educación los nuevos contenidos mínimos y las sólidamente fundada sobre una transmisión de conoci- competencias que deben adquirir los jóvenes al salir mientos muy estructurados, organizados en disciplinas del sistema educativo. escolares que se dedican la mayor parte a transmitir Este documento pretende averiguar cuáles son los conocimientos teóricos. La pedagogía es a menudo se- conocimientos y las competencias indispensables que cundaria, aunque los profesores disfrutan de una ver- deben dominar para terminar con éxito su escolaridad, dadera libertad pedagógica en sus clases. El trabajo seguir su formación y construir su futuro personal y crítico sobre los medios que estaba aún en elaboración profesional. Siete competencias diferentes han sido te- necesitaba este empuje para hacerse oficial. nidas en cuenta y en cada una de ellas, el trabajo con Aunque el trabajo de educación en los medios no los medios es reconocido frecuentemente. Para citar esté reconocido como disciplina, no está ausente de un ejemplo, la competencia sobre el dominio de la len- gua francesa definen las capa- cidades para expresarse oral- La metodología elaborada en el marco de la educación en mente que pueden adquirirse con la utilización de la radio e, medios parece incluso permitir la inclinación de la sociedad incluso, se propone fomentar de la información hacia una sociedad del conocimiento, como defiende la UNESCO. En Francia, se necesitaría unir el interés por la lectura a través de la lectura de la prensa. La educación en los medios las fuerzas dispersas en función de los soportes mediáticos y orientarse más hacia la educación en medios que al dominio adquiere pleno derecho y entidad en la sección sexta titulada «competencias sociales y cívi- técnico de los aparatos. cas» que indica que «los alum- nos deberán ser capaces de juz- gar y tendrán espíritu crítico, lo que supone ser educados en los las programaciones oficiales, ya que, a lo largo de un medios y tener conciencia de su lugar y de su influencia estudio de los textos, los documentalistas del CLEMI en la sociedad». han podido señalar más de una centena de referencias a la educación de los medios en el seno de disciplinas 4. Un entorno positivo como el francés, la historia, la geografía, las lenguas, Si nos atenemos a las cifras, el panorama de la las artes plásticas : trabajos sobre las portadas de educación en medios es muy positivo. Una gran ope- prensa, reflexiones sobre temas mediáticos, análisis de ración de visibilidad como la «Semana de la prensa y publicidad, análisis de imágenes desde todos los ángu- de los medios en la escuela», coordinada por el CLE- los, reflexión sobre las noticias en los países europeos, MI, confirma año tras año, después de 17 convocato- información y opinión rias, el atractivo que ejerce sobre los profesores y los Esta presencia se constata desde la escuela mater- alumnos. Concebida como una gran operación de nal (2 a 6 años) donde, por ejemplo, se le pregunta a complementariedad source of circulating FGF-21. The lack of association between circulating and muscle-expressed FGF-21 also suggests that muscle FGF-21 primarily works in a local manner regulating glucose metabolism in the muscle and/or signals to the adipose tissue in close contact to the muscle. Our study has some limitations. The number of subjects is small and some correlations could have been significant with greater statistical power. Another aspect is that protein levels of FGF-21 were not determined in the muscles extracts, consequently we cannot be sure the increase in FGF-21 mRNA is followed by increased protein expression. In conclusion, we show that FGF-21 mRNA is increased in skeletal muscle in HIV patients and that FGF-21 mRNA in muscle correlates to whole-body (primarily reflecting muscle) insulin resistance. These findings add to the evidence that FGF-21 is a myokine and that muscle FGF-21 might primarily work in an autocrine manner. Acknowledgments We thank the subjects for their participation in this study. Ruth Rousing, Hanne Willumsen, Carsten Nielsen and Flemming Jessen are thanked for excellent technical help. The Danish HIV-Cohort is thanked for providing us HIV-related data. 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