RGFP966 inactivation of the YAP pathway attenuates cardiac dysfunction induced by prolonged hypothermic preservation / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Oxidative stress and apoptosis are the key factors that limit the hypothermic preservation time of donor hearts to within 4-6 h. The aim of this study was to investigate whether the histone deacetylase 3 (HDAC3) inhibitor RGFP966 could protect against cardiac injury induced by prolonged hypothermic preservation. Rat hearts were hypothermically preserved in Celsior solution with or without RGFP966 for 12 h followed by 60 min of reperfusion. Hemodynamic parameters during reperfusion were evaluated. The expression and phosphorylation levels of mammalian STE20-like kinase-1 (Mst1) and Yes-associated protein (YAP) were determined by western blotting. Cell apoptosis was measured by the terminal deoxynucleotidyl-transferase (TdT)-mediated dUTP nick-end labeling (TUNEL) method. Addition of RGFP966 in Celsior solution significantly inhibited cardiac dysfunction induced by hypothermic preservation. RGFP966 inhibited the hypothermic preservation-induced increase of the phosphorylated (p)-Mst1/Mst1 and p-YAP/YAP ratios, prevented a reduction in total YAP protein expression, and increased the nuclear YAP protein level. Verteporfin (VP), a small molecular inhibitor of YAP-transcriptional enhanced associate domain (TEAD) interaction, partially abolished the protective effect of RGFP966 on cardiac function, and reduced lactate dehydrogenase activity and malondialdehyde content. RGFP966 increased superoxide dismutase, catalase, and glutathione peroxidase gene and protein expression, which was abolished by VP. RGFP966 inhibited hypothermic preservation-induced overexpression of B-cell lymphoma protein 2 (Bcl-2)-associated X (Bax) and cleaved caspase-3, increased Bcl-2 mRNA and protein expression, and reduced cardiomyocyte apoptosis. The antioxidant and anti-apoptotic effects of RGFP966 were cancelled by VP. The results suggest that supplementation of Celsior solution with RGFP966 attenuated prolonged hypothermic preservation-induced cardiac dysfunction. The mechanism may involve inhibition of oxidative stress and apoptosis via inactivation of the YAP pathway.

The m-rna, expression of serca2 and ncx1 in the process of pharmacological cell protection in experimental acute pancreatitis induced by taurocholate / Expressão do rna-m, da serca2 e do ncx1 no processo de proteção celular farmacológica na pancreatite aguda experimental induzida por taurocolato

ABSTRACT Background: Intracellular calcium overload is known to be a precipitating factor of pancreatic cell injury in acute pancreatitis (AP). Intracellular calcium homeostasis depends of Plasmatic Membrane Calcium ATPase (PMCA), Sarcoplasmic Endothelial Reticulum Calcium ATPase 2 (SERCA 2) and the Sodium Calcium Exchanger (NCX1). The antioxidant melatonin (Mel) and Trisulfate Disaccharide (TD) that accelerates NCX1 action could reduce the cell damage determined by the AP. Aim: To evaluate m-RNA expressions of SERCA2 and NCX1 in acute pancreatitis induced by sodium taurocholate in Wistar rats pre-treated with melatonin and/or TD. Methods: Wistar rats were divided in groups: 1) without AP; 2) AP without pre-treatment; 3) AP and Melatonin; 4) AP and TD; 5) AP and Melatonin associated to TD. Pancreatic tissue samples were collected for detection of SERCA2 and NCX1 m-R NA levels by polymerase chain reaction (PCR). Results: Increased m-RNA expression of SERCA2 in the melatonin treated group, without increase of m-RNA expression of the NCX1. The TD did not affect levels of SERCA2 and NCX1 m-RNA expressions. The combined melatonin and TD treatment reduced the m-RNA expression of SERCA2. Conclusions: The effect of melatonin is restricted to increased m-RNA expression of SERCA2. Although TD does not affect gene expression, its action in accelerating calcium exchanger function can explain the slightest expression of SERCA2 m-RNA when associated with Melatonin, perhaps by a joint action of drugs with different and but possibly complementary mechanisms.

RESUMO Racional: A lesão celular da pancreatite aguda (PA) envolve sobrecarga de cálcio, regulada pela atividade da Cálcio ATPase de membrana (PMCA), Cálcio ATPase do Retículo (SERCA2) e pelo Trocador Sódio Cálcio (NCX1). A melatonina (antioxidante) e o Dissacarídeo Trissulfatado (acelerador do NCX1) poderiam reduzir a lesão celular na PA. Objetivo: Avaliar a expressão do RNAm da SERCA2 e NCX1 em modelo animal de pancreatite aguda tratados com melatonina e/ou dissacarídeo trissulfatado (DT). Método: Ratos Wistar foram divididos em grupos: 1) sem pancreatite aguda; 2) com pancreatite aguda por taurocolato; 3) PA e Melatonina; 4) PA e DT; 5) PA e Melatonina com DT. Amostras de tecido foram colhidas para detecção dos níveis de RNAm da SERCA2 e NCX1 por PCR. Resultados: Houve aumento da expressão do RNAm da SERCA2 no grupo com PA tratados com Melatonina, porém sem aumento de expressão do NCX1. O DT não afetou os níveis de SERCA2 e NCX1. O tratamento conjunto com Melatonina e DT diminuiu a expressão da SERCA2. Conclusões: O efeito da Melatonina é restrito ao aumento da expressão da SERCA2. O DT não tem ação na expressão gênica, porém sua ação na aceleração do trocador na retirada do cálcio pode explicar a menor expressão da SERCA2 quando associado à Melatonina, pela ação conjunta de drogas com mecanismos diferentes e possivelmente complementares.

Comparative experimental study of myocardial protection with crystalloid solutions for heart transplantation / Estudo comparativo experimental da proteção miocárdica com soluções cristalóides para transplante cardíaco

BACKGROUND: There is a growing need to improve myocardial protection, which will lead to better performance of cardiac operations and reduce morbidity and mortality. Therefore, the objective of this study was to compare the efficacy of myocardial protection solution using both intracellular and extracellular crystalloid type regarding the performance of the electrical conduction system, left ventricular contractility and edema, after being subjected to ischemic arrest and reperfusion. METHODS: Hearts isolated from male Wistar (n=32) rats were prepared using Langendorff method and randomly divided equally into four groups according the cardioprotective solutions used Krebs-Henseleit-Buffer (KHB), Bretschneider-HTK (HTK), St. Thomas-1 (STH-1) and Celsior (CEL). After stabilization with KHB at 37ºC, baseline values (control) were collected for heart rate (HR), left ventricle systolic pressure (LVSP), maximum first derivate of rise left ventricular pressure (+dP/dt), maximum first derivate of fall left ventricular pressure (-dP/dt) and coronary flow (CF). The hearts were then perfused at 10ºC for 5 min and kept for 2 h in static ischemia at 20ºC in each cardioprotective solution. Data evaluation was done using analysis of variance in completely randomized One-Way ANOVA and Tukey's test for multiple comparisons. The level of statistical significance chosen was P<0.05. RESULTS: HR was restored with all the solutions used. The evaluation of left ventricular contractility (LVSP, +dP/ dt and -dP/dt) showed that treatment with CEL solution was better compared to other solutions. When analyzing the CF, the HTK solution showed better protection against edema. CONCLUSION: Despite the cardioprotective crystalloid solutions studied are not fully able to suppress the deleterious effects of ischemia and reperfusion in the rat heart, the CEL solution had significantly higher results followed by HTK>KHB>STH-1.

INTRODUÇÃO: Existe crescente necessidade de aprimorar a proteção miocárdica, para melhor desempenho das operações cardíacas e diminuição da morbimortalidade. Portanto, o objetivo deste estudo foi comparar a eficácia da proteção miocárdica usando tanto solução cristaloide tipo intracelular como extracelular quanto ao desempenho do sistema de condução elétrica, contratilidade do ventrículo esquerdo e edema, após parada isquêmica e posterior reperfusão. MÉTODOS: Corações isolados de ratos Wistar foram montados em Langendorff e aleatoriamente divididos em quatro grupos. de acordo com as soluções cardioprotetoras utilizadas Krebs-Henseleit-Buffer (KHB), Bretschneider-HTK (HTK), St. Thomas-1(STH-1) e Celsior (CEL). Após a estabilização com KHB a 37ºC, valores basais (controle) foram coletados para frequência cardíaca (FC), pressão sistólica do ventrículo esquerdo (PSVE), derivada máxima de aumento da pressão ventricular esquerda (+dP/dt), derivada máxima de queda da pressão ventricular esquerda (-dP/dt) e fluxo coronariano (FCo). Os corações foram então perfundidos a 10ºC por 5 min e mantidos por 2 h em isquemia estática a 20ºC em cada solução cardioprotetora. Avaliação dos dados foi por análise de variância inteiramente casualizados em One-Way ANOVA e teste de Tukey para comparações múltiplas. O nível de significância estatística escolhido foi P<0,05. RESULTADOS: Houve recuperação da FC com todas as soluções utilizadas. A avaliação da contratilidade ventricular esquerda (PSVE, +dP/dt e -dP/dt) demonstrou que o tratamento com a solução CEL foi melhor em comparação às outras soluções. Ao analisar o CF, a solução HTK indicou melhor proteção contra edema. CONCLUSÃO: Apesar das soluções cristaloides cardioprotetoras estudadas não serem capazes de suprimir os efeitos deletérios da isquemia e reperfusão no coração de ratos, a solução CEL apresentou resultado superior seguido por HTK>KHB>STH-1.

Effects of ischemic preconditioning associated to different preservation solutions in protecting the intestinal graft / Efeitos do precondicionamento isquêmico associado a diferentes soluções de preservação na proteção do enxerto intestinal

PURPOSE: To evaluate the effects of ischemic preconditioning (IPC) associate with different preservation solutions, in the protecting of gut. METHODS: Four groups of 14 rats underwent laparotomy and collecting 20 cm of ileum, for preservation, at 4ºC, in Belzer (Belz), Ringer (RL), Celsior (Cs) and Custodiol (Cust) solutions, for 24 hours. Prior to collection, half of the animals in each group were subjected to IPC. During preservation, in the periods of zero, 12, 18 and 24 hours, were conducted evaluating the degree of mucosal injury and dosage of malondialdehyde acid (MDA). RESULTS: In all periods the RL group, with and without IPC, presented MDA values higher than the Belz and Cs. The degree of mucosal injury in the non-ipc RLgroup with 12h preservation was higher than the others; with 18 and 24h, the RL and Cust had higher degrees of damage than Cs and Belz. With IPC, in all periods, the group Cs and Belz had lower degrees of injury. CONCLUSION: The Celsior and Belzer solutions had better protective effects on the gut and these effects were enhanced by IPC.

OBJETIVO: Avaliar os efeitos do precondicionamento isquêmico (PCI) associado a diferentes soluções de preservação, na proteção do intestino delgado. MÉTODOS: Quatro grupos de 14 ratos Wistar, foram submetidos à laparotomia e coleta de 20 cm de íleo, para preservação, a 4ºC, nas soluções de Belzer (Belz), Ringer (RL), Celsior (Cs) e Custodiol (Cust) por 24 horas. Previamente à coleta, em metade dos animais de cada grupo, o intestino foi submetido ao PCI. Durante a preservação, nos períodos de Zero, 12, 18 e 24 horas, foram realizados avaliação do grau de lesão da mucosa e dosagem do ácido malondialdeído (MDA). RESULTADOS: Em todos os períodos o grupo RL, sem e com pci, apresentou valores maiores de MDA do que o Belz e Cs. O grau de lesão da mucosa nos grupos sem pci com preservação de 12h, no grupo RL, foi maior que nos demais; com 18h e 24h o grupo RL e Cust apresentaram maiores graus de lesão do que Cs e Belz. Com o pci, em todos os períodos, os grupos Belz e Cs apresentaram menores graus de lesão CONCLUSÃO: As Soluções Celsior e Belzer tiveram melhores efeitos na proteção do intestino e estes efeitos foram incrementados pelo precondicionamento isquêmico.

The influence of developmental stages and protective additives on cryopreservation of surf clam (Spisula sachalinensis) larvae.

This study was performed to find out the optimal larval stage and the most desirable protective additives for cryopreservation of surf clam, Spisula sachalinensis larvae. The survival rates of frozen-thawed larvae increased with post developmental stage. The highest value of 96.1 +/- 1.0% was achieved using umbo stage larva as developmental stage and 0.2 M sucrose as protective additive.