Evaluation of Reference Genes for Quantitative PCR in Four Tissues from Rabbits with Hypercholesterolaemia

Abstract Rabbit with hypercholesterolaemia is an important model for studying cholesterol metabolism disease. This study aimed to evaluate the expression stability of nine reference genes for quantitative PCR (qPCR) analysis in adrenal gland, liver, spleen, and kidney tissue from rabbits with hypercholesterolaemia. In total, 30 male Harbin Large White (HLW) rabbits were fed a normal feed (n = 15) or a high cholesterol feed (n = 15) for 8 weeks to induce hypercholesterolaemia. Nine reference genes were verified by qPCR using cDNA extracted from rabbit tissue samples. For qPCR analysis, reference genes were evaluated using the RefFinder and GeNorm algorithms. Overall, seven rabbits with hypercholesterolaemia were identified based on body weight and total cholesterol measurements. Combining the results of the RefFinder and GeNorm algorithms, the most stable reference genes were hypoxanthine phosphoribosyltransferase 1 (Hprt1) and eukaryotic translation elongation factor 1 alpha 1 (Eef1a1) in the adrenal gland, β-2-microglobulin (B2m) and glyceraldehyde-3-phosphate dehydrogenase (Gapdh) in the liver, tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (Ywhaz) and Gapdh in the spleen, and peptidylprolyl isomerase (Ppia), β-actin (Actb), succinate dehydrogenase complex subunit A flavoprotein (Sdha), and B2m in the kidney. Taken together, our results confirmed that Hprt1 and Eef1a1, B2m and Gapdh, Ywhaz and Gapdh, and Ppia, Actb, Sdha, and B2m were the best reference genes for qPCR analyses in adrenal gland, liver, spleen, and kidney tissue, respectively, of rabbits with hypercholesterolaemia.

Polineuropatia secundária ao uso crônico de estatinas / Polineuropathy due to chronic use of statins

Os inibidores da 3-hidroxi-3-metilglutaryl coenzima-A (HMG-CoA)redutase têm eficácia comprovada em reduzir os níveis de colesterole prevenir a inflamação do endotélio coronariano, cerebral e periférico.Os efeitos adversos devem ser conhecidos, pois sua suspensão pode levar à completa reversibilidade dos sintomas. São descritas complicações musculares, entre elas, mialgia, miosite e rabdomiólise, além de complicações hepáticas, neuropatias e outras. Foram revistos 1 estudo experimental, 6 estudos populacionais, 25 relatos de casos e 2 revisões sobre o tema, a maioria apontando para a real existência dessa complicação. A neuropatia induzida por estatinas tem incidência aproximada de 12 por 100.000 pessoas-ano. Apresenta-se mais comumente como polineuropatia sensitivo-motora axonal de predomínio sensitivo. Em alguns casos, agravam neuropatias periféricas preexistentes. A fisiopatologia parece convergir para o comprometimento da cadeia respiratória mitocondrial. O diagnóstico baseia-se na relação temporal entre o uso ou suspensão da droga e o surgimento ou melhora dos sinais e sintomas. Os exames laboratoriais são fundamentais para excluir causas de neuropatias periféricas bem estabelecidas. O prognóstico relaciona-se com o momento de suspensão da droga, com relatos desde melhora completa até irreversibilidade.

Inhibitors of coenzyme A 3-hydroxy-3-metilglutaryl (HMG-CoA) reductaseinhibitors have proven to reduce cholesterol levels and prevent inflammation of the coronary, cerebral and peripheral endothelium. Adverse effects should be known, for its suspension can lead to complete reversibility of symptoms. Muscle complications are described, among them, myalgia, myositis and rhabdomyolysis, besides hepatic, neuropathies, and others. One experimental study and 6 population studies, 25 cases reports, and other 2 reviews were reviewed, most pointing to the actual existence of this complication. Statin induced neuropathy has an approximate incidence of 12 per 100,000 persons-year. It most commonly is presented as a sensorimotor axonal polyneuropathy predominantly sensory. In some cases it aggravates pre-existing peripheral neuropathies. The pathophysiology seems to converge to impairment of the mitochondrial respiratory chain. The diagnosis is based on the temporal relationship between the use or discontinuation of the drug and the emergence or improvement of signs and symptoms. Laboratory tests are essential to exclude well established causes of peripheral neuropathies. The prognosis is related to the moment of drug suspension, with reports from complete recovery to irreversibility.

Pharmacological activity of the hydroalcoholic extract from Hovenia dulcis thunberg fruit and the flavonoid dihydromyricetin during hypercholesterolemia induced in rats

Cerebrovascular accidents and coronary artery disease are the leading causes of cardiovascular mortalities in Brazil and high levels of LDL cholesterol are one of the main risk factors. In this context, several plant extracts and natural substances have shown promise as cholesterol-lowering. The objective of this study was to evaluate the potential of the hydroalcoholic extract of the fruit of H. dulcis and of dihydromyricetin in cholesterol reduction in hypercholesterolemic rats. Forty-two Wistar male rats were distributed into seven groups of six animals that received diets supplemented with 1% cholesterol and 0.3% cholic acid, with the exception of the control group, which received conventional diets. Animals were treated with oral suspensions containing: atorvastatin 1.0 mg/kg; H. dulcis extract at 50.0 and 100.0 mg/kg and dihydromyricetin at 25.0 and 50.0 mg/kg vehicle (control group). The following biochemical markers were evaluated; total cholesterol, HDL-C, LDL-C, triglycerides, AST, ALT, and alkaline phosphatase. The hypercholesterolemic diet was effective in inducing hypercholesterolemia, increasing total cholesterol by 112.7% relative to the control group. The treatments with two doses of the extract proved to be promising hypocholesterolemic agents, as they were able to substantially reduce total cholesterol and LDL-C, without significantly altering triglycerides, hepatic transaminases, and alkaline phosphatase, thereby encouraging the studies with the plant H. dulcis. The groups treated with the flavonoid dihydromyricetin, although they showed a significant reduction in total cholesterol and LDL-C, and found increases in triglycerides and hepatic transaminases, which is unwanted in the context of hypercholesterolaemia.

No Brasil, o acidente vascular cerebral e a doença arterial coronariana constituem as principais causas de mortalidade cardiovascular, sendo os altos níveis de colesterol LDL um dos principais fatores de risco. Nesse contexto, diversos extratos vegetais e substâncias naturais isoladas têm se mostrado promissoras como hipocolesterolemiantes. O objetivo do trabalho foi avaliar o potencial do extrato hidroalcoólico dos frutos de Hovenia dulcis e do flavonóide diidromiricetina na redução do colesterol em ratos hipercolesterolêmicos. Quarenta e dois ratos Wistar machos, foram distribuídos em 7 grupos de 6 animais, que receberam dieta suplementada com 1% de colesterol e 0,3% de ácido cólico, à exceção do grupo controle, que recebeu ração convencional. Posteriormente, os animais foram tratados com suspensões orais contendo: atorvastatina 1,0 mg/kg; extrato de H. dulcis de 50,0 e 100,0 mg/kg; diidromiricetina de 25,0 e 50,0 mg/kg e veículo (grupo controle). Avaliaram-se os parâmetros bioquímicos: colesterol total, HDL-C, LDL-C, triglicérides, AST, ALT e fosfatase alcalina. A dieta hipercolesterolêmica foi efetiva na indução da hipercolesterolemia, aumentando o colesterol total em 112,7% em relação ao controle. Os tratamentos com as duas doses do extrato mostraram-se promissores como agentes hipocolesterolemiantes, já que foram capazes de reduzir substancialmente o colesterol total e LDL-C, sem alterar significativamente triglicérides, as transaminases hepáticas e a fosfatase alcalina, incentivando, assim, a continuidade de estudos com a planta H. dulcis. Já os grupos tratados com o flavonóide diidromiricetina, apesar de apresentarem redução significativa do colesterol total e de LDL-C, apresentaram elevações nos triglicérides e nos parâmetros hepáticos, resultado indesejável no âmbito das hipercolesterolemias.

Avaliação do efeito do alho (Allium sativum L. ) sobre o colesterol plasmático em coelhos com hipercolesterolemia induzida / Evaluation of garlic (Allium sativum L. ) effects on the serum cholesterol of rabbits with induced hypercholesterolemia

A utilização de plantas no tratamento de doenças ou como meio curativo é uma tradição popular e altamente difundida, sendo que muitos trabalhos abordam as propriedades terapêuticas e farmacológicas do alho na redução das hiperlipidemias. O objetivo deste estudo foi investigar a influência do extrato aquoso (E.A.) do alho (Allium sativum L.) no tratamento do colesterol plasmático em coelhos com hipercolesterolemia experimental. Os animais foram divididos em G1 (grupo controle) e G2 (grupo tratado com alho). O experimento foi desenvolvido em três fases: na 1ª fase os animais receberam dieta comercial de coelhos para avaliar o nível basal de colesterol nos animais; na 2ªfase, todos os animais passaram a receber dieta suplementada com gema de ovo, até o final do experimento, para desenvolver hipercolesterolemia, e; na 3ª fase os animais do grupo G2 receberam o tratamento com E. A. de alho. O colesterol na 1ª fase foi de 39,94 ± 9,57 mg dL-1. Na 2ª fase houve elevação significativa (p<0,05) no nível de colesterol plasmático nos dois grupos -acima de 100 mg dL-1. Com relação ao tratamento, o alho não promoveu redução no colesterol plasmático dos coelhos, contrapondo os dados da literatura.

The use of plants to treat diseases or even to cure them is a high diffused popular tradition, and several studies discuss the therapeutic and pharmacological properties of garlic in the reduction of hyperlipidaemias. The objective of the present study was to investigate the influence of the aqueous extract of garlic (Allium sativum) to treat the serum cholesterol of rabbits with experimental hypercholesterolemia. The animals were divided into G1 (control group) and G2 (group treated with garlic). The experiment was developed according to 3 phases: during the 1st phase, all animals were provided with a regular diet to evaluate the basal cholesterol; during the 2nd phase, all animals received a supplemented diet until the end of the experiment, in order to develop hypercholesterolemia and, in the 3rd phase, the animals on G2 received the garlic treatment. The cholesterol registered on the 1st phase was 39.94 ± 9.57 mg dL-1. On the 2nd phase, there was an increase on the serum cholesterol level in both groups - higher than 100 mg dL-1. Concerning the treatment, the garlic did not reduce the serum cholesterol in rabbits.

Sirolimus en trasplante de órgano sólido pediátrico: Experiencia en 5 casos / Use of Sirolimus in five pediatric patients undergoing solid organ transplantation

Sirolimus (SRL) is an immunosuppressive drug increasingly used in children undergoing solid organ transplantation. SRL does not cause glucose intolerance, hypertension, nephrotoxicity or neurotoxicity offering significant potential advantages over calceneurin inhibitors (CM). Aim: To report five children treated with SRL. Material and methods: A retrospective review of four children undergoing orthotopic liver transplantation (OLT) and one undergoing renal transplantation with recurrent acute rejection (RAR), chronic rejection (CR) or toxicity due to CM, treated with SRL between June 2001 and November 2006. Results: As primary immunosuppressive therapy, all patients received 3 drugs: CM (Tacrolimus (FK) or Cyclosporine), mycophenolate mofetil and steroids. Mean age at treatment with SRL was 98 months. Children undergoing OLT had a ¡ate introduction of SRL (mean time after OLT: 37 months), and mean follow-up was 24 months. In this group rescue indications of SRL were RAR in one, CR in one, thrombotic thrombocytopenic purpura (TTP) in one, food allergy in one and other CM toxicity in three. Only one did not experience adverse events due to SRL, but no one required discontinuation of SRL. There were remissions of RAR, CR, TTP and food allergy. The patient with RT was switched from FK to SRL at day 18th after RT, but he had severe neutropenia that led to discontinuation of SRL. Conclusions: SRL may be useful in pediatric solid organ transplant recipients suffering from RAR, CR, TTP, food allergy and CM toxicity. Careful attention should be directed to detect side effects and avoid severe complications.

Achados laboratoriais associados à acitretina e ao metotrexato no tratamento da psoríase: uma coorte histórica / Laboratorial findings of acitretine and methotrexate on the psoriasis treatment: an historic cohort study

Objetivos: Quantificar e avaliar os efeitos adversos causados pelo uso de acitretina e metotrexato o tratamento da psoríase. Material e métodos: Foi realizada uma coorte histórica para avaliação de intervenção em pacientes com uso de metotrexato e acitretina para tratamento de psoríase. Resultados: Foram revisados os prontuários de 101 pacientes, com um total de 127 tratamentos, tendo sido avaliadas as variáveis sexo, idade, tipo clínico de psoríase, medicação utilizada, dose média, tempo de uso, hepatotoxicidade, mielotoxicidade, nefrotoxicidade, alterações nos lipídeos e comorbidades. A incidência de hepatotoxicidade no grupo todo foi de 8,7% (11/127), sendo de 9% (4/44) no grupo do acitretina e 10,5% (6/58) no grupo do metotrexato. Mielotoxicidade teve incidência de 6% (8/127) em todo o grupo, 2% (1/44) no grupo do acitretina e 10,5% (6/58) no do metotrexato. Hiperlipidemia apresentou incidência de 34% (15/44) nos pacientes em uso de acitretina e 7% (4/58) nos que utilizaram metotrexato, sendo este o efeito adverso mais importante da acitretina (p=0,002). Alterações renais não foram encontradas. Conclusão: A principal diferença encontrada pelo estudo foi entre hiperlipidemia causada por acitretina em relação ao metotrexato. Na amostra estudada não foi observada diferença estatisticamente significativa entre as drogas quanto à hepatotoxicidade e à mielotoxicidade. Os resultados do estudo permitem inferir que o metotrexato, medicação acessível, de baixo custo e bem conhecida do meio médico, demonstra perfil de toxicidade aceitável quando usado em determinadas doses, podendo ter seu uso preferencial cogitado, especialmente em contexto de assistência no setor público

Objectives: To quantify and evaluate the adverse effects caused by acitretine and methotrexate when treating psoriasis. Methods: A retrospective cohort study was performed to evaluate an intervention in patients using methotrexate and acitretine to treat psoriasis. Results: The records of 101 patients were reviewed, with a total of 127 treatments, and the following variables were evaluated: sex, age, clinical type of psoriasis, medication used, mean dose, time of use, hepatotoxicity, myelotoxicity, nephrotoxicity, alterations in the lipids and comorbidities. The rate of occurrence of hepatotoxicity in the group as a whole was 8.7% (11/127), 9% (4/44) in the acitretine group and 10.5% (6/58) in the methotrexate group. Myelotoxicity had a rate of occurrence of 6% (8/127) in the group as a whole, 2% (1/44) in the acitretine group and 10.5% (6/58) in the methotrexate one. The presence of hyperlipidemia was observed in 34% (15/44) of the patients who used acitretine, and in 7% (4/58) of those who used methotrexate, and this was the most important adverse effect of acitretine (p=0.002). No renal alterations were found. Conclusion: Themain difference found by the study was between hyperlipidemia caused by acitretine compared to methotrexate. In the sample studied no statistically significant difference was observed between the drugs as to hepatotoxicity and myelotoxicity. The results of the study allow the inference that methotrexate, a low-cost medication, well-known in the medical world, shows an acceptable toxicity profile, and its preferential use can be considered, especially in a context of public care

A point mutant of apolipoprotein A-I, V156K, exhibited potent anti-oxidant and anti-atherosclerotic activity in hypercholesterolemic C57BL/6 mice

In our previous study, two point mutants of apolipoprotein A-I, designated V156K and A158E, revealed peculiar characteristics in their lipid-free and lipid-bound states. In order to determine the putative therapeutic potential of these mutants, several in vitro and in vivo evaluations were conducted. In the lipid-free state, V156K showed more profound antioxidant activity against LDL oxidation than did the wildtype (WT) or A158E variants in an in vitro assay. In the lipid-bound state, V156K-rHDL showed an enhanced cholesterol delivery activity to HepG2 cells in a time-dependent manner, as compared to WT-rHDL, A158E-rHDL, and R173C-rHDL. We assessed the physiological activities of the mutants in circulation, using hypercholesterolemic mice (C57BL6/J). Palmitoyloleoyl phosphatidylcholine (POPC)-rHDL preparations containing each of the apoA-I variants were injected into the mice at a dosage of 30 mg of apoA-I/kg of body weight. Forty eight hours after injection, the sera of the V156K-rHDL injected group showed the most potent antioxidant abilities in the ferric acid removal assay. The V156K-rHDL- or R173C-rHDL-injected mice showed no atherosclerotic lesions and manifested striking increases in their serum apo-E levels, as compared to the mice injected with WT-rHDL or A158E-rHDL. In conclusion, V156K-rHDL exhibited the most pronounced antioxidant activity and anti-atherosclerotic activity, both in vitro and in vivo. These results support the notion that HDL-therapy may prove beneficial due to its capacity to induce accelerated cholesterol excretion, as well as its enhanced antioxidant and anti-inflammatory effects and lesion regression effect.

Somatic, Biochemical and Hepatic Alterations in Wild Type Mice Chronically Fed High Fat Diet / Alteraciones Somáticas Bioquímicas y hepáticas en un Tipo de Ratas Alimentadas Crónicamente con Dieta Alta en Grasas

This study evaluated whether a high fat diet (HFC group) induces overweight, hepatic steatosis and plasma lipoproteins level alteration compared to standard chow diet (SC group). Female mice were submitted to each diet over 6 months. Body mass and food intake were evaluated weekly throughout the experiment. Total cholesterol, TG, LDL-c, HDL-c and VLDL-c were analyzed. Mice were sacrificed to remove liver, spleen, heart and intestine. The volume of the organs was determined according to the submersion method. Fixed livers were embedded in paraffin and stained with hematoxylin and eosin and Masson's trichrome. The analysis used a video microscope system and a test-system with 42 test-points. The volume density was estimated for hepatocytes, steatosis and sinusoids. Animals fed HFC had smaller chow intake than SC group. HFC group presented body mass greater than SC. Animals fed HFC showed heavier liver and spleen and lighter intestine than SC (p<0.05), heart mass was not significant between groups. Plasma lipoproteins differed between groups (p<0.05) except VLDL-c and TG fractions. The liver structure was without major alteration in SC group however, HFC mice group showed different degrees of fatty degeneration with micro- and macrovesicular steatosis dispersed in all liver with typical peri-cellular/peri-sinusoidal fibrosis. The quantitative study showed significant (p<0.05) volume density reduction for hepatocytes and sinusoids. In conclusion, our results clearly show that hepatic steatosis can be induced in mouse by such a fat-rich diet without any toxin ingestion, alimentary deficiency and genes depletion.

Este estudio evaluó cómo una dieta de alta densidad energética (grupo ADE) induce sobrepeso, esteatosis hepática y altera los niveles de las lipoproteínas plasmáticas cuando son comparados con la dieta patrón (grupo SC). Hembras de camundongos fueron sometidas a cada una de las dietas durante 6 meses. La masa corporal y la ingestión de alimento fueron evaluadas semanalmente durante el experimento. Además fueron medidos el colesterol total, TG, LDL-c, HDL-c e VLDL-c. Los animales fueron sacrificados y el hígado, bazo, corazón e intestinos fueron removidos para estudio. El volumen de los órganos fue medido por el método de la sumersión. Fragmentos de hígado fueron preparados para el estudio en microscopía de luz, teñidos con hematoxilina-eosina y tricrómico de Masson. El análisis fue realizado con video microscopía y sistema test M42. La densidad de volumen fue estimada para hepatocitos, esteatosis y sinusoides. Los animales alimentados con dieta ADE presentaron menor ingestión de alimento y tuvieron masa corporal mayor que los animales con dieta patrón. Animales ADE mostraron también hígado y bazo más pesados e intestino más liviano que animales SC (p<0.05). Para la masa del corazón no hubo diferencia significativa entre los dos grupos. Las lipoproteínas plasmáticas fueron diferentes entre los grupos (p<0.05) excepto VLDL-c y fracciones de TG. La estructura hepática no presentó grandes alteraciones en el grupo SC; sin embargo, animales del grupo ADE presentaron diferentes grados de degeneración adiposa con esteatosis macro y microvesicular dispersas en todo el hígado con típica fibrosis pericelular y perisinusoidal, y significativa reducción de la densidad de volumen de hepatocitos y sinosoides. En conclusión, los resultados muestran que la esteatosis hepática puede ser inducida experimentalmente en camundongos, a través de dieta ADE, sin ingestión de cualquier toxina, deficiencia alimentaria o depleción genética.

Aterosclerosis experimental: cambios bioquímicos y vasculares / Experimental atherosclerosis: biochemical and vascular changes

Se estudió el desarrollo de placas ateros-clerósicas en conejos Nueva Zelanda alimentados con una dieta rica en colesterol. Para ello se comparó el contenido sérico de lípidos y glucosa en conejos sanos y conejos alimentados con 1 y 10 por ciento de colesterol por 10 semanas. Además, se hicieron estudios histológicos de las aortas de dichos animales para evaluar las lesiones ateromatosas. En los animales que recibían una dieta con 10 por ciento de colesterol, los niveles séricos de éste aumentaron significativamente de 26.3 ñ 8.1 mg/dL a 1485 ñ 26.8 mg/dL (p < 0.05). El colesterol asociado con LDL también se incrementó, de 15.9 ñ 5.9 a 1383.8 ñ 58.9 (p < 0.5); y los triglicéridos de 88.3 ñ 35.6 a 411 ñ 154.5. Se encontraron lesiones ateros-clerósicas solamente en los conejos alimentados con 10 por ciento de colesterol. Este modelo es reproducible y puede ser útil en el estudio de la aterosclerosis per se y de la aterogénesis asociada con enfermedades como la diabetes mellitus