Rheum khorasanicum. Hydroalcoholic root extract induces cell death in human colorectal adenocarcinoma: An in vitro and in silico study.

Colorectal cancer (CRC) is the second greatest cause of cancer-related death in the world and chemotherapy, as an important part of CRC treatment, has some drawbacks, including systemic toxicity. Therefore, it is crucial to discover new and more effective CRC treatment plans. Rheum khorasanicum (R. khorasanicum) is a medicinal plant with high flavonoids, stilbenes, and anthraquinone contents, so it can be a potential source of antioxidants and can be used for therapeutic purposes and trigger apoptosis in cancer cells. In this study, we investigated the effects of hydroalcoholic root extract of R. khorasanicum treatment on inducing mitochondrial apoptosis of HT-29 and Caco-2 human colorectal adenocarcinoma cells. Firstly, the total phenolic and flavonoid content was determined. Then, the cytotoxic effects of R. khorasanicum on cells of three different types, including HT-29 and Caco-2 colon cancer cells as well as normal 3T3 cells were assessed using the MTT assay. To investigate the characteristics of cellular death, flow cytometry, and western blotting were performed. The results of this study indicated considerable phenolic (356.4±9.4 GAE/gDW) and flavonoid (934.55±17.1 QE/gDW) contents in R. khorasanicum. MTT assay's finding indicated that 100, 60, and 30µg/mL concentrations of R. khorasanicum reduce cell viability in HT-29 and Caco-2 cell lines significantly (P<0.05). It has been also revealed that R. khorasanicum extract induces apoptosis rather than necrosis in these cell lines. Moreover, Bcl-2 expression was significantly reduced in both HT-29 and Caco-2 cell lines, while Bax and cleaved caspase-3 expression soared considerably in the groups under R. khorasanicum treatment (P<0.05). In conclusion, our findings have suggested that high phenol and flavonoid contents of R. khorasanicum root extract possibly play an important role in cell cytotoxicity and apoptosis induction in HT-29 and Caco-2 colon cancer cells.

Delta-tocotrienol enhances the anti-tumor effects of interferon alpha through reactive oxygen species and Erk/MAPK signaling pathways in hepatocellular carcinoma cells.

The complexity of hepatocellular carcinoma (HCC) signaling and the failure of pharmacological therapeutics reveal the significance of establishing new anti-cancer strategies. Interferon alpha (IFN-α) has been used as adjuvant therapy for reducing HCC recurrence and improving survival. Delta-tocotrienol (δ-tocotrienol), a natural unsaturated isoform of vitamin E, is a promising candidate for cancer treatment. In this study, we evaluated whether the combination of δ-tocotrienol with IFN-α displays significant advantages in the treatment of HCC cells. Results showed that the combination significantly decreased cell viability, migration and invasion of HCC cells compared with single therapies. Combining δ-tocotrienol and IFN-α enhanced the decrease in proliferating cell nuclear antigen (PCNA) and matrix metalloproteinase (MMP) 7 and MMP-9. The combination also produced an enhancement of apoptosis together with increased Bax/Bcl-xL ratio and reactive oxygen species (ROS) generation. δ-tocotrienol induced Notch1 activation and changes in Erk and p38 MAPK signaling status. Blocking experiments confirmed that ROS and Erk are involved, at least in part, in the anti-cancer effects of the combined treatment. In conclusion, the combination of δ-tocotrienol with IFN-α therapy showed promising results for HCC cell treatment, which makes the combination of cytokine-based immunotherapy with natural products a potential strategy against liver cancer.

Estrategias para la evaluación de extractos de polifenoles en modelos in vitro de cáncer de vías digestivas / Strategies for Evaluation of Polyphenol Extracts on in vitro Models of Digestive Tract Cancer / Estratégias para a avaliação de extratos de polifenóis em modelos in vitro de câncer do sistema digestivo

Introducción: Los polifenoles son compuestos que se encuentran naturalmente en alimentos como frutas, verduras, té, vino y chocolates, a los que se les atribuyen beneficios a la salud humana por su capacidad antioxidante. El cáncer de las vías digestivas se encuentra entre la tercera y quinta causas de muerte para la población, por lo que se ha incrementado el interés por realizar los estudios dirigidos a encontrar compuestos polifenólicos que ayuden en su prevención o tratamiento. Objetivo: Identificar las estrategias disponibles para la evaluación de polifenoles en células de cáncer de vías digestivas. Metodología: Búsqueda de literatura en bases de datos como Ovid, Pubmed, Science Direct y Elsevier Journal. Se seleccionaron artículos en los cuales se reporta el efecto biológico de los polifenoles sobre líneas celulares de cáncer de vías digestivas publicados entre 2012 y 2022. Resultados: Varios estudios han reportado el uso de un buen número de líneas celulares como modelos in vitropara estudios de polifenoles en cáncer y han resaltado las líneas AGS y HT-29, además de técnicas para la caracterización de los polifenoles, como el ensayo 2,2-Difenil-I-Picril Hidrazilo (DPPH). Sin embargo, para evaluar el efecto biológico se identifican diversas pruebas que deben analizarse antes de su implementación. Conclusiones: En la literatura se identifica que existen varias alternativas y estrategias para la evaluación de extrac-tos vegetales en cultivos in vitro de cáncer de vías digestivas; no obstante, antes de pasar al diseño experimental, deben tenerse en cuenta una serie de consideraciones para garantizar la utilidad de los resultados.

Introduction: Polyphenols are compounds naturally found in foods such as fruits, vegetables, tea, wine and chocolates, and it was attributed with benefits to human health due to their antioxidant capacity. Cancer of the digestive tract is between the third and fifth cause of death for the population, increasing the interest in carrying out studies aimed at finding polyphenolic compounds that help in their prevention or treatment. Objective: Identify the available strategies for the evaluation of polyphenols in digestive tract cancer cells. Method: A literature search was performed in databases such Ovid, Pubmed, Science Direct and Elsevier Journal and selected articles reporting the biological effect of polyphenols on digestive tract cancer cell lines, published between 2012 and 2022. Results: Currently studies report the use of a good number of cell lines as in vitro models for poly-phenol studies in cancer highlighting the AGS and HT-29 lines, in addition to techniques for the characterization of polyphenols such as the α, α-diphenyl-ß-picrylhydrazyl DPPH assay, however, to evaluate the biological effect various tests are identified that should be analyzed before implemen-tation. Conclusions: The literature identifies that there are many alternatives and strategies for the evaluation of plant extracts in in vitro cultures of digestive tract cancer, however, before moving on to the experimental design, a number of considerations should be taken into account to ensure the usability of the results

Introdução: Os polifenóis são compostos encontrados naturalmente em alimentos como frutas, legumes, chá, vinho e chocolates, aos quais são atribuídos benefícios para a saúde humana devido à sua capacidade antioxidante. O câncer do sistema digestivo está entre a terceira e a quinta principais causas de morte na população, o que levou a um interesse crescente em estudos destinados a encon-trar compostos polifenólicos que ajudem a prevenir ou tratar esse tipo de câncer. Objetivo: Identificar as estratégias disponíveis para a avaliação dos polifenóis nas células cancerosas do sistema digestivo. Metodologia: Pesquisa bibliográfica em bases de dados como Ovid, Pubmed, Science Direct e Elsevier Journal. Foram selecionados artigos que relatam o efeito biológico dos polifenóis em linhas celulares de câncer do sistema digestivo, publicados entre 2012 e 2022. Resultados: Vários estudos relataram a utilização de várias linhas celulares como modelos in vitro para estudos de polifenóis no câncer destacando as linhas AGS e HT-29, bem como técnicas para a ca-racterização de polifenóis, como o ensaio 2,2-Difenil-I-Picril Hidrazil (DPPH). No entanto, para avaliar o efeito biológico, são identificados vários testes que devem ser analisados antes da sua aplicação. Conclusões: A literatura identifica que existem várias alternativas e estratégias para a avaliação de extratos de plantas em culturas in vitro de câncer do sistema digestivo; no entanto, antes de passar à concepção experimental, é necessário ter em conta uma série de considerações para garantir a uti-lidade dos resultados

The effects of high-intensity interval training and saffron aqueous extract supplementation on alterations of body weight and apoptotic indices in skeletal muscle of 4T1 breast cancer-bearing mice with cachexia.

Exercise training and antioxidant supplementation may improve unintentional weight loss and programmed cell death associated with cancer cachexia. The aim of this study was to examine the alterations of body weight and apoptotic indices in skeletal muscle of 4T1 breast cancer-bearing mice with cachexia following 4 weeks of high-intensity interval training (HIIT) and saffron aqueous extract (SAE) supplementation. Female BALB/c mice following induction of breast cancer were divided into (i) controls, (ii) HIIT, (iii) SAE, (iv) HIIT+SAE, and (v) sham groups. Mice were euthanized and gastrocnemius muscle was collected after intervention. The control group elicited a significant weight reduction during third and fourth weeks of tumor injection, while other treatments such as HIIT and SAE, but not HIIT+SAE, showed that they counteracted this adverse event. Furthermore, HIIT and SAE treatments (not HIIT+SAE) demonstrated reduced caspase-3 and Bax levels compared with the control group. The level of Bcl-2 was elevated following both HIIT and SAE treatments compared with the control group. Finally, the ratio of Bcl-2 to Bax was significantly higher in both HIIT and SAE groups, but was lower in HIIT+SAE group compared with sham group. It is likely that either HIIT or SAE intervention alone (not HIIT+SAE) represents a readily applicable approach in the regulation of muscle wasting and apoptosis in cancer cachexia. Novelty HIIT is associated with a reduced risk of cancer-related muscle wasting. SAE enhances the improvement of muscle loss and apoptotic indices. Combination of HIIT and SAE does not improve cancer-related loss of muscle mass and mediate apoptotic activation.

Salvadora persica protects mouse intestine from eimeriosis / Salvadora persica protege o intestino do rato da eimeriose

Abstract Eimeriosis is a global poultry health problem. In the current study, we investigated the role of Salvadora persica leaf extracts (SE) against murine eimeriosis induced by Eimeria papillata. The infection induced an oocyst output of 6242 ± 731 oocysts/g feces. After treatment with 300 mg⁄kg SE, the oocysts expelled in feces decreased by approximately 3-fold. In addition, the total number of E. papillata in the parasitic stage decreased in the jejunum of mice after treatment with SE. In addition, SE significantly reduced the number of apoptotic cells by approximately 2-fold in the infected jejunum. SE ameliorated the changes in glutathione, malondialdehyde, and catalase due to E. papillata infection. Finally, SE regulated the cytokine genes, interleukin (IL)-1β, IL-6, interferon-γ, and tumor necrosis factor-α, and the apoptotic genes, B-cell lymphoma-2, Bax, and Caspase-3. SE protects the jejunum from E. papillata induced injury and may have potential therapeutic value as a food additive during eimeriosis.

Resumo A eimeriose é um problema global de saúde avícola. No presente estudo, investigou-se o papel dos extratos de folhas de Salvadora persica (SE) contra a eimeriose murina induzida por Eimeria papillata. A infecção induziu uma produção de oocistos de 6242 ± 731 oocistos/g de fezes. Após o tratamento com 300 mg⁄kg SE, os oocistos eliminados nas fezes diminuíram em aproximadamente 3 vezes. Além disso, o número total de E. papillata no estágio parasitário diminuiu nos jejunos de camundongos após o tratamento com SE. Da mesma forma, o SE reduziu significativamente o número de células apoptóticas em aproximadamente 2 vezes no jejuno infectado. O estudo mostrou que o SE melhorou as alterações na glutationa, malonaldeído e catalase devido à infecção por E. papillata. Finalmente, o SE regulou os genes das citocinas, interleucina (IL) -1β, IL-6, interferon-γ e fator de necrose tumoral α, e os genes apoptóticos, linfoma-2, Bax e Caspase-3. Assim, o SE protegeu os jejunos das lesões induzidas por E. papillata e pode ter potencial valor terapêutico como aditivo alimentar durante a eimeriose.

A suplementação com quercetina previne mudanças em células imunorreativas à serotonina e caspase-3 do jejuno de ratos diabéticos / Quercetin supplementation prevents changes in the serotonin and caspase-3 immunoreactive cells of the jejunum of diabetic rats

ABSTRACT BACKGROUND: Serotonin (5-HT) is present in the epithelial enterochromaffin cells (EC), mast cells of the lamina propria and enteric neurons. The 5-HT is involved in regulating motility, secretion, gut sensation, immune system and inflammation. OBJECTIVE: Evaluate the effects of diabetes and quercetin supplementation on serotoninergic cells and its cell loss by apoptosis in jejunal mucosa of streptozotocin-induced diabetic rats (STZ-rats). METHODS: Twenty-four male Wistar rats were divided into four groups: normoglycemic (C), normoglycemic supplemented with 40 mg/day quercetin (Q), diabetic (D) and diabetic supplemented with 40 mg/day quercetin (DQ). After 120 days, the jejunum was collected and fixated in Zamboni's solution for 18 h. After obtaining cryosections, immunohistochemistry was performed to label 5-HT and caspase-3. Quantification of 5-HT and caspase-3 immunoreactive (IR) cells in the lamina propria, villi and crypts were performed. RESULTS: The diabetic condition displayed an increase of the number of 5-HT-IR cells in villi and crypts, while decreased number of these cells was observed in lamina propria in the jejunum of STZ-rats. In the diabetic animals, an increased density of apoptotic cells in epithelial villi and crypts of the jejunum was observed, whereas a decreased number of caspase-3-IR cells was observed in lamina propria. Possibly, quercetin supplementation slightly suppressed the apoptosis phenomena in the epithelial villi and crypts of the STZ-rats, however the opposite effect was observed on the 5-HT-IR cells of the lamina propria. Quercetin supplementation on healthy animals promoted few changes of serotoninergic function and apoptotic stimuli. CONCLUSION: These results suggest that quercetin supplementation mostly improved the serotonergic function affected by diabetes maybe due to antioxidant and anti-inflammatory properties of quercetin.

RESUMO CONTEXTO: A serotonina (5-HT) está presente nas células epiteliais enterocromafins (CE), nos mastócitos da lâmina própria e nos neurônios entéricos. A 5-HT está envolvida na regulação da motilidade, secreção, nocepção intestinal, sistema imunológico e inflamação. Objetivo: Avaliar os efeitos do diabetes e da suplementação de quercetina sobre a função serotoninérgica e a perda celular por apoptose na mucosa jejunal de ratos diabéticos induzidos por estreptozotocina (ratos STZ). MÉTODOS: Vinte e quatro ratos Wistar machos foram divididos em quatro grupos: normoglicêmico (C), normoglicêmico suplementado com quercetina 40 mg/dia (Q), diabético (D) e diabético suplementado com quercetina 40 mg/dia (DQ). Após 120 dias, o jejuno foi coletado e fixado na solução de Zamboni por 18 horas. Após a obtenção de cortes em criostato, a imuno-histoquímica foi realizada para marcar 5-HT e caspase-3. A quantificação de células imunorreativas (IR) à 5-HT e caspase-3 foram realizadas na lâmina própria, vilosidades e criptas. RESULTADOS: A condição diabética ocasionou um aumento do número de células 5-HT-IR nas vilosidades e criptas, enquanto que na lâmina própria houve uma redução dessas células, no jejuno de ratos STZ. Nos animais diabéticos, foi observada uma densidade aumentada de células apoptóticas no epitélio do jejuno, tanto nas vilosidades quanto nas criptas, por outro lado um número reduzido de células caspase-3-IR foi observado na lâmina própria. Possivelmente, a suplementação de quercetina suprimiu ligeiramente os fenômenos de apoptose no epitélio de vilosidades e criptas do jejuno de ratos STZ, no entanto, o efeito oposto foi observado nas células 5-HT-IR da lâmina própria. A suplementação com quercetina em animais saudáveis promoveu poucas alterações na função serotoninérgica e nos estímulos apoptóticos. CONCLUSÃO: Estes resultados sugerem que a suplementação de quercetina melhorou principalmente a função serotoninérgica afetada pelo diabetes, talvez devido às propriedades antioxidantes e anti-inflamatórias da quercetina.

Blueberry extract attenuates doxorubicin-induced damage in H9c2 cardiac cells 1.

The objective of this study was to analyze the cardioprotective roles of 3 wild blueberry genotypes and one commercial blueberry genotype by measuring markers of oxidative stress and cell death in H9c2 cardiac cells exposed to doxorubicin. Ripe berries of the 3 wild blueberry genotypes were collected from a 10-year-old clearcut forest near Nipigon, Ontario, Canada (49°1'39″N, 87°52'21″W), whereas the commercial blueberries were purchased from a local grocery store. H9c2 cardiac cells were incubated with 15 µg gallic acid equivalent/mL blueberry extract for 4 h followed by 5 µM doxorubicin for 4 h, and oxidative stress and active caspase 3/7 were analyzed. The surface area as well as total phenolic content was significantly higher in all 3 wild blueberry genotypes compared with the commercial species. Increase in oxidative stress due to doxorubicin exposure was attenuated by pre-treatment with all 3 types of wild blueberries but not by commercial berries. Furthermore, increase in caspase 3/7 activity was also attenuated by all 3 wild genotypes as well. These data demonstrate that wild blueberry extracts can attenuate doxorubicin-induced damage to H9c2 cardiomyocytes through reduction in oxidative stress and apoptosis, whereas the commercial blueberry had little effect.

Antiproliferative and pro-apoptotic activities of wild asparagus (Asparagus acutifolius L.), black bryony (Tamus communis L.) and butcher's broom (Ruscus aculeatus L.) aqueous extracts against T24 and A549 cancer cell lines.

The aim of this study was to determine the phenolic profile, antiproliferative, and pro-apoptotic activities of Asparagus acutifolius, Tamus communis, and Ruscus aculeatus aqueous extracts against human bladder (T24) and lung cancer (A549) cell lines. Antiproliferative activity of the extracts at the concentration range from 0.5 to 4 mg/ml was measured by MTT test, after the incubation period of 4, 24, 48, or 72 hr. Pro-apoptotic activity of selected plant extracts against T24 and A549 cells was analyzed by flow cytometry at the concentrations of 0.5, 1, and 2 mg/ml. The highest antiproliferative activity against both cancer cell lines was detected by T. communis aqueous extract. The highest pro-apoptotic effect was achieved by A. acutifolius aqueous extract and its effect was similar against A549 and T24 cells. PRACTICAL APPLICATIONS: Wild edible plants, such as wild asparagus, black bryony, and butcher's broom represent important aspect of the Mediterranean diet. They are rich source of various natural bioactive compounds which express different biological activities (antioxidative, antiproliferative, antibacterial, anti-inflammatory…). The current trend for natural and healthy diet, such as the Mediterranean diet, is in relation to the rapidly growing problem of chronic diseases in a modern society. Wild edible plants have a high direct-use value using them in dishes containing mixed vegetables, in soup preparation, as natural additives in fermented natural products containing cheese or fish, in water infusions (natural drinks).

Acetoacetate extract of pleione bulbocodioides (franch. ) rolfe induces apoptosis of human leukemia thp-1cells through a mitochondria-regulated intrinsic apoptotic pathway / Extrato de acetoacetato de Pleione bulbocodioides (Franch. ) Rolfe induz a apoptose de células THP-1 de leucemia humana através de uma via apoptótica intrínseca regulada por mitocôndrias

The tubers of three orchidaceous plants, includingPleione bulbocodioides (Franch.) Rolfe, have been used as 'Shan-Ci-Gu' in traditional Chinese medicine for the treatment of bacterial infections and cancers for thousands of years. In this study, the effects of an acetoacetate (EtOAc) extract of P. bulbocodioides on the cell viability and apoptosis of THP-1 (human acute monocytic leukemia cell line) cells and its interaction with possible apoptotic pathways were investigated. THP-1 cells were treated with the EtOAc extract of P. bulbocodioides at different concentrations. The results showed that THP-1 cell viability was significantly inhibited by the EtOAc extract ofP. bulbocodioides with an IC50 of 51.37±2.68 µ g/ mL at 24 h. The examination of cytotoxic effects on healthy cells showed that the EtOAc extract of P. bulbocodioidesdid not show any effect on healthy Vero cells. Selectivity indexes were greater than 15.57, suggesting that the EtOAc extract of P. bulbocodioides had selective toxicity against THP-1 cells. The results of annexin V-FITC/PI and DAPI staining showed that the EtOAc extract of P. bulbocodioides induced cell apoptosis in a dose-dependent manner. The apoptotic rate was increased in the treatment groups compared with that in the control group (P<0.05). The distribution of cells in the G2 phase of the cell cycle increased along with typical cell apoptosis-induced morphological changes. The levels of the pro-apoptotic proteins Bax, cleaved PARP and cleaved caspase-3 increased with increasing concentration of acetoacetate extract of P. bulbocodioides, while the anti-apoptosis protein Bcl-2 was downregulated. Cyt c and AIF, which are characteristic proteins of the mitochondria-regulated intrinsic apoptosis pathway, also increased in the cytosol with increasing concentrations of the EtOAc extract of P. bulbocodioides. These results showed that the EtOAc extract of P. bulbocodioidessignificantly inhibits cell viability and induces cell apoptosis in the human leukemia cell line THP-1 through a mitochondria-regulated intrinsic apoptotic pathway

Os tubérculos de três plantas orquidáceas, incluindo Pleione bulbocodioides (Franch.) Rolfe, têm sido usados como "Shan-Ci-Gu" na medicina tradicional chinesa para o tratamento de infecções bacterianas e cânceres por milhares de anos. Neste estudo, os efeitos de um extrato de acetoacetato (EtOAc) de P. bulbocodioides na viabilidade celular e apoptose de células THP-1 (linhagem celular de leucemia monocítica aguda humana) e sua interação com possíveis vias apoptóticas foram investigados. As células THP-1 foram tratadas com o extrato EtOAc de P. bulbocodioides em diferentes concentrações. Os resultados mostraram que a viabilidade das células THP-1 foi significativamente inibida pelo extrato EtOAc de P. bulbocodioides com IC50 de 51,37 ± 2,68 µ g/mL às 24 h. O exame dos efeitos citotóxicos em células saudáveis mostrou que oextrato de EtOAc de P. bulbocodioides não mostrou nenhum efeito sobre células Vero saudáveis. Os índices de seletividade foram maiores que 15,57, sugerindo que o extrato de EtOAc de P. bulbocodioides teve toxicidade seletiva contra as células THP-1. Os resultados da coloração da anexina V-FITC/PI e DAPI mostraram que o extrato de EtOAc de P. bulbocodioides induziu a apoptose celular de maneira dose-dependente. A taxa de apoptose foi aumentada nos grupos de tratamento em comparação com o grupo controle (P <0,05). A distribuição de células na fase G2 do ciclo celular aumentou juntamente com alterações morfológicas típicas induzidas pela apoptose celular. Os níveis das proteínas pró-apoptóticas Bax, PARP clivada e caspase-3 clivada aumentaram com o aumento da concentração do extrato acetoacetato de P. bulbocodioides, enquanto a proteína anti-apoptose Bcl-2 foi regulada negativamente. Cyt c e AIF, que são proteínas características da via de apoptose intrínseca regulada por mitocôndrias, também aumentaram no citosol com concentrações crescentes do extrato de EtOAc de P. bulbocodioides. Estes resultados mostraram que o extrato de EtOAc de P. bulbocodioides inibe significativamente a viabilidade celular e induz a apoptose na linha celular de leucemia humana THP-1 através de uma via apoptótica intrínseca regulada por mitocôndrias.

Vitamin D3 supplementation ameliorates ovariectomy-induced cardiac apoptotic and structural changes in adult albino rats.

The effect of vitamin D on cardiac dysfunction after menopause is still under investigation. Therefore, we investigated the effect of vitamin D3 on cardiac apoptotic and structural changes in ovariectomized rats. Forty adult female albino rats were divided into 4 equal groups: sham rats, sham rats treated with vitamin D3, ovariectomized rats, and ovariectomized rats treated with vitamin D3 (500 IU/kg per day for 6 weeks, orally). Body mass, blood pressure, heart rate, and whole heart mass (WHM) were measured. Serum soluble receptors of advanced glycation end products (sRAGE), C-reactive protein, malondialdehyde, and total antioxidant capacity were estimated. Cardiac sections were stained with haematoxylin-eosin and Masson's trichrome stain. Fas and FasL apoptosis-related proteins were detected by immunohistochemistry. Vitamin D3 treatment significantly decreased ovariectomy-induced cardiac Fas and FasL apoptosis-related proteins, whole heart mass, body mass, C-reactive protein, and malondialdehyde accompanied by decreased inflammation and reduced collagen deposition between cardiac muscle fibres. However, vitamin D3 significantly increased total antioxidant capacity and sRAGE in ovariectomized and sham treated groups. Our findings suggest that vitamin D3 treatment can prevent ovariectomy-induced cardiac structural and apoptotic changes in rats via increasing sRAGE and antioxidant activity. Our results suggest that vitamin D3 has therapeutic effect against postmenopausal cardiovascular disease.

DNA damage and primordial follicle activation after in vitro culture of sheep ovarian cortex in Morus nigra leaf extract / Dano ao DNA e ativação do folículo primordial após a cultivo in vitro do córtex ovariano de ovelha em extrato de folha de Morus nigra

This study evaluated the effect of Morus nigra leaf extract, with or without supplementation, on morphology, activation and DNA damage of preantral follicles cultured within sheep ovarian tissue. Ovaries were collected and divided into fragments, being one fixed for histological and Terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) analysis (fresh control). The remaining fragments were cultured for 7 days in alpha minimum essential media (α-MEM) supplemented with bovine serum albumin (BSA), insulin, transferrin, selenium, glutamine, hypoxanthine and ascorbic acid (α-MEM+; control medium) or into medium composed of M. nigra extract without supplements (0.1; 0.2 or 0.4mg/mL) or supplemented with the same substances described above for α-MEM+ (MN 0.1+; 0.2+ or 0.4+mg/mL). Then, tissues were destined to histological and TUNEL analysis. The α-MEM+ treatment had more morphologically normal follicles than all M. nigra extract treatments. However, α-MEM+ treatment also showed signs of atresia because the percentage of TUNEL positive cells was similar in α-MEM+ and in 0.1mg/mL M. nigra without and with supplements. Moreover, a reduction in the primordial follicles and an increase in the growing ones were observed in all treatments, except 0.2mg/mL M. nigra. In conclusion, the follicles cultured at 0.1mg/mL M. nigra extract were in good condition and able to continue their development, as demonstrated by the same rates of DNA damage and follicular activation as the control medium.(AU)

Este estudo avaliou o efeito do extrato das folhas de Morus nigra, com ou sem suplementos, sobre a morfologia, a ativação e o dano ao DNA de folículos pré-antrais cultivados inclusos em tecido ovariano. Os ovários foram coletados e divididos em fragmentos, sendo um fixado para análise histológica e ensaio de marcação de terminações dUTP mediada por desoxinucleotidil transferase terminal (TUNEL) (controle fresco). Os fragmentos restantes foram cultivados durante 7 dias em meio essencial mínimo alfa (α-MEM) suplementado com albumina sérica bovina (BSA), insulina, transferrina, selênio, glutamina, hipoxantina e ácido ascorbico (α-MEM+; meio controle) ou em meio composto de extrato de M. nigra sem suplementos (0,1; 0,2 or 0,4mg/mL) ou suplementado com as mesmas substâncias descritas para α-MEM+ (MN 0,1+; 0,2+ or 0,4+mg/mL). Então, os tecidos foram destinados à análise histológica e TUNEL. O tratamento do α-MEM+ apresentou mais folículos morfologicamente normais que todos os tratamentos do extrato de M. nigra. No entanto, o tratamento com α-MEM+ também mostrou sinais de atresia, pois a porcentagem de células TUNEL positivas foi semelhante em α-MEM+ e em 0,1mg/mL M. nigra sem e com suplementos. Além disso, observou-se uma redução nos folículos primordiais e um aumento nos folículos em crescimento em todos os tratamentos, exceto 0,2mg/mL M. nigra. Em conclusão, os folículos cultivados com 0,1mg/mL de extrato de M. nigra estavam em boas condições e aptos a continuar seu desenvolvimento, como demonstrado pelas taxas de dano ao DNA e de ativação folicular semelhantes ao meio controle.(AU)

Direct inhibitory effect of flaxseed on porcine ovarian granulosa cell functions.

Flaxseed is useful as a functional food and alternative medicine owing to its beneficial health effects. Its action on ovarian cell functions and interrelationships with the upstream hormonal regulators remain unknown. Our aim was to examine the direct influence of flaxseed extract on basal porcine ovarian functions (proliferation, apoptosis), leptin release, and response to insulin-like growth factor I (IGF-I). First, we examined the effect of flaxseed extract on the accumulation of proliferation (PCNA) and apoptosis (Bax) markers and on leptin release in cultured porcine ovarian granulosa cells. Next, granulosa cells were cultured with IGF-I with and without flaxseed extract and analyzed for PCNA and Bax accumulation by quantitative immunocytochemistry and for leptin release by radioimmunoassay. Flaxseed decreased the accumulation of PCNA and increased that of Bax at all doses and reduced leptin output at 100 µg/mL. In contrast, IGF-I promoted PCNA accumulation and suppressed Bax. Flaxseed did not modify IGF-I action on these parameters. Thus, we showed that flaxseed influences porcine reproductive processes, having a direct effect on the ovary and the ability to affect ovarian cell proliferation, apoptosis, and leptin release. Furthermore, we confirmed the pro-proliferative and antiapoptotic actions of IGF-I but showed that flaxseed action on ovarian cell proliferation and apoptosis is not due to changes in the cell response to IGF-I. The potential direct anti-reproductive action of flaxseed needs to be considered during its application in nutrition, medicine, and animal production.

Does posttreatment thymoquinone reverse high-dose atorvastatin-induced hepatic oxidative injury in rats?

Atorvastatin (ATO) was commonly used to lower blood cholesterol, but it caused harmful effects to organs, including the liver. Thymoquinone (TQ), a prominent constituent of Nigella sativa, has antioxidant, antiinflammatory, antiapoptotic, antimicrobial, and anticancer activity. The current study investigated the mechanism of ATO-induced hepatotoxicity, whether posttreatment TQ could reverse ATO-induced hepatic injury, and the mechanism of action of TQ as a hepatoprotective agent. Forty adult male Sprague Dawley rats were divided into four equal groups: control, TQ-treated, ATO-treated, and combined ATO/TQ-treated. Rats were treated for 8 weeks and 10 days and euthanized by cervical dislocation 3 days after the last treatment. Blood samples and livers were tested for liver enzymes, oxidative stress, and apoptosis markers and used for histopathological and ultrastructural examination. The ATO-treated group showed an increase in liver enzymes, decreases in reduced glutathione and catalase, and increases in the malondialdehyde lipid peroxidation marker, protein carbonylation, and caspase 3 activity. Posttreatment TQ in the ATO/TQ-treated group seemed to reverse these changes. Histopathological and ultrastructural examination supported these data. Results from the current study suggested that posttreatment TQ may reverse oxidative stress injury in rat liver produced by ATO, suggesting a potential clinical application of using TQ to prevent ATO-induced hepatic injury.

Anti-Inflammatory, Antiapoptotic and Proproliferative Effects of Vitis vinifera Seed Ethanolic Extract in the Liver of Streptozotocin-Nicotinamide-Induced Type 2 Diabetes in Male Rats.

OBJECTIVES: Consumption of Vitis vinifera seed has been reported to ameliorate liver pathology in diabetes mellitus; however, the mechanisms underlying its effects remain unknown. In this study, the anti-inflammatory, anti-apoptotic and pro-proliferative effects of the ethanolic seed extract of V. vinifera (VVSEE) in the liver in cases of diabetes were identified. METHODS: Adult male rats with streptozotocin-nicotinamide-induced diabetes were given 50, 100 or 200 mg/kg body weight VVSEE orally for 28 days. At the end of the treatment, body weights were determined, and the blood was collected for analyses of fasting blood glucose, insulin and liver enzyme levels. Following sacrifice, livers were harvested and their wet weights and glycogen contents were measured. Histologic appearances of the livers were observed under light microscopy, and the expression and distribution of inflammatory, apoptosis and proliferative markers in the livers were identified by molecular biologic techniques. RESULTS: Treatment of rats with diabetes by VVSEE attenuates decreased body weight, liver weight and liver glycogen content. Additionally, increases in fasting blood glucose levels and liver enzyme levels and decreases in serum insulin levels were ameliorated. Lesser histopathologic changes were also observed: decreased inflammation and apoptosis, as indicated by decreased levels of inflammatory markers (TNF-α, NF-Kß, IKK-ß, IL-6, IL-1ß) and apoptosis markers (caspase-3, caspase-9 and Bax). VVSEE treatment induces increase in hepatocyte regeneration, as indicated by increased PCNA and Ki-67 distribution in the livers of rats with diabetes. Several molecules identified in VVSEE via gas chromatography mass spectrometry might contribute to these effects. CONCLUSIONS: The anti-inflammatory, anti-apoptotic and pro-proliferative effects of VVSEE could account for its hepatoprotective actions in diabetes.

Danshen attenuates osteoarthritis-related cartilage degeneration through inhibition of NF-κB signaling pathway in vivo and in vitro.

Danshen (Salvia miltiorrhiza) is a traditional Chinese medicine herb that can alleviate the symptoms of osteoarthritis (OA) (Söder et al. 2006) in animals. However, the underlying mechanisms remain poorly understood and require further investigation. In this study, rabbits with experimentally induced OA were given an intra-articular injection of danshen (0.7 mL/day) for 5 weeks. In addition to attenuating the cartilage degeneration of OA in the rabbits, danshen decreased the expression and activity of matrix metalloproteinase 9 (MMP-9) and MMP-13, and increased the expression of their natural inhibitors: tissue inhibitor of matrix metalloproteinase 1 (TIMP-1) and TIMP-2. Apoptosis in osteoarthritic cartilage tissues was attenuated by danshen, accompanied with increased expression of B cell lymphoma 2 (Bcl-2) and decreased levels of Bcl-2-associated X protein (Bax). Further, danshen inhibited the nuclear accumulation of nuclear factor kappa-B (NF-κB) p65 in osteoarthritic cartilage. The therapeutic effects of danshen in vivo were comparable to that of sodium hyaluronate, which is a drug used clinically for the treatment OA. In vitro, sodium nitroprusside (SNP) was used to stimulate apoptosis in primary rabbit chondrocytes. We found that the SNP-induced apoptosis was mitigated by danshen. BAY11-7028, an inhibitor of the NF-κB pathway, augmented danshen's anti-apoptotic effects in cells exposed to SNP. When these results are considered together, they indicate that danshen alleviates the cartilage injury in rabbit OA through inhibition of the NF-κB signaling pathway.

Apoptosis of rat hepatic stellate cells induced by diallyl trisulfide and proteomics profiling in vitro.

Diallyl trisulfide (DATS), a major garlic derivative, inhibits cell proliferation and triggers apoptosis in a variety of cancer cell lines. However, the effects of DATS on hepatic stellate cells (HSCs) remain unknown. The aim of this study was to analyze the effects of DATS on cell proliferation and apoptosis, as well as the protein expression profile in rat HSCs. Rat HSCs were treated with or without 12 and 24 µg/mL DATS for various time intervals. Cell proliferation and apoptosis were determined using tetrazolium dye (MTT) colorimetric assay, bromodeoxyuridine (5-bromo-2'-deoxyuridine; BrdU) assay, Hoechst 33342 staining, electroscopy, and flow cytometry. Protein expression patterns in HSCs were systematically studied using 2-dimensional electrophoresis and mass spectrometry. DATS inhibited cell proliferation and induced apoptosis of HSCs in a time-dependent manner. We observed clear morphological changes in apoptotic HSCs and dramatically increased annexin V-positive - propidium iodide negative apoptosis compared with the untreated control group. Twenty-one significant differentially expressed proteins, including 9 downregulated proteins and 12 upregulated proteins, were identified after DATS administration, and most of them were involved in apoptosis. Our results suggest that DATS is an inducer of apoptosis in HSCs, and several key proteins may be involved in the molecular mechanism of apoptosis induced by DATS.

Excessive training is associated with endoplasmic reticulum stress but not apoptosis in the hypothalamus of mice.

Downhill running-based overtraining model increases the hypothalamic levels of IL-1ß, TNF-α, SOCS3, and pSAPK-JNK. The aim of the present study was to verify the effects of 3 overtraining protocols on the levels of BiP, pIRE-1 (Ser724), pPERK (Thr981), pelF2α (Ser52), ATF-6, GRP-94, caspase 4, caspase 12, pAKT (Ser473), pmTOR (Ser2448), and pAMPK (Thr172) proteins in the mouse hypothalamus. The mice were randomized into the control, overtrained by downhill running (OTR/down), overtrained by uphill running (OTR/up), and overtrained by running without inclination (OTR) groups. After the overtraining protocols (i.e., at the end of week 8), hypothalamus was removed and used for immunoblotting. The OTR/down group exhibited increased levels of all of the analyzed endoplasmic reticulum stress markers in the hypothalamus at the end of week 8. The OTR/up and OTR groups exhibited increased levels of BiP, pIRE-1 (Ser724), and pPERK (Thr981) in the hypothalamus at the end of week 8. There were no significant differences in the levels of caspase 4, caspase 12, pAKT (Ser473), pmTOR (Ser2448), and pAMPK (Thr172) between the experimental groups at the end of week 8. In conclusion, the 3 overtraining protocols increased the endoplasmic reticulum stress at the end of week 8.

The cardioprotection of dihydrotanshinone I against myocardial ischemia-reperfusion injury via inhibition of arachidonic acid ω-hydroxylase.

Arachidonic acid (AA) is a precursor that is metabolized by several enzymes to many biological eicosanoids. Accumulating data indicate that the ω-hydroxylation metabolite of AA, 20-hydroxyeicosatetraenoic acid (20-HETE), is considered to be involved in the myocardial ischemia-reperfusion injury (MIRI). The inhibitors of AA ω-hydroxylase, however, are demonstrated to exhibit protective effects on MIRI. Dihydrotanshinone I (DI), a bioactive constituent of danshen, is proven to be a potent inhibitor of AA ω-hydroxylase by our preliminary study in vitro. The purpose of the present study was to investigate the cardioprotection of DI against MIRI and its effects on the concentrations of 20-HETE in vivo. Rats subjected to 30 min of ischemia followed by 24 h of reperfusion were assigned to intravenously receive vehicle (sham and ischemia-reperfusion), low (1 mg/kg), middle (2 mg/kg), or high (4 mg/kg) doses of DI before reperfusion. The results demonstrated that DI treatment could improve cardiac function, reduce infarct size, ameliorate the variations in myocardial zymogram and histopathological disorders, decrease 20-HETE generation, and regulate apoptosis-related protein in myocardial ischemia-reperfusion rats. These findings suggested DI could exert considerable cardioprotective action on MIRI by the attenuation of 20-HETE generation, subsequent myocardial injury, and apoptosis through inhibition on AA ω-hydroxylase.

Pinocembrin attenuates gentamicin-induced nephrotoxicity in rats.

Oxidative stress mediated apoptosis of renal tubular cells is a major pathology of gentamicin-induced nephrotoxicity, which is one of the prevailing causes of acute renal failure. Pinocembrin is a major flavonoid found in rhizomes of fingerroot (Boesenbergia pandurata). It has pharmacological and biological activities including antimicrobial, anti-inflammatory, and antioxidant effects. Preclinical studies have suggested that pinocembrin protects rat brain and heart against oxidation and apoptosis induced by ischemia-reperfusion. The aim of the current study was to investigate the mechanisms of renoprotection elicited by pinocembrin in gentamicin-induced nephrotoxicity. Nephrotoxicity was induced in rats by intraperitoneal injection (i.p.) of gentamicin, and pinocembrin was administered via i.p. 30 min before gentamicin treatment for 10 days. Gentamicin-induced nephrotoxicity was indicated by the reduced renal function and renal Oat3 function and expression. Gentamicin treatment also stimulated Nrf2, HO-1, and NQO1, as well as the pro-apoptotic proteins Bax and caspase-3, concomitant with the attenuation of Bcl-XL expression in the renal cortical tissues. Pinocembrin pretreatment improved renal function and renal Oat3 function and reduced oxidative stress and apoptotic conditions. These findings indicate that pinocembrin has a protective effect against gentamicin-induced nephrotoxicity, which may be due in part to its antioxidant and anti-apoptotic effects, subsequently leading to improved renal function.

Modified citrus pectin stops progression of liver fibrosis by inhibiting galectin-3 and inducing apoptosis of stellate cells.

Modified citrus pectin (MCP) is a pH modified form of the dietary soluble citrus peel fiber known as pectin. The current study aims at testing its effect on liver fibrosis progression. Rats were injected with CCl4 (1 mL/kg, 40% v/v, i.p., twice a week for 8 weeks). Concurrently, MCP (400 or 1200 mg/kg) was administered daily in drinking water from the first week in groups I and II (prophylactic model) and in the beginning of week 5 in groups III and IV (therapeutic model). Liver function biomarkers (ATL, AST, and ALP), fibrosis markers (laminin and hyaluronic acid), and antioxidant biomarkers (reduced glutathione (GSH) and superoxide dismutase (SOD)) were measured. Stained liver sections were scored for fibrosis and necroinflammation. Additionally, expression of galectin-3 (Gal-3), α-smooth muscle actin (SMA), tissue inhibitor metalloproteinase (TIMP)-1, collagen (Col)1A1, caspase (Cas)-3, and apoptosis related factor (FAS) were assigned. Modified pectin late administration significantly (p < 0.05) decreased malondialdehyde (MDA), TIMP-1, Col1A1, α-SMA, and Gal-3 levels and increased levels of FAS, Cas-3, GSH, and SOD. It also decreased percentage of fibrosis and necroinflammation significantly (p < 0.05). It can be concluded that MCP can attenuate liver fibrosis through an antioxidant effect, inhibition of Gal-3 mediated hepatic stellate cells activation, and induction of apoptosis.