Gynura segetum induces hepatic sinusoidal obstruction syndrome in mice by impairing autophagy

ABSTRACT Purpose: To investigate the underlying mechanism of hepatic sinusoidal obstruction syndrome (HSOS) induced by Gynura segetum by measuring autophagy in mouse models. Methods: The model group was administered G. segetum (30 g/kg/d) by gavage, while the normal control group was administered an equal volume of saline daily for five weeks. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), hepatic histopathological examinations, and Masson staining were performed to evaluate liver injury. Liver intercellular adhesion molecule-1 (ICAM-1) and P-selectin were evaluated by immunohistochemistry. Hepatocellular apoptosis was assessed using the terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) assay. Protein expression levels of autophagy markers were measured using Western blot analysis. Results: Gynura segetum was found to significantly induce liver injury compared with control mice, as evidenced by the increase of serum transaminases, a decrease in triglyceride levels, and histopathological changes in mice. Gynura segetum remarkably induced hepatocellular apoptosis and upregulated the expressions of ICAM-1 and P-selectin and also downregulated the protein expression levels of LC3, Atg12 and cytoplasmic polyadenylation element binding protein. Conclusions: Our results suggested that G. segetum induced liver injury with HSOS, and it was partly due to its ability to impair the autophagy pathway.

Hydrogen-rich saline alleviates early brain injury through regulating of ER stress and autophagy after experimental subarachnoid hemorrhage

ABSTRACT Purpose: Subarachnoid hemorrhage (SAH) is a common complication of cerebral vascular disease. Hydrogen has been reported to alleviate early brain injury (EBI) through oxidative stress injury, reactive oxygen species (ROS), and autophagy. Autophagy is a programmed cell death mechanism that plays a vital role in neuronal cell death after SAH. However, the precise role of autophagy in hydrogen-mediated neuroprotection following SAH has not been confirmed. Methods: In the present study, the objective was to investigate the neuroprotective effects and potential molecular mechanisms of hydrogen-rich saline in SAH-induced EBI by regulating neural autophagy in the C57BL/6 mice model. Mortality, neurological score, brain water content, ROS, malondialdehyde (MDA), and neuronal death were evaluated. Results: The results show that hydrogen-rich saline treatment markedly increased the survival rate and neurological score, increased neuron survival, downregulated the autophagy protein expression of Beclin-1 and LC3, and endoplasmic reticulum (ER) stress. That indicates that hydrogen-rich saline-mediated inhibition of autophagy and ER stress ameliorate neuronal death after SAH. The neuroprotective capacity of hydrogen-rich saline is partly dependent on the ROS/Nrf2/heme oxygenase-1 (HO-1) signaling pathway. Conclusions: The results of this study demonstrate that hydrogen-rich saline improves neurological outcomes in mice and reduces neuronal death by protecting against neural autophagy and ER stress.

Estudo dos processos de autofagia e apoptose no córtex cerebral de camundongos Swiss Webster altamente agressivos / Study of the autophagy and apoptosis processes in the cerebral cortex of highly aggressive Swiss Webster mice

A violência é um grave problema de saúde pública. O desenvolvimento de modelos experimentais para o estudo do comportamento agressivo torna-se altamente relevante. Através da aplicação do Modelo Espontâneo de Agressividade (MEA), observamos que alguns camundongos machos adultos Swiss Webster apresentaram comportamento altamente agressivo, enquanto em outros grupos os animais demonstraram uma interação social harmônica. O objetivo deste estudo foi dar continuidade à investigação da capacidade de respiração celular através da análise dos processos de autofagia e morte celular por apoptose no córtex frontal cerebral de camundongos. O MEA foi estruturado, basicamente, pelo agrupamento de camundongos jovens e o reagrupamento dos mesmos indivíduos na idade adulta. Este reagrupamento promove uma situação de estresse social onde observamos indivíduos altamente agressivos (Agg), subordinados (Sub) e harmônicos (Har). Nossas análises basearam-se na avaliação dos processos de autofagia e apoptose através dos métodos de imunofluorescência por LC3-II e ensaio TUNEL, respectivamente. Na imunofluorescência por autofagia, os animais reagrupados Har e Agg apresentaram aumento moderado da quantidade de células autofágicas em comparação aos NR. Além disso, foi notório que no córtex dos animais Sub, a autofagia ocorreu de forma significativamente mais intensa que nas outras categorias comportamentais. O estresse também promoveu aumento no percentual de células que sofreram apoptose, sendo que este resultado foi mais evidente nos animais Agg. Nosso estudo demonstrou que há uma possível conexão entre a condição de estresse, o metabolismo energético mitocondrial, a influência nos processos de autofagia e apoptose que em conjunto poderia influenciar no comportamento altamente agressivo.

Violence is a serious public health problem. The development of experimental models for the study of aggressive behavior becomes highly relevant. Through the application of the Spontaneous Aggression Model (MEA), we observed that some Swiss Webster male mice presented a highly aggressive behavior, while in other groups the animals showed a harmonic social interaction. The aim of this study was to continue the inves-tigation of the cellular respiration capacity, autophagy and cell death by apoptosis in the cerebral frontal cortex of mice. MEA was structured, basically, by grouping young mice and regrouping the same individuals in adulthood. This regrouping promotes a social stress situation where we observe highly aggressive (Agg), subordinate (Sub) and harmonic (Har) individuals. Our analyzes were based on the evaluation of mitochondrial energy metabolism, quantification of mitochondrial protein expression, autophagy and apoptosis through LC3-II immunofluorescent methods and TUNEL assay, respectively. In immunofluorescence by autophagy, Har and Agg regrouped animals showed a moderate increase in the number of autophagic cells compared to NR. In addition, it was noted that in the sub-cortex, autophagy occurred significantly more strongly than in other behavioral categories. Stress also promoted an increase in the percentage of cells that underwent apoptosis, and this result was more evident in Agg animals. Our study demonstrated that there is a possible connection between stress condition, mitochondrial energy metabolism, influence on autophagy and apoptosis processes that together could influence the highly aggressive behavior.

Estudo dos processos de autofagia e apoptose no córtex cerebral de camundongos Swiss Webster altamente agressivos / Study of the autophagy and apoptosis processes in the cerebral cortex of highly aggressive Swiss Webster mice

A violência é um grave problema de saúde pública. O desenvolvimento de modelos experimentais para o estudo do comportamento agressivo torna-se altamente relevante. Através da aplicação do Modelo Espontâneo de Agressividade (MEA), observamos que alguns camundongos machos adultos Swiss Webster apresentaram comportamento altamente agressivo, enquanto em outros grupos os animais demonstraram uma interação social harmônica. O objetivo deste estudo foi dar continuidade à investigação da capacidade de respiração celular através da análise dos processos de autofagia e morte celular por apoptose no córtex frontal cerebral de camundongos. O MEA foi estruturado, basicamente, pelo agrupamento de camundongos jovens e o reagrupamento dos mesmos indivíduos na idade adulta. Este reagrupamento promove uma situação de estresse social onde observamos indivíduos altamente agressivos (Agg), subordinados (Sub) e harmônicos (Har). Nossas análises basearam-se na avaliação dos processos de autofagia e apoptose através dos métodos de imunofluorescência por LC3-II e ensaio TUNEL, respectivamente. Na imunofluorescência por autofagia, os animais reagrupados Har e Agg apresentaram aumento moderado da quantidade de células autofágicas em comparação aos NR. Além disso, foi notório que no córtex dos animais Sub, a autofagia ocorreu de forma significativamente mais intensa que nas outras categorias comportamentais. O estresse também promoveu aumento no percentual de células que sofreram apoptose, sendo que este resultado foi mais evidente nos animais Agg. Nosso estudo demonstrou que há uma possível conexão entre a condição de estresse, o metabolismo energético mitocondrial, a influência nos processos de autofagia e apoptose que em conjunto poderia influenciar no comportamento altamente agressivo.(AU)

Violence is a serious public health problem. The development of experimental models for the study of aggressive behavior becomes highly relevant. Through the application of the Spontaneous Aggression Model (MEA), we observed that some Swiss Webster male mice presented a highly aggressive behavior, while in other groups the animals showed a harmonic social interaction. The aim of this study was to continue the inves-tigation of the cellular respiration capacity, autophagy and cell death by apoptosis in the cerebral frontal cortex of mice. MEA was structured, basically, by grouping young mice and regrouping the same individuals in adulthood. This regrouping promotes a social stress situation where we observe highly aggressive (Agg), subordinate (Sub) and harmonic (Har) individuals. Our analyzes were based on the evaluation of mitochondrial energy metabolism, quantification of mitochondrial protein expression, autophagy and apoptosis through LC3-II immunofluorescent methods and TUNEL assay, respectively. In immunofluorescence by autophagy, Har and Agg regrouped animals showed a moderate increase in the number of autophagic cells compared to NR. In addition, it was noted that in the sub-cortex, autophagy occurred significantly more strongly than in other behavioral categories. Stress also promoted an increase in the percentage of cells that underwent apoptosis, and this result was more evident in Agg animals. Our study demonstrated that there is a possible connection between stress condition, mitochondrial energy metabolism, influence on autophagy and apoptosis processes that together could influence the highly aggressive behavior.(AU)

Effect of varying hypoxia reoxygenation times on autophagy of cardiomyocytes

Purpose: To investigate the impact of different hypoxia reoxygenation (HR) times on autophagy of rat cardiomyocytes (H9C2). Methods: Rat cardiomyocytes were randomly divided into normal control group (group A), hypoxia group (group B), 2 h HR group (group C), 12 h HR group (group D), and 24 h HR group (group E). LC3 II/LC3 I was determined via western blotting, and cell viabilities of cardiomyocytes were measured using methyl thiazolyl tetrazolium (MTT) assay. Results: Cell viabilities in HR model groups were significantly lower than those of group A (P<0.05). LC3 II/LC3 I levels in groups B to D were significantly higher than those of group A (P<0.05), and group D showed the highest LC3 II/LC3 I levels. Cell viabilities in groups B to D were significantly lower than those of group A (P<0.05), with group D showing the lowest cell viabilities (P<0.05). Conclusions: Hypoxia can induce autophagy in rat cardiomyocytes, which can be further activated by reoxygenation; most notable after 12 h. Hypoxia-induced cell injury can be aggravated by reoxygenation. The lowest cell viability was observed at 12 h after reoxygenation; however, cell viability can be recovered after 24 h.(AU)

Evaluation of programmed cell death processes on the lens epithelium of older dogs with diabetic and hypermature cataracts / Avaliação dos processos de morte celular programada no epitélio da lente de cães idosos com catarata diabética e hipermadura

It is well known that posterior capsule opacification (PCO), one of the most common late postoperative complications of cataract surgery, is mainly caused by proliferation and differentiation of remaining lens epithelial cells (LECs) on the posterior lens capsule. Many authors suggest that alterations induced by the pathophysiology of cataracts, its metabolism and the use of 0.1% trypan blue (TB) must cause some degree of cellular damage on these cells, wicht would help to prevent and/or reduce the incidence of PCO after cataract surgery in humans. Therefore, the aim of this study was to evaluate the expression of cell death markers on LECs of older dogs with diabetic and hypermature cataracts, after capsulorhexis, both using 0.1% TB. Twenty samples collected from 13 dogs of different breeds, with ages varying from 8 to 12 years-old, with diabetic and hypermature cataracts, which had been subjected to phacoemulsification surgery (Phaco) using 0.1% TB for staining were studied. Animals were classified as dogs with diabetic (DC) and hypermature cataracts (HC), and expression of molecular markers for apoptosis and autophagy (caspase-3 and beclin-1) on LECs were obtained by immunofluorescence technique. The expression of caspase-3 and beclin-1 was observed in every studied sample and did not differ between groups. In conclusion, our findings suggest that apoptosis and autophagy processes occur to LECs in older dogs presenting diabetic and hypermature cataracts after Phaco utilizing 0.1% TB. Our results may be helpful to future studies of PCO in post-phacoemulsification surgery patients.(AU)

A opacificação da cápsula posterior da lente do globo ocular é a complicação mais observada após a remoção da lente. Essa patologia é causada principalmente pela proliferação e diferenciação das células do epitélio anterior da lente em sua cápsula posterior. Muitos autores sugerem que alterações induzidas pelo metabolismo e/ou patofisiologia da catarata e o uso do corante de azul de tripan a 0,1% devam causar algum dano a essas células, o que supostamente ajudaria a prevenir e reduzir a incidência de tal complicação em humanos. Este trabalho avaliou a expressão de marcadores de morte celular no epitélio anterior da lente de cães idosos com catarata diabética e hipermadura, após capsulorrexe realizada com o emprego do azul de tripan a 0,1%. Foram estudadas vinte amostras colhidas de treze cães de diferentes raças, com idades variando de oito a doze anos, que apresentavam catarata diabética ou hipermadura e que foram submetidos à facoemulsificação utilizando corante de azul de tripan a 0,1%. Foram designados dois grupos: com catarata diabética (DC) e com catarata hipermadura (HC). A expressão molecular dos marcadores de morte celular por apoptose a autofagia (caspase-3 e beclina-1) no epitélio anterior da lente foi avaliada pela técnica de imunofluorescência. Observou-se que a expressão de caspase-3 e beclina-1 ocorreu em todas as amostras e não foi diferente entre os grupos. Os achados deste estudo sugerem que o processo de morte celular por apoptose e autofagia ocorre no epitélio anterior da lente de cães idosos com catarata diabética e hipermadura submetidos à facoemulsificação com o corante de azul de tripan a 0,1%. Este resultado pode ser útil para estudos futuros da opacidade da cápsula posterior da lente em cães submetidos à facoemulsificação.(AU)

Evaluation of programmed cell death processes on the lens epithelium of older dogs with diabetic and hypermature cataracts / Avaliação dos processos de morte celular programada no epitélio da lente de cães idosos com catarata diabética e hipermadura

It is well known that posterior capsule opacification (PCO), one of the most common late postoperative complications of cataract surgery, is mainly caused by proliferation and differentiation of remaining lens epithelial cells (LECs) on the posterior lens capsule. Many authors suggest that alterations induced by the pathophysiology of cataracts, its metabolism and the use of 0.1% trypan blue (TB) must cause some degree of cellular damage on these cells, wicht would help to prevent and/or reduce the incidence of PCO after cataract surgery in humans. Therefore, the aim of this study was to evaluate the expression of cell death markers on LECs of older dogs with diabetic and hypermature cataracts, after capsulorhexis, both using 0.1% TB. Twenty samples collected from 13 dogs of different breeds, with ages varying from 8 to 12 years-old, with diabetic and hypermature cataracts, which had been subjected to phacoemulsification surgery (Phaco) using 0.1% TB for staining were studied. Animals were classified as dogs with diabetic (DC) and hypermature cataracts (HC), and expression of molecular markers for apoptosis and autophagy (caspase-3 and beclin-1) on LECs were obtained by immunofluorescence technique. The expression of caspase-3 and beclin-1 was observed in every studied sample and did not differ between groups. In conclusion, our findings suggest that apoptosis and autophagy processes occur to LECs in older dogs presenting diabetic and hypermature cataracts after Phaco utilizing 0.1% TB. Our results may be helpful to future studies of PCO in post-phacoemulsification surgery patients.(AU)

A opacificação da cápsula posterior da lente do globo ocular é a complicação mais observada após a remoção da lente. Essa patologia é causada principalmente pela proliferação e diferenciação das células do epitélio anterior da lente em sua cápsula posterior. Muitos autores sugerem que alterações induzidas pelo metabolismo e/ou patofisiologia da catarata e o uso do corante de azul de tripan a 0,1% devam causar algum dano a essas células, o que supostamente ajudaria a prevenir e reduzir a incidência de tal complicação em humanos. Este trabalho avaliou a expressão de marcadores de morte celular no epitélio anterior da lente de cães idosos com catarata diabética e hipermadura, após capsulorrexe realizada com o emprego do azul de tripan a 0,1%. Foram estudadas vinte amostras colhidas de treze cães de diferentes raças, com idades variando de oito a doze anos, que apresentavam catarata diabética ou hipermadura e que foram submetidos à facoemulsificação utilizando corante de azul de tripan a 0,1%. Foram designados dois grupos: com catarata diabética (DC) e com catarata hipermadura (HC). A expressão molecular dos marcadores de morte celular por apoptose a autofagia (caspase-3 e beclina-1) no epitélio anterior da lente foi avaliada pela técnica de imunofluorescência. Observou-se que a expressão de caspase-3 e beclina-1 ocorreu em todas as amostras e não foi diferente entre os grupos. Os achados deste estudo sugerem que o processo de morte celular por apoptose e autofagia ocorre no epitélio anterior da lente de cães idosos com catarata diabética e hipermadura submetidos à facoemulsificação com o corante de azul de tripan a 0,1%. Este resultado pode ser útil para estudos futuros da opacidade da cápsula posterior da lente em cães submetidos à facoemulsificação.(AU)

Cell death induced by tamoxifen in human blood lymphocytescultivated in vitro / Morte celular induzida pelo tamoxifeno em linfócitos humanos cultivados in vitro

Many chemotherapeutic agents with a potential against solid tumors or leukemiacan cause lymphopenia. Tamoxifen (TAM) is a synthetic non-steroidal anti-estrogen drugemployed in female breast cancer treatment. The present study investigated the capacity of TAMto induce cell death in human lymphocytes cultivated in vitro. Lymphocytes were obtained fromyoung (25-30 years; n = 3) and elderly women (58-77 years; n = 3) and cultivated for 24 or 48h,with or without TAM (20 μM). After the culture, cell viability, immunocytochemical responseand ultrastructure were evaluated. TAM affected lymphocytes in a time- dependent manner, andcells obtained from elderly women were the most sensitive to TAM. Immunocytochemicalanalysis evidenced higher frequency of apoptosis in treated cells, and the ultrastructural studyrevealed autophagic vacuoles, differing from the controls. In summary, the treated lymphocyteswere affected by TAM, leading to cell death by apoptosis and autophagy.(AU)

Muitos agentes quimioterápicos com potencial contra tumores sólidos ou leucemiaspodem causar linfopenia. O Tamoxifeno (TAM) é um agente antiestrógeno não-esteroidalempregado no tratamento de câncer de mama feminino. O presente trabalho investigou acapacidade do TAM em induzir morte celular em linfócitos humanos cultivados in vitro. Oslinfócitos foram obtidos de mulheres jovens (25-30 anos; n = 3) e idosas (58-77 anos; n = 3) ecultivados por 24 ou 48h, com ou sem TAM (20 μM). Após a cultura, foram analisadas aviabilidade celular, a resposta imunocitoquímica e a ultraestrutura. Os resultados indicam que oTamoxifeno induziu morte celular em linfócitos de ambos os grupos, entretanto, as células dasmulheres idosas apresentaram-se mais sensíveis ao tratamento. A análise imunocitoquímicamostrou maior frequência de apoptose nas células tratadas e o estudo ultraestrutural revelouvacúolos autofágicos nos linfócitos expostos ao Tamoxifeno.(AU)