The protective ability and cellular mechanism of Koelreuteria henryi Dummer flower extract against hydrogen peroxide-induced cellular oxidative damage

BACKGROUND: Koelreuteria henryi Dummer is an indigenous plant in Taiwan. The species has been used in traditional folk medicine for the promotion of liver functions and for treating malaria and urethritis. The present study investigated the antioxidant activity of the flower extract of Koelreuteria henryi Dummer. The extraction conditions were optimized by the contents of total phenolic acids and total flavonoids, and antioxidant activity assays. Moreover, an in vitro study for investigating antioxidant activity of K. henryi flower extract was demonstrated by hydrogen peroxide (H2O2)-induced apoptosis. RESULTS: K. henryi flower extracted for 150 min showed high contents of total phenolic acids and total flavonoids. In an in vitro model, L929 cells were pretreated with K. henryi flower extract, and then treated with H2O2 to induce oxidative damage. Results demonstrated that H2O2-induced apoptosis was inhibited by the treatment of 200 µg/ml K. henryi flower extract through the mitochondria-mediated pathway and mitogen-activated protein kinase (MAPK) pathway. The caspase 8/9 activity and expression of p-p38 and pERK were repressed by K. henryi flower extract. In addition, the prevention of H2O2-induced apoptosis by K. henryi flower extract activated the nuclear factor-erythroid 2-related factor (Nrf2) stress response pathway to transcript heme oxygenase 1 (HO-1). Also, K. henryi flower extract prevented H2O2-induced apoptosis through HO-1 production, as evident by the use of HO-1 inhibitor. CONCLUSIONS: The present study demonstrated that K. henryi flower extract could inhibit the H2O2-induced apoptosis in L929 cells through the activation of the Nrf2/HO-1 pathway.

Morphometric analysis and pattern of protein and gene expression of apoptosis in focal cerebral ischemia in rats and the neuroprotetive action of hypothermia and ketoprofen / Análisis morfométrico y modelo de expresión de proteína y genes de apoptosis en isquemia cerebral focal en ratas y la acción neuroprotectora de hipotermia y ketoprofeno

This study aimed to investigate the morphometric and the pattern of protein and gene expression related to the extrinsic apoptotic pathway in experimental focal cerebral ischemia and the hole of neuroprotection with hypothermia and ketoprofen. For this analysis, 120 rats were randomly divided into 3 groups (20 animals each): control - no surgery (20 animals); sham - simulation of surgery (20 animals); ischemic - focal ischemia for 1 hour, without reperfusion (80 animals) and divided into four subgroups with 20 animals each: ischemic + intraischemic hypothermia; ischemic + previous intravenous ketoprofen, and ischemic + hypothermia and ketoprofen. The infarct volume was measured using morphometric analysis of infarct areas defined by triphenyl tetrazolium chloride and the patterns of expression of the apoptosis genes (Fas, c-Flip, caspase-8 and caspase-3) and the apoptosis protein caspase-3 were evaluated by quantitative real-time PCR and immunohistochemistry, respectively. Hypo expression of genes of extrinsic pathway of apoptosis was observed: Fas receptor, c-Flip and caspase-8 in the ischemics areas. Increases in the gene and protein caspase-3 in the ischemic areas were also observed, and these increases were reduced by hypothermia and ketoprofen, also noted in the morphometric study. The caspases-3 increase suggests that this gene plays an important role in apoptosis, probably culminating in cell death and that the neuroprotective effect of hypothermia and ketoprofen is involved.

Este estudio tuvo como objetivo investigar la morfometría y el patrón de expresión de proteínas y genes relacionados con la vía apoptótica extrínseca en la isquemia cerebral focal experimental y el agujero de neuroprotección con hipotermia y ketoprofeno. Se dividieron aleatoriamente 120 ratas en 3 grupos (20 animales cada uno): control - sin cirugía (20 animales); simulación - simulación de cirugía (20 animales); isquemia isquemia focal durante 1 hora, sin reperfusión (80 animales) y dividida en cuatro subgrupos con 20 animales cada uno: isquemia + hipotermia intraisquémica; isquemia + ketoprofeno intravenoso previo, e isquemia + hipotermia y ketoprofeno. El volumen del infarto se midió utilizando un análisis morfométrico de áreas de infarto definidas por cloruro de trifenil tetrazolio y los patrones de expresión de los genes de apoptosis (Fas, c-Flip, caspase-8 y caspase-3) y la proteína de apoptosis caspase-3 fueron evaluados por PCR cuantitativa en tiempo real e inmunohistoquímica, respectivamente. Se observó hipoexpresión de genes de la vía extrínseca de la apoptosis: receptor Fas, c-Flip y caspasa-8 en las áreas isquémicas. También se observaron aumentos en el gen y la proteína caspasa-3 en las áreas isquémicas y estos aumentos se redujeron por hipotermia y ketoprofeno, también observado por estudio morfométrico. El aumento de caspasas-3 sugiere que este gen tiene un papel importante en la apoptosis, y probable causa de muerte celular, involucrando el efecto neuroprotector de la hipotermia y el ketoprofeno.

Investigating the effect of intracameral cefuroxime on oxidative stress and apoptosis in the rat cornea / Investigando o efeito de cefuroxima intracameral sobre o estresse oxidativo e a apoptose na córnea de ratos

ABSTRACT PURPOSE: We examined the effect of intracameral administration of cefuroxime on oxidative stress and endothelial apoptosis in rat corneal tissue. METHODS: In total, 30 rats were divided into 3 groups of 10 rats each (intracameral administration of cefuroxime 0.1 mg/0.01 mL (cefuroxime group); intracameral administration of balanced salt solution 0.01 mL (control group); or absence of intracameral injection (sham group). Corneal endothelial apoptosis was assessed by immunohistochemical analysis using caspase-3 and caspase-8. Total oxidant status, total antioxidant status, oxidative stress index, and paraoxonase and arylesterase levels were examined in corneal endothelial tissue and serum. RESULTS: Paraoxonase levels in the serum were significantly different between the sham and cefuroxime groups (p=0.027). A significant difference was also observed in total oxidant status levels between the cefuroxime and balanced salt solution groups (p=0.023). In addition, there were significant differences in total antioxidant status levels in corneal tissue between the cefuroxime and sham groups (p<0.001) and between the cefuroxime and balanced salt solution groups (p<0.001). Furthermore, significant differences were also observed in oxidative stress index levels between the cefuroxime and balanced salt solution groups (p=0.001) and between the cefuroxime and sham groups (p=0.026). According to the immunohistochemical staining results, a significant association with caspase-3 activity existed between the cefuroxime and balanced salt solution groups (p=0.007), while no significant difference was found with caspase-8 activity (p=0.541). Caspase-3 activity exhibited a significant relationship between the sham and balanced salt solution groups (p=0.018), but no relationship was found with caspase-8 activity (p=0.623). CONCLUSION: Immunohistochemical examination revealed that intracameral cefuroxime increased apoptosis when compared to the sham and balanced salt solution groups. Moreover, intracameral cefuroxime increased oxidative stress in the cornea and simultaneously induced apoptosis.

RESUMO OBJETIVO: Examinamos o efeito da administração intracameral da cefuroxima sobre o estresse oxidativo e a apoptose endotelial no tecido corneano de ratos. MÉTODOS: No total, 30 ratos foram divididos em 3 grupos de 10 ratos cada (administração intracameral de cefuroxima 0,1 mg/0,01 mL (grupo cefuroxima), administração intracameral de solução salina balanceada 0,01 mL (grupo controle) ou ausência de injeção intracameral (grupo sham)). A apoptose endotelial da córnea foi avaliada por análise imuno-histoquimica usando caspase-3 e -8. O status oxidante total, o status antioxidante total, o índice de estresse oxidativo e os níveis de a paraoxonase e arilesterase foram investigados no tecido endotelial da córnea e no soro. RESULTADOS: Os níveis de paraoxonase no soro foram significativamente diferentes entre os grupos sham e cefuroxima (p=0,027). Foi também observada uma diferença significativa nos níveis de estado oxidante total entre os grupos cefuroxima e solução salina balanceada (p=0,023). Além disso, houve diferenças significativas nos níveis de status antioxidante total no tecido da córnea entre os grupos cefuroxima e sham (p<0,001) e entre os grupos cefuroxima e solução salina balanceada (p<0,001). Diferenças significativas também foram observadas nos níveis do índice de estresse oxidativo entre os grupos cefuroxima e solução salina balanceada (p=0,001) e entre os grupos cefuroxima e sham (p=0,026). De acordo com os resultados de coloração imuno-histoquimica, houve associação significativa com a atividade da caspase-3 entre os grupos cefuroxima e solução salina balanceada (p=0,007), enquanto não houve diferença significativa com a atividade da caspase-8 (p=0,541). A atividade da caspase-3 exibiu uma relação significativa entre os grupos sham e solução salina balanceada (p=0,018), mas nenhuma relação foi encontrada com a atividade da caspase-8 (p=0,623). CONCLUSÃO: O exame imuno-histoquímico revelou que a cefuroxima intracameral aumentou a apoptose quando comparada com os grupos sham e solução salina balanceada. Além disso, a cefuroxima intracameral aumentou o estresse oxidativo na córnea e induziu simultaneamente a apoptose.

Necroptosis occurs in osteoblasts during tumor necrosis factor-α stimulation and caspase-8 inhibition

Necroptosis is a regulated cell death mechanism. However, it is unknown whether necroptosis is involved in the death of tumor necrosis factor-α (TNF-α)-treated osteoblasts. Therefore, we conducted the study with TNF-α, Nec-1 (a specific inhibitor of necroptosis), and Z-IETD-FMK (a specific inhibitor of apoptosis) to determine whether necroptosis plays a role in the death of TNF-α-treated osteoblast cell line MC3T3-E1. Cell viability, cell death, and lactate dehydrogenase (LDH) release were assayed to evaluate cytotoxicity. Specific marker proteins receptor interacting protein kinase (RIPK3) and phosphorylated mixed lineage kinase domain-like protein (p-MLKL) for necroptosis, and cleaved caspase 3 for apoptosis were detected by western blot, and mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). We found that TNF-α inhibited cell proliferation in a dose- and time-dependent manner. Nec-1 plus Z-IETD-FMK restored cell viability and significantly decreased LDH release. In addition, TNF-α alone increased the cell population of AV+PI−, while Z-IETD-FMK caused a shift in the cell population from AV+PI− to AV+PI+. Furthermore, TNF-α significantly increased protein cleaved caspase 3. TNF-α plus Z-IETD-FMK significantly increased the proteins RIPK3 and MLKL phosphorylation in MC3T3-E1 cells, while the changes in mRNA levels of RIPK3, MLKL, and caspase 3 were not consistent with the changes in the corresponding protein expression levels. In conclusion, TNF-α induced preferentially apoptosis in osteoblast cell line and necroptosis played a decisive role when TNF-α-induced death was inhibited by the inhibitor of apoptosis. Combined treatment with Nec-1 and Z-IETD-FMK protected mouse osteoblasts from death induced by TNF-α.

Gene Polymorphisms in FAS (Rs3740286 and Rs4064) Are Involved in Endometriosis Development in Brazilian Women, but not those in CASP8 (rs13416436 and rs2037815) / Polimorfismos do gene FAS (rs3740286 e rs4064) estão envolvidos no desenvolvimento de endometriose em mulheres brasileiras, mas não os no CASP8 (rs13416436 e rs2037815)

Abstract Objective The present study aims to investigate the association between caspase-8 (CASP8) (rs13416436 and rs2037815) and Fas cell surface death receptor (FAS) (rs3740286 and rs4064) polymorphisms with endometriosis in Brazilian women. Methods In the present case-control study, 45 women with a diagnosis of endometriosis and 78 normal healthy women as a control group were included. The genotyping was determined by real-time polymerase chain reaction (PCR) with Taqman hydrolysis probes (Thermo Fisher Scientific, Darmstadt, Germany). Genotypic and allelic frequencies were analyzed using Chi-squared (χ2) test. In order to determine the inheritance models and haplotypes ,SNPStats (Institut Català d'Oncologia, Barcelona, Spain) was used. Levels of 5% (p = 0.05) were considered statistically significant. Results No significant difference was observed in genotypic or allelic frequencies between control and endometriosis groups for rs13416436 and rs2037815 (CASP8 gene). On the other hand, a significant difference between rs3740286 and rs4064 (FAS gene) was found. Regarding polymorphisms in the FAS gene, a statistically significant differencewas found in co-dominant and dominantmodels. Only the haplotype containing the rs3740286A and rs4064G alleles in the FAS gene were statistically significant. Conclusion The polymorphisms in the CASP8 gene were not associated with endometriosis. The results indicate an association between FAS gene polymorphisms and the risk of developing endometriosis.

Resumo Objetivo Investigar a associação entre os polimorfismos dos genes caspase-8 (CASP8) (rs13416436 e rs2037815) e FAS (rs3740286 e rs4064) em mulheres brasileiras com endometriose. Métodos Trata-se de um estudo do tipo caso-controle, no qual foram incluídas 45 mulheres com diagnóstico de endometriose e 78 controles. A genotipagem das amostras foi determinada usando a reação em cadeia de polimerase em tempo real com sondas de hidrólise TaqMan (Thermo Fisher Scientific, Darmstadt, Germany). As frequências genotípicas e alélicas foram analisadas usando o teste do qui-quadrado. O SNPStats (Institut Català d'Oncologia, Barcelona, Espanha) foi usado para determinar os modelos de herança e os haplótipos. Os níveis de significância estatística considerados foram de 5% (p = 0,05). Resultados Não foi observada diferença significativa nas frequências genotípicas ou alélicas entre os grupos de controle e de endometriose para os polimorfismos rs13416436 e rs2037815 (gene CASP8). Por outro lado, foi encontrada uma diferença significativa entre os polimorfismos rs3740286 e rs4064 (gene FAS). Em relação aos polimorfismos do gene FAS, foi encontrada uma diferença estatisticamente significativa nos modelos codominante e dominante. Apenas o haplótipo contendo os alelos rs3740286A e rs4064G no gene FAS foi estatisticamente significativo. Conclusão Não há associação entre os polimorfismos do gene CASP8 e endometriose. Entretanto, há associação entre os polimorfismos do gene FAS e o risco de desenvolver endometriose.

MicroRNA-122 regulates caspase-8 and promotes the apoptosis of mouse cardiomyocytes

Cardiomyocyte apoptosis plays key roles in the pathogenesis of heart diseases such as myocardial infarction. MicroRNAs are important regulators of gene expression, which are also involved in the regulation of cardiomyocyte apoptosis. However, cardiomyocyte apoptosis regulated by microRNA (miR)-122 is largely unexplored. The aim of this study focused on the role of miR-122 in cardiomyocyte apoptosis. Cardiomyocytes were isolated from neonatal mice and primarily cultured. MiR-122 mimic and inhibitor were transfected to cardiomyocytes and verified by qRT-PCR. Cell viability and apoptosis post-transfection were assessed by MTT assay and flow cytometry, respectively. Changes in expression of caspase-8 were quantified by qRT-PCR and western blot. Results showed that miR-122 mimic and inhibitor successfully induced changes in miR-122 levels in cultured cardiomyocytes (P<0.01). MiR-122 overexpression suppressed viability and promoted apoptosis of cardiomyocytes (P<0.05), and miR-122 knockdown promoted cell viability and inhibited apoptosis (P<0.05). The mRNA and protein levels of caspase-8 were elevated by miR-122 overexpression (P<0.01) and reduced by miR-122 knockdown (P<0.001). These results suggest an inductive role of miR-122 in cardiomyocyte apoptosis, which may be related to its regulation on caspase-8.

In vitro assessment of a computer-designed potential anticancer agent in cervical cancer cells

BACKGROUND: Computer-based technology is becoming increasingly essential in biological research where drug discovery programs start with the identification of suitable drug targets. 2-Methoxyestradiol (2ME2) is a 17ß-estradiol metabolite that induces apoptosis in various cancer cell lines including cervical cancer, breast cancer and multiple myeloma. Owing to 2ME2's poor in vivo bioavailability, our laboratory in silico-designed and subsequently synthesized a novel 2ME2 analogue, 2-ethyl-3-O-sulphamoyl-estra-1,3,5(10),15-tetraen-17-ol (ESE-15-ol), using receptor- and ligand molecular modeling. In this study, the biological effects of ESE-15-ol (180 nM) and its parent molecule, 2ME2 (1 µM), were assessed on morphology and apoptosis induction in cervical cancer cells. RESULTS: Transmission electron microscopy, scanning electron microscopy and polarization-optical transmitted light differential interference contrast (PlasDIC) images demonstrated morphological hallmarks of apoptosis including apoptotic bodies, shrunken cells, vacuoles, reduced cell density and cell debris. Flow cytometry analysis showed apoptosis induction by means of annexin V-FITC staining. Cell cycle analysis showed that ESE-15-ol exposure resulted in a statistically significant increase in the G2M phase (72%) compared to 2ME2 (19%). Apoptosis induction was more pronounced when cells were exposed to ESE-15-ol compared to 2ME2. Spectrophotometric analysis of caspase 8 activity demonstrated that 2ME2 and ESE-15-ol both induced caspase 8 activation by 2- and 1.7-fold respectively indicating the induction of the apoptosis. However, ESE-15-ol exerted all of the above-mentioned effects at a much lower pharmacological concentration (180 nM) compared to 2ME2 (1 µM physiological concentration). CONCLUSION: Computer-based technology is essential in drug discovery and together with in vitro studies for the evaluation of these in silico-designed compounds, drug development can be improved to be cost effective and time consuming. This study evaluated the anticancer potential of ESE-15-ol, an in silico-designed compound in vitro. Research demonstrated that ESE-15-ol exerts antiproliferative activity accompanied with apoptosis induction at a nanomolar concentration compared to the micromolar range required by 2ME2. This study is the first study to demonstrate the influence of ESE-15-ol on morphology, cell cycle progression and apoptosis induction in HeLa cells. In silico-design by means of receptor- and ligand molecular modeling is thus effective in improving compound bioavailability while preserving apoptotic activity in vitro.