RESUMO
Acute Kidney Injury (AKI) is associated with high morbidity and mortality. Ischemia and reperfusion (I/R) are events that lead to AKI through hypoxia, reactive oxygen species (ROS) production, oxidative stress and apoptosis. We aimed to evaluate the mechanism of nephroprotection mediated by Bisabolol in human tubular kidney cells after injury by I/R in vitro. HK2 cells were exposed to I/R and treated with Bisabolol. Cell viability was accessed by MTT assay. Cells were submitted to flow cytometry to evaluate necrotic/apoptotic cells, reactive oxygen species production and mitochondrial transmembrane depolarization. TBARS and GSH were used as parameters of redox balance. Also, KIM-1 supernatant levels were measured. In order to identify an interaction between bisabolol and NOX4, molecular docking and enzymatic assays were performed. Expression of isoform NOX4 on treated cells was examined by western-blot. Finally, cells were visualized by scanning electron microscopy. Bisabolol improved cell viability and prevented cell death by apoptosis, indicated also by the decreased levels of KIM-1. It was observed a decrease on reactive oxygen species production and mitochondrial depolarization, with antioxidant regulation by increased GSH and decreased lipid peroxidation. It was also demonstrated that bisabolol treatment can inhibit NOX4. Finally, SEM images showed that bisabolol reduced I/R-induced cell damage. Bisabolol treatment protects HK2 cells against oxidative damage occasioned by I/R. This effect is related to inhibition of apoptosis, decrease on KIM-1 release, reactive oxygen species accumulation and mitochondrial dysfunction. Bisabolol inhibited NOX4 activity in the tubular cells, impairing reactive oxygen species synthesis.
Assuntos
Túbulos Renais/efeitos dos fármacos , Túbulos Renais/patologia , Sesquiterpenos Monocíclicos/farmacologia , NADPH Oxidases/metabolismo , Oxigênio/metabolismo , Traumatismo por Reperfusão/prevenção & controle , Apoptose/efeitos dos fármacos , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Citoproteção/efeitos dos fármacos , Glutationa/metabolismo , Receptor Celular 1 do Vírus da Hepatite A/metabolismo , Humanos , Túbulos Renais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Substâncias Reativas com Ácido Tiobarbitúrico/metabolismoRESUMO
Previous studies have reported the anti-obesity effects of α, ß-Amyrin in high fat-fed mice. This study aimed to evaluate whether α, ß-Amyrin has an anti-adipogenic effect in 3T3-L1 murine adipocytes and to explore the possible underlying mechanisms. 3T3-L1 pre-adipocytes were differentiated in a medium containing insulin, dexamethasone, and 1-methyl-3-isobutylxanthine. Cytotoxicity of α, ß-Amyrin was assessed by MTT assay. Lipid content in adipocytes was determined by Oil-Red O staining. In addition, the protein expression levels of peroxisome proliferator-activated receptor gamma (PPARγ), CCAAT/enhancer binding proteins alpha (C/EBPα), beta (C/EBPß), and delta (C/EBPδ) and glucose transporter 4 (GLUT4) were determined by qRT-PCR and western blot analysis. Oil-Red O staining revealed markedly reduced fat accumulation by α, ß-Amyrin (6.25-50 µg/mL) without affecting cell viability. Furthermore, our results indicate that α, ß-Amyrin can significantly suppress the adipocyte differentiation by downregulating the expression levels of adipogenesis-related key transcription factors such as PPARγ and C/EBPα, but not C/EBPß or C/EPBδ. In addition, the protein expression of membrane GLUT4 in 3T3- L1 adipocytes treated with α, ß-Amyrin was significantly higher than in control cells, indicating that α, ß-Amyrin augments glucose uptake. These findings suggest that α, ß-Amyrin exerts an anti-adipogenic effect principally via modulation of lipid and carbohydrate metabolism in 3T3-L1cells. The present in vitro findings, taken together with our earlier observation of the anti-obesity effect in vivo, suggest that α, ß-Amyrin can be developed as a new therapeutic agent for treatment and prevention of obesity.
Assuntos
Adipócitos/efeitos dos fármacos , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Ácido Oleanólico/análogos & derivados , PPAR gama/metabolismo , Triterpenos Pentacíclicos/farmacologia , Células 3T3-L1 , Adipogenia/efeitos dos fármacos , Animais , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Proteínas de Ligação a Ácido Graxo/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Camundongos , Obesidade/tratamento farmacológico , Obesidade/metabolismo , Ácido Oleanólico/farmacologia , Extratos Vegetais/farmacologiaRESUMO
Recent research has shown broad antifungal activity of the classic antidepressants selective serotonin reuptake inhibitors (SSRIs). This fact, combined with the increased cross-resistance frequency of the genre Candida regarding the main treatment today, fluconazole, requires the development of novel therapeutic strategies. In that context, this study aimed to assess the antifungal potential of fluoxetine, sertraline, and paroxetine against fluconazole-resistant Candida spp. planktonic cells, as well as to assess the mechanism of action and the viability of biofilms treated with fluoxetine. After 24 h, the fluconazole-resistant Candida spp. strains showed minimum inhibitory concentration (MIC) in the ranges of 20-160 µg/mL for fluoxetine, 10-20 µg/mL for sertraline, and 10-100.8 µg/mL for paroxetine by the broth microdilution method (M27-A3). According to our data by flow cytometry, each of the SSRIs cause fungal death after damaging the plasma and mitochondrial membrane, which activates apoptotic signaling pathways and leads to dose-dependant cell viability loss. Regarding biofilm-forming isolates, the fluoxetine reduce mature biofilm of all the species tested. Therefore, it is concluded that SSRIs are capable of inhibit the growth in vitro of Candida spp., both in planktonic form, as biofilm, inducing cellular death by apoptosis.
Assuntos
Antifúngicos/farmacologia , Biofilmes/efeitos dos fármacos , Candida/efeitos dos fármacos , Farmacorresistência Fúngica/efeitos dos fármacos , Fluconazol/farmacologia , Inibidores Seletivos de Recaptação de Serotonina/farmacologia , Animais , Apoptose/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Candida/citologia , Candida/genética , Candida/crescimento & desenvolvimento , Contagem de Células , Morte Celular/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Dano ao DNA/efeitos dos fármacos , DNA Fúngico/efeitos dos fármacos , Fibroblastos/microbiologia , Citometria de Fluxo , Técnicas In Vitro , Potenciais da Membrana , Camundongos , Testes de Sensibilidade Microbiana , Viabilidade Microbiana/efeitos dos fármacos , Membranas Mitocondriais/efeitos dos fármacos , Paroxetina/farmacologia , Plasma/efeitos dos fármacos , Inibidores Seletivos de Recaptação de Serotonina/administração & dosagem , Sertralina/farmacologiaRESUMO
The incidence of fungal infections and, in particular, the incidence of fungal antibiotic resistance, which is associated with biofilm formation, have significantly increased, contributing to morbidity and mortality. Thus, new therapeutic strategies need to be developed. In this context, natural products have emerged as a major source of possible antifungal agents. Berberine is a protoberberine-type isoquinoline alkaloid isolated from the roots, rhizomes, and stem bark of natural herbs, such as Berberis aquifolium, Berberis vulgaris, Berberis aristata, and Hydrastis canadensis, and of Phellodendron amurense Berberine has been proven to have broad antibacterial and antifungal activity. In the present study, the potential antifungal effect of berberine against fluconazole-resistant Candida and Cryptococcus neoformans strains, as well as against the biofilm form of Candida spp., was assessed. The antifungal effect of berberine was determined by a broth microdilution method (the M27-A3 method of the Clinical and Laboratory Standards Institute) and flow cytometry techniques, in which the probable mechanism of action of the compound was also assessed. For biofilm assessment, a colorimetric 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay was used to determine the susceptibility of sessile cells. The isolates used in the study belonged to the Laboratory of Bioprospection and Experiments in Yeast (LABEL) of the Federal University of Ceará. After 24 and 72 h, fluconazole-resistant Candida and Cryptococcus neoformans strains showed berberine MICs equal to 8 µg/ml and 16 µg/ml, respectively. Cytometric analysis showed that treatment with berberine caused alterations to the integrity of the plasma and mitochondrial membranes and DNA damage, which led to cell death, probably by apoptosis. Assessment of biofilm-forming isolates after treatment showed statistically significant reductions in biofilm cell activity (P < 0.001).