RESUMO
Understanding how excitable cells work in health and disease and how that behavior can be altered by small molecules or genetic manipulation is important. Genetically encoded calcium indicators (GECIs) with multiple emission windows can be combined (e.g., for simultaneous observation of distinct subcellular events) or used in extended applications with other light-dependent actuators in excitable cells (e.g., combining genetically encoded optogenetic control with spectrally compatible calcium indicators). Such approaches have been used in primary or stem cell-derived neurons, cardiomyocytes, and pancreatic beta-cells. However, it has been challenging to increase the throughput, or duration of observation, of such approaches due to limitations of the instruments, analysis software, indicator performance, and gene delivery efficiency. Here, a high-performance green GECI, mNeonGreen-GECO (mNG-GECO), and red-shifted GECI, K-GECO, is combined with optogenetic control to achieve all-optical control and visualization of cellular activity in a high-throughput imaging format using a High-Content Imaging System. Applications demonstrating cardiotoxicity testing and phenotypic drug screening with healthy and patient-derived iPSC-CMs are shown. In addition, multi-parametric assessments using combinations of spectral and calcium affinity indicator variants (NIR-GECO, LAR-GECO, and mtGCEPIA or Orai1-G-GECO) are restricted to different cellular compartments are also demonstrated in the iPSC-CM model.
Assuntos
Cálcio , Células-Tronco Pluripotentes Induzidas , Cálcio/análise , Avaliação Pré-Clínica de Medicamentos , Humanos , Indicadores e Reagentes , Células-Tronco Pluripotentes Induzidas/química , Miócitos Cardíacos/química , OptogenéticaAssuntos
Abscesso/cirurgia , Doenças do Ânus/cirurgia , Misturas Complexas/farmacologia , Fitoterapia , Extratos Vegetais/farmacologia , Infecção da Ferida Cirúrgica/tratamento farmacológico , Cicatrização/efeitos dos fármacos , Adulto , Animais , Bandagens , Feminino , Humanos , Masculino , Ratos , Ferida Cirúrgica/terapiaRESUMO
Propionibacterium acnes is a key pathogen involved in acne inflammation. Wild bitter melon (WBM, Momordica charantia L. var. abbreviate Seringe) is consumed as both a vegetable and as folk medicine in Taiwan. We examined the inhibitory activity of the total phenolic extract (TPE) of WBM leaf on P. acnes-induced inflammatory responses in vivo and in vitro. Our data showed that TPE significantly attenuated P. acnes-induced ear swelling in mice along with microabscess. Flow cytometry analysis revealed that TPE treatment significantly decreased the migration of neutrophils and interleukin (IL)-1ß(+) populations in vivo. In P. acnes-stimulated human monocytic THP-1 cells, TPE suppressed the mRNA levels and production of IL-8, IL-1ß, and tumor necrosis factor (TNF)-αin vitro. In addition, TPE suppressed P. acnes-induced matrix metalloproteinase-9 levels. TPE blocked nuclear factor-κB (NF-κB) activation and inactivated mitogen-activated protein kinases (MAPK); these actions may partially account for its inhibitory effect on cytokine production. The quantitative HPLC analysis revealed gallic, chlorogenic, caffeic, ferulic, and cinnamic acids, myricetin, quercetin, luteolin, apigenin, and thymol in TPE. All these phenolics significantly suppressed P. acnes-induced IL-8 production in vitro. Our results suggest that WBM leaf extract effectively inhibits P. acnes-induced inflammatory responses and may be useful to relieve the inflammation of acne.
Assuntos
Acne Vulgar/tratamento farmacológico , Acne Vulgar/imunologia , Momordica charantia/química , Extratos Vegetais/administração & dosagem , Propionibacterium acnes/fisiologia , Acne Vulgar/genética , Acne Vulgar/microbiologia , Animais , Humanos , Interleucina-1beta/genética , Interleucina-1beta/imunologia , Interleucina-8/genética , Interleucina-8/imunologia , Masculino , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/imunologia , Camundongos , Camundongos Endogâmicos ICR , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/imunologia , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta/química , Taiwan , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/imunologiaRESUMO
In this study, we aimed to evaluate the inhibitory effect of wild bitter melons (WBM; Momordica charantia Linn. var. abbreviata Ser.) on Propionibacterium acnes-induced inflammation and to identify the bioactive components. Our results showed that ethyl acetate (EA) extract of WBM fruit in vitro potently suppressed pro-inflammatory cytokine and matrix metalloproteinase (MMP)-9 levels in P. acnes-stimulated THP-1 cells. Furthermore, concomitant intradermal injection of WBM EA extract in mice effectively attenuated P. acnes-induced ear swelling and granulomatous inflammation. To further investigate the bioactive components, we found that both saponifiable (S) and nonsaponifiable (NS) fractions of WBM EA extract significantly suppressed pro-inflammatory cytokine and MMP-9 levels. Phytol and lutein, identified in the NS fraction, also inhibited cytokine production. Moreover, S and NS fractions of EA extract, phytol and lutein, activated peroxisome proliferator-activated receptor (PPAR) α and ß in the transactivation assay. Our results suggested that PPARα or PPARγ signalling may contribute, at least in part, to the anti-inflammatory activity of WBM.