RESUMO
Human immunodeficiency virus (HIV) encodes a transcription trans-activator (Tat) with an essential role in the transcriptional elongation of viral RNA based on the viral promoter long terminal repeat (LTR). Tat-mediated transcription is conserved and can be distinguished from host transcription, so it is a therapeutic target for combating HIV replication. Traditional screening assays for Tat-mediated transcriptional inhibitors are based on the biochemical properties of Tat and transactivation-responsive RNA. We developed an inducible system based on two lentiviral expression cassettes for doxycycline (Dox)-inducible Tat and Renilla luciferase (R-Luc) using TZM-bl cells harboring LTR-driven firefly luciferase (F-Luc). The cells simultaneously expressed both Tat-induced F-Luc and R-Luc, so it was possible to recognize off-target effects in the presence of Dox. The system was validated with known inhibitors: CYC202 obtained high sensitivity and specificity, whereas 6Bio and DRB had off-target effects. The MTT-based cytotoxicity test indicated the resistance of the system even at concentrations with off-target effects. The specificity of the system was confirmed using antiretroviral drugs. Our dual reporter system can simply detect Tat inhibitory effects, as well as precisely discriminate between the inhibitory and off-target effects of inhibitors, and may be useful for the development of a therapeutic anti-HIV drug.
Assuntos
Fármacos Anti-HIV/farmacologia , HIV-1/efeitos dos fármacos , Transcrição Gênica/efeitos dos fármacos , Produtos do Gene tat do Vírus da Imunodeficiência Humana/antagonistas & inibidores , Doxiciclina/farmacologia , Avaliação Pré-Clínica de Medicamentos , Regulação Viral da Expressão Gênica , Infecções por HIV/tratamento farmacológico , Infecções por HIV/virologia , HIV-1/genética , Células HeLa , Humanos , Luciferases/genética , Luciferases/metabolismo , Purinas/farmacologia , RNA Viral/genética , Roscovitina , Sensibilidade e Especificidade , Produtos do Gene tat do Vírus da Imunodeficiência Humana/genéticaRESUMO
Traditional herbal medicines may be viable alternatives to corticosteroid therapy for treatment of asthma. However, the therapeutic mechanisms of herbal compounds remain a matter of considerable debate. This study was performed to evaluate the effects of Chung-Sang-Bo-Ha-Tang (CSBHT), a herbal compound administrated therapeutically to asthma patients for centuries, on airway inflammation and remodeling in a murine model of chronic asthma. BALB/c mice sensitized to ovalbumin (OVA) were chronically challenged with aerosolized OVA for 6 weeks. During the last 2 weeks, some mice were treated daily with CSBHT by intragastric feeding. Dexamethasone (Dex)-treated, phosphate-buffered saline (PBS)-treated, and naive mice served as controls. The effects of CSBHT on airway inflammation, lung pathology, and cytokine production were evaluated. Mice exposed to recurrent airway challenge with OVA had chronic inflammation and characteristics of airway remodeling, including subepithelial fibrosis, epithelial hypertrophy, and goblet cell hyperplasia. CSBHT was as effective as Dex at moderately reducing these changes compared to the PBS-treated mice. In addition, IL-5 and IFN-gamma levels in supernatants of Concanavalin A (Con A)-activated splenocyte cultures were reduced in mice treated with CSBHT. Treatment with CSBHT during the last 2 weeks of challenge modulated airway inflammation and remodeling in a murine model of chronic asthma. Thus, CSBHT may effectively delay the progression of airway inflammation and remodeling.