ABSTRACT
Epithelial cell migration is an essential response to enteric pathogens such as enteropathogenic Escherichia coli (EPEC). This study aimed to investigate the effects of EPEC infection on intestinal epithelial cell migration in vitro, as well as the involvement of type III secretion system (T3SS) and Rho GTPases. Crypt intestinal epithelial cells (IEC-6) were infected with EPEC strains (E2348/69, ΔescF, and the LDI001 strain isolated from a malnourished Brazilian child) and commensal E. coli HS. Wound migration and cell death assays were performed at different time-points. Transcription and expression of Rho GTPases were evaluated using real-time PCR and western blotting. Overall, EPEC E2348/69 reduced migration and increased apoptosis and necrosis levels compared to EPEC LDI001 and E. coli HS strains. Moreover, EPEC LDI001 impaired cell migration at a higher level than E. coli HS and increased necrosis after 24 hours compared to the control group. The different profiles of virulence genes between the two wild-type EPEC strains, characterized by the absence of espL and nleE genes in the LDI001, might explain the phenotypic results, playing significant roles on cell migration impairment and cell death-related events. Moreover, the type III secretion system is determinant for the inhibition of intestinal epithelial cell migration by EPEC 2348/69, as its deletion prevented the effect. Active Rac1 concentrations were increased in E2348/69 and LDI001-infected cells, while the T3SS-deficient strain did not demonstrate this activation. This study contributes with valuable insight to characterize the mechanisms involved in the impairment of intestinal cell migration induced by EPEC.
Subject(s)
Humans , Cell Movement/physiology , rho GTP-Binding Proteins/physiology , Virulence Factors/genetics , Epithelial Cells/microbiology , Enteropathogenic Escherichia coli/pathogenicity , Type III Secretion Systems/physiology , Blotting, Western , Apoptosis , Virulence Factors/physiology , Real-Time Polymerase Chain Reaction , Flow CytometryABSTRACT
Plectranthus sp. pertence à família Lamiaceae e inclui cerca de 300 espécies. Duas delas, P. barbatus (PB) e P. ornatus (PO) destacam-se pelo interesse medicinal. As principais substâncias encontradas em Plectranthus sp. são os diterpenóides, óleos essenciais e compostos fenólicos. A melhor forma de utilizar as folhas de PB e PO é a fresco; porém, na comercialização há necessidade de recorrer a processos de secagem. Entretanto, isto pode interferir no princípio ativo, desta forma, estudos para determinar o melhor método gravimétrico são importantes. O objetivo deste estudo foi avaliar qual o melhor método de secagem, onde haja maior perda de água possível, e calcular os rendimentos dos extratos visando avaliar se o método gravimétrico interfere na quantidade de extrato produzido. Desta forma, utilizou-se quatro métodos de secagem e após evaporação do solvente, fez-se o cálculo do rendimento dos extratos secos. O percentual de água foi superior em PB e PO (média geral de 90,12±8,22 por cento). Dentre as secagens, em estufa de ventilação forçada (EVF) foi mais eficiente em PB (88,87 por cento) e em estufa convencional (ECO) foi melhor em PO (95,13 por cento). O maior rendimento do extrato seco em PB foi na secagem em camada delgada (CD) (20,96 por cento), enquanto que em PO foi ECO (41,34 por cento). Não houve correlação entre o método de secagem e o rendimento de extrato obtido.
Plectranthus sp. belongs to the family Lamiaceae and includes about 300 species. Two of them, P. barbatus (PB) and P. ornatus (PO), have medicinal interest. The main substances found in Plectranthus sp. are the diterpenoids, essential oils and phenolic compounds. The best way to use the leaves of PB and PO is cool; but for marketing there is a need of resort to drying processes. However, this may interfere on the values of active principle; thus, studies to determine the best gravimetric methods are important. The objective of this study was to evaluate the best method of drying, where there the greater loss of water possible, and calculate the percentage of the extracts to assess whether the gravimetric method interferes with the amount of extract produced. To this was done by drying four different methods ventilated dry (EVF), conventional drying (ECO), microwave (MCO) and layer thing (CD) and after evaporation of the solvent, was made the calculation the percentage of the dried extract. The water content was higher in PB and PO (overall average of 90.12±8.22 percent). Among the drying, EVF was more efficient for PB (88.87 percent) and ECO was better for PO (95.13 percent). The highest content of dry extract was for PB in to CD (20.96 percent), whereas for PO was in to ECO (41.34 percent). There is no relationship between the drying method and in the quantity of extract obtained.