Avaliação dos mecanismos de proliferação e tipos de morte celular na lesão induzida pela Escherichia coli enteroagregativa e sua modulação por alanil-glutamina e betacaroteno

• Ano de defesa: 2013
• Autor(a) principal: Prata, Mara de Moura Gondim
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Palavras-chave em Português: Escherichia coli; beta Caroteno; Apoptose
• Link de acesso: http://www.repositorio.ufc.br/handle/riufc/7181

Enteroaggregative Escherichia coli (EAEC) is among the most important agents associated with persistent diarrhea (DP) and was prevalent in studies in children in poor communities in the city of Fortaleza. EAEC cause intestinal injury and inflammation leading to DP and, when combined with malnutrition, can cause a cognitive deficit and infant growth impairment. The current study examined in vitro (IEC-6 and HEp-2) intestinal pathophysiology of three strains: EAEC wild type, EAEC 042 (positive control) and non-pathogenic E.coli HS as well as the role of alanyl-glutamine (AG) and beta-carotene in the mechanisms of proliferation, apoptosis and necrosis in response to the injury caused by EAEC strains. The wild type strain was isolated from a malnourished child. Intestinal cells viability assay in showed a significant reduction (p<0.05) after post-infection with EAEC strains at concentrations of 105UFC/mL in 12, 24 and 48 hours. However, the E.coli HS only changed the intestinal cell viability after 48 hours. EAEC post-infected intestinal cells presented mRNA transcription decrease (p<0.05) of c-jun and c-fos at the period of zero, 6 and 12 hours after infection was terminated, but E.coli HS showed this decrease only after 12h of infection. Intestinal cell apoptosis increased after infection by all strains 24h of infection. However, the cell damage remained intense in the cells treated only with EAEC strains. EAEC 042 increased cell necrosis (p<0.05) in all evaluated periods, but the wild type strain caused damage with only at the period of 24h. All infected cells had a mRNA transcription increase of caspase 8 gene (p<0.05) in12h. NF-kB mRNA transcription increase (p<0.05) was seen in infected cells only in the period of 12h. While transcription levels of IL-8 were extremely high immediately after the infection was interrupted (0h), that was a drastic reduction at 12h after the end of infection. The wild type strain caused a reduction in cell viability linked apoptosis. However, EAEC 042 induced this decrease as also cellular necrosis. E. coli HS showed a different infection profile probably because its lack of virulence genes. AG 1mM supplementation was able to enhance cell proliferation (p<0.05) associated with apoptosis and necrosis reduction (p<0.05) in post-infected cells at the periods of 12, 24 and 48h. However, the presence of AG 1mM in infected cells did not affect the mRNA transcription low levels of c-jun and c-fos as seen after EAEC infection, and failed to block caspase 8 mRNA transcription increase (p<0.05). The IL-8 mRNA transcription temporal reduction was not associated with AG treatment in post-infected cells. Supplementation with AG had positive effects on epithelial protection against damage caused by both EAEC strains in proliferation assay and inhibition of cell death. However, since caspase 8 mRNA transcription decrease was not observed after AG treatment, AG anti-apoptosis feature could be probably related to another mechanism. Beta-carotene cell damage reversal on cell viability assay was statistical significant (p<0.05) 24 hours after infection was ended. Beta-carotene caused 48h apoptosis and necrosis (p<0.05) in wild type strain post-infected cells. Infected cells treated with beta-carotene could not block c-jun and c-fos mRNA transcription reduction and also failed to inhibit caspase 8 mRNA transcription, but beta-carotene itself increased levels of caspase 3 at the period of 12h after infection. Beta-carotene was shown to be potentially harmful to intestinal cells causing cell death probably related to its pro-oxidant effects.