Dieta deficiente em doadores de metil altera a dinâmica da célula tronco intestinal e aumenta a profundidade de cripta no intestino delgado: estudo em modelo in vivo e em organóides intestinais

Abstract

In child populations of the developing world, physical and cognitive development impairment are highly prevalent and are accompanied by altered small intestinal (SI) morphology. Such problems occur in an environment of malnutrition, a context where micronutrient deficiency plays a major role. Based on the above, the objective of this study was to investigate the effect of the deficiency of methyl donors (folate and choline) in vivo on the structure of the small intestine, verifying the impact of those nutrients on the intestinal stem cell (ISC) dynamics in vitro. We randomized pregnant dams to one of four different diets: control diet CD- and CD+; isocaloric methyl donor deficient diet, MDD- and MDD+. +/- means presence or absence of sulfamethoxazole in the formulation. We weaned pups to their dam's diet and measured growth, folate status and SI crypt morphology. Following the sacrifice, enteroids were cultured from the isolated intestinal crypt to assess ISC dynamics. Our results reveal a prominent reduction in weight and growth of the animals exposed to the MDD as well as significant increase of crypt depth in the jejunum and ileum segments of those animals. Such alterations were associated with significant reductions in serum folate and increase in red blood cell size mainly in animals exposed to a deficient antibiotic diet (MDD+). Enteroids from animals exposed to deficient diet showed reduced number of crypt domain and lower survival rate associated with reduced amount of EdU-tagged proliferative cells. PCR-arrray showed a significant increase of Atoh1 suggesting expansion of the pool of progenitors compromised with the secretory lineage. Reduction of the transcriptional of Lrig1, Ascl2 and Sox9, a subset of Wnt signaling target genes suggest that pathway is impaired. The impaired ISC function found in the enteroids from MDD- can be explained by the reduction of the quiescent ICS markers while Lgr5, the main marker for high cycling ICS, was intact. In comparison to the control, methylation of DNA in the ISC showed that enteroids cultivated in medium with low levels of methyl donors presented lower percentile of methylation of the genes involved in the production of intestinal mucin B4Galnt1 and Phospho1 in the 3'CGI, an epigenetic alteration associated to lower expression of those genes. From the above, we conclude that in the model used the deficiency of methyl donors is a sufficient causal condition to induce at the tissue level intestinal histological alterations associated with weight and growth impairment. At the cellular level, such alterations are related to impaired ISC function, also altering the cellular composition in the crypt-villus axis with greater predominance of the secretory progenitor lineage. At the molecular level the alterations found were related to the reduction of DNA methylation in genes involved with mucin production. In general, all the changes found harmonize with the well-delineated role of folate and other methyl donors as essential cofactors for synthesis, maturation and methylation of DNA.