Effect of early weaning on molecular markers and intestinal mucosal development.

Author(s)

Aline Vasques da Costa

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Instituto de Ciências Biomédicas (ICB/SDI)
• Defense date: 2021-11-25

Examining board members

Patricia Gama; Carla Roberta de Oliveira Carvalho; Patricia Castelucci; Thereza Christina Barja Fidalgo Coelho

• Advisor: Patricia Gama

Abstract

Considering breastfeeding as an important food source during babyhood and its role in regulating the growth and development of intestinal mucosa, early weaning (EW) could promote changes in cell proliferation and differentiation processes, with impairment of intestinal function. Therefore, the aim of this study was to evaluate markers of cell proliferation, differentiation, and growth factors in the intestinal epithelium, observing the immediate and late effects triggered by EW. Wistar rats were submitted to EW at 15 postnatal days, when animals were isolated from their dams (control suckling group was weaned at 21 days). Jejunum samples were collected at 18, 60 and 120 d. We evaluated morphological parameters and their correlation with expression of genes and proteins related to growth, stem cell niche, signaling, epigenetic modification and metabolic markers. We observed that the villus height was significantly decreased at 18 and 60 days after EW, whereas the crypt depth was not affected by treatment, by grew throughout development. The proliferative index was significantly reduced at 18 and 60 days. After counting the differentiated cells, we found EW decreased the number of enterocytes and globlet cells (villi) at 18 days; at 60 days the distribution of goblet cells was reduced along villus, and at 120 days this population increased in the crypt-villus axis. Paneth cells, which are solely in the crypt, increased at 18 and 60 days in EW group. Among the target genes, the expression of sucrase, mucin 2 and defensin augmented at 18 days in EW and, conversely, Chga gene (enteroendocrine cells) and Lactase were reduced by EW at 18 days. From growth factor markers, EW triggered the decrease of Tgfb1 at 18 days, and of Egfr at 60 days. Interestingly, among cell cycle markers, Cdkn1b (p27) expression was higher in EW vs. control at all ages. From stem cell markers, Lgr5 and Ascl2 decreased at 18 days (EW) and augmented at 60 days. Of note, only Ascl2 was reduced by EW at 120 days. We studied the main signaling pathways in the crypt, and we found that after EW, Wnt3A, Notch 1 and Atoh1 showed a reduction at 18 days, whereas BMP2 increased at the same age. At 60 days, we found higher levels of Wnt3A and Atoh1 and a reduced expression of Notch 1, Notch 2 and BMP2. As for protein levels, EW decreased Hes1 at 60 days. Epigenetic changes were inconclusive, but wer detected decreased expression of Dnmt 3a and Dnmt 3b enzymes at 18 days in EW. At 60 days these changes were seen only in Dnmt1 and at 120 days the Dnmt1 and Dnmt3b were reduced in EW. In terms of metabolism, we observed that EW triggered a reduction of serum glycemia at 18 days, and conversely, at 60 days, there was a significant increase after (glucose tolerance test -GTT). Our results demonstrated that early weaning influenced cell proliferation in the small intestine, affecting the expression of target genes in the stem cell niche, and cell differentiation, changing enterocyte, globlet and Paneth cell populations immediately after breastfeeding interruption at 18 days. Part of the effects were maintained into adulthood with phenotypic and metabolic modifications. We suggest that cellular responses to EW can be permanent in the intestinal mucosa, indicating that breastfeeding is essential to the development of small intestine and its maintenance in adult life. (AU)

• FAPESP's process: 18/07409-5 - Effect of early weaning on molecular markers and development of intestinal mucosa
• Grantee: Aline Vasques da Costa
• Support Opportunities: Scholarships in Brazil - Doctorate