The Role Of AFP In Glucocorticoid Induced Premature In Rat Colon Of Development

Among physiological factors involved in gastrointestinal maturation, glucocorticoids (GCs) are the most potent regulators. Exogenous GCs induce precocious changes in the gastrointestinal maturation in the first and second postnatal weeks. Although this regulation seems to be mediated by the GCs receptor pathway, the precise regulatory mechanism has not yet been documented. Some results have indicated that the effect of GCs may require interaction with other tissues or cell types, such as mesenchyme. Mammalian alpha-fetoprotein (AFP) is a single-chain glycoprotein with molecular mass ranging from 37 to 72 kDa and 3%–5% carbohydrate (glycan) content. Resent reports showed that the AFP was expressed and produced in mesenchymal cells and was considered as an important growth factor with a specific function in gastrointestinal development, including pancreas. Thus, AFP seems to play a role in GCs induced precocious gastrointestinal maturation. To address this issue, we undertook the following research works: 1. To investigate whether the AFP expresses in the rat colon during development; 2. To investigate whether there is a correlation between the expression of AFP and the precocious colon maturation induced by GCs.Part one: Colons from Sprague-Dawley rat fetuses (embryonic day 18.5 and 20.5), young (postnatal day 0, 7, 14 and 21) and adult (8 wk old) animals were used in this study. Expression levels of AFP were detected by reverse-transcriptase PCR (RT-PCR) and Western blotting. To identify the cell location of AFP in the developing rat colons, double-immunofluorescent staining was performed. Only 72 kDa isoform of AFP was detected by Western blotting. The highest levels of AFP mRNA and protein were detected in colons of rats at embryonic day 18.5. Then the expression of AFP was significantly decreased during rat development and undetected in adult rats. The positive staining for both AFP and vimentin was overlapped in mesenchymal cells at each stage tested.Part two: Young rats were treated with GCs by subcutaneous injection at the 7th, 9th and 11th days after birth. All animals were randomly divided into 4 groups: control group, administered with equal volume of normal saline; hydrocortisone group, administered with 100μg hydrocortisone per gram of body weight(100μg/g); hydrocortisone + mifepristone (a specific antagonist of hydrocortisone) group, administered with 100μg/g hydrocortisone and 100μg/g mifepristone together; mifepristone group, administered with 100μg/g mifepristone alone. Then the animals were killed at the 8th, 10th and 14th day of life and their colons were extracted. Routine hematoxylin-eosin staining was used to observe the morphological changes and the spontaneous contractile activity were recorded to determine the functional changes of colon. Expression levels of AFP in colons were also detected by RT-PCR and Western blot. Double-immunofluorescent staining was used to identify the cell location of AFP, like those in the Part One. Exogenous hydrocortisone induced significantly precocious changes in colon morphology and contractile function in suckling rats. Meanwhile, the levels of AFP mRNA and protein were increased by hydrocortisone administration. These results were confirmed by the proliferation of AFP positive cells. Mefipristone could significantly converse this effect.Our study has for the first time demonstrated that AFP is localized in the mesenchyme of rat colon. Exogenous GCs can elicit precocious maturation of the rat colon in both morphology and contractile function, in which accompanied by an increase in expression of AFP. AFP seems to play a role in GCs induced precocious rat colon maturation.