Mammalian Target of Rapamycin Signaling Pathway Changes with Intestinal Epithelial Cells Renewal Along Crypt-Villus Axis

Huansheng Yang, Xia Xiong, Xiaocheng Wang, Yulong Yin
2016 Cellular Physiology and Biochemistry  
Background/Aims: Understanding the mechanism that involves in regulating epithelial cells renewal is the fundamental of regulating intestinal mucosa development and functions and related diseases. The mechanistic target of rapamycin (mTOR) signaling pathway involves in controlling various major processes by integrating intracellular and extracellular cues. The present experiment was conducted to test the correlation between the mTOR signaling pathway and intestinal epithelial cells renewal
more » ... cells renewal along crypt-villus axis (CVA). Methods: Intestinal epithelial cells were sequentially isolated from the jejunum of piglets along CVA, and the amount or phosphorylation level of proteins involved in cell cycle, mTOR signaling pathway, gene expression, and the antioxidant capacity in the isolated cells were measured. Results: The results showed that the amount of proteins involved in cell cycle decreased from crypt to villus tip. The amount or phosphorylation level of proteins related to mTOR signaling pathway in intestinal epithelial cells mainly decreased during maturation along CVA. The amount of proteins involved in gene expression and the antioxidant capacity also decreased from crypt to the top of villi. Conclusions: These results indicate that the mTOR signaling pathway may be involved in regulating the intestinal epithelial cells renewal along CVA and it may partly through affecting the antioxidant capacity and gene expression of intestinal epithelial cells. Further histological verification is needed to confirm the results of the present experiments. H. Yang and X. Xiong contribute equally to this work. μm particles (Phenomenex, USA) and then by Strata X C18 chromatography (Phenomenex, USA). A splitless nanoACQuity (Waters, USA) system coupled to Triple TOF was used for analytical separation. Mascot software (version 2.3.02, Matrix Science) was used to simultaneously identify and quantify proteins. Searches were made against the NCBI non-redundant database consisting of mammalian proteins. In order to select differentially expressed proteins, we used the following criteria: 1) the proteins must contain at least two unique high-scoring peptides (peptide confidence > 95%) and 2) proteins must have ratios higher than 1.2 or lower than 0.8, with the relative quantified p-values below 0.05 [12] . The WEGO program was used to classify and group the differentially expressed proteins [16] . The proteins related to gene expression were clustered using Cluster 3.0 [17]. Statistical analysis All data were subjected to t test using the SAS version 9.2 Program. Data were presented as means ± SEM, and probability values < 0.05 were taken to indicate statistical significance. Fig. 3 . Relative expression of proteins involved in gene expression along CVA. Relative total protein levels of proteins involved in gene expression were quantified using iTRAQ. The differentially expressed proteins were classified and selected by WEGO program. The proteins involved in gene expression were clustered using Cluster 3.0. Fig. 4 . Antioxidant capacity along CVA. (A) total antioxidant capacity in each cell fraction was quantified (n = 6). Significant differences: relative total protein levels of total antioxidant capacity compared to F1, P < 0.05. All results are expressed as the mean ± SEM. (B) the contents of malondialdehyde in each cell fraction were quantified (n = 6). Significant differences: relative total protein levels of total antioxidant capacity compared to F1, #relative total protein levels of total antioxidant capacity compared to F2, P < 0.05. All results are expressed as the mean ± SEM. (C) the activity of catalase in each cell fraction was quantified (n = 6). Significant differences: * relative total protein levels of total antioxidant capacity compared to F1, P < 0.05. All results are expressed as the mean ± SEM. (D) the activity of superoxide dismutase in each cell fraction was quantified (n = 6). Significant differences: * relative total protein levels of total antioxidant capacity compared to F1, P < 0.05. All results are expressed as the mean ± SEM.
doi:10.1159/000445665 pmid:27459644 fatcat:trokshh4yzcszjfz753hg3slhm