The Changes And Significance Of Autophagy In Hypoxic Intestinal Epithelial Cells

Objective : Autophagy is an intracellular degradation process by which cytoplasmic proteins,other substances and organelles are degraded by lysosomes in turn so as to meet the demands of the cell metabolism and organelles constantly updated. Autophagy plays a vital role in cell growth, development and Ischemia or hypoxia. Severe trauma and burns can cause hypovolemic shock,which can effectively reduce blood volume,leading to ischemia and hypoxia of the whole body. To ensure the blood-supply of brain, heart and other important organs, the blood redistribution happens, which means the vasoconstriction in skin, abdominal viscera and skeletal muscle, but not in the heart and brain. The intestinal tract is the most commonly affected organ in response to blood redistribution, leading to the impairment of both structure and function in intestinal mucosa because of the hypoperfusion in intestinal tissue, increased demand for oxygen and hypermetabolism. So far, the schemia and hypoxia-induced autophagy of gastrointestinal epithelial cells has not yet been clarified. In order to determine the effect of hypoxia on the autophagy of intestinal epithelial cells, this study investigated the dynamic changes of autophagy in human intestinal epithelial cell Caco-2 by using the model of hypoxia in vitro to imitate the ischemia and hypoxia in the gastrointestinal tract, laying the foundation for the study on the role of autophagy in intestinal epithelial cell damage after hypoxia. Methods:1.The normal cultured human intestinal epithelial cell lines Caco-2 were divided into normoxia group(normal control group) and hypoxia group. In hypoxia group, cells were exposed to hypoxia for 0.5,1,2,6,12 and 24 h. The cells total protein were extracted, and the protein concentration was measured.2.The protein expresssions of Beclin1, LC3 and P62 were assayed by Western blot to reflect the dynamic changes of autophagy-related molecular level in each time point under hypoxia condition.3.The cellular autophagic lysosomals were detected by transmission electron microscopy before hypoxia and after hypoxia for 6 hour.4.The GFP-LC3 B fusion protein was assessed to visualize autophagosome formation in intestinal epithelial cells by the Laser Scanning Confocal Microscope before hypoxia and after hypoxia for 6 hour. Results:1.The protein expressions of both Beclin1 and LC3 began to increase at 0.5h in hypoxic intestinal epithelial cells when compared with those in normal control, peaked at 12 h, but reduced at 24 h and still significantly higher than in normal control. The expressions of P62 decreased gradully after hypoxia, with the lowest at 24 h..2.Under transmission electron microscopy, it was observed that the numbers of autophagy lysosomal were increased significantly at 6h in hypoxic intestinal epithelial cells. The results show that autophagy was increased in intestinal epithelial cells.3.GFP-LC3 B fusion proteintracer also showed that Intracellular green fluorescent spots increased significantly and autophagosome formation was quite evident at 6h in hypoxic intestinal epithelial cells. Conclusions:1.The autophagy is significantly increased in intestinal epithelial cells after hypoxia, which, to a certain extent, goes up gradually as the hypoxic time extended.2.The protein expressions of both Beclin1 and LC3 increase significantly in hypoxic intestinal epithelial cells, whereas the expression of P62 protein decreases significantly in hypoxic intestinal epithelial cells. The autophagy may contribute to the regulation of hypoxia-induced damage of intestinal epithelial cells.