| Objects:Milk fat globule-epidermal growth factor 8(MFG-E8), a glycoprotein secreted from macrophages and dendritic cells,can bind to phosphatidylserine and integrin receptors. The expression of intestinal MFG-E8 decreased in the severe period and increased in the recovery phase in a variety of intestinal injury models such as intestinal ischemia reperfusion(I/R) injury,sepsis,colitis.Treatment by recombinant murine MFG-E8(rmMFG-E8) accelerates intestinal mucosa damage repair dramaticlly.MFG-E8 plays a crucial role in maintenance and repair of murine intestinal epithelium,which makes it become a great potential in the prevention and treatment of intestinal injury in patients with severe systemic inflammatory response.However,the research about MFG-E8 mainly concentrated on the intestinal inflammatory injury at present, if it can be used as a nutritional agent has not been reported.First,we observed the change of intestinal villi and MFG-E8 expression of fasted mice to investigate the role of MFG-E8 in intestinal injury after long-term starvation.Glutamine is the most abundant amino acid in the human body and considered to be conditionally essential to the body during severe injury and illness. It will produce many seriously complications and affect the prognosis of patients if the lack of body glutamine is not corrected in time.A large number of literature has reported the intestinal protection of glutamine in mice after starvation. But the specific mechanism remains unkown.Hence,we speculated that whether glutamine can play the role of intestinal protection by changing the expression of MFG-E8. and observe the role of MFG-E8 in intestine injury of hungry mice. Finally,MFG-E8 mRNA was expressed by macrophages in the lamina propria of murine small intestinal.We cultivate RAW264.7 cells using different concentrations of glutamine to detection the expression of MFG-E8 in vitro.Methods:Part 1:To verify whether starvation can change the expression of intestinal MFG-E8.Through an pre-experiment and the related reports, we learn that mice show obvious stress behavior after fasting for 24 h,significantly decreased activity after 48 h, become very weak after 72 h and begin to die after 84 h.The weight loss of hungry mice begins to be flatten in 72 h after fasting, so we choose mice fasted for 72 h to research. Twenty C57BL/6 male mice were randomly divided into two groups: normal control group(NC,n=10) and starvation group(S,n=10).S group drank water only and no food supply.The mice were sacrificed in 72 hours after starvation.The intestine specimens were sampled for histological examination by HE staining and cell apoptosis detection by TUNEL assay. The expression of MFG-E8 mRNA and protein was measured by Real-Time PCR and Western Blot.Part 2: To investigate the effect of glutamine on intestinal MFG-E8 expression in hungry mice.Twenty-five C57BL/6 male mice were randomly divided into five groups: normal control group(NC,n=5), starvation group(S,n=5)and glutamine group(G). G group received Glu 1.0 or 3.0 or 5.0 g/kg by gastric feeding once a day from the day of starvation and named G1 or G3 or G5 group respectively. NC group ate food and drank water freely.S and G group drank water only and no food supply.The mice were sacrificed in 72 hours after starvation.The intestinal villus development,cell apoptosis and the expression of MFG-E8 mRNA and protein were measured just as part2. Part3:To investigate the effect of different concentration of glutamine on RAW264.7 cell proliferation and of MFG-E8 expression in vitro.The concentration of glutamine in normal rodents and human serum is approximately 0.6mM.The concentration of normal cells cultivation and long-term storage of glutamine is 2mM. Greater than or equal to 8mM of glutamine concentration can induce a variety of cells and tissues to express HSPs and inhibit cells inflammatory responses.So we choose 0, 0.6, 2, 10 mM glutamine to incubate RAW264.7 cell to measure the cell proliferation and the influence of expressing MFG-E8.Results: Part 1: The intestinal mucosa became atrophic after starvation. The intestinal mucosa was sparse and thin,and the villus heights,crypt depths and villus areas were significantly reduced after fasting for 72 h. At the same time,the starvation increased apoptosis of intestine epithelial. In addition,we observed that the expression of MFG-E8 protein and mRNA was increased after starvation.Part2:Glutamine,especially the concentration of 3g/kg/d,ameliorated villous atrophy and epithelial apoptosis caused by fasting.Furthermore,we found that glutamine decreased the MFG-E8 protein and mRNA expression compared to starvation group.Interestingly,we could see that the decrease of MFG-E8 mRNA and the concentration of glutamine were dose dependent.Part3:In vitro,glutamine played an important role to the proliferation of RAW264.7 cells.Higher glutamine concentration increased RAW264.7 cell proliferation. RAW264.7 cells showed no growth or even negative growth in the case of lacking glutamine.While the relationship between MFG-E8 expression and glutamine concentration was negative.Conclusion: Glutamine improved the atrophy of small intestinal villus and reduced the intestinal epithelial cell apoptosis of fasting mice.The finding confirms that glutamine has a protective effect on intestine. Meanwhile, we found the MFG-E8 protein and mRNA expression was increased after starvation and glutamine made the change small.This phenomenon suggests that MFG-E8, a kind of protein which closely related to intestinal mucosa damage repair, can be used as a sign of intestinal compensatory response after starvation. The rise of MFG-E8 may be a self protective response after injury of small intestine starvation.Therefore, focusing on MFG-E8 expression can acquaintance the intestinal function so that people can take appropriate intervention measures timely.In vitro, low concentration glutamine inhibited RAW264.7 cells proliferation but expressed MFG-E8 highly.we can draw a conclusion that glutamine do not effect the MFG-E8 expression through macrophage number. |
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