| The antioxidant enzymes system of intestine plays an important role in the intestinal oxidative stress. Firstly, we investigated the spatio-temporal expression pattern of antioxidant enzymes system, and the relationship between absorptive efficiency and antioxidant enzymes system in intestine; Secondly, we established a method of culturing the intestinal epithelial cells from chicken embryo, and established a oxidative stress model in vitro by treating the intestinal epithelial cells with hydrogen peroxide, and the involvement of Nrf2 and MAPK signaling pathway in regulation of oxidative damage was studied. This research could provide a theoretical foundation for the intestine development and intestinal damage.Experiment 1.The spatio-temporal expression of antioxidant enzymes system of the intestine in chicken. One hundred twenty 1-day-old Arbor Acres broilers were randomly assigned into 8 replicate with 15 birds per replicate. 8 birds from each replicate were killed, and their duodenum,jejunum,ileum, heart, liver and kidney tissue were collected at 1, 3, 7, 11, 14, 21 and 35 days for further detection. The results showed that antioxidant enzymes activity in intestine is lower than heart, liver and kidney; During intestine development, the activity of CAT and GPx in intestine significantly declined,and the expression levels of antioxidant enzymes system related genes and Nrf2 were also reduced(P <0.05). Activity of antioxidant enzymes and gene expression were different between intestine and intestinal mucosa. Result from immunohistochemistry showed intestinal antioxidant enzymes mainly expression in intestinal epithelial cells of villus. These results indicates that the expression of antioxidant enzymes reduced during intestinal development and Nrf2 may play a regulatory role in this process.Experiment 2. Analysis of intestinal antioxidant enzymes system in broiler chickens with different absorption efficiency.Selecting broilers with different absorption efficiency by using D-xylose absorption test. Antioxidant enzymes activity, intestinal morphology and expression of nutrient transporters of two groups in broilers were compared. At 24 days and 28 days of age, D-xylose absorption test was used to select serous D-xylose content of broilers and the content values were significant different(P <0.05), though the body weight and intestinal index were not different between the two groups; Serous T-SOD activity(P <0.05) and MDA(P =0.10) in the high absorption efficiency broilers were lower than that of the low absorption of broilers; Activity of CAT in the ileum were significantly different(P <0.05); The villus height and the villus: crypts ratio of ileum in high absorption efficiency group were significantly higher than the low absorption efficiency group(P <0.05); The expression of SGLT1, CAT1, CAT2(B) and b0, + AT m RNA in two groups were different(P <0.05). These results suggests that D-xylose absorption test could be used to select in broilers with different absorption efficiency. Intestinal morphology and expression of nutrient transporters may cause this difference, but the absorption efficiency were not relationship with intestinal antioxidant enzymes system.Experiment 3. Isolation, culture, and verification of chicken intestine epithelial cells. The SPF chicken embryo with 18 embryonic age was selected to separate duodenum, and the intestinal epithelial cell were obtained by using mechanical squeezing and digestion with type ? collagenase. These cells grew with a cobblestone-like monolayer pattern and could be observed by microscope after 48 h. Epithelial shape could be identified by Giemsa staining and H.E staining. The microstructure of microvillus, desmosomes and tight junctions could be observed by electron microscopy. Furthermore,cultured cells were identified by specific protein expression of alkaline phosphatase, keratin 18 and Claudin-1. These results showed that intestinal epithelial cells were able to obtained and cultured succesfully.Experiment 4. Effect of oxidative stress on intestinal epithelial cells. 5 mmol/m L and 10 mmol/m L of hydrogen peroxide were used to stimulate chicken primary intestinal epithelial cells for 1h. The results showed that hydrogen peroxide decresed cellular antioxidant enzymes expression significantly, and oxidative damage significantly increased in dose dependent manner(P <0.05); Phosphorylated protein level of ERK1/2, JNK and p38 were up-regulated by oxidative stress in intestinal epithelial cells(P <0.05). The expression of Nrf2 and its downstream genes were significantly down-regulated by oxidative stress in intestinal epithelial cells(P <0.05). These results indicated that oxidation- antioxidant balance was destroyed in intestinal epithelial cells. MAPK signaling pathway was activated and Nrf2 signaling pathway was inhibited during acute oxidative damage. |
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