| objective: To simulating neonatal asphyxia,a model of intestinal mucosal injury in young rats with acute hypoxia was established,glutamine(Gln)and autophagy inhibitor 6-amino-3-methylindole(3-MA)were used to detect the expression of autophagy-related proteins Beclin 1 and LC3-II in the small intestinal mucosal injury model,the protective effect of Gln on intestinal mucosal epithelial cells in young rats with acute hypoxia-induced intestinal mucosal injury was observed and whether the protective effect of Gln on intestinal mucosal epithelial cells in young rats with acute hypoxia-induced intestinal mucosal injury was related to the change the level of autophagy was explored.Methods:120 SD rats of 6 days old are randomly divided into four groups: A,B,C and D,all of which were fed freely by the mother.Group A was the model control group,group B was Gln group,group C was Gln+ 3-MA group,and group D was 3-MA group,30 ratd in each group.30 minutes before the model was established,group A and group B were given intraperitoneal injection of 1 ml 0.9% normal saline,group C and group D were intraperitoneally injected with 3-MA(dose of 20 mg/kg,dissolved in 1ml of normal saline).The suckling rats were placed in a simple anoxic tank with an oxygen concentration of 3%,and nitrogen was continuously ventilated for 10 min at a flow rate of 1.5 L/min.After the model was established,rats ingroups B and C were simultaneously intragastrically administered with Gln0.3g/kg(concentration concentration: 20 g/L,dissolved in physiological saline),and then intragastrically administered once a day.At the time points 0h,6h,24 h,48h,and 72 h of each experiment,6 young rats were randomly selected and the rats were sacrificed by cervical dislocation immediately after the blood was taken.The suckling rats were quickly dissected,and the small intestine was observed.The small intestine tissue specimens were taken.The blood taken out at each time point was allowed to stand at room temperature for 2 hours,and then centrifuged(3000 rpm,15 min)to take the supernatant,and the expression of IL-1,TNF-?,and HIF-1a was detected by ELISA;the sample of the small intestine was taken to prepare paraffin.The pathological changes of small intestinal mucosa were observed under microscope by sectioning and HE staining.The expressions of Beclin 1 and LC3-II protein in small intestine were detected by immunohistochemistry and Western blotting.Results:1.Pathological changes of small intestinal mucosa: Group D(3MA)The degree of swelling was the heaviest.Group B(Gln)was the lightest,A(control group)and C(Gln+3MA)were between the two.Under the HE staining microscope,the intestinal epithelial structure began to appear pathologically in the four groups.The pathological changes were most severe at 24h;the pathological changes gradually decreased at 48h;the pathological changes gradually recovered at 72h;the pathological scores of group B(Gln)at each time point were lower than those of group A(control group),group D(3MA).The score was significantly higher than that of group A(control group),and the pathological score of group C(Gln+3MA)was higher than that of group B(Gln).The expression of IL-1,TNF-? and HIF-1a in serum: IL-1,TNF-? and HIF-1a in the four groups had the highest concentration at 6h,began to decrease at 24 h,and gradually recovered at 48h-72 h.Except for 0h,the expressions of IL-1,TNF-? and HIF-1a in group B(Gln)were significantly lower than those in group A(control group),group D(3MA)IL-1,TNF-? and HIF-The expression of 1a was significantly higher than that of group A(control group).The expression levels of pro-inflammatory factors IL-1,TNF-? and HIF-1a in group C(Gln+3MA)were lower than those in group D(3MA).The expressions of Beclin 1 and LC3-II protein in small intestine were detected by immunohistochemistry.The differences of immunohistochemical evaluations of LC3-II and Beclin 1 in the four groups except for 0h were statistically significant(P<0.05).The expression of Beclin 1 and LC3-II in intestinal epithelial cells was minimal at 0h in each group,and increased at 6h and 24 h,and decreased at 48 h and 72 h.Except for 0h,the staining of Beclin 1 and LC3-II at each time point of group D(3MA)was weaker than that of group A(control group),and the average optical density value was lower than that of group A(control group);The staining of Beclin 1 and LC3-II in group B(Gln)was stronger than that of group A(control group)at every time point,and the average optical density value was higher than that of group A(control group);each time point The staining of Beclin 1 and LC3-II in group B(Gln)and group C(Gln+3MA)were was stronger than that of group D(3MA),and the average optical density value was higher than that of group D(3MA).The expression of Beclin 1 and LC3-II protein in small intestine was detected by Western Blot:the gray scale ratios of Beclin 1 and LC3-II protein in the four groups were statistically significant(P<0.0001).The gray value of Beclin 1 and LC3-II protein began to rise at 6h,and began to fall after 24 h,and gradually recovered to the basal level at 72 h.Compared with group A(control group),group D(3MA)had less expression of LC3-II and Beclin 1 in small intestine tissue of group D(3MA);group B(Gln)compared with group A(control group)at each time point,group C(Gln+3MA)Compared with the D(3MA)group,the expression levels of LC3-II and Beclin 1 in the small intestine were higher than those in the latter.Conclusion:1.In the environment of oxygen concentration of 3% and hypoxia for 10 minutes ntestinal mucosal epithelial cells damage in young rats can be caused i,and the expression of autophagy-related proteins Beclin 1 and LC3-II in small intestine increases.After the autophagy inhibitor3-MA was applied.,the expression of autophagy-related proteins was attenuated.2.Gln can reduce the levels of proinflammatory cytokines IL-1 and TNF-? in the serum of acute hypoxic young rats,and reduce the pathological damage of intestinal mucosa,showing the role of protecting intestinal epithelial cells.Gln can promote the expression of autophagy-associated proteins Beclin1 and LC3-II in small intestinal mucosa induced by acute hypoxia,suggesting that Gln is involved in the protection of injured intestinal mucosa and autophagy. |
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