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Objective: To investigate the expression and role of programmed death ligand-1 (PD-L1) in a mouse model of necrotizing enterocolitis (NEC)., Methods: A total of 20 wild-type C 57 BL /6 J mice were randomly assigned to the control and the model groups. Mice in the control group were breastfed, while mice in the model group were given lipopolysaccharide, formula feeding, hypoxia, and cold stimulation for NEC induction. Then, the intestines of the mice were collected in order to assess the pathological changes through HE staining, to examine PD-L1 expression and localization with immunofluorescence co-localization, and to evaluate intestinal PD-L1 expression with Western blot. Peripheral blood was collected for flow cytometry to examine leukocyte subpopulations and their PD-L1 expression. On the other hand, 14 PD-L 1 (+/+) mice and 14 PD-L 1 (-/-) mice were randomly divided into their respective genotype control groups and model groups. The same induction method as was already mentioned was adopted for the model groups. The intestines of the mice were collected for HE staining to evaluate the pathological change and peripheral blood was collected to examine the expression of inflammatory factors., Results: The NEC mouse model was successfully constructed. PD-L1 was widely expressed in enterocytes and inflammatory cells in the mouse intestines and in T cells, monocytes, and neutrophils in peripheral blood. The expression of PD-L1 in NEC mouse intestines increased in comparison with that of the control group. In the peripheral blood of NEC mice, the proportion of T cells and monocytes and their PD-L1 expression showed no significant changes compared with those of the control group, while the proportion of neutrophils and their PD-L1 expression increased by about 140% and 150%, respectively, in comparison with those of the control group ( P <0.05). According to the results of the PD-L 1 gene mouse experiment, the control groups of PD-L 1 (+/+) mice and PD-L 1 (-/-) mice showed no significant difference in their intestinal conditions and serum inflammatory factor levels, while the PD-L 1 (-/-) NEC mouse had worse intestinal pathological changes and increased mean pathological scores compared with those of PD-L 1 (+/+) NEC mouse ( P <0.05). In addition, serum interleukin (IL)-10 in PD-L 1 (-/-) NEC mouse decreased by about 44% compared with that of PD-L 1 (+/+) NEC mice, and chemokine (C-X-C motif) ligand 1/IL-6/IL-1β all increased by more than 25% (all P <0.05)., Conclusion: PD-L1 is widely expressed in inflammatory cells and enterocytes in mice. Knocking out PD-L 1 aggravates the degree of NEC inflammation and intestinal pathological changes. PD-L1 plays a protective role by reducing inflammation in the pathogenesis of NEC, the mechanism of which may be related to the regulation of neutrophils/enterocytes., (Copyright© by Editorial Board of Journal of Sichuan University (Medical Sciences).)

Objective: To establish an appropriate neonatal rat model of necrotizing enterocolitis (NEC) and to investigate the protective effects of glycomacropeptide (GMP) on the gut from injury in neonatal rats with NEC., Method: A total of 36 neonatal SD rats were randomly divided into 3 groups: NEC model group (Group M), NEC + GMP group (Group G) and normal control group (Group N), each group had 12 rats. All the neonatal rats were fed with breast milk in the first 3 days after birth. During the second 3 days after birth, the rats of Group N were still maternal breast-fed, but the rats of Group M and Group G were separated from their mothers and lived in incubator and began to be formula fed, and were subjected to cold exposure shortly after hypoxic-reoxygenation treatment. After being fed in such means for 6 days, all the neonatal rats were placed into the incubator and fasted for 24 hours. Then all the rats were sacrificed by cervical dislocation. Intestinal tissue located at the boundary of ileum and cecum was obtained for: (1) histological examination after HE staining, (2) TUNEL detection, (3) electron microscopic observation; and the tissue homogenate was obtained for checking TNF-α and IL-1β levels by ELISA and platelet activating factor (PAF) mRNA expression by quantitative fluorescence (QF)-PCR., Result: (1) The pathological scores of the 3 groups were 2.17 ± 0.83 (Group M), 0.92 ± 0.79 (Group G) and 0.17 ± 0.39 (Group N) separately. There was significant difference between Group M and Group G (H = 8.819, P = 0.003). (2) TNF-α levels of 3 groups were (41.94 ± 13.51) pg/ml (Group M), (31.69 ± 11.68) pg/ml (Group G) and (17.42 ± 7.18) pg/ml (Group N) separately, and TNF-α level in Group G was significantly lower than that of Group M (F = 3.959, P = 0.030). (3) IL-1β levels of 3 groups were (150.33 ± 36.41) pg/ml (Group M), (118.36 ± 33.00) pg/ml (Group G) and (28.44 ± 15.04) pg/ml (Group N) separately, and IL-1β level in Group G was lower than that of Group M (F = 5.080, P = 0.013). (4) Expression levels of intestinal PAF mRNA (2(-ΔΔCt) value): 3.01 ± 0.96 (Group M), 1.56 ± 0.29 (Group G), 1.01 ± 0.13 (Group N), the level of Group G was significantly lower than that of Group M (F = 25.251, P = 0.000). (5)Electron microscopy: Group N showed that its cell volume was mostly occupied by the nucleus, the structure was clear, nuclear membrane existed, suggesting the normal phase of cell; Group M showed that apoptotic body existed, suggesting that the advanced stage phase of apoptosis; Group G showed that condensed chromatin marginated around the nuclear envelope, nuclear pores expanded, suggesting the early phase of apoptosis. (6) The apoptosis rate of intestinal epithelial cells by TUNEL detection: 38.79 ± 9.79 (Group M), 29.54 ± 7.30 (Group G), 6.37 ± 1.96 (Group N); the apoptosis rate of intestinal epithelial cells of Group G was significantly lower than that of Group M (F = 6.888, P = 0.003)., Conclusion: GMP has protective effects on guts of neonatal rats with NEC, which may probably work by reducing TNF-α, IL-1β and PAF expression, inhibiting the apoptosis of intestinal epithelial cells and reducing intestinal tissue injury.

Objective: To study the expression of Toll-like receptor 4 (TLR-4) and caspase-3 in the intestine of neonatal rats with necrotizing enterocolitis (NEC), and explore the protective effects and possible regulatory mechanisms of glutamine (Gln) in NEC., Methods: Sixty premature rats were randomly divided into three groups (n=20 each): control, NEC model and Gln intervention group. NEC model was prepared by formula feeding, hypoxia and cold stress. The Gln intervention group was also subjected to hypoxia and cold stress but was fed with formula containing Gln (0.3 g/kg). Two days later, the rats were sacrificed and the intestine tissues were obtained. The histological changes of ileal tissues were observed by hemetoxylin and eosin staining. The expression of caspase-3 and TLR-4 protein in the jejunum, ileum and colon were detected by inmunohistochemistry. The expression of TLR-4 mRNA in the jejunum, ileum and colon were detected by RT-PCR., Results: Compared with the control group, the histological score of ileal tissues, and the expression of caspase-3, TLR-4 protein and TLR-4 mRNA in the NEC model group increased significantly (P<0.01). Gln intervention decreased significantly the histological score of ileal tissues, and the expression of caspase-3, TLR-4 protein and TLR-4 mRNA compared with the NEC model group (P<0.05)., Conclusions: TLR-4 might be involved in the pathogenesis of NEC. Gln may provide protective effects on intestine possibly through reducing the TLR-4 expression and then decreasing the apoptosis of intestinal epithelial cells.