Glutamine downregulates TLR-2 and TLR-4 expression and protects intestinal tract in preterm neonatal rats with necrotizing enterocolitis.

BACKGROUND/PURPOSE: Toll-like receptor (TLR)-4 and TLR-2 play an essential role in the pathogenesis of necrotizing enterocolitis (NEC). In this study, we investigated the protective effect of glutamine (Gln) in an NEC neonatal rat model, and the potential association with TLR-4 and TLR-2 expression in local intestinal tissues. METHODS: Preterm neonatal rats were randomly divided into 3 groups: normal control; NEC model; and NEC plus Gln intervention. NEC was induced by feeding with artificial milk substitutes, plus exposure to hypoxia and cold stress. All preterm rats were sacrificed at 3 days after birth. The intestinal tissues were taken for pathological analysis. Protein and mRNA expression of TLR-2, TLR-4, and caspase-3 was examined by immunohistochemistry and real-time RT-PCR, respectively. RESULTS: Compared with the normal control, the NEC neonatal rats showed mucosal injury and upregulated mRNA and protein expression of TLR-2, TLR-4, and caspase-3 in ileum and colon. Gln intervention significantly reduced the mucosal injury and suppressed the upregulated expression of TLR-2, TLR-4, and caspace-3 in the ileum and colon of NEC neonatal rats. CONCLUSIONS: Gln protects the intestinal tract of NEC neonatal rats, which may be associated with the reduction of TLR-2 and TLR-4 expression in intestines.

[Effects of glycomacropeptide on damage to intestinal tissue and apoptosis of intestinal epithelial cells in neonatal rats with necrotizing enterocolitis].

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-1ß 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-1ß 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-1ß 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-1ß and PAF expression, inhibiting the apoptosis of intestinal epithelial cells and reducing intestinal tissue injury.

Establishment and evaluation of three necrotizing enterocolitis models in premature rats.

Necrotizing enterocolitis (NEC) is a gastrointestinal disease that usually affects premature infants and has high morbidity and mortality rates. Reliable animal models aid further study of the etiological factors, pathogenesis, prevention and treatment of NEC. The present study aimed to establish NEC models in premature rats using three common methods, and to determine the optimal model establishment method. The study consisted of six groups; in group A, rats were raised with simulated milk and subjected to inhalation of 100% nitrogen gas (hypoxia) for 90 sec, followed by exposure to cold ambient conditions (4˚C) for 10 min twice daily for 3 days. In group B, rats were exposed to 100% nitrogen gas for 5 min and 100% oxygen for 5 min twice daily for 3 days. Group C rats were intraperitoneally administered 5 mg/kg lipopolysaccharide. Group D and E rats did not receive any intervention. Group F rats were intraperitoneally administered 1 ml/kg physiological saline. Groups D-F served as the control groups corresponding to groups A-C, respectively. Following hematoxylin and eosin staining, intestinal tract, liver, lung and kidney tissues were observed under optical microscopy and were scored. Successful NEC induction was measured by a score of ≥2. Rats from groups A-C exhibited reduced movement, abdominal distention, diarrhea, intestinal tract expansion, and congestion to varying degrees. The pathological scores of intestinal injury in groups A-F were 3.13±0.64, 1.40±0.52, 2.00±0.42, 0.30±0.48, 0.30±0.48, and 0.40±0.52 points, respectively. Significant differences were found between the model groups and their corresponding control groups (p<0.01). Among the model groups, the histological score of group A was higher than that of groups B (p<0.01) and C (p<0.05). The morbidity rate of NEC in groups A-C was 75, 20 and 50%, respectively. There was no morbidity in groups D-F. Compared with groups A and B, injury to the liver, kidney and lung was more severe in group C. Similar symptoms were not observed in groups D-F. Compared with methods of simple hypoxia-reoxygenation or intraperitoneal administration of lipopolysaccharide, the combination of artificial feeding and hypoxia plus cold stimulation most resembles the pathological causes of neonatal NEC. This method resulted in high morbidity, reproducibility and specificity, and was therefore considered an ideal model for establishing NEC.

[Regulatory effects of glutamine on Toll-like receptor 4 in neonatal rats with necrotizing enterocolitis].

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.