| Background:Necrotizing enterocolitis (NEC) is the most common inflammatory gastrointestinal disease in neonates with high morbidity and mortality. Terminal ileum and proximate colon are mainly involved in it. Clinically, abdominal distension, emesis and hemafecia are the main symptoms. In severe cases, there may accompany with intestinal perforation, peritonitis, shock, sepsis and other complications. Abdominal X-ray is characterized by cystic pneumatosis in the intestinal wall. Currently the exact causes and pathogenesis are not completely understood, there haven't been any specific prevention and therapeutic methods. Nowadays, it is considered that premature, improper feeding, infection and intestinal hypoxia-ischemia are the high risk factors of NEC, and NEC is considered to be the result of interaction of multiple factors. Under the effect of various risk factors, the epithelial permeability of intestine increases, then intestinal barrier is demolished, bacterial products such as LPS is invaded, which activating the immune system and then triggering a cascade of inflammation and focal necrosis. Glycomacropeptide (GMP) is a multi-functional polypeptide fragment generated by hydrolysis of chymosin toκ-casein protein in milk. It has various biological activities, such as anti-infection, immunity regulation, anti-inflammation, nutrition and health care. In view of this, GMP attracts extensive attention in the following fields: food, medicine and health care products, particularly in the field of infant food. Currently the research on GMP of boving milk has been widely reported, but for GMP of human milk is rarely reported. In addition, since GMP has special effect on the gut, it is possible to provide a new idea for the prevention and treatment of NEC.Therefore, the following studies will be carried out: (1) to detect GMP content in human milk, and take it as reference of infant formula milk powder for optimizing the nutritional content; (2) to establish an appropriate neonatal rat NEC model and to administrate GMP as intervention, then observe the items as follows:①whether GMP can reduce the intestinal injury in neonatal rats of NEC;②effects of GMP on the expression of PAF, TNF-αand IL-1β;③effects of GMP on intestinal epithelial cells apoptosis. All these studies will provide a theoretical basis for prevention and treatment of NEC by administration of GMP.Objective:1. To detect GMP content of human milk, and take it as reference of infant formula milk powder for optimizing the nutritional content.2. To establish an appropriate neonatal rat NEC model and to administrate GMP as intervention, then to discuss whether GMP can protect the gut from injury in neonatal rats with NEC, and to provide an experimental basis for prevention and treatment of NEC by administration of GMP.Methods:1. Selected 30 primipara fit the following conditions: healthy, no special diet habit, living stable, having adequate milk, age between 25 to 39 years old, term delivery. Then they were divided into 2 groups, the colostrum group and mature milk group, 15 cases in each group, each case had been collected breast milk 5ml (front milk). The gathering time of colostrum group was the 2nd day postpartum and mature milk group was the 42nd day postpartum. Before collecting samples, make sure that the nipple and the hands of collectors had been disinfected, then squeezed the breast milk directly into the sterile centrifuge tube artificially and labeled. Hydrolyze the breast milk by chymosin at 37℃, then centrifuged the hydrolysis products after 120 minutes, extract the supernatant liquids, at last the sialic acid content in the supernatant was detected by a sialic acid detection kit, and GMP content was represent by sialic acid content. In addition, 6 brands of formula milk powder (Wyeth, Dumex, Nestle, Abbott, MeadJohnson and Beingmate) were made standard liquid milk, which were detected in the same way as breast milk.2. 36 neonatal SD rats were randomly divided into 3 groups: group M (NEC model), group G (NEC+GMP) and group N (Normal control), each group including 12 rats. All the neonatal rats were fed with breast milk in the first 3 days after birth. During the second 3 days, the rats of group N were still maternal breast-fed, but the rats of group M and group N were separated with their mothers and lived in incubator began formula fed ( once every 4 hours, the rats of group M were fed with simulated milk only, the rats of group G were fed with glycomacropeptide and simulated milk), and were given cold exposure shortly after hypoxic-reoxygen treatment (once every 8 hours). Weights of all the rats were weighed at the same time during the course everyday. After feeding of 6 days, all the neonatal rats were placed into the incubator and fasting for 24 hours. Then all the rats were killed by cervical dislocation. Intestine tissue located at the boundary of ileum and cecum were obtained for: 1) histological examination by HE staining, 2) TUNEL detection, 3) electronmicroscope observation; the tissue homogenate was obtained for: 1) checking PAF mRNA expression by QF-PCR, 2) checking TNF-αand IL-1βlevel by ELISA. Results:1. Determination of GMP content of human milk and formula milk,and comparison between each other.1.1 Best conditions for enzymolysis are: chymosin concentration 0.25mg/ml, hydrolysis time 120min.1.2 GMP content of colostrum group was 3486.98±406.70 mg/L, mature milk group was 2706.38±344.83 mg/L, the former was significantly higher than the latter (P<0.01), but the differences between individuals within each group were small (CVcolostrum =0.12,CVmature milk =0.14).1.3 The average level of GMP content of various of infant formula was 1271.75±802.42mg/L, and was significantly lower than in colostrum and mature milk (P<0.01); GMP contents of various brands of formula were as follows: Wyeth SMA GOLD 2854.58mg/L, Dumex Nutrifant 1279.08mg/L, Dumex for LBW 1114.77mg/L, Nestle Pre NAN 558.19mg/L, Nestle NAN 611.35mg/L, Nestle LACTOGEN 655.65mg/L, Abbott Similac 898.90mg/L, MeadJohnson Enfamil A+ 940.79mg/L, Beingmate for Infant 1130.88mg/L, the difference among these brands was relatively big (CV=0.63).2. Effects of GMP on neonatal rats with NEC.2.1 Each group got weight increase, the increase in group G(2.02±0.14g) was more than group M[(1.57±0.39g), P<0.01], but both in Group M and Group G were less than group N[(3.53±0.46g), P<0.01].2.2 The pathological scores of 3 groups were 2.17±0.83(M), 0.92±0.79(G) and 0.17±0.39(N) separately. There was significantly difference between group M and group G (P<0.01).2.3 Expression levels of Intestinal PAF mRNA (2-ΔΔCt value): 3.01±0.96 (M) , 1.56±0.29 (G) , 1.01±0.13 (N), the level of group G was significantly lower than that of group M (P <0.01). 2.4 TNF-αlevels of 3 groups were 41.94±13.51pg/ml(M), 31.69±11.68pg/ml(G) and 17.42±7.18pg/ml(N) separately, and TNF-αlevel in group G was significantly lower than that of group M (P<0.05).2.5 IL-1βlevels of 3 groups were 150.33±36.41pg/ml(M), 118.36±33.00pg/ml(G) and 28.44±15.04pg/ml(N) separately, and IL-1βlevel in group G was significantly lower than that of group M (P<0.05).2.6 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.2.7 The apoptosis rate of intestinal epithelial cells by TUNEL detection: 38.79±9.79 (M), 29.54±7.30 (G), 6.37±1.96 (N); the apoptosis rate of intestinal epithelial cells of Group G was significantly lower than that of group M (P<0.05).Conclusion:1. GMP content of breast milk was higher than that of infant formula, and the content in colostrum was higher than that in mature milk; the nutritional content of infant formula should be further optimized.2. GMP has some protective effects on guts of neonatal rats with NEC, which may probably work by reducing PAF, TNF-αand IL-1βexpression, inhibiting the apoptosis of intestinal epithelial cells and reducing intestinal tissue injury. |
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