Effects of glycomacropeptide in human milk and formula milk on proliferation ofbiifdobacterium infantis in vitro / 中华围产医学杂志

ObjectiveTo explore the effects of glycomacropeptide (GMP) in human milk and formula milk on proliferation ofbifidobacterium infantis and their dose-response relationship.Methods Casein was isolated from the milk of 30 healthy postpartum women from Guangzhou Women and Children's Medical Center in September 2014, and hydrolyzed by rennet to obtain GMP, which was then purified by ultrafiltration and ion exchange chromatography. Human milk GMP and cow milk GMP (0, 250, 500, 1 000, 1 500, 2 000 and 3 000 mg/L) were added tobifidobacterium infantis liquid medium, and cultured under anaerobic conditions. Concentration of bacteria was measured by turbidimetric microplate assay (detection of OD600 nmvalue of medium). Difference of proliferative activities ofbiifdobacterium infantis in human milk GMP and cow milk GMP was compared with independent samplest-test.ResultsPurified human milk GMP concentration was 1 712.20 mg/L, with a purity of 80.3%. Increasing the cow milk GMP initial concentration in the culture medium at 250-2 000 mg/L could increase the concentration and proliferation rate ofbiifdobacteria infantis. When cultured at 36 h with GMP of various concentrations, the proliferation ofbiifdobacteria infantis maintained at a logarithmic phase. Therefore, 36 h was chosen as the test time point to compare the proliferation ofbifidobacterium infantis. At 36 h, when GMP in the medium was 1 000, 1 500, 2 000 and 3 000 mg/L, concentrations ofbiifdobacteria infantis in human milk GMP were 2.255±0.036, 2.583±0.088, 2.877±0.080 and 3.219±0.081, respectively, which were significantly higher than those in cow milk GMP (2.115±0.053, 2.312±0.064, 2.542±0.090 and 2.894±0.076;t=4.867, 5.569, 6.192 and 6.516; allP<0.01).Conclusions Both human milk GMP and cow milk GMP can promote the proliferation ofbiifdobacterium infantisin vitro, and the proliferative activity in human milk is greater than in cow milk at the same concentration of GMP.

Regulation of bifidobacterium on Wnt/β-Catenin signal pathway of intestinal tissue in newborn rats with ne-crotizing enterocolitis / 中华实用儿科临床杂志

Objective To study the effect of bifidobacterium on intestinal tissue of necrotizing enterocolitis (NEC)in newborn rats and its regulation of Wnt/β-Catenin signal pathway.Methods Seventy -five newborn SD rats were randomly divided into 5 groups,and each group had 1 5 rats.Group A was artificial feeding control group;group B was NEC model group;group C was bifidobacterium treatment group;group D was artificial feeding +bifidobacterium control group;group E was rat breast feeding control group.The localization expression of Toll -like re-ceptor 4(TLR4)of ileocecal ileum tissue was detected by immunohistochemical detection,and also the equivalen-tileum tissues were detected for the contents of glycogen synthase kinase -3β(GSK3β)and β-Catenin expression by Wes-tern blot.Comparing the differences of these indicators between the groups,in addition,the data of TLR4,GSK3βandβ-Catenin were analyzed by Bivariate correlations.Results The levels of TLR4 in ileum tissue of 5 groups were 0.36 ±0.03,0.48 ±0.05,0.34 ±0.03,0.37 ±0.04,0.35 ±0.02.The levels of GSK3βin ileum tissue of 5 groups were 0.98 ±0.23,1 .48 ±0.42,0.99 ±0.20,0.56 ±0.1 7,0.60 ±0.1 5.The levels of β-Catenin in ileum tissue of 5 groups were 1 .48 ±0.22,0.64 ±0.55,1 .27 ±0.36,1 .72 ±0.51 ,1 .82 ±0.44.The levels of TLR4 and GSK3βin ileum tissue of group B were significantly increased compared with group E (P <0.05).The levels of β-Catenin sig-nificantly decreased compared with group E (P <0.05).The levels of TLR4 and GSK3βin ileum tissue of group C were significantly decreased compared with group B (P <0.05).The levels of β-Catenin significantly increased com-pared with group B (P <0.05).Negative correlation was observed between the levels of GSK3βand β-Catenin(r =-0.592,P <0.05),while positive correlation was observed between the levels of TLR4 and GSK3β(r =0.295,P <0.05),and negative correlation was observed between the levels of TLR4 and β-Catenin(r =-0.426,P <0.05). Conclusions Bifidobacterium has certain protective effect on the NEC newborn rat intestines,which can reduce the in-cidence of experimental NEC and the severity of intestinal injury.Its effect may be achieved by regulating the Wnt/β-Catenin signal pathway,which decreases the expression of the level of GSK3βand increases the level of repair fac-tor β-Catenin.

Protective Effects of Biifdobacterium on intestinal tissue of newborn rats with necrotizing enterocolitis and its regulation / 中华围产医学杂志

Objective To discuss the possible molecular mechanisms involved in the protective effects of Biifdobacterium on intestinal tissue of necrotizing enterocolitis (NEC) newborn rats. Methods Seventy-five newborn Sprague-Dawley rats (born within 2 h) were randomly divided into five groups, each group with 15 rats. Group A was the NEC model group, and the rats were fed lipopolysaccharide (LPS) and formula. Group B was the Biifdobacterium treatment group, and the rats were fed LPS and formula and Biifdobacterium micro-capsule. Group C was the artificial feeding control group, and the rats were fed formula. Group D was the Biifdobacterium control group, and the rats were fed formula and Biifdobacterium micro-capsule. Group E was the breastfeeding control group, and the rats were fed rat breast milk by mothers. LPS 30 mg/kg was administered by gavage once per day for 3 days. Bifidobacterium micro-capsules were given as 1×1010 colony forming units/ml by gavage with formula once per day. After fed for 72 h and fasted for 12 h, the five groups of rats were killed by decapitation. Morphological changes in the terminal ileum tissue were observed under a light microscope and intestinal injury was scored. The expression of Toll-like receptor (TLR) 2, TLR4, and nuclear transcription factor (NF)-κB p65 was detected by immunohistochemical methods. Kruskal-Wallis test, analysis of variance, corrected Chi-square test and Fisher's exact test were used for statistics. Results The morbidity of NEC in group A to E was 11/15, 4/15, 3/15, 2/15 and 0/15, respectively;the intestinal injury score in group A to E was 3.37±0.27, 1.53±0.44, 1.75±0.37, 0.92±0.39 and 0.30±0.18, respectively; the expression level of TLR2 in group A to E was 0.35±0.05, 0.30±0.03, 0.32±0.04, 0.30±0.02 and 0.29±0.03, respectively;the expression level of TLR4 in group A to E was 0.48±0.05, 0.34±0.03, 0.36±0.03, 0.37±0.04 and 0.35±0.02, respectively;the expression level of NF-κB p65 in group A to E was 0.43±0.03, 0.29±0.03, 0.35±0.02, 0.32±0.02 and 0.30±0.02, respectively. The differences in NEC morbidity, intestinal injury score, and the expression levels of TLR4, TLR2 and NF-κB p65 among the five groups were all statistically significant (χ2, H or F=23.863, 70.290, 8.803, 38.599 and 75.076, respectively, all P 0.05). The intestinal injury score in the Bifidobacterium treatment group was significantly higher than that in the Bifidobacterium control group and the breastfeeding control group (both P 0.05). The expression levels of TLR4 and NF-κB p65 in the Biifdobacterium treatment group were significantly lower than those in the artificial feeding control group and the Biifdobacterium control group (all P 0.05). The expression level of TLR2 in the Biifdobacterium treatment group compared with the three control groups was not significantly different (all P > 0.05). Conclusions Biifdobacterium may inhibit pathogenic bacteria or regulate the negative feedback of TLR2 to reduce the expression of TLR2 and TLR4 in intestinal mucosa cells, inhibit the NF-κB pathway, attenuate the inflammatory reaction, and play a role in the prevention and control of NEC.

Effects of bifidobacterium on mRNA expression of tumor necrosis factor receptor-associated factor 6, glycogen synthase kinase-3β and miRNA-146a in intestinal epithelial cells induced by lipopolysaccharide in rats / 中华实用儿科临床杂志

Objective To detect the effects of bifidobacterium or bifidobacterium cultured supernatant on the mRNA expression of tumor necrosis factor receptor-associated factor 6 (TRAF6),glycogen synthase kinase-3β (GSK-3β) and the miRNA-146a in rat small intestinal epithelial cell(IEC-6) induced by lipopolysaccharide (LPS).Methods IEC-6 in logarithmic phase were randomly divided into LPS group,cultured supernatant group and inactivated bacteria group.All the 3 groups were exposed to 5 mg/L LPS for 5 hours,and then 1 mL sterile saline was added in LPS group and culturing continued for 24 hours ; 1 mL bifidobacterium cultured supernatant was added in cultured supernatant group and culturing continued for 24 hours;1 mL inactivated bifidobacterium 1 x 1010 CFU/L added in inactivated bacteria group and continued culturing for 24 hours.The mRNA expressions of TRAF6,GSK-3 β and miRNA-146a were detected by reverse transcription-polymerase chain reaction (RT-PCR).Results The level of TRAF6,GSK-3 β of culture supematant group (0.72 ± 0.05,0.46 ± 0.14) were all lower than LPS group (1.01 ± 0.14,1.02 ± 0.25),but the level of miRNA-146a(3.05 ± 0.40) was higher than that in LPS group(1.01 ± 0.12),and there were significant differences between them (t =5.278,6.316,13.218,P =0.000).The level of GSK-3 β of inactivated bacteria group(0.59 ±0.13) was significantly lower than that in LPS group(t =4.837,P =0.000).The levels of TRAF6 and miRNA-146a of inactivated bacteria group(1.05 ±0.11,0.78 ±0.22) had no significant differences with LPS group (t =0.732,1.463,P ＞ 0.05).The level of TRAF6 of cultured supernatant group was lower than that in inactivated bacteria group,and the level of miRNA-146a was higher than that in inactivated bacteria group,and there were significant differences between 2 groups (t =6.009,14.687,P =0.000).Conclusions Bifidobacterium cultured supernatant and inactivated bacteria both have certain protective effect on the IEC-6 induced by LPS.One of the protective mechanisms of bifidobacterium cultured supernatant may be achieved by elevating the expression of miRNA-146a,and decreasing the levels of inflammation related factor TRAF6 and damage related factor GSK-3β.The protective effects of inactivated bifidobacterium may be achieved by decreasing the level of damage related factor GSK-3β.

Effects of lipopolysaccharides in different concentrations on the proliferation and interleukin-6, interleukin-1β and tumor necrosis factor-α secretion of intestinal epithelial cell of rats in vitro / 中华实用儿科临床杂志

Objective To investigate the effects of lipopolysaccharides (LPS) in different concentrations on the proliferation and interleukin(IL)-6,IL-1 β and tumor necrosis factor-α(TNF-o) secretion of intestinal epithelial cells (IEC-6) of rats in vitro.Methods IEC-6 of rats were divided into normal group (0 mg/L,group A),0.1 mg/L group (group B),0.5 mg/L group (group C),1.0 mg/L group (group D),5.0 mg/L group (group E) and 10.0 mg/L group(group F).Different groups cells were exposed to LPS with different concentrations for 3 h,5 h and 7 h.Thiazolyl blue(MTT) was performed to investigate the proliferation of IEC-6.The concentrations of IL-6,IL-1 β and TNF-α in culture supernatant were detected by enzyme linked immunosorbent assay(ELISA).Results The proliferation rate of IEC-6 was gradually lower while the concentration of LPS increased.After co-culture with LPS 3h and 5 h,the proliferation rates of group B,group C,group D,group E and group F had no significant difference with those of group A (all P ＞ 0.05);after co-culture with LPS 7 h,the proliferation rates of group B,group C,and group D had no significant difference with those of group A (all P ＞ 0.05);the proliferation rates of group E and group F had significant difference with those of group A(t =4.216,P =0.014;t =14.991,P =0.000).The proliferation rates of group E and group F were lower after co-culture with LPS 5 h than 7 h,and there were significant differences (t =2.576,P =0.033;t =2.975,P =0.018);but there was no significant differences between group E and group A after co-culture with LPS 5 h (P ＞ 0.05).Group B,group C,group D,group E and group F all had a significant higher level of IL-6 than group A after co-culture with LPS 3 h,5 h and 7 h(all P ＜0.01).In addition,group E had the highest level of IL-6 at all time points.And the peak level of IL-6 rose after co-culture with LPS 5 h.The TNF-α level and IL-1 β level of group B,group C,group D,group E and group F all had no significant differences than that of group A after co-culture with LPS 3 h,5 h and 7 h (all P ＞ 0.05).Conclusions In a certain concentration,incubation time range,the proliferation rates of IEC-6 cells were gradually lower while the concentration of LPS increased.Co-cultured IEC-6 cells with LPS(0-10.0 mg/L) can stimulate them secrete to IL-6.The highest level of IL-6 was of group E after 5 h co-culture.LPS had no effects on TNF-α and IL-1 β level of IEC-6 cells cultural supernatant.So 5.0 mg/L concentration of LPS stimulating IEC-6 cells for 5 h can be chosen to build the IEC-6 inflammatory models.

Relationship between myeloid differentiation 2 gene polymorphisms and necrotizing enterocolitis in neonates / 中华围产医学杂志

Objective To investigate the relationship between myeloid differentiation (MD-2) gene polymorphisms and necrotizing enterocolitis (NEC) in neonates.Methods A gene-sequencing method was used to re-sequence the exons and the promoter functional polymorphism region (rs11465996) of MD-2 gene of 42 NEC neonates admitted in neonatal intensive care unit of Women and Children's Medical Center,Guangzhou Medical University from June 1,2011 to May 31,2012.These functional polymorphism loci were compared with 83 non NEC cases.The Chi square test was used for statistical analysis.Results No polymorphism was detected in the exons of MD-2 gene in any of the 42 cases of NEC.The C-1625G polymorphism [rs11465996 (C＞G)] was identified in both the NEC and control groups,and there were two genotypes,C/C and C/G.The frequency ofC/G genotype in the NEC group (38.1％,16/42) did not differ significantly compared to that in the control group (30.1％,25/83) (x2=0.805,P=0.370).However,the frequency of C/G genotype in severe NEC cases (operation group) (55.0％,11/20) was significantly higher than that in the control group (x2=4.388,P=0.036).Among the NEC group,the frequency of C/G genotype in operation cases and term infants was higher than that of the non-operation cases and preterm infants,although the differences were not significant (x2=3.343,P=0.067; xx2=0.913,P=0.339).Conclusions The polymorphisms in the exons of MD-2 gene are not associated with the development of NEC.The rs 1 1465996 polymorphism (G allele) in the promoter region may be related to the severity of NEC.

Effects of heparin-binding epidermal growth factor-like growth factor on mitochondrial pathway of apoptosis in neonatal rats with necrotizing enterocolitis / 中华围产医学杂志

Objective To investigate the effect of heparin-binding epidermal growth factor-like growth factor (HB-EGF) on mitochondrial pathway of apoptosis in rats with neonatal necrotizing enterocolitis (NEC).Methods Sprague-Dawley neonatal rats were randomly divided into three groups with ten in each.NEC group rats were formula fed,and hypoxia exposed by 100％ N2 for 90 s and cold stress at 4 ℃ for 10 min twice a day for three days.Additionally,rats in HB-EGF group received HB-EGF 800μg/kg by gavage four times a day for three days.Rats in control group were given breast milk feeding for three days without any interventions.Seventy-two hours after born,all neonatal rats were sacrificed after fasting for 12 h,from which the terminal ileum was removed.HE-staining was done for histologic evaluation.Mitochondrial ultrastructure was observed under electron microscopy.Cytochrome C was detected by immunohistochemical analysis and apoptosis inducing factor (AIF) and apoptotic protease activating factor-1 (APAF-1) were measured by Western blot.Analysis of variance and q-test were used to compare the difference among groups.Results (1) The incidence of NEC in HB-EGF group was lower than that in NEC group (2/10 vs 9/10,x2 =7.27,P＜0.01).(2) In NEC group,mitochondria in epithelial cells and muscle cells of intestine were significantly swelling,appearing many electron-lucent zones in matrix.Ultrastructure of mitochondria were severely damaged.In HB-EGF group,mitochondria were less swelling and showed milder damage than those in NEC group.(3) The expression of cytochrome C in ileal tissue in NEC group was higher than that in control group (0.030±0.018 vs 0.002±0.001,q=6.15,P＜0.01).The expression of cytoehrome C in ileal tissue in HB-EGF group was lower than that in NEC group (0.014±0.018 vs 0.030±0.018,q=3.53,P＜0.05).The expression of APAF-1 and AIF in NEC group was higher than those in control group (1.364±0.299 vs 0.215±0.033,q=15.31,P＜0.05;0.181±0.050 vs0.127±0.045,q=3.71,P＜0.05).Compared to NEC group,the expression of APAF-1 was lower (0.455±0.123 vs 1.364±0.299,q=4.04,P＜0.05) and the expression of AIF was higher (0.289±0.045 vs 0.181±0.050,q=7.32,P＜0.05) in HB-EGF group.Conclusions HB-EGF could reduce the incidence of NEC in neonatal rats by inhibiting the mitochondrial pathway related apoptosis through down regulation of APAF-1.

Expression of Toll-like receptor-2 in premature rats with necrotizing enterocolitis and regulatory effects of glutamine / 中华围产医学杂志

Objective To study the expression of Toll-like receptor-2 (TLR-2) and caspase-3 in the intestine of premature rats with necrotizing enterocolitis (NEC),and to explore the protective effects and possible regulatory mechanism of glutamine (Gln) in the NEC.Methods Sixty premature rats (gestational age 21 d) were divided into three groups (n = 20 each) according to the random number table: control group,model group and Gln intervention group.Rats in model group were given formula feeding,hypoxia and cold stress.Rats in Gln intervention group were given Gln 0.3 g/kg to the formula feeding,hypoxia and cold stress.All the premature rats were sacrificed and the intestine tissues were obtained on the third day after birth.The histological changes of ileal tissues were scored after HE staining.The expression of TLR-2 and caspase-3 in jejunum,ileum and colon were detected by inmunohistochemistry,and the expression of TLR-2 mRNA in jejunum,ileum and colon were detected by real-time fluorescence quantitative reverse transcription-polymerase chain reaction.Results Pathology score of ileum in model group,Gln intervention group and control group were 3.10 ±0.99,2.40 ± 0.69 and 0.30 ±0.48,respectively.The expressions of TLR-2 protein in ileum were 2.53±0.94,2.15±0.82 and 1.57 ± 0.62 in the three groups respectively,and the expression of caspase-3 protein were 2.83 ± 0.45,2.70 ± 0.04 and 0.91 ± 0.29.The content of TLR2 mRNA in model group was 1.46 times higher than that of Gln intervention group and was 2.10 times higher than that of control group.Compared with the control group,the pathology score,expression of TLR-2 and caspase-3 protein,and TLR-2 mRNA in model group were significantly higher,P＜0.01.However,compared with the model group,those changes were improved in Gln intervention group,P＜0.05.Expression of TLR-2 mRNA positively correlated to the expression of caspase-3 protein (r=0.71,P＜0.01) and pathology score (r = 0.69,P＜ 0.01).Expression of caspase-3 protein positively correlated to the intestine injury pathology score (r=0.81,P＜0.01).Conclusions TLR-2 may be involved in the pathogenesis of NEC.Gln might reduce the expression of TLR-2 in the intestine,and decrease the apoptosis of intestinal epithelial cells to protect the intestine of preterm birth rats.

Comparison of different methods in the establishment of necrotizing enterocolitis models in premature rats / 中华围产医学杂志

Objective To establish and evaluate three different necrotizing enterocolitis models,established by combination of formula feeding, hypoxia and cold exposure, hypoxia-reoxygenation, and intraperitoneal injection of lipopolysaccharides (LPS) in premature rats. Methods Group A was given formula feeding, hypoxia by exposing to 100% N2 for 90 s and 4 ℃ cold stress for 10 minutes, the hypoxia and cold stress were given twice a day for 2 d. Group B was put into 100% N2 for 5 min and then 100% O2for 5 min, twice a day for 3 d. Group C was injected intraperitoneally 5 mg/kg LPS. Group D, E and F were served as the corresponding controls for group A, B and C. Ileocecal junction, liver, kidney and lung tissues were harvested and evaluated by HE staining for histological analysis, histological changes of ileal tissues were scored, and rats with score higher than two were diagnosed with NEC. Results Premature rats in group A, B and C showed various degrees of decreasing activity, abdominal distention, diarrhea,intestinal dilatation and congestion. Histological score in group A to 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, respectively. There were significant differences between model groups and their corresponding control groups (P＜0. 01 ). Among the model groups, the histological score of group A was higher than group B (P＜0.01) and group C (P＜0.05). The incidences of NEC in group A, B and C were 6/8, 20% (5/10) and 4/8, respectively, while of zero in all control groups. Liver, kidney and lung injures were more serious in group C compared with the other groups.Conclusions Compared with the single-factor modeling approaches of intraperitoneal injection of LPS and hypoxiareoxygenation, the NEC animal model in preterm rats established by formula feeding, repeated hypoxia and cold exposure, is more similar to the etiological factors of neonatal NEC in human, with higher incidence, better reproducibility and specificity.

Progress in biological function of glycomacropeptide / 国际儿科学杂志

Glycomacropeptide(GMP) is a polypeptide fragment derived from κ-casein after rennin treatment.At present,the structures of GMP in human beings,cows and goats have been established.The sialic acid structure contained in the polypeptide chain of GMP is very important for the exertion of the biological function.GMP has many biological functions,such as anti-infection,regulating immunity,anti-inflammation,nourishing and maintaining health,and so on.As a new-type bio-functional protein,it will be more and more widely used in the areas of medicine and foodstuff.