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To revise GBZ 188 Technical Specification for Occupational Health Surveillance based on national laws, regulations, standards, specifications and legal documents of occupational disease, and combination with the actual situation in China. The main modifications are as follows: the occupational health surveillance for workers exposed to toluene (xylene may implement by reference), bromopropane, methyl iodide, ethylene oxide, chloroacetic acid, indium and its compounds, coal tar, coal tarasphalt, asphalt, β-naphthylamine, dust of metal and its compounds(tin, iron, antimony, barium and its compounds), hard metal dust, erionite dust, low temperature, laser, tick-borne encephalitis virus, Borrelia burgdorferi, and human immunodeficiency virus, for scraper or grind operators, and underground workers using squatting or kneeling position, crawling position, side-lying position, or shoulder position for a long period of time are included. The emergency health screening for workers exposed to arsenic, fluorine and its inorganic compounds, and acrylamide are included. The occupational medical examination (OME) for workers exposed to amino and nitro compounds of benzene, phosgene, monomethylamine, organic fluorine and dimethyl sulfate has been adjusted and made mandatory, with corresponding assessments required upon leaving the job. The special occupational health surveillance for workers exposed to mycobacterium tuberculosis and hepatitis virus is removed. The OME conclusion of reexamination is removed, and standardize recheck/additional inspection requirements. The optional items in OME performed before, during and after leaving post are removed, but the optional items in emergency medical examination are retained. Additional OME items are added. The Guideline for OME Summary Reports is added as informative appendix, and so on. The revised GBZ 188 Technical Specification for Occupational Health Surveillance is more scientific and practical.
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Background Equivalent continuous A-weighted sound pressure level is not appropriate for evaluating the risk of non-steady noise exposure, and need to be corrected by noise time-domain structure, but the correction method and its applicability need to be discussed. Objective To validate the application of the kurtosis-adjusted normalization of equivalent continuous A-weighted sound pressure level to a normal 8 h working day ( LAeq,8 h) in assessing noise-induced hearing loss (NIHL), and to improve the methods for assessing occupational hearing loss associated with different types of noise. Methods Audiometric and shift-long noise exposure data were acquired from a population(n=2 466) of screened workers exposed to noise between 70 dB(A) and 95 dB(A) from 6 industries in China. The cohort data were collapsed into 1 dB(A) bins, and the average kurtosis and noise-induced permanent threshold shifts at 3 kHz, 4 kHz, and 6 kHz (NIPTS346) within 1 dB(A) were calculated respectively. According to the existing correction method, the adjustment coefficient λ was calculated by multiple regression, and LAeq,8 h was corrected by λ (L'Aeq,8 h). The entire cohort was divided into K1 (≤10; steady noise), K2 (10~50; non-steady noise), and K3 (＞50; non-steady noise) groups based on mean kurtosis levels. Predicted NIPTS346 was calculated using the ISO 1999 model for each participant and the actual measured NIPTS346 was corrected for age and gender. The underestimated NIPTS346 was the difference between the values of estimated NIPTS346 and the corresponding actual NIPTS346. To validate the applicability of L′Aeq,8 h in evaluating NIHL, the correlation between L′Aeq,8 h and HFNIHL, and the mean difference between real NIPTS346 and estimated NIPTS346 were analyzed. Results The adjustment coefficient λ was determined at 5.43. The results of multiple logistic regression analysis showed that the relationship between L'Aeq,8 h and HFNIHL increased from 6.6% to 9.6% after the kurtosis adjustment. The DRR of LAeq,8 h and HFNIHL showed that the percentage of HFNIHL decreased after the adjustment of kurtosis in the non-steady noise groups, and the regression lines of the non-steady noise groups approached that of the steady noise group. The R2 of the K2 group increased from 0.935 3 to 0.986 3, and the R 2 of the K3 group increased from 0.905 6 to 0.951 6. Under the un-adjusted condition, the NIPTS346 underestimation for the K3 group was significantly higher than that for the steady noise group (t=−3.23, P=0.001). After the LAeq,8 h was adjusted by kurtosis, the NIPTS346 underestimation decreased significantly in the three kurtosis groups (K1: t=6.78, P＜0.001; K2: t=14.31, P＜0.001; K3: t=11.06, P＜0.001). There was no significant difference in the degree of underestimation between the three kurtosis groups (K1 vs K2: t=−0.22, P=0.830; K1 vs K3: t=−1.40, P=0.205) as the curves of the three kurtosis groups were nearly overlapped. Conclusion The kurtosis-adjusted LAeq,8 h can effectively estimate the hearing loss associated with non-steady state noise.
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Objective:To study the neurodevelopmental status of very/extremely low birth weight preterm infants with gestational age less than 32 weeks at the corrected age of 1 year.Methods:Preterm infants admitted to the Neonatology Department after birth at Guangzhou Women and Children′s Medical Center from January 2015 to December 2018 and followed up regularly to the high-risk infants clinic for at least 1 year after discharge were selected as the research subjects.According to the birth weight(BW), preterm infants were divided into very/extremely low birth weight group(BW<1 500 g), low birth weight group(1 500 g<BW<2 500 g)and normal birth weight group(2 500 g≤BW<4 000 g). The neurodevelopment of preterm infants with different BW at the corrected age of 1 year were compared and the influence of perinatal risk factors on neurodevelopment of very/extremely low birth weight preterm infants were analyzed.Results:A total of 270 preterm infants were included in the study, including 95 in the very/extremely low birth weight group, 124 in the low birth weight group, and 51 in the normal birth weight group.At the corrected age of 1 year, adaptability, gross motor, fine motor, language, individual sociability and development quotient(DQ) in the very/extremely low birth weight group were lower than those in the other two groups( P<0.05). The incidence of neurodevelopmental abnormality(DQ<85)in very/extremely low birth weight preterm infants was 42.1%(40/95) at the corrected age of 1 year.The incidence of intracranial hemorrhage in neurodevelopmental abnormality group(85.0%, 34/40) was higher than that in the control group(29.1%, 16/55)( P<0.05). Conclusion:Very/extremely low birth weight preterm infants are at high risk of neurodevelopmental abnormality at the age of 1 year, and intracranial hemorrhage may be a related potential risk factor.Perinatal follow-up care and early intervention should be emphasized to strengthen neurodevelopmental monitoring.
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Objective To explore the clinical features and risk factors of poor prognosis in neonatal necrotizing enterocolitis(NEC).Methods A retrospective study was carried out in the infants with NEC admitted to 6 cooperative hospitals in Guangdong Province between January 2005 and December 2014.The clinical features and risk factors of poor prognosis in preterm and full-term infants diagnosed NEC,early onset and late onset NEC were analyzed.Results A total of 449 cases who met the criteria were admitted during the study time.The mortality was 23.6％ (106/449 cases),of which the preterm group was 24.6％ (58/238 cases) while the full-term group was 22.7％ (48/211 cases),the early onset group was 22.1％ (45/204 cases) while the late onset group was 24.3％ (57/235 cases).The median number of NEC onset in preterm group was 11 d after birth while the number of the full-term group was 6 d.Full-term infants who diagnosed NEC were more likely to manifest themselves as abdominal distension (52.1％ vs.42.0％,x2 =4.597,P =0.032),vomiting(36.5％ vs.17.2％,x2 =21.428,P =0.000) and bloody stool(30.3％ vs.21.4％,x2 =4.653,P =0.031);but in the onset of NEC,preterm infants more likely to have feeding intolerance (21.0％ vs.12.8％,x2=5.309,P =0.021).The early onset group of full-term NEC was much common in twins or multiplets(9.4％ vs.1.1％,x2 =6.226,P =0.013),which rate of surgical therapy was much higher (41.0％ vs.27.0％,P =0.036) and the breast-feeding rate before NEC was lower than the late onset group(14.5％ vs.32.6％,x2 =9.500,P =0.002),the differences were statistically significant.The gestational age and birth weight were bigger in the early onset group of preterm NEC[(33.8 ±2.5) weeks vs.(32.2 ±2.8) weeks,t =4.261,P =0.000;(2.1 ±0.5) kg vs.(1.7 ± 0.5) kg,t =4.735,P =0.000)],but length of stay was shorter than the late onset group (18.0 d vs.26.5 d,P =0.000).Logistic regression analysis showed that the risk factors of poor prognosis of full-term NEC were shock,peritonitis and sepsis;while risk factors of poor prognosis of preterm NEC were small for gestational age infant,pulmonary hemorrhage,shock,intestinal perforation and sepsis;the risk factors of poor prognosis of the early onset group of full-term NEC was shock;while those of the late onset group were shock and peritonitis;the risk factors of poor prognosis in the early onset group of preterm NEC were shock and sepsis,while those in the late onset group were pulmonary hemorrhage,shock,intestinal perforation and sepsis.Conclusions Compared to the preterm NEC,the onset time of full-term NEC was earlier and the clinical manifestations were more typical.Early identification and management of shock,peritonitis,intestinal perforation,sepsis and pulmonary hemorrhage can reduce the risk of poor prognosis of neonate NEC.
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Objective To study the relationship between serum neutrophil gelatinase-associated lipocalin (NGAL),kerbs von lungren 6 antigen (KL-6) and bronchopulmonary dysplasia (BPD) in preterm infants.Method From Jan.2015 to Dec.2015,preterm infants admitted to NICU of Guangzhou Women and Childrem's Medical Center with gestational age less than 32 weeks and birth weight less than 1 500 g were enrolled.The serum levels of procalcitonin (PCT),NGAL and KL-6 protein were detected at 24 h,7 d and 14 d after birth.At the 28 d after birth,according to the presence of BPD or not,the infants were assigned into BPD group and non-BPD group.The differences of the serum levels of PCT,NGAL and KL-6 between the two groups were compared.Result A total of 55 cases were included in the study (BPD group 20 cases,non-BPD group 35 cases).No significant differences existed in gender,birth weight and gestational age between the two groups (P ＞ 0.05).The incidence of respiratory distress syndrome were siginificantly higher in the BPD group (P ＜ 0.05) and the duration of mechanical ventilation and oxygen therapy were siginifantly longer in the BPD group (P ＜ 0.05).No significant difference between the two groups in the level of PCT at 24 h after birth (P ＞ 0.05).The levels of serum PCT at 7 d and 14 d after birth in BPD group were significantly higher than non-BPD group [7 d:(1.5 ± 1.7) ng/ml vs.(0.4 ± 0.2)ng/ml,14 d:(0.8 ± 0.7) ng/ml vs.(0.2 ± 0.1) ng/ml] (P ＜ 0.001).The levels of serum NGAL at 24 h,7 d and 14 d were significantly higher than non-BPD group [24 h:(1.6 ± 0.3) pg/ml vs.(0.8 ±0.2) pg/ml,7 d:(2.3 ±0.5) pg/ml vs.(0.7 ±0.2) pg/ml,14 d:(2.5 ±0.3) pg/ml vs.(0.8 ±0.2)pg/ml] (P ＜0.001).The levels of serum NGAL at 7 d and 14 d after birth in BPD group were significantly higher than 24 h in BPD group (P ＜ 0.05).No significant difference between KL-6 at 24 h after birth in BPD group and non-BPD group (P ＞0.05).The levels of serum KL-6 at 7 d and 14 d after birth in BPD group were significantly higher than non-BPD group [7 d:(1.2 ± 0.2) ng/ml vs.(0.8 ± 0.1) ng/ml,14 d:(1.3 ±0.2) ng/ml vs.(0.8 ±0.9) ng/ml] (P ＜0.001).The level of serum KL-6 at 7 d and 14 d after birth in BPD group were significantly higher than 24 h in BPD group (P ＜ 0.05).Conclusion Respiratory distress syndrome and prolonged mechanical ventilation were risk factors of BPD.The rising of serum NGAL and KL-6 early after birth might be involved in the development of BPD,which had predictive value of BPD.
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Objective To study the composition of gut microbiome in neonates with severe bilinebinemia (serum total bilirubin ＞ 342 μmol/L),and to explore the relationship between gut microbiome and bilirubin brain injury.Methods A prospective study was conducted.The neonates with serum total bilirubin ＞ 342 μmol/L from September 2016 to March 2017 in Guangzhou Women and Children's Medical Center,Guangzhou Medical University,were enrolled in the study and 16S rDNA sequence analysis technology was used to detect the composition of gut microbiome in all subjects.According to the results of brain magnetic resonance imaging (MRI),brain stem auditory evoked potential (BAEP) and clinical manifestations,the subjects were divided into the brain injury group (26 cases) and no brain injury group (28 cases).The differences of the composition of gut microbiome between the 2 groups were compared,and the levels of unconjugated bilirubin in serum and cerebrospinal fluid were also compared.Results The level of unconjugated bilirubin in serum of the brain injury group was (463.51 ± 110.62) μmol/L,but in no brain injury group was(364.18 ±63.13) μmol/L,and there was significant difference between the 2 groups(t =4.090,P =0.000 1).The level of unconjugated bilirubin in the cerebrospinal fluid of the brain injury group was (9.53 ± 2.68) μmol/L,but in no brain injury group was (6.94 ± 2.31) μmol/L,and there was significant difference between the 2 groups (t =3.812,P =0.000 3).There was no correlation between the level of unconjugated bilirubin in the cerebrospinal fluid and serum between the 2 groups(r =0.137,0.081,all P ＞0.05).The abundance of gut microbiome in the brain injury group was lower than that in no brain injury group in genus level,among which Fusobacterium,Catabacter,Succinivibrio,Clostridium and Bacteroides were significantly different (all P ＜ 0.05).Conclusions The occurrence of bilirubin brain injury depends on the level of unconjugated bilirubin in serum cerebrospinal fluid,but it may be more directly dependent on the level of bilirubin in the cerebrospinal fluid.The diversity of gut microbiome in neonates with bilirubin brain injury was significantly lower than that in no brain injury group.The level of unconjugated bilirubin in cerebrospinal fluid may be related to the different blood-brain barrier permeability caused by different composition of gut microbiome.
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Objective To establish an appropriate neonatal rat model with necrotizing enterocolitis (NEC) and to investigate the protective effects of interleukin-1 receptor associated kinase (IRAK) 1/4 inhibitor on intestinal tissue of neonatal rats with NEC.Methods Thirty-six newborn SD rats were randomly divided into normal control group,NEC model group and IRAK1/4 inhibitor group,12 rats in each group.The rats in normal control group were raised by their mother and they did not receive any intervention.The rats of NEC model group were given artificial feeding,under hypoxia and cold stress.The IRAK1/4 inhibitor group were given IRAK1/4 inhibitor intervention,and also given artificial feeding,under hypoxia and cold stress.Three days later,all rats were sacrificed and intestinal tissues were obtained.The histopathological changes in ileocecal tissues were evaluated by pathological score after hematoxylin-eosin staining.The levels of interleukin (IL)-1β,IL-6 and tumor necrosis factor (TNF)-α in intestinal tissues were detected by enzyme-linked immunosorbent assay.Results The rats of NEC model group presented abdominal distention,diarrhea (partly with mucous bloody stool),decreased activity,poor response,lethargy and other symptoms,and the extent was gradually worsened;the rats in IRAK1/4 inhibitor group also had abdominal distention,diarrhea,decreased activity,poor response and other symptoms,but the symptoms emerged later and milder.The histopathological score of intestinal tissues of normal control group was (0.33 ± 0.49) scores,NEC model group was (3.08 ± 0.99) scores,and IRAK1/4 inhibitor group was (1.75 ±0.96) scores.The histopathological scores of NEC model group and IRAK1/4 inhibitors group were significantly higher than those in normal control group (all P ＜ 0.01),and the histopathological scores of IRAK1/4 inhibitor group were significantly lower than those in the NEC model group (P ＜ 0.01).The levels of IL-1β,IL-6 and TNF-α in normal control group separately were (128.76 ± 27.25) ng/L,(0.41 ± 0.10) ng/L,(6.93 ± 1.79) ng/L,respectively;the levels of NEC model group separately were (410.99 ± 44.16) ng/L,(1.79 ± 0.18) ng/L,(44.39 ± 6.00) ng/L;the levels of IRAK1/4 inhibitor group separately were (256.23 ±41.29) ng/L,(1.05 ±0.19) ng/L,(21.45 ± 6.36) ng/L,respectively.The levels of IL-1β,IL-6 and TNF-α of NEC model group had significant differences compared with those of normal control group,respectively (all P ＜0.01);the levels of IL-1β3,IL-6 and TNF-α of IRAK1/4 inhibitor group had significant differences compared with those of normal control group,respectively (all P ＜ 0.01);the levels of IL-1β,IL-6 and TNF-α of IRAK1/4 inhibitor group had significant differences compared with those of NEC model group (all P ＜ 0.01),respectively.Conclusions IRAK1/4 inhibitor has some protective effects on the intestinal tissues of neonatal rats with NEC,which can reduce the damage to the intestinal tissues,and decrease the secretion of inflammatory cytokines like IL-1β,IL-6 and TNF-α.
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ObjectiveTo analyze the differential expression of microRNA (miRNA) and its significance in patients with neonatal necrotizing enterocolitis (NEC).MethodsTwenty-five patients diagnosed with NEC with Bell stage≥Ⅱ, and 25 non-NEC patients as control group admitted to Guangzhou Women and Children's Medical Center between October 2014 and November 2015 were collected. White blood cells were extracted from the peripheral blood. Five samples were selected randomly each from NEC group and control group, and sequenced by second-generation Illumina high-throughput sequencing, screened for differentially expressed miRNA and analyzed for target genes prediction and biological function. The rest samples of the two groups were detected by real-time fluorescent quantitative polymerase chain reaction technology (RT-qPCR), the results were used to validate the results of high-throughput sequencing. Differentially expressed miRNA in the two groups of data was analyzed using DEGseq software.P<0.05 was considered statistically significant.P<0.01,q<0.001 and丨Log2 Ratio丨≥1 were taken as criteria for screening the differential expression. The differential expressions of miRNA in NEC group and control group were analyzed by cluster analysis using MeV4.6 software.ResultsA total of 482 miRNAs were differentially expressed in the two groups, with significant difference (P<0.05). Among them, 126 were known miRNAs with significantly differential expression in the two groups, with 58 being up-regulated and 68 being down-regulated. The results of up-regulated miRNAs (hsa-miR-223-5p,-183-3p,-222-5p) and down-regulated miRNAs (hsa-miR-23b-5p,-150-5p,-146a-3p,-1298-5p) were confirmed to be consistent with the results of sequencing. Bioinformatics analysis showed that the target genes with differential miRNA expression mainly involved Toll-like receptor signal transduction pathway, mitogen-activated protein kinase pathway, JAK-STAT and other signal transduction pathways.ConclusionsThere are significantly differential expressions of miRNAs in peripheral white blood cells of NEC neonates. These miRNs may be involved in the occurrence and development of NEC via adjusting different target genes to regulate the signal pathway.
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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.
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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.
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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.
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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.
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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.
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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.
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Neonatal necrotizing enterocolitis (NEC) is still a disease with high mortality and morbidity in neonates.The pathogenesis of NEC is not quite clear.Inflammatory cascade,innate immune response and intestinal blood flow are thought to play significant roles in the machinism of NEC.Single nuleotide polymorhism (SNP) is characterized by single nucleotide mutation in the genecome consequences.They may alter the susceptibility to some kinds of diseases by changing the efficiency of transcription or the structure of protein coded by the related gene.This review summarizes the progress of the research on genetic polymorphisms in neonatal necrotizing enterocolitis.