Objective To determine the dynamic changes in adenosine monophosphate-activated protein kinase (AMPK) in neurons of neonatal rats suffering from hypoxic ischemic brain injury.Methods Twenty-four-hour old and seven-day old neonatal rats were used in this study.A classic primary cortical neuron oxygen glucose deprivation (OGD) model and neonatal rat hypoxic ischemic encephalopathy (HIE) model were employed.Lactic dehydrogenase (LDH) and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were used to evaluate neuron viability and damage.The expression of phosphorylated adenosine monophosphate-activated protein kinase (P-AMPK),phosphorylated activated protein kinase (P-Akt) and Cleaved Caspase-3 in neurons and brain tissue was measured by Western blot at different time points after OGD or HIE.The Student-t test was used for statistical analysis.Results (1) Compared with the control group,LDH levels at 2,4,8 and 24 h after OGD were higher (all P＜0.05) and optical absorption levels of MTT were lower (all P＜0.05).(2) Levels of P-AMPK in the OGD group were higher than those in the control group,and showed a time-dependent increase at 30 min and 2,4,8 and 24 h (all P＜0.05).The expression levels of P-AMPK in the HIE group were higher than those in the control group (0.345 ± 0.038,0.387 ± 0.112 and 0.618 ± 0.075 at 1,3 and 7 days after HIE,and 0.132±0.032 in the control group,all P＜0.05).(3) The levels of P-Akt increased above the control levels at 30 min (0.991 ±0.134 vs 0.304±0.050),reached a maximum level at 2 h (1.183± 0.107),and then gradually declined,whereas the levels of Cleaved Caspase-3 started to increase at 30 min,and remained elevated at 24 h (all P＜0.05).Conclusion Following hypoxic ischemic brain damage,the expression of P-AMPK is significantly increased in both in vivo and in vitro studies in a time-dependent manner.

Objective To investigate the efficacy and safety of aminophylline,caffeine citrate and aminophylline combined with naloxone in prevention of apnea of prematurity(AOP).Methods Ninety-four infants with a birth weight ＜ 1 500 g and gestational age ＜ 34 weeks admitted to Department of Pediatrics,Tongji Hospital Affiliated to Tongji Medical College,Huazhong University of Science and Technology between Jan.2010 and Jan.2012 were randomly divided into 3 groups.(1) Aminophylline group (n =30):30 infants received a loading dose of 4-5 mg/kg of aminophylline and then maintained by a dose of 2 mg/kg,with intravenous drip q12 h.(2) Caffeine citrate group(n =32):a loading dose of 20 mg/kg of caffeine citrate was followed by a daily maintained dose of 5 mg/kg,with intravenous drip per day.(3) Aminophylline combined naloxone group (observation group,n =32):32 infants were treated with Aminophylline combined with naloxone.After 6 hours of the first dose of aminophylline,a dose of 0.1 mg/kg naloxone was injected,q12 h.Then the two drugs were used alternately.The mortality and incidence of AOP,bronchopulmonary dysplasia(BPD),retinopathy of prematurity (ROP) and brain injury were evaluated,and drug-related side effects were recorded.Results 1.There was no significant difference in gender,gestational age,birth weight,maternal antenatal glucocorticoid application,pregnancy (including multiple pregnancy) and delivery,5 min Apgar score,oxygen therapy,and the application of positive airway pressure as well as pulmonary surfactant among the 3 groups(all P ＞0.05).2.Compared with aminophylline group,the incidence of apnea of caffeine group and observation group were significantly lower (F =6.704,P ＜ 0.05),but there was no significant difference between caffeine group and observation group (P ＞0.05).3.There was no statistically significant difference in mortality,duration of oxygen therapy,the incidence of ROP,brain injury and hearing loss,postmenstrual age,body weight at discharge,the duration and cost of hospitalization among the 3 groups(all P ＞0.05).4.The BPD incidence in caffeine group[9.4％ (3/32 cases)] and observation group [12.5％ (4/32 cases)] were lower than that in Aminophylline group [20.0％ (6/30 cases)],but there was no statistical significance among the 3 groups(P ＞ 0.05).5.No drug-related side effects were recorded in the 3 groups.Conclusions It is safe and effective to use aminophylline combined with naloxone in prevention of AOP,and its efficiency is similar to caffeine citrate.

Objective To investigate the diagnosis,treatment and prognosis of neonates with severe acute respiratory distress syndrome (ARDS) according to Berlin definition.Methods A retrospective study was carried to analyze the clinical features about diagnosis,treatment,chest X-ray findings,mortality,complications and ventilator parameters in 86 neonates with severe ARDS admitted in the NICU from January 2005 to December 2013.Results (1)Among the 86 cases,55 were cured,and 31 died with 36.0％ mortality.(2) Chest X-ray showed there was decreased lucency of bilateral lungs with ground-glass appearance,lung texture with thick chaos or dot flakes or patchy shadows in 36 neonates; diffuse infiltrates and extensive confluent consolidation shadows in bilateral lungs along with peripheral air brornchograms in 26 cases; heart shadow and diaphragmatic surface disappeared like a white lung change in 24 cases.(3) Persistent pulmonary hypertension of newborn as a complication occurred in 68 cases with 79.1％ incidence.(4) Eighty-six cases were categorized into survival group and death group.The results showed compared with the survival group,the neonates in death group required higher FiO2,and PaO2,and lower PaO2/FiO2 before mechanical ventilation (P ＜ 0.01),but needed higher initial PIP of mechanical ventilation (P ＜ 0.01).Conclusions Neonatal ARDS is still a kind of critical condition with high mortality and lack of evidence-based diagnostic criteria so far.The therapeutic strategy for neonatal ARDS should be a comprehensive measures in addition to appropriate respiratory support.

Objective To investigate the neuroprotective effects and mechanisms of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/ IMP cyclohydrolase(AICAR) supplement (AMPK activator) in different stages of neonatal rats sufferring from hypoxia-ischemia encephalopathy ( HIE). Methods Neonatal rat hypoxia-ischemia brain injury model was employed in this study. A total of 160 neonatal rats were distributed into five groups: sham, model control,AICAR30 min, AICAR24 h and AICAR72 h. The neuroprotective effects of AICAR supplement (30 min, 24 h, 72 h post operation) were compared by cresyl violet staining; Expressions of P-AMPK,AMPK in the brain tissue were measured by Western blotting.Foot-faults method was used to evaluate the long-term prognosis of the rats. Results Compared with the sham group, the survival of rats brain in model control group was significantly decreased [(100.0± 0.1)% and (45.3± 6.3)%, P< 0.05]. AICAR had neuroprotective effects when treated at 30 min and 24 h post operation,while the protective effects disappeared when treated later (72 h post operation) (P>0.05). Compared with the sham group, the expression of P-AMPK significantly increased about three times, while ATP level decreased close to the same. Conclusion Early AICAR treatment can protect hypoxia-ischemia brain injury by increasing AMPK-ATP level.

ObjectiveTo investigate the change of gene polymorphorism of surfactant protein-B (SP-B) intron 4 in infants with bronchopulmonary dysplasia (BPD).MethodsForty-five infants with BPD (BPD group) and ninety-nine infants without lung diseases (control group) who admitted into Tongji Hospital,Tongji Medical College,Huazhong University of Science and Technology from July 2008 to July 2011 were selected into this study.Genotyping for fragment length polymorphism of SP-B intron 4 was performed by polymerase chain reaction (PCR),agarose gel electrophoresis,cloning and sequencing methods in both groups.Differences of allele frequencies (invariant allele and variant allele) and genotype frequencies (invariant genotype and variant genotype) between BPD group and control group were analyzed.The differences of gestational age and birth weight between the two groups were compared with Independent-Samples t test.The gender composition and differences of allele or genotype frequencies between the two groups were compared with Chi-square test.Results Invariant allele frequencies in BPD group and control group were 83.3％ (75/90) and 92.0％ (182/198),and variant allele frequencies were 16.7％ (15/90,including eight insertion alleles and seven deletion alleles) and 8.1％ (16/198,including eight insertion alleles and eight deletion alleles).There were significant differences between the two groups (x2 =4.75,P =0.029).In BPD group,there were 32 cases (71.1 ％,32/45) invariant genotypes and 13 cases (28.9 ％,13/45,including seven cases insertions and six cases deletions) variant genotypes; in the control group,there were 85 cases invariant genotypes (85.8％,85/99) and 14 cases (14.1％,14/99,six insertions and eight deletions) variant genotypes.Significant difference was found between the two groups (x2=4.42,P＜0.036). ConclusionsVariations of SP-B intron 4 were more in BPD infants,and the variation of SP-B intron 4 might be associated with BPD.

Objective To construct human surfactant protein B (SP-B) gene promoter luciferase reporter plasmids and detect their transcriptional activities in H441 cells.Methods (1)The fragment of SP-B promoter (-218/+ 435 bp) was acquired from human genome DNA by polymerase chain reaction (PCR) amplification and then was inserted into pGM-T vector by the T4 DNA ligase.The vector was transfected into TOP10 E.coli.The positive clone was identified by DNA sequencing.The identified target SP-B promoter sequence was cloned into pGL3-basic vector to construct the recombinant vector pGL3-basic-SP-B-promoter and was identified by enzyme digestion and sequencing; (2)The pGL3-basic-SP-B-promoter vector was converted into pGL4.17-SP-B-promoter vector through enzyme digestion.The identified recombinant vectors and control plasmid pRL-TK were transfected into H441 cells by lipofectamine 2000,and luciferase assays was performed using the dual-luciferase reporter assay system.Results The sequences of SP-B promoter in the recombinant luciferase reporter plasmids were consistent with the one published on Genebank.The firefly/renilla luciferase activity ratio of pGL3-basic/pGL4.17-SP-B-promoter vector (2.8 ± 1.1,66.5±3.8) was significantly higher than pGL3-Basic,pGL4.17 control vector (0.2 ±0.1,4.3 ±0.4) with statistical significance (t =4.182,27.419,P =0.000),respectively.The SP-B promoter activity of pGL4.17-SP-B-promoter vector was significantly higher than pGL3-basic-SP-B-promoter vector (t =27.712,P =0.000).Conclusions The pGL3-basic/pGL4.17-SP-B-promoter vectors are successfully constructed with SP-B promoter activity in H441 cells and pGL4.17-SP-B-promoter vector is the better choice for further study with higher luciferase activity.

Objective To construct two kinds of eukaryotic ccll expression vcctors pIRES2-EGFP-SP-B-C/T 1580 and evaluate their expressions in 293T cells,for the further study of relationship between polymorphism of surfactant protein B (SP-B) gene and bronchopulmonary dysplasia (BPD).Methods The eukaryotic pIRES2-EGFP-SP-B-C/T 1580 expression vectors were constructed by gene recombination,and identified by gene sequencing.The recombinant expression vectors were transfected into 293T cells by lipofectamine2000.The expression of green fluorescence protein in 293T cells was observed by fluorescence microscopy.The mRNAs and proteins of SP-B-C/T 1580 were tested and identified by reverse transcriptionpolymerase chain reaction-restriction fragment length polymorphism(RT-PCR-RFLP) and western blot.Results Two recombinant plasmids contained the complete cDNA of SP-B with the same sequence as in gene bank.The base of SP-B 1580 gene of pIRES2-EGFP-SP-B-C 1580 was C,that of pIRES2-EGFP-SP-B-T 1580 was T.After being transfected into 293T cells,highly efficient expression of SP-B-C/T 1580 gene was detected at mRNA and protein levels.Conclusions The pIRES2-EGFP-SP-B-C/T 1580 eukaryotic cell expression vectors were successfully constructed.

Objective:To investigate the effects of different concentrations of hydrogen peroxide (H 2O 2) on the expression of histone deacetylase-2(HDAC-2) and mitogen activated protein kinases phosphatases-1(MKP-1) in A549 cells. Methods:The experimental models of A549 cells were established by intervening of different concentrations of H 2O 2(0, 100, 200, 400, 600, 800 μmol/L)for 2 h, 4 h, 8 h, 12 h and 24 h, respectively.The survival rate of A549 cells was detected by thiazolyl blue(MTT) assay, and the apoptosis of A549 cells was tested by lactic dehydrogenase(LDH) assay.The expression of HDAC-2 and MKP-1 protein was detected by Western blot. Results:(1)MTT results showed that compared with the control group (H 2O 2 was 0 μmol/L), the survival rate of A549 cells was decreased with the increase of H 2O 2 concentration at 2 h, 4 h, 8 h, 12 h, 24 h and 48 h. (2)LDH results showed that, compared with the control group, the inhibition rate of A549 cells increased with the increase of H 2O 2 concentration at 2 h, 4 h, 8 h, 12 h, 24 h and 48 h. (3)The expression of HDAC-2 protein decreased ( F=14.588, P<0.01), and the expression of MKP-1 protein increased ( F=64.297, P<0.01) in a concentration-dependent manner with the increase of H 2O 2 concentration at 12 h after H 2O 2 treatment. Conclusions:H 2O 2 participates in oxidative stress injury of lung cells by regulating the expression of HDAC-2 and MKP-1.

Objective:To investigate the effect of laparoscopic splenectomy and azygoportal disconnection (LSD) on liver synthesis and development of liver cirrhosis.Methods:This is a prospective case series study.The clinical data of liver cirrhotic patients with portal hypertension who received LSD at the Department of Hepatobiliary Surgery of Northern Jiangsu People′s Hospital Affiliated to Yangzhou University from September 2014 to January 2016 were included. This study analyzed the diameter of the portal vein, the velocity of portal blood flow, the routine blood parameters, the liver function, the synthetic proteins of liver (antithrombin Ⅲ (AT-Ⅲ), protein S, protein C), and the serum content of liver fibrotic markers(collagen type Ⅳ, procollagen type Ⅲ, laminin, hyaluronidase). Repeated measures ANOVA was used for comparison between multiple groups, and least significance difference was used for post-hoc multiple comparison.Results:A total of 106 patients were included in the study, including 70 males and 36 females, aged (51.8±9.8) years(range: 28 to 75 years).Compared with the preoperative results, the diameter of portal vein and the velocity of portal vein decreased after surgery ( F=14.03, 12.15, respectively, both P<0.01). Compared with the preoperative results, the total bilirubin, albumin, prothrombin time, international normalized ratio, Child-Pugh score and classification were improved ( F=17.96, 56.01, 66.63, 35.83, 33.49, and 27.50, respectively, all P<0.01), and the AT-Ⅲ, protein S, protein C,collagen type Ⅳ, procollagen type Ⅲ, laminin and hyaluronidase levels were also improved ( F=47.87, 36.26, 18.02, 2.79, 14.58, 44.35, and 14.38, respectively, all P<0.01). Compared with the preoperative period, the diameter of portal vein was reduced from the first week to the 24 th month after surgery ( t=5.45 to 9.39, all P<0.01). Compared with the preoperative period, the velocity of portal vein blood from the first week after surgery to the 24 th month after surgery was decreased ( t=4.02 to 8.43, all P<0.01). Compared with the preoperative period, routine blood parameters (white blood count, hemoglobin, platelet count), liver function (total bilirubin, albumin, prothrombin time, international normalized ratio, Child-Pugh score), liver synthetic protein (AT-Ⅲ, protein S, protein C) and liver fibrotic markers (collagen type Ⅳ, procollagen type Ⅲ, laminin, hyaluronidase) were improved to varying degrees at the 24th month after surgery ( t=-20.46 to 11.93, all P<0.01). Conclusion:Preliminary findings show that LSD can reduce portal vein pressure, restore blood cell number, and improve liver synthesis function and the degree of liver fibrosis in patients with portal hypertension in liver cirrhosis.

Objective:To investigate the effect of laparoscopic splenectomy and azygoportal disconnection (LSD) on liver synthesis and development of liver cirrhosis.Methods:This is a prospective case series study.The clinical data of liver cirrhotic patients with portal hypertension who received LSD at the Department of Hepatobiliary Surgery of Northern Jiangsu People′s Hospital Affiliated to Yangzhou University from September 2014 to January 2016 were included. This study analyzed the diameter of the portal vein, the velocity of portal blood flow, the routine blood parameters, the liver function, the synthetic proteins of liver (antithrombin Ⅲ (AT-Ⅲ), protein S, protein C), and the serum content of liver fibrotic markers(collagen type Ⅳ, procollagen type Ⅲ, laminin, hyaluronidase). Repeated measures ANOVA was used for comparison between multiple groups, and least significance difference was used for post-hoc multiple comparison.Results:A total of 106 patients were included in the study, including 70 males and 36 females, aged (51.8±9.8) years(range: 28 to 75 years).Compared with the preoperative results, the diameter of portal vein and the velocity of portal vein decreased after surgery ( F=14.03, 12.15, respectively, both P<0.01). Compared with the preoperative results, the total bilirubin, albumin, prothrombin time, international normalized ratio, Child-Pugh score and classification were improved ( F=17.96, 56.01, 66.63, 35.83, 33.49, and 27.50, respectively, all P<0.01), and the AT-Ⅲ, protein S, protein C,collagen type Ⅳ, procollagen type Ⅲ, laminin and hyaluronidase levels were also improved ( F=47.87, 36.26, 18.02, 2.79, 14.58, 44.35, and 14.38, respectively, all P<0.01). Compared with the preoperative period, the diameter of portal vein was reduced from the first week to the 24 th month after surgery ( t=5.45 to 9.39, all P<0.01). Compared with the preoperative period, the velocity of portal vein blood from the first week after surgery to the 24 th month after surgery was decreased ( t=4.02 to 8.43, all P<0.01). Compared with the preoperative period, routine blood parameters (white blood count, hemoglobin, platelet count), liver function (total bilirubin, albumin, prothrombin time, international normalized ratio, Child-Pugh score), liver synthetic protein (AT-Ⅲ, protein S, protein C) and liver fibrotic markers (collagen type Ⅳ, procollagen type Ⅲ, laminin, hyaluronidase) were improved to varying degrees at the 24th month after surgery ( t=-20.46 to 11.93, all P<0.01). Conclusion:Preliminary findings show that LSD can reduce portal vein pressure, restore blood cell number, and improve liver synthesis function and the degree of liver fibrosis in patients with portal hypertension in liver cirrhosis.