Ellagic acid attenuates hypoxic-ischemic brain injury by alleviating autophagy / 中国病理生理杂志

AIM:To investigate whether ellagic acid (EA) attenuates hypoxic-ischemic encephalopathy (HIE) by down-regulating autophagy.METHODS:In vivo, Sprague-Dawley rats (n=17) were randomly divided into 3groups:5 rats for sham group, 6 rats for HIE group and 6 rats for HIE+EA pretreatment group.The rats in HIE+EA pretreatment group were treated with EA (10 mg/kg, 10 m L/kg, suspended in corn oil, ig).After 24 h of operation, the rats from each group were sacrificed and their brains were collected.TTC staining and HE staining were used to define the infarct areas and brain structure.The autophagy-related proteins beclin-1, P62, LC3-II/-I and Atg5 in the cortex in each group were compared by Western blot.In vitro, PC12 cells were divided into 3 groups:control group, Coand CoEA pretreatment group.Co800μmol/L was added to the PC12 cells to induce an anoxic environment.The PC12 cells were pretreated with EA at 8μmol/L and the cell viability was measured by CCK-8 assay.The production of reactive oxidative species (ROS) in the cells was detected by flow cytometry with DCFH-DA staining.MDC staining and TM-RE staining were applied to reflect the extent of autophagy and the state of apoptosis, respectively.The autophagy-related proteins in PC12 cells were also investigated.RESULTS:In HIE group, 7-day-old rats were given the operations and the their large infarct areas in the hemisphere were observed by TTC staining.HE staining displayed the injured hemispheres which contained few neurons, and exhibited edema status and serious structural damage.EA pretreatment decreased the infarct area and alleviated the damage to hemisphere with more visible neurons, compared with HIE group.Compared with sham group, the levels of autophagy-related proteins Atg5, beclin-1 and LC3-II/-I in the cortex were increased (P<0.01) , and P62 protein expression was decreased (P<0.01) in HIE group.Compared with HIE group, the protein expression of Atg5, beclin-1 and LC3-II/-I was decreased (P<0.01) and P62 protein expression was increased in HIE+EA pretreatment group (P<0.01).In vitro, compared with CoPC12 cells in CoEA pretreatment group showed a lower ROS level.Moreover, the cells in CoEA pretreatment group exhibited higher mitochondrial membrane potential than that in CoMDC staining in Coshowed high value of fluorescence and increased number of autophagosomes.EA pretreatment reduced the number of autophagosomes and the extent of autophagy to protect PC12cells.Furthermore, the protein levels of Atg5, beclin-1 and LC3-II/-I in Cowere higher (P<0.01) , and the protein expression of P62 was lower (P<0.01) than those in control group.In CoEA pretreatment group, the protein levels of Atg5, beclin-1 and LC3-II/-I were decreased (P<0.01) and the protein expression of P62 was increased as compared with Co (P<0.01).CONCLUSION:EA pretreatment attenuates autophagy to protect the neurons against HIE injury.

Expression rhythm of autophagic gene in neurons of neonatal rats with hypoxia/ischemia and its regulatory mechanism / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To investigate the expression of autophagic gene and circadian gene in the neurons of neonatal rats after hypoxic-ischemic brain damage and the mechanism of nerve injury induced by hypoxia/ischemia.</p><p><b>METHODS</b>Twelve Sprague-Dawley (SD) rats were randomly divided into hypoxic-ischemic (HI) group and sham-operation group, with 6 rats in each group. Ligation of the right common carotid artery and hypoxic treatment were performed to establish a model of hypoxic-ischemic brain damage. Western blot was used to measure the expression of the circadian protein Clock in the cortex and hippocampus. The neurons of the rats were cultured in vitro and randomly divided into oxygen glucose deprivation (OGD) group and control group. The neurons in the OGD group were treated with DMEM medium without glucose or serum to simulate ischemic state, and hypoxic treatment was performed to establish an in vitro model of hypoxic-ischemic brain damage. Western blot was used to measure the expression of autophagy-related proteins Beclin1 and LC3 and Clock protein at different time points. The changes in the expression of Beclin1 and LC3 were measured after the expression of Clock protein in neurons was inhibited by small interfering RNA technique.</p><p><b>RESULTS</b>The expression of autophagy-related proteins Beclin1 and LC3Ⅱ in neurons cultured in vitro displayed a rhythmic fluctuation; after OGD treatment, the expression of Beclin1 and LC3Ⅱ gradually increased over the time of treatment and no longer had a rhythmic fluctuation. Compared with the sham-operation group, the HI group had a significant reduction in the expression of Clock protein in the cortex and hippocampus (P<0.05). After OGD treatment, the neurons cultured in vitro had a significant reduction in the expression of Clock protein (P<0.05). Compared with the negative control group, the Clock gene inhibition group had significant reductions in the expression of Beclin1 and LC3Ⅱ (P<0.05).</p><p><b>CONCLUSIONS</b>Hypoxia/ischemia induces the disorder in the expression rhythm of autophagy-related proteins Beclin1 and LC3, and the mechanism may be associated with the fact that the circadian protein Clock participates in the regulation of the expression of Beclin1 and LC3.</p>

Effects of PINK1 gene on cell apoptosis and cell autophagy in neonatal mice with hypoxic-ischemic brain damage / 中国当代儿科杂志

<p><b>OBJECTIVE</b>To study the effect of PINK1 (phosphatase and tensin homolog deleted on chromosome ten induced putative kinase 1) gene on cell apoptosis and cell autophagy in neonatal mice with hypoxic-ischemic brain damage (HIBD).</p><p><b>METHODS</b>Seventy-two wild-type C57BL/6 mice and 72 PINK1 gene knockout neonatal C57BL/6 mice were randomly divided into four groups: sham-operated wild-type (SWT), HIBD model wild-type (MWT), sham-operated knockout (SKO) and HIBD model knockout (MKO). HIBD model was prepared by low oxygen exposure for 2.5 hours after right carotid artery ligation. After 24 hours of hypoxia-ischemia treatment, TTC (2,3,5-triphenyl four azole nitrogen chloride) staining was used to measure brain infarct volume. The immunohistochemical staining was used to measure the expression of cell apoptosis protein cleaved-caspase-3 (CC3) in brain tissues. The TUNEL method was used to measure cell apoptosis. The immunofluorescence staining and Western blot were used to measure the expression of cell autophagy protein LC3.</p><p><b>RESULTS</b>Compared with the MWT group, the infarct volume of brain tissues was markedly reduced in the MKO group (P<0.05), the number of apoptotic cells and the cell apoptosis index were markedly decreased in the MKO group (P<0.05), the expression of apoptosis protein CC3 was significantly reduced in the MKO group (P<0.05), the expression of cell autophagy protein LC3 was significantly decreased in the MKO group, and the autophagy indicator LC3II/LC3I was also markedly reduced in the MKO group (P<0.05).</p><p><b>CONCLUSIONS</b>PINK1 gene knockout can protect neonatal mice from HIBD.</p>

Clinical analysis of 31 cases of neonatal purulent meningitis caused by Escherichia coli / 中国当代儿科杂志

<p><b>OBJECTIVE</b>Neonatal purulent meningitis is a severe infection responsible for high mortality and disabling sequelae. Escherichia coli is the main pathogen of neonatal purulent meningitis. This study explored the clinical characteristics and antibiotic resistance of Escherichia coli-induced neonatal meningitis.</p><p><b>METHODS</b>A retrospective chart review was performed. A total of 31 cases of neonatal purulent meningitis caused by Escherichia coli were identified in the neonatal intensive care unit between January 1, 2001 and December 31, 2011. The clinical characteristics and antibiotic sensitivity test results were analyzed.</p><p><b>RESULTS</b>Fever, poor feeding, lethargy and seizure were common clinical signs of neonatal purulent meningitis caused by Escherichia coli. Acute complications mainly included hyponatremia (17 cases), hydrocephalus (8 cases), subdural collection (2 cases), ventriculitis (2 cases) and cerebral infarction (1 case). Thirty neonates (97%) had increased CRP levels. Of the 31 patients, 14 cases were cured and 12 had adverse outcomes (5 patients died during hospitalization). Escherichia coli strains were resistant (>50%) to commonly used penicillins and cephalosporins between 2007 and 2011, presenting significantly higher resistance rates than between 2001 and 2006. The detection rate of extended spectrum β-lactamases (ESBLs)-producing strains between 2007 and 2011 increased significantly compared with between 2001 and 2006 (57% vs 0).</p><p><b>CONCLUSIONS</b>The clinical manifestations of neonatal purulent meningitis caused by Escherichia coli are non specific. The outcome is poor. Monitoring of CRP levels is valuable for the early diagnosis of neonatal purulent meningitis. The antimicrobial resistance rates of Escherichia coli are increasing, especially to cephalosporins. The percentage of ESBLs-producing strains is increasing over the years.</p>

Comparative analysis of the pathogens responsible for hospital acquired and community acquired late onset neonatal septicemia / 中华儿科杂志

<p><b>OBJECTIVE</b>Late onset neonatal septicemia (systemic infection after 72 hours of life) remains a major cause of neonatal morbidity and mortality. Early treatment with appropriate antibiotics is critical since infected infants can deteriorate rapidly. The aim of this study was to review the pathogens responsible for late onset neonatal septicemia (LONS) and their antimicrobial susceptibilities in order to guide the initial selection of appropriate antibiotics for infants with suspected LONS.</p><p><b>METHODS</b>A retrospective chart review of all cases with LONS seen in the neonatal intensive care unit (NICU) of Yuying Children's Hospital of Wenzhou Medical College from January 1, 2002 to December 31, 2005 was conducted. All cases were selected based on the clinical presentation and at least one positive result of blood culture. The basic clinical characteristics and the results of blood culture and antimicrobial susceptibilities were analyzed.</p><p><b>RESULTS</b>A total of 102 cases with LONS were identified. Among those 102 cases, 80 were community acquired (infants admitted from home and the blood culture was done on admission) and 22 were hospital acquired (infants became sick while in the NICU and the blood culture was done prior to use of antibiotics). The clinical presentations were non-specific. Compared to the infants with community acquired LONS, infants with hospital acquired LONS were usually born more prematurely (mean gestational age 33 +/- 3 vs 39 +/- 2 wks, t = 2.255, P < 0.01), with lower weight (mean weight 1.79 +/- 0.70 vs 3.23 +/- 0.67 kg, t = 8.818, P < 0.01) and with younger age (mean age 12 +/- 6 vs 16 +/- 7 days, t = 7.581, P < 0.05). Of the 102 cases, a total of 103 strains of bacteria were isolated. Among the pathogenic bacteria isolated, the most common were coagulase-negative Staphylococcus (CoNS) (50/103, 48.5%), followed by Klebsiella pneumoniae (16/103, 15.5%). The main pathogens for community acquired LONS were Staphylococcus species and Escherichia coli. The most important pathogen responsible for hospital acquired LONS was Klebsiella pneumoniae. Most (> 80%) of the Staphylococcus especially CoNS were resistant to common antibiotics such as penicillin, erythromycin and cefazolin. Significant numbers (6/9) of Staphylococcus aureus isolated were methicillin-resistant Staphylococcus aureus (MRSA). However, all of the Staphyloccus isolates were sensitive to vancomycin. Almost all (15/16) of the Klebsiella pneumoniae isolated were multi-drug resistant due to production of extended-spectrum beta-lactamases (ESBLs). They were sensitive only to a few antibiotics such as carbapenems, aminoglycosides and quinolones. There was also one strain of vancomycin-resistant Enterococcus (VRE). Furthermore, there was no a single case of late onset neonatal sepsis due to infection with group B Streptococcus (GBS).</p><p><b>CONCLUSIONS</b>The clinical manifestations of late onset neonatal sepsis are usually non-specific. GBS is not a significant pathogen responsible for community acquired LONS in the Wenzhou area. There are increasing numbers of multi-drug resistant bacterial species isolated from the newborn infants with late onset neonatal septicemia, which is most likely due the non-restricted use of antibiotics in the hospitals as well as in the communities. A routine blood culture should be taken from any newborn infant who is suspected of LONS and empirical use of appropriate antibiotics should be initiated as soon as the blood specimen for culture has been drawn. To reduce the occurrence of multi-drug resistant bacteria, the use of antibiotics especially the third generation cephalosporins in neonates should be restricted as much as possible.</p>