肠三叶因子对新生鼠NEC模型PI3K和caspase-3/9的影响

Objective To study the effects of ITF on PI3K (phosphatidylinositol 3-kinase) and caspases-3/9 (cysteinyl aspartate-specific protea-ses) in neonatal rat NEC model. Methods NEC model of neonatal rats was established. Asphyxia stress was accomplished by exposure to l00% nitrogen for 60s, followed by exposure to coldness (4 ℃ ) for 10 min twice daily. Neonatal rats were fed formula (200 kcal/kg/day) every 3 h via an gavage tube. The feeding volume began at 0. 1 cc every 3 h and was increased incrementally. This procedure is done once everyday and continued for 3 days. On the 4th day,all the subjects were sacrificed. Fifty neonatal rats were randomized into five groups: A) NEC + NS0. 2mi, (B) NEC + ITF 0. 2 mg, (C) NEC(Wortmannin (0. 1 mg/kg) (D) NEC + ITF 0. 2 mg + Wortmannin (0. 1 mg/kg), (E) control. The intestinal tissue located at the boundary of ileum and cecum was sampled for histology. The remaining intestinal tissue was homogenized. After the homogenate was centrifuged,supernatants were used to assay PI3K and Caspase-3 and caspase-9. Results The pathological lesions showed that intestinal necrosis was severe in group A、C and D, which was graded as 3points. They were significantly decreased in group B,which was graded 1 point. The level of PI3K(pg/ml)in Group A was higher than those in group E (P<0. 05), the latter had difference with those in group D(P>0. 05). The level of PI3K(pg/ml) were the strongest in group B and were lowest in group C. The activity of Caspase-3 and Caspase-9 were significantly increased in group A compared to those in group B and E (P<0. 01 ),and was significantly increased in Group C(P<0. 01 ). Caspase-3 and Caspase-9 levels of group A and D,group B and E showed no significant difference(P>0. 05). Conclusions Intestinal inflammation was ameliorated by intraperitoneal ITF. ITF may provide a new therapy for NEC; PI3K/Akt signal pathways might play important roles in signal transduction during NEC;ITF may protect the intestinal injury of neonatal Wistar rat by activation of PI3K/Akt signal transduction pathway, and down regulation of caspase-9.     

高氧致慢性肺疾病新生大鼠肺泡损伤及肺泡上皮细胞的变化

Objective To explore the changes of alveolar morphology and alveolar epithelial cells in rats with hyperoxia-induced chronic lung diseases (CLD). Methods CLD model in neonatal rats was established by inhalation of high concentration oxygen(85%～90% ). Eighty neonatal rats were randomly exposed to hyperoxia (model group) and to room air (control group) (n =40 each). Radical alveolar counts and the alveolar septum thickness were used to evaluate alveolar development. The site and intensity of expression of SPC,AQP5 protein were detected by immunohistochemical staining,the dynamic expression of SPC mRNA,AQP5mRNA was detected by RT-PCR on day 1,3,7,14 and 21 after exposure. Results There were no significant differences about alveolar wall thickness and RAC between experimental groups and control group on day 1～3 ( P > 0. 05 ). But there was significant difference between the model group and the control groups on day 7 and 14 (P <0. 01 ). For model group,alveolar septum thickness peaked on day 21, the difference was significant compared with control group ( 10. 62±5.01 vs 3.62±0. 88, P < 0. 001 ), but RAC decreased to the lowest level, the difference was significant compared with control group ( 3.57±1.24 vs 10. 47±0. 88,P <0. 001 ). The expression of SPC decreased on day 3 manifestedly but increased on day 7 and the levels of SPC were higher than that in the control group. Experimental group showed gradual decrease in AQP5 expression as the lung impairment devastated. Conclusion Alveolar development was delayed and alveolar epithelial cell (AEC) was damaged in the neonatal CLD rats. The changes of SPC,AQP5 expression suggested AECI was severely damaged and failed in full recovery, meanwhile the quantity of AEC Ⅱ was increased but the ability of its differentiation and transformation was decreased.     

高氧致慢性肺疾病新生大鼠肺泡损伤及肺泡上皮细胞的变化

Objective To explore the changes of alveolar morphology and alveolar epithelial cells in rats with hyperoxia-induced chronic lung diseases (CLD). Methods CLD model in neonatal rats was established by inhalation of high concentration oxygen(85%～90% ). Eighty neonatal rats were randomly exposed to hyperoxia (model group) and to room air (control group) (n =40 each). Radical alveolar counts and the alveolar septum thickness were used to evaluate alveolar development. The site and intensity of expression of SPC,AQP5 protein were detected by immunohistochemical staining,the dynamic expression of SPC mRNA,AQP5mRNA was detected by RT-PCR on day 1,3,7,14 and 21 after exposure. Results There were no significant differences about alveolar wall thickness and RAC between experimental groups and control group on day 1～3 ( P > 0. 05 ). But there was significant difference between the model group and the control groups on day 7 and 14 (P <0. 01 ). For model group,alveolar septum thickness peaked on day 21, the difference was significant compared with control group ( 10. 62±5.01 vs 3.62±0. 88, P < 0. 001 ), but RAC decreased to the lowest level, the difference was significant compared with control group ( 3.57±1.24 vs 10. 47±0. 88,P <0. 001 ). The expression of SPC decreased on day 3 manifestedly but increased on day 7 and the levels of SPC were higher than that in the control group. Experimental group showed gradual decrease in AQP5 expression as the lung impairment devastated. Conclusion Alveolar development was delayed and alveolar epithelial cell (AEC) was damaged in the neonatal CLD rats. The changes of SPC,AQP5 expression suggested AECI was severely damaged and failed in full recovery, meanwhile the quantity of AEC Ⅱ was increased but the ability of its differentiation and transformation was decreased.     

电针可下调缺氧缺血性脑损伤幼鼠大脑皮层硫化氢含量

Objective To establish hypoxic-ischemic brain damaged (HIBD) rat model,investigate whether H2S and cystathionine-β-synthase (CBS), the key enzyme for its generation, may be a mediator of electro-acupuncture(EA) stimulation treatment for HIBD. Methods Thirty-two healthy Sprague-Dawley neonatal rats were divided into four groups randomly: sham control group ( n = 8 ); sham + EA group ( n =8); HIBD control group ( n = 8); and HIBD + EA group ( n = 8 ). HIBD rat models were established on their 7-day-old. From the next day ,rats of sham + EA group and HIBD + EA group were electric stimulated 30 min daily for 14 d,BAIHUI and DAZHUI as the acupoints. Control ones were just fixed at the same time,without acupuncture. The rats were sacrificed on the 22 nd day, one day after the treatment course. Cortical H2S concentration was measured by sensitive sulphur electrode assay. The CBS protein expression was measured by western blot analysis and immunohistochemistry for localization. Results The concentration of cortical H2S in HIBD control group was (26. 83 ± 4. 31 ) nmol/mg protein, which was significantly higher than that of sham control group[(22. 78 ± 1.54) nmol/mg protein]( P ＜ 0. 01 ). The H2 S levels in HIBD + EA group and sham + EA group were ( 18.08 ± 2.71 ) nmol/mg protein and ( 18.91 ± 2. 78 ) nmol/mg protein, respectively. Compared with corresponding control group, they were much lower( P ＜ 0. 01 ). The expression of CBS protein in rats with EA stimulation decreased in cortex compared to corresponding control group( P ＜0. 05 ).Conclusion EA can down-resulate H2S/CBS pathway. This may be one of the mechanisms of how EA contributes to the recovery of brain damage.     

先天性甲状腺功能减退症新生大鼠大脑蛋白质差异表达的研究

目的 应用蛋白质组学方法筛选新生先天性甲状腺功能减退症大鼠大脑差异表达蛋白质,为阐明CH致脑发育障碍发病机制提供有价值的线索.方法 制作CH仔鼠动物模型,于出生时称重后处死仔鼠,取大脑,提取大脑皮质总蛋白,Bradford法检测蛋白质浓度.应用双向电泳(2-DE)技术分析新生正常仔鼠与CH仔鼠大脑蛋白质差异表达情况.选择重复性、分辨率好且表达差异明显的蛋白质点进行质谱分析.RIA法检测各组大鼠血清FT3、FT4水平.结果 新生甲减组仔鼠体重、FT3、FT4水平均低于正常组仔鼠(t体重=-8.07,tFT3=5.39,tFT4=7.62,P＜0.01).建立了较稳定的正常与CH仔鼠大脑2-DE图谱,筛选出7个重复性、分辨率好且表达差异明显的蛋白质点进行质谱分析.MALDI-TOF-MS质谱分析鉴定了相关的7个差异表达蛋白,包括塌陷反应介导蛋白2、肌动蛋白相关蛋白2/3复合物第5亚单位、泛素结合酶E2-25K、ATP合酶D亚单位、Cu-Zn超氧化物歧化酶、突触核蛋白α、核苷二磷酸激酶.结论 神经元突触形成异常、ROS产生异常增多、细胞凋亡等多条途径可能参与了CH致脑发育障碍,本研究为探讨CH致脑发育障碍机制提供了重要线索.

Abstract：

Objective To screen differentially expressed brain proteins with proteomic method in cerebral cortex of neonatal rats with congenital hypothyroidism. Method From the 13th day of gestation,pregnant Wistar rats from the experimental group were given intragastrically with 2. 5 ml of 1%propylthiouracil daily. Cerebral cortex specimens were collected from the control and hypothyroidism neonatal rats. Two-directional electrophoresis (2-DE) was applied to analyze protein expression diversities between the euthyroid and hypothyroidism neonatal rat cerebral cortex. Protein spots with significantly different expression were screened and identified by mass spectrometry. Radioimmunoassay (RIA) was used to analyze serum FT3 , FT4 levels of each groups. Result The body weight of hypothyroid neonatal rats were lower than those in the corresponding control group (t = -8.07, P ＜0. 01 ). The FT3 levels of hypothyroid neonatal rats were lower than those in the corresponding control group ( t = 5. 39, P ＜ 0. 01 ). The FT4 levels of hypothyroid neonatal rats were lower than those in the corresponding control group (t = 7.62, P ＜ 0. 01 ).Stable 2-DE maps of normal and CH neonatal rat were constantly obtained. The maps were analyzed by software. Seven protein spots with high reproducibility, high resolution and significantly different expression were chosen and identified by mass spectrometry, including collapsing response mediator protein 2, actin related protein 2/3 complex subunit 5, ubiquitin-conjugating enzyme E2-25K, ATP synthase subunit d, CuZn superoxide dismutase, synuclein alpha, and nucleoside diphosphate kinase. Conclusion The value of this research is demonstrated here by the identification of several proteins known to be associated with nerve synapse structures formation, cell survival, metabolism, cell signal transduction, neural differentiation and nerve growth in the central nervous system. Furthermore this study identified several proteins except for collapsing response mediator protein 2 and Cu-Zn superoxide dismutase that have not previously been described in the literature and which may play an important role as either sensitive biomarkers of brain dysfunction caused by congenital hypothyroidism. In congenital hypothyroidism, brain development retardation may be related with some important processes, including abnomal synaptic formation, excess ROS production and apoptosis. The above-mentioned proteins may play critical roles in the processes, which provide valuable clues to clarify the pathogenesis of brain developmental disorders induced by congenital hypothyroidism.     

白藜芦醇甙对缺氧缺血性脑损伤新生大鼠学习记忆和海马突触素表达的影响

Objective To explore the protective effects and possible mechanisms of Polydatin (PD)on hypoxic-ischemia brain injury(HIBD) in neonatal rat by means of spatial learning memory and the expression of synaptophysin in hippocampal CA1. Methods Thirty-seven neonatal SD rats were divided into 3 groups at random: normal sham-operated group( no hypoxia and ischemia); HIBD group( no medication) ;PD treatment group. 7-old-day rat' s model of HIBD was established by left carotid artery ligation and 2 h hypoxia. Morris water maze test was used to evaluate cognitive function in the rats after 28-day-old( 21-day later after HI). Immunohistochemical method was used to measure the expression of synaptophysin after the end of Morris water maze test. Results Morris water maze results showed that the mean escape latency of the shamgroup (SG) ,HIBD group (HIBD) and PD treatment group (PD) were (39. 55 ±8. 08) s, (52. 37 ±8.03) s and (43.29 ± 7. 63 ) s respectirely. For PD and SG, the mean escape latency was significantly shorter than the HIBD (P ＜0.05). After training,the mean escape latency in the three groups of rats was shortened gradually. The frequency of platform crossings were 5. 29 ±2.62、2. 36 ± 1.80、4. 25 ± 1. 66 in the SG,HIBD and PD respectirely. The frequency of platform crossings in PD was higher than that of HIBD ( P ＜ 0. 05 ). The swimming time in target quadrant were ( 15.74 ± 3.85) s, ( 10. 63 ± 3.66) s and ( 14. 32 ± 2. 52 ) s in SG, HIBD and PD respectirely. For HIBD ,the swimming time in target quadrant was significantly shorter comparing to SG and PD ( P ＜ 0. 05 ). The expression of synaptophys in hippocampal CA1 in PD ( 0. 295 2 ± 0. 044 3 )were evidently higher than that in the HIBD group (0.261 2 ±0.032 3) at 3 week after operation (P ＜0. 05). Conclusion Spatial learning memory deficits and the decrease of synaptophys in hippocampal CA1 could be induced by hypoxic-ischemia. Polydatin could improve the learning and memory ability in neonatal rats following hypoxic-ischemia brain damage. The mechanisms of improvement with Polydatin treatment is associated with the enhancement of expression of synaptophys.     

单侧隐睾大鼠对侧睾丸组织中SCF/c-kit基因表达变化及意义

Objective To investigate the pathologic changes and expressions of SCF and c-kit in the contralateral testes in rat model of unilateral cryptorchidism. Methods Thirty male SD rats were maintained under controlled temperature and constant photoperiodic conditions with access to food and water. The rats were randomly assaigned to the control group and the experimental unilateral cryptorchidism group. The left testis of the rats in the unilateral cryptorchidism group was placed into the abdominal cavity. The control group rats were subjected to sham surgery. Three months later, the rats were sacrificed and their right testes were harvested. The pathological changes were observed under microscope. The mRNA and protein expression of SCF and c-kit were also investigated using quantitative Real-time RT-PCR, Western blotting and immunohistochemical staining. Apoptotic germ cells were detected by TUNEL staining. Results All rats survived to the endpoints. The right testes of rats of the unilateral cryptorichid group were smaller than those of the control group. Normal testes of the control group rats manifested active spermatogenesis and orderly arrangement of germ cells.However, disordered and sloughed germinal cells with less distinct seminiferous tubule borders were observed in the contralateral testes of rats with experimental unilateral cryptorichid under the microscope. Cmpared with the control testes, the mRNA and protein expression of SCF and c-kit of the contralateral testes of rats with experimental unilateral cryptorichid was decreased (P＜0.05). The apoptotic germ cells were increased (19.7±3.83 vs 5. 4 ± 1.02, P＜0. 05). The SCF/c-kit expression was positively correlated with the germ cell apoptosis (P＜0. 01 ). Conclusions The decreased expression of SCF and c-kit and increased germ cell apoptosis are found in the contralateral testes, which may contribute to thc infertility in the rat model of experimental unilateral cryptorchidism.     

生长抑素对缺氧缺血性脑病肠损伤的治疗

Objective To explore the therapeutical effect of somatostatin(SST) on the damaged bowel barrier of 7-day-old SD rats with hypoxic-ischemic encephalopathy(HIE). Methods Total 7-day-old SD rats were randomly divided into untreated control group(n=6), sham-operated group(n = 6), HIE model group(n = 8) and SST-treated group(n = 8). After 6 hours of operation, abdominal aorta blood of the rats was collected to measure the level of D-lactic acid, the ileum was taken for pathological analysis and immunohistochemistry staining of SP. Results The level of D-lactic acid in HIE model group was obviously increased[(10.30 ± 1.70)mg/L], and there were significant differences compared to untreated control group, sham-operated group as well as SST-treated group respectively(P < 0.05, respectively). The cillia of ileum became widen and shorten, and the number of it decreased. At the same time, in the stratum mucosa and the neurofibrillary tangle, the expression of SP was increased (average optical density: 12.67 ± 5.46), and there were significant differences compared to untreated control group, sham-operated group as well as SST-treated group raspectively (P < 0.05,respectively).Compared with HIE model group,the level of D-lactic aeid decreased obviously[(7.35 ± 1.55) mg/L] in SST-treated group,and there were no differences compared to untreated control group and sham-operated group respectively. The intestinal villi became thinner and higher in the SST-treated group, even the number increased. Besides that, the expression of SP decreased(average optical density: 0.73 ± 0.09), and there were no differences compared to untreated control group and sham-operated group respectively. Conclusion The therapeutical effect of SST on the bowel barrier damage of 7-day-old SD rats with HIE is clear.     

生长抑素对缺氧缺血性脑病肠损伤的治疗

Objective To explore the therapeutical effect of somatostatin(SST) on the damaged bowel barrier of 7-day-old SD rats with hypoxic-ischemic encephalopathy(HIE). Methods Total 7-day-old SD rats were randomly divided into untreated control group(n=6), sham-operated group(n = 6), HIE model group(n = 8) and SST-treated group(n = 8). After 6 hours of operation, abdominal aorta blood of the rats was collected to measure the level of D-lactic acid, the ileum was taken for pathological analysis and immunohistochemistry staining of SP. Results The level of D-lactic acid in HIE model group was obviously increased[(10.30 ± 1.70)mg/L], and there were significant differences compared to untreated control group, sham-operated group as well as SST-treated group respectively(P < 0.05, respectively). The cillia of ileum became widen and shorten, and the number of it decreased. At the same time, in the stratum mucosa and the neurofibrillary tangle, the expression of SP was increased (average optical density: 12.67 ± 5.46), and there were significant differences compared to untreated control group, sham-operated group as well as SST-treated group raspectively (P < 0.05,respectively).Compared with HIE model group,the level of D-lactic aeid decreased obviously[(7.35 ± 1.55) mg/L] in SST-treated group,and there were no differences compared to untreated control group and sham-operated group respectively. The intestinal villi became thinner and higher in the SST-treated group, even the number increased. Besides that, the expression of SP decreased(average optical density: 0.73 ± 0.09), and there were no differences compared to untreated control group and sham-operated group respectively. Conclusion The therapeutical effect of SST on the bowel barrier damage of 7-day-old SD rats with HIE is clear.     

亚低温治疗新生儿缺氧缺血性脑损伤的近期疗效分析

Objective To assess short-time effect of selective head cooling on neonatal hypoxic-ischemic encephalopathy induced by perinatal asphyxia. Methods Twenty-three infants of moderate/severe hypoxic ischemic encephalopathy (HIE) were divided into treatment group ( 14 cases) and control group (9 cases). The head hypothermia in the treatment group [rectal temperature ( 34. 0 ～ 35.0) ℃, nasopharyngeal temperature (34. 0 ± 0. 5 ) ℃]was induced by circulating water cooling cap for up to 72 h. Control group were treated routinely. All newborns were monitored and analyzed for blood pressure, heart rate, neurological function. Neonatal behavioral neurological assessment (NBNA) was conducted for assessment of neurobehavioral development on day 28 after birth. Results Treatment group showed slower hear rate as nasopharyngeal temperature decreased and there was significant difference as compared with the control group ( P ＜ 0.05 ). Apnea and arrhythmia were not found in either group. As compared with the control group, treatment group showed better results in seizure incidence, recovery of neonatal reflex( embracing reflection, sucking reflex and grasp reflex), and improvement of limb muscle tension. The NBNA score on day 28 was much higher in hypothermia group(35.00 ± 1.41 ) than that of control group(30. 67 ± 1.58) and there was significant difference (P ＜ 0.05). Conclusion Selective head cooling is safe for newborns with moderate/severe HIE if the nasopharyngeal temperature maintains at (34 :± 0. 5 ) ℃, rectal temperature at 34 ～ 35 ℃. Selective head cooling can effectively improve the short-term nervous symptoms and the neurobehavioral score,but the long-term efficacy remains to be further studied.     

3-hydroxy-3-methylglutaraciduria (case report of a female Turkish sisters with 3-hydroxy-3- methylglutaryl-Coenzyme A lyase deficiency

3-Hydroxy-3-methylglutaric aciduria is a rare inborn error of metabolism, caused by reduced enzyme activity of the intramitochondrial 3-hydroxy-3-methylglutaryl-CoA lyase. We describe two turkish sisters with this disease. In the older sister clinical symptoms with lethargy, convulsions, metabolic acidosis, hypoglycemia and hyperammonemia lead to the diagnosis. The younger sister was diagnosed prenatally. The clinical course of our patients is compared with those reported in the literature with respect to clinical symptoms, differential diagnosis and therapeutic regimens.     

A case of biotin-responsive 3-methylcrotonylglycin- and 3-hydroxyisovaleric aciduria

During selective screening for organic acidurias, a 10-week-old girl with muscular hypotonia and recurrent fits was shown to be excreting 3-methylcrotonylglycin and 3-hydroxyisovaleric acid. Besides these metabolites of leucine the presence of small but pathological amounts of propionic and methylcitric acids were demonstrable in her urine, pointing to a defect in the metabolism of biotin.On treatment with biotin (2×5 mg/day) the convulsions stopped at once, her clinical condition improved gradually, and the abnormal metabolites disappeared from the urine. Within 6 weeks the child was discharged in a good general condition without apparent signs of neurological damage.     

关于脓毒症3.0的争议

感染诱发的脓毒症是ICU最常见的疾病之一.为了统一认识,利于早期诊断和治疗,降低重症感染的病死率,1991年首次提出了脓毒症的概念.近20余年,伴随脓毒症病理生理研究的深入,其定义和诊断标准也不断更新和完善.2016年,欧洲危重病学会指定专家组重新修订了脓毒症的定义和诊断标准,即脓毒症3.0,主要强调感染导致宿主内环境稳态失衡、潜在致命性风险的器官功能障碍.每次更新都融入许多新的理论或观点,同时也充满了争议.本文主要介绍脓毒症的定义和诊断标准,以及脓毒症3.0存在的争议.     

3-HYDROXY-3-METHYLGLUTARYL COENZYME A LYASE DEFICIENCY

ABSTRACT. Shilkin, R., Wilson, G. and Owles, E. (Princess Margaret Hospital for Children, Perth, Western Australia). 3-Hydroxy-3-methylglutaryl Coenzyme A lyase deficiency: follow-up of first described case. Acta Paediatr Scand, 70:265, 1981. –We report the progress of a child with a defect in leucine metabolism due to a deficiency of 3-hydroxy-3-methylglutaryl Coenzyme A lyase activity. This child was reported briefly in 1976 when the abnormality was first suspected at which time he was 7 months old. He is now aged 4 years 7 months and appears to be well and developing satisfactorily. His diet has been difficult to control and the biochemical defect is extremely sensitive to small amounts of leucine in the diet.     

3-Methylglutaconic and 3-methylglutaric aciduria in a patient with suspected 3-methylglutaconyl-CoA hydratase deficiency

A girl suffering from marked muscular hypotonia, severe statomotor and mental retardation, bilateral optic atrophy with chorioretinal degeneration, convulsions and a moderate compensated metabolic acidosis is described. Screening for metabolic disorders revealed massive 3-methylglutaconic with 3-methylglutaric aciduria leading to the tentative diagnosis of 3-methylglutaconyl-CoA hydratase deficiency. Metabolite excretion was correlated with variation of leucine intake. 3-methyl-3-hydroxyglutaryl-CoA lyase activity in cultured fibroblasts was normal. The suspected metabolic defect was not demonstrable in cultured skin fibroblasts, however.Abbreviation MSUD Maple syrup urine disease     

胰岛素样因子3研究进展

胰岛素样因子3属于胰岛素超家族松弛素亚族,是睾丸间质细胞分泌的主要产物,其合成完全依赖睾丸间质细胞的分化状态.胰岛素样因子3除了在睾丸下降方面起重要作用,对于评估睾丸间质细胞功能也很敏感,有很大的临床价值.胰岛素样因子3在生殖细胞存活、骨代谢调控中发挥重要作用.