OBJECTIVETo study the expression of Toll-like receptor 4 (TLR-4) and caspase-3 in the intestine of neonatal rats with necrotizing enterocolitis (NEC), and explore the protective effects and possible regulatory mechanisms of glutamine (Gln) in NEC.

METHODSSixty premature rats were randomly divided into three groups (n=20 each): control, NEC model and Gln intervention group. NEC model was prepared by formula feeding, hypoxia and cold stress. The Gln intervention group was also subjected to hypoxia and cold stress but was fed with formula containing Gln (0.3 g/kg). Two days later, the rats were sacrificed and the intestine tissues were obtained. The histological changes of ileal tissues were observed by hemetoxylin and eosin staining. The expression of caspase-3 and TLR-4 protein in the jejunum, ileum and colon were detected by inmunohistochemistry. The expression of TLR-4 mRNA in the jejunum, ileum and colon were detected by RT-PCR.

RESULTSCompared with the control group, the histological score of ileal tissues, and the expression of caspase-3, TLR-4 protein and TLR-4 mRNA in the NEC model group increased significantly (P<0.01). Gln intervention decreased significantly the histological score of ileal tissues, and the expression of caspase-3, TLR-4 protein and TLR-4 mRNA compared with the NEC model group (P<0.05).

CONCLUSIONSTLR-4 might be involved in the pathogenesis of NEC. Gln may provide protective effects on intestine possibly through reducing the TLR-4 expression and then decreasing the apoptosis of intestinal epithelial cells.

Animals ; Animals, Newborn ; Caspase 3 ; analysis ; Enterocolitis, Necrotizing ; metabolism ; pathology ; Female ; Glutamine ; pharmacology ; Immunohistochemistry ; Pregnancy ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley ; Toll-Like Receptor 4 ; analysis ; genetics

BACKGROUNDAngiopoietin-1 (Ang-1) is an endothelial-specific growth factor that can promote angiogenesis. Studies demonstrated that Ang-1 can inhibit apoptosis of umbilical endothelial cells, but so far little is known about its effects on apoptosis of microvascular endothelial cells. With the apoptotic model of murine-cerebral-derived microvascular endothelial cells (bEnd.3) induced by serum-free culture, we attempted to clarify the molecular mechanism of bEnd.3 apoptosis, particularly its relation to cytochrome C (Cyt C).

METHODSThe cultured microvascular endothelial cell strain, bEnd.3 cell, was employed. An apoptotic model of bEnd.3 was established by serum-free culture. Flow cytometry after Annexin labeling and PI staining were used to assess the apoptotic effects of Ang-1 on bEnd.3, and the expression of Bax/Bcl-2, caspase 8, caspase 3, and Cyt C were detected with Western blotting and ELISA.

RESULTSThe apoptotic rate of bEnd.3 cells after stimulation with Ang-1 (100 ng/L) in serum-free medium was significantly higher than that in control group. Ang-1 inhibited early-stage apoptosis more than late-stage apoptosis provided by propidium iodide (PI) and AnnexinV double staining. The inhibition of Ang-1 on bEnd.3 cell apoptosis was strengthened with the increase in concentration (0 - 400 ng/ml). Ang-1 could decrease the expression of Bax, caspase3 and 8, and increase that of Bcl-2. The results of ELISA indicated that Ang-1 significantly decreased CytC content in cytoplasm and increase that in mitochondria.

CONCLUSIONSAng-1 could inhibit bEnd.3 apoptosis induced by serum-free medium culture. The apoptosis was associated with decreased Bax expression, increased Bcl-2 expression, which result in Cyt C transferring from mitochondria to cytoplasm, and then caspases activation are reduced and cell apoptosis is suppressed.

Angiopoietin-1 ; pharmacology ; Animals ; Annexin A5 ; analysis ; Apoptosis ; drug effects ; Caspase 3 ; Caspase 8 ; Caspases ; analysis ; Cells, Cultured ; Cytochromes c ; physiology ; Endothelial Cells ; cytology ; drug effects ; Mice ; Microcirculation ; drug effects ; bcl-2-Associated X Protein ; analysis

BACKGROUNDNeuroblastoma, one of the common tumors in children, possesses the feature of natural regression that might be related to apoptosis caspase-3 and survivin are believed to respectively induce and inhibit apoptosis. We investigated the expression of caspase-3 and survivin in pediatric neuroblastoma and the role that these genes played in apoptosis.

METHODSThe expression of caspase-3 and survivin in pediatric neuroblastoma tissue samples was detected using in situ hybridization, ter mintuesal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL), and immunohistochemical staining. The role that these genes played in apoptosis was then evaluated.

RESULTSA converse correlation was observed between the expression of survivin and caspase-3. When survivin was expressed at high levels in neuroblastoma samples, caspase-3 expression was downregulated, and the apoptotic index decreased simultaneously.

CONCLUSIONThere is a converse correlation between the expression of caspase-3 and the expression of survivin in neuroblastoma cells, indicating that caspase-3 might induce apoptosis, and survivin may inhibit this process.

Apoptosis ; Caspase 3 ; Caspases ; analysis ; physiology ; Female ; Humans ; Immunohistochemistry ; Infant ; Infant, Newborn ; Inhibitor of Apoptosis Proteins ; Male ; Microtubule-Associated Proteins ; analysis ; physiology ; Neoplasm Proteins ; Neuroblastoma ; chemistry ; pathology

OBJECTIVETo study the effects of perinatal recurrent infection on the brain development in immature mice.

METHODSSix pregnant C57BL6 mice were randomly assigned to three groups: intrauterine infection, perinatal recurrent infection and control. The intrauterine infection group was intraperitoneally injected with LPS (0.5 mg/kg) on the 18th day of pregnancy. The perinatal recurrent infection group was injected with LPS (0.5 mg/kg) on the 18th day of pregnancy and their offsprings were intraperitoneally injected with the same dose of LPS daily from postnatal day 3 to 12. The control group was administered with normal saline at the same time points as the recurrent infection group. The short-time neurobehaviors were assessed on postnatal day 13. The mice were then sacrificed to measure brain weights and neuropathological changes using cresyl violet staining. Western blot was used to evaluate the expression of TNF-α, Caspase-3 and myelin basic protein (MBP).

RESULTSThe brain weights of the recurrent infection group were significantly lower than the control and intrauterine infection groups (P<0.05) and the recurrent infection group displayed significant neuropathological changes. Perinatal recurrent infection resulted in increased expression levels of TNF-α and Caspase-3, and decreased expression level of MBP compared with the intrauterine infection and control groups (P<0.01). The neurobehavior test showed that the recurrent infection group used longer time in gait reflex, right reflex and geotaxis reflex compared with the control and intrauterine infection groups on postnatal day 13 (P<0.05).

CONCLUSIONSPerinatal recurrent infection may exacerbate inflammatory response and cell death in the immature brain, which may be one of the important factors for perinatal brain injury.

Animals ; Animals, Newborn ; Bacterial Infections ; physiopathology ; Body Weight ; Brain ; growth & development ; pathology ; Caspase 3 ; analysis ; Female ; Mice ; Mice, Inbred C57BL ; Myelin Basic Protein ; analysis ; Pregnancy ; Recurrence ; Reflex

BACKGROUND: Caspase-3 is a cysteine protease that plays a major role in the process of apoptotic cell death. The dysregulated expression of c-myc contributes to the tumorigenesis in a variety of human cancers. The aim of this study was to investigate the expressions of caspase-3 and c-myc and their significances as prognosis markers in patients with completely resected non-small cell lung cancer (NSCLC). MATERIAL AND METHOD: A total 130 consecutive patients who had undergone complete resection without pre-operative radio-therapy or chemotherapy between May 1996 and December 2003 for NSCLC were retrospectively reviewed. The median follow-up period of the patients was 50 months (range: 3~128 months). The expressions of caspase-3 and c-myc were immunohistochemically examined, and these were correlated with the clinico-pathologic data. RESULT: The prevalence of caspase-3 and c-myc expressions in the patients was 68% (88/130) and 59% (77/130), respectively. Significant association was found between the frequency of the expressions of caspase-3 and c-myc (p=0.025). The caspase-3 and c-myc expressions were not significantly associated with the prognosis in all the patients. However, according to stages, a positive caspase-3 expression was significantly correlated with a favorable prognosis for patients with stage IIIa disease (median survival period: 35 months vs. 10 months, p=0.021). Multivariate analysis showed the pathologic stage to be significantly correlated with a good prognosis in all the patients (p=0.024), and with a positive caspase-3 expression, well differentiated tumor and negative neuronal invasion in the patients with stage IIIa disease (p=0.005, p=0.003, p=0.004, respectively). CONCLUSION: Caspase-3 and c-myc were frequently expressed in NSCLC, suggesting its possible involvement in tumor development. The caspase-3 expression, as determined with performing immunohistochemical staining, may be a favorable prognostic indicator in patients with completely resected NSCLC of an advanced stage (IIIa).
Carcinoma, Non-Small-Cell Lung ; Caspase 3 ; Cell Death ; Cell Transformation, Neoplastic ; Cysteine Proteases ; Follow-Up Studies ; Humans ; Multivariate Analysis ; Neurons ; Prevalence ; Prognosis ; Retrospective Studies

OBJECTIVETo investigate the effects of mild hypothermia on sequential events of neuronal apoptosis following hypoxic-ischemic brain damage (HIBD) in neonatal rats.

METHODSA model of HIBD was prepared by ligating the left common carotid artery in 7-day-old rats, followed by 8% hypoxia exposure. HIBD rats were randomly assigned into a hypothermia group (rectal temperature = 33 centi-degrees) and a normothermia group (rectal temperature = 36 centi-degrees). TUNEL, Haematoxylin and Eosin, and Nissl staining were used to detect neuronal apoptosis. Western blotting, RT-PCR and enzyme activity measurement were used to evaluate the changes of plasma and mitochondrial cytochrome c (Cyt c), caspase-3 mRNA expression and caspase-3 enzyme activity, respectively.

RESULTSThe number of apoptotic cells in the ipsilateral hemisphere of the hypothermia group was significantly reduced compared with that of the normothermia group at 72 hrs post-HI (6.4 +/- 1.7% vs 25.3 +/- 1.5%) (P < 0.01). Analysis of Western blotting showed that Cyt c levels increased in the cytosolic fraction, but decreased significantly in the mitochondrial fraction in the ipsilateral hemisphere of the hypothermia group at 24, 48 and 72 hrs of HI insult compared with the normothermia group (P < 0.05). Caspase-3 mRNA increased significantly after 24 hrs post-HI in the normothermia group, and this change became more pronounced with time. Mild hypothermia treatment decreased significantly caspase-3 mRNA expression at 24, 48 and 72 hrs post-HI (P < 0.05). Caspase-3 activity gradually increased 2 hrs after HI insult and peaked at 24 hrs in the normothermia group. Mild hypothermia treatment resulted in a significant reduction in caspase-3 activity in the ipsilateral hemisphere, with an optimal effect produced at 24 hrs post-HI (2.42 +/- 0.5 RFU vs 34.7 +/- 3.2 RFU; P < 0.01).

CONCLUSIONSMild hypothermia treatment attenuates neuronal apoptosis following HIBD, possibly through a reduction in Cyt c release from mitochondria and an inhibition of caspase-3 mRNA expression and its enzyme activity.

Animals ; Apoptosis ; Brain ; pathology ; Caspase 3 ; genetics ; metabolism ; Cytochromes c ; secretion ; Female ; Hypothermia, Induced ; Hypoxia-Ischemia, Brain ; pathology ; therapy ; Male ; RNA, Messenger ; analysis ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the single nucleotide polymorphisms (SNPs) and the distribution of their haplotypes in caspase-3 gene in Zhejiang Han nationality in China.

METHODSDenaturing high-performance liquid chromatography (DHPLC) and DNA sequencing were used to detect the SNPs in the regulatory region and the exons 2-7 and their flanking sequences in caspase-3 gene. Expectation maximization (EM) algorithm was used for haplotype frequencies analysis and pairwise linkage disequilibrium test.

RESULTS(1) Three SNPs were identified in caspase-3 gene; the three sites C829A, A17532C and C20541T were located in 5' regulatory region, intron 4 and 3' regulatory region, respectively. (2) Strong linkage disequilibrium was found among these SNPs; site A17532C and C20541T were in complete linkage disequilibrium. (3) C-829/A-17532/C-20541 (54.3%) was the main haplotype of Zhejiang Han nationality.

CONCLUSIONThe above findings indicated there is strong linkage disequilibrium among the three SNPs in caspase-3 gene in Han nationality of Zhejiang province and the main haplotype of Han nationality is obviously different from that of North American.

Asian Continental Ancestry Group ; genetics ; Caspase 3 ; Caspases ; genetics ; China ; ethnology ; DNA Mutational Analysis ; Gene Frequency ; Genotype ; Haplotypes ; Humans ; Introns ; Linkage Disequilibrium ; Polymorphism, Single Nucleotide

To evaluate expression of caspase-3 protein in CD34(+) cells from cord blood (CB) and its significance during culture in vitro with various growth factors, RT-PCR, Western blot and flow cytometry were used to determine cell proliferation, apoptosis and expression of caspase-3 in CD34(+) cells during culture in vitro. The results demonstrated that caspase-3 mRNA was constitutively expressed at a low level in freshly isolated CD34(+) cells. Expression of caspase-3 mRNA and protein was upregulated when these cells were expanded in suspension culture with growth factors for 3 days. However, only the 32 kD inactive caspase-3 proenzyme was detected in the freshly isolated CD34(+) cells and those cells after 3 days expansion with cytokines. Along with the culture time extension (7 days) in vitro, especially without early-effective cytokines SCF or FL existence, caspase-3 was activated and a cleavage 20 kD was detected. It is concluded that the caspase-3 is involved in apoptosis of primitive CD34(+) cells during expansion in vitro, and early-effective cytokines, SCF and FL, could conserve hematopoietic stem cells and suppress apoptosis.
Antigens, CD34 ; blood ; Apoptosis ; Caspase 3 ; Caspases ; analysis ; physiology ; Cells, Cultured ; Fetal Blood ; cytology ; Hematopoietic Stem Cells ; enzymology ; physiology ; Humans ; Infant, Newborn ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate Caspase-3 expression and its role in neuronal apoptosis.

METHODSThe T13-L2 spinal cord of rats was injured by traction after the amplitude of P1-N1 wave, monitored by a cortical somatosensory evoked potential (CSEP) monitor, decreased to seventy percent of that before operation. Then rats were killed in 6 h, 1 d, 4 d, 7 d, 14 d and 21 d respectively after operation. Flow cytometer terminal deoxynucleotldyl transferease-mediated biotinylated deoxynuridine triphosphate nick end labeling (TUNEL), Caspase-3 activity assay and immunohistochemical method were applied to investigate Caspase-3 expression in the spinal cord tissue and to study neuronal apoptosis in rats.

RESULTSAfter spinal cord injury, apoptotic cells detected by flow cytometry and TUNEL-positive cells were significantly more, and positive immunohistochemical staining of Caspase-3 and Caspase-3 activity were significantly higher in Group injury than in Groups control and laminectomy, respectively (P > 0.05, P > 0.01). Similar trend of changes was noticed in apoptotic cells, TUNEL-positive cells and positive immunohistochemical staining of Caspase-3, all of which reached their respective peak 7 days after operation. Caspase-3 activity reached its peak, however, 4 days postoperatively.

CONCLUSIONSIncreased expression and activity of Caspase-3 protein in neurons after tractive spinal cord injury is the biochemical signal of early spinal cell apoptosis. It is of great significance for understanding the mechanism of spinal cord injury.

Animals ; Apoptosis ; Caspase 3 ; Caspases ; analysis ; physiology ; Flow Cytometry ; Immunohistochemistry ; In Situ Nick-End Labeling ; Rats ; Rats, Sprague-Dawley ; Spinal Cord Injuries ; enzymology ; pathology

OBJECTIVE: To study the anti- scarring effect of rapamycin in rabbits receiving glaucoma filtering surgery. METHODS: Ninety-six Chinchilla rabbits were randomized equally into 3 rapamycin treatment groups and one control group. All the rabbits underwent trabeculectomy, after which the rabbits in the 3 rapamycin groups were treated with eye drops containing 1%, 3%, or 5% rapamycin in the operated eyes, and those in the control groups were given castor oil 4 times a day. The intraocular pressure (IOP) and inflammatory reaction in the treated eyes were observed, and the PCNA-positive cells in the filtering bleb were detected using immunohistochemistry. RTFs isolated from the Tenon's capsule of the rabbits were cultured , and the expressions of caspase-3, caspase-8, and caspase-9 in the fibroblasts were detected after treatment with different concentrations of rapamycin. RESULTS: The IOP was significantly lower in rapamycin-treated group than in the control group after the surgery ( < 0.05). The counts of the PCNA-positive cells were significantly lower in rapamycin-treated rabbits than in the control group ( < 0.05). Rapamycin treatment dose-dependently increased the expressions of caspase-3 and caspase- 9 at both the mRNA ( < 0.001) and protein ( < 0.001) levels without causing significant changes in the expressions of caspase-8. CONCLUSIONS Rapamycin can inhibit excessive proliferation of the fibroblasts in the filtering bleb to reduce scar formation after glaucoma filtration surgery in rabbits. Rapamycin also increases the expressions of caspase-3 and caspase-9 to induce apoptosis of the RTFs.
Animals ; Caspase 3 ; metabolism ; Caspase 9 ; metabolism ; Cell Proliferation ; drug effects ; Cicatrix ; prevention & control ; Filtering Surgery ; adverse effects ; Glaucoma ; surgery ; Intraocular Pressure ; Postoperative Complications ; enzymology ; prevention & control ; Proliferating Cell Nuclear Antigen ; analysis ; Rabbits ; Random Allocation ; Sirolimus ; therapeutic use ; Trabeculectomy