TdT) dUTP Nick-end Labeling Research Articles

Therapeutic effects of Dioscorea on post-menopause-induced cardiac apoptosis in rats.

To evaluate the effects of Dioscorea on bilateral ovariectomies-induced cardiac Fasdependent and mitochondria-dependent apoptotic pathways. Forty-eight female Wistar rats at 6-7 months of age were equally divided into a sham-operated group, and a bilateral ovariectomized (OVX) group for 2 months, and the rats in the OVX group were further fed with 0, 250 or 750 mL/kg Dioscorea spp. daily in the 2nd month. The excised hearts were measured by positive terminal deoxynucleotidyltransferase UTP nick end labeling (TUNEL) assays, western blotting and reverse transcription-polymerase chain reaction. Dioscorea spp. decreased OVX-induced cardiac TUNEL-positive apoptotic cells; decreased OVX-induced TNF-alpha, Fas ligand, Fas death receptors, Fas-associated death domain, activated caspase-8, and -3 (Fas pathways); decreased OVX-induced Bad, Bax, Bax-to-Bcl2 ratio, activated caspase-9, and -3 (mitochondria pathway). Dioscorea spp. prevented ovariectomy-induced cardiac Fas-dependent and mitochondriadependent apoptotic pathways in rat models. The fifi ndings may provide possible therapeutic effects of dioscorea for potentially preventing cardiac apoptosis after ovariectomy or post-menopause.

Fabrication of novel vesicles of triptolide for antirheumatoid activity with reduced toxicity in vitro and in vivo.

Triptolide (TP) displays a strong immunosuppression function in immune-mediated diseases, especially in the treatment of rheumatoid arthritis. However, in addition to its medical and health-related functions, TP also exhibits diverse pharmacological side effects, for instance, liver and kidney toxicity and myelosuppression. In order to reduce the side effects, a nano drug carrier system (γ-PGA-l-PAE-TP [PPT]), in which TP was loaded by a poly-γ-glutamic acid-grafted l-phenylalanine ethylester copolymer, was developed. PPT was characterized by photon scattering correlation spectroscopy and transmission electron microscopy, which demonstrated that the average diameter of the drug carrier system is 98±15 nm, the polydispersity index is 0.18, the zeta potential is −35 mV, and the TP encapsulation efficiency is 48.6% with a controlled release manner. The methylthiazolyldiphenyl-tetrazolium bromide assay and flow cytometry revealed that PPT could decrease toxicity and apoptosis induced by free TP on RAW264.7 cells, respectively. The detection of reactive oxygen species showed that PPT could decrease the cellular reactive oxygen species induced by TP. Compared with the free TP-treated group, PPT improved the survival rate of the mice (P<0.01) and had no side effects or toxic effects on the thymus index (P>0.05) and spleen index (P>0.05). The blood biochemical indexes revealed that PPT did not cause much damage to the kidney (blood urea nitrogen and creatinine), liver (serum alanine aminotransferase and aspartate aminotransferase), or blood cells (P>0.05). Meanwhile, hematoxylin and eosin staining and terminal-deoxynucleotidyl transferase dUTP nick-end labeling staining indicated that PPT reduced the damage of free TP on the liver, kidney, and spleen. Our results demonstrated that PPT reduced free TP toxicity in vitro and in vivo and that it is a promising fundamental drug delivery system for rheumatoid arthritis treatment.

Subclinical concentrations of sevoflurane reduce oxidative stress but do not prevent hippocampal apoptosis.

Sevoflurane is generally considered a pro-apoptotic agent in the neonatal brain. However, recent studies have suggested that low levels of sevoflurane anesthesia may be neuroprotective and have a memory enhancing effect. The present study aimed to investigate whether sevoflurane exerts a neuroprotective effect at subclinical concentrations, with regard to oxidative state. In the current study, postnatal day 7 (P7) Sprague-Dawley rats were continuously exposed to 0.3, 1.3, or 2.3% sevoflurane for 6 h. ELISA was used to quantify the levels of superoxide dismutase, glutathione peroxidase (GSH-px) and malondialdehyde (MDA) in the plasma and the hippocampus. Terminal deoxynucleotidyl-transferase dUTP nick-end labeling staining was used to observe hippocampal neuronal apoptosis. Altered object exploration tests for recognition memory were employed to investigate long-term behavioral effects at postnatal day 28. The results demonstrated that a single 6 h exposure to a subclinical concentration (1.3%) of sevoflurane at P7 reduces MDA and GPH-px production in rats. Sevoflurane induced hippocampal apoptosis in a dose-dependent manner and altered recognition memory testing indicated no differences among the groups. Although early exposure to a subclinical concentration of sevoflurane reduced oxidative stress, it did not prevent the process of sevoflurane-induced hippocampal apoptosis. These changes did not affect subsequent recognition memory in juvenile rats.

Developmental Potential of Vitrified Mouse Testicular Tissue after Ectopic Transplantation.

Cryopreservation of immature testicular tissue should be considered as an important factor for fertility preservation in young boys with cancer. The objective of this study is to investigate whether immature testicular tissue of mice can be successfully cryopreserved using a simple vitrification procedure to maintain testicular cell viability, proliferation, and differentiation capacity. In this experimental study, immature mice testicular tissue fragments (0.5-1 mm²) were vitrified-warmed in order to assess the effect of vitrification on testicular tissue cell viability. Trypan blue staining was used to evaluate developmental capacity. Vitrified tissue (n=42) and fresh (control, n=42) were ectopically transplanted into the same strain of mature mice (n=14) with normal immunity. After 4 weeks, the graft recovery rate was determined. Hematoxylin and eosin (H&E) staining was used to evaluate germ cell differentiation, immunohistochemistry staining by proliferating cell nuclear antigen (PCNA) antibody, and terminal deoxynucleotidyl transferase (TdT) dUTP Nick- End Labeling (TUNEL) assay for proliferation and apoptosis frequency. Vitrification did not affect the percentage of cell viability. Vascular anastomoses was seen at the graft site. The recovery rate of the vitrified graft did not significantly differ with the fresh graft. In the vitrified graft, germ cell differentiation developed up to the secondary spermatocyte, which was similar to fresh tissue. Proliferation and apoptosis in the vitrified tissue was comparable to the fresh graft. Vitrification resulted in a success rates similar to fresh tissue (control) in maintaining testicular cell viability and tissue function. These data provided further evidence that vitrification could be considered an alternative for cryopreservation of immature testicular tissue.

White matter apoptosis is increased by delayed hypothermia and rewarming in a neonatal piglet model of hypoxic ischemic encephalopathy

Therapeutic hypothermia is widely used to treat neonatal hypoxic ischemic (HI) brain injuries. However, potentially deleterious effects of delaying the induction of hypothermia and of rewarming on white matter injury remain unclear. We used a piglet model of HI to assess the effects of delayed hypothermia and rewarming on white matter apoptosis. Piglets underwent HI injury or sham surgery followed by normothermic or hypothermic recovery at 2h. Hypothermic groups were divided into those with no rewarming, slow rewarming at 0.5°C/h, or rapid rewarming at 4°C/h. Apoptotic cells in the subcortical white matter of the motor gyrus, corpus callosum, lateral olfactory tract, and internal capsule at 29h were identified morphologically and counted by hematoxylin & eosin staining. Cell death was verified by terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay. White matter neurons were also counted, and apoptotic cells were immunophenotyped with the oligodendrocyte marker 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (CNPase). Hypothermia, slow rewarming, and rapid rewarming increased apoptosis in the subcortical white matter relative to normothermia (p<0.05). The number of white matter neurons was not lower in groups with more apoptosis after hypothermia or rapid rewarming, indicating that the apoptosis occurred among glial cells. Hypothermic piglets had more apoptosis in the lateral olfactory tract than those that were rewarmed (p<0.05). The promotion of apoptosis by hypothermia and rewarming in these regions was independent of HI. In the corpus callosum, HI piglets had more apoptosis than shams after normothermia, slow rewarming, and rapid rewarming (p<0.05). Many apoptotic cells were myelinating oligodendrocytes identified by CNPase positivity. Our results indicate that delaying the induction of hypothermia and rewarming are associated with white matter apoptosis in a piglet model of HI; in some regions these temperature effects are independent of HI. Vulnerable cells include myelinating oligodendrocytes. This study identifies a deleterious effect of therapeutic hypothermia in the developing brain.

Increased Oxidative Damage and Reduced DNA Repair Enzyme XPD Involvement in High Glucose-Mediated Enhancement of Levobupivacaine-Induced Neurotoxicity.

Levobupivacaine is one of the major clinical local anesthetics, but it can cause neuron toxic damage. Hyperglycemia can cause neuronal DNA oxidative damage and inhibit expression of the DNA repair gene Xeroderma pigmentosum complementation group D (XPD). This study was designed to determine whether high glucose levels inhibit XPD expression and enhance levobupivacaine-induced DNA damage. We evaluated XPD mRNA and protein expression in SH-SY5Y cells after glucose and levobupivacaine exposure. We next investigated cells reactive oxygen species (ROS) levels, DNA damage and apoptosis with redox-sensitive fluorescent dye DCFH-DA (2',7'-dichlorofluorescein diacetate), comet assays, flow cytometry, and TUNEL (terminal deoxynucleotidyl transferased UTP nick end labeling) assays. XPD expression was inhibited in cells exposed to prolonged high glucose with a concomitant increase in ROS production and more severe DNA damage compared to control culture conditions, and these changes were further exacerbated by levobupivacaine. Our findings indicate that subjects with diabetes may experience more detrimental effects following local anesthetic use.

Abstract 26: Suppression of E-cadherin mediates gallotannin-induced apoptosis in Hep G2 hepatocelluar carcinoma cells

Abstract Though gallotannin was known to have anti-oxidant and antitumor activity, the underlying antitumor mechanism of gallotannin still remains unclear. Thus, in the present study, antitumor mechanism of gallotannin was elucidated in hepatocellular carcinoma cells. Gallotannin significantly exerted cytotoxicity against Hep G2 and Chang hepatocellular carcinoma cells with the accumulation of the sub-G1 population and increase of terminal deoxynucleotidyltransferasedUTP nick end labeling (TUNEL) positive cells as an apoptotic feature. Also, gallotannin attenuated the expression of pro-caspase9, pro-caspase3, Bcl2 and integrin β1 and cleaved poly(ADP)-ribose polymerase (PARP) in Hep G2 and Chang cancer cells. Furthermore, gallotannin suppressed cell repair motility by wound healing assay and also inhibited cell adhesion in Hep G2 cells. Of note, gallotannin attenuated the expression of epithelial cadherin (E-cadherin) to form cell-cell adhesion from the early stage, and also beta-catenin at late phase in Hep G2 cells. Consistently, Immunofluorescence assay showed that E-cadherin or β-catenin expression was suppressed in a time dependent manner by gallotannin. Furthermore, silencing of E-cadherin by siRNA transfection method enhanced PAPR cleavage, caspase 3 activation and sub G1 population and attenuated the cell adhesion induced by gallotannin in Hep G2 cells. Overall, our findings demonstrate that the disruption of cell adhesion junction by suppression of E-cadherin mediates gallotannin enhanced apoptosis in Hep G2 liver cancer cells. Citation Format: Hee Young Kwon, Ji Hoon Jung, Hyun Joo Lee, Myoung Seok Jeong, Deok-Beom Jung, Bonglee Kim, Hyemin Lee, Sung-Hoon Kim. Suppression of E-cadherin mediates gallotannin-induced apoptosis in Hep G2 hepatocelluar carcinoma cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 26. doi:10.1158/1538-7445.AM2015-26

Calpain-Dependent ErbB4 Cleavage Is Involved in Brain Ischemia-Induced Neuronal Death.

Disturbance of neuregulin-1β/ErbB4 signaling is considered to be associated with brain ischemia, but the mechanisms of this disruption are largely unknown. In the present study, we provide evidence that degradation of ErbB4 is involved in neuronal cell death in response to ischemia. Our data showed that the application of neuregulin-1β provided significant protection against oxygen-glucose deprivation (OGD)-induced neuronal death as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, annexin V/propidium iodide flow cytometry analysis and terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) staining. Furthermore, neuregulin-1β treatment significantly reduced the infarct volume of ischemic mice, and this result was not seen in the ErbB4 knockout mice. We found that brain ischemia induced the breakdown of ErbB4 in a time-dependent manner in vivo, but not that of ErbB2. In vitro studies further indicated that recombinant calpain induced the cleavage of ErbB4 in a dose-dependent way, whereas the calpain inhibitor significantly reduced the OGD-induced ErbB4 breakdown. Additionally, OGD-induced apoptosis was partially abolished by transfection with the ErbB4E872K mutant. Taken together, neuregulin-1β elicits its neuroprotective effect in an ErbB4-dependent manner, and the cleavage of ErbB4 by calpain contributes to a neuronal cell death cascade during brain ischemia.

Detection of end-stage apoptosis by ApopTag® TUNEL technique.

DNA fragmentation, the end stage of apoptosis, is the measure of ultimate demise of the cell. A convenient method for examining apoptosis via DNA fragmentation is by the Terminal deoxynucleotidyl transferase (Tdt) dUTP Nick-End Labeling (TUNEL) assay where the DNA strand breaks are detected by enzymatically labeling the free 3'-OH termini with modified nucleotides. ApopTag(®) kits detect single-stranded and double-stranded breaks associated with apoptosis. This technique is also helpful to distinguish between apoptotic and necrotic cell death where the latter is associated with random DNA fragment lengths producing a DNA smear. Apoptotic cells stained positive with ApopTag(®) kits are easier to detect and their identification is more certain, as compared to the examination of simply histochemically stained tissues. In addition, quantitative results can be obtained using flow cytometry and apoptotic cells can be differentiated from necrotic cells with greater than tenfold sensitivity.

Potent effects of flavonoid-rich extract from Rosa laevigata Michx fruit against hydrogen peroxide-induced damage in PC12 cells via attenuation of oxidative stress, inflammation and apoptosis.

Oxidative stress-induced neuronal death has an important role in the pathogenesis of neurodegenerative disorders. The effects and mechanisms of action of the total flavonoids (TFs) from Rosa laevigata Michx fruit against hydrogen peroxide (H2O2)-induced oxidative injury in PC12 cells were investigated in this study. The results demonstrated that the TFs protected against cell apoptosis, DNA and mitochondrial damage caused by H2O2 based on single cell gel electrophoresis, in situ terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL), flow cytometry and transmission electron microscope (TEM) assays. In addition, the TFs notably decreased cytochrome C release from mitochondria into the cytosol and intracellular Ca2+ levels, and diminished intracellular generation of reactive oxygen species (ROS). Furthermore, the TFs inhibited the phosphorylation levels of JNK, ERK and p38 MAPK as well as down-regulated the expressions of IL-1, IL-6, TNF-α, Fas, FasL, CYP2E1, Bak, caspase-3, caspase-9, p53, COX-2, NF-κB, AP-1, and up-regulated the expressions of Bcl-2 and Bcl-xl. In conclusion, these results suggest that the TFs from R. laevigata Michx fruit show good effects against H2O2-induced oxidative injury in PC12 cells by adjusting oxidative stress, and suppression of apoptosis and inflammation, and could be developed as a potential candidate to prevent oxidative stress in the future.

Ethanolic extract of Azadirachta indica (A. Juss.) causing apoptosis by ROS upregulation in Dirofilaria immitis microfilaria

Dirofilaria immitis is the causative agent of cardiopulmonary dirofilariasis in the Canine family. The aim of the study was to evaluate the efficacy of the ethanolic extract of Azadirachta indica leaves (EEA) against the microfilaria (mf) of D. immitis in vitro. EEA was evaluated for different compound classes through HPTLC. Relative motility, mortality and morphological alterations were observed in the mf after exposure to EEA. The effect of EEA on redox status in the treated mf was evaluated by some key enzymatic and non-enzymatic parameters. An enhanced reactive oxygen species (ROS) level in the treated mf along with altered redox status was evident. DNA fragmentation and terminal-deoxynucleotidyl-transferase dUTP nick end labeling (TUNEL) confirmed apoptosis. In addition, western blotting revealed down-regulation of anti-apoptotic protein and up-regulation of pro-apoptotic proteins. Taken together, the microfilaricidal activity of EEA can be attributed to its capacity to inflict oxidative stress culminating in apoptosis.

Nickel ions from a corroded cardiovascular stent induce monocytic cell apoptosis: Proposed impact on vascular remodeling and mechanism

Monocytes play important roles in inflammatory responses and vascular remodeling after vascular stenting. This research focused on impacts of nickel (Ni) ions released from a corroded cardiovascular stent on cytotoxicity and monocyte activation. A human promonocytic (macrophage-like) cell line (U937) was exposed to graduated concentrations of Ni(2+)in vitro. Cells were observed and harvested at indicated times to determine the effects using histological and biochemical methods. Ni caused U937 cell death in dose- and time-dependent manners. In vitro, high concentrations of Ni(2+) (>240 μM) significantly induced cell apoptosis and increased terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL)-positive cells according to flow cytometric surveillance and triggered apoptotic cell death. Although no significant changes in Bcl-2 or Bax expressions were detected after 24 hours of Ni(2+) treatment, increasing cleavage of caspase-3 and -8 was present. Results showed that cleavage of caspase-8 was inhibited by the presence of the inhibitor, Z-IETD-FMK, and this suggested the presence of Ni(2+)-induced U937 cell death through a death receptor-mediated pathway. Simultaneously, when treated with a high concentration of Ni(2+) ions, expressions of the vascular remodeling factors, matrix metalloproteinases (MMP)-9 and -2, were activated in dose- and time-dependent manners. Secretion of the proliferative factor, monocyte chemoattractant protein (MCP)-1, significantly increased during the first 6 hours of incubation with 480 μM Ni(2+)-treated medium. Our results demonstrated that a high concentration of Ni ions causes apoptotic cell death of circulating monocytes. They may also play different roles in vascular remodeling during the corrosion process following implantation of Ni alloy-containing devices.

Cytotoxicity and Apoptotic Activity of &lt;i&gt;Ficus pseudopalma&lt;/i&gt; Blanco Leaf Extracts Against Human Prostate Cancer Cell Lines

Purpose: To investigate the cytotoxic and apoptotic activities of Ficus pseudopalma (FP) Blanco leaf extracts against normal human FSE cells and human prostate PRST2 cancer cell line.Methods: FP leaves were extracted with 95 % ethanol, and partitioned with chloroform, ethylacetate, and water. The presence of terpenoid lupeol and flavonoid quercetin was determined through high performance liquid chromatography (HPLC). The cytotoxic and apoptotic effects of differentconcentrations of FP extracts on PRST2 cells and on non-cancerous human foreskin surface epithelial (hFSE) cells were determined by 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyltetrazolium bromide (MTT) assay, tryphan blue exclusion assay, Live/Dead viability assay and terminaldeoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay.Results: Ethylacetate, chloroform, and crude ethanol extracts were significantly cytotoxic to PRST2 cell lines (p &lt; 0.001) in a concentration-dependent manner with a 50 % cell growth inhibitory concentration of 4.16, 4.83 and 44.53 ìg/mL, respectively. A significant decrease in the viability ofPRST2-treated cells (p &lt; 0.001) in a concentration-dependent manner was observed in the tryphan blue exclusion assay and in the Live/Dead® viability assay. Using APO-BrdU TUNEL assay, apoptotic activities of the extracts increased in a concentration-dependent manner. All the extractsdid not show significant cytotoxic effect on hFSE cells (p = 0.064).Conclusion: The cytotoxic and apoptotic activities of FP extracts may be due to the presence of lupeol and quercetin. This study suggests that apoptotic mechanisms may be involved in the growth inhibitory activity of FP leaf extracts against human PRST 2 cell lines.Keywords: Ficus pseudopalma, Cytotoxicity, Apopotic, human prostate PRST2 cancer cell, Lupeol, Quercetin.

Prognostic value of p53, c-ErbB2 and tunel data in upper urothelial carcinoma associated with Balkan nephropathy

A characteristic tumor suppressor protein 53 (p53) mutational profile of genotoxic action of aristolochic acid was identified in the upper urothelial carcinoma (UUTT) associated with Balkan nephropathy (BEN). In the present study, we examined the prognostic value of tissue-based molecular markers in overall-survival (OS) risk after surgical treatment of UUTT, adjusted for gender, age and urological characteristics in 32 patients with BEN. Immunohistochemical examination of p53, the proliferation cell nuclear antigen (PCNA), the human epidermal growth factor receptor 2 (c-ErbB2; also known as HER-2/neu) proto-oncogene and the in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay for apoptosis detection were used to examine serial tumor sections. The median OS-time was 60 months for UUTT operation; the mortality rate (18.7%) was related to (new) disease (re)occurrence or invasion in 12-216 months. High-grade (p=0.029), TUNEL&gt;0.36%+ cells (p=0.010), and c-ErbB2+ cells (p=0.014) can define the risk of tumor invasion. Patients with Balkan nephropathy that develop UUTT at a stage greater than pT1 (with apoptosis TUNEL+ cells &gt;0.36% and p53+ cells greater than 10%) were at high risk of poor-OS after the tumor surgery (h(x)=6.35; p=0.045). The obtained data present evidence for p53, cErbB2 and apoptosis deregulation, as a result of environmental toxin action. This is the first report of molecular biomarker linkage with OS for BEN-associated UUTT.

Suppression of E-cadherin mediates gallotannin induced apoptosis in Hep G2 hepatocelluar carcinoma cells.

Though gallotannin was known to have anti-oxidant and antitumor activity, the underlying antitumor mechanism of gallotannin still remains unclear. Thus, in the present study, antitumor mechanism of gallotannin was elucidated in hepatocellular carcinoma cells. Gallotannin significantly exerted cytotoxicity against Hep G2 and Chang hepatocellular carcinoma cells with the accumulation of the sub-G1 population and increase of terminal deoxynucleotidyltransferasedUTP nick end labeling (TUNEL) positive cells as an apoptotic feature. Also, gallotannin attenuated the expression of pro-caspase9, pro-caspase3, Bcl2 and integrin β1 and cleaved poly(ADP)-ribose polymerase (PARP) in Hep G2 and Chang cancer cells. Furthermore, gallotannin suppressed cell repair motility by wound healing assay and also inhibited cell adhesion in Hep G2 cells. Of note, gallotannin attenuated the expression of epithelial cadherin (E-cadherin) to form cell-cell adhesion from the early stage, and also beta-catenin at late phase in Hep G2 cells. Consistently, Immunofluorescence assay showed that E-cadherin or β-catenin expression was suppressed in a time dependent manner by gallotannin. Furthermore, silencing of E-cadherin by siRNA transfection method enhanced PAPR cleavage, caspase 3 activation and sub G1 population and attenuated the cell adhesion induced by gallotannin in Hep G2 cells. Overall, our findings demonstrate that the disruption of cell adhesion junction by suppression of E-cadherin mediates gallotannin enhanced apoptosis in Hep G2 liver cancer cells.

Abnormal sperm motility and morphology predict sperm DNA damage and density gradient preparation improves sperm DNA damage compared to sperm wash in patients with abnormal semen parameters

A standard semen analysis does not reliably predict pregnancy outcome and an ideal sperm processing technique should be gentle and one that recovers a highly functional sperm population with the least DNA fragmentation. Our objective is to evaluate if semen motility and morphology predicts the level of DNA fragmentation and to compare the effects of density gradient and sperm wash processing techniques on DNA integrity using the TUNEL assay and flow cytometry. Prospective, Cohort study. Two groups of patients were included. Group 1 (N=11) included men with normal semen parameters and group II (N=9) included men with abnormal semen motility and/or morphology. All patients had normal semen concentrations. Each semen sample was divided into three tubes. The first tube was unprocessed semen, the second tube was processed by sperm wash and third tube was processed by double density gradient. DNA fragmentation of the sperm in all three tubes was assessed with terminal deoxynucleotidyltransferase dUTP nick end labeling (TUNEL) followed by flow cytometric analysis. Statistical analysis using t-test and ANOVA was done to evaluate the prediction of sperm DNA fragmentation by sperm parameters and to evaluate if sperm preparation using density gradient decreased the level of DNA damage compared to sperm wash. 1. Abnormal semen samples showed higher levels of DNA fragmentation with mean ±SD of 28.1±3.1 vs 11.8±4.4 in normal semen samples, p<0.0001. 2. Density gradient sperm processing significantly decreased the amount of DNA fragmentation (mean ±SD of 4.84±4.3) vs sperm wash (12.8 ± 5.9), p<0.01. Abnormal sperm motility and morphology is associated with abnormal DNA fragmentation. Sperm preparation by density gradient decreases DNA damage compared to sperm wash and may mprove the reproductive outcome in couples with infertility.

CD14 Contributes to Warm Hepatic Ischemia-Reperfusion Injury in Mice

Ischemia/reperfusion (I/R) of the liver contributes to the pathobiology of liver injury in transplantation, liver surgery, and hemorrhagic shock. Ischemia/reperfusion induces an inflammatory response that is driven, in part, by Toll-like receptor 4 (TLR) signaling. CD14 is known to participate in the function of TLR4. We hypothesized that CD14 would be involved in the pathobiology of warm hepatic I/R. Using a 70% liver inflow inclusion model, CD14 knockout and wild-type (WT) mice were subjected to 1-h warm ischemia followed by reperfusion. CD14 mRNA, circulating transaminase, interleukin 6, soluble CD14, and high-mobility group box 1 (HMGB1) levels were measured. CD14 neutralizing antibody or isotype control antibody was given before ischemia or reperfusion for CD14 blockade in WT mice. Recombinant HMGB1 was given before reperfusion in some experiments to test if liver injury worsens. There was an upregulation of CD14 mRNA in reperfused livers together with increased soluble CD14 levels in the circulation. Compared with WT control mice, CD14 knockout mice had much lower alanine aminotransferase and interleukin 6 levels at 6 and 24 h following I/R, and much less liver necrosis by histology. TUNEL (terminal deoxynucleotidyl-transferase dUTP nick end labeling) staining displayed less apoptosis at 24 h in the absence of CD14. CD14 blockage by neutralizing antibody also attenuated liver injury and the inflammatory response in C57BL/6 mice following I/R, but did not provide additional protection to TLR4 mutant C3H/Hej mice. CD14 deficiency did not change circulating HMGB1 levels following I/R (6 h). A dose of recombinant HMGB1, which worsened hepatic injury when given before reperfusion in WT mice, did not increase liver damage in CD14-deficient mice. CD14 is actively involved in hepatic I/R injury. Its deficiency or blockade ischemia attenuates liver injury and inflammatory response. CD14 mediates liver damage and inflammatory responses in the setting of warm hepatic I/R in mice.

BDNF protects human vascular endothelial cells from TNFα-induced apoptosis

Brain-derived neurotrophic factor (BDNF) enhances periodontal tissue regeneration. Tissue regeneration is characterized by inflammation that directs the quality of tissue repair. In this study, we investigated the anti-apoptotic effect of BDNF against the toxicity of tumor necrosis factor α (TNFα), which is known for its pro-apoptotic action in human microvascular endothelial cells (HMVECs). We demonstrate that BDNF attenuates TNFα-increased Annexin V-positive cells, lactic dehydrogenase (LDH) release, and intercellular adhesion molecule 1 (ICAM-1) mRNA and cleaved caspase-3 expression. In addition, biochemical analyses indicate that TNFα increases phosphatase and tensin homolog (PTEN) expression; however, it decreases phosphorylated PTEN. BDNF did not affect PTEN expression, but it did increase the phosphorylation of PTEN. BDNF-induced Akt phosphorylation was inhibited by TNFα. Terminal deoxynucleotidyl transferase (TdT) dUTP nick end labeling (TUNEL) assay showed that the PTEN inhibitor bpV(pic) rescues HMVECs from TNFα-induced apoptosis. In conclusion, BDNF protects HMVECs from toxicity of TNFα through the regulation of the PTEN/Akt pathway.

The beneficial effects of tadalafil on left ventricular dysfunction in doxorubicin-induced cardiomyopathy

BackgroundIt is not clear yet how tadalafil affects nonischemic cardiomyopathy, although its beneficial effects on acute myocardial infarction are well-known. We investigated tadalafil's beneficial effects on nonischemic cardiomyopathy and the specific mechanisms of its effects. MethodsCardiomyopathy was induced in mice by a single intraperitoneal injection of doxorubicin (15mg/kg). In some cases, tadalafil (4mg/kg/day, p.o., 14 days) was started simultaneously. After two weeks, cardiac function was evaluated by echocardiography and cardiac catheterization, then all of the mice were killed and cardiac specimens were subjected for hemotoxylin and eosin staining, Masson's trichrome staining, terminal deoxynucleotidyltransferase dUTP nick-end labeling assay, enzyme-linked immunosorbent assay, and Western blot. ResultsTwo weeks later, left ventricular dilatation and dysfunction were apparent in mice given doxorubicin but were significantly attenuated by tadalafil treatment. Tadalafil also protected hearts against doxorubicin-induced cardiomyocyte atrophy/degeneration and myocardial fibrosis. No doxorubicin-induced apoptotic effects were seen between groups. Cardiac cGMP level was lower in the doxorubicin-treated group, however it was significantly increased with tadalafil treatment. Compared to the control group, the myocardial expression of 3 sarcomeric proteins, myosin heavy chain, troponin I, and desmin were significantly decreased in the doxorubicin-treated group, which were restored by the tadalafil treatment. ConclusionsThe present study indicates a protective effect of tadalafil mainly through cGMP signaling pathway against doxorubicin-induced nonischemic cardiomyopathy.

Follicular development and expression of nuclear respiratory factor-1 and peroxisome proliferator-activated receptor γ coactivator-1 alpha in ovaries of fetal and neonatal doelings1

In livestock, the ovarian reserve of follicles is established during the fetal stage. However, at least two-thirds of the oocytes present in the reserve die because of apoptosis before birth. Notably, mitochondria have been reported to play a crucial role in the fate (life/death) of oocytes. In this study, mitochondrial regulators nuclear respiratory factor-1 (NRF-1) and PPAR γ coactivator-1 alpha (PGC-1α) were examined during this period of follicle development to investigate their effects on follicular development and apoptosis. Fetal and neonatal Capra haimen were used, ranging in age from 60 d postcoitum (dpc) to 30 d postpartum (dpp). Our data demonstrated that egg nests were the earliest recognizable gamete cells in ovaries of fetal and neonatal doelings. Proportions of egg nests decreased from 92.68 to 25.08% whereas single follicles increased from 7.32 to 74.92% between 60 and 120 dpc. Subsequently, between 90 and 120 dpc, the proportion of primordial follicles increased from 9.98 to 61.56% (P < 0.01). However, it did not change between 1 and 30 dpp (P = 0.12). The proportion of primary follicles increased from 1.23 to 37.93% between 90 dpc to 1 dpp (P = 0.01) but did not change between 1 and 30 dpp (P = 0.11). Meanwhile, proportions of secondary and tertiary follicles increased in an age-dependent manner. In addition, results of this study suggested that NRF-1 and PGC-1α proteins are mainly localized in germ cells of egg nests, cytoplasm of oocytes, and granulosa cells of follicles ranging from primordial to tertiary follicles. The transcript abundance of NRF-1 mRNA was up-regulated in 60-dpc-old ovaries compared with 1-dpp-old ovaries (P < 0.05), but the PGC-1α mRNA expression pattern did not change (P = 0.05). Nevertheless, the number of terminal deoxynucleotidyltransferase UTP nick-end labeling (TUNEL) positive cells and caspase-3 activity in 60-dpc-old ovaries was less than those in 1-dpp-old ovaries (P < 0.01, P = 0.01). In conclusion, our results demonstrate that the key stage of primordial follicle formation is between 90 and 120 dpc in Capra haimen. Also, this study suggests that NRF-1 and PGC-1α might have roles in cell apoptosis during ovarian development of fetal and neonatal Capra haimen. These results improve our understanding of apoptotic mechanisms in oogenesis and folliculogenesis.