Deoxynucleotide Transferase-mediated dUTP Nick-end Research Articles

Effect of propofol on lung metastasis of circulating tumor cells after colorectal cancer surgery


 
 
 
 Purpose: To investigate the effect of propofol on lung metastasis of circulating tumor cells (CTCs) after colorectal cancer (CRC) surgery, and the mechanism involved.
 Methods: Specific-pathogen-free (SPF) male BALB/c mice (n = 30; mean age = 7 ± 1 weeks; mean weight = 19 ± 3 g) were used for this study. To establish mouse model of CRC, 100 μL of CRC (CT26) cells was injected into the caudal vein of each mouse. Three groups of 10 mice were used: control, 5 μL/mL propofol, and 10 μL/mL propofol groups. Changes in pulmonary superficial nodules of mice lungs were determined. Colorectal cancer cell (CT26) proliferation and apoptosis were measured using Ki-67 immunohistochemical staining and in situ terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays, respectively.
 Results: The number of pulmonary nodules and proportion of proliferating cells (Ki-67 expression level) were significantly higher in 5 μL/mL propofol-treated mice, relative to control, but markedly lower in 10 μL/mL propofol group than in 5 μL/mL propofol group (p < 0.05). Although apoptotic index increased in 5 μL/mL propofol group, cell apoptosis was comparable among the groups (p > 0.05).
 Conclusion: These results suggest that propofol promotes pulmonary metastasis of CTCs after CRC surgery via stimulation of CTC proliferation in the lungs. Therefore, caution should be exercised in the use of propofol in colorectal cancer surgery.
 
 
 

Tanshinone IIA inhibits Leu27IGF-II-induced insulin-like growth factor receptor II signaling and myocardial apoptosis via estrogen receptor-mediated Akt activation.

Different stress condition stimulates the expression level of insulin-like growth factor receptor II (IGF-IIR) in cardiomyoblasts that lead to apoptosis. Tanshinone IIA (TSN), a pharmacologically active component from Danshen, has been shown cardioprotective effects against cardiac apoptosis induced by several stress conditions. Therefore, this study was conducted to assess the cardioprotective effects of TSN IIA mediated through the estrogen receptor (ER) in order to inhibit the Leu27IGF-II-enhanced IGF-IIR-mediated cardiac apoptosis. The estrogenic activity of TSN IIA was examined after myocardial cells were pretreated with the ER antagonist, and inhibited the phospho-inositide-3 kinase (PI3K). Here, we found that TSN IIA significantly induced ER that phosphorylated Akt. Further, Akt activation considerably suppressed the Leu27IGF-II induced IGF-IIR expression level and the downstream effectors, including Gαq and calcineurin as well as mitochondrial dependent apoptosis proteins including Bad, cytochrome c, and active caspase-3 that result in cardiac apoptosis resistance. However, the western blot analysis, JC-1 staining, and terminal deoxynucleotide transferase-mediated dUTP nick end labeling assay revealed that TSN IIA attenuated Leu27IGF-II-induced IGF-IIR mediated cardiac apoptosis was reversed by an ER antagonist such as ICI 182780, and PI3K inhibition. All these findings demonstrate that TSN IIA exerts estrogenic activity, which can activate PI3K-Akt pathway, and thereby inhibits Leu27IGFII induced IGF-IIR mediated cardiac apoptosis. Thus, TSN IIA can be considered as an effective therapeutic strategy against IGF-IIR signaling cascade to suppress cardiac apoptosis.

Long Noncoding RNA TYKRIL Plays a Role in Pulmonary Hypertension via the p53-mediated Regulation of PDGFRβ.

Rationale: Long noncoding RNAs (lncRNAs) are emerging as important regulators of diverse biological functions. Their role in pulmonary arterial hypertension (PAH) remains to be explored.Objectives: To elucidate the role of TYKRIL (tyrosine kinase receptor–inducing lncRNA) as a regulator of p53/ PDGFRβ (platelet-derived growth factor receptor β) signaling pathway and to investigate its role in PAH.Methods: Pericytes and pulmonary arterial smooth muscle cells exposed to hypoxia and derived from patients with idiopathic PAH were analyzed with RNA sequencing. TYKRIL knockdown was performed in above-mentioned human primary cells and in precision-cut lung slices derived from patients with PAH.Measurements and Main Results: Using RNA sequencing data, TYKRIL was identified to be consistently upregulated in pericytes and pulmonary arterial smooth muscles cells exposed to hypoxia and derived from patients with idiopathic PAH. TYKRIL knockdown reversed the proproliferative (n = 3) and antiapoptotic (n = 3) phenotype induced under hypoxic and idiopathic PAH conditions. Owing to the poor species conservation of TYKRIL, ex vivo studies were performed in precision-cut lung slices from patients with PAH. Knockdown of TYKRIL in precision-cut lung slices decreased the vascular remodeling (n = 5). The number of proliferating cell nuclear antigen–positive cells in the vessels was decreased and the number of terminal deoxynucleotide transferase–mediated dUTP nick end label–positive cells in the vessels was increased in the LNA (locked nucleic acid)-treated group compared with control. Expression of PDGFRβ, a key player in PAH, was found to strongly correlate with TYKRIL expression in the patient samples (n = 12), and TYKRIL knockdown decreased PDGFRβ expression (n = 3). From the transcription factor–screening array, it was observed that TYKRIL knockdown increased the p53 activity, a known repressor of PDGFRβ. RNA immunoprecipitation using various p53 mutants demonstrated that TYKRIL binds to the N-terminal of p53 (an important region for p300 interaction with p53). The proximity ligation assay revealed that TYKRIL interferes with the p53–p300 interaction (n = 3) and regulates p53 nuclear translocation.Conclusions: TYKRIL plays an important role in PAH by regulating the p53/PDGFRβ axis.

Naringin alleviates H2O2-induced apoptosis via the PI3K/Akt pathway in rat nucleus pulposus-derived mesenchymal stem cells

ABSTRACT Purpose: To investigate the protective effect of naringin (Nar) on H2O2-induced apoptosis of nucleus pulposus-derived mesenchymal stem cells (NPMSC) and the potential mechanism in this process. Methods: Rat NPMSC were cultured in MSC culture medium or culture medium with different concentrations of H2O2. Nar or the combination of Nar and LY294002 was added into the culture medium to investigate the effects of Nar. Cell viability was evaluated by cell counting kit-8 (CCK-8) assay. The apoptosis rate was determined using Annexin V/PI dual staining and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assays. Additionally, the levels of reactive oxygen species (ROS) and mitochondrial membrane potential (MMP) were analyzed by flow cytometry. ATP level in NPMSC was analyzed via ATP detection kit. Mitochondrial ultrastructure change was observed through transmission electron microscope (TEM). Levels of apoptosis-associated molecules (cleaved caspase-3, Bax and Bcl-2) were evaluated via RT-PCR and western blot, respectively. Results: The cells isolated from NP met the criteria for MSC. H2O2 significantly promoted NPMSC apoptosis in a dose and time-dependent manner. Nar showed no cytotoxicity effect on NPMSC up to a concentration of 100 μM for 24 h. Nar exhibited protective effects against H2O2-induced NPMSC apoptosis including apoptosis rate, expressions of proapoptosis and antiapoptosis related genes and protein. Nar could also alleviate H2O2-induced mitochondrial dysfunction of increased mitochondrial ROS production, reduced MMP, decreased intracellular ATP and mitochondrial ultrastructure change. However, these protected effects were inhibited after LY294002 treatment. Conclusions: Our results demonstrated that Nar efficiently attenuated H2O2-induced NPMSC apoptosis and mitochondrial dysfunction. The activation of ROS-mediated PI3K/Akt pathway may be the potential mechanism in this process.

Influence of temperature, serum, and gonadotropin supplementation in short- and long-term organotypic culture of human immature testicular tissue

To study how temperature, serum, and gonadotropin supplementation affect the organotypic culture of human immature testicular tissue (ITT) invitro. Experimental basic science study. Reproductive biology laboratory. ITT from 4 boys with cancer that had testicular tissue cryopreserved as part of their fertility preservation treatment. Invitro organotypic culture of ITT, exposed to different temperatures (37°C vs. 34°C), serum (fetal bovine serum [FBS] vs. Knockout Serum Replacement [KOS]), and gonadotropin supplementation (with and without FSH and LH). Characterization of the tissue was performed at days 0, 14, and 70 with the use of reverse-transcription quantitative polymerase chain reaction, terminal deoxynucleotide transferase-mediated dUTP nick-end labeling, histologic analysis by means of hematoxylin-eosin staining, and immunohistochemical staining. Hormonal secretion was determined at days 3, 14, 28, and 70by means of immunofluorescent assay. The 37°C conditions showed an accelerated loss of tubular morphology and higher intratubular apoptosis. KOS supplementation triggered the up-regulation of STAR, SOX9, DAZL, DDX4, PLZF, and UTF1, the percentage of SOX9+/androgen receptor (AR)-positive mature Sertoli cells at day 14, and testosterone secretion. Gonadotropin supplementation increased the numbers of both undifferentiated UTF1+ spermatogonia and premeiotic VASA+/SYCP3+ spermatogonia at day 14, and the number of SOX9+ Sertoli cells at day 70. The low SOX9+/AR+ colocalization, the disorganized pattern of ZO-1, and the progressive decrease of antimüllerian hormone secretion indicated inefficient Sertoli cell maturation invitro. The 34°C condition in KOS showed the best results for the survival of both spermatogonia and Sertoli cells. FSH/LH supplementation also improved long-term survival of Sertoli cells and the maturation of spermatogonia up to meiotic initiation in short-term culture.

Ulinastatin Alleviates Neurological Deficiencies Evoked by Transient Cerebral Ischemia via Improving Autophagy, Nrf-2-ARE and Apoptosis Signals in Hippocampus

Ulinastatin [or called as urinary trypsin inhibitor (UTI)] plays a role in regulating neurological deficits evoked by transient cerebral ischemia. However, the underlying mechanisms still need to be determined. The present study was to examine the effects of UTI on autophagy, Nrf2-ARE and apoptosis signal pathway in the hippocampus in the process of neurological functions after cerebral ischemia using a rat model of cardiac arrest (CA). CA was induced by asphyxia followed by cardiopulmonary resuscitation (CPR) in rats. Western blot analysis was employed to determine the expression of representative autophagy (namely, Atg5, LC3, Beclin 1), p62 protein (a maker of autophagic flux), and Nrf2-ARE pathways. Neuronal apoptosis was assessed by determining expression levels of Caspase-3 and Caspase-9, and by examining terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL). The modified neurological severity score (mNSS) and spatial working memory performance were used to assess neurological deficiencies in CA rats. Our results show that CA amplified autophagy and apoptotic Caspase-3/Caspase-9, and downregulated Nrf2-ARE pathway in the hippocampus CA1 region. Systemic administration of UTI attenuated autophagy and apoptosis, and largely restored Nrf2-ARE signal pathway following cerebral ischemia and thereby alleviated neurological deficits with increasing survival of CA rats. Our data suggest that UTI improves the worsened protein expression of autophagy and apoptosis, and restores Nrf2-ARE signals in the hippocampus and this is linked to inhibition of neurological deficiencies in transient cerebral ischemia. UTI plays a beneficial role in modulating neurological deficits induced by transient cerebral ischemia via central autophagy, apoptosis and Nrf2-ARE mechanisms.

Alpha-lipoic acid reduces retinal cell death in diabetic mice

Oxidative stress plays an important role in the development of diabetic retinopathy. Here, we examined whether α-lipoic acid (α-LA), a natural antioxidant, attenuated retinal injury in diabetic mice. The α-LA was orally administered to control mice or mice with streptozotocin-induced diabetes. We found that α-LA reduced oxidative stress, decreased and increased retinal 4-hydroxy-2-nonenal and glutathione peroxidase, respectively, and inhibited retinal cell death. Concomitantly, α-LA reversed the decreased activation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase, and increased the levels of peroxisome proliferator-activated receptor delta and sirtuin3 in diabetic mouse retinas, similar to results shown after metformin treatment of retinal pigment epithelial cells (RPE) exposed to high glucose. Moreover, α-LA lowered the levels of O-linked β-N-acetylglucosamine transferase (OGT) and thioredoxin-interacting protein (TXNIP) in diabetic retinas that were more pronounced after metformin treatment of RPE cells. Importantly, α-LA lowered interactions between AMPK and OGT as shown by co-immunoprecipitation analyses, and this was accompanied by less cell death as measured by double immunofluorescence staining by terminal deoxynucleotide transferase-mediated dUTP nick-end labelling and OGT or TXNIP in retinal ganglion cells. Consistently, α-LA lowered the levels of cleaved poly(ADP-ribose) polymerase and pro-apoptotic marker cleaved caspase-3 in diabetic retinas. Our results indicated that α-LA reduced retinal cell death partly through AMPK activation or OGT inhibition in diabetic mice.

Reactive oxygen species-mediated phosphorylation of p38 signaling is critically involved in apoptotic effect of Tanshinone I in colon cancer cells.

Though Tanshinone I (Tan I), a phenolic compound from Salvia miltiorrhiza, is known to have anticancer activity in several cancers, its anticancer mechanisms are not fully understood in colon cancer cells. Thus, in the present study, the underlying molecular mechanism of Tan I was explored in HCT116 and HT29 colorectal cancer cells (CRCs). Here, Tan I suppressed viability in HCT116 and HT29 cells in a time- and dose-dependent manner. Also, Tan I increased the number of terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and sub-G1 population in HCT116 and HT29 cells. Consistently, Tan I cleaved poly (adenosine diphosphate-ribose) polymerase (PARP) and caspase-8, caspase-3, attenuated the expression of Bid and activated tBid as a caspase-8 substrate and activated phosphorylation of p38 MAPK in HCT116and HT29 cells. Of note, Tan I generated reactive oxygen species (ROS) and conversely an ROS scavenger, N-acetyl-L -cysteine, reversed ROS production, PARP cleavage, caspase-3 activation, and p38 MAPK phosphorylation induced by Tan I in HCT116 cells. Furthermore, p38 MAPK inhibitor SB203580 reduced cytotoxicity, increase of TUNEL-positive cells, cleavages of PARP and caspase-3 induced by Tan I in HCT116 cells. Overall, our findings for the first time suggest that ROS-dependent activation of p38 MAPK and caspase-3 is critically involved in Tan I induced apoptosis in CRCs as a potent anticancer agent.

A potential antiapoptotic regulation: The interaction of heat shock protein 70 and apoptosis-inducing factor mitochondrial 1 during heat stress and aestivation in sea cucumber.

The sea cucumber (Apostichopus japonicus) has become a good model organism for studying environmentally induced aestivation in marine invertebrates. A characteristic feature of aestivation in this species is the degeneration of the intestine. In the current study, we hypothesized that energy conservation and cytoprotective strategies need to be coordinated in the intestine to ensure long-term survival during aestivation, and there was potential relationship between heat shock protein 70 (HSP70) and apoptosis-inducing factor mitochondrial 1 (AIFM1) during extreme environmental stress. AIFM1 is a bifunctional flavoprotein that is involved in the caspase-independent activation of apoptosis. The gene and protein expression profiles of AjAIFM1 and AjHSP70 in intestinal tissue during aestivation were analyzed and results showed an inverse correlation between them, AjAIFM1 being suppressed during aestivation whereas AjHSP70 was strongly upregulated. Comparable responses were also seen when intestinal cells were isolated and analyzed in vitro for responses to heat stress at 25°C (a water temperature typical during aestivation), compared with 15°C control cells. Combined with co-immunoprecipitation studies in vivo and in vitro, our results suggested that AjHSP70 protein may have potential interaction with AjAIFM1. To determine the influence of heat stress on apoptotic rate of intestinal cells, we also assessed the DNA fragmentation by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay, and results also supported a potential antiapoptotic response in sea cucumber during heat stress. This type of cytoprotective mechanism could be used to preserve the existing cellular components during long-term aestivation in sea cucumber.

The cardioprotective effect of interleukin-11 against ischemia-reperfusion injury in a heart donor model.

Previously, we have demonstrated the cardioprotective effect of interleukin (IL)-11 in animal models of acute coronary syndrome. In this study, we sought to evaluate its cardioprotective potential during prolonged hypothermic global ischemia and subsequent reperfusion using a rat heart donor model. IL-11 was administered intravenously 10 minutes before harvesting the rat heart. The hearts were preserved in cold (4 °C) Krebs-Henseleit buffer for 6 hours, and then attached to a Langendorff perfusion apparatus and reperfused with an oxygenated Krebs-Henseleit solution containing IL-11. Normal saline was used instead of IL-11 in the control group. Functional recovery of the reperfused heart was observed by using a left ventricular balloon. Myocardial cell injury was quantified by measuring the biomarkers collected from the coronary effluent. Apoptotic cells were identified and counted using the terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL) staining technique. IL-11 administration improved myocardial function after 6 hours of cold ischemia. Although there were no significant differences in any of the baseline-measured values between the two groups, left ventricular developed pressure (LVDP) and changes in left ventricular pressures (dP/dt) were significantly higher in the IL-11 group at 120-minute reperfusion. The number of TUNEL-labeled cardiomyocytes was also significantly smaller in the IL-11 group. The administration of IL-11 showed a significant recovery of cardiac contractile function after 6 hours of cold ischemia. Our data suggest that it may have significant therapeutic potential for maintaining the functional viability of the heart exposed to prolonged hypothermic global ischemia.

Metformin protects against retinal cell death in diabetic mice

Retinal degeneration is an early feature of diabetic retinopathy, the major cause of blindness in the developed world. Here we investigated how the widely used antidiabetic drug metformin reduces retinal injury in diabetic mice. Metformin was orally administered to control mice or mice with streptozotocin-induced diabetes. Western blot analysis showed that levels of O-linked β-N-acetylglucosamine (O-GlcNAc) transferase (OGT) and other related proteins such as carbohydrate-responsive element-binding protein (ChREBP) and thioredoxin-interacting protein (TXNIP) were significantly increased, and nuclear factor kappaB (NF-κB) and poly (ADP-ribose) polymerase (PARP) were activated in the diabetic retinas or retinal pigment epithelial (RPE) cells exposed to high glucose compared to controls. More importantly, RPE cells exposed to high glucose and treated with thiamet-G had higher levels of those proteins, demonstrating the role of elevated O-GlcNAcylation. Double immunofluorescence analysis revealed increased co-localization of terminal deoxynucleotide transferase-mediated dUTP nick-end labelling (TUNEL)-positive ganglion cells and OGT, ChREBP, TXNIP, or NF-κB in diabetic retinas compared to control retinas. Co-immunoprecipitation analysis showed that interaction between OGT and ChREBP or NF-κB was increased in diabetic retinas compared to control retinas, and this was accompanied by more cell death. Notably, metformin attenuated the increases in protein levels; reduced co-localization of TUNEL-positive ganglion cells and OGT, ChREBP, TXNIP, or NF-κB; and reduced interaction between OGT and ChREBP or NF-κB. Our results indicate that OGT inhibition might be one of the mechanisms by which metformin decreases retinal cell death.

β Cells Persist in T1D Pancreata Without Evidence of Ongoing β-Cell Turnover or Neogenesis.

The cellular basis of persistent β-cell function in type 1 diabetes (T1D) remains enigmatic. No extensive quantitative β-cell studies of T1D pancreata have been performed to test for ongoing β-cell regeneration or neogenesis. We sought to determine the mechanism of β-cell persistence in T1D pancreata. We studied T1D (n = 47) and nondiabetic control (n = 59) pancreata over a wide range of ages from the Juvenile Diabetes Research Foundation Network of Pancreatic Organ Donors with Diabetes via high-throughput microscopy. We quantified β-cell mass, β-cell turnover [via Ki-67 and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL)], islet ductal association, and insulin/glucagon coexpression in T1D and control pancreata. Residual insulin-producing β cells were detected in some (but not all) T1D cases of varying disease duration. Several T1D pancreata had substantial numbers of β cells. Although β-cell proliferation was prominent early in life, it dramatically declined after infancy in both nondiabetic controls and T1D individuals. However, β-cell proliferation was equivalent in control and T1D pancreata. β-cell death (assessed by TUNEL) was extremely rare in control and T1D pancreata. Thus, β-cell turnover was not increased in T1D. Furthermore, we found no evidence of small islet/ductal neogenesis or α-cell to β-cell transdifferentiation in T1D pancreata, regardless of disease duration. Longstanding β-cell function in patients with T1D appears to be largely a result of β cells that persist, without any evidence of attempted β-cell regeneration, small islet/ductal neogenesis, or transdifferentiation from other islet endocrine cell types.

Nafamostat Mesilate Attenuates Ischemia-Reperfusion-Induced Renal Injury.

It has been reported that nafamostat mesilate (NM) inhibits inflammatory injury via inhibition of complement activation in ischemic heart, liver, and intestine. However, it is unclear if NM also inhibits apoptosis in ischemia-reperfusion (IR)-injured kidney. We therefore investigated whether NM attenuates IR renal injury that involves inhibition of apoptosis. HK-2 cells and male C57BL/6 mice were used for this study. C57Bl/6 mice were divided into 4 groups: sham, NM (2mg/kg)+ sham, IR injury (IR injury; reperfusion 27 minutes after clamping of both the renal artery and vein), and NM+ IR injury. Kidneys were harvested 24 hours after IR injury, and functional and molecular parameters were evaluated. For invitro studies, HK-2 cells were incubated for 6 hours with mineral paraffin oil to induce hypoxic injury, and then treated with various doses of NM to evaluate the antiapoptotic effects. Blood urea nitrogen, serum creatinine levels, and renal tissue injury scores in NM+ IR-injured mice were significantly lower than those of control IR mice (all P< .01). NM significantly improved cell survival in hypoxic HK-2 cells (P< .01), significantly decreased renal Bax expression (P< .05), and increased renal Bcl-2 protein levels in IR kidneys and hypoxic HK-2 cells compared with those of the sham and control groups. The numbers of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling- and 8-oxo-2'-deoxyguanosine-positive cells were significantly lower in NM+ IR-injured kidneys compared with those in control IR-injured mice (P< .05); NM treatment decreased the expression of inducible and endothelial nitric oxide synthase in IR-injured mice (P< .05). NM ameliorates IR renal injury via inhibition of apoptosis by, at least in part, lowering nitric oxide overproduction, reducing Bax, and increasing Bcl-2.

MicroRNA-29 regulates high-glucose-induced apoptosis in human retinal pigment epithelial cells through PTEN.

Hyperglycemia or high-glucose (HG)-induced apoptosis in human retinal pigment epithelial (RPE) cells is a characteristic process in diabetic retinopathy. In our study, we examined whether microRNA-29 (miR-29) may regulate HG-induced RPE cell apoptosis. Human RPE cell line, ARPE-19 cells, was treated with various high concentration of glucose in vitro. HG-induced RPE cell apoptosis was examined by terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay and miR-29 gene expression by quantitative RT-PCR (qRT-PCR). miR-29 was then downregulated in RPE cells, and its effect on HG-induced apoptosis was examined by TUNEL assay and western blot assay on caspase-7 protein. Association of miR-29 on its downstream target, PTEN, in HG-induced RPE cell apoptosis was evaluated by dual-luciferase assay and qRT-PCR. PTEN was silenced in RPE cells. The effects of PTEN downregulation on miR-29-mediated HG-induced RPE cell apoptosis were also examined by TUNEL and western blot assays. HG induced significant apoptosis in RPE cells in a dose-dependent manner. miR-29 was upregulated by HG in RPE cells. miR-29 downregulation protected HG-induced apoptosis and reduced the production of caspase-7 protein in RPE cells. PTEN was shown to be directly downregulated by HG and then upregulated by miR-29 downregulation in RPE cells. Small interfering RNA (siRNA)-mediated PTEN downregulation reversed the protective effect of miR-29 downregulation on HG-induced RPE cell apoptosis. This study demonstrates that miR-29, through inverse association of PTEN, plays an important role in the process of HG-induced apoptosis in RPE cells.

Effect of superoxide anion scavenger on rat hearts with chronic intermittent hypoxia

Only very limited information regarding the protective effects of the superoxide anion scavenger on chronic intermittent hypoxia-induced cardiac apoptosis is available. The purpose of this study is to evaluate the effects of the superoxide anion scavenger on cardiac apoptotic and prosurvival pathways in rats with sleep apnea. Forty-two Sprague-Dawley rats were divided into three groups, rats with normoxic exposure (Control, 21% O2, 1 mo), rats with chronic intermittent hypoxia exposure (Hypoxia, 3-7% O2vs. 21% O2per 40 s cycle, 8 h per day, 1 mo), and rats with pretreatment of the superoxide anion scavenger and chronic intermittent hypoxia exposure (Hypoxia-O2 (-)-Scavenger, MnTMPyP pentachloride, 1 mg/kg ip per day; 3-7% O2vs. 21% O2per 40 s cycle, 8 h per day, 1 mo) at 5-6 mo of age. After 1 mo, the protein levels and apoptotic cells of excised hearts from three groups were measured by Western blotting and terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) assay. The superoxide anion scavenger decreased hypoxia-induced myocardial architecture abnormalities, left ventricular hypertrophy, and TUNEL-positive apoptosis. The superoxide anion scavenger decreased hypoxia-induced Fas ligand, Fas death receptors, Fas-associated death domain (FADD), activated caspase-8, and activated caspase-3 (Fas-dependent apoptotic pathway) as well as Bad, activated caspase-9 and activated caspase-3 (mitochondria-dependent apoptotic pathway), endonuclease G (EndoG), apoptosis-inducing factor (AIF), and TUNEL-positive apoptosis. The superoxide anion scavenger increased IGF-1, IGF-1R, p-PI3k, p-Akt, p-Bad, Bcl-2, and Bcl-xL (survival pathway). Our findings imply that the superoxide anion scavenger might prevent cardiac Fas-mediated and mitochondrial-mediated apoptosis and enhance the IGF-1-related survival pathway in chronic intermittent hypoxia. The superoxide anion scavenger may prevent chronic sleep apnea-enhanced cardiac apoptotic pathways and enhances cardiac survival pathways.

DNA fragmentation in brighter sperm predicts male fertility independently from age and semen parameters

To evaluate whether sperm DNA fragmentation (sDF), measured in brighter, dimmer, and total populations, predicts natural conception, and to evaluate the intra-individual variability of sDF. Prospective study. Outpatient clinic and diagnostic laboratory. A total of 348 unselected patients and 86 proven fertile men. None. sDF was revealed with the use of terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL)/propidium iodide (PI). Receiver operating characteristic (ROC) curves were built before and after matching fertile men to patients for age (76:152) or semen parameters (68:136) or both (49:98). Intra-individual variability of sDF was assessed over 2years. Brighter (area under ROC curve [AUC] 0.718 ± 0.54), dimmer (AUC 0.655 ± 0.63), and total (AUC 0.757 ± 0.54) sDF predict male fertility in unmatched and age- or semen parameters-matched subjects. After matching for both age and semen parameters, only brighter (AUC 0.711 ± 0.83) and total (AUC 0.675 ± 0.92) sDF predict male fertility. At high values of total sDF, brighter predicts natural conception better than total sDF. Intra-individual coefficients of variation of sDF were 9.2 ± 8.6% (n = 25), 12.9 ± 12.7% (n = 53), and 14.0 ± 12.6% (n = 70) over, respectively, 100-day and 1- and 2-year periods, appearing to be the most stable of the evaluated semen parameters. The predictive power of total sDF partially depends on age and semen parameters, whereas brighter sDF independently predicts natural conception. Therefore, brighter sDF is a fraction of sDF that adds new information to the routine semen analysis. At high levels of sDF, distinguishing the two sperm populations improves the predictive power of sDF. Overall, our results support the idea that TUNEL/PI can be of clinical usefulness in the male fertility workup.

Nucleostemin exerts anti-apoptotic function via p53 signaling pathway in cardiomyocytes.

Cardiomyocytes apoptosis following reperfusion injury causes irreversible damage to cardiac function. Understanding the mechanisms underlying cardiomyocytes death under these conditions can be helpful to identify strategies to abrogate such detrimental effects. Stem cell-specific proteins and regulatory pathways become important in understanding reparative processes in the myocardium. One such regulatory protein named nucleostemin (NS) has vital roles in cardiac ischemia. Although the relationship between NS and cell apoptosis has been studied, it is unknown how NS is controlled and how it participates in cardiomyocytes apoptosis induced by ischemia reperfusion (I/R). In the present study, we aimed to investigate the direct role of NS in myocardial I/R. In vivo, NS was highly expressed in cardiac tissues after I/R. Double immunofluorescent staining showed that NS located in the nucleolar of cardiomyocytes and correlated with cardiomyocytes apoptosis. Furthermore, in vitro primary rat cardiomyocytes increased NS expression induced by hypoxia-reoxygenation (H/R) treatment, in line with results in vivo. Suppression of NS expression by siNS promoted the expression of terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL)-positive cells p53 and cleaved caspase-3, which demonstrates I/R may require increased expression of NS to suppress p53 activation and maintain cardiomyocytes survival.

In vivo mechanisms of uterine myoma volume reduction with ulipristal acetate treatment

To study the in vivo mechanisms of action of ulipristal acetate (UPA) on uterine myomas. Retrospective histologic and immunohistochemical (IHC) study of myomas. Academic research unit. Among 59 women with symptomatic myomas who underwent myomectomy, 42 were treated preoperatively with UPA, while 17 were not. Histology and IHC were analyzed on tissue microarrays obtained from surgical specimens. Proliferation, apoptosis, extracellular matrix (ECM) remodeling, and matrix metalloproteinase 2 (MMP-2) expression. Proliferation was low in all conditions, with no statistical difference between groups. Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay showed an increase in cell death in UPA-treated myomas compared with untreated myomas, but only after short-term treatment; this was not associated with elevated levels of cleaved caspase-3. After long-term treatment, cell density was higher and the ECM volume fraction lower in UPA-treated myomas than in untreated myomas. MMP-2 expression was found to be increased after treatment, showing the highest level after long-term treatment, compared with untreated myomas. Regarding sustained clinical volume reduction of myomas, this study strongly points to multifactorial mechanisms of action of UPA, involving: 1) a persistently low cell proliferation rate; 2) a limited period of cell death; and 3) ECM remodeling concomitant with stimulation of MMP-2 expression.

Effects of Cryopreservation on the Molecular Integrity of Ovarian Grafts

INTRODUCTION: The objective of this study was to compare the effects of slow freezing and vitrification on the molecular integrity of ovarian grafts. METHODS: Fresh bovine ovaries were processed and divided into three groups (fresh control, slow freezing, and vitrification) before transplantation. For slow freezing and vitrification, 1.5 M dimethylsulfoxide-based cryoprotectant and a two-step vitrification method were used, respectively. Two pieces of ovarian tissue were transplanted onto the back muscle of the severe combined immunodeficient mouse (six animals for each group). Ovarian grafts were recovered on day 7 and day 28 after transplantation to assess DNA damage, apoptosis, and autophagy by histology, immunohistochemistry, and Western blot analysis. RESULTS: The slow freezing group showed significantly higher normal follicle count compared with the vitrification group. There was no significant difference in apoptosis (terminal deoxynucleotide transferase-mediated dUTP nick-end labeling, caspase 3) among three groups; however, autophagy (LC3B) was decreased in both slow freezing and vitrification groups. On day 28, a significant decrease in LC3B in the vitrification group was noted compared with the slow freezing group. There was no difference in expression of DNA damage and repair markers (γH2AX, Rad51, PCNA) and vascular density makers (CD31). CONCLUSION: The expression pattern of biochemical markers for DNA damage and repair and apoptosis in ovarian grafts was similar between three groups. There is not yet strong evidence that vitrification is better than slow freezing. Therefore, further improved vitrification protocols are needed.

C/EBP Homologous Protein-Mediated Endoplasmic Reticulum Stress-Related Renal Apoptosis Is Involved in Rats With Brain Death

BackgroundC/EBP homologous protein (CHOP) is an important marker in endoplasmic reticulum stress (ERS)-associated cell apoptosis. The role of CHOP-induced renal apoptosis remains unclear in rats with brain death (BD). The present study aims to investigate the possible implication of CHOP-mediated, ERS-related BD-induced apoptosis in rats. Materials and MethodsForty male Sprague–Dawley rats were divided randomly into 4 experimental groups. We examined activation of ERS and apoptosis-related protein expression using Western blot and immunohistochemical staining. In addition, apoptosis is assessed by terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay. ResultsKidneys harvested after BD show increased ERS- and apoptosis-related protein markers compared with kidneys of non-BD rats. Salubrinal (Sal) significantly increased levels of p-eIF2a and decreased the activity of CHOP at 6 hours after BD compared with vehicle-treated dimethylsulfoxide. ConclusionsTreating the BD donor with Sal influences renal apoptosis compared with vehicle-treated BD rats. Our results indicate that targeting the CHOP pathway provides a promising therapeutic approach for kidney injury associated with donor BD.