Triphosphate Nick-end Labeling Research Articles

Noninvasive Whole-Body Imaging of Phosphatidylethanolamine as a Cell Death Marker Using 99mTc-Duramycin During TNF-Induced SIRS.

Systemic inflammatory response syndrome (SIRS) is an inflammatory state affecting the whole body. It is associated with the presence of pro- and antiinflammatory cytokines in serum, including tumor necrosis factor (TNF). TNF has multiple effects and leads to cytokine production, leukocyte infiltration, and blood pressure reduction and coagulation, thereby contributing to tissue damage and organ failure. A sterile mouse model of sepsis, TNF-induced SIRS, was used to visualize the temporal and spatial distribution of damage in susceptible tissues during SIRS. For this, a radiopharmaceutical agent, 99mTc-duramycin, that binds to exposed phosphatidylethanolamine on dying cells was longitudinally visualized using SPECT/CT imaging. Methods: C57BL/6J mice were challenged with intravenous injections of murine TNF or vehicle, and necrostatin-1 was used to interfere with cell death. Two hours after vehicle or TNF treatment, mice received 99mTc-duramycin intravenously (35.44 ± 3.80 MBq). Static whole-body 99mTc-duramycin SPECT/CT imaging was performed 2, 4, and 6 h after tracer injection. Tracer uptake in different organs was quantified by volume-of-interest analysis using PMOD software and expressed as SUVmean After the last scan, ex vivo biodistribution was performed to validate the SPECT imaging data. Lastly, terminal deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed to correlate the obtained results to cell death. Results: An increased 99mTc-duramycin uptake was detected in mice injected with TNF, when compared with control mice, in lungs (0.55 ± 0.1 vs. 0.34 ± 0.05), intestine (0.75 ± 0.13 vs. 0.56 ± 0.1), and liver (1.03 ± 0.14 vs. 0.64 ± 0.04) 4 h after TNF and remained significantly elevated until 8 h after TNF. The imaging results were consistent with ex vivo γ-counting results. Significantly increased levels of tissue damage were detected via TUNEL staining in the lungs and intestine of mice injected with TNF. Interestingly, necrostatin-1 pretreatment conferred protection against lethal SIRS and reduced the 99mTc-duramycin uptake in the lungs 8 h after TNF (SUV, 0.32 ± 0.1 vs. 0.51 ± 0.15). Conclusion: This study demonstrated that noninvasive 99mTc-duramycin SPECT imaging can be used to characterize temporal and spatial kinetics of injury and cell death in susceptible tissues during TNF-induced SIRS, making it useful for global, whole-body assessment of tissue damage during diseases associated with inflammation and injury.

A morphological and biochemical evaluation of the effects of quercetin on experimental sciatic nerve damage in rats.

The present study evaluated the neuroprotective and antioxidant effects of quercetin in a rat model of sciatic nerve crush injury using histopathological, morphometric and biochemical methods. A total of 48 male Sprague Dawley rats, aged 10–12 weeks old were randomly divided into eight groups, consisting of two sham groups (S-7, S-28), three quercetin-treated groups (Q-7, Q-28; 200 mg/kg/7 days), trauma (T-7, T-28; 1 min sciatic nerve crush injury) and three trauma+quercetin groups (T+Q-7, T+Q-28; trauma+quercetin 200 mg/kg/7 days). Rats were sacrificed on day 7 or 28. Oxidant-antioxidant biochemical parameters in nerve tissues from all groups were analyzed using histopathological staining with toluidine blue and Masson's trichrome. DNA fragmentations were identified using terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling in cells from each tissue sample. Degeneration of the axons and myelin sheath, the breakdown of the concentric lamellar structure of the myelin sheath and axonal swelling were observed in groups T-7 and T-28. Myelin sheath thicknesses, nerve fiber diameters and the number of myelinated nerve fibers decreased, while the apoptotic index (AI) increased in the T-7 and T-28 groups. However, it was observed that nerve regeneration began in the T+Q-7 and T+Q-28 groups compared with the sham groups, together with the healing of cellular damage and axonal structure and a decrease in the AI. Malondialdehyde and superoxide dismutase activity did not differ significantly between the T-7 and S-7 groups. However, catalase activity significantly decreased in the T-28 group when compared with the sham 7 day group. Tissue malondialdehyde levels significantly increased, while serum catalase activity increased in the T+Q-7 group compared with the T-7 group. These results suggest that quercetin has beneficial effects on nerve regeneration and may shorten the healing period in crush-type sciatic nerve injuries.

Sperm DNA and detection of DNA fragmentations in sperm.

The questionable effectiveness of routine sperm parameters in determining male factor infertility problems and increasing the success rates of assisted reproductive techniques have led to the investigation of more detailed sperm parameters that could affect the male fertility and reproduction. Thus, the effects of different sperm parameters such as sperm DNA integrity was started to be investigated thanks to the previously described methods such as single cell gel electrophoresis (COMET) assay, sperm chromatin structure assay (SCSA), acridine orange test (AOT), terminal deoxynucleotidyl transferase-mediated deoxyuridine (TdT) triphosphate (dUTP) nick end labeling (TUNEL) assay and sperm chromatin dispersion (SCD) test. However, studying on sperm DNA might be very complex because the sperm DNA differs from the somatic cell DNA with its unique structure. Also, the sperm DNA undergoes many changes during spermatogenesis and it is condensed by being packaged tightly with different types and numbers of protamines in different species. Despite all these difficulties, these methods provide important information about the reasons and consequences of DNA damages in sperm and the effects of these damages on reproduction.

Expression of diaphragmatic myostatin and correlation with apoptosis in rats with chronic obstructive pulmonary disease.

Chronic obstructive pulmonary disease (COPD) is characterized by progressive airflow limitation and loss of lung function. The present study aimed to investigate the diaphragmatic protein expression of myostatin and its correlation with apoptosis in a rat model of CPOD. Sprague Dawley rats were randomly divided into a control group and a COPD group, the latter of which were exposed to cigarette smoke to build a rat model of COPD. The validity of the COPD model was evaluated by assessment of lung function and histopathological analysis. Diaphragmatic myostatin expression and apoptosis were measured by western blot and terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling, respectively. The rat model of COPD was efficiently established by cigarette smoke exposure. Diaphragmatic myostatin expression and apoptotic index in COPD rats were obviously increased as compared with that in the control animals. A positive correlation between diaphragmatic myostatin expression and apoptotic index was identified (r=0.857). Diaphragmatic myostatin overexpression in rats with COPD may promote diaphragmatic apoptosis and atrophy, leading to diaphragm weakness and respiratory muscle dysfunction, which is involved in the pathology of COPD.

The effect of beta glucan on MTX induced testicular damage in rats

We investigated the histopathological effects of methotrexate (MTX), a chemotherapeutic agent, and beta glucan (BG), an antioxidant, on rat testis. We used four groups of Sprague-Dawley male rats: MTX, MTX + BG, BG, and control. The MTX group was exposed to a single dose of MTX on the first day of experiment. The MTX + BG group was exposed to a single dose of MTX and BG on the first day of experiment followed by BG for 4 additional days. The BG group was exposed to BG for 5 days. The control group was given saline for 5 days. On day five, all animals were sacrificed and testicular tissue was evaluated for histopathology and the terminal deoxynucleotidyl transferase (TdT) deoxyuridine triphosphate nick-end labeling assay (TUNEL) was used to detect apoptosis. The apoptotic index (AI) and testicular damage increased in the MTX group compared to the other three groups. Histopathology was reduced in the MTX + BG group compared to the MTX group. Seminiferous tubule diameter was reduced in the MTX group compared to the BG group; we found no difference between control and BG groups. The thickness of th e germinal epithelium was reduced in the MTX group compared to the other groups. We found no difference in testicular weight among the groups. We compared body weight before and after the experiment; weights in the MTX and MTX + BG groups were significantly reduced compared to controls. In the control groups, we found a statistically significant increase in body weight, whereas there was no change in the BG group. We found that MTX causes deleterious effects on testicular tissue and that beta glucan may be protective.

Calpain inhibition improves erectile function in diabetic mice via upregulating endothelial nitric oxide synthase expression and reducing apoptosis.

Calpain activation contributes to hyperglycemia-induced endothelial dysfunction and apoptosis. This study was designed to investigate the role of calpain inhibition in improving diabetic erectile dysfunction (ED) in mice. Thirty-eight-week-old male C57BL/6J mice were divided into three groups: (1) nondiabetic control group, (2) diabetic mice + vehicle group, and (3) diabetic mice + MDL28170 (an inhibitor of calpain) group. Type 1 diabetes was induced by intraperitoneal injection of streptozotocin at 60 mg kg−1 body weight for 5 consecutive days. Thirteen weeks later, diabetic mice were treated with MDL28170 or vehicle for 4 weeks. The erectile function was assessed by electrical stimulation of the cavernous nerve. Penile tissues were collected for measurement of calpain activity and the endothelial nitric oxide synthase (eNOS)-nitric oxide (NO)-cyclic guanosine monophosphate (cGMP) pathway. Terminal deoxynucleotidyl transferase 2'-deoxyuridine 5'-triphosphate nick end labeling (TUNEL) staining was used to evaluate apoptosis. Caspase-3 expression and activity were also measured to determine apoptosis. Our results showed that erectile function was enhanced by MDL28170 treatment in diabetic mice compared with the vehicle diabetic group. No differences in calpain-1 and calpain-2 expressions were observed among the three groups. However, calpain activity was increased in the diabetic group and reduced by MDL28170. The eNOS-NO-cGMP pathway was upregulated by MDL28170 treatment in diabetic mice. Additionally, MDL28170 could attenuate apoptosis and increase the endothelium and smooth muscle levels in corpus cavernosum. Inhibition of calpain could improve erectile function, probably by upregulating the eNOS-NO-cGMP pathway and reducing apoptosis.

Shaofu Zhuyu Decoction Regresses Endometriotic Lesions in a Rat Model.

The current therapies for endometriosis are restricted by various side effects and treatment outcome has been less than satisfactory. Shaofu Zhuyu Decoction (SZD), a classic traditional Chinese medicinal (TCM) prescription for dysmenorrhea, has been widely used in clinical practice by TCM doctors to relieve symptoms of endometriosis. The present study aimed to investigate the effects of SZD on a rat model of endometriosis. Forty-eight female Sprague-Dawley rats with regular estrous cycles went through autotransplantation operation to establish endometriosis model. Then 38 rats with successful ectopic implants were randomized into two groups: vehicle- and SZD-treated groups. The latter were administered SZD through oral gavage for 4 weeks. By the end of the treatment period, the volume of the endometriotic lesions was measured, the histopathological properties of the ectopic endometrium were evaluated, and levels of proliferating cell nuclear antigen (PCNA), CD34, and hypoxia inducible factor- (HIF-) 1α in the ectopic endometrium were detected with immunohistochemistry. Furthermore, apoptosis was assessed using the terminal deoxynucleotidyl transferase (TdT) deoxyuridine 5′-triphosphate (dUTP) nick-end labeling (TUNEL) assay. In this study, SZD significantly reduced the size of ectopic lesions in rats with endometriosis, inhibited cell proliferation, increased cell apoptosis, and reduced microvessel density and HIF-1α expression. It suggested that SZD could be an effective therapy for the treatment and prevention of endometriosis recurrence.

Apoptosis in the cochlear nucleus and inferior colliculus upon repeated noise exposure.

The time course of apoptosis and the corresponding neuronal loss was previously shown in central auditory pathway of mice after a single noise exposure. However, repeated acoustic exposure is a major risk factor for noise-induced hearing loss. The present study investigated apoptosis by terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick end labeling (TUNEL) assay after a second noise trauma in the ventral and dorsal cochlear nucleus and central nucleus of the inferior colliculus. Mice [Naval Medical Research Institute (NMRI) strain] were noise exposed [115 dB sound pressure level, 5–20 kHz, 3 h) at day 0. A double group received the identical noise exposure a second time at day 7 post-exposure and apoptosis was either analyzed immediately (7-day group-double) or 1 week later (14-day group-double). Corresponding single exposure groups were chosen as controls.No differences in TUNEL were seen between 7-day or 14-day single and double-trauma groups. Interestingly, independent of the second noise exposure, apoptosis increased significantly in the 14-day groups compared to the 7-day groups in all investigated areas.It seems that the first noise trauma has a long-lasting effect on apoptotic mechanisms in the central auditory pathway that were not largely influenced by a second trauma. Homeostatic mechanisms induced by the first trauma might protect the central auditory pathway from further damage during a specific time slot. These results might help to understand the underlying mechanisms of different psychoacoustic phenomena in noise-induced hearing loss.

GYY4137, an Extended-Release Hydrogen Sulfide Donor, Reduces NMDA-Induced Neuronal Injury in the Murine Retina.

We previously reported that systemic administration with sodium hydrogen sulfide, a rapid-release donor compound of hydrogen sulfide (H2S), protected retinal neurons against N-methyl-D-aspartic acid (NMDA)-induced injury. For clinical application of H2S donors for retinal neurodegeneration, topical administration with an extended-release donor compound will be better. In the present study, we histologically investigated whether GYY4137, an extended-release hydrogen sulfide donor, had a protective effect on NMDA-induced retinal injury in the mice in vivo. Male and female B6.Cg-Tg(Thy1-CFP)23Jrs/J and C57BL/6J mice anesthetized with a mixture of ketamine and xylazine were subjected to intravitreal NMDA injection (80 nmol/eye). GYY4137 was intravitreally administered with NMDA simultaneously. Morphometric evaluation was carried out seven days after NMDA injection. Intravitreal NMDA induced retinal ganglion cell loss. GYY4137 (1, 10 and 100 nmol/eye) significantly reduced retinal ganglion cell loss seven days after NMDA injection. GYY4137 (10 nmol/eye) decreased the numbers of terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL)-positive and 8-hydroxy-2'-deoxyguanosine (8-OHdG)-positive cells 12 h after NMDA injection. These results suggest that extended release donor compounds of H2S protect retinal neurons against excitotoxicity induced by intravitreal NMDA in the mice in vivo through its anti-oxidative activity.

Quercetin induces cell death in cervical cancer by reducing O-GlcNAcylation of adenosine monophosphate-activated protein kinase.

Hyper-O-GlcNAcylation is a general feature of cancer which contributes to various cancer phenotypes, including cell proliferation and cell growth. Quercetin, a naturally occurring dietary flavonoid, has been reported to reduce the proliferation and growth of cancer. Several reports of the anticancer effect of quercetin have been published, but there is no study regarding its effect on O-GlcNAcylation. The aim of this study was to investigate the anticancer effect of quercetin on HeLa cells and compare this with its effect on HaCaT cells. Cell viability and cell death were determined by MTT and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling assays. O-GlcNAcylation of AMP-activated protein kinase (AMPK) was examined by succinylated wheat germ agglutinin pulldown and immunoprecipitation. Immunofluorescence staining was used to detect the immunoreactivitiy of O-linked N-acetylglucosamine transferase (OGT) and sterol regulatory element binding protein 1 (SREBP-1). Quercetin decreased cell proliferation and induced cell death, but its effect on HaCaT cells was lower than that on HeLa cells. O-GlcNAcylation level was higher in HeLa cells than in HaCaT cells. Quercetin decreased the expression of global O-GlcNAcylation and increased AMPK activation by reducing the O-GlcNAcylation of AMPK. AMPK activation due to reduced O-GlcNAcylation of AMPK was confirmed by treatment with 6-diazo-5-oxo-L-norleucine. Our results also demonstrated that quercetin regulated SREBP-1 and its transcriptional targets. Furthermore, immunofluorescence staining showed that quercetin treatment decreased the immunoreactivities of OGT and SREBP-1 in HeLa cells. Our findings demonstrate that quercetin exhibited its anticancer effect by decreasing the O-GlcNAcylation of AMPK. Further studies are needed to explore how quercetin regulates O-GlcNAcylation in cancer.

Interleukin-17A Promotes Parietal Cell Atrophy by Inducing Apoptosis.

Background & AimsAtrophic gastritis caused by chronic inflammation in the gastric mucosa leads to the loss of gastric glandular cells, including acid-secreting parietal cells. Parietal cell atrophy in a setting of chronic inflammation induces spasmolytic polypeptide expressing metaplasia, a critical step in gastric carcinogenesis. However, the mechanisms by which inflammation causes parietal cell atrophy and spasmolytic polypeptide expressing metaplasia are not well defined. We investigated the role of interleukin-17A (IL-17A) in causing parietal cell atrophy.MethodsA mouse model of autoimmune atrophic gastritis was used to examine IL-17A production during early and late stages of disease. Organoids derived from corpus glands were used to determine the direct effects of IL-17A on gastric epithelial cells. Immunofluorescent staining was used to examine IL-17A receptors and the direct effect of signaling on parietal cells. Mice were infected with an IL-17A-producing adenovirus to determine the effects of IL-17A on parietal cells in vivo. Finally, IL-17A neutralizing antibodies were administered to mice with active atrophic gastritis to evaluate the effects on parietal cell atrophy and metaplasia.ResultsIncreased IL-17A correlated with disease severity in mice with chronic atrophic gastritis. IL-17A caused caspase-dependent gastric organoid degeneration, which could not be rescued with a necroptosis inhibitor. Parietal cells expressed IL-17A receptors and IL-17A treatment induced apoptosis in parietal cells. Overexpressing IL-17A in vivo induced caspase-3 activation and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling staining in parietal cells. Finally, IL-17A neutralizing antibody decreased parietal cell atrophy and metaplasia in mice with chronic atrophic gastritis.ConclusionsThese data identify IL-17A as a cytokine that promotes parietal cell apoptosis during atrophic gastritis, a precursor lesion for gastric cancer.

A Novel Peptide from Vespa ducalis Induces Apoptosis in Osteosarcoma Cells by Activating the p38 MAPK and JNK Signaling Pathways.

Osteosarcoma (OS) is a typical bone cancer, and most frequently used cancer treatments for OS are limited due to severe drug-related toxicities. Wasp venoms contain functional components that may offer pharmaceutical components for the treatment of cancers. This study aimed to isolate and characterize a novel peptide (venom anti-cancer peptide 1, VACP1) derived from the wasp venom of Vespa ducalis SMITH. Toxins from Vespa ducalis crude venom were separated by gel filtration and purified by C18 reverse-phase HPLC. As examined by Edman degradation, the amino acid sequence of VACP1 is AQKWLKYWKADKVKGFGRKIKKIWFG. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays revealed that VACP1 inhibited the cell proliferation of MG-63, U-2 OS and Saos-2 cells. Furthermore, annexin V and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining revealed that VACP1 could induce the apoptosis of OS cell lines. In addition, VACP1 increased the protein levels of cleaved poly ADP-ribose polymerase (PARP), caspase 3, but decreased B-cell lymphoma 2 (Bcl-2). Apoptotic signaling pathway screening in MG-63 cells via an antibody array revealed that VACP1 activated the p38 mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) pathways. The present study demonstrates that VACP1 potently suppressed cell proliferation and induced the cell apoptosis of OS cells by inducing the activation of the p38 MAPK and JNK signaling pathways, suggesting that VACP1 is a promising agent for OS therapy.

Terminal Deoxynucleotidyl Transferase-Mediated Deoxyuridine Triphosphate Nick End Labeling (TUNEL) Assay to Characterize Histopathologic Changes Following Thermal Injury.

BackgroundDespite the wide application of lasers and radiofrequency (RF) surgery in dermatology, it is difficult to find studies showing the extent of damage dependent on cell death.ObjectiveWe evaluated histopathologic changes following in vivo thermal damage generated by CO2 laser, 1,444 nm long-pulsed neodymium:yttrium-aluminum-garnet (LP Nd:YAG) laser and RF emitting electrosurgical unit.MethodsThermal damage was induced by the above instruments on ventral skin of rat. Specimens were stained with hematoxylin and eosin, along with a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay, to highlight the degree of irreversible cellular injury.ResultsThe volume of vaporization was largest with the CO2 laser. Area of cell death area identified by TUNEL assay, when arranged from widest to narrowest, was 1,444 nm LP Nd:YAG laser, CO2 laser, and RF emitting electrosurgical unit.ConclusionThis histopathologic evaluation of the acute characterization of injury across devices may be advantageous for attaining better treatment outcomes.

Combined liver-kidney perfusion enhances protective effects of normothermic perfusion on liver grafts from donation after cardiac death.

It has been shown that combined liver-kidney normothermic machine perfusion (NMP) is able to better maintain the circuit's biochemical milieu. Nevertheless, whether the combined perfusion is superior to liver perfusion alone in protecting livers from donation after circulatory death (DCD) is unclear. We aimed to test the hypothesis and explored the mechanisms. Livers from 15 DCD pig donors were subjected to either static cold storage (group A), liver-alone NMP (group B), or combined liver-kidney NMP (group C). Livers were preserved for 6 hours and reperfused ex vivo for 2 hours to simulate transplantation or were transplanted in situ. During perfusion, group C showed an improved acid-base and biochemical environment in the circuit over group B. After reperfusion, the architecture of the liver grafts was best preserved in group C, followed by group B, then group A, as shown by the histology and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining of both hepatocytes and biliary epithelium. Ki-67 staining showed substantial hepatocyte proliferation and biliary epithelial regeneration after perfusion in group B and group C. Group C produced more bile in the reperfusion phase than those in group A and group B, with more physiological bile composition and less severe biliary epithelium injury. Von Willebrand factor-positive endothelial cells and E-selectin expression decreased in both group B and group C. Combined liver-kidney NMP not only produced more adenosine triphosphate, protected the nitric oxide signaling pathway, but also diminished oxidative stress (high mobility group box-1 protein and 8-hydroxy-2-deoxy guanosine levels) and inflammatory cytokine (IL6 and IL8) release when compared with liver-alone NMP and CS. In addition, the 7-day survival rate of liver transplant recipients was higher in group C than that in groups A and B. In conclusion, combined liver-kidney NMP can better protect DCD livers from warm ischemia and reperfusion injury probably by maintaining the stability of the internal environment and by abolishing oxidative stress injury. Liver Transplantation 24 67-79 2018 AASLD.

The Japanese Angelica acutiloba root and yokukansan increase hippocampal acetylcholine level, prevent apoptosis and improve memory in a rat model of repeated cerebral ischemia

Ethnopharmacological relevanceJapanese Angelica acutiloba root (Angelica root) is included in several Kampo medicines including Yokukansan (YKS). Angelica root and YKS are used for the treatment of a variety of psychological and neurodegenerative disorders. Development of safe and effective therapeutic agents against cerebrovascular disorders will improve the treatment of patients with dementia. Aim of the studyThe effect of Angelica root and YKS on ischemia-impaired memory has not yet been fully investigated. The present study investigated whether Angelica root is also involved in memory improving and neuroprotective effect of YKS in a model of cerebrovascular ischemia. Materials and methodsMale Wistar rats grouped into sham rats received saline, and other three groups subjected to repeated cerebral ischemia induced by 4-vessel occlusion (4-VO), received a 7-day oral administration of either saline, Angelica root or YKS. Memory was evaluated by eight-arm radial maze task. Acetylcholine release (ACh) in the dorsal hippocampus was investigated by microdialysis-HPLC. Apoptosis was determined by terminal deoxynucleotidyl transferase (TdT)-mediated fluorescein-deoxyuridine triphosphate (dUTP) nick-end labeling. ResultsIschemia induced apoptosis, reduced release of ACh, and impaired the memory (increased error choices and decreased correct choices). Angelica root and YKS improved the memory deficits, upregulated the release of ACh and prevented 4-VO-induced hippocampal apoptosis. ConclusionThe dual ACh-increasing and neuroprotective effect of Angelica root could make it a promising therapeutic agent useful for the treatment of symptoms of cerebrovascular dementia. Angelica root could be one of the components contributing to the memory-improving and neuroprotective effects of YKS.

72 Protective Effects of C-Phycocyanin on the Developmental Competence of Porcine Parthenotes

C-Phycocyanin (CP) is a biliprotein enriched in blue-green algae that is known to possess antioxidant, anti-apoptosis, anti-inflammatory, and radical-scavenging properties in somatic cells. However, the protective effect of CP on porcine embryo developmental competence in vitro remains unclear. In the present study, we investigated the effect of CP on the development of porcine early embryos as well as its underlying mechanisms exposing them to H2O2 to induce oxidative stress. The levels of reactive oxygen species, mitochondrial membrane potential, apoptosis, DNA damage, and autophagy in the blastocysts were observed by staining with 2′,7′-dichlorodihydrofluorescein diacetate (H2DCF-DA), 5,5′,6,6’-tetrachloro-1,1′,3,3′-tetraethyl-imidacarbocyanine iodide (JC-1), terminal deoxynucleotidyl transferase-mediated 2′-deoxyuridine 5′-triphosphate (dUTP) nick-end labelling (TUNEL), anti-cytochrome c, and anti-γH2A.X (Ser139), respectively. Colocalization assay of mitochondria and cytochrome c of blastocysts were staining with MitoTracker Red CMXRos and anti-cytochrome c. All data were subjected to one-way ANOVA. Different concentrations of CP (1, 2, 5, 8, 10 µg mL−1) were added to porcine zygote medium 5 (PZM-5, l-glutamine concentration of PZM-3 was modified from 1 to 2 mM) during in vitro culture. The results showed that 5 µg mL−1 CP significantly increased blastocyst formation (62.5 ± 2.1 v. 52.7 ± 2.4; P < 0.05) and hatching rate (10.9 ± 1.9 v. 36.6 ± 5.2; P < 0.05) compared with controls. Blastocyst formation (53.1 ± 2.3 v. 40.1 ± 2.3; P < 0.05) and quality were significantly increased in the 50 µM H2O2 treatment group following 5 µg mL−1 CP addition. C-Phycocyanin prevented the H2O2-induced compromise of mitochondrial membrane potential, release of cytochrome c from the mitochondria, and generation of reactive oxygen species. Furthermore, apoptosis, DNA damage level, and autophagy in the blastocysts were attenuated by supplementation of CP in the H2O2-induced oxidative injury group compared with that in controls. These results suggest that CP has beneficial effects on the development of porcine parthenotes by attenuating mitochondrial dysfunction and oxidative stress.

The Orphan Nuclear Receptor Liver Homolog Receptor-1 (Nr5a2) Regulates Ovarian Granulosa Cell Proliferation.

In mouse ovaries, liver receptor homolog-1 [nuclear receptor subfamily 5, group A, member 2 (Nr5a2)] expression is restricted to granulosa cells. Mice with Nr5a2 depletion in this cell population fail to ovulate. To determine whether Nr5a2 is essential for granulosa cell proliferation during follicular maturation, we generated granulosa-specific conditional knockout mice (genotype Nr5a2 floxed Cre-recombinase driven by the anti-Müllerian type II receptor, hereafter cKO) with Nr5a2 depletion from primary follicles forward. Proliferation in cKO granulosa cells was substantially reduced relative to control (CON) counterparts, as assessed by bromodeoxyuridine incorporation, proliferative cell nuclear antigen expression, and fluorescent-activated cell sorting. Microarray analysis revealed >2000 differentially regulated transcripts between cKO and CON granulosa cells. Major gene ontology pathways disrupted were proliferation, steroid biosynthesis, female gamete formation, and ovulatory cycle. Transcripts for key cell-cycle genes, including Ccnd1, Ccnd2, Ccne1, Ccne2, E2f1, and E2f2, were in reduced abundance. Transcripts from other cell-cycle-related factors, including Cdh2, Plagl1, Cdkn1a, Prkar2b, Gstm1, Cdk7, and Pts, were overexpressed. Although the follicle-stimulating hormone and estrogen receptors were overexpressed in the cKO animals, in vivo treatment with estradiol-17β failed to rescue decreased proliferation. In vitro inactivation of Nr5a2 using the ML180 reverse agonist similarly decreased cell-cycle-related gene transcripts and downstream targets, as in cKO mice. Pharmacological inhibition of β-catenin, an Nr5a2 cofactor, decreased cyclin gene transcripts and downstream targets. Terminal deoxynucleotidyltransferase-mediated deoxyuridine triphosphate nick end labeling immunofluorescence and quantitative polymerase chain reaction of pro/antiapoptotic and autophagic markers showed no differences between cKO and CON granulosa cells. Thus, Nr5a2 is essential for granulosa cell proliferation, but its depletion does not alter the frequency of apoptosis nor autophagy.

Intravesical Immunomodulatory Imiquimod Enhances Bacillus Calmette-Guérin Downregulation of Nonmuscle-invasive Bladder Cancer.

The Toll-like receptor (TLR)2/4 agonist bacillus Calmette-Guérin (BCG), although not failure proof, has been the most efficient immunomodulatory treatment of immunogenic nonmuscle-invasive bladder cancer (NMIBC) for > 40 years. We investigated the role of the immunomodulatory molecule TLR7 agonist imiquimod through the BCG key receptors TLR2/4 and the main downstream molecules of the mammalian target of rapamycin pathway in NMIBC treatment. A total of 40 Fischer-344 rats, 7 weeks old, received 4 doses of 1.5 mg/kg N-methyl-N-nitrosourea intravesically on weeks 0, 2, 4, and 6 for cancer induction. At week 8, the rats were randomized into 4 groups (10 per group) and treated intravesically once a week for 6 weeks: control (0.2 mL of vehicle); BCG (2× 106 colony-forming units Connaught strain in 0.2 mL); imiquimod (20 mg/kg in 0.2 mL), and associated treatment BCG plus imiquimod in 0.2 mL. The bladders were extracted and analyzed for histopathology, immunohistochemistry, cell proliferation (Ki-67), apoptosis (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling [TUNEL]), and immunoblotting for TLR2, TLR4, p-P70S6K, and p-4E-BP1 proteins. The histopathology results showed that BCG and imiquimod decreased bladder tumorigenesis compared with the control group, with a proliferation decrease (Ki-67) and an apoptosis increase (TUNEL). BCG upregulated TLR2/4, imiquimod upregulated TLR4, and both downregulated P70S6K1. Imiquimod is able to efficiently decrease bladder carcinogenesis through upregulation of TLR7/4 and downregulation of P70S6K1 protein, generating new perspectives to boost BCG effects in the future.

Alpha1-adrenergic receptor antagonist tamsulosin ameliorates aging-induced memory impairment by enhancing neurogenesis and suppressing apoptosis in the hippocampus of old-aged rats

ABSTRACTAge-related memory decline is closely associated with decreased neurogenesis and increased apoptosis in the hippocampus. Noradrenaline exerts its effect by selectively binding to and activating adrenergic receptors (ARs). Tamsulosin, α1-AR antagonist, is reported to have access to the brain and interact with α1-AR. In this study, the effects of tamsulosin on short-term and spatial learning memory in terms of neurogenesis and apoptosis were investigated using rats. Step-down avoidance test for short-term memory and radial 8-arm maze test for spatial learning memory were conducted. Neurogenesis was detected by 5-bromo-2’-deoxyuridine (BrdU) immunohistochemistry and apoptosis was evaluated by caspase-3 immunohistochemisty and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNE) staining. Western blot for protein kinase C (PKC), cAMP-responsive element-binding protein (CREB), brain-derived neurotrophic factor (BDNF), tyrosine kinase B (TrkB), phosphatidylinositol 3-kinase (PI 3-kinase), Akt, Bcl-2, and Bax was conducted. In the aged rats, short-term and spatial learning memory was declined. Hippocampal nerogenesis was suppressed and hippocampal apoptosis was enhanced in the aged rats. In addition, phosphorylation of PKCα, CREB, PI-3 kinase, and Akt was decreased in the hippocampus of old-aged rats. Tamsulosin activated PKC/CREB and PI-3 kinase/Akt pathways. With these pathways, BDNF-TrkB signaling enhanced hippocampal neurogenesis and suppressed apoptosis in the old-aged rats. As the results, tamsulosin improved performance of short-term and spatial learning memory in the aged rats.

Effects of Rosiglitazone on the Outcome of Experimental Periapical Lesions in Mice

IntroductionThe purpose of this study was to evaluate a protocol for systemic administration of rosiglitazone in mice in order to stimulate apoptosis of osteocytes in the jaws and to evaluate the effect of osteocyte apoptosis induced by rosiglitazone in the progression of periapical lesions in mice at 7, 21, and 42 days. MethodsC57BL/6 mice at 4–5 weeks of age were used. In phase 1, mice (n = 24) were treated with rosiglitazone (gavage, 10 mg/kg dose) or without (phosphate-buffered saline + 10% dimethyl sulfoxide) for 1, 2, or 3 weeks. We used the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and 4′-6-diamidino-2-phenylindole methods for quantification of apoptotic cells. In phase 2, mice (n = 30) received rosiglitazone for 2 weeks or just vehicle for 1 week (n = 30), and periapical lesions were induced for 7, 21, or 42 days. We performed the measurement of periapical lesions, tartrate-resistant acid phosphatase staining, dual-energy x-ray absorptiometry for the evaluation of bone mineral density (BMD) in long bone, and gene evaluation using real time quantitative polymerase chain reaction of osteocyte markers (Sost, Hyou1, and Dmp1) and receptor activator of nuclear factor kappa-B ligand (RANKL) (Tnfsf11). ResultsIt was observed that systemic administration of rosiglitazone for 2 weeks showed apoptosis of osteocytes in a more expressive manner. In phase 2, in the groups that received rosiglitazone, a trend toward larger periapical lesions was observed (P > .05). Rosiglitazone also induced a greater number of osteoclasts and a greater expression of Sost and Hyou1 at 21 days of lesions. Moreover, there were no statistically significant differences in RANKL and Dmp1 expression or in the BMD of femurs. ConclusionsRosiglitazone stimulated apoptosis of osteocytes, interfering in the progression of periapical lesions in mice.