Transferase-mediated fluorescein-dUTP Nick End Labeling Research Articles

Acupotomy relieves pain and improves motor function by regulating autophagy and apoptosis of cartilage in rabbits with knee osteoarthritis apoptosis

To observe the effect of acupotomy on the expression of Beclin-1, Bcl-2 and Caspase-3 in the cartilage tissue in rabbits with knee osteoarthritis (KOA), so as to explore its mechanism underling improvement of KOA. Twenty-four healthy male New Zealand rabbits were randomly and equally divided into blank control, model and acupotomy groups, with 8 rabbits in each group. By using the modified Videman's methods, the KOA model was established by left hind limb immobilization with a plaster cast for 6 weeks. The severity of KOA (knee pain, swelling and motor function) was assessed using Lequesne score, and the rabbits with a score below 4 were excluded. The acupotomy was applied to "Hedingci" (the attachment of the quadriceps tendon to the patella at the upper edge), "Binneixia" (the medial patellar supporting band attachment of medial inferior patellar margin), "Binwaixia" (the lateral patellar supporting band attachment of the lower lateral patellar margin), "Chengfeijian" (the lateral collateral ligament of the knee passes over the lateral joint space), "Weiyangci" (the medial margin of biceps femoris at the lateral end of popliteus), "Yinlingci" (the medial tibial attachment of anserinus tendon) on the left hind limb once a week for 4 weeks. One week after the last intervention, the left knee joint dysfunction severity(pain, maximum walking distance, and some activities of daily living) was evaluated by using modified Lequesne score. Histopathological changes of the cartilage were observed under light microscope after H.E. staining. The apoptosis of chondrocytes was observed after terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick-end labeling (TUNEL) staining. The autophagolysosomes of chondrocytes were observed using transmission electron microscopy. The expression levels of Beclin-1, Bcl-2 and Caspase-3 (related factors of autophagy and apoptosis) were detected using Real-time PCR and Western blot separately. In comparison with the blank control group, the Lequesne score, apoptosis rate, expression levels of Caspase-3 mRNA and protein were significantly increased (P<0.001), and the number of autophagolysosomes, expression levels of Beclin-1 and Bcl-2 mRNAs and proteins considerably decreased (P<0.001) in the model group. Relevant to the model group, the acupotomy group had an obvious decrease in Lequesne score, rate of apoptosis, and expression levels of Caspase-3 mRNA and protein (P<0.001) and an apparent increase in the number of autophagolysosomes and expression levels of Beclin-1 and Bcl-2 mRNAs and proteins (P<0.001). Findings of H.E. staining showed severe damaged cartilage surface, with a large number of exfoliation defects, few chondrocytes on the surface and disordered arrangement of transitional cells in the model group, which was relatively milder in the acupotomy group. Acupotomy can mitigate knee-joint pain and improve functional activity in KOA rabbits, which may be associated with its functions in promoting autophagy and suppressing apoptosis by up-regulating expressions of Beclin-1 and Bcl-2 mRNAs and proteins and down-regulation of Caspase-3 mRNA and protein.

Disease-Modifying Anti-rheumatic Drug Prescription Baihu-Guizhi Decoction Attenuates Rheumatoid Arthritis via Suppressing Toll-Like Receptor 4-mediated NLRP3 Inflammasome Activation.

As a traditional Chinese medicine-originated disease-modifying anti-rheumatic drug prescription, Baihu-Guizhi decoction (BHGZD) is extensively used for the treatment of rheumatoid arthritis (RA) with a satisfying therapeutic efficacy. Mechanically, our previous data indicated that BHGZD may ameliorate RA partially by restoring the balance of the “inflammation-immune” system through regulating the TLR4-c-Fos-IL2-TNF-alpha axis. Toll-like receptor 4 (TLR4) has been revealed to be involved in the activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome complex. Thus, the aim of the current study was to determine the regulatory effects of BHGZD on the TLR4–mediated inflammasome activation during RA progression based on the modified adjuvant-induced arthritis model (AIA-M) and the lipopolysaccharide/adenosine triphosphate (LPS/ATP)–induced pyroptosis cellular models. As a result, oral administration of BHGZD exhibited prominent improvement in the disease severity of AIA-M rats, such as reducing the redness and swelling of joints, arthritis incidence, arthritic scores, and diameter of the limb and increasing pain thresholds. In line with the in vivo findings, BHGZD treatment effectively inhibited the LPS/ATP–induced pyroptosis of both Raw264.7 macrophage and MH7A cells in vitro by reducing pyroptotic cell death morphology (swollen cells) and decreasing propidium iodide–positive and terminal deoxynucleotidyl transferase–mediated dUTP-fluorescein nick end labeling (TUNEL)–positive cells. Notably, the increased expression levels of TLR4, NLRP3, interleukin 1β, and interleukin 18 proteins and the elevated activities of caspase-1 and lactic dehydrogenase in in vivo and in vitro disease models were markedly reversed by the treatment with BHGZD. In conclusion, the above findings proved the immunomodulatory and anti-inflammatory activities of BHGZD, especially in pyroptosis, which may be attributed to the activation of TLR4–mediated NLRP3 inflammasome signaling.

Mangiferin Inhibits Apoptosis in Doxorubicin-Induced Vascular Endothelial Cells via the Nrf2 Signaling Pathway.

Doxorubicin increases endothelial permeability, hence increasing cardiomyocytes’ exposure to doxorubicin (DOX) and exposing myocytes to more immediate damage. Reactive oxygen species are major effector molecules of doxorubicin’s activity. Mangiferin (MGN) is a xanthone derivative that consists of C-glucosylxanthone with additional antioxidant properties. This particular study assessed the effects of MGN on DOX-induced cytotoxicity in human umbilical vein endothelial cells’ (HUVECs’) signaling networks. Mechanistically, MGN dramatically elevated Nrf2 expression at both the messenger RNA and protein levels through the upregulation of the PI3K/AKT pathway, leading to an increase in Nrf2-downstream genes. Cell apoptosis was assessed with a caspase-3 activity assay, transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) staining was performed to assess DNA fragmentation, and protein expression was determined by Western blot analysis. DOX markedly increased the generation of reactive oxygen species, PARP, caspase-3, and TUNEL-positive cell numbers, but reduced the expression of Bcl-2 and antioxidants’ intracellular concentrations. These were effectively antagonized with MGN (20 μM), which led to HUVECs being protected against DOX-induced apoptosis, partly through the PI3K/AKT-mediated NRF2/HO-1 signaling pathway, which could theoretically protect the vessels from severe DOX toxicity.

Activation of \u03b2-catenin signaling in aggrecan-expressing cells in temporomandibular joint causes osteoarthritis-like defects

β-Catenin plays a critical role in cartilage formation and development. To further understand the role of β-catenin in osteoarthritis (OA) development in temporomandibular joint (TMJ), we have generated β-catenin conditional activation mice (β-cat(ex3)Agc1CreER) by breeding Agc1-CreER mice with β-cateninflox(ex3)/+ mice. Results of histologic analysis showed the progressive TMJ defects in 3- and 6-month-old β-cat(ex3)Agc1CreER mice (tamoxifen induction was performed at 2 weeks of age), including decreased chondrocyte numbers in the superficial layer associated with less Alcian blue staining, increased numbers of hypertrophic chondrocytes in deep layers, and rough articular surface. Compared to the TMJ phenotype of β-cat(ex3)Col2CreER mice, β-cat(ex3)Agc1CreER mice showed much severe morphological defects in the superficial layer of TMJ. This may reflect that Agc1-CreER mice could efficiently target cells in the superficial layer of TMJ. Results of immunostaining showed significantly increased expression of MMP13, Col-X, Adamts4, and Adamts5 in TMJ of β-cat(ex3)Agc1CreER mice. Results of proliferating cell nuclear antigen (PCNA), Ki67, and terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL) staining further demonstrated that cell proliferation was decreased and cell apoptosis was increased in condylar cartilage of β-cat(ex3)Agc1CreER mice. Our findings indicate that abnormal upregulation of β-catenin in TMJ leads to defects assembling to OA-like phenotype, further demonstrating that β-catenin plays a critical role in TMJ pathogenesis.

Saffron supplementation ameliorates oxidative damage to sperm DNA following a 16-week low-to-intensive cycling training in male road cyclists

The effects of the natural food saffron (90 mg/d) on exercise induced sperm DNA damage, antioxidative and peroxidative biomarkers and seminological profile in male road cyclists were evaluated. Twenty-four healthy nonprofessional cyclists (aged 17–26 years) were randomly assigned to exercise + Saffron (EX + SAF, n = 12) and exercise (EX, n = 12) groups for an experimental period of 16 weeks. After the intervention, the number of terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL)-positive sperm cells and peroxidative biomarkers increased, while antioxidative biomarkers and seminological profile decreased in the EX group. These changes were significantly attenuated in the EX + SAF group. Moreover, for both groups the observed changes in peroxidative and antioxidative biomarkers could be correlated positively and negatively, respectively, with sperm DNA fragmentation. Saffron, rich in carotenoids, flavonoids and vitamins, is therefore a potentially potent functional food for preventing exercise-induced sperm DNA damage, at least in part, through optimizing oxidant/antioxidant equilibrium and attenuation of oxidative stress.

Adenovirus-delivered wwox inhibited lung cancer growth in vivo in a mouse model.

Lung cancer is the most prevalent and deadly malignancy worldwide. This study investigated the possibility of inhibiting lung cancer in vivo with adenovirus-delivered WW domain-containing oxidoreductase (wwox). The lung cancer model was established by inoculating A549 lung cancer cells into the pleural space of nude mice. The control or wwox adenovirus was injected into the pleural space 7 days after cell inoculation and 14 days after first injection. The tumor number and burdens were measured 2 weeks after second virus injection. The carcinoembryonic antigen (CEA) and alpha-feto protein (AFP) levels in pleural effusion were analyzed by enzyme-linked immunosorbent assay. Apoptosis, proliferation and angiogenesis of tumor cells were assessed by terminal deoxinucleotidyl transferase-mediated dUTP-fluorescein nick end labeling assay, proliferating cell nuclear antigen (PCNA) and CD31 staining, respectively. Ectopic wwox significantly reduced both the number and size of lung tumors accompanied by substantially lower CEA and AFP levels in pleural effusion. The expression levels of Bcl2, Bcl-xL, vascular endothelial growth factor, PCNA-positive and CD31-positive cells in the tumors were significantly decreased, whereas levels of p21 and p73 and apoptotic cells markedly increased in mice receiving the wwox virus. These data demonstrated that wwox delivered by adenovirus was able to inhibit the growth of lung cancer in vivo, indicating the potential of using wwox as a gene therapy agent for lung cancer.

Immunoliposome co-delivery of bufalin and anti-CD40 antibody adjuvant induces synergetic therapeutic efficacy against melanoma.

Liposomes constitute one of the most popular nanocarriers for improving the delivery and efficacy of agents in cancer patients. The purpose of this study was to design and evaluate immunoliposome co-delivery of bufalin and anti-CD40 to induce synergetic therapeutic efficacy while eliminating systemic side effects. Bufalin liposomes (BFL) conjugated with anti-CD40 antibody (anti-CD40-BFL) showed enhanced cytotoxicity compared with bufalin alone. In a mouse B16 melanoma model, intravenous injection of anti-CD40-BFL achieved smaller tumor volume than did treatment with BFL (average: 117 mm3 versus 270 mm3, respectively); the enhanced therapeutic efficacy through a caspase-dependent pathway induced apoptosis, which was confirmed using terminal deoxynucleotidyl transferase-mediated dUTP-Fluorescein nick end labeling and Western blot assay. Meanwhile, anti-CD40-BFL elicited unapparent body-weight changes and a significant reduction in serum levels of tumor necrosis factor-α, interleukin-1β, interleukin-6, interferon-γ, and hepatic enzyme alanine transaminase, suggesting minimized systemic side effects. This may be attributed to the mechanism by which liposomes are retained within the tumor site for an extended period of time, which is supported by the following biodistribution and flow cytometric analyses. Taken together, the results demonstrated a highly promising strategy for liposomal vehicle transport of anti-CD40 plus bufalin that can be used to enhance antitumor effects via synergetic systemic immunity while blocking systemic toxicity.

Lipopolysaccharide-induced cross-tolerance against renal ischemia–reperfusion injury is mediated by hypoxia-inducible factor-2α-regulated nitric oxide production

Although the protective effect of lipopolysaccharide (LPS) pretreatment on renal ischemia/reperfusion injury is known, a link to hypoxia-inducible factors (HIFs) has not been established. Here we show that LPS treatment led to HIF-2α accumulation in mouse kidneys and endothelial cells, a result of nuclear factor-κB activation. Inactivation of HIF-2α, rather than HIF-1α, completely negated LPS-mediated protection against renal ischemia/reperfusion injury. LPS-stimulated renoprotection was related to inducible/endothelial nitric oxide synthase (iNOS/eNOS) expression, increased production of nitric oxide, and enhanced postischemic microcirculatory recovery. All these effects were lost in HIF-2α knockout mice. Preischemic administration of a nitric oxide donor, rather than erythropoietin, restored the lost preconditioning effect of LPS in HIF-2α knockout mice. In vitro and in vivo studies demonstrated that HIF-2α in endothelial cells, rather than myeloid cells or hepatocytes, was responsible for the LPS-mediated effects. Thus, our results demonstrated that LPS preconditioning protected against renal ischemia/reperfusion injury by HIF-2α activation in endothelial cells that subsequently improved renal microvascular perfusion and reduced ischemic tubular damage.

Inhibition of dendritic cell apoptosis by Leishmania mexicana amastigotes

Macrophages (Mφ) and dendritic cells are the major target cell populations of the obligate intracellular parasite Leishmania. Inhibition of host cell apoptosis is a strategy employed by multiple pathogens to ensure their survival in the infected cell. Leishmania promastigotes have been shown to protect Mφ, neutrophils, and dendritic cells from both natural and induced apoptosis. Nevertheless, the effect of the infection with Leishmania amastigotes in the apoptosis of these cell populations has not been established, which results are very important since amastigotes persist in cells for many days and are responsible for sustaining infection in the host. As shown in this study, apoptosis of monocyte-derived dendritic cells (moDC) induced by treatment with camptothecin was downregulated by infection with L. mexicana amastigotes from 42.48 to 36.92% as detected by Annexin-V binding to phosphatidylserine. Also, the infection of moDC with L. mexicana amastigotes diminished the fragmentation of DNA as detected by terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay, and changes in cell morphology were analyzed by electron microscopy. The observed antiapoptotic effect was found to be associated with an 80% reduction in the presence of active caspase-3 in infected moDC. The capacity of L. mexicana amastigotes to delay apoptosis induction in the infected moDC may have implications for Leishmania pathogenesis by favoring the invasion of its host and the persistence of the parasite in the infected cells.

Differential expression of the P2X7 receptor in ovarian surface epithelium during the oestrous cycle in the mouse

Purinergic signalling has been proposed as an intraovarian regulatory mechanism. Of the receptors responsible for purinergic transmission, the P2X7 receptor is an ATP-gated cationic channel that displays a broad spectrum of cellular functions ranging from apoptosis to cell proliferation and tumourigenesis. In the present study, we investigated the functional expression of P2X7 receptors in ovarian surface epithelium (OSE). P2X7 protein was detected in the OSE layer of the mouse, both in situ and in primary cultures. In cultures, 2'(3')-O-(4-Benzoylbenzoyl)adenosine-5'-triphosphate (BzATP) activation of P2X7 receptors increased [Ca(2+)]i and induced apoptosis. The functionality of the P2X7 receptor was investigated in situ by intrabursal injection of BzATP on each day of the oestrous cycle and evaluation of apoptosis 24h using the terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end-labelling (TUNEL) assay. Maximum effects of BzATP were observed during pro-oestrus, with the effects being blocked by A438079, a specific P2X7 receptor antagonist. Immunofluorescence staining for P2X7 protein revealed more robust expression during pro-oestrus and in OSE regions behind the antral follicles, strongly supporting the notion that the differences in apoptosis can be explained by increased receptor expression, which is regulated during the oestrous cycle. Finally, P2X7 receptor expression was detected in the OSE layer of human ovaries, with receptor expression maintained in human ovaries diagnosed with cancer, as well as in the human ovarian carcinoma SKOV3 cell line.

Effects of PEG-liposomal oxaliplatin on apoptosis, and expression of Cyclin A and Cyclin D1 in colorectal cancer cells

Oxaliplatin is one of the agents used against colorectal cancer. Using PEG-liposome encapsulated oxaliplatin may enhance the accumulation of drugs in tumor cells, inducing apoptosis. However, the mechanism of action of PEG-liposome encapsulated oxaliplatin remains unclear. SW480 human colorectal cancer cells were treated with empty PEG-liposomes, free oxaliplatin or PEG-liposomal oxaliplatin. Cell cycle and apoptosis were assessed using fluorescence confocal microscopy and terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick-end-labeling (TUNEL). Western blotting was used to analyze the expression of pro-apoptotic, anti-apoptotic and cyclin proteins. We found that PEG-liposomal oxaliplatin induced a stronger apoptotic response than empty PEG-liposomes or free oxaliplatin. Moreover, expression of Cyclin D1 increased, whereas expression of Cyclin A decreased after treatment with PEG-liposomal oxaliplatin. Furthermore, the cell cycle was arrested in the G1 phase. The results presented here indicate that PEG-liposome entrapment of oxaliplatin enhances the anticancer potency of the chemotherapeutic agent. The effect of PEG-liposomal oxaliplatin on apoptosis of SW480 human colorectal cancer cells may be through regulation of expression of Cyclin A or Cyclin D1, as well as pro-apoptotic and anti-apoptotic proteins.

Testing sperm DNA damage by tunel assay in specific cases of male infertility

OBJECTIVE: The TUNEL (terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling) assay is a direct assay measuring single and double strand DNA breaks. Our objective was to standardize the TUNEL assay by: 1) establishing inter- intra observer variability, inter assay variability, cutoff value, sensitivity, specificity of the assay and 2) study the distribution of the DNA damage in a population of infertile men referred to the clinical andrology laboratory.DESIGN: Sperm DNA damage by TUNEL and flow cytometry.MATERIALS AND METHODS: Seminal ejaculates from 43 controls and 114 infertile men referred to andrology lab were tested for sperm DNA damage by TUNEL assay using Apo-Direct kit. Negative and positive controls were included in each run. Flow cytometric analysis was performed and percentage of cells positive for TUNEL was calculated using the flow cytometer software. Receiver operating characteristic (ROC) curve, cutoff value, sensitivity and specificity was calculated.RESULTS: Inter- and intra-observer and interassay variability was <10%. Higher DNA damage was seen in patients (29.5% ± 18.7% compared with controls (11.9% ± 6.8%; P<0.001). The cutoff value of 19.3% showed 64.9% sensitivity and 100 % specificity. When the DNA damage was categorized into 0-10%, 10-20%, 20-30%, 30-40% and >40%, in the patients, 14.9% (29 of 194) had DNA damage between 0-10%; 22.7% (44 of 194) between 10-20%, 8.8% (17 of 194) between 20-30%, 17.5% (34 of 194) between 30-40% and 27.3% (53 of 194) had TUNEL values >40%.CONCLUSION: We report a detailed stepwise standardization of the TUNEL assay for clinical use. A cutoff of 19.5% with 100% specificity can differentiate infertile men with DNA damage from healthy men. High sensitivity and 100% specificity in our program makes it an ideal test. Each program must establish their threshold values. This test can be offered to infertile men who are idiopathic or who have oxidative stress and are considering assisted reproductive options. OBJECTIVE: The TUNEL (terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling) assay is a direct assay measuring single and double strand DNA breaks. Our objective was to standardize the TUNEL assay by: 1) establishing inter- intra observer variability, inter assay variability, cutoff value, sensitivity, specificity of the assay and 2) study the distribution of the DNA damage in a population of infertile men referred to the clinical andrology laboratory. DESIGN: Sperm DNA damage by TUNEL and flow cytometry. MATERIALS AND METHODS: Seminal ejaculates from 43 controls and 114 infertile men referred to andrology lab were tested for sperm DNA damage by TUNEL assay using Apo-Direct kit. Negative and positive controls were included in each run. Flow cytometric analysis was performed and percentage of cells positive for TUNEL was calculated using the flow cytometer software. Receiver operating characteristic (ROC) curve, cutoff value, sensitivity and specificity was calculated. RESULTS: Inter- and intra-observer and interassay variability was <10%. Higher DNA damage was seen in patients (29.5% ± 18.7% compared with controls (11.9% ± 6.8%; P<0.001). The cutoff value of 19.3% showed 64.9% sensitivity and 100 % specificity. When the DNA damage was categorized into 0-10%, 10-20%, 20-30%, 30-40% and >40%, in the patients, 14.9% (29 of 194) had DNA damage between 0-10%; 22.7% (44 of 194) between 10-20%, 8.8% (17 of 194) between 20-30%, 17.5% (34 of 194) between 30-40% and 27.3% (53 of 194) had TUNEL values >40%. CONCLUSION: We report a detailed stepwise standardization of the TUNEL assay for clinical use. A cutoff of 19.5% with 100% specificity can differentiate infertile men with DNA damage from healthy men. High sensitivity and 100% specificity in our program makes it an ideal test. Each program must establish their threshold values. This test can be offered to infertile men who are idiopathic or who have oxidative stress and are considering assisted reproductive options.

Small Variations in Crucial Steps of TUNEL Assay Coupled to Flow Cytometry Greatly Affect Measures of Sperm DNA Fragmentation

Techniques for assessing sperm DNA damage are numerous and various. There are 2 main types of assay: direct and indirect. The former directly detects the amount of sperm DNA damage, whereas the latter reveals the effects of an exogenous insult on sperm chromatin. In addition, even considering the same type of technique, different strategies to reveal or quantify sperm DNA damage, or both, are used. Finally, these techniques, except for sperm chromatin structure assay (SCSA), lack standardized protocols to which all users can adhere to minimize interlaboratory variations. In this study, we investigated the effects of some of the many ways the terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL) assay is performed when measuring sperm DNA fragmentation by flow cytometry. In addition, by using an established procedure, we determined the precision of the technique by calculating intra-assay coefficients of variation (CVs). We found that concentration of the fixative, the time of storage of fixed samples, the fluorochrome used to label DNA breaks, and the method used to analyze flow cytometric data all greatly affect the measures of sperm DNA fragmentation. In particular, we found that treatment with paraformaldehyde produced additional damage in most samples, suggesting that TUNEL also can be considered an indirect assay when performed in semen samples treated with such a fixative reagent. We also showed that 2 different methods used to analyze data yielded results that, albeit correlating, were different and associated differently to semen quality. On the contrary, the TUNEL assay, as measured here, showed low intraassay CVs, resulting in a quite precise technique when performed in established conditions.

Substance P accelerates intestinal tissue regeneration after γ‐irradiation–induced damage

Radiation therapy causes varying degrees of damage to biological systems. Many groups are investigating the mechanism underlying radiation-induced cellular damage but there are limited therapeutic solutions for affected patients. Recent studies show that substance P (SP) participates in cell proliferation. In the present study, we characterized the mechanism underlying SP-induced cellular signaling in radiation-induced damage of the intestine. Exposure of Caco-2 cells to SP increases cell proliferation and Erk phosphorylation in a time- and dose-dependent manner. The proliferation of cells exposed to gamma-irradiation is also stimulated by exposure to SP, a phenomenon that may result from inhibition of apoptosis because SP activates Akt and inhibits the cleavage of caspase-3. The effect of SP on cell proliferation and protection was confirmed by investigations in mice. Proliferating cell nuclear antigen staining shows that cell proliferation in radiation-damaged mouse intestine increases significantly upon exposure to SP. Furthermore, terminal deoxynucleotidyl transferase-mediated dUTP-fluorescein nick end labeling assay reveals fewer cells stained in SP-treated mice compared with untreated controls. These findings show the potential for SP-induced acceleration of intestinal wound healing and reveal that the mechanism underlying this process involves activation of Erk and Akt and inhibition of caspase-3 cleavage.

Characterization of a Bcl-XL-Interacting Protein FKBP8 and Its Splice Variant in Human RPE Cells

The immunophilin protein FKBP8 interacts with Bcl2/Bcl-XL and is essential for mouse eye development. The purpose of this study was to define the expression of the FKBP8 gene in cultured human RPE cells and explore its involvement in the control of apoptosis. Rapid amplification of cDNA ends (RACE) was performed on RNA isolated from human RPE cells. The existence of FKBP8 and a splice variant was confirmed by RT-PCR. The interaction between Bcl-XL and FKBP8 was measured by coimmunoprecipitation. ARPE-19 cells stably overexpressing FKBP8 and its splice variant were established. Their responses to thapsigargin and t-butyl hydroperoxide-induced cell death were measured by flow cytometry. Apoptosis was determined by terminal deoxyribonucleotidyl transferase-mediated fluorescein-dUTP nick-end labeling (TUNEL) assay. The activities of the nuclear factor of activated T cells (NFAT) were measured by reporter assay after transient transfection. RACE and RT-PCR identified a splice variant of FKBP8 lacking exons 3, 4, and 5 in human RPE cells. Both the full-length and the short form of FKBP8 proteins showed mitochondrial distribution and directly interacted with Bcl-XL. Overexpression of FKBP8 caused increased sensitivity to apoptosis induced by thapsigargin. The transcriptional activity of NFAT was not affected by FKBP8. FKBP8 and its novel splice variant are Bcl-XL-interacting proteins and regulate the apoptotic signaling pathways in the RPE.

Nuclear staining identifies two populations of human sperm with different DNA fragmentation extent and relationship with semen parameters

Sperm DNA fragmentation is a possible predictive parameter for male fertility status. The occurrence of M540 bodies in semen of subfertile subjects affects flow cytometric investigations in sperm. We set up a new method to evaluate DNA fragmentation excluding M540 bodies. DNA fragmentation was evaluated by flow cytometry in semen of 75 subjects both by terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling (TUNEL, traditional method) and by double staining with TUNEL and propidium iodide (PI, new method). The use of the new method revealed that TUNEL underestimates sperm DNA fragmentation in flow cytometry and showed two sperm populations stained with low (PI(dim)) and high (PI(br)) avidity for PI. The PI(dim) population is entirely composed of DNA fragmented sperm and its incidence shows highly significant negative correlations with morphology, motility, sperm count and concentration (respectively, r = -0.51, -0.52, -0.46 and -0.32, n = 75). DNA fragmentation in the PI(br) sperm population is independent from semen quality. The correlations between sperm DNA breakage and semen quality previously reported are mainly driven by the occurrence of the PI(dim) population. DNA fragmented sperm in this population are more likely to have poorer morphology, reduced motility and thus a reduced chance to fertilize an oocyte than DNA damaged sperm in PI(br) population. Distinguishing between the two types of sperm DNA fragmentation appears to be important in clinical investigations.

Blood micromolar concentrations of kaempferol afford protection against ischemia/reperfusion-induced damage in rat brain

The slow time course of neurodegeneration after brain ischemia/reperfusion opened a realistic time window for the application of protective therapies to prevent spreading of brain damage. In this work, we studied the ability of micromolar concentrations of this flavonoid in the blood to protect against brain damage induced by transient focal cerebral ischemia in rats. Transient focal cerebral ischemia was induced by middle cerebral artery occlusion in adult rats and brain damage has been monitored by 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (H-E) staining, 'in situ' terminal deoxyribonucleotidyl transferase-mediated dUTP-fluorescein nick end labeling (TUNEL), 'in situ' metalloproteinase activity using DQ-gelatin and loss of anti-laminin staining. Intravenous injections of kaempferol, at a dose of 10-15 mumol/L of blood 30 min before the induction of a 60 min ischemia-episode and just after reperfusion, led to >90% and 70-80% (TTC, H-E, TUNEL) decrease of brain damage in the temporal-frontal areas of neocortex and striatum, respectively, but only 40-50% decrease of brain damage was observed in the hippocampus and vicinal caudal areas of the striatum. This treatment with kaempferol also produced a similar reduction of metalloproteinase activation and loss of anti-laminin staining in cortical and striatum infarct areas. Kaempferol treatment efficiently protected against nitrosative-oxidative stress after ischemia/reperfusion, as shown by nearly complete protection against the increase of protein nitrotyrosines, and also afforded strong protection against the increase of apoptotic cell death (TUNEL) and biochemical markers of apoptosis, such as caspase-9 activity and poly-(ADP-ribose) polymerase degradation. On these grounds, a potential new therapeutic role of kaempferol to acute treatment of ischemic stroke is suggested.

The effect of melatonin on endotoxemia-induced intestinal apoptosis and oxidative stress in infant rats

To evaluate the effect of melatonin on the intestinal apoptosis along with oxidative damage in endotoxemic infant rats. Prospective animal study in a university-based experimental research laboratory. Wistar albino 7-day-old rat pups (n=21). The animals were randomized into three experimental groups: (1) controls; (2) endotoxemia; (3) endotoxemia treated with melatonin (10mg/kg). Endotoxemia was induced in rats by intraperitoneal injection of lipopolysaccharide (Escherichia coli serotype 0111:B4; 3 mg/kg). Four hours after LPS injection, the antioxidant enzyme activities, including superoxide dismutase (SOD), glutathione peroxidase (GPx), and thiobarbituric acid reactive substance (TBARS) levels as an indicator of lipid peroxidation, were determined. Intestinal apoptosis was assessed by hematoxylin-eosin staining and terminal deoxynucleotide transferase-mediated fluorescein-dUTP nick end labeling. The administration of melatonin into endotoxemic rats prevented the increase in the TBARS levels, and increased the activities of antioxidant enzymes and attenuated apoptotic cell death in both intestinal epithelium and lamina propria. Melatonin diminished the intestinal oxidative stress and apoptotic damage induced by endotoxemia in infant rats.

Erythropoietin attenuates lipopolysaccharide-induced splenic and thymic apoptosis in rats

Apoptosis of lymphoid tissues during sepsis is well documented and linked to the pathobiology of organ failure and death. In this study, we evaluated the effect of a single dose of recombinant erythropoietin (EPO) on thymic and splenic apoptosis in an endotoxic sepsis model. Young male Wistar rats were divided into 3 groups and administered intraperitoneally (IP) either normal saline; lipopolysaccharide (LPS) 10 mg/kg; or EPO (5000 U/kg) 30 min before lipopolysaccharide. Six hours following LPS administration animals were sacrificed. Apoptosis was assessed by hematoxylin-eosin staining, terminal deoxynucleotide transferase-mediated fluorescein-dUTP nick end labeling (TUNEL), and caspase-3 immunostaining. When compared with animals given LPS, animals pretreated with EPO displayed reduced splenic and thymic TUNEL positivity of 44+/-3 (p<0.05) and 143+/-4 (p<0.05) nuclei per high power field (hpf), respectively. Caspase-3 positivity was also significantly reduced in the spleen and thymus, with 31+/-4 (p<0.05) and 93+/-3 (p<0.05) positive stained nuclei per hpf, respectively. Serum nitrite levels were elevated in animals given lipopolysaccharide. Pretreatment with EPO attenuated the increase in nitrite levels; however, this did not reach statistical significance. We conclude that a single dose of recombinant erythropoietin can reduce thymic and splenic apoptosis associated with lipopolysaccharide administration.

Selection of Nonapoptotic Spermatozoa As a New Tool for Enhancing Assisted Reproduction Outcomes: An In Vitro Model1

Magnetic cell sorting (MACS) using annexin V-conjugated microbeads eliminates apoptotic spermatozoa based on the externalization of phosphatidylserine residues. The procedure delivers two sperm fractions: annexin V-negative (nonapoptotic) and annexin V-positive (apoptotic). Our aim was to determine whether the sperm fertilizing potential can be improved by selecting a nonapoptotic fraction using MACS. Semen samples (n = 35) were subjected to separation on a density gradient followed by MACS. Extent of apoptosis was assessed by measuring levels of activated caspase 3 using fluorescein-labeled inhibitors of caspase, alterations in mitochondrial membrane potential (MMP) using a lipophilic cationic dye, and DNA fragmentation using terminal deoxynucleotidyl transferase-mediated fluorescein-dUTP nick end labeling assay. The sperm fertilization potential was assessed using hamster oocyte penetration assay and hamster oocyte-intracytoplasmic sperm injection (ICSI). Annexin V-negative sperm displayed superior quality in terms of high motility, low caspase 3 activation, MMP integrity, and small extent of DNA fragmentation. Annexin V-negative sperm demonstrated higher oocyte penetration capacity but comparable sperm chromatin decondensation (SCD) following ICSI. Conversely, the annexin V-positive sperm presented with poor quality and fertilization potential. The oocyte penetration rate was negatively correlated with apoptotic marker expression, whereas SCD following ICSI was only associated with apoptosis on sperm-damaged membranes. We conclude that apoptosis appears to impact sperm-oocyte penetration rate; however, it does not seem to affect early stages of fertilization such as SCD in spermatozoa of healthy donors. The selection of nonapoptotic sperm by MACS may be used to enhance results of in vitro fertilization by increasing sperm-oocyte penetration.