We question whether, in men with an abnormal rate of sperm DNA fragmentation, the magnetic-activated cell sorting (MACS) could select spermatozoa with lower rates of DNA fragmentation as well as spermatozoa with unbalanced chromosome content. Cryopreserved spermatozoa from six males were separated into nonapoptotic and apoptotic populations. We determined the percentages of spermatozoa with (i) externalization of phosphatidylserine (EPS) by annexin V-Fluorescein isothiocyanate (FITC) labeling, (ii) DNA fragmentation by TdT-mediated-dUTP nick-end labeling (TUNEL), and (iii) numerical abnormalities for chromosomes X, Y, 13, 18, and 21 by fluorescence in situ hybridization (FISH), on the whole ejaculate and selected spermatozoa in the same patient. Compared to the nonapoptotic fraction, the apoptotic fraction statistically showed a higher number of spermatozoa with EPS, with DNA fragmentation, and with numerical chromosomal abnormalities. Compared to the whole ejaculate, we found a significant decrease in the percentage of spermatozoa with EPS and decrease tendencies of the DNA fragmentation rate and the sum of disomy levels in the nonapoptotic fraction. Conversely, we observed statistically significant higher rates of these three parameters in the apoptotic fraction. MACS may help to select spermatozoa with lower rates of DNA fragmentation and unbalanced chromosome content in men with abnormal rates of sperm DNA fragmentation.
Annexin A5 ; Chromosome Aberrations ; DNA Fragmentation ; Humans ; In Situ Hybridization, Fluorescence ; In Situ Nick-End Labeling ; Male ; Pilot Projects ; Semen ; Spermatozoa

BACKGROUND AND OBJECTIVES: The feature of chronic kidney failure (CKF) is loss of kidney functions due to erosion of healthy tissue and fibrosis. Recent studies showed that Mesenchymal stem cells (MSCs) differentiated into tubular epithelial cells thus renal function and structures renewed. Furthermore, MSCs protect renal function in CKF. Therefore, we aimed to investigate whether human amnion-derived mesenchymal stem cells (hAMSCs) can repair fibrosis and determine the effects on proliferation and apoptosis mechanisms in chronic kidney failure. METHODS AND RESULTS: In this study, rat model of CKF was constituted by applying Aristolochic acid (AA). hAMSCs were isolated from term placenta amnion membrane and transplanted into tail vein of rats. At the end of 30 days and 60 days of recovery period, we examined expressions of PCNA, p57 and Parp-1 by western blotting. Immunoreactivity of PCNA, Ki67, IL-6 and Collagen type I were detected by immunohistochemistry. Besides, apoptosis was detected by TUNEL. Serum creatinine and urea were measured. Expressions of PCNA and Ki67 increased in hAMSC groups compared with AA group. Furthermore, expressions of PARP-1 apoptosis marker and p57 cell cycle inhibitory protein increased in AA group significantly according to control, hAMSC groups and sham groups. IL-6 proinflammatory cytokine increased in AA group significantly according to control, hAMSCs groups and sham groups. Expressions of Collagen type I protein reduced in hAMSCs groups compared to AA group. After hAMSC treatment, serum creatinine and urea levels significantly decreased compared to AA group. After injection of hAMSC to rats, Masson’s Trichrome and Sirius Red staining showed fibrosis reduction in kidney. CONCLUSIONS: According to our results hAMSCs can be ameliorate renal failure.
Amnion ; Animals ; Apoptosis ; Blotting, Western ; Cell Cycle ; Collagen Type I ; Creatinine ; Epithelial Cells ; Fibrosis ; Humans ; Immunohistochemistry ; In Situ Nick-End Labeling ; Interleukin-6 ; Kidney ; Kidney Failure, Chronic ; Membranes ; Mesenchymal Stromal Cells ; Models, Animal ; Placenta ; Proliferating Cell Nuclear Antigen ; Rats ; Renal Insufficiency ; Renal Insufficiency, Chronic ; Tail ; Urea ; Veins

The complement cascade is a central component of innate immunity which plays a critical role in brain inflammation. Complement C3a receptor (C3aR) is a key mediator of post-ischemic cerebral injury, and pharmacological antagonism of the C3a receptor is neuroprotective in stroke. Cerebral ischemia injures brain endothelial cells, causing blood brain barrier (BBB) disruption which further exacerbates ischemic neuronal injury. In this study, we used an in vitro model of ischemia (oxygen glucose deprivation; OGD) to investigate the protective effect of a C3aR antagonist (C3aRA, SB290157) on brain endothelial cells (bEnd.3). Following 24 hours of reperfusion, OGD-induced cell death was assessed by TUNEL and Caspase-3 staining. Western blot and immunocytochemistry were utilized to demonstrate that OGD upregulates inflammatory, oxidative stress and antioxidant markers (ICAM-1, Cox-2, Nox-2 and MnSOD) in endothelial cells and that C3aRA treatment significantly attenuate these markers. We also found that C3aRA administration restored the expression level of the tight junction protein occludin in endothelial cells following OGD. Interestingly, OGD/reperfusion injury increased the phosphorylation of ERK1/2 and C3aR inhibition significantly reduced the activation of ERK suggesting that endothelial C3aR may act via ERK signaling. Furthermore, exogenous C3a administration stimulates these same inflammatory mechanisms both with and without OGD, and C3aRA suppresses these C3a-mediated responses, supporting an antagonist role for C3aRA. Based on these results, we conclude that C3aRA administration attenuates inflammation, oxidative stress, ERK activation, and protects brain endothelial cells following experimental brain ischemia.
Blood-Brain Barrier ; Blotting, Western ; Brain Ischemia ; Brain ; Caspase 3 ; Cell Death ; Complement C3a ; Complement System Proteins ; Encephalitis ; Endothelial Cells ; Glucose ; Immunity, Innate ; Immunohistochemistry ; In Situ Nick-End Labeling ; In Vitro Techniques ; Inflammation ; Ischemia ; Neurons ; Occludin ; Oxidative Stress ; Phosphorylation ; Reperfusion ; Stroke ; Tight Junctions

Swertiamarin (STM) is an iridoid compound that is present in the Gentianaceae swertia genus. Here we investigated antiapoptotic effects of STM on carbon tetrachloride (CCl₄)-induced liver injury and its possible mechanisms. Adult male Sprague Dawley rats were randomly divided into a control group, an STM 200 mg/kg group, a CCl₄ group, a CCl₄+STM 100 mg/kg group, and a CCl₄+STM 200 mg/kg group. Rats in experimental groups were subcutaneously injected with 40% CCl₄ twice weekly for 8 weeks. STM (100 and 200 mg/kg per day) was orally given to experimental rats by gavage for 8 consecutive weeks. Hepatocyte apoptosis was determined by TUNEL assay and the expression levels of Bcl-2, Bax, and cleaved caspase-3 proteins were evaluated by western blot analysis. The expression of TGF-β1, collagen I, collagen III, CTGF and fibronectin mRNA were estimated by qRT-PCR. The results showed that STM significantly reduced the number of TUNEL-positive cells compared with the CCl₄ group. The levels of Bax and cleaved caspase-3 proteins, and TGF-β1, collagen I, collagen III, CTGF, and fibronectin mRNA were significantly reduced by STM compared with the CCl₄ group. In addition, STM markedly abrogated the repression of Bcl-2 by CCl₄. STM also attenuated the activation of the PI3K/Akt pathway in the liver. These results suggested that STM ameliorated CCl₄-induced hepatocyte apoptosis in rats.
Adult ; Animals ; Apoptosis* ; Blotting, Western ; Carbon Tetrachloride ; Carbon* ; Caspase 3 ; Collagen ; Fibronectins ; Gentianaceae ; Hepatocytes ; Humans ; In Situ Nick-End Labeling ; Liver ; Male ; Rats* ; Rats, Sprague-Dawley ; Repression, Psychology ; RNA, Messenger ; Swertia

BACKGROUND: Colorectal cancer is the third most common cancer worldwide and still lack of effective therapy so far. Petasin, a natural product found in plants of the genus Petasites, has been reported to possess anticancer activity. The present study aimed to investigate the anticolon cancer activity of petasin both in vitro and in vivo. The molecular mechanism of petasin was also further explored. METHODS: Caco-2, LoVo, SW-620, and HT-29 cell lines were used to detect the inhibitory effect of petasin on colon cancer proliferation. Cell viability was determined using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay. Cell apoptosis was analyzed by flow cytometry. Hoechst 33258 staining was used to visualize morphological changes. Cell migration was assessed using a wound-healing migration assay, and cell invasion was investigated using Transwell chambers. Western blotting assays were employed to evaluate the expression levels of proteins in the protein kinase B/mammalian target of rapamycin (Akt/mTOR) signaling pathway. Finally, in vivo activity of petasin was evaluated using the SW-620 subcutaneous tumor model established in Balb/c nude mice. Twelve rats were randomly divided into control group and 10 mg/kg petasin group. The tumor volume was calculated every 7 days for 28 days. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) assay was performed to assess the apoptotic effect of petasin. Differences between two groups were assessed by analysis of independent-sample t tests. RESULTS: Petasin significantly inhibited the proliferation of human colon carcinoma cell lines, induced apoptosis, and suppressed migration and invasion in SW-620 cells. Western blotting results showed that petasin decreased the phosphorylation of Akt (1.01 ± 0.16 vs. 0.74 ± 0.06, P = 0.042), mTOR (0.71 ± 0.12 vs. 0.32 ± 0.11, P = 0.013), and P70S6K (1.23 ± 0.21 vs. 0.85 ± 0.14, P = 0.008), elevated the expression of caspase-3 (0.41 ± 0.09 vs. 0.74 ± 0.12, P = 0.018) and caspase-9 (1.10 ± 0.27 vs. 1.98 ± 0.22, P = 0.009), decreased the Bcl-2 protein (2.75 ± 0.47 vs. 1.51 ± 0.36, P = 0.008), downregulated the expression of matrix metalloproteinase (MMP)-3 (1.51 ± 0.31 vs. 0.82 ± 0.11, P = 0.021) and MMP-9 (1.56 ± 0.32 vs. 0.94 ± 0.15, P = 0.039) in SW-620 cell. In vivo, 10 mg/kg petasin inhibited tumor growth in Balb/c nude mice (924.18 ± 101.23 vs. 577.67 ± 75.12 mm at day 28, P = 0.001) and induced apoptosis (3.6 ± 0.7% vs. 36.0 ± 4.9%, P = 0.001) in tumor tissues. CONCLUSIONS Petasin inhibits the proliferation of colon cancer SW-620 cells via inactivating the Akt/mTOR pathway. Our findings suggest petasin as a potential candidate for colon cancer therapy.
Animals ; Antineoplastic Agents ; therapeutic use ; Apoptosis ; drug effects ; Caco-2 Cells ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; HT29 Cells ; Humans ; In Situ Nick-End Labeling ; Matrix Metalloproteinase 3 ; metabolism ; Matrix Metalloproteinase 9 ; metabolism ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Phosphorylation ; drug effects ; Proto-Oncogene Proteins c-akt ; genetics ; metabolism ; Sesquiterpenes ; therapeutic use ; Signal Transduction ; drug effects ; TOR Serine-Threonine Kinases ; genetics ; metabolism

OBJECTIVE: The usual seminal profile has been customarily used for diagnosing male infertility based on an examination of semen samples. However, sperm DNA fragmentation has also been causally linked to reproductive failure, suggesting that it should be evaluated as part of male infertility assessments. To compare the ability of the five most widely utilized methodologies of measuring DNA fragmentation to predict male infertility and reactive oxygen species by Oxisperm kit assay. METHODS: In this case-control study, which received ethical committee approval, the participants were divided into fertile and infertile groups (50 patients in each group). RESULTS: The alkaline comet test showed the best ability to predict male infertility, followed by the terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, the sperm chromatin dispersion (SCD) test, and the sperm chromatin structure assay (SCSA), while the neutral comet test had no predictive power. For our patient population, the projected cut-off point for the DNA fragmentation index was 22.08% using the TUNEL assay, 19.90% using SCSA, 24.74% using the SCD test, 48.47% using the alkaline comet test, and 36.37% using the neutral comet test. Significant correlations were found between the results of the SCD test and those obtained using SCSA and TUNEL (r =0.70 and r =0.68, respectively; p<0.001), and a statistically significant correlation was also found between the results of SCSA and the TUNEL assay (r =0.77, p<0.001). Likewise, the results of the alkaline comet test showed significant correlations with those of the SCD, SCSA, and TUNEL tests (r =0.59, r =0.57, and r =0.72, respectively; p<0.001). CONCLUSION: The TUNEL assay, SCSA, SCD, and the alkaline comet test were effective for distinguishing between fertile and infertile patients, and the alkaline comet test was the best predictor of male infertility.
Case-Control Studies ; Chromatin ; DNA Fragmentation ; DNA Nucleotidylexotransferase ; DNA ; Humans ; In Situ Nick-End Labeling ; Infertility ; Infertility, Male ; Male ; Male ; Methods ; Reactive Oxygen Species ; Semen ; Sensitivity and Specificity ; Spermatozoa

Lack of standardized, reproducible protocols and reference values is among the challenges faced when using new or upgraded versions of instruments in reproductive laboratories and flow cytometry. Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay combined with flow cytometry routinely used for diagnostic measurement of sperm DNA fragmentation (SDF) is a unique example. Any change in the setting of the standard instrument, including upgrades of hardware or software, can lead to different results and may affect clinicians' decision for treatment. Therefore, we compared TUNEL results of SDF obtained from a standard (C6) flow cytometer with a newer version of the same instrument (C6 Plus) and examined the cutoff, sensitivity, and specificity without calibration (adjustment) and after adjustment. Identical sperm preparation and matched acquisition settings were used to examine the performance of two flow cytometers. The strength of agreement of the results between the two observers was also assessed. After adjustment of the settings, overall concordance became high and the two cytometers showed 100% positive and negative predictive value with 100% area under the curve. The overall correlation coefficient observed between C6 and C6 Plus was highly significant (P < 0.0001; r = 0.992; 95% confidence interval [CI]: 0.982-0.997). After adjustment, the two cytometers showed very high precision of 98% and accuracy of >99%. The interobserver agreement on C6 flow cytometer for the two observers was 0.801 ± 0.062 and 0.746 ± 0.044 for C6 Plus. We demonstrated a strong agreement between the samples tested on the two flow cytometers after calibration and established the robustness of both instruments.
Adult ; Calibration ; DNA Fragmentation ; Flow Cytometry/instrumentation* ; Humans ; In Situ Nick-End Labeling ; Male ; Observer Variation ; Reference Values ; Reproducibility of Results ; Semen Analysis/methods* ; Sensitivity and Specificity ; Spermatozoa/chemistry*

The retinal degeneration resulting from elevated intraocular pressure was evaluated through functional and morphological analyses, for better understanding of the pathophysiology of glaucoma. Ocular hypertension was induced via unilateral episcleral venous cauterization in rats. Experimental time was set at 1 and 3 days, and 1, 2, 4, and 8 weeks post-operation. Retinal function was analyzed using electroretinography. For morphological analysis, retinal tissues were processed for immunochemistry by using antibodies against the calcium-sensing receptor and calcium-binding proteins. Apoptosis was analyzed using the TUNEL method and electron microscopy. Amplitudes of a- and b-wave in scotopic and photopic responses were found to be reduced in all glaucomatous retinas. Photopic negative response for ganglion cell function significantly reduced from 1-day and more significantly reduced in 2-week glaucoma. Calcium-sensing receptor immunoreactivity in ganglion cells remarkably reduced at 8 weeks; conversely, protein amounts increased significantly. Calcium-binding proteins immunoreactivity in amacrine cells clearly reduced at 8 weeks, despite of uneven changes in protein amounts. Apoptosis appeared in both photoreceptors and ganglion cells in 8-week glaucomatous retina. Apoptotic feature of photoreceptors was typical, whereas that of ganglion cells was necrotic in nature. These findings suggest that elevated intraocular pressure affects the sensitivity of photoreceptors and retinal ganglion cells, and leads to apoptotic death. The calcium-sensing receptor may be a useful detector for alteration of extracellular calcium levels surrounding the ganglion cells.
Amacrine Cells ; Animals ; Antibodies ; Apoptosis ; Calcium ; Calcium-Binding Proteins ; Cautery ; Electroretinography ; Ganglion Cysts ; Glaucoma ; Immunochemistry ; In Situ Nick-End Labeling ; Intraocular Pressure ; Methods ; Microscopy, Electron ; Ocular Hypertension ; Rats ; Receptors, Calcium-Sensing ; Retina ; Retinal Degeneration ; Retinal Ganglion Cells ; Retinaldehyde

BACKGROUND/AIMS: Several lines of evidence from epidemiologic and laboratory studies have shown that the consumption of Artemisia or green tea extracts (MPGT) is inversely associated with the risk of alcohol-induced damage and other chronic diseases. Supported by previous studies showing that the combined extract of Artemisia and green tea, MPGT, exerted significantly either antioxidative or anti-inflammatory actions against Helicobacter pylori-associated gastric diseases, it was hypothesized that MPGT can offer protection against alcoholic gastritis. METHODS: Ethanol was administered to induce gastric damage in Wistar rats, which had been pretreated with various doses of MPGT, to measure the rescuing action of a MPGT pretreatment against ethanol-induced gastric damage. In addition, the molecular mechanisms for the preventive effects were examined. RESULTS: The MPGT pretreatment (100, 300, and 500 mg/kg) alleviated the ethanol-induced gastric damage, which was evidenced by the significant decrease in calcium-dependent phospholipase A2, MAPKs, and NF-κB levels compared to ethanol alone. Furthermore, the MPGT pretreatment preserved 15-prostaglandin dehydrogenase, whereas cyclooxygenase-2 was decreased significantly. All of these biochemical changes led to the significant alleviation of alcohol-associated gastric mucosal damage. Ethanol significantly increased the TUNEL positivity in the stomach, but MPGT decreased the apoptotic index significantly, which was associated with significantly lower pathological scores of ethanol-induced mucosal ulcerations. The significant protective changes observed alcoholic gastritis with MPGT were related to the increased expression of cytoprotective genes, such as heat-shock protein (HSP)27, HSP60, and PDGF. CONCLUSIONS: The efficient anti-inflammatory, anti-apoptotic, and regenerative actions of MPGT make it a potential nutrient phytoceutical to rescue the stomach from alcoholic gastritis.
Alcoholics* ; Artemisia* ; Chronic Disease ; Cyclooxygenase 2 ; Ethanol ; Gastritis* ; Heat-Shock Proteins* ; Helicobacter ; HSP27 Heat-Shock Proteins* ; Humans ; In Situ Nick-End Labeling ; Oxidoreductases ; Phospholipases A2 ; Rats, Wistar ; Stomach ; Stomach Diseases ; Tea* ; Ulcer

Wet wipes are being increasingly used because of their convenience. Particularly, oral wet wipes are useful for regular cleaning of a baby's mouth after birth. Therefore, the consumption of oral wet wipes has increased over the past few years and a variety of products are commercially available. However, product information on safety is not sufficiently provided and still raises doubts regarding adverse effects. To confirm the safety of wet wipes as an oral hygiene item and provide information for their use, we investigated the cytotoxicity of oral wet wipes and verified the underlying mechanism. The anti-bacterial effect of oral wet wipes was analyzed using the disk diffusion method. The cytotoxic effects of oral wet wipes were observed based on morphological changes using microscopy and determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay in gingival epithelial cells and gingival fibroblasts. Evaluation of apoptosis by oral wet wipes was explored using propidium iodide flow cytometric analysis and a terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) assay. Apoptosis-related molecules were also analyzed using western blotting. Five types of oral wet wipes were tested, and two products from Fisher-Price and Dr. Kennedy revealed strong cytotoxic effects on gingiva epithelial cells and gingiva fibroblasts, although they also showed intense anti-bacterial effects on oral bacteria. Cell cycle arrest in the G2/M phase and apoptosis were observed based on treatment of extracts from Fisher-Price and Dr. KENNEDY. Relatively high TUNEL levels, reduction of proliferating cell nuclear antigen and cyclin-dependent kinase 4 expression, and fragmentation of poly (ADP-ribose) polymerase were also elucidated. These results suggest that commercial oral wet wipes could exert cytotoxic influences on oral tissue, although there are anti-bacterial effects, and careful attention is required, especially for infants and toddlers.
Apoptosis ; Bacteria ; Blotting, Western ; Cell Cycle ; Cell Cycle Checkpoints ; Cell Survival ; Cyclin-Dependent Kinase 4 ; Deoxyuridine ; Diffusion ; DNA Nucleotidylexotransferase ; Epithelial Cells ; Fibroblasts ; Gingiva ; Humans ; In Situ Nick-End Labeling ; Infant ; Methods ; Microscopy ; Mouth ; Oral Hygiene ; Parturition ; Proliferating Cell Nuclear Antigen ; Propidium