Transferase-mediated Deoxyuridine Triphosphate Nick-end Labeling Research Articles

Biphasic Regulation of Apoptosis Following Gastric Irreversible Electroporation Using Tissue Immunohistochemistry of Activated Caspase-3 with TUNEL Method.

The regulation of apoptosis is the primary goal of ablation therapy. Irreversible electroporation (IRE) is a promising non-thermal tissue ablation-based therapy that induces apoptosis by manipulating electrical conditions. This study aimed to investigate IRE-induced gastric tissue apoptosis in response to changes in the electric field intensity, followed by the repair process. Among the 52 rats used in this study, 24 were used to explore apoptosis, and 28 were used to study regeneration. The apoptosis-to-necrosis ratio of the electrical field strength was evaluated using terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling and caspase-3 immunohistochemistry. The size of IRE-induced ulcers in the gastric tissue continuously increased with increasing electrical intensity (r2 = 0.830, p < 0.001). The level of apoptosis gradually decreased after peaking at 200 V (1000 V/cm). The size of the 400 V-ablated ulcers continued to decrease, and they were not visible by day 14. The proliferation and migration of epithelial cells with fibroblasts were observed on day 3 and augmented on day 7 post-ablation. This investigation demonstrated the biphasic activation of apoptosis with respect to the electrical field strength. Visually and histologically, IRE-induced gastric ulcers demonstrated complete tissue regeneration after two weeks.

Colitis-Induced Small Intestinal Hypomotility Is Dependent on Enteroendocrine Cell Loss in Mice

The abdominal discomfort experienced by patients with colitis may be attributable in part to the presence of small intestinal dysmotility, yet mechanisms linking colonic inflammation with small-bowel motility remain largely unexplored. We hypothesize that colitis results in small intestinal hypomotility owing to a loss of enteroendocrine cells (EECs) within the small intestine that can be rescued using serotonergic-modulating agents. Male C57BL/6J mice, as well as mice that overexpress (EECOVER) or lack (EECDEL) NeuroD1+ enteroendocrine cells, were exposed to dextran sulfate sodium (DSS) colitis (2.5% or 5% for 7 days) and small intestinal motility was assessed by 70-kilodalton fluorescein isothiocyanate-dextran fluorescence transit. EEC number and differentiation were evaluated by immunohistochemistry, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining, and quantitative reverse-transcriptase polymerase chain reaction. Mice were treated with the 5-hydroxytryptamine 4 agonist prucalopride (5 mg/kg orally, daily) to restore serotonin signaling. DSS-induced colitis was associated with a significant small-bowel hypomotility that developed in the absence of significant inflammation in the small intestine and was associated with a significant reduction in EEC density. EEC loss occurred in conjunction with alterations in the expression of key serotonin synthesis and transporter genes, including Tph1, Ddc, and Slc6a4. Importantly, mice overexpressing EECs revealed improved small intestinal motility, whereas mice lacking EECs had worse intestinal motility when exposed to DSS. Finally, treatment of DSS-exposed mice with the 5-hydroxytryptamine 4 agonist prucalopride restored small intestinal motility and attenuated colitis. Experimental DSS colitis induces significant small-bowel dysmotility in mice owing to enteroendocrine loss that can be reversed by genetic modulation of EEC or administering serotonin analogs, suggesting novel therapeutic approaches for patients with symptomatic colitis.

Protective effects of hesperidin in gastric damage caused by experimental ischemia-reperfusion injury model in rats.

This study evaluated the protective effect of hesperidin on injury induced by gastric ischemia-reperfusion. Fifty male Sprague Dawley rats (250-300g) were divided into five groups: control (C), sham (S), ischemia (I), ischemia-reperfusion (I/R) and hesperidin + ischemia-reperfusion (Hes + I/R). Hesperidin was injected intraperitoneally at the dose of 100mg/kg one hour before the experimental stomach ischemia-reperfusion. Celiac artery was ligated. After 45minutes ischemia and 60minutes reperfusion period, blood samples were obtained under anesthesia. Then, animals were sacrificed, stomach tissues were excised for biochemical, and histopathological analyses were performed. Malondialdehyde levels and superoxide dismutase, glutathione peroxidase activities and total antioxidant status (TAS), total oxidant status (TOS), protein, total thiol parameters were measured in plasma, and tissue homogenate samples. H + E, periodic acid-Schiff, hypoxia inducible factor, terminal deoxynucleotidyl transferase mediated deoxyuridine triphosphate nick end-labeling (TUNEL), and proliferating cell nuclear antigen (PCNA) for cell proliferation as immunohistochemical parameters were determined. Upon biochemical and histopathological assessment, hesperidin decreased stomach tissue changes in comparison with IR group. Ischemia-reperfusion injury led to a considerably increase in malondialdehyde, protein, and TOS levels (p < 0.001) in stomach tissue. Hesperidin treatment significantly decreased malondialdehyde, protein, and TOS levels (p < 0.001). Hesperidin increased superoxide dismutase, TAS, total thiol and glutathione peroxidase activities in comparison with IR group. Hesperidin reduced damage and also increased TUNEL and PCNA immunoreactivity in stomach tissue. Hesperidin was able to decrease I/R injury of the stomach tissue due to inhibition of lipid peroxidation and protein oxidation, duration of antioxidant, and free radical scavenger properties. Consequently, hesperidin can provide a beneficial therapeutic choice for preventing stomach tissue ischemia-reperfusion injury in clinical application.

NCX 470 Exerts Retinal Cell Protection and Enhances Ophthalmic Artery Blood Flow After Ischemia/Reperfusion Injury of Optic Nerve Head and Retina.

The purpose of this study was to assess the retinal protective activity and ocular hemodynamics after NCX 470 (0.1%) compared to bimatoprost administered as the US Food and Drug Administration (FDA)-approved drug (Lumigan - 0.01% ophthalmic solution, LUM) and at an equimolar dose (0.072%, BIM) to that released by NCX 470. Endothelin-1 (ET-1) induced ischemia/reperfusion injury model in rabbits was used. ET-1 was injected nearby the optic nerve head (ONH) twice/week for 6 weeks. Starting on week 3, the animals received vehicle (VEH), NCX 470, LUM, or BIM (30 µL/eye, twice daily, 6days/week) until the end of ET-1 treatment. Intraocular pressure (IOP), ophthalmic artery resistive index (OA-RI), and electroretinogram (ERG) data were collected prior to dosing and at different time points postdosing. Reduced glutathione, 8-Hydroxy 2-deoxyguanosine, and Caspase-3 were determined in the retina of treated eyes. DNA fragmentation was determined by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining. ET-1 increased IOP (VEHIOP_Baseline = 20.5 ± 0.8 and VEHIOP_Week6 = 24.8 ± 0.3mmHg) and OA-RI (VEHOA-RI_Baseline = 0.36 ± 0.02 and VEHOA-RI_Week6 = 0.55 ± 0.01) and reduced rod/cone responses over time. Oxidative stress, inflammation, and apoptotic markers increased in ET-1-treated eyes. NCX 470 prevented IOP (NCX 470IOP_Week6 = 18.1 ± 0.6mmHg) and OA-RI changes (NCX 470OA-RI_Week6 = 0.33 ± 0.01) and restored ERG amplitude leaving unaltered the respective latency; these effects were only partially demonstrated by LUM or BIM. Additionally, NCX 470 reduced oxidative stress, inflammation, and apoptosis in the retinas of treated eyes. BIM and LUM were numerically less effective on these parameters. NCX 470 repeated ocular dosing ameliorates ocular hemodynamics and retinal cell dysfunction caused by ischemia/reperfusion via nitric oxide- and bimatoprost-mediated mechanisms. If confirmed in clinical setting our data may open new therapeutic opportunities to reduce visual field loss in glaucoma.

Ononin Relieves the Thyroid Cancer Progression through Targeting the Caspase 3 and CD274 Expression Levels.

Thyroid cancer (TC) is the most common malignant tumor of endocrine system and head and neck. Ononin is an isoflavone component, which exhibited great antioxidant and anti-inflammatory activities. This study was conducted to explore the functions of ononin in the TC progression. The cell counting kit-8 (CCK8) assay was applied for the cell viability determination. The cell death and apoptosis rate were analyzed by terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining and flow cytometry. The quantitative real-time PCR (qRT-PCR) and Western blot assays were performed for the relative expressions determination. Lactate dehydrogenase (LDH) release assay was used to assess cytotoxicity. Ononin treatment prominently inhibited the cell viability and induced the cell apoptosis of the TC cells. Besides, caspase 3 (CASP3) was down-regulated and CD274 was up-regulated in TC. Ononin treatment prominently decreased the CD274 levels and increased the CASP3 levels in the TC cells. Additionally, ononin treatment dramatically enhanced the LDH release of the cytotoxicity of T cells. What is more, CASP3 overexpression or CD274 knockdown promoted the role of ononin in TC cells. Ononin treatment induced the cell death of the TC cells through regulating the CASP3 and CD274 expressions.

Hyperoside alleviates doxorubicin-induced myocardial cells apoptosis by inhibiting the apoptosis signal-regulating kinase 1/p38 pathway.

Cardiotoxicity is a side effect of the anthracycline broad-spectrum anti-tumor agent, doxorubicin (DOX). Hyperoside, a flavonoid glycoside extracted from many herbs, has anti-apoptotic and anticancer properties. However, its impact on the alleviation of DOX-induced apoptosis in cardiomyocytes remains elusive. The HL-1 cell line was treated with 100 µ M hyperoside for 1 h prior to treatment with 100 µ M hyperoside and 1 µ M DOX for 24 h. The cell counting kit-8 (CCK-8) assay was used to detect cell viability; DCFH-DA fluorescent probe was used to detect (reactive oxygen species) ROS; biochemical methods were used to detect the activity of glutathione (GSH), catalase (CAT), superoxide dismutase (SOD), malondialdehyde (MDA); the degree of apoptosis following DOX insult was assessed using immunofluorescence staining and terminal deoxynucleotidyl transferase mediated deoxy uridine triphosphate nick end labeling (TUNEL) assay; the change in protein expression of apoptosis signal-regulating kinase 1 (ASK1), p38, and apoptosis markers was determined using western blot. Hyperoside ameliorated DOX-induced oxidative stress in HL-1 cells, up-regulated GSH, SOD and CAT activity, reduced ROS production and inhibited MDA overproduction. Moreover, in addition to promoting HL-1 cell apoptosis, DOX administration also increased B-cell lymphoma (Bcl)-2-associated X-protein and cleaved caspase-3 protein levels and decreased Bcl-2 protein level. Hyperoside therapy, however, significantly reversed the impact of DOX on the cardiomyocytes. Mechanically, DOX treatment increased the phosphorylation of the ASK1/p38 axis whereas hyperoside treatment attenuated those changes. In a further step, hyperoside synergizes with DOX to kill MDA-MB-231 cells. Hyperoside protects HL-1 cells from DOX-induced cardiotoxicity by inhibiting the ASK1/p38 signaling pathway. Meanwhile, hyperoside maintained the cytotoxicity of DOX in MDA-MB-231 cells.

Levistolide A and periplogenin inhibit the growth of gastric cancer cells in vitro and in vivo

Aim: In the present study, the natural products levistolide A (LA) and periplogenin (PPG) were studied for their growth inhibitory effects on the development of gastric cancer cells in vitro and, more critically, in vivo, alone or in combination with the therapeutic medication 5-fluorouracil (5-FU). Methods: Methyl thiazolyl tetrazolium (MTT), also known as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assays were used for the cell viability study. Apoptosis was detected by western blot to detect the cleavage of caspase substrate poly (ADP-ribose) polymerase (PARP) and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate (dUTP) nick-end labelling (TUNEL) assays. The nude mice bearing gastric cancer cells were used for the anti-cancer activity detection of LA and its combinational treatment effect with 5-FU. Results: The results in the present study shown that the two compounds were able to inhibit the viability of the cancer cells in a dose- and time-dependent manner by MTT method. They could trigger apoptosis when used alone, and more potently, in combination with 5-FU detected by TUNEL positivity and the cleavage of caspase substrate PARP. In nude mice bearing gastric cancer cells, injection (i.p.) of LA or PPG alone inhibited the growth of the cancer cells. The treatment using one of the compounds in combination with 5-FU inhibited the cancer cell growth at a higher level than the treatment by a compound alone. Conclusions: LA and PPG could inhibit the growth of the cancer cells, alone or in combination with 5-FU, in vitro and in vivo, suggesting that they are promising investigational drugs for therapeutic development.

Incomplete Caspase 3 Activation and Mitigation of Apoptosis in Hibernating Ground Squirrels, Spermophilus lateralis.

AbstractHibernating golden-mantled ground squirrels, Spermophilus [Callospermophilus] lateralis, tolerate proapoptotic conditions, such as low body temperature, anorexia, acidosis, and ischemia/reperfusion. Avoiding widespread apoptosis is critical for hibernator survival. Caspase 3, the key executioner of apoptosis, cleaves a majority of apoptotic targets. Under proapoptotic conditions, inactive procaspase 3 (32 kDa) is activated when cleaved into 17- and 12-kDa fragments (p32, p17, and p12, respectively). Caspase 3 activation results in extreme enzymatic activation. Activity increases >10,000-fold followed by apoptotic execution. Is widespread apoptosis occurring during the proapoptotic hibernation season? Western blots showed p17 increased ∼2-fold during hibernation, indicating caspase 3 activation. However, in vitro caspase 3 activity assays found no extreme increases in activity. Downstream caspase 3 targets ICAD (inhibitor of caspase-activated deoxyribonuclease) and PARP (poly (ADP-ribose) polymerase) did not experience elevated cleavage during hibernation, which is inconsistent with caspase 3 activation. TUNEL (terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling) assays from multiple tissues found only 0.001%-0.009% of cells were TUNEL positive during winter, indicating negligible apoptosis during hibernation. Typically, caspase 3 activation generates a strong commitment toward apoptosis. We found that despite a ∼2-fold increase in active caspase 3, hibernators experience no downstream caspase 3 activity or widespread apoptosis. A systems-level approach suggests an incomplete signaling cascade wherein some caspase 3 activation during hibernation does not necessarily lead to bona fide apoptosis.

Proliferative and apoptotic evaluations of renal preventive effects of coenzyme Q10 in radioiodine-131 induced renal damage

The aim of this study was to investigated anti-proliferative and anti-apoptotic effects of coenzyme Q10 (CoQ10) in the prevention of radioiodine-131 (RAI) (I131) induced kidney damage. A total of 24 Wistar albino rats were separated into equal three groups (n = 8/group): Group 1 (control): untreated group; Group 2 (RAI): 3 mCi/kg RAI oral route; Group 3 (RAI+CoQ10): 3 mCi/kg RAI oral route and intraperitoneally 30 mg/kg/day CoQ10. CoQ10 treatment was started two days before RAI administration and was continued five days once daily after RAI. Pathomorphological parameters of kidneys were measured using hematoxylin–eosin and Masson’s trichrome staining. Immunohistochemically; proliferating cell nuclear antigen (PCNA), caspase 8, caspase 9 and terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick end labeling (TUNEL) were used to determine proliferation and apoptosis. With the exception of the control group, varying degrees of inflammation, degeneration, necrosis, and interstitial/perivascular fibrosis were detected in the kidneys of all rats. This histopathological damage was found to be significantly less in CoQ10 group versus RAI group (P&amp;lt;0.05). The all immunohistochemical examinations demonstrated that administration of CoQ10 had reduced proliferation and apoptosis (P&amp;lt;0.05). The results of kidney histopathology and immunohistochemistry demonstrated that administration of CoQ10 had reduced inflammation, proliferation, and apoptosis. These findings show CoQ10 can play an important role in the radioprotection of kidneys against RAI-induced damage

Fluoxetine attenuates apoptosis in early brain injury after subarachnoid hemorrhage through Notch1/ASK1/p38 MAPK signaling pathway

ABSTRACT Subarachnoid hemorrhage (SAH) is a severe brain condition associated with a significantly high incidence and mortality. As a consequence of SAH, early brain injury (EBI) may contribute to poor SAH patient outcomes. Apoptosis is a signaling pathway contributing to post-SAH early brain injury and the diagnosis of the disease. Fluoxetine is a well-studied serotonin selective reuptake inhibitor (SSRI). However, its role in apoptosis has not been clearly understood. The present investigation assessed the effects of Fluoxetine in apoptosis and the potential Notch1/ASK1/p38 MAPK signaling pathway in EBI after SAH. Adult C57BL/6 J mice were subjected to SAH. Study mice (56) were randomly divided into 4 groups: the surgery without SAH (sham (n = 8), SAH+ vehicle; (SAH+V) (n = 16), surgery+ Fluoxetine (Fluox), (n = 16) and SAH+ Fluoxetine (n = 16). Various parameters were investigated 12, 24, 48, and 72 h after induction of SAH. Western blot analysis, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining, Immunohistochemistry (IHC), and flow cytometry were carried out in every experimental group. According to the findings, the SAH downregulated NOTCH1 signaling pathway, Jlk6 inhibited Notch1, Notch1 inactivation increased apoptotic protein expression and suppressed Bax, and cytochrome C. Fluoxetine reversed the effects of notch1 inhibition in SAH. The Neuroprotective Fluoxetine effects involved suppression of apoptosis post-SAH. In summary, early Fluoxetine treatment significantly attenuates apoptosis and the expression of apoptosis-related proteins after 72 h post-SAH. Fluoxetine may ameliorate early brain injury after subarachnoid hemorrhage through anti-apoptotic effects and Notch1/ASK1/p38 MAPK signaling pathway.

Safety Evaluation of Photoacoustic Tomography System for Intraocular Tumors.

PurposePhotoacoustic tomography (PAT) has demonstrated the ability to characterize molecular components and architectural heterogeneities of intraocular tumors in enucleated human globes and in animals in vivo. Although laser safety levels have been established for illumination through the cornea, the safety limit for PAT illumination through the sclera has not been investigated. The purpose of this study is to examine if the energy level used in intraocular PAT results in ocular damage.MethodsRabbit eyes were exposed to pulsed laser illumination at 20 mJ/cm2 at the scleral surface. Eyes were examined at 1, 7, and 28 days after the laser exposure. Examination procedures included white light and fluorescence fundus imaging, optical coherence tomography (OCT), electroretinography (ERG), and histology with hematoxylin and eosin (H&E) staining as well as terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL staining).ResultsFundus imaging and OCT of rabbit eyes at 1, 7, and 28 days following exposure of the laser illumination of the PAT system did not reveal any damage to the retinal structures. ERG showed no significant difference between the experimental and control eyes. Similarly, H&E histology did not show abnormalities in either the scleral tissue where the laser illumination was delivered or in the retinal layers. No sign of apoptosis in the layers of the retina, choroid, or optic nerve was found on TUNEL staining.ConclusionsSimilar to the application of PAT to other organs, the proposed laser illumination energy level at 20 mJ/cm2 does not impose detectable harm to the ocular tissue.Translational RelevanceThis study addresses illumination safety issues for PAT.

Irinotecan (CPT-11) Treatment Induces Mild Gonadotoxicity.

Background:Gonadal toxicity following chemotherapy is an important issue among the population of young cancer survivors. The inhibitor of DNA topoisomerase I, irinotecan (CPT-11), is widely used for several cancer types. However, little is known about the effect of irinotecan on the fertility of both genders. Thus, the aim of the present study was to evaluate irinotecan gonadotoxicity, using a mouse model.MethodsMature male and female mice were injected intraperitoneally with either saline (), irinotecan (100 mg/kg) or cyclophosphamide (100 mg/kg); and sacrificed one week or three months later for an acute or long-term toxicity assessment, respectively. We used thorough and advanced fertility assessment by already established methods: Gonadal and epididymal weights, as well as sperm count and sperm motility were determined; serum anti-Müllerian hormone (AMH) was measured by ELISA. Immunohistochemistry (Ki-67), immunofluorescence (PCNA, CD34), terminal transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) and computerized analysis were performed to examine gonadal proliferation, apoptosis and vascularization. qPCR was used to assess the amount of testicular spermatogonia (Id4 and Gafra1 mRNA) and ovarian primordial oocytes reserves (Sohlh2, Nobox and Figla mRNA).ResultsFemales: Irinotecan administration induced acute ovarian apoptosis and decreased vascularity, as well as a mild, statistically significant, long-term decrease in the number of growing follicles, ovarian weight, and ovarian reserve. Males: Irinotecan administration caused an acute testicular apoptosis and reduced testicular spermatogenesis, but had no effect on vascularity. Irinotecan induced long-term decrease of testicular weight, sperm count and testicular spermatogonia and caused elevated serum AMH.ConclusionOur findings imply a mild, though irreversible effect of irinotecan on mice gonads.

Sevoflurane attenuates hepatic ischemia reperfusion injury by the miR-122/Nrf2 pathway

BackgroundSevoflurane can protect organs from ischemia-reperfusion (IR) injury, but the mechanism is still unclear. MicroRNA-122 (miR-122) is a liver-specific microRNA (miRNA) and regulates liver function. Therefore, this study aims to elucidate the relationship between the protective effect of sevoflurane and miR-122 in liver IR injury.MethodsWistar rats were divided into the following groups: sham, IR, IR + sevoflurane, IR + miR-122 antagomir, and IR + miR-122 antagomir + sevoflurane. Hematoxylin and eosin (H&E) staining and Suzuki score were used to evaluate the pathological damage of the liver. The levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH), tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, and IL-10 in the serum and the levels of malondialdehyde (MDA), superoxide dismutase (SOD), and nitric oxide (NO) in the liver homogenate supernatant were detected by using the corresponding kit. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) and flow cytometry was applied to evaluate the apoptosis of liver tissues. The expression of nuclear factor E2-related factor 2 (Nrf2), miR-122, p53, and HO-1 in liver tissue was evaluated by using immunohistochemistry, qRT-PCR, and western blot as needed.ResultsCompared to the IR group, the sevoflurane post-treatment or miR-122 antagomir groups showed improved liver injury, decreased Suzuki score, inhibited the levels of AST, ALT, LDH, MDA, NO, TNF-α, IL-1β, and IL-6, increased levels of SOD, IL-10, and inhibited hepatocyte apoptosis. Regarding the molecular mechanism, sevoflurane post-treatment fostered the expression of HO-1, promoted the transport of Nrf2 from cytoplasm to the nucleus, and decreased the expression of miR-122 and p53. The combined use of miR-122 antagomir and sevoflurane enhanced the protective effect of miR-122 antagomir in liver injury in IR rats.ConclusionsSevoflurane protected the liver from IR damage by regulating the miR-122/Nrf2/HO-1 pathway.

Pretreatment with gonadotropin-releasing hormone antagonist protects against chemotherapy-induced testicular damage 'in mice.

Background:Testicular toxicity following chemotherapy is of increasing importance with the continuous improvement of survival rates. Gonadotropin-releasing hormone (GnRH) was suggested to protect testis against such toxicity; however, its suppressive quality and mechanism of action are still unclear. We examined whether and how pretreatment with GnRH antagonist protects against the testicular damage caused by chemotherapy.Methods:Mature male mice were injected subcutaneously eight times in 2-day intervals with either saline or GnRH antagonist (Cetrotide; 1 g/mg), followed by an intraperitoneal injection with either saline or cyclophosphamide (CTX;100 mg/kg BW) and sacrificed 2 weeks or 3 months later. Testicular weight, epididymis weight, epididymal sperm count and sperm motility were measured. Serum anti-Müllerian hormone (AMH) was measured by enzyme-linked immunosorbent assay. Immunohistochemistry (Ki-67), immunofluorescence (PCNA, CD34), terminal transferase-mediated deoxyuridine 5-triphosphate nick-end labeling (TUNEL) and computerized analysis were performed to examine testicular proliferation, apoptosis and vascularization. Quantitative real-time PCR was used to assess the amount of spermatogonial reserve (Id4 and Gfra1 mRNAs).Results:Pretreatment with GnRH antagonist transiently reduced testicular weight, epididymal weight, germinal proliferation and sperm count; it also abolished the permanent long-term effect of CTX on these parameters and prevented cyclophosphamide-induced testicular toxicity characterized by apoptosis and serum AMH increase and irreversible loss of spermatogonial reserve.Conclusions:Our findings imply that pretreatment with GnRH antagonist temporarily reduces spermatogenesis and may be used as pretreatment for reducing chemotherapeutic testicular toxicity.

Effect of Ginsenoside Rb2 on a Myocardial Cell Model of Coronary Heart Disease through Nrf2/HO-1 Signaling Pathway.

The ginsenoside Rbs are the primary active compounds of Panax ginseng and ginsenoside Rb2 is a renowned component among the Rbs. This study aimed to investigate the potential effects of ginsenoside Rb2 on coronary heart disease (CHD). H9c2 cells were exposed to H2O2 to establish CHD model in vitro. Gene expression was determined by quantitative realtime PCR (qPCR) and Western blot. Cellular functions were detected by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), flow cytometry, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assays. We found that Ginsenoside Rb2 promoted cell proliferation while suppressed oxidative stress and apoptosis of H9c2 cells induced by H2O2 exposure. Mechanistically, Ginsenodise Rb2 involves in the regulation of nuclear factor, erythroid 2 like 2 (Nrf2)/heme oxygenase (HO)-1 signaling pathway. Inactivation of Nrf2/HO-1 signaling pathway reversed the effects of ginsenoside Rb2 on H9c2 cells. Taken together, ginsenoside Rb2 exhibited a cardioprotective effect in vitro. The underlying mechanism of ginsenoside Rb2 in H9c2 cells could be standardized to Nrf2/HO-1 signaling pathway, inhibiting cell apoptosis and regaining cell proliferation. The present study has proposed a novel mechanism of ginsenoside Rb2 in the cardioprotective effect.

Redundant Functions of ERK1 and ERK2 Maintain Mouse Liver Homeostasis Through Down-Regulation of Bile Acid Synthesis.

Activation of extracellular signal–regulated kinase (ERK) 1/2 promotes hepatocyte proliferation in response to growth stimuli, but whether constitutive hepatocyte ERK1/2 signaling functions in liver physiology is unknown. To examine the role of ERK1/2 in hepatic homeostasis, the effects of a knockout of Erk1 and/or Erk2 in mouse liver were examined. The livers of mice with a global Erk1 knockout or a tamoxifen‐inducible, hepatocyte‐specific Erk2 knockout were normal. In contrast, Erk1/2 double‐knockout mice developed hepatomegaly and hepatitis by serum transaminases, histology, terminal deoxynucleotide transferase‐mediated deoxyuridine triphosphate nick end‐labeling, and assays of hepatic inflammation. Liver injury was associated with biochemical evidence of cholestasis with increased serum and hepatic bile acids and led to hepatic fibrosis and mortality. RNA sequencing and polymerase chain reaction analysis of double‐knockout mouse livers revealed that the rate‐limiting bile acid synthesis gene Cyp7a1 (cholesterol 7α‐hydroxylase) was up‐regulated in concert with decreased expression of the transcriptional repressor short heterodimer partner. Elevated bile acids were the mechanism of liver injury, as bile acid reduction by SC‐435, an inhibitor of the ileal apical sodium–dependent bile acid transporter, prevented liver injury. Conclusion: Constitutive ERK1 and ERK2 signaling has a redundant but critical physiological function in the down‐regulation of hepatic bile acid synthesis to maintain normal liver homeostasis.

Pirfenidone attenuates acetaminophen-induced liver injury via suppressing c-Jun N-terminal kinase phosphorylation

Acetaminophen (APAP)-induced liver injury is the most frequent cause of acute liver failure in Western countries. Pirfenidone (PFD), an orally bioavailable pyridone derivative, is clinically used for idiopathic pulmonary fibrosis treatment and has antifibrotic, anti-inflammatory, and antioxidant effects. Here we examined the PFD effect on APAP-induced liver injury.In a murine model, APAP caused serum alanine aminotransferase elevation attenuated by PFD treatment. We performed terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) and vital propidium iodide (PI) stainings simultaneously. APAP induced TUNEL-positive/PI-negative necrosis around the central vein and subsequent TUNEL-negative/PI-positive oncotic necrosis with hemorrhage and caused the upregulation of hypercoagulation- and hypoxia-associated gene expressions. PFD treatment suppressed these findings. Western blotting revealed PFD suppressed APAP-induced c-Jun N-terminal kinase (JNK) phosphorylation despite no effect on JNK phosphatase expressions.In conclusion, simultaneous TUNEL and vital PI staining is useful for discriminating APAP-induced necrosis from typical oncotic necrosis. Our results indicated that PFD attenuated APAP-induced liver injury by suppressing TUNEL-positive necrosis by directly blocking JNK phosphorylation. PFD is promising as a new option to prevent APAP-induced liver injury.

Curcumin represses lipid accumulation through inhibiting ERK1/2-PPAR-γ signaling pathway and triggering apoptosis in porcine subcutaneous preadipocytes.

ObjectiveExcessive lipid accumulation in adipocytes results in prevalence of obesity and metabolic syndrome. Curcumin (CUR), a naturally phenolic active ingredient, has been shown to have lipid-lowering effects. However, its underlying mechanisms have remained largely unknown. Therefore, the study aims to determine the effect of CUR on cellular lipid accumulation in porcine subcutaneous preadipocytes (PSPA) and to clarify novel mechanisms.MethodsThe PSPA were cultured and treated with or without CUR. Both cell counting Kit-8 and lactate dehydrogenase release assays were used to examine cytotoxicity. Intracellular lipid contents were measured by oil-red-o staining extraction and triglyceride quantification. Apoptosis was determined by flow cytometry and the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-nick end labelling assay. Adipogenic and apoptosis genes were analyzed by quantitative polymerase chain reaction and Western blot.ResultsThe CUR dose-dependently reduced the proliferation and lipid accumulation of PSPA. Noncytotoxic doses of CUR (10 to 20 μM) significantly inhibited extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation and expression of adipogenic genes peroxisome proliferation-activity receptor-γ (PPAR-γ), CCAAT/enhancer binding protein-α, sterol regulatory element-binding protein-1c, adipocyte protein-2, glucose transporter-4 as well as key lipogenic enzymes fatty acid synthase and acetyl-CoA carboxylase, while ERK1/2 activation significantly reversed CUR-reduced lipid accumulation by increasing PPAR-γ. Furthermore, compared with differentiation induced media treated cells, higher dose of CUR (30 μM) significantly decreased the expression of AKT and B-cell lymphoma-2 (BCL-2), while increased the expression of BCL-2-associated X (BAX) and the BAX/BCL-2 expression ratio, suggesting triggered apoptosis by inactivating AKT and increasing BAX/BCL-2 ratio and Caspase-3 expression. Moreover, AKT activation significantly rescued CUR inhibiting lipid accumulation via repressing apoptosis.ConclusionThese results demonstrate that CUR is capable of suppressing differentiation by inhibiting ERK1/2-PPAR-γ signaling pathway and triggering apoptosis via decreasing AKT and subsequently increasing BAX/BCL-2 ratio and Caspase-3, suggesting that CUR provides an important method for the reduction of porcine body fat, as well as the prevention and treatment of human obesity.

Fraxetin suppresses reactive oxygen species-dependent autophagy by the PI3K/Akt pathway to inhibit isoflurane-induced neurotoxicity in hippocampal neuronal cells.

Isoflurane, a common volatile anesthetic, has been widely used to provide general anesthesia in operations. However, exposure to isoflurane may cause widespread neurotoxicity in the developing animal brain. Fraxetin, a natural coumarin derivative extracted from the bark of Fraxinus rhynchophylla, possesses versatile pharmacological properties including anti-oxidative, anti-inflammatory, and neuroprotective effects. However, the effect and action mechanism of fraxetin on neurotoxicity induced by isoflurane are unknown. Reactive oxygen species (ROS) generation, cell viability, lactate dehydrogenase (LDH) release, and apoptosis were estimated by 2',7'-dichlorofluorescin-diacetate (DCFH-DA) staining, MTT, LDH release, and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) staining assays, respectively. The protein levels of light chain 3 (LC3)-I, LC3-II, p62, protein kinase B (Akt), and phosphorylated Akt (p-Akt) were detected by western blot analysis. Isoflurane induced ROS, LDH release, apoptosis, and autophagy, but inhibited the viability in HT22 cells, which were overturned by fraxetin or ROS scavenger N-acetyl-L-cysteine. Fraxetin suppressed isoflurane-induced PI3K/Akt inactivation in HT22 cells. PI3K/Akt inactivation by LY294002 resisted the effects of fraxetin on isoflurane-induced autophagy and autophagy-modulated neurotoxicity in HT22 cells. In conclusion, fraxetin suppressed ROS-dependent autophagy by activating the PI3K/Akt pathway to inhibit isoflurane-induced neurotoxicity in hippocampal neuronal cells.

Clinical Guidelines for Sperm DNA Fragmentation (SDF) is Associated with Male Infertility from Reactive Oxygen Species (ROS)

Sperm DNA fragmentation is common in infertile male. Besides, sperm DNA integrity is essential for fertilization and healthy offspring development. Numerous genetic and environmental elements are associated with impacting sperm DNA integrity negatively. Such as lifestyle, ageing, industrial toxins, and infection. The mechanisms behind SDF are many, but apoptosis and reactive oxygen species are considered the main SDF mechanisms. The management of male infertility has led to the desire for more advanced SDF diagnostic tools to diagnose sperm DNA. Numerous sperm DNA damage assays such as terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labelling (TUNEL) assay and in situ nick translation (ISNT) are available to enhance SDF diagnosing and ultimately to better SDF management. Clinical SDF can lead to a low pregnancy rate, defects in embryo development and impaired offspring health. Moreover, SDF can impact the effectiveness of assisted reproductive technology through transfer genetics impartment to the embryo b in vitro fertilization or intracytoplasmic sperm injection. SDF can be mange through lifestyle changing, treating existing infection in the male reproductive tract and reactive oxygen species.