Increase In TUNEL-positive Cells Research Articles

Apoptosis in Postmortal Tissues of Goat Spinal Cords and Survival of Resident Neural Progenitors.

Growing demand for therapeutic tissue repair recurrently focusses scientists' attention on critical assessment of postmortal collection of live cells, especially stem cells. Our study aimed to assess the survival of neuronal progenitors in postmortal spinal cord and their differentiation potential. Postmortal samples of spinal cords were obtained from human-sized animals (goats) at 6, 12, 24, 36, and 54 h after slaughter. Samples were studied by immunohistology, differentiation assay, Western blot and flow cytometry for the presence and location of GD2-positive neural progenitors and their susceptibility to cell death. TUNEL staining of the goat spinal cord samples over 6-54 h postmortem revealed no difference in the number of positive cells per cross-section. Many TUNEL-positive cells were located in the gray commissure around the central canal of the spinal cord; no increase in TUNEL-positive cells was recorded in either posterior or anterior horns of the gray matter where many GD2-positive neural progenitors can be found. The active caspase 3 amount as measured by Western blot at the same intervals was moderately increasing over time. Neuronal cells were enriched by magnetic separation with antibodies against CD24; among them, the GD2-positive neural progenitor subpopulation did not overlap with apoptotic cells having high pan-caspase activity. Apoptotic cell death events are relatively rare in postmortal spinal cords and are not increased in areas of the neural progenitor cell's location, within measured postmortal intervals, or among the CD24/GD2-positive cells. Data from our study suggest postmortal spinal cords as a valuable source for harvesting highly viable allogenic neural progenitor cells.

Decreased eggshell strength caused by impairment of uterine calcium transport coincide with higher bone minerals and quality in aged laying hens

BackgroundDeteriorations in eggshell and bone quality are major challenges in aged laying hens. This study compared the differences of eggshell quality, bone parameters and their correlations as well as uterine physiological characteristics and the bone remodeling processes of hens laying eggs of different eggshell breaking strength to explore the mechanism of eggshell and bone quality reduction and their interaction. A total of 240 74-week-old Hy-line Brown laying hens were selected and allocated to a high (HBS, 44.83 ± 1.31 N) or low (LBS, 24.43 ± 0.57 N) eggshell breaking strength group.ResultsA decreased thickness, weight and weight ratio of eggshells were observed in the LBS, accompanied with ultrastructural deterioration and total Ca reduction. Bone quality was negatively correlated with eggshell quality, marked with enhanced structures and increased components in the LBS. In the LBS, the mammillary knobs and effective layer grew slowly. At the initiation stage of eggshell calcification, a total of 130 differentially expressed genes (DEGs, 122 upregulated and 8 downregulated) were identified in the uterus of hens in the LBS relative to those in the HBS. These DEGs were relevant to apoptosis due to the cellular Ca overload. Higher values of p62 protein level, caspase-8 activity, Bax protein expression and lower values of Bcl protein expression and Bcl/Bax ratio were seen in the LBS. TUNEL assay and hematoxylin-eosin staining showed a significant increase in TUNEL-positive cells and tissue damages in the uterus of the LBS. Although few DEGs were identified at the growth stage, similar uterine tissue damages were also observed in the LBS. The expressions of runt-related transcription factor 2 and osteocalcin were upregulated in humeri of the LBS. Enlarged diameter and more structural damages of endocortical bones and decreased ash were observed in femurs of the HBS.ConclusionThe lower eggshell breaking strength may be attributed to a declined Ca transport due to uterine tissue damages, which could affect eggshell calcification and lead to a weak ultrastructure. Impaired uterine Ca transport may result in reduced femoral bone resorption and increased humeral bone formation to maintain a higher mineral and bone quality in the LBS.Graphical

Dexamethasone Induced Osteocyte Apoptosis in Steroid-Induced Femoral Head Osteonecrosis through ROS-Mediated Oxidative Stress.

Glucocorticoid (GC) overuse is strongly associated with steroid-induced osteonecrosis of the femoral head (SINFH). However, the underlying mechanism of SINFH remains unclear. This study aims to investigate the effect of dexamethasone (Dex)-induced oxidative stress on osteocyte apoptosis and the underlying mechanisms. Ten patients with SINFH and 10 patients with developmental dysplasia of the hips (DDH) were enrolled in our study. Sixty rats were randomly assigned to the Control, Dex, Dex + N-Acetyl-L-cysteine (NAC), Dex + Dibenziodolium chloride (DPI), NAC, and DPI groups. Magnetic resonance imaging (MRI) was used to examine edema in the femoral head of rats. Histopathological staining was performed to assess osteonecrosis. Immunofluorescence staining with TUNEL and 8-OHdG was conducted to evaluate osteocyte apoptosis and oxidative damage. Immunohistochemical staining was carried out to detect the expression of NOX1, NOX2, and NOX4. Viability and apoptosis of MLO-Y4 cells were measured using the CCK-8 assay and TUNEL staining. 8-OHdG staining was conducted to detect oxidative stress. 2',7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) staining was performed to measure reactive oxygen species (ROS). The expression of NOX1, NOX2, and NOX4 in MLO-Y4 cells was analyzed by Western blotting. Multiple comparisons were performed using one-way analysis of variance (ANOVA). In patients and the rat model, hematoxylin-eosin (HE) staining revealed a significantly higher rate of empty lacunae in the SINFH group than in the DDH group. Immunofluorescence staining indicated a significant increase in TUNEL-positive cells and 8-OHdG-positive cells in the SINFH group compared to the DDH group. Immunohistochemical staining demonstrated a significant increase in the expression of NOX1, NOX2, and NOX4 proteins in SINFH patients compared to DDH patients. Moreover, immunohistochemical staining showed a significant increase in the proportion of NOX2-positive cells compared to the Control group in the femoral head of rats. In vitro, Dex significantly inhibited the viability of osteocyte cells and induced apoptosis. After Dex treatment, the intracellular ROS level increased. However, Dex treatment did not alter the expression of NOX proteins in vitro. Additionally, NAC and DPI inhibited the generation of intracellular ROS and partially alleviated osteocyte apoptosis in vivo and in vitro. This study demonstrates that GC promotes apoptosis of osteocyte cells through ROS-induced oxidative stress. Furthermore, we found that the increased expression of NOXs induced by GC serves as an important source of ROS generation.

Environmentally relevant atrazine exposure leads to increases in DNA damage and changes in morphology in the hepatopancreas of crayfish (Faxonius virilis)

The herbicide atrazine is widely used for controlling broad leaf weeds and increasing crop yields in agricultural areas. Atrazine enters aquatic environments through runoff, ground water discharge and seepage where concentrations have been recorded above 300 ppb. Exposure to the herbicide atrazine at environmentally relevant concentrations has been shown to negatively impact aquatic organisms, including crayfish. Because xenobiotics are concentrated in the crayfish hepatopancreas (digestive gland), we examined changes in morphology and DNA damage in hepatopancreatic tissue structure and cells following a 10-day exposure to atrazine (0, 10, 40, 80, 100 and 300 ppb). We found that there were marked morphological changes, post-exposure, for all atrazine concentrations tested. Hepatopancreatic tissue exhibited degenerated tubule epithelium with necrosis of microvilli, tubule lumen dilation, changes in tubular epithelium height and vacuolization of the epithelium. Likewise, we also performed a terminal deoxynucleotidyl transferase (TdT) mediated dUTP nick-end labeling (TUNEL) assay which showed the percentage of cells with DNA damage increased following atrazine exposure. Crayfish hepatopancreatic tissue displayed significant increases in TUNEL-positive cells following exposure to atrazine at 100 ppb and above. Overall, exposure to atrazine at environmentally relevant concentrations damages hepatopancreatic tissue. This impairment could lead to changes in biotransformation, detoxification, digestion and molting, subsequently reducing crayfish populations and negatively impacting the aquatic ecosystem.

Valproic acid induced selective apoptosis of ocular fibrous tunic in mice fetuses.

Valproic acid (VPA), a prescribed drug commonly used for various neurological perturbations, has been implicated in teratogenic inflictions on developing fetuses during pregnancy. The purpose of this research was to delineate the gross morphological and histological effects of VPA in the developing eye tunics and lens. A time-dependent administration of 500 mg/kg VPA to BALB/c groups of female mice was coordinated during organogenesis (gestational days 7, 8, and 9) and compared to controls that received normal saline. Seized fetuses were checked for macroscopic eye anomalies, histological malformations with Azan trichrome staining, and levels of apoptotic activity with the terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay. Histochemical analysis showed that VPA-treated groups exhibited collagen deficiency (2.5-50% decrease in aniline blue intensity) and a marked increase in TUNEL-positive cells (p < .05) in corneal stroma and sclera/choroid layers while less was detected in retina and lens, when compared to controls. Since the evaluation of the inner structures did not manifest major differences, we conclude that VPA teratogenic influence display eclectic toxicity, as seen by increased apoptosis to eye layers with high degree fibrous context, particularly the outer tunics.

Gasdermin D Deficiency Limits the Transition of Atherosclerotic Plaques to an Inflammatory Phenotype in ApoE Knock-Out Mice.

Gasdermin D (GSDMD) is the key executor of pyroptotic cell death. Recent studies suggest that GSDMD-mediated pyroptosis is involved in atherosclerotic plaque destabilization. We report that cleaved GSDMD is expressed in macrophage- and smooth muscle cell-rich areas of human plaques. To determine the effects of GSDMD deficiency on atherogenesis, ApoE−/− Gsdmd−/− (n = 16) and ApoE−/− Gsdmd+/+ (n = 18) mice were fed a western-type diet for 16 weeks. Plaque initiation and formation of stable proximal aortic plaques were not altered. However, plaques in the brachiocephalic artery (representing more advanced lesions compared to aortic plaques) of ApoE−/− Gsdmd−/− mice were significantly smaller (115 ± 18 vs. 186 ± 16 × 103 µm2, p = 0.006) and showed features of increased stability, such as decreased necrotic core area (19 ± 4 vs. 37 ± 7 × 103 µm2, p = 0.03) and increased αSMA/MAC3 ratio (1.6 ± 0.3 vs. 0.7 ± 0.1, p = 0.01), which was also observed in proximal aortic plaques. Interestingly, a significant increase in TUNEL positive cells was observed in brachiocephalic artery plaques from ApoE−/− Gsdmd−/− mice (141 ± 25 vs. 62 ± 8 cells/mm2, p = 0.005), indicating a switch to apoptosis. This switch from pyroptosis to apoptosis was also observed in vitro in Gsdmd−/− macrophages. In conclusion, targeting GSDMD appears to be a promising approach for limiting the transition to an inflammatory, vulnerable plaque phenotype.

A Plant-Derived Antioxidant Supplement Prevents the Loss of Retinal Ganglion Cells in the Retinas of NMDA-Injured Mice.

PurposeTo investigate the effect of plant-derived antioxidant compounds, identified with primary culture screening, on retinal ganglion cell (RGC) survival in mice under excitotoxic conditions. Additionally, to determine the effect of these compounds on the involvement of calpain inactivation.Materials and MethodsPlant-derived antioxidant compounds including hesperidin, crocetin, and Tamarindus indica were administrated orally to C57BL/6J mice. The levels of lipid oxidation and calpain activation were assessed with a TBARS assay and western blotting. RGC survival was evaluated with a TUNEL assay and RBPMS immunostaining after intravitreal injection of NMDA.ResultsPlant-derived antioxidant compounds significantly ameliorated the increase in the level of MDA in the retinas after NMDA injury. Cleaved α-fodrin fragments were detected in the NMDA-injured retinas, and these fragments were significantly lower in mice that received the plant-derived antioxidant compounds. The plant-derived antioxidants also ameliorated increases in TUNEL-positive cells and RGC death after NMDA injection.ConclusionThese results indicate that oral administration of plant-derived antioxidant compounds such as hesperidin, crocetin, and Tamarindus indica suppressed RGC death. This oral supplementation decreased lipid oxidation and excessive calpain activation in NMDA-injured retinas. Thus, our newly developed antioxidant supplement has a potential role in neuroprotective treatment for retinal diseases, such as glaucoma.

Reaction of the Liver upon Long-Term Treatment of Fluoxetine and Atorvastatin Compared with Alcohol in a Mouse Model.

Background Alcoholism is known to cause liver toxicity and is extensively researched. On the other hand, stress, depression, and obesity are interrelated conditions with alcoholism, and their medications would affect the liver itself. In this study, we investigated the effects of the drugs fluoxetine and atorvastatin on the liver and compared with those of alcohol in a mouse model. Methods Comparisons of animals treated with the three drugs were carried out: serum aspartate transaminase (AST), alanine transaminase (ALT), and albumin were measured; liver tumor necrosis factor alpha (TNF alpha) and transforming growth factor beta (TGF beta-1) levels were evaluated; proliferative cells were detected via immunohistochemistry (IHC) targeting on proliferating cell nuclear antigen (PCNA) and minichromosome maintenance complex component 2 (MCM2); for apoptosis, IHC targeting on activated caspase-3 and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were employed; and histopathology was also documented in all groups. Results For ALT, AST, albumin, and liver TNF alpha, only the ethanol group surged to significantly higher levels. For TGF beta-1, both ethanol and atorvastatin groups reached a significantly higher level. PCNA and MCM2 showed increased proliferation in the livers of all three groups, with the ethanol group having the highest number of positive cells followed by atorvastatin and then the fluoxetine group. As for cell death, both ethanol and fluoxetine groups showed significantly more apoptosis than control in TUNEL and activated caspase-3, while in the atorvastatin group, activated caspase-3 positive cells increased significantly, but the increase in TUNEL-positive cells did not reach statistical significance.

Post Stroke Safinamide Treatment Attenuates Neurological Damage by Modulating Autophagy and Apoptosis in Experimental Model of Stroke in Rats.

Exploring and repurposing a drug have become a lower risk alternative. Safinamide, approved for Parkinson's disease, has shown neuroprotection in various animal models of neurological disorders. The present study aimed to explore the potential of safinamide in cerebral ischemia/reperfusion (I/R) in rats. Sprague-Dawley rats were used in middle cerebral artery occlusion model of stroke. The effective dose of safinamide was selected based on the results of neurobehavioral parameters and reduction in infarct size assessed 24h post-reperfusion. For sub-acute study, the treatment with effective dose was extended for 3days and effects on neurobehavioral parameters, infarct size (TTC staining and MRI), oxidative stress parameters (MDA, GSH, SOD, NOX-2), inflammatory cytokines (TNF-α, IL-1β, IL-10), apoptosis (Bax, Bcl-2, cleaved caspase-3 expression, and TUNEL staining), and autophagy (pAMPK, Beclin-1, LC3-II expression) were studied. The results of dose selection study showed significant reduction (p < 0.05) in infarct size and improvement in neurobehavioral parameters with safinamide (80mg/kg). In sub-acute study, safinamide showed significant (p < 0.05) improvement in motor coordination and infarct size reduction. Additionally, safinamide treatment significantly normalized altered redox homeostasis and inflammatory cytokine levels. However, no change was observed in expression of NOX-2 in I/R or safinamide treatment group when compared with sham. I/R induced deranged expression of apoptotic markers and increased TUNEL positive cells in cortex were significantly normalized with safinamide treatment. Further, safinamide significantly (p < 0.05) induced the expressions of autophagic proteins (Beclin-1 and LC3-II) in cortex. Overall, the results demonstrated neuroprotective potential of safinamide via anti-oxidant, anti-inflammatory, anti-apoptotic, and autophagy inducing properties. Thus, safinamide can be explored for repurposing in ischemic stroke after further exploration.

Anti-apoptotic and Matrix Remodeling Actions of a Small Molecule Agonist of the Human Relaxin Receptor, ML290 in Mice With Unilateral Ureteral Obstruction.

Diseases, such as diabetes and hypertension, often lead to chronic kidney failure. The peptide hormone relaxin has been shown to have therapeutic effects in various organs. In the present study, we tested the hypothesis that ML290, a small molecule agonist of the human relaxin receptor (RXFP1), is able to target the kidney to remodel the extracellular matrix and reduce apoptosis induced by unilateral ureteral obstruction (UUO). UUO was performed on the left kidney of humanized RXFP1 mice, where the right kidneys served as contralateral controls. Mice were randomly allocated to receive either vehicle or ML290 (30 mg/kg) via daily intraperitoneal injection, and kidneys were collected for apoptosis, RNA, and protein analyses. UUO significantly increased expression of pro-apoptotic markers in both vehicle- and ML290-treated mice when compared to their contralateral control kidneys. Specifically, Bax expression and Erk1/2 activity were upregulated, accompanied by an increase of TUNEL-positive cells in the UUO kidneys. Additionally, UUO induced marked increase in myofibroblast differentiation and aberrant remodeling on the extracellular matrix. ML290 suppressed these processes by promoting a reduction of pro-apoptotic, fibroblastic, and inflammatory markers in the UUO kidneys. Finally, the potent effects of ML290 to remodel the extracellular matrix were demonstrated by its ability to reduce collagen gene expression in the UUO kidneys. Our data indicate that daily administration of ML290 has renal protective effects in the UUO mouse model, specifically through its anti-apoptotic and extracellular matrix remodeling properties.

Transcriptomic and Histopathological Effects of Bifenthrin to the Brain of Juvenile Rainbow Trout (Oncorhynchus mykiss).

The increased global use of pyrethroids raises concern for non-target aquatic species. Bifenthrin, among the most predominantly detected pyrethroids in the environment, is frequently measured in water samples above concentrations reported to induce neuroendocrine and neurotoxic effects to several threatened and endangered fish species, such as the Chinook salmon and steelhead trout. To better characterize the neurotoxic effect of bifenthrin to salmonids, rainbow trout were treated with environmentally relevant concentrations of bifenthrin (15 and 30 ng/L) for two weeks and assessed for changes in transcriptomic profiles and histopathological alterations. The top bioinformatic pathways predicted to be impaired in bifenthrin-exposed trout were involved in gonadotropin releasing hormone signaling, the dysregulation of iron homeostasis, reduced extracellular matrix stability and adhesion, and cell death. Subsequent histopathological analysis showed a significant increase in TUNEL positive cells in the cerebellum and optic tectum of bifenthrin-treated trout, relative to controls (p < 0.05). These findings suggest that low, ng/L concentrations of bifenthrin are capable of dysregulating proper neuroendocrine function, impair the structural integrity of the extracellular matrix and cell signaling pathways in the brain, and induce apoptosis in neurons of juvenile salmonids following bifenthrin treatment, which is consistent with metabolomic profiles demonstrating a common target and mechanism.

Pemafibrate prevents retinal neuronal cell death in NMDA-induced excitotoxicity via inhibition of p-c-Jun expression

Excitotoxicity is involved in the retinal neuronal cell death in diabetic retinopathy. Although fenofibrate has been shown to ameliorate the progression of diabetic retinopathy, the effect of pemafibrate, which is highly selective for peroxisome proliferator-activated receptor α on retinal neuronal cell death has not been documented. Here, we investigated whether pemafibrate exerts a beneficial effect against retinal ganglion cell (RGC) death induced by N-methyl-D-aspartate (NMDA) in rats. Experiments were performed on adult male Wistar rats that received an intravitreal injection of 20 nmol NMDA. Fluoro-Gold labeled RGC morphometry showed that oral intake of pemafibrate once a day for 7 days resulted in significant protection on RGC death induced by NMDA. Phosphorylated c-Jun protein, which is involved in apoptosis, was upregulated after NMDA exposure, and this increase was significantly lessened by the systemic pemafibrate treatment. Phosphorylated c-Jun immunopositive cells were colocalized with Thy-1 immunopositive cells, and the increased these cells were ameliorated by the pemafibrate treatment. An increase in TUNEL-positive cells was significantly suppressed by the pemafibrate treatment. Phosphorylated c-Jun immunopositive cells were colocalized with TUNEL-positive cells, and they were decreased by pemafibrate treatment. These results suggest that the RGC protection achieved with pemafibrate appears to be associated with inhibition of phosphorylated c-Jun and its anti-apoptotic effect.

MFG-E8-derived peptide attenuates inflammation and injury after renal ischemia-reperfusion in mice

BackgroundRenal ischemia-reperfusion (renal I/R) injury may lead to acute kidney injury (AKI). After renal I/R, proinflammatory mediators cause immune cell infiltration and further injury. Milk fat globule-epidermal growth factor-factor 8 (MFG-E8) is a protein involved in cell-cell and cell-matrix interactions. MSP68 is an MFG-E8-derived peptide that inhibits neutrophil adhesion and migration. Here, we evaluated whether MSP68 attenuates renal I/R injury. Materials and methodsAdult C57BL/6 mice were subjected to bilateral renal ischemia for 30 min followed by reperfusion and intraperitoneal administration of saline (vehicle) or MSP68 (5 mg/kg). Sham animals underwent laparotomy without renal I/R. The blood collected and studied for BUN, creatinine, and LDH by colorimetry. The kidneys were analyzed for IL-6 and TNFα by qPCR, ELISA, histological injury, and apoptosis by TUNEL. ResultsAt 24 h after surgery, serum levels of BUN, creatinine, and LDH were markedly higher in vehicle-treated renal I/R mice than in sham mice, but significantly lower in MSP68-treated renal I/R mice. Similarly, compared to sham, renal levels of IL-6 mRNA and protein and TNFα protein were markedly higher in vehicle-treated renal I/R mice, but significantly lower in MSP68-treated renal I/R mice. Vehicle-treated renal I/R mice also had severe renal tubular histological injury, which was significantly lower in MSP68-treated renal I/R mice. Additionally, the kidneys of vehicle-treated renal I/R mice had a 93-fold increase in TUNEL-positive cells, which were reduced by 35% in mice treated with MSP68. ConclusionMSP68 has the potential to be developed as novel therapeutic agent for patients with AKI.

Remote Ischemic Post-Conditioning Therapy is Protective in Mouse Model of Traumatic Optic Neuropathy.

Traumatic optic neuropathy (TON) is characterized by visual dysfunction after indirect or direct injury to the optic nerve following blunt head trauma. TON is associated with increased oxidative stress and inflammation resulting in retinal ganglion cell (RGC) death. Remote ischemic post-conditioning (RIC) has been shown to enhance endogenous protective mechanisms in diverse disease models including stroke, vascular cognitive impairment (VCI), retinal injury and optic nerve injury. However, the protective mechanisms underlying the improvement of retinal function and RGC survival after RIC treatment remain unclear. Here, we hypothesized that RIC therapy may be protective following TON by preventing RGC death, oxidative insult and inflammation in the mouse retina. To carry out the study, mice were divided in three different groups (Control, TON and TON + RIC). We harvested retinal tissue 5days after TON induction for western blotting and histochemical analysis. We observed increased TON-induced retinal cell death compared with controls by cleaved caspase-3 immunohistochemistry. Furthermore, the TON cohort demonstrated increased TUNEL positive cells which were significantly attenuated by RIC. Immunofluorescence data showed that oxidative stress markers dihydroethidium (DHE), NOX-2 and nitrotyrosine expression were elevated in the TON group relative to controls and RIC therapy significantly reduced the expression level of these markers. Next, we found that the proinflammatory cytokine TNF-α was increased and anti-inflammatory IL-10 was decreased in plasma of TON animals, and RIC therapy reversed this expression level. Interestingly, western blotting of retinal tissue showed that RGC marker Brn3a and tight junction proteins (ZO-1 and Occludin), and AMPKα1 expression were downregulated in the TON group compared to controls. However, RIC significantly increased the expression levels of these proteins. Together these data suggest that RIC therapy activates endogenous protective mechanisms which may attenuate TON-induced oxidative stress and inflammation, and improves BRB integrity.

Encapsulation of Hydrophobic Drugs in Shell-by-Shell Coated Nanoparticles for Radio-and Chemotherapy-An In Vitro Study.

Our research objective was to develop novel drug delivery vehicles consisting of TiO2 and Al2O3 nanoparticles encapsulated by a bilayer shell that allows the reversible embedment of hydrophobic drugs. The first shell is formed by covalent binding of hydrophobic phosphonic acid at the metal oxide surface. The second shell composed of amphiphilic sodium dodecylbenzenesulfonate emerges by self-aggregation driven by hydrophobic interactions between the dodecylbenzene moiety and the hydrophobic first shell. The resulting double layer provides hydrophobic pockets suited for the intake of hydrophobic drugs. The nanoparticles were loaded with the anticancer drugs quercetin and 7-amino-4-methylcoumarin. Irradiation with X-rays was observed to release the potential anticancer drugs into the cytoplasm. In Michigan Cancer Foundation (MCF)-10 A cells, quercetin and 7-amino-4-methylcoumarin acted as antioxidants by protecting the non-tumorigenic cells from harmful radiation effects. In contrast, these agents increased the reactive oxygen species (ROS) formation in cancerous MCF-7 cells. Quercetin and 7-amino-4-methylcoumarin were shown to induce apoptosis via the mitochondrial pathway in cancer cells by determining an increase in TUNEL-positive cells and a decrease in mitochondrial membrane potential after irradiation. After X-ray irradiation, the survival fraction of MCF-7 cells with drug-loaded nanoparticles considerably decreased, which demonstrates the excellent performance of the double-layer stabilized nanoparticles as drug delivery vehicles.

Epigenetic modulation of the MAPK pathway prevents isoflurane-induced neuronal apoptosis and cognitive decline in aged rats.

Isoflurane is a broadly used inhalation anesthetic that causes cognitive impairment in rodent models as well as humans. Although previous studies suggested an association between isoflurane exposure and neuro-inflammation, apoptosis and mitochondrial dysfunction, the pathogenesis of isoflurane-induced cognitive decline remains elusive. In the present study, 22-month-old male Sprague-Dawley male rats (n=96) were divided into three groups: Control (Cont), isoflurane (ISO) and MS-275 pre-treated groups. The rats were sacrificed following exposure to isoflurane and a cognitive test. The hippocampus of each animal was harvested for quantitative PCR, TUNEL staining and western blot analysis. Histone deacetylases (HDAC)-1, -2 and -3 exhibited a significant increase at the gene and protein expression levels, whereas negligible mRNA expressions were observed for genes HDAC 4-11 (P>0.05; compared with Cont). Pre-treatment with the HDAC inhibitor MS-275 significantly inhibited the increase in TUNEL-positive cells induced by isoflurane exposure (70.72% decrease; P<0.001; compared with ISO). Furthermore, MS-275 significantly decreased caspase-3 and Bax expression levels while increasing Bcl-2 protein expression. The isoflurane-induced changes in the MAPK pathway signaling proteins ERK1/2, JNK and p38 were also reversed with MS-275 pre-treatment. Finally, in a Morris water maze test, the time to find a hidden platform was reduced in MS-275 pre-treated rats, compared with the ISO group. Therefore, the present study provided insight into the effect of isoflurane exposure on neuronal apoptosis pathways, as well as cognitive decline via epigenetic programming of MAPK signaling in aged rats.

Linagliptin affects IRS1/Akt signaling and prevents high glucose-induced apoptosis in podocytes

Diabetes-induced podocyte apoptosis is considered to play a critical role in the pathogenesis of diabetic kidney disease (DKD). We proposed that hyperglycaemia can induce podocyte apoptosis by inhibiting the action of podocyte survival factors, thus inactivating the cellular effects of insulin signalling. In this study, we aimed to determine the effects of linagliptin on high glucose-induced podocyte apoptosis. Linagliptin reduced the increase in DNA fragmentation as well as the increase in TUNEL-positive cells in podocytes induced by high-glucose condition. Furthermore, linagliptin improved insulin-induced phosphorylation of insulin receptor substrate 1 (IRS1) and Akt, which was inhibited in high-glucose conditions. Adenoviral vector-mediated IRS1 overexpression in podocytes partially normalised DNA fragmentation in high-glucose conditions, while downregulation of IRS1 expression using small interfering RNA increased DNA fragmentation even in low-glucose conditions. Because reactive oxygen species inhibit glomerular insulin signalling in diabetes and Kelch-like ECH-associated protein 1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) pathway is one of the most important intrinsic antioxidative systems, we evaluated whether linagliptin increased Nrf2 in podocytes. High-glucose condition and linagliptin addition increased Nrf2 levels compared to low-glucose conditions. In summary, linagliptin offers protection against DKD by enhancing IRS1/Akt insulin signalling in podocytes and partially via the Keap1/Nrf2 pathway. Our findings suggest that linagliptin may induce protective effects in patients with DKD, and increasing IRS1 levels could be a potential therapeutic target in DKD.

A pyruvate dehydrogenase kinase inhibitor prevents retinal cell death and improves energy metabolism in rat retinas after ischemia/reperfusion injury

We aimed to assess the neuroprotective effect of a pyruvate dehydrogenase kinase (PDK) inhibitor, Nov3r after ischemia/reperfusion (IR) injury in rats. IR injury was induced by applying 150 mmHg of intraocular pressure for 50 min. Nov3r was orally administered (100 mg/kg) 3 h before and 24 h after IR injury. TUNEL-positive cells increased and immunoreactive RBPMS-positive cells decreased in the rat retinas after IR injury. Administration of Nov3r significantly ameliorated the increase in TUNEL-positive cells and prevented the RBPMS-positive cell decrease. Similarly, the number of IR-induced Iba1-positive microglial cells was significantly reduced with Nov3r treatment. Among metabolic parameters, IR damage induced the elevation of lactate and pyruvate, and the reduction of ATP. Oral administration of Nov3r ameliorated these changes. Our data suggest that the Nov3r had a retinal neuroprotective effect in IR injury in rats. This finding suggests that the regulation of pyruvate dehydrogenase (PDH) activity has potential therapeutic value by enabling metabolic reprograming in diseases associated with ischemic retinal damage, such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion, ischemic optic neuropathy and glaucoma.

NOSH-aspirin (NBS-1120) inhibits pancreatic cancer cell growth in a xenograft mouse model: Modulation of FoxM1, p53, NF-κB, iNOS, caspase-3 and ROS

Pancreatic cancer has poor survival rates and largely ineffective therapies. Aspirin is the prototypical anti-cancer agent but its long-term use is associated with significant side effects. NOSH-aspirin belongs to a new class of anti-inflammatory agents that were designed to be safer alternatives by releasing nitric oxide and hydrogen sulfide. In this study we evaluated the effects of NOSH-aspirin against pancreatic cancer using cell lines and a xenograft mouse model. NOSH-aspirin inhibited growth of MIA PaCa-2 and BxPC-3 pancreatic cancer cells with IC50s of 47 ± 5, and 57 ± 4 nM, respectively, while it did not inhibit growth of a normal pancreatic epithelial cell line at these concentrations. NOSH-aspirin inhibited cell proliferation, caused G0/G1 phase cycle arrest, leading to increased apoptosis. Treated cells displayed increases in reactive oxygen species (ROS) and caspase-3 activity. In MIA PaCa-2 cell xenografts, NOSH-aspirin significantly reduced tumor growth and tumor mass. Growth inhibition was due to reduced proliferation (decreased PCNA expression) and induction of apoptosis (increased TUNEL positive cells). Expressions of ROS, iNOS, and mutated p53 were increased; while that of NF-κB and FoxM1 that were high in vehicle-treated xenografts were significantly inhibited by NOSH-aspirin. Taken together, these molecular events and signaling pathways contribute to NOSH-aspirin mediated growth inhibition and apoptotic death of pancreatic cancer cells in vitro and in vivo.

Suicide Gene Therapy with a CD33 Targeted AAV6 Vector Expressing an Inducible Caspase-9 Suicide Gene Is Therapeutic in a Xenotransplantation Model of Acute Myeloid Leukemia

Suicide gene therapy for Acute myeloid leukemia (AML) can offer a high therapeutic index if the pro-apoptotic genes can be selectively introduced into cancer cells with high efficiency with no apparent vector-related toxicities in the recipient. Since hematopoietic cells or myeloid cells are particularly intransigent to transduction with gene delivery vectors such as Adeno-associated virus (AAV), we reasoned that a cell-receptor specific AAV that can target the CD33 antigen, over-expressed in leukemic cells may be beneficial.For designing a targeting peptide, we used bioinformatic tools to identify antibody binding region (ABR) from a monoclonal antibody (M195) available against CD33. After further validation, a consensus antigen-binding region comprising a heptamer sequence was selected and inserted into the loop IV region of the best infecting AAV serotype (AAV1-rh10) in leukemic cells, the AAV6 by standard cloning techniques. The AAV6-CD33 vectors thus developed was packaged with a inducible caspase 9 based suicide gene (iCasp9) and further verified for its capsid integrity and functionality. In the next set of studies, we wished to determine if the AAV6-CD33 vectors can enhance suicide gene transfer in AML cells. We evaluated their cytotoxic effect by a luminescence based ATP assay, in a panel of cell lines which were of hematopoietic origin, that either over-express the CD33 antigen (U937) or that do not express CD33 antigen (CEM) or a control hepatic cell line, Huh7. Interestingly, the targeted AAV6 vectors were highly cytotoxic (1.6 fold, 41 vs 67 % viability, p&lt; 0.0004) when compared to AAV6- wild type (WT) vectors in the CD33 positive U937 cells. However, in cells that do not express CD33 antigen, such as CEM or Huh7, AAV6-CD33 vectors showed no advantage over WT-AAV6 vectors in terms of their cytotoxicity. These data demonstrated the specificity of CD33 receptor targeted AAV6 vectors in AML cells in vitro.To further validate the novel AAV6 vector, we developed a Zebrafish xenograft model of AML. Briefly, ~1×105 fluorescently labelled U937 cells per recipient were transplanted into busulfan (20mg/kg bodyweight) conditioned Zebrafish. Four days after engraftment of leukemic cells, fish were randomised to: mock-treated, AAV6-WT iCasp9 vector administered either systemically (retro-orbital,RO) or locally (intra-tumoral,IT) and the receptor targeted AAV vector (AAV6-CD33) administered systemically through the retro-orbital plexus. Ten days later, the rate of tumor growth in vector treated fish was significantly lower in vector treated fishes when compared to mock-treated Zebrafish. The survival was significantly higher in the treatment group that received AAV6-iCasp9 vectors as compared to mock-treated control fishes (~80% vs 15%, p&lt;0.0001). Moreover, Zebrafish in the AAV6-CD33 treatment arm showed better survival as compared to untreated fishes (~100% vs 15%, p&lt;0.0001) or to those treated with wild type AAV6 vectors administered either by RO or IT route (100% vs 77% or 80%). A morphological characterisation and a TUNEL based apoptosis assay further revealed that fishes treated with CD33 targeted AAV6-iCasp9 vectors by systemic administration, had a significantly higher (~39 fold) increase in TUNEL positive cells in comparison to the control group. Interestingly, the receptor targeted AAV6 vectors augmented the in vivocytotoxicity of U937 tumors by at least 3-fold (p&lt;0.0001) when administered retro-orbitally in comparison to AAV6-WT vector administered group. Taken together, our work demonstrates the efficacy and translational potential of CD33-targeted AAV6 vectors for cytotoxic gene therapy in AML. Disclosures No relevant conflicts of interest to declare.