HuR-dependent SOD2 protein synthesis is an early adaptation to anchorage-independence.

During metastasis cancer cells must adapt to survive loss of anchorage and evade anoikis. An important pro-survival adaptation is the ability of metastatic tumor cells to increase their antioxidant capacity and restore cellular redox balance. Although much is known about the transcriptional regulation of antioxidant enzymes in response to stress, how cells acutely adapt to alter antioxidant enzyme levels is less well understood. Using ovarian cancer cells as a model, we demonstrate that an increase in mitochondrial superoxide dismutase SOD2 protein expression is a very early event initiated in response to detachment, an important step during metastasis that has been associated with increased oxidative stress. SOD2 protein synthesis is rapidly induced within 0.5-2 h of matrix detachment, and polyribosome profiling demonstrates an increase in the number of ribosomes bound to SOD2 mRNA, indicating an increase in SOD2 mRNA translation in response to anchorage-independence. Mechanistically, we find that anchorage-independence induces cytosolic accumulation of the RNA binding protein HuR/ELAVL1 and promotes HuR binding to SOD2 mRNA. Using HuR siRNA-mediated knockdown, we show that the presence of HuR is necessary for the increase in SOD2 mRNA association with the heavy polyribosome fraction and consequent nascent SOD2 protein synthesis in anchorage-independence. Cellular detachment also activates the stress-response mitogen-activated kinase p38, which is necessary for HuR-SOD2 mRNA interactions and induction of SOD2 protein output. These findings illustrate a novel translational regulatory mechanism of SOD2 by which ovarian cancer cells rapidly increase their mitochondrial antioxidant capacity as an acute stress response to anchorage-independence.

Radon inhalation decreases DNA damage induced by oxidative stress in mouse organs via the activation of antioxidative functions.

Radon inhalation decreases the level of lipid peroxide (LPO); this is attributed to the activation of antioxidative functions. This activation contributes to the beneficial effects of radon therapy, but there are no studies on the risks of radon therapy, such as DNA damage. We evaluated the effect of radon inhalation on DNA damage caused by oxidative stress and explored the underlying mechanisms. Mice were exposed to radon inhalation at concentrations of 2 or 20 kBq/m3 (for one, three, or 10 days). The 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels decreased in the brains of mice that inhaled 20 kBq/m3 radon for three days and in the kidneys of mice that inhaled 2 or 20 kBq/m3 radon for one, three or 10 days. The 8-OHdG levels in the small intestine decreased by approximately 20-40% (2 kBq/m3 for three days or 20 kBq/m3 for one, three or 10 days), but there were no significant differences in the 8-OHdG levels between mice that inhaled a sham treatment and those that inhaled radon. There was no significant change in the levels of 8-oxoguanine DNA glycosylase, which plays an important role in DNA repair. However, the level of Mn-superoxide dismutase (SOD) increased by 15-60% and 15-45% in the small intestine and kidney, respectively, following radon inhalation. These results suggest that Mn-SOD probably plays an important role in the inhibition of oxidative DNA damage.

Sulfur dioxide-induced exacerbation of airway inflammation via reactive oxygen species production and the toll-like receptor 4/nuclear factor-κB pathway in asthmatic mice.

Sulfur dioxide (SO2) is a common air pollutant that can exacerbate asthmatic airway inflammation. The mechanisms underlying these effects are not yet fully understood. In this study, we investigated the effects of SO2 exposure (10 mg/m3) on asthmatic airway inflammation in ovalbumin-induced asthmatic mice. Our results showed that SO2 exposure alone induced slight airway injury, decreased superoxide dismutase activity, and increased nuclear factor-κB (NF-κB) expression in the lungs of mice. Moreover, SO2 exposure in asthmatic mice induced marked pathological damage, significantly increased the counts of inflammatory cells (e.g., macrophages, lymphocytes, and eosinophils) in bronchoalveolar lavage fluid, and significantly enhanced malondialdehyde and glutathione levels in the lungs. Moreover, the expression of toll-like receptor 4 (TLR4), NF-κB, pro-inflammatory cytokines (e.g., tumor necrosis factor α and interleukin-6), and type II T-helper cell (Th2) cytokines was found to be elevated in the mice exposed to SO2 and ovalbumin compared to those exposed to ovalbumin alone. These results suggest that SO2 amplifies Th2-mediated inflammatory responses, which involve reactive oxygen species and TLR4/NF-κB pathway activation; these can further enhance Th2 cytokine expression and eosinophilic inflammation. Thus, our findings provide important evidence to understand a potential mechanism through which SO2 may exacerbate airway asthmatic inflammation.

Moderate Hydrogen Peroxide Postconditioning Ameliorates Ischemia/Reperfusion Injury in Cardiomyocytes via STAT3-Induced Calcium, ROS, and ATP Homeostasis.

INTRODUCTION: Moderate hydrogen peroxide postconditioning (H2O2PoC) activates signal transducer and activator of transcription 3 (STAT3) to alleviate mitochondrial calcium overload during cardiac ischemia/reperfusion (I/R). However, the initial time window of STAT3-induced calcium hemostasis, the production of reactive oxygen species (ROS) and adenosine triphosphate (ATP) in H2O2PoC, and its regulated mechanism remain unknown. This study aimed to investigate H2O2PoC-induced homeostasis of calcium, ROS and ATP, and the role of STAT3 in the regulation. METHODS: Isolated rat cardiomyocytes were exposed to H2O2PoC and Janus kinase 2 (JAK2)/STAT3 inhibitor AG490 during I/R. Ca2+ transients, cell contraction, intracellular calcium concentration, ROS production, ATP contents, phosphorylation of STAT3, gene and protein expression of manganese superoxide dismutase (MnSOD), metallothionein 1 (MT1) and metallothionein 2 (MT2), as well as activities of mitochondrial complex I and complex II were detected. RESULTS: Moderate H2O2PoC improved post-ischemic Ca2+ transients and cell contraction recovery as well as alleviated cytosolic and mitochondrial calcium overload, which were abrogated by AG490 in rat cardiomyocytes. Moderate H2O2PoC increased ROS production and rate of ROS production at early reperfusion in rat I/R cardiomyocytes, and this phenomenon was also abrogated by AG490. Notably, the expression of phosphorylated nuclear STAT3; gene and protein expression of MnSOD, MT1, and MT2; and activities of mitochondrial complex I and complex II were upregulated by moderate H2O2PoC but downregulated by AG490. CONCLUSION: These findings indicated that the cardioprotection of moderate H2O2PoC against cardiac I/R could be associated with activated STAT3 at early reperfusion to maintain calcium, ROS, and ATP homeostasis in rat cardiomyocytes.

Tomentosin inhibit cerebral ischemia/reperfusion induced inflammatory response via TLR4/ NLRP3 signalling pathway - in vivo and in vitro studies.

Stoke is a global threat, leading to 50 % of deaths worldwide and it causes permanent disability to about 5 million individuals globally each year. In this study, we assessed the potency of tomentosin to inhibit the neuroinflammation in in vivo and in vitro models. The Sprague Dawley rats were pretreated with 25 mg/kg bodyweight (b.wt) and 50 mg/kg b.wt of tomentosin for seven days followed by induction of cerebral ischemic reperfusion. The brain edema and cerebral infractions were analyzed. The levels of antioxidants and the interleukins were measured by standard methods. The NLRP3 signaling proteins expression was evaluated using qPCR analysis. In vitro studies were performed in SH-SY5Y-cells pretreated with tomentosin and subjected to OGD-R treatment. Our results depicts tomentosin scavenges the free radicals, enhances antioxidant system, inhibits the NLRP3 signaling. In vitro results substantiates with in vivo results. To conclude, our in vivo and in vitro results confirm tomentosin may be potent alternative for existing antistroke drugs.

Therapeutic effect of phycocyanin on acute liver oxidative damage caused by X-ray.

1) BACKGROUND: Phycocyanin (PC) is a type of natural protein in algae with antioxidant and anti-inflammatory properties. However, the protective effect of PC on hepatic damage induced by X-ray remains unknown. 2) METHODS: Male C57BL/6 mice were gavaged with 200mg/kg PC for consecutive 7 days before or after radiation. The blood samples and tissues were collected on days 1 and 7 after radiation exposure. 3) RESULTS: Pretreatment or treatment with PC decreased significantly the levels of alanine aminotransferase (ALT), aspartate aminotransferase(AST) in the plasma. Histological evaluation further confirmed the protection of PC against radiation-induced hepatotoxicity. PC-treatment also increased the relative mRNA expression of superoxide dismutase (SOD) and glutathione (GSH-PX), and descended the ROS in the liver. Moreover, the expression of H2AX, an indicator of DNA damage in mice, of the PC-intervention group was much smaller than that of the radiation group. In vivo, PC-treatment markedly up-regulated NF-E2-related factor 2(Nrf2) expression and downstream gene such as hemeoxygenase-1 (HO-1), NQO1. 4) Conclusion: PC could attenuate the radiation-induced oxidative stress damage by activating Nrf2/ HO-1 signaling pathway, and reduce the radiation-induced DNA damage. Therefore, PC is a protective agent against radiation-induced liver damage.

LncRNA ANRIL Silencing Alleviates High Glucose-Induced Inflammation, Oxidative Stress, and Apoptosis via Upregulation of MME in Podocytes.

Diabetic nephropathy (DN), characterized by glomerular injury, is a common complication of both type 1 and type 2 diabetes, accompanied by massive proteinuria. Podocytes are reported to play pivotal roles in maintaining the glomerular filtration barrier. In addition, the expression of long non-coding RNAs (lncRNAs) ANRIL was upregulated in type 2 diabetes patients. Hence, the aim of this study was to investigate the underlying mechanisms implicated the role of LncRNA ANRIL in podocyte injury in DN. The concentration of inflammatory cytokines was quantified by the corresponding enzyme-linked immunosorbent assay (ELISA) kits. The mRNA levels of the target gene were determined by reverse transcription and real-time quantitative PCR (RT-qPCR). The expressions of proteins were evaluated by Western blot. The activities of lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA) level were measured by corresponding commercial kits. Finally, the apoptosis of podocytes was analyzed by TUNEL assay. In our study, LncRNA ANRIL was highly expressed in high glucose (HG)-induced podocytes. Moreover, LncRNA ANRIL silencing attenuated HG-induced inflammation, oxidative stress, and apoptosis and induced MME overexpression in podocytes. Interestingly, MME knockdown abolished the suppressive effect of LncRNA ANRIL silencing on HG-induced inflammation, oxidative stress, and apoptosis in podocytes. LncRNA ANRIL silencing alleviates HG-induced inflammation, oxidative stress, and apoptosis via upregulation of MME in podocytes. Hence, LncRNA ANRIL may be a novel and effective target to ameliorate podocyte injury in DN.

Therapeutic Role of Neuregulin 1 Type III in SOD1-Linked Amyotrophic Lateral Sclerosis.

Amyotrophic lateral sclerosis (ALS) is a devastating motoneuron (Mn) disease without effective cure currently available. Death of MNs in ALS is preceded by failure of neuromuscular junctions and axonal retraction. Neuregulin 1 (NRG1) is a neurotrophic factor highly expressed in MNs and neuromuscular junctions that support axonal and neuromuscular development and maintenance. NRG1 and its ErbB receptors are involved in ALS. Reduced NRG1 expression has been found in ALS patients and in the ALS SOD1G93A mouse model; however, the expression of the isoforms of NRG1 and its receptors is still controversial. Due to the reduced levels of NRG1 type III (NRG1-III) in the spinal cord of ALS patients, we used gene therapy based on intrathecal administration of adeno-associated virus to overexpress NRG1-III in SOD1G93A mice. The mice were evaluated from 9 to 16 weeks of age by electrophysiology and rotarod tests. At 16 weeks, samples were harvested for histological and molecular analyses. Our results indicate that overexpression of NRG1-III is able to preserve neuromuscular function of the hindlimbs, improve locomotor performance, increase the number of surviving MNs, and reduce glial reactivity in the treated female SOD1G93A mice. Furthermore, the NRG1-III/ErbB4 axis appears to regulate MN excitability by modulating the chloride transporter KCC2 and reduces the expression of the MN vulnerability marker MMP-9. However, NRG1-III did not have a significant effect on male mice, indicating relevant sex differences. These findings indicate that increasing NRG1-III at the spinal cord is a promising approach for promoting MN protection and functional improvement in ALS.

SIRT3 retards intervertebral disc degeneration by anti-oxidative stress by activating the SIRT3/FOXO3/SOD2 signaling pathway.

OBJECTIVE: The objective of this paper is to determine whether SIRT3 could retard intervertebral disc degeneration and study the mechanism. MATERIALS AND METHODS: We chose the 3-month mice to establish intervertebral disc degeneration model and study the effect of SIRT3 on the intervertebral disc by Western blotting, quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), immunohistochemistry. Mouse nucleus pulposus cells were cultured to study the exact mechanism. RESULTS: The expression of SIRT3 was decreased in degenerated human nucleus pulposus. Intervertebral discs of mice treated with theacrine expressed more collagen II and less collagen X. In addition, nucleus pulposus cells stimulated with interleukin-1ß (IL-1ß) expressed less SIRT3 than that in the control group and nucleus pulposus cells with SIRT3 overexpress vectors expressed more collagen II FOXO3a and superoxide dismutase 2 (SOD2), indicating that SIRT3 could improve the intervertebral disc degeneration by anti-oxidative stress. CONCLUSIONS: SIRT3 is a protective factor for intervertebral discs and can reduce oxidative stress in the intervertebral disc.

Taraxasterol from Taraxacum prevents concanavalin A-induced acute hepatic injury in mice via modulating TLRs/NF-κB and Bax/Bc1-2 signalling pathways.

Immune hepatic injury is a liver disease closely related to an immune imbalance of T cells and macrophages. Our previous series of studies have demonstrated that taraxasterol isolated from Taraxacum possesses great anti-inflammatory and immunomodulatory effects in vivo and in vitro. In this study, we explored the preventive effects of taraxasterol and its underlying mechanisms on concanavalin A (Con A)-induced acute hepatic injury in mice. It was found that treatment with taraxasterol significantly decreased the Con A-induced increase of liver index, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) and hepatic malondialdehyde (MDA) levels, and increased the Con A-induced decrease of hepatic glutathione (GSH) and superoxide dismutase (SOD) production. Taraxasterol also significantly inhibited the release of pro-inflammatory cytokines tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6), IL-1ß, interferon-γ (IFN-γ) and IL-4. In addition, treatment with taraxasterol alleviated the hepatic histopathological injury and apoptosis induced by Con A. Furthermore, taraxasterol dramatically down-regulated the expressions of T toll-like receptor (TLR2), TLR4 and nuclear factor-κappaB (NF-κB) p65, and decreased the expression ratio of Bax/Bc1-2 in hepatic tissues. These findings suggest that taraxasterol prevents Con A-induced acute hepatic injury in mice by inhibiting TLRs/NF-κB inflammatory signalling pathway and promoting Bax/Bc1-2 anti-apoptotic signalling pathway.

Anti-inflammatory and antioxidant effects of muscarinic acetylcholine receptor (mAChR) activation in the rat hippocampus.

Recently we found that acute treatment with Oxotremorine (Oxo), a non-selective mAChRs agonist, up-regulates heat shock proteins and activates their transcription factor heat shock factor 1 in the rat hippocampus. Here we aimed to investigate: a) if acute treatment with Oxo may regulate pro-inflammatory or anti-inflammatory cytokines and oxidative stress in the rat hippocampus; b) if chronic restraint stress (CRS) induces inflammatory or oxidative alterations in the hippocampus and whether such alterations may be affected by chronic treatment with Oxo. In the acute experiment, rats were injected with single dose of Oxo (0.4 mg/kg) and sacrificed at 24 h, 48 h and 72 h. In the CRS experiment, the rats were exposed for 21 days to the CRS and then were treated with Oxo (0.2 mg/kg) for further 10 days. The acute Oxo treatment showed an ability to significantly reduce reactive oxygen species (ROS), singlet oxygen (1O2), pro-inflammatory cytokines levels (IL-1ß and IL-6) and phosphorylated NF-κB-p65. Acute Oxo treatment also increased superoxide dismutase (SOD)-2 protein levels and stimulated SOD activity. No differences were detected in the anti-inflammatory cytokine levels, including IL-10 and TGF-ß1. In the group of rats exposed to the CRS were found increased hippocampal IL-1ß and IL-6 levels, together with a reduction of SOD activity level. These changes produced by CRS were counteracted by chronic Oxo treatment. In contrast, the upregulation of ROS and 1O2 levels in the CRS group was not counteracted by chronic Oxo treatment. The results revealed a hippocampal anti-inflammatory and antioxidant effect of Oxo treatment in both basal conditions and anti-inflammatory in the CRS rat model.

Isoliquiritigenin attenuates lipopolysaccharide-induced cognitive impairment through antioxidant and anti-inflammatory activity.

BACKGROUND: Oxidative stress and neuroinflammation are central pathogenic mechanisms common to many neurological diseases. Isoliquiritigenin (ISL) is a flavonoid in licorice with multiple pharmacological properties, including anti-inflammatory activity, and has demonstrated protective efficacy against acute neural injury. However, potential actions against cognitive impairments have not been examined extensively. We established a rat model of cognitive impairment by intracerebroventricular injection of lipopolysaccharide (LPS), and examined the effects of ISL pretreatment on cognitive function, hippocampal injury, and hippocampal expression of various synaptic proteins, antioxidant enzymes, pro-inflammatory cytokines, and signaling factors controlling anti-oxidant and pro-inflammatory responses. RESULTS: Rats receiving LPS alone demonstrated spatial learning deficits in the Morris water maze test as evidenced by longer average escape latency, fewer platform crossings, and shorter average time in the target quadrant than untreated controls. ISL pretreatment reversed these deficits as well as LPS-induced decreases in the hippocampal expression levels of synaptophysin, postsynaptic density-95, brain-derived neurotrophic factor, superoxide dismutase, glutathione peroxidase, and BCL-2. ISL pretreatment also reversed LPS-induced increases in TUNEL-positive (apoptotic) cells, BAX/BCL-2 ratio, and expression levels of tumor necrosis factor-α, interleukin (IL)-1ß, IL-6, and C-C motif chemokine ligand 3. Pretreatment with ISL increased the expression levels of phosphorylated (p)-GSK-3ß, nuclear NRF2, HO-1 mRNA, and NQO1 mRNA, and reversed LPS-induced nuclear translocation of nuclear factor (NF)-κB. CONCLUSIONS: ISL protects against LPS-induced cognitive impairment and neuronal injury by promoting or maintaining antioxidant capacity and suppressing neuroinflammation, likely through phosphorylation-dependent inactivation of GSK-3ß, enhanced expression of NRF2-responsive antioxidant genes, and suppression of NF-κB-responsive pro-inflammatory genes.

TNF­α­mediated upregulation of SOD­2 contributes to cell proliferation and cisplatin resistance in esophageal squamous cell carcinoma.

Intrinsic and acquired resistance of cancer to radio­and chemotherapy is one of the major challenges in the treatment of esophageal squamous cell carcinoma (ESCC). Elevated reactive oxygen species (ROS) play an important role in the resistance to cisplatin in ESCCs. Super dismutase [Mn], mitochondrial (SOD­2), an important primary antioxidant enzyme located in mitochondria, could regulate ROS production. Our previous study showed that tumor necrosis factor­α (TNF­α)­mediated SOD­2 through NF­κB was involved in epithelial­mesenchymal transition and migration in A549 cells. Therefore, the present study aimed to identify if TNF­α mediated SOD­2 upregulation is involved in cisplatin resistance in ESCC. It was identified that a higher expression of SOD­2 in human ESCC samples was associated with TNF­α expression and poor overall survival in patients with ESCC, suggesting that SOD­2 may act as an oncogene in ESCC. To further confirm if TNF­α could upregulate SOD­2 to contribute to cell proliferation, the human ESCC cell line Eca­109 was treated with TNF­α in vitro. TNF­α could upregulate SOD­2 and induce cell proliferation in Eca109 cells, while blocking SOD­2 using small interfering RNA (siRNA) inhibited TNF­α­induced cell proliferation. Upregulation of SOD­2 by TNF­α was inhibited by blocking the NF­κB pathway, which suggested that SOD­2 by TNF­α/NF­κB contributes to cell proliferation in Eca109 cells. Furthermore, it was observed that TNF­α could induce cisplatin resistance in Eca109 cells, while transfection with SOD­2 siRNA could significantly increase the chemosensitivity of ESCC to cisplatin. Therefore, the present results suggested that SOD­2 may serve as an oncogene, and the upregulation of SOD­2 by TNF­α/NF­κB may contribute to cisplatin resistance in ESCC.

Membralin deficiency dysregulates astrocytic glutamate homeostasis leading to ALS-like impairment.

Mechanisms underlying motor neuron degeneration in amyotrophic lateral sclerosis (ALS) are yet unclear. Specific deletion of the ER-component membralin in astrocytes manifested postnatal motor defects and lethality in mice, causing the accumulation of extracellular glutamate through reducing the glutamate transporter EAAT2. Restoring EAAT2 levels in membralin KO astrocytes limited astrocyte-dependent excitotoxicity in motor neurons. Transcriptomic profiles from mouse astrocytic membralin KO motor cortex indicated significant perturbation in KEGG pathway components related to ALS, including downregulation of Eaat2 and upregulation of Tnfrsf1a. Changes in gene expression with membralin deletion also overlapped with mouse ALS models and reactive astrocytes. Our results shown that activation of TNF receptor (TNFR1)-NFκB pathway known to suppress Eaat2 transcription was upregulated with membralin deletion. Further, reduced membralin and EAAT2 levels correlated with disease progression in spinal cord from SOD1-mutant mouse models, and reductions in membralin/EAAT2 were observed in human ALS spinal cord. Importantly, overexpression of membralin in SOD1G93A astrocytes decreased TNFR1 levels and increased EAAT2 expression, and improved motor neuron survival. Importantly, upregulation of membralin in SOD1G93A mice significantly prolonged mouse survival. Together, our study provided a mechanism for ALS pathogenesis where membralin limited glutamatergic neurotoxicity, suggesting that modulating membralin had potentials in ALS therapy.

Does Seipin Play a Role in Oxidative Stress Protection and Peroxisome Biogenesis? New Insights from Human Brain Autopsies.

Seipin is a widely expressed protein but with highest levels found in the brain and testes. Seipin function is not yet completely understood, therefore the aim of this study was to evaluate the expression of BSCL2 transcripts in the central nervous system (CNS) of humans and investigate the effect of their overexpression on a neuron model and their relationship with oxidative stress protection, as well as shed light on the pathogenic mechanisms of Celia's Encephalopathy. We analyzed the expression of BSCL2 transcripts using real-time RT-PCR in samples across the brain regions of subjects who underwent necropsy and from a case with Celia's Encephalopathy. The transcript encoding the long seipin isoform (BSCL2-203, 462 aa) is expressed primarily in the brain and its expression is inversely correlated with age in the temporal lobe, amygdala, and hypothalamus. Strong positive correlations were found between BSCL2 expression and some genes encoding protective enzymes against oxidative stress including SOD1 and SOD2, as well as peroxisome proliferator-activated receptor gamma (PPARG) in the amygdala. These results were experimentally corroborated by overexpressing BSCL2 transcripts in SH-SY5Y cells with lentiviral transduction and assessing their effects on neuron differentiated cells. Confocal microscopy studies showed that both seipin and PEX16 are closely expressed in the hypothalami of healthy human brains, and PEX16 was absent in the same region of the PELD case. We hypothesize that seipin has specific CNS functions and may play a role in peroxisome biogenesis.

Estrogen and progesterone dependent expression of visfatin/NAMPT regulates proliferation and apoptosis in mice uterus during estrous cycle.

Visfatin is an adipokine which has an endocrine effect on reproductive functions and regulates ovarian steroidogenesis. There is scant information about the expression, regulation, and functions of visfatin in the mammalian uterus. The present study examined expression and localization of visfatin in the mouse uterus at various stages of the natural estrous cycle, effects of estrogen and progesterone on localization and expression of visfatin in the ovariectomised mouse uterus and effect of visfatin inhibition by a specific inhibitor, FK866 on proliferation and apoptosis in the uterus. Western blot analysis of visfatin showed high expression in proestrus and metestrus while it declined in estrus and diestrus. Immulocalization study also showed strong immunostaining in the cells of endometrium, myometrium, luminal and glandular epithelium during proestrus and metestrus that estrus and diestrus. The uterine visfatin expression closely related to the increased estrogen levels in proestrus and suppressed when progesterone rose to a high level in diestrus. The treatment with estrogen to ovariectomised mice up-regulates visfatin, PCNA, and active caspase3 whereas progesterone up-regulates PCNA and down-regulates visfatin and active caspase3 expression in mouse uterus. The co-treatment with estrogen and progesterone up-regulates visfatin and down-regulates PCNA and active caspase3. In vitro study showed endogenous visfatin inhibition by FK866 increased expression of PCNA and BCL2 increased catalase activity while FK866 treatment decreased expression of active caspase3 and BAX with decreased SOD and GPx activity. BrdU labeling showed that inhibition of visfatin modulates the uterine proliferation. This study showed that expression of visfatin protein is steroid dependent in mouse uterus which is involved in the regulation of proliferation and apoptosis via modulating antioxidant system in the uterus of mice during the reproductive cycle.

Toxic and Genotoxic Effects of Silver Nanoparticles in Drosophila.

The in vivo model Drosophila melanogaster was used here to determine the detrimental effects induced by silver nanoparticles (AgNPs) exposure. The main aim was to explore its interaction with the intestinal barrier and the genotoxic effects induced in hemocytes. The observed effects were compared with those obtained by silver nitrate, as an agent acting via the release of silver ions. Larvae were fed in food media containing both forms of silver. Results indicated that silver nitrate was more toxic than AgNPs when the viability "egg-to-adult" was determined. Depigmentation was observed in adults including those exposed to nontoxic concentrations, as indicative of exposure action. Interestingly, AgNPs were able to cross the intestinal barrier affecting hemocytes that show significant increases in the levels of intracellular reactive oxygen species. Additionally, significant levels of genotoxic damage, as determined by the comet assay, were also induced. When the expression of different stress-response genes was determined, for both AgNPs and silver nitrate, significant upregulation of Sod2 and p53 genes was observed. Our results confirm for the first time that in an in vivo model as Drosophila, AgNPs are able to cross the intestinal barriers and produce primary DNA damage (comet assay) via oxidative stress induction. In general, the effects induced by silver nitrate were more pronounced than those induced by AgNPs what would emphasize the role of silver ions in the observed effects. Environ. Mol. Mutagen. 60:277-285, 2019. © 2018 Wiley Periodicals, Inc.

Inhibition of Neointima Hyperplasia, Inflammation, and Reactive Oxygen Species in Balloon-Injured Arteries by HVJ Envelope Vector-Mediated Delivery of Superoxide Dismutase Gene.

Extracellular superoxide dismutase (EC-SOD) has been implicated in regulation of vascular function but its underlying molecular mechanism is largely unknown. These two-step experiments investigate whether hemagglutinating virus of Japan envelope (HVJ-E) vector-mediated EC-SOD gene delivery might protect against neointima formation, vascular inflammation, and reactive oxygen species (ROS) generation, and also explore cell growth signaling pathways. The first in-vitro experiment was performed to assess the transfection efficacy and safety of HVJ-E compared to lipofectamine®. Results revealed that HVJ-E has higher transfection efficiency and lower cytotoxicity than those of lipofectamine®. Another in-vivo study initially used balloon denudation to rat carotid artery, then delivered EC-SOD cDNA through the vector of HVJ-E. Arterial section with H&E staining from the animals 14 days after balloon injury showed a significant reduction of intima-to-media area ratio in EC-SOD transfected arteries when compared with control (empty vector-transfected arteries) (p < 0.05). Arterial tissue with EC-SOD gene delivery also exhibited lower levels of ROS, as assessed by fluorescent microphotography with dihydroethidium staining. Quantitative RT-PCR revealed that EC-SOD gene delivery significantly diminished mRNA expression of tumor necrosis factor (TNF)-α and interleukin (IL)-1ß (p < 0.05 in all comparisons). An immunoblotting assay from vascular smooth muscle cell (VSMC) cultures showed that the EC-SOD transfected group attenuated the activation of MEK1/2, ERK1/2, and Akt signaling significantly. In conclusion, EC-SOD overexpression by HVJ-E vector inhibits neointima hyperplasia, inflammation, and ROS level triggered by balloon injury. The modulation of cell growth-signaling pathways by EC-SOD in VSMCs might play an important role in these inhibitory effects.

Enzymatic gene expression by Pleurotus tuoliensis (Bailinggu): differential regulation under low temperature induction conditions.

Pleurotus tuoliensis is a valuable, rare and edible mushroom that is been commercially cultivated and is rapidly developing in China markets. Low temperatures are required to induces primordia initiation for the successful production of fruiting bodies (basidiomes) during commercial cultivation. In this work, we investigated the enzymatic activities and performed transcription profiling analysis of enzymatic genes under different low temperature conditions. The results suggest that the enzymatic activities and transcription levels decrease or increase significantly at 4 and 13 °C. Lacc10 and mnp6 seems to play a dominant role during nutrition growth. Furthermore, the expression of laccase and peroxidase genes was highly correlated to the detected extracellular enzymatic activity. Cold stress genes expression profiles were upregulated under 4 °C/13 °C (3 days), while only the Hsp70 gene was downregulated (at the stage of fruiting bodies production) at 13 °C (12 days). Our results showed that the transcriptional regulation of laccase and ligninolytic peroxidase genes plays an important role in the fruiting bodies of Bailinggu under low temperature induction (4 °C). Induction at low temperatures was a highly important cultivation condition in Bailinggu.

HZ08 suppresses RelB-activated MnSOD expression and enhances Radiosensitivity of prostate Cancer cells.

BACKGROUND: The development of radioresistance is one of main causes for therapeutic failure of prostate cancer (PCa). The present study aims to investigate the function and the related mechanism by which HZ08 sensitizes radiotherapeutic efficiency to treat aggressive PCa cells. METHODS: PCa cells were pretreated with HZ08 (6,7-dimethoxy-1-(3,4-dimethoxy) benzyl-2-(N-n-octyl-N'-cyano) guanyl-1,2,3,4-tetrahydroisoquinoline) and followed by ionizing radiation (IR) treatment. Cytotoxicity in the treated cells was analyzed to assess the radiosensitization capacity of HZ08 by flow cytometry, MTT and colony survival assays. The cellular levels of reactive oxygen species (ROS) and oxygen consumption rates (OCR) were measured using specific ROS detection probes and a Seahorse XF96 Analyzer, respectively. RelB binding to the NF-κB intronic enhancer region of the human SOD2 gene was determined using a ChIP assay. The levels of phosphorylation of PI3K, Akt and IKKα were quantified and further confirmed using a PI3K inhibitor. Finally, the synergistic effect of HZ08 on radiosensitization of PCa cells was validated using a mouse xenograft tumor model. RESULTS: HZ08 enhanced radiosensitivity of PCa cells through increasing ROS and declining mitochondrial respiration due to suppression of mitochondrial antioxidant enzyme MnSOD. Mechanistically, HZ08 appeared to inhibit PI3K/Akt/IKKα signaling axis, resulting in transcriptional repression of MnSOD expression by preventing RelB nuclear translocation. CONCLUSIONS: HZ08 can serve as a useful radiosensitizing agent to improve radiotherapy for treating aggressive PCa cells with high level of constitutive RelB. The present study suggests a promising approach for enhancing radiotherapeutic efficiency to treat advanced PCa by inhibiting antioxidant defense function.