Reduction of Superoxide Dismutase 1 Delays Regeneration of Cardiotoxin-Injured Skeletal Muscle in KK/Ta-Ins2Akita Mice with Progressive Diabetic Nephropathy.

Superoxide dismutase (SOD) is a major antioxidant enzyme for superoxide removal, and cytoplasmic SOD (SOD1) is expressed as a predominant isoform in all cells. We previously reported that renal SOD1 deficiency accelerates the progression of diabetic nephropathy (DN) via increasing renal oxidative stress. To evaluate whether the degree of SOD1 expression determines regeneration capacity and sarcopenic phenotypes of skeletal muscles under incipient and advanced DN conditions, we investigated the alterations of SOD1 expression, oxidative stress marker, inflammation, fibrosis, and regeneration capacity in cardiotoxin (CTX)-injured tibialis anterior (TA) muscles of two Akita diabetic mouse models with different susceptibility to DN, DN-resistant C57BL/6-Ins2Akita and DN-prone KK/Ta-Ins2Akita mice. Here, we report that KK/Ta-Ins2Akita mice, but not C57BL/6-Ins2Akita mice, exhibit delayed muscle regeneration after CTX injection, as demonstrated by the finding indicating significantly smaller average cross-sectional areas of regenerating TA muscle myofibers relative to KK/Ta-wild-type mice. Furthermore, we observed markedly reduced SOD1 expression in CTX-injected TA muscles of KK/Ta-Ins2Akita mice, but not C57BL/6-Ins2Akita mice, along with increased inflammatory cell infiltration, prominent fibrosis and superoxide overproduction. Our study provides the first evidence that SOD1 reduction and the following superoxide overproduction delay skeletal muscle regeneration through induction of overt inflammation and fibrosis in a mouse model of progressive DN.

Pyrroloquinoline Quinone Inhibits Oxidative Stress in Rats with Diabetic Nephropathy.

BACKGROUND Diabetic nephropathy (DN) is one of the chronic microvascular complications of diabetes. This study focused on the protective effects of pyrroloquinoline quinone (PQQ) on oxidative stress (OS) in DN. MATERIAL AND METHODS Thirty Sprague Dawley rats were randomly selected for this study; 10 rats were randomly selected as the control group. The other 20 rats were established for the DN model. After establishment of the successful model, the DN model rats were randomly divided into a DN group and a PQQ group. The PQQ group was fed with a PQQ diet. Blood urea nitrogen (BUN), serum creatinine (SCr), and blood glucose levels were measured in each group, and OS-related protein expression and AMPK pathway were detected by western blot and quantitative real-time polymerase chain reaction (qRT-PCR). At the same time, we constructed a DN model by culturing NRK-52E cells with high glucose to detect the molecular mechanisms. RESULTS The kidney function of the DN group was significantly decreased, SCr and BUN levels were significantly increased, and the renal structure under the microscope was disordered, and interstitial edema was obvious. The expression of SOD1, SOD2, GPX1, and GPX3 were significantly decreased, and the level of reactive oxygen species (ROS) was significantly increased. PQQ treatment can effectively alleviate renal function, improve structural damage, and inhibit OS. In vivo, PQQ can effectively inhibit high glucose-induced OS damage and activate the AMPK/FOXO3a signaling pathway. CONCLUSIONS PQQ improves renal structural damage and functional damage, and protects kidney cells in DN by inhibiting OS, which may be related to activating the AMPK/FOXO3a pathway.

Quercetin and Baicalein Act as Potent Antiamyloidogenic and Fibril Destabilizing Agents for SOD1 Fibrils.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that has been associated with the deposition of aggregates of superoxide dismutase 1 (SOD1). Effective therapeutics against SOD1 fibrillation is still an area of active research. Herein, we demonstrate the potential of two naturally occurring flavonoids (quercetin and baicalein) to inhibit fibrillation of wild-type SOD1 with the aid of a series of biophysical techniques. Our seeding experiments reveal that both of these flavonoids significantly affect the fibril elongation. Interestingly, our ThT binding assay, TEM, and SDS-PAGE experiments suggest that these flavonoids also disintegrate the fibrils into shorter fragments but do not completely depolymerize them into monomers. Binding parameters obtained from the analysis of UV-vis spectra suggest that these flavonoids bind moderately to native SOD1 dimer and have different binding sites. Docking of these flavonoids with a non-native monomer, non-native trimer, and oligomer derived from the 11-residue segment of SOD1 indicates that both quercetin and baicalein can bind to these species and thus can arrest the elongation of fibrils by blocking the fibrillar core regions on the intermediate species formed during aggregation of SOD1. MTT assay data revealed that both the flavonoids reduced the cytotoxicity of SOD1 fibrils. Experimental data also show the antiamyloidogenic potential of both flavonoids against A4V SOD1 mutant fibrillation. Thus, our findings may provide a direction for designing effective therapeutic agents against ALS which can act as promising antiamyloidogenic and fibril destabilizing agents.

Ebselen as template for stabilization of A4V mutant dimer for motor neuron disease therapy.

Mutations to the gene encoding superoxide dismutase-1 (SOD1) were the first genetic elements discovered that cause motor neuron disease (MND). These mutations result in compromised SOD1 dimer stability, with one of the severest and most common mutations Ala4Val (A4V) displaying a propensity to monomerise and aggregate leading to neuronal death. We show that the clinically used ebselen and related analogues promote thermal stability of A4V SOD1 when binding to Cys111 only. We have developed a A4V SOD1 differential scanning fluorescence-based assay on a C6S mutation background that is effective in assessing suitability of compounds. Crystallographic data show that the selenium atom of these compounds binds covalently to A4V SOD1 at Cys111 at the dimer interface, resulting in stabilisation. This together with chemical amenability for hit expansion of ebselen and its on-target SOD1 pharmacological chaperone activity holds remarkable promise for structure-based therapeutics for MND using ebselen as a template.

Combined intramuscular and intraspinal transplant of bone marrow cells improves neuromuscular function in the SOD1G93A mice.

BACKGROUND: The simultaneous contribution of several etiopathogenic disturbances makes amyotrophic lateral sclerosis (ALS) a fatal and challenging disease. Here, we studied two different cell therapy protocols to protect both central and peripheral nervous system in a murine model of ALS. METHODS: Since ALS begins with a distal axonopathy, in a first assay, we performed injection of bone marrow cells into two hindlimb muscles of transgenic SOD1G93A mice. In a second study, we combined intramuscular and intraspinal injection of bone marrow cells. Fluorescence-activated cell sorting was used to assess the survival of the transplanted cells into the injected tissues. The mice were assessed from 8 to 16 weeks of age by means of locomotion and electrophysiological tests. After follow-up, the spinal cord was processed for analysis of motoneuron survival and glial cell reactivity. RESULTS: We found that, after intramuscular injection, bone marrow cells were able to engraft within the muscle. However, bone marrow cell intramuscular injection failed to promote a general therapeutic effect. In the second approach, we found that bone marrow cells had limited survival in the spinal cord, but this strategy significantly improved motor outcomes. Moreover, we also found that the dual cell therapy tended to preserve spinal motoneurons at late stages of the disease and to reduce microgliosis, although this did not prolong mice survival. CONCLUSION: Overall, our findings suggest that targeting more than one affected area of the motor system at once with bone marrow cell therapy results in a valuable therapeutic intervention for ALS.

Anti-oxidative and anti-adipogenic effects of caffeine in an in vitro model of Graves' orbitopathy.

Oxidative stress and adipogenesis play key roles in the pathogenesis of Graves' orbitopathy (GO). In this study, the therapeutic effects of caffeine on the reduction of oxidative stress and adipogenesis were evaluated in primary cultured GO orbital fibroblasts in vitro. Orbital fibroblasts were cultured from orbital connective tissues obtained from individuals with GO. Intracellular reactive oxygen species (ROS) levels induced by hydrogen peroxide or cigarette smoke extract and the expression of anti-oxidative enzymes were measured after caffeine treatment. After adipogenic differentiation and caffeine treatment, cells were stained with Oil Red O and the levels of peroxisome proliferator activator γ (PPARγ), C/EBPα, and C/EBPß were determined by western blot analysis. Hydrogen peroxide and cigarette smoke extract increased the levels of intracellular ROS and anti-oxidative enzymes, which decreased in a dose-dependent manner upon pretreatment with caffeine in GO orbital fibroblasts. Oil Red-O staining results revealed a decrease in lipid droplets; furthermore, PPARγ, C/EBPα, and C/EBPß protein expression levels were inhibited upon treatment with caffeine during adipocyte differentiation. In conclusion, caffeine decreased oxidative stress and adipogenesis in GO orbital fibroblasts in vitro. These findings may contribute to the development of new types of caffeine-containing pharmacological agents for use in the management of GO.

Low-dose alcohol ameliorated homocysteine-induced anxiety-related behavior via attenuating oxidative stress in mice.

Recent studies showed that homocysteine (Hcy) levels were obviously elevated in patients with anxiety, furthermore, oxidative stress and inflammation were closely linked with Hcy-related damage. Despite alcohol exposure has differential effects on different forms of anxiety, the role of alcohol on anxiety-related behavior induced by high Hcy levels is still not entirely clear. The present study investigated the protective potential of low-dose alcohol against homocysteine-induced anxiety-related behavior and explored the possible underlying mechanisms. Mice were administered intragastrically with methionine (2.0 g/kg/day) or alcohol (0.6 g/kg/day). After 21 days of administration, the anxiety-related behavior was evaluated through open field (OF) and elevated plus maze (EPM) tests, and the variations of oxidative stress and inflammation levels were measured. The results of OF and EPM tests showed that the anxiety-related behavior in mice was prevented by alcohol treatment. Alcohol lowered the elevated serum Hcy levels and alleviated the damage of hippocampal tissues in hyperhomocysteinemia (HHcy) mice. Meanwhile, the superoxide dismutase (SOD) activity of the hippocampal tissues enhanced, and the malondialdehyde (MDA) concentration of the hippocampal tissues and the serum interleukin-1ß (IL-1ß) level decreased. In addition, after administering alcohol, the increase of superoxide dismutase 1 (SOD1), heme oxygenase 1 (HO-1) protein expression and the decrease of IL-1ß protein expression were also detected in HHcy mice hippocampal tissues. Taken together, low-dose alcohol significantly ameliorated the Hcy-induced anxiety-related behavior in mice, which might be related to SOD1 and HO-1 upregulation and IL-1ß downregulation.

The psychoactive cathinone derivative pyrovalerone alters locomotor activity and decreases dopamine receptor expression in zebrafish (Danio rerio).

INTRODUCTION: Pyrovalerone (4-methyl-ß-keto-prolintane) is a synthetic cathinone (beta-keto-amphetamine) derivative. Cathinones are a concern as drugs of abuse, as related street drugs such as methylenedioxypyrovalerone have garnered significant attention. The primary mechanism of action of cathinones is to inhibit reuptake transporters (dopamine and norepinephrine) in reward centers of the central nervous system. METHODS: We measured bioenergetic, behavioral, and molecular responses to pyrovalerone (nM-µM) in zebrafish to evaluate its potential for neurotoxicity and neurological impairment. RESULTS: Pyrovalerone did not induce any mortality in zebrafish larvae over a 3- and 24-hr period; however, seizures were prevalent at the highest dose tested (100 µM). Oxidative phosphorylation was not affected in the embryos, and there was no change in superoxide dismutase 1 expression. Following a 3-hr treatment to pyrovalerone (1-100 µM), larval zebrafish (6d) showed a dose-dependent decrease (70%-90%) in total distance moved in a visual motor response (VMR) test. We interrogated potential mechanisms related to the hypoactivity, focusing on the expression of dopamine-related transcripts as cathinones can modulate the dopamine system. Pyrovalerone decreased the expression levels of dopamine receptor D1 (~60%) in larval zebrafish but did not affect the expression of tyrosine hydroxylase, dopamine active transporter, or any other dopamine receptor subunit examined, suggesting that pyrovalerone may regulate the expression of dopamine receptors in a specific manner. DISCUSSION: Further studies using zebrafish are expected to reveal new insight into molecular mechanisms and behavioral responses to cathinone derivates, and zebrafish may be a useful model for understanding the relationship between the dopamine system and bath salts.

Effects of low-dose oleuropein diet supplementation on the oxidative status of skeletal muscles and plasma hormonal concentration of growing broiler chickens.

1. Oleuropein (Ole) is a major phenolic compound in Olea europaea, with anti-oxidative, anti-obesity, and anti-inflammatory properties. To explore the effect of Ole on the physiology and metabolism of poultry, this study, evaluated the effects of feeding low-dose Ole on the growth performance, metabolic hormonal status, muscle oxidative status in growing broiler chickens.2. Thirty-two 8-day-old chickens were assigned to four different treatments, and fed either 0 (control), 0.1, 0.5, or 2.5 ppm Ole-supplemented diets for 2 weeks.3. There were no differences in the body weight gain, feed consumption, and feed efficiency during the feeding periods between the groups tested. Birds fed Ole 0.5- and 2.5 ppm-supplemented diets exhibited a significant decrease in muscle carbonyl content compared to the control group. In the group fed Ole 0.5 ppm, the mRNA expression levels of mitochondrial ROS-reducing factors: avian uncoupling protein and manganese superoxide dismutase, as well as peroxisome proliferator-activated receptor γ coactivator 1-α, sirtuin-1 and -3 (each of which co-ordinately induce the transcription of the previous two factors) were upregulated compared to the control group, and the changes were independent of plasma noradrenaline and thyroid hormone levels. The group fed Ole-2.5 ppm did not show such transcriptional changes, but exhibited a higher corticosterone concentration.4. This study demonstrates that ingesting a low dose of Ole can reduce muscle oxidative damage, and that the suppression machinery may differ depending on the amount of Ole ingested by growing broiler chickens.

Rational design of linear tripeptides against the aggregation of human mutant SOD1 protein causing amyotrophic lateral sclerosis.

Formation of protein aggregation is considered a hallmark feature of various neurological diseases. Amyotrophic lateral sclerosis is one such devastating neurodegenerative disorder characterized by mutation in Cu/Zn superoxide dismutase protein (SOD1). In our study, we contemplated the most aggregated and pathogenic mutant A4V in a viewpoint of finding a therapeutic regime by inhibiting the formation of the aggregates with the aid of tripeptides since new perspectives in the field of drug design in the current era are being focused on peptide-based drugs. Reports from the experimental study have stipulated that the SOD1 derived peptide, "LSGDHCIIGRTLVVHEKADD" was found to have the inhibitory activity against aggregated SOD1 protein. Moreover, it was determined that the hexapeptide, "LSGDHC" was the key factor in inhibiting the aggregates of SOD1. Accordingly, we utilized the computerized algorithms and programs on determining the binding efficiency and inhibitory activity of hexapeptide on mutant SOD1. Following that, we incorporated a cutting-edge methodology with the use of molecular docking, affinity predictions, alanine scanning, steered molecular dynamics (SMD) and discrete molecular dynamics (DMD) in designing the de novo tripeptides, which could act against the aggregated mutant SOD1 protein. Upon examining the results from the various conformational studies, we identified that CGH had an enhanced binding affinity and inhibitory activity against the aggregated mutant SOD1 protein than other tripeptides and hexapeptide. Thus, our study could be a lead for state-of-the-art design in peptide-based drugs for doctoring the cureless ALS disorder.

Edaravone reduces Aß-induced oxidative damage in SH-SY5Y cells by activating the Nrf2/ARE signaling pathway.

AIMS: Edaravone potentially alleviates cognitive deficits in a mouse model of Alzheimer's disease (AD). However, the mechanism of edaravone in suppressing AD progression remains unclear. We aim to investigate the mechanism of edaravone in suppressing oxidative stress-mediated AD progression in vitro. MAIN METHODS: Human neuroblastoma SH-SY5Y cells were pretreated with different concentrations of edaravone prior to the induction by Aß25-35. Cell viability, apoptosis, reactive oxygen species, and expression of antioxidative response elements (ARE) including Nrf2, SOD, and HO-1 were assessed. KEY FINDINGS: The results showed that apoptosis and reactive oxygen species levels significantly increased in Aß25-35-treated cells, whereas the mRNA and protein levels of Nrf2, SOD and HO-1 decreased. The opposite changes were observed in cells that were pre-treated with edaravone, particularly at a concentration of 40 µM. Aß25-35-treatment suppressed Nrf2 expression and nuclear translocation were rescued by Edaravone. Genetic inhibition of Nrf2 greatly decreased the protective effect of edaravone against cell apoptosis and cytotoxicity induced by Aß25-35, accompanied by decreases in SOD and HO-1 expression. SIGNIFICANCE: Activation of the Nrf2/ARE signaling pathway may underlie the protective effects of edaravone against the oxidative damage associated with Alzheimer's disease.

Modulating P1 Adenosine Receptors in Disease Progression of SOD1G93A Mutant Mice.

Amyotrophic lateral sclerosis (ALS) is a fatal progressing neurodegenerative disease; to date, despite the intense research effort, only two therapeutic options, with very limited effects, are available. The purinergic system has been indicated as a possible new therapeutic target for ALS, but the results are often contradictory and generally confused. The present study was designed to determine whether P1 adenosine receptor ligands affected disease progression in a transgenic model of ALS. SOD1G93A mice were chronically treated, from presymptomatic stage, with a selective adenosine A2A receptor agonist (CGS21680), antagonist (KW6002) or the A1 receptor antagonist DPCPX. Body weight, motor performance and survival time were evaluated. The results showed that neither the stimulation nor the blockade of adenosine A2A receptors modified the progressive loss of motor skills or survival of mSOD1G93A mice. Conversely, blockade of adenosine A1 receptors from the presymptomatic stage significantly attenuated motor disease progression and induced a non-significant increase of median survival in ALS mice. Our data confirm that the modulation of adenosine receptors can elicit very different (and even opposite) effects during the progression of ALS course, thus strengthens the importance of further studies to elucidated their real therapeutic potential in this pathology.

Arsenic enhances cell death and DNA damage induced by ultraviolet B exposure in mouse epidermal cells through the production of reactive oxygen species.

BACKGROUND: Ultraviolet (UV)B radiation has long been considered a carcinogen in both epidemiological surveys and experimental studies. However, recent work has suggested that different dosages of UVB exert different influences on cells. There are also co-carcinogenesis factors such as arsenic that affect the role of UVB. AIM: To explore the co-carcinogenesis effect of UVB and arsenic on the mouse epidermal cell line JB6 and the mechanism underlying it. METHODS: Growth of JB6 cells was measured by MTT assay. We carried out a comet assay to determine the DNA damage caused by UVB and arsenic, and tested the expression of DNA repair protein by western blotting. Reactive oxygen species (ROS) were measured using DCF and DHE staining, and changes in antioxidant enzymes were assessed using western blotting. RESULTS: Viability assays showed that arsenic increased the UVB-induced death rate. Arsenic enhanced DNA damage caused by UVB both directly by injury to double-stranded DNA and indirectly by reducing the capability of DNA repair in JB6 cells. All of these effects are the results of increased ROS generation and reduced expression of the antioxidant enzyme superoxide dismutase (SOD)1. CONCLUSION: Arsenic was found to enhance UVB-induced production of ROS and to downregulate SOD1 expression, leading to DNA damage and apoptosis in mouse skin cells. The combination of arsenic and UVB exposure was found to differentially regulate the expression of SOD1 and SOD2.

Oxidant stress induction and signalling in xenografted (human breast cancer-tissues) plus estradiol treated or N-ethyl-N-nitrosourea treated female rats via altered estrogen sulfotransferase (rSULT1E1) expressions and SOD1/catalase regulations.

N-ethyl-N-nitrosourea (ENU) is highly used in rodent models of tumerogenesis/carcinogenesis. Xenografting human-cancer tissues/cells with estradiol (E2) treatment is also used to generate rodent-models of gynaecological cancers. The altered metabolic-redox environment leading to establishment of pre-tumorigenesis condition and their mechanism are less studied. Here, female Wister rats were treated with these drugs at their pre-tumerogenic dosage (one group ENU single intra-peritoneal dose of 90 mg/kg b.w. and another group were implanted with human breast tumor (stage-IIIB) and fed with 2.5 mg of 17ß-estradiol once in a week for 4 months). After 4 months, animals were sacrificed; their serum and liver tissues were tested. A brief comparison was made with a rat model (regarded as positive control) of toxicity induced by mutagenic environmental pollutant arsenic (0.6 ppm daily/4 weeks). The increase in serum alkaline phosphatase and glutamate-pyruvate transaminase suggests the possible organ toxicity is favoured by the increase in hepatic/systemic free radicals and oxidative stress in all drug application models. But the increase in the serum E2 level as noted in the ELISA data with impairment in the hepatic estrogen sulfotransferase (SULT1E1) protein expression (immuno-blot data) were noticed with interfered hepatic free-thiols only in ENU and xenograft-E2 group compared to arsenic group. It is also evident in the in vitro result from E2/GSH/NAC added hepatic slices with altered antioxidant regulations. Moreover, impairment in hepatic SOD1, catalase and glutathiole peroxidase activities (PAGEzymographic data), especially in the ENU-treated group makes them more vulnerable to the oxidative threat in creating pre-tumerogenic microenvironment. This is evident in the result of their higher DNA-damage and histological abnormalities. The Bioinformatics study revealed an important role of rSULT1E1 in the regulations of E2 metabolism. This study is important for the exploration of the pre-tumerogenic condition by ENU and E2 by impairing SULT1E1 expression and E2 regulations via oxidant-stress signalling. The finding may help to find new therapeutic-targets to treat gynaecological-cancers more effectively.

Post-exposure distribution of selenium and aluminum ions and their effects on superoxide dismutase activity in mouse brain.

The study was conducted to determine how aluminum (Al) and selenium (Se) ions alone and in combination affect superoxide dismutase (SOD) activity and to evaluate the distribution of these elements in the blood and the brain of laboratory mice. SOD activity in mouse brain was evaluated after single-time (within 24 h) and repeated (over 14 days) intraperitoneal (IP) injections of SeO32-, Al3+, and (SeO32-+Al3+) solutions. The control mice received IP injections of the same volume of saline. Aluminum concentration in mouse blood increased both after single-time and repeated injections of AlCl3 and the combined (AlCl3 + Na2SeO3) solutions. The concentration of Se increased in blood after single-time and repeated injections of Na2SeO3 and the combined (AlCl3 + Na2SeO3) solutions. After the single-time injection of the experimental solutions, the concentrations of both Al and Se in mouse brain remained at baseline, but after repeated injections of (AlCl3 + Na2SeO3) solutions increased aluminum concentration. A single IP injection of Al did not change SOD activity in mouse brain, while a single injection of Se or the Se + Al mixture decreased it. After 14 days, IP injections of Al or Se alone did not affect SOD activity, while their combination decreased it. Our results showed that Se ions decreased SOD activity in mouse brain after both single-time and repeated IP injections of selenium-containing solutions. The study failed to show a direct or linear effect of increased Al or Se concentrations on SOD activity in mouse brain.

Intravenous immunoglobulin ameliorates motor and cognitive deficits and neuropathology in R6/2 mouse model of Huntington's disease by decreasing mutant huntingtin protein level and normalizing NF-κB signaling pathway.

Huntington's disease (HD) is a fatal neurodegenerative disorder characterized by progressive movement disorders and cognitive deficits, which is caused by a CAG-repeat expansion encoding an extended polyglutamine (polyQ) tract in the huntingtin protein (HTT). Reduction of mutant HTT levels and inhibition of neuroinflammation has been proposed as a major therapeutic strategy in treating HD. Intravenous immunoglobulin (IVIg) therapy has been firmly established for the treatment of several autoimmune or inflammatory neurological diseases, either as adjunctive treatment or as first-line therapy. However, whether IVIg has therapeutic potential on HD remains unclear. Here we for the first time demonstrated that IVIg treatment remarkably rescued motor and cognitive deficits, prevented synaptic degeneration, attenuated neuroinflammation and oxidative stress in R6/2 mouse model. Further investigation showed that the beneficial effects of IVIg resulted from the reduced levels of mutant HTT and inhibition of NF-κB signalling pathway. These findings suggest that IVIg is a promising therapeutic potential for HD.

EZH2 Inhibition Ameliorates Transverse Aortic Constriction-Induced Pulmonary Arterial Hypertension in Mice.

Background: EPZ005687 is a selective inhibiter of methyltransferase EZH2. In this article, we investigated the protective role and mechanism of EPZ005687 in transverse aortic constriction-induced pulmonary arterial hypertension in mice. Methods: We assigned 15 (6-8 weeks old) male balb/c mice to 3 groups randomly: Sham control + DMSO group, TAC + DMSO group, and TAC + EPZ005687 group (10 mg kg-1, once a week for 4 weeks). On day 28 following TAC operation, the right ventricular systolic blood pressure (RVSBP) was measured, and lung tissues were collected for laboratory examinations (DHE, Western blot, real-time PCR, and ChIP). Results: Murine PAH model was successfully created by TAC operation as evidenced by increased RVSBP and hypertrophic right ventricle. Compared with the sham control, TAC-induced PAH markedly upregulated the expression of EZH2 and ROS deposition in lungs in PAH mice. The inhibiter of methyltransferase EZH2, EPZ005687 significantly inhibits the development of TAC-induced PAH in an EZH2-SOD1-ROS dependent manner. Conclusion: Our data identified that EZH2 serves a fundamental role in TAC-induced PAH, and administration of EPZ005687 might represent a novel therapeutic target for the treatment of TAC-induced PAH.

Modulation of nuclear factor-κB signaling and reduction of neural tube defects by quercetin-3-glucoside in embryos of diabetic mice.

BACKGROUND: Diabetes mellitus in early pregnancy increases the risk of birth defects in infants. Maternal hyperglycemia stimulates the expression of nitric oxide synthase 2, which can be regulated by transcription factors of the nuclear factor-κB family. Increases in reactive nitrogen species generate intracellular stress conditions, including nitrosative, oxidative, and endoplasmic reticulum stresses, and trigger programmed cell death (or apoptosis) in the neural folds, resulting in neural tube defects in the embryo. Inhibiting nitric oxide synthase 2 can reduce neural tube defects; however, the underlying mechanisms require further delineation. Targeting nitric oxide synthase 2 and associated nitrosative stress using naturally occurring phytochemicals is a potential approach to preventing birth defects in diabetic pregnancies. OBJECTIVE: This study aims to investigate the effect of quercetin-3-glucoside, a naturally occurring polyphenol flavonoid, in reducing maternal diabetes-induced neural tube defects in an animal model, and to delineate the molecular mechanisms underlying quercetin-3-glucoside action in regulating nitric oxide synthase 2 expression. STUDY DESIGN: Female mice (C57BL/6) were induced to develop diabetes using streptozotocin before pregnancy. Diabetic pregnant mice were administered quercetin-3-glucoside (100 mg/kg) daily via gavage feeding, introduction of drug to the stomach directly via a feeding needle, during neurulation from embryonic day 6.5-9.5. After treatment at embryonic day 10.5, embryos were collected and examined for the presence of neural tube defects and apoptosis in the neural tube. Expression of nitric oxide synthase 2 and superoxide dismutase 1 (an antioxidative enzyme) was quantified using Western blot assay. Nitrosative, oxidative, and endoplasmic reticulum stress conditions were assessed using specific biomarkers. Expression and posttranslational modification of factors in the nuclear factor-κB system were investigated. RESULTS: Treatment with quercetin-3-glucoside (suspended in water) significantly decreased neural tube defect rate and apoptosis in the embryos of diabetic mice, compared with those in the water-treated diabetic group (3.1% vs. 24.7%; P < .001). Quercetin-3-glucoside decreased the expression of nitric oxide synthase 2 and nitrosative stress (P < .05). It also increased the levels of superoxide dismutase 1 (P < .05), further increasing the antioxidative capacity of the cells. Quercetin-3-glucoside treatment also alleviated of endoplasmic reticulum stress in the embryos of diabetic mice (P < .05). Quercetin-3-glucoside reduced the levels of p65 (P < .05), a member of the nuclear factor-κB transcription factor family, but augmented the levels of the inhibitor of κBα (P < .05), which suppresses p65 nuclear translocation. In association with these changes, the levels of inhibitor of κB kinase-α and inhibitor of κBα phosphorylation were elevated (P < .05). CONCLUSION: Quercetin-3-glucoside reduces the neural tube defects rate in the embryos of diabetic dams. Quercetin-3-glucoside suppresses nitric oxide synthase 2 and increases superoxide dismutase 1 expression, leading to alleviation of nitrosative, oxidative, and endoplasmic reticulum stress conditions. Quercetin-3-glucoside may regulate the expression of nitric oxide synthase 2 via modulating the nuclear factor-κB transcription regulation system. Quercetin-3-glucoside, a naturally occurring polyphenol that has high bioavailability and low toxicity, is a promising candidate agent to prevent birth defects in diabetic pregnancies.

Pyrimethamine significantly lowers cerebrospinal fluid Cu/Zn superoxide dismutase in amyotrophic lateral sclerosis patients with SOD1 mutations.

OBJECTIVE: Cu/Zn superoxide dismutase (SOD1) reduction prolongs survival in SOD1-transgenic animal models. Pyrimethamine produces dose-dependent SOD1 reduction in cell culture systems. A previous phase 1 trial showed pyrimethamine lowers SOD1 levels in leukocytes in patients with SOD1 mutations. This study investigated whether pyrimethamine lowered SOD1 levels in the cerebrospinal fluid (CSF) in patients carrying SOD1 mutations linked to familial amyotrophic lateral sclerosis (fALS/SOD1). METHODS: A multicenter (5 sites), open-label, 9-month-duration, dose-ranging study was undertaken to determine the safety and efficacy of pyrimethamine to lower SOD1 levels in the CSF in fALS/SOD1. All participants underwent 3 lumbar punctures, blood draw, clinical assessment of strength, motor function, quality of life, and adverse effect assessments. SOD1 levels were measured in erythrocytes and CSF. Pyrimethamine was measured in plasma and CSF. Appel ALS score, ALS Functional Rating Scale-Revised, and McGill Quality of Life Single-Item Scale were measured at screening, visit 6, and visit 9. RESULTS: We enrolled 32 patients; 24 completed 6 visits (18 weeks), and 21 completed all study visits. A linear mixed effects model showed a significant reduction in CSF SOD1 at visit 6 (p < 0.001) with a mean reduction of 13.5% (95% confidence interval [CI] = 8.4-18.5) and at visit 9 (p < 0.001) with a mean reduction of 10.5% (95% CI = 5.2-15.8). INTERPRETATION: Pyrimethamine is safe and well tolerated in ALS. Pyrimethamine is capable of producing a significant reduction in total CSF SOD1 protein content in patients with ALS caused by different SOD1 mutations. Further long-term studies are warranted to assess clinical efficacy. Ann Neurol 2017;81:837-848.