Superoxide dismutase 3 prevents early stage diabetic retinopathy in streptozotocin-induced diabetic rat model.

PURPOSE: To identify the effects of superoxide dismutase (SOD)3 on diabetes mellitus (DM)-induced retinal changes in a diabetic rat model. METHODS: Diabetic models were established by a single intraperitoneal injection of streptozotocin (STZ) in Sprague-Dawley rats. After purification of the recombinant SOD3, intravitreal injection of SOD3 was performed at the time of STZ injection, and 1 and 2 weeks following STZ injection. Scotopic and photopic electroretinography (ERG) were recorded. Immunofluorescence staining with ɑ-smooth muscle actin (SMA), glial fibrillary acidic protein (GFAP), pigment epithelium-derived factor (PEDF), Flt1, recoverin, parvalbumin, extracellular superoxide dismutase (SOD3), 8-Hydroxy-2'deoxyguanosine (8-OHdG) and tumor necrosis factor-ɑ (TNF-ɑ) were evaluated. RESULTS: In the scotopic ERG, the diabetic group showed reduced a- and b-wave amplitudes compared with the control group. In the photopic ERG, b-wave amplitude showed significant (p < 0.0005) reduction at 8 weeks following DM induction. However, the trend of a- and b-wave reduction was not evident in the SOD3 treated group. GFAP, Flt1, 8-OHdG and TNF-ɑ immunoreactivity were increased, and ɑ-SMA, PEDF and SOD3 immunoreactivity were decreased in the diabetic retina. The immunoreactivity of these markers was partially recovered in the SOD3 treated group. Parvalbumin expression was not decreased in the SOD3 treated group. In the diabetic retinas, the immunoreactivity of recoverin was weakly detected in both of the inner nuclear layer and inner plexiform layer compared to the control group but not in the SOD3 treated group. CONCLUSIONS: SOD3 treatment attenuated the loss of a/b-wave amplitudes in the diabetic rats, which was consistent with the immunohistochemical evaluation. We also suggest that in rod-dominant rodents, the use of blue on green photopic negative response (PhNR) is effective in measuring the inner retinal function in animal models of diabetic retinopathy. SOD3 treatment ameliorated the retinal Müller cell activation in diabetic rats and pericyte dysfunction. These results suggested that SOD3 exerted protective effects on the development of diabetic retinopathy.

Novel pharmacological effects of lecithinized superoxide dismutase on ischemia/reperfusion injury in the kidneys of mice.

Renal ischemia/reperfusion (I/R) injury is a major clinical problem because it can cause acute kidney injury (AKI) or lead to the transition from AKI to chronic kidney disease (CKD). Oxidative stress, which involves the production of reactive oxygen species (ROS), plays an important role in the development and exacerbation of I/R-induced kidney injury. However, we have previously reported that lecithinized superoxide dismutase (PC-SOD), a SOD derivative with high tissue affinity and high stability in plasma, has beneficial effects in various disease models because of its inhibitory effect on ROS production. Therefore, we aimed to determine the effects of intravenous PC-SOD administration in a mouse model of renal injury induced by I/R. PC-SOD markedly ameliorated the I/R-induced increases in markers of renal damage (urea nitrogen, creatinine, neutrophil gelatinase-associated lipocalin, and interleukin-6) and tubular necrosis 48 h after the intervention. We also found that PC-SOD significantly ameliorated the I/R-induced increase in ROS production, using an ex vivo imaging system. Furthermore, PC-SOD inhibited the increases in expression of markers of fibrosis (α-smooth muscle actin and collagen 1A1) 96 h after, and renal fibrosis 25 days after I/R was induced. Finally, we found that PC-SOD ameliorated the I/R-induced AKI in mice with high-fat diet-induced prediabetes. These results suggest that PC-SOD inhibits AKI and the transition from AKI to CKD through the inhibition of ROS production. Therefore, we believe that PC-SOD may represent an effective therapeutic agent for I/R-induced renal injury.

Antioxidant status and their enhancements strategies for water stress tolerance in chickpea / Status antioxidante e suas estratégias de aprimoramento para tolerância ao estresse hídrico no grão-de-bico

Water stress executes severe influences on the plant growth and development through modifying physio-chemical properties. Therefore, a field experiment was designed to evaluate the antioxidant status and their enhancements strategies for water stress tolerance in chickpea on loam and clay loam soils under agro-ecological conditions of Arid Zone Research Institute, Bahawalpur (29.3871 °N, 71.653 °E) and Cholistan farm near Derawer (28.19°N, 71.80°E) of Southern Punjab, Pakistan during winter 2014-15. Experimental treatments comprised of two chickpea cultivars i.e. Bhakhar 2011 (drought tolerant) and DUSHT (drought sensitive), two water stress levels i.e. water stress at flowering stage and water stress at flowering + pod formation + grain filling stage including well watered (control) and two exogenous application of osmoprotectants i.e. glycine betaine (GB) 20 ppm and proline 10 uM including distilled water (control). Results indicated that water stress at various growth stages adversely affects the growth, yield and quality attributes of both chickpea cultivars. Exogenous application of GB and proline improved the growth, yield and quality parameters of both chickpea cultivars even under water stress conditions. However, superior results were obtained with exogenously applied GB on Bhakhar 2011 under well-watered conditions. Similarly, foliar spray of GB on chickpea cultivar Bhakhar 2011 under stress at flowering + pod formation + grain filling stage produced maximum superoxide dismutase, peroxidase and catalase contents. These results suggested that application of GB mitigates the adverse effects of water stress and enhanced tolerance in chickpea mainly due to higher antioxidant enzymes activity, demonstrating the protective measures of plant cells in stress condition. Hence, antioxidants status might be a suitable method for illustrating water stress tolerance in chickpea.

O estresse hídrico exerce fortes influências no crescimento e no desenvolvimento das plantas, modificando as propriedades físico-químicas. Portanto, a presente atividade de pesquisa foi projetada para avaliar o status antioxidante e suas estratégias de aprimoramento para tolerância ao estresse hídrico no grão-de-bico em condiçõesa groecológicas, no Instituto de Pesquisa da Zona Árida, Bahawalpur (29.3871 ° N, 71.653 ° E) e fazenda do Cholistan, perto de Derawer (28.19 ° N, 71,80 ° E), no sul de Punjab, Paquistão, durante Rabi 2014-15. Tratamentos experimentais compostos de dois genótipos de grão-de-bico, como Bhakhar 2011 (tolerante à seca) e DUSHT (sensível à seca), dois níveis de estresse hídrico, ou seja, estresse hídrico no estágio de floração, estresse hídrico na fase de floração e estresse hídrico na fase de floração + formação de vagem + estágio de enchimento de grãos, incluindo água bem controlada (controle) e duas aplicações exógenas de osmoprotetores, isto é, glicina betaína 20 ppm e prolina 10 uM, incluindo água destilada (controle). Os resultados indicaram que o estresse hídrico em vários estágios de crescimento afeta negativamente os atributos de crescimento, rendimento e qualidade de ambas as cultivares de grão-de-bico. A aplicação exógena de glicina betaína e prolina melhorou os parâmetros de crescimento, rendimento e qualidade de ambos os genótipos de grão- de-bico, mesmo sob condições de estresse hídrico. No entanto, resultados superiores foram obtidos com glicina betaína aplicada exogenamente em Bhakhar 2011, em condições bem regadas. Além disso, o spray foliar de glicina betaína na cultivar de grão-de-bico Bhakhar 2011, sob estresse na fase de floração + formação de vagem + enchimento de grãos, produziu o máximo de superóxido dismutase, peroxidase e catalase. Esses resultados sugeriram que a aplicação de glicina betaína atenua os efeitos adversos do estresse hídrico e aumenta a [...].

The Effect of Superoxide Dismutase on Inhibition of Acute Kidney Injury Induced by Sepsis Based on Kidney Tissue Histology and Murine Sepsis Score.

Sepsis is one of the leading causes contributing to the incidence of acute kidney injury (AKI). Oxidative stress can be used as the main approach against sepsis-induced AKI. One of the primary antioxidants that plays a role in warding off oxidative stress is superoxide dismutase (SOD). This research aimed to observe the effect of antioxidant SOD in inhibiting sepsis in AKI based on kidney tissue histopathology. The research method was an experimental laboratory with a post-test-only control group design. Twenty-five adult male rats aged 12-16 weeks, weighing between 200 and 250 g, were randomly divided into five groups: Group I, as a positive control, where rats were injected with lipopolysaccharides (LPS); Group II, as a negative control; Group III, as treatment 1, where rats were injected with LPS and administered orally with SOD (Glisodin®) 250 IU daily; Group IV, as treatment 2, where rats were injected with LPS and administered orally with SOD (Glisodin®) 500 IU daily; and Group V, as treatment 2, where rats were injected with LPS and administered orally with SOD (Glisodin®) 1000 IU daily. Rats were administered with SOD (Glisodin®) by oral gavage with a flexible feeding tube for 16 weeks, given once daily in the morning, and then injected with LPS of 10 mg/kg body weight. Glisodin SOD had a significant effect on murine sepsis score (MSS). MSS influenced the tubular injury score linearly. We conclude that the optimal dose of SOD at 1000 IU for inhibiting sepsis-induced AKI incidence is compared to SOD at a dose of 250 and 500 IU. The antioxidant effect of SOD can prevent sepsis-induced AKI with oxidative stress events.

Construction and potential application of bacterial superoxide dismutase expressed in Bacillus subtilis against mycotoxins.

Oxidative stress, which could be evoked by numerous inducements including mycotoxins like deoxynivalenol (DON), cause severe damages to organisms. Antioxidants are promising protectants against oxidative stress that could be applied in pharmaceutical, cosmetic, and food and feed industries. In this study, a thermostable and acidophilic superoxide dismutase (AaSOD) was used to develop an antioxidant product that can potentially protect organisms from oxidative stress related damages. The enzyme was successfully expressed as an extracelluar protein in Bacillus subtilis with a high yield. To obtain a feasible protocol for industrial production of AaSOD, the fermentation mediums that are commonly used for culturing B. subtilis were screened, the feasibility of expressing AaSOD without antibiotic as selection pressure was confirmed, and the effect of using lactose as an inducer instead of isopropyl-ß-d-thiogalactoside (IPTG) was investigated. Batch fermentation was conducted to validate the optimized conditions for AaSOD production, and 6530 U mL-1 of SOD activity was obtained in the fermentation broth. The dry powder product of AaSOD with an activity of 22202 U g-1 was prepared by spray-drying and was administrated on zebrafish to test its function as a protectant against DON, and thus gained a significant redress of the reactive oxygen species (ROS) accumulation induced by DON. Taken together, this study provides a feasible protocol to prepare the AaSOD-based antioxidant product that is potentially applied in livestock industry.

Effects of 60-Day Saccharomyces boulardii and Superoxide Dismutase Supplementation on Body Composition, Hunger Sensation, Pro/Antioxidant Ratio, Inflammation and Hormonal Lipo-Metabolic Biomarkers in Obese Adults: A Double-Blind, Placebo-Controlled Trial.

In animals it has been demonstrated that Saccharomyces boulardii and Superoxide Dismutase (SOD) decrease low-grade inflammation and that S. boulardii can also decrease adiposity. The purpose of this study was to evaluate the effect of a 60-day S. boulardii and SOD supplementation on circulating markers of inflammation, body composition, hunger sensation, pro/antioxidant ratio, hormonal, lipid profile, glucose, insulin and HOMA-IR, in obese adults (BMI 30-35 kg/m2). Twenty-five obese adults were randomly assigned to intervention (8/4 women/men, 57 ± 8 years) or Placebo (9/4 women/men, 50 ± 9 years). Intervention group showed a statistically significant (p < 0.05) decrease of body weight, BMI, fat mass, insulin, HOMA Index and uric acid. Patients in intervention and control groups showed a significant decrease (p < 0.05) of GLP-1. Intervention group showed an increase (p < 0.05) of Vitamin D as well. In conclusion, the 60-day S. boulardii-SOD supplementation in obese subjects determined a significant weight loss with consequent decrease on fat mass, with preservation of fat free mass. The decrease of HOMA index and uric acid, produced additional benefits in obesity management. The observed increase in vitamin D levels in treated group requires further investigation.

Efficacy and Safety of the Combination of Superoxide Dismutase, Alpha Lipoic Acid, Vitamin B12, and Carnitine for 12 Months in Patients with Diabetic Neuropathy.

AIM: To investigate the efficacy of Superoxide Dismutase, Alpha Lipoic Acid, Acetyl L-Carnitine, and Vitamin B12 (B12) in one tablet in Diabetic Neuropathy (DN). PATIENTS-METHODS: In this prospective, double-blind, placebo-controlled study, 85 patients with Diabetes Mellitus Type 2 (DMT2) were randomly assigned, either to receive the combination of four elements (active group, n = 43), or placebo (n = 42) for 12 months. We used the Michigan Neuropathy Screening Instrument Questionnaire and Examination (MNSIQ and MNSIE), measured the vibration perception threshold (BIO), and Cardiovascular Autonomic Reflex Tests (CARTs). Nerve function was assessed by DPN Check [sural nerve conduction velocity (SNCV) and amplitude (SNAP)]. Pain (PS) and quality of life (QL) questionnaires were administered. RESULTS: At follow-up, BIO, MNSIQ, QL, PAIN, and SNCV, SNAP, and B12 levels had significantly improved inactive group (p <0.001, p <0.001, p <0.001, p <0.001, p = 0.027, p = 0.031, and p <0.001 respectively), whereas the inplacebo group MCR (mean circular resultant) and PAIN deteriorated (p <0.001, p <0.001). The changes in MNSIQ, QL, SNCV, BIO, and PAIN differed significantly between groups (p <0.001, p <0.001, p = 0.031, p <0.001, and p <0.001 respectively). CONCLUSIONS: The combination of the four elements in one tablet for 12 months in patients with DMT2 improved all indices of peripheral neuropathy, including SNAP and SNCV, pain, and Quality of Life perception, except CARTs and MNSIE.

Advanced biomimetic nanoreactor for specifically killing tumor cells through multi-enzyme cascade.

Although the enzyme catalytic nanoreactors reported so far have achieved excellent therapeutic efficacy, how to accurately exert enzyme activity in the tumor microenvironment to specifically kill tumor cells and avoid systemic oxidative damage would be an inevitable challenge for catalytic nanomedicine. At the present study, we fabricate an advanced biomimetic nanoreactor, SOD-Fe0@Lapa-ZRF for tumor multi-enzyme cascade delivery that combined specifically killing tumor cells and protect cells from oxidative stress. Methods: We first synthesized the FeNP-embedded SOD (SOD-Fe0) by reduction reaction using sodium borohydride. Next, SOD-Fe0 and Lapa cargo were encapsulated in ZIF-8 by self-assembly. In order to protect the cargo enzyme from digestion by protease and prolong blood circulating time, SOD-Fe0@Lapa-Z was further cloaked with RBC membrane and functionalized with folate targeting, resulting in the final advanced biomimetic nanoreactor SOD-Fe0@Lapa-ZRF. Results: Once internalized, ZIF-8 achieves pH-triggered disassembly in weakly acidic tumor microenvironment. The released SOD-Fe0 and Lapa were further endocytosed by tumor cells and the Lapa produces superoxide anion (O2-•) through the catalysis of NQO1 that is overexpressed in tumor cells, while O2-• is converted to H2O2 via SOD. At this time, the released ferrous ions from SOD-Fe0 and H2O2 are further transformed to highly toxic hydroxyl radicals (•OH) for specifically killing tumor cells, and there was no obvious toxicological response during long-term treatment. Importantly, SOD-Fe0@Lapa-ZRF enhanced the normal cell's anti-oxidation ability, and thus had little effect on the secretion of TNF-α, IL-6 and IL-1ß pro-inflammatory cytokines, while effectively reversed the decreased activity of T-SOD and GSH-Px and remained stable MDA content after tumor treatment. In vitro and in vivo results indicate that the tumor microenvironment-responsive release multi-enzyme cascade have high tumor specificity and effective anti-tumor efficacy, and can protect cells from oxidative stress damage. Conclusion: The biomimetic nanoreactor will have a great potential in cancer nanomedicine and provide a novel strategy to regulate oxidative stress.

Poly(N-Isopropyl-Acrylamide)/Poly(γ-Glutamic Acid) Thermo-Sensitive Hydrogels Loaded with Superoxide Dismutase for Wound Dressing Application.

INTRODUCTION: Chronic trauma repair is an important issue affecting people's healthy lives. Thermo-sensitive hydrogel is injectable in situ and can be used to treat large-area wounds. In addition, antioxidants play important roles in promoting wound repair. METHODS: The purpose of this research was to prepare a novel thermo-sensitive hydrogel-poly(N-isopropyl-acrylamide)/poly(γ-glutamic acid) (PP) loaded with superoxide dismutase (SOD) to improve the effect for trauma treatment. The micromorphology of the hydrogel was observed by scanning electron microscope and the physical properties were measured. The biocompatibility of hydrogel was evaluated by MTT experiment, and the effect of hydrogel on skin wound healing was evaluated by in vivo histological staining. RESULTS: Gelling behavior and differential scanning calorimeter outcomes showed that the PP hydrogels possessed thermo-sensitivity at physiological temperature and the phase transformation temperature was 28.2°C. The high swelling rate and good water retention were conducive to wound healing. The activity of SOD in vitro was up to 85% at 10 h, which was advantageous to eliminate the superoxide anion. MTT assay revealed that this hydrogel possessed good biocompatibility. Dressings of PP loaded with SOD (SOD-PP) had a higher wound closure rate than other treatments in vivo in diabetic rat model. DISCUSSION: The SOD-PP thermo-sensitive hydrogels can effectively promote wound healing and have good application prospects for wound repair.

Superoxide Dismutase 3 Inhibits LL-37/KLK-5-Mediated Skin Inflammation through Modulation of EGFR and Associated Inflammatory Cascades.

The expressions of LL-37 and KLK-5 were found to be altered in various dermatoses, including atopic dermatitis, psoriasis, and rosacea. However, the downstream inflammatory effect of LL-37 and KLK-5 is not as well studied. In addition, there is little high-quality evidence for the treatment of LL-37- and KLK-5-mediated inflammation. In this study, we investigated the effect of superoxide dismutase 3 (SOD3) on LL-37- or KLK-5-induced skin inflammation in vitro and in vivo and its underlying anti-inflammatory mechanisms. Our data showed that SOD3 significantly reduced both LL-37- and KLK-5-induced expression of pro-inflammatory mediators and suppressed the activation of EGFR, protease-activated receptor 2, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3, and p38/extracellular signal-regulated kinase signaling pathways in human keratinocytes. Moreover, SOD3 suppressed LL-37-induced expression of inflammatory mediators, reactive oxygen species production, and p38/extracellular signal-regulated kinase activation in mast cells. In addition, subcutaneous injection of KLK-5 in SOD3 knockout mice exhibited erythema with increased epidermal thickness, mast cell and neutrophil infiltration, expression of inflammatory mediators, and activation of EGFR, protease-activated receptor 2, nucleotide-binding domain, leucine-rich-containing family, pyrin domain-containing-3, and downstream mitogen-activated protein kinase pathways. However, treatment with SOD3 in SOD3 knockout mice rescued KLK-5-induced inflammatory cascades. Similarly, KLK-5-induced inflammation in wild-type mice was also ameliorated when treated with SOD3. Taken together, our data suggest that SOD3 is a potentially effective therapy for both LL-37-and KLK-5-induced skin inflammation.

Effects of TAT-SOD at Acupoints on Essential Hypertension by Monitoring Meridians Electrical Potential.

OBJECTIVES: To investigate the effect on essential hypertension of the topical application of TAT-Cu, Zn-superoxide dismutase (TAT-SOD) at left acupoint Zusanli (ST 36), and to observe whether the change of electrical potential difference (EPD) can be related to the change of blood pressure. METHODS: Sixteen patients with essential hypertension and 16 healthy subjects were included in the study. EPD between the left acupoints of Yanglingquan (GB 34) and Qiuxu (GB 40) was firstly screened out for the EPD detection. An intracellular superoxide quenching enzyme, TAT-SOD, was topically applied to the acupoint ST 36 within an area of 1 cm2 once a day, and the influence on EPD was investigated. The dosage applied to TAT-SOD group (n=8) was 0.2 mL of 3000 U/mL TAT-SOD cream prepared by adding purified TAT-SOD to a vehicle cream, while placebo group (n=8) used the vehicle cream instead. The left acupoints of Yanglingquan (GB 34) and Qiuxu (GB 40) were selected for EPD measurement after comparing EPD readings between 5 acupoints on each of all 12 meridians. RESULTS: EPDs between the left acupoints of GB 34 and GB 40 for 16 patients of essential hypertension and 16 healthy subjects were 44.9±6.4 and 5.6±0.9 mV, respectively. Daily application of TAT-SOD for 15 days at ST 36 of essential hypertension patients significantly decreased systolic blood pressure (SBP) and diastolic blood pressure (DBP) of 179.6 and 81.5 mm Hg to 153.1 and 74.1 mm Hg, respectively. Responding to the change in blood pressure, EPD between the left acupoints of GB 34 and GB 40 also declined from 44.4 to 22.8 mV with the same trend. No change was observed with SBP, DBP and EPD between the left acupoints of GB 34 and GB 40 with the daily application of the placebo cream. CONCLUSION: Enzymatic scavenging of the intracellular superoxide at ST 36 proved to be effective in decreasing SBP and DBP. The results reconfirm the involvement of superoxide anions and its transportation along the meridians, and demonstrate that EPD between acupoints may be an indicator to reflect its functioning status. Moreover, preliminary results suggest a close correlation between EPD and blood pressure readings, implying a possibility of using EPD as a sensitive parameter for blood pressure and to monitor the effect of antihypertensive treatment.

A new recombinant MS-superoxide dismutase alleviates 5-fluorouracil-induced intestinal mucositis in mice.

Intestinal mucositis is a common side effect of anticancer regimens that exerts a negative impact on chemotherapy. Superoxide dismutase (SOD) is a potential therapy for mucositis but efficient product is not available because the enzyme is degraded following oral administration or induces an immune reaction after intravascular infusion. Multi-modified Stable Anti-Oxidant Enzymes® (MS-AOE®) is a new recombinant SOD with better resistance to pepsin and trypsin. We referred it as MS-SOD to distinguish from other SODs. In this study we investigated its potential to alleviate 5-FU-induced intestinal injury and the mechanisms. An intestinal mucositis model was established in C57/BL6 mice by 5-day administration of 5-FU (50 mg/kg every day, ip). MS-SOD (800 IU/10 g, ig) was given once daily for 9 days. 5-FU caused severe mucositis with intestinal morphological damage, bodyweight loss and diarrhea; MS-SOD significantly decreased the severity. 5-FU markedly increased reactive oxygen species (ROS) and inflammatory cytokines in the intestine which were ameliorated by MS-SOD. Furthermore, MS-SOD modified intestinal microbes, particularly reduced Verrucomicrobia, compared with the 5-FU group. In Caco2 cells, MS-SOD (250-1000 U/mL) dose-dependently decreased tBHP-induced ROS generation. In RAW264.7 cells, MS-SOD (500 U/mL) had no effect on LPS-induced inflammatory cytokines, but inhibited iNOS expression. These results demonstrate that MS-SOD can scavenge ROS at the initial stage of injury, thus play an indirect role in anti-inflammatory and barrier protein protection. In conclusion, MS-SOD attenuates 5-FU-induced intestinal mucositis by suppressing oxidative stress and inflammation, and influencing microbes. MS-SOD may exert beneficial effect in prevention of intestinal mucositis during chemotherapy in clinic.

Neutral Sphingomyelinase Modulation in the Protective/Preventive Role of rMnSOD from Radiation-Induced Damage in the Brain.

Studies on the relationship between reactive oxygen species (ROS)/manganese superoxide dismutase (MnSOD) and sphingomyelinase (SMase) are controversial. It has been demonstrated that SMase increases the intracellular ROS level and induces gene expression for MnSOD protein. On the other hand, some authors showed that ROS modulate the activation of SMase. The human recombinant manganese superoxide dismutase (rMnSOD) exerting a radioprotective effect on normal cells, qualifies as a possible pharmaceutical tool to prevent and/or cure damages derived from accidental exposure to ionizing radiation. This study aimed to identify neutral SMase (nSMase) as novel molecule connecting rMnSOD to its radiation protective effects. We used a new, and to this date, unique, experimental model to assess the effect of both radiation and rMnSOD in the brain of mice, within a collaborative project among Italian research groups and the Joint Institute for Nuclear Research, Dubna (Russia). Mice were exposed to a set of minor γ radiation and neutrons and a spectrum of neutrons, simulating the radiation levels to which cosmonauts will be exposed during deep-space, long-term missions. Groups of mice were treated or not-treated (controls) with daily subcutaneous injections of rMnSOD during a period of 10 days. An additional group of mice was also pretreated with rMnSOD for three days before irradiation, as a model for preventive measures. We demonstrate that rMnSOD significantly protects the midbrain cells from radiation-induced damage, inducing a strong upregulation of nSMase gene and protein expression. Pretreatment with rMnSOD before irradiation protects the brain with a value of very high nSMase activity, indicating that high levels of activity might be sufficient to exert the rMnSOD preventive role. In conclusion, the protective effect of rMnSOD from radiation-induced brain damage may require nSMase enzyme.

Combined addition of superoxide dismutase, catalase and glutathione peroxidase improves quality of cooled stored stallion semen.

During cold storage stallion spermatozoa experience undergo oxidative stress, which can impair sperm function and fertilizing capacity. Superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPX) are the main endogenous enzymatic antioxidants in stallion seminal plasma, and counteract reactive oxygen species. Semen dilution reduces the endogenous antioxidant concentrations. The aim of this study was to investigate whether addition of 15 IU/mL each of SOD, CAT, and GPX to diluted stallion semen would ameliorate a reactive oxygen-mediated decrease in semen quality during 72 h of storage at 5 °C. Ejaculates (n = 7) were divided in two aliquots and diluted in INRA 96 without (control) or with addition of antioxidants. Semen analysis was performed at the time of dilution and every 24 h during chilled storage. Antioxidant supplementation completely inhibited the storage-dependent increase in activated caspase 3 (P < 0.05). Concomitantly, the antioxidant-supplemented samples had a greater percentage of viable, motile and rapidly moving sperm than control samples after 72 h storage (P < 0.05). The DNA damage, as evaluated by TUNEL assay and SCSA, increased with storage time (P < 0.05). Antioxidant supplementation did not prevent, but did significantly reduce the increase in DNA strand breakage. The results indicate part of the intrinsic apoptotic pathway leading to effector caspase activation was inhibited, although an activation of molecules with endonuclease activity still occurred. In conclusion, adding equal concentrations of SOD, CAT and GPX to a semen extender suppressed caspase-3 activation and improved preservation of stallion sperm motility and viability during 72 h of storage at 5 °C.

Enrichment of superoxide dismutase 2 in glioblastoma confers to acquisition of temozolomide resistance that is associated with tumor-initiating cell subsets.

BACKGROUND: Intratumor subsets with tumor-initiating features in glioblastoma are likely to survive treatment. Our goal is to identify the key factor in the process by which cells develop temozolomide (TMZ) resistance. METHODS: Resistant cell lines derived from U87MG and A172 were established through long-term co-incubation of TMZ. Primary tumors obtained from patients were maintained as patient-derived xenograft for studies of tumor-initating cell (TIC) features. The cell manifestations were assessed in the gene modulated cells for relevance to drug resistance. RESULTS: Among the mitochondria-related genes in the gene expression databases, superoxide dismutase 2 (SOD2) was a significant factor in resistance and patient survival. SOD2 in the resistant cells functionally determined the cell fate by limiting TMZ-stimulated superoxide reaction and cleavage of caspase-3. Genetic inhibition of the protein led to retrieval of drug effect in mouse study. SOD2 was also associated with the TIC features, which enriched in the resistant cells. The CD133+ specific subsets in the resistant cells exhibited superior superoxide regulation and the SOD2-related caspase-3 reaction. Experiments applying SOD2 modulation showed a positive correlation between the TIC features and the protein expression. Finally, co-treatment with TMZ and the SOD inhibitor sodium diethyldithiocarbamate trihydrate in xenograft mouse models with the TMZ-resistant primary tumor resulted in lower tumor proliferation, longer survival, and less CD133, Bmi-1, and SOD2 expression. CONCLUSION: SOD2 plays crucial roles in the tumor-initiating features that are related to TMZ resistance. Inhibition of the protein is a potential therapeutic strategy that can be used to enhance the effects of chemotherapy.

The effects of superoxide dismutase-rich melon pulp concentrate on inflammation, antioxidant status and growth performance of challenged post-weaning piglets.

Piglets can often suffer impaired antioxidant status and poor immune response during post-weaning, especially when chronic inflammation takes place, leading to lower growth rates than expected. Oral administration of dietary antioxidant compounds during this period could be a feasible way to balance oxidation processes and increase health and growth performance. The aim of the trial was to study the effects of an antioxidant feed supplement (melon pulp concentrate) that contains high concentration of the antioxidant superoxide dismutase (SOD) on inflammation, antioxidant status and growth performance of lipopolysaccharide (LPS) challenged weaned piglets. In total, 48 weaned piglets were individually allocated to four experimental groups in a 2×2 factorial design for 29 days. Two different dietary treatments were adopted: (a) control (CTR), fed a basal diet, (b) treatment (MPC), fed the basal diet plus 30 g/ton of melon pulp concentrate. On days 19, 21, 23 and 25 half of the animals within CTR and MPC groups were subjected to a challenge with intramuscular injections of an increasing dosage of LPS from Escherichia coli (serotype 0.55:B5) (+) or were injected with an equal amount of PBS solution (-). Blood samples were collected at the beginning of the trial and under the challenge period for interleukin 1ß, interleukin 6, tumour necrosis factor α, haptoglobin, plasma SOD activity, total antioxidant capacity, reactive oxygen species, red blood cells and plasma resistance to haemolysis, and 8-oxo-7, 8-dihydro-2'-deoxyguanosine. Growth performance was evaluated weekly. A positive effect of melon pulp concentrate was evidenced on total antioxidant capacity, half-haemolysis time of red blood cells, average daily gain (ADG) and feed intake, while LPS challenge increased pro-inflammatory cytokines and haptoglobin serum concentrations, with a reduced feed intake and gain : feed (G : F). The obtained results show that oral SOD supplementation with melon pulp concentrate ameliorates the total antioxidant capacity and the half-haemolysis time in red blood cell of post-weaning piglets, with positive results on growing performance.

Liquid chromatography-tandem mass spectrometry of recombinant human extracellular superoxide dismutase (rhSOD3) in mouse plasma and its application to pharmacokinetic study.

The antioxidant enzyme human extracellular superoxide dismutase (SOD3) is a promising biopharmaceutical candidate for the treatment of various diseases. To support the early development of SOD3 as a biopharmaceutical, a simple, sensitive, and rapid liquid chromatography tandem mass spectrometry procedure was developed and validated for the determination of SOD3 levels in the plasma of ICR mice. After purification with Ni-NTA magnetic beads and digestion with trypsin, SOD3 signature peptides and internal standard signature peptide (ISP) were separated via high performance liquid chromatography using a Zorbax C18 column (2.1 × 50 mm, 3.5 µm) and a mobile phase consisting of 10 mM ammonium formate, 0.1% formic acid, and acetonitrile. The analyte and ISP were detected via a tandem mass spectrometer in electrospray ionization and multiple reaction monitoring modes to select both the signature peptide for SOD3 at m/z 669 to 969 and the ISP at m/z 655 to 941 in the positive ion mode. The calibration curves were linear (r > 0.99) between 5 and 1000 µg/mL with a lower limit of quantification of 5 µg/mL. The relative standard deviation ranged from 3.08 to 8.84% while the relative error ranged from -0.13 to -9.56%. This method was successfully applied to a preclinical pharmacokinetic study of SOD3 in male ICR mice.

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.

MnSOD is implicated in accelerated wound healing upon Negative Pressure Wound Therapy (NPWT): A case in point for MnSOD mimetics as adjuvants for wound management.

Negative Pressure Wound Therapy (NPWT), a widely used modality in the management of surgical and trauma wounds, offers clear benefits over conventional wound healing strategies. Despite the wide-ranging effects ascribed to NPWT, the precise molecular mechanisms underlying the accelerated healing supported by NPWT remains poorly understood. Notably, cellular redox status-a product of the balance between cellular reactive oxygen species (ROS) production and anti-oxidant defense systems-plays an important role in wound healing and dysregulation of redox homeostasis has a profound effect on wound healing. Here we investigated potential links between the use of NPWT and the regulation of antioxidant mechanisms. Using patient samples and a rodent model of acute injury, we observed a significant accumulation of MnSOD protein as well as higher enzymatic activity in tissues upon NPWT. As a proof of concept and to outline the important role of SOD activity in wound healing, we replaced NPWT by the topical application of a MnSOD mimetic, Mn(III) meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP5+, MnE, BMX-010, AEOl10113) in the rodent model. We observed that MnE is a potent wound healing enhancer as it appears to facilitate the formation of new tissue within the wound bed and consequently advances wound closure by two days, compared to the non-treated animals. Taken together, these results show for the first time a link between NPWT and regulation of antioxidant mechanism through the maintenance of MnSOD activity. Additionally this discovery outlined the potential role of MnSOD mimetics as topical agents enhancing wound healing.

Spatially controlled assembly of affinity ligand and enzyme cargo enables targeting ferritin nanocarriers to caveolae.

One of the goals of nanomedicine is targeted delivery of therapeutic enzymes to the sub-cellular compartments where their action is needed. Endothelial caveolae-derived endosomes represent an important yet challenging destination for targeting, in part due to smaller size of the entry aperture of caveolae (ca. 30-50 nm). Here, we designed modular, multi-molecular, ferritin-based nanocarriers with uniform size (20 nm diameter) for easy drug-loading and targeted delivery of enzymatic cargo to these specific vesicles. These nanocarriers targeted to caveolar Plasmalemmal Vesicle-Associated Protein (Plvap) deliver superoxide dismutase (SOD) into endosomes in endothelial cells, the specific site of influx of superoxide mediating by such pro-inflammatory signaling as some cytokines and lipopolysaccharide (LPS). Cell studies showed efficient internalization of Plvap-targeted SOD-loaded nanocarriers followed by dissociation from caveolin-containing vesicles and intracellular transport to endosomes. The nanocarriers had a profound protective anti-inflammatory effect in an animal model of LPS-induced inflammation, in agreement with the characteristics of their endothelial uptake and intracellular transport, indicating that these novel, targeted nanocarriers provide an advantageous platform for caveolae-dependent delivery of biotherapeutics.