Expression Levels Of Superoxide Dismutase Research Articles

Identification and validation of stable reference genes for RT-qPCR analyses of Kobresia littledalei seedlings

BackgroundKobreisa littledalei, belonging to the Cyperaceae family is the first Kobresia species with a reference genome and the most dominant species in Qinghai-Tibet Plateau alpine meadows. It has several resistance genes which could be used to breed improved crop varieties. Reverse Transcription Quantitative Real-Time Polymerase Chain Reaction (RT-qPCR) is a popular and accurate gene expression analysis method. Its reliability depends on the expression levels of reference genes, which vary by species, tissues and environments. However, K.littledalei lacks a stable and normalized reference gene for RT-qPCR analysis.ResultsThe stability of 13 potential reference genes was tested and the stable reference genes were selected for RT-qPCR normalization for the expression analysis in the different tissues of K. littledalei under two abiotic stresses (salt and drought) and two hormonal treatments (abscisic acid (ABA) and gibberellin (GA)). Five algorithms were used to assess the stability of putative reference genes. The results showed a variation amongst the methods, and the same reference genes showed tissue expression differences under the same conditions. The stability of combining two reference genes was better than a single one. The expression levels of ACTIN were stable in leaves and stems under normal conditions, in leaves under drought stress and in roots under ABA treatment. The expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression was stable in the roots under the control conditions and salt stress and in stems exposed to drought stress. Expression levels of superoxide dismutase (SOD) were stable in stems of ABA-treated plants and in the roots under drought stress. Moreover, RPL6 expression was stable in the leaves and stems under salt stress and in the stems of the GA-treated plants. EF1-alpha expression was stable in leaves under ABA and GA treatments. The expression levels of 28 S were stable in the roots under GA treatment. In general, ACTIN and GAPDH could be employed as housekeeping genes for K. littledalei under different treatments.ConclusionThis study identified the best RT-qPCR reference genes for different K. littledalei tissues under five experimental conditions. ACTIN and GAPDH genes can be employed as the ideal housekeeping genes for expression analysis under different conditions. This is the first study to investigate the stable reference genes for normalized gene expression analysis of K. littledalei under different conditions. The results could aid molecular biology and gene function research on Kobresia and other related species.

A novel histone deacetylase inhibitor Se-SAHA attenuates isoproterenol-induced heart failure via antioxidative stress and autophagy inhibition

Heart failure is associated with histone deacetylase (HDAC) regulation of gene expression, the inhibition of which is thought to be beneficial for heart failure therapy. Here, we explored the cardioprotective effects and underlying mechanism of a novel selenium-containing HDAC inhibitor, Se-SAHA, on isoproterenol (ISO)-induced heart failure. We found that pretreatment with Se-SAHA attenuated ISO-induced cardiac hypertrophy and fibrosis in neonatal rat ventricular myocytes (NRVMs). Se-SAHA significantly attenuated the generation of ISO-induced reactive oxygen species (ROS) and restored the expression levels of superoxide dismutase 2 (SOD2) and heme oxygenase-1 (HO-1) in vitro. Furthermore, Se-SAHA pretreatment prevented the accumulation of autophagosomes. Se-SAHA reversed the high expression of HDAC1 and HDAC6 induced by ISO incubation. However, after the addition of the HDAC agonist, the effect of Se-SAHA on blocking autophagy was inhibited. Using ISO-induced mouse models, cardiac ventricular contractile dysfunction, hypertrophy, and fibrosis was reduced treated by Se-SAHA. In addition, Se-SAHA inhibited HDAC1 and HDAC6 overexpression in ISO-treated mice. Se-SAHA treatment significantly increased the activity of SOD2 and improved the ability to eliminate free radicals. Se-SAHA hindered the excessive levels of the microtubule-associated protein 1 light chain 3 (LC3)-II and Beclin-1 in heart failure mice. Collectively, our results indicate that Se-SAHA exerts cardio-protection against ISO-induced heart failure via antioxidative stress and autophagy inhibition.

WTAP regulates the production of reactive oxygen species, promotes malignant progression, and is closely related to the tumor microenvironment in glioblastoma.

RNA modifications have been substantiated to regulate the majority of physiological activities in the organism, including the metabolism of reactive oxygen species (ROS), which plays an important role in cells. As for the effect of RNA modification genes on ROS metabolism in glioblastoma (GBM), it has not been studied yet. Therefore, this study aims to screen the RNA modification genes that are most related to ROS metabolism and explore their effects on the biological behavior of GBM in vitro. Here, an association between WTAP and ROS metabolism was identified by bioinformatics analysis, and WTAP was highly expressed in GBM tissue compared with normal brain tissue, which was confirmed by western blotting and immunohistochemical staining. When using a ROS inducer to stimulate GBM cells in the WTAP overexpression group, the ROS level increased more significantly and the expression levels of superoxide dismutase 1 (SOD1) and catalase (CAT) also increased. Next, colony formation assay, wound healing assay, and transwell assay were performed to investigate the proliferation, migration, and invasion of GBM cells. The results showed that WTAP, as an oncogene, promoted the malignant progression of GBM cells. Functional enrichment analysis predicted that WTAP was involved in the regulation of tumor/immune-related functional pathways. Western blotting was used to identify that WTAP had a regulatory effect on the phosphorylation of PI3K/Akt signaling. Finally, based on functional enrichment analysis, we further performed immune-related analysis on WTAP. In conclusion, this study analyzed WTAP from three aspects, which provided new ideas for the treatment of GBM.

Imperatorin inhibits oxidative stress injury and neuroinflammation via the PI3K/AKT signaling pathway in the MPTP-induced Parkinson's disease mouse.

Parkinson's disease (PD) is a disorder of neurodegeneration. Imperatorin is an active natural furocoumarin characterized by antioxidant, anti-inflammatory, and potent vasodilatory properties. Therefore, we aimed to investigate the biological functions of imperatorin and its mechanisms against PD progression. C57BL/6 mice were intraperitoneally injected with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP; 30 mg/kg) daily for 5 consecutive days to mimic PD conditions in vivo. The MPTP-induced PD model mice were intraperitoneally injected with imperatorin (5 mg/kg) for 25 consecutive days after MPTP administration. The motor and cognitive functions of mice were examined by rotarod test, hanging test, narrow beam test and Morris water maze test. After analysis of MWM test, the expression levels of tyrosine hydroxylase and Iba-1 in the substantia nigra pars compacta were measured by immunohistochemistry staining, immunofluorescence staining and western blotting. The expression levels of striatal dopamine and its metabolite 3,4-dihydroxyphenylacetic acid were also measured. The protein levels of inducible nitric-oxide synthase, cyclooxygenase-2, phosphorylated phosphatidylinositol 3-kinase (PI3K) and phosphorylated protein kinase B (Akt) in the mouse striatum were estimated by western blotting. The expression levels of proinflammatory cytokines including tumor necrosis factor, interleukin (IL)-1β and IL-6 in the mouse striatum were measured by ELISA kits. The expression levels of superoxide dismutase, malondialdehyde and glutathione in the mouse midbrains were measured with commercially available kits. TUNEL staining was performed to identify the apoptosis of midbrain cells. Histopathologic changes in the mouse striata were assessed by hematoxylin-eosin staining. Imperatorin treatment markedly improved spatial learning and memory abilities of MPTP-induced PD mice. The MPTP-induced dopaminergic neuron loss in the mouse striata was inhibited by imperatorin. Imperatorin also suppressed neuroinflammation and neuronal oxidative stress in the midbrains of MPTP-induced PD mice. Mechanistically, imperatorin treatment inhibited the MPTP-induced reduction in the PI3K/Akt pathway. Imperatorin treatment can prevent dopaminergic neuron degeneration and improve cognitive functions via its potent antioxidant and anti-inflammatory properties in an MPTP-induced PD model in mice by regulating the PI3K/Akt pathway.

The role of the SIRT1-BMAL1 pathway in regulating oxidative stress in the early development of ischaemic stroke

Oxidative stress is the primary cause of ischaemic stroke and is closely related to circadian rhythm. However, the mechanism by which circadian rhythm regulates oxidative stress in ischaemic stroke remains elusive. The Silent Information Regulator 1 (SIRT1) controls circadian rhythm by activating the transcription of the circadian clock core protein Basic Helix-Loop-Helix ARNT Like 1 (BMAL1) through deacetylation. Studies have shown that the SIRT1-BMAL1 pathway can regulate oxidative stress. To investigate its correlation with oxidative stress, we examined the expression levels and influencing factors of SIRT1-BMAL1 at different times in ischaemic stroke patients and analyzed their clinical indexes, oxidative stress, and inflammatory factor indicators. The expression levels of oxidative stress and inflammatory factor indicators, including malondialdehyde (MDA), superoxide dismutase (SOD), interleukin-6 (IL-6), and tumor necrosis factor-a (TNF-α), SIRT1, and BMAL1, were detected in ischaemic stroke patients within 4.5 h of onset and in non-stroke patients. Patients were divided into four subgroups based on onset time: subgroup 1 (0:00–05:59); subgroup 2 (06:00–11:59); subgroup 3 (12:00–17: 59); and subgroup 4 (18:00–23:59). Our results showed higher MDA, IL-6, and TNF-α levels, and lower SOD, SIRT1, and BMAL1 levels in ischaemic stroke patients compared to control patients (P < 0.05). Among the four subgroups, the content of MDA, IL-6, and TNF-α was highest in patients with ischaemic stroke onset from subgroup 2 (06:00–11:59), while the expression levels of SOD, BMAL1, and SIRT1 were lowest in patients with ischaemic stroke in subgroup 2. Additionally, myeloperoxidase (MPO) reached the highest value showing the same trends consistent with MDA, IL-6, and TNF-ɑ and opposite trends consistent with SOD, BMAL1, and SIRT1. However, triglycerides (TGs), total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), immediate blood glucose, immediate diastolic blood pressure, immediate systolic blood pressure, and homocysteine (HCY) did not show any statistically significant circadian rhythm changes (P > 0.05). Our findings suggest that the SIRT1-BMAL1 pathway may be involved in early oxidative stress in ischaemic stroke, which may be related to MPO.

5-Aminolevulinic acid treatment mitigates pesticide stress in bean seedlings by regulating stress-related gene expression and retrotransposon movements

Overdoses of pesticides lead to a decrease in the yield and quality of plants, such as beans. The unconscious use of deltamethrin, one of the synthetic insecticides, increases the amount of reactive oxygen species (ROS) by causing oxidative stress in plants. In this case, plants tolerate stress by activating the antioxidant defense mechanism and many genes. 5-Aminolevulinic acid (ALA) improves tolerance to stress by acting exogenously in low doses. There are many gene families that are effective in the regulation of this mechanism. In addition, one of the response mechanisms at the molecular level against environmental stressors in plants is retrotransposon movement. In this study, the expression levels of superoxide dismutase (SOD), ascorbate peroxidase (APX), catalase (CAT), glutathione reductase (GR), and stress-associated protein (SAP) genes were determined by Q-PCR in deltamethrin (0.5 ppm) and various doses (20, 40, and 80 mg/l) of ALA-treated bean seedlings. In addition, one of the response mechanisms at the molecular level against environmental stressors in plants is retrotransposon movement. It was determined that deltamethrin increased the expression of SOD (1.8-fold), GPX (1.4-fold), CAT (2.7-fold), and SAP (2.5-fold) genes, while 20 and 40 mg/l ALA gradually increased the expression of these genes at levels close to control, but 80 mg/l ALA increased the expression of these genes almost to the same level as deltamethrin (2.1-fold, 1.4-fold, 2.6-fold, and 2.6-fold in SOD, GPX, CAT, and SAP genes, respectively). In addition, retrotransposon-microsatellite amplified polymorphism (REMAP) was performed to determine the polymorphism caused by retrotransposon movements. While deltamethrin treatment has caused a decrease in genomic template stability (GTS) (27%), ALA treatments have prevented this decline. At doses of 20, 40, and 80 mg/L of ALA treatments, the GTS ratios were determined to be 96.8%, 74.6%, and 58.7%, respectively. Collectively, these findings demonstrated that ALA has the utility of alleviating pesticide stress effects on beans.

Rutin improves salt stress tolerance in Zea mays L. by modulating osmolytes accumulation and antioxidant capacity

Maize seedlings were grown hydroponically to study the effect of exogenous rutin (Rut) on changes in the stress parameters (thiobarbituric acid reactive substances (TBARS), hydrogen peroxide (H2O2), total chlorophyll), water status (leaf relative water content (RWC), osmolytes such as proline, total soluble sugar), and activities of the main antioxidant enzymes (superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD)) in seedlings under salt stress. After 21 days of growth, plants were applied with Rut as foliar spray pretreatments for 24 hours, and after that, seedlings were exposed to osmotic stress induced by 100 and 200 mM NaCI in the Hoagland’s Solution for 72 hours. Six treatments with three replicates were tested, including a control (basal nutrients without NaCl or Rut), 150 mM NaCl, 200 mM NaCI, Rut, Rut+150 mM NaCl, and Rut+200 mM NaCl. Plant leaves were harvested 25 days after treatments and assayed for the above analysis. Exogenous Rut significantly decreased TBARS and H2O2 contents in leaves of salt-stressed seedlings compared to salt stresses, enhanced the level of osmolytes, leaf RWC, activities of SOD, CAT, APX, and POD, and relative expression levels of SOD, CAT1, and APX1. Besides, the effects were more hopeful under 150 mM NaCl stress. As a result, findings from the study present reveal the effect of Rut on salt stress tolerance in maize seedlings under different osmotic stress. Here, it was clear that Rut played an active role in stress-alleviating. This application under salt stress can be useful in developing salt stress tolerance in crops for the agriculture sector.

Growth performance, hematological and immune parameters, and mRNA levels of cytokines and antioxidant-related genes in rainbow trout (Oncorhynchus mykiss) fed on Pediocuccus pentosaceus and/or ferulic acid

The present study investigated the growth performance, blood parameters, and mRNA levels of growth, cytokines and antioxidant-related factors in rainbow trout (Oncorhynchus mykiss) that were fed with Pediocuccus pentosaceus (PP) and/or ferulic acid (FA). For this aim, 180 fish with initial body weight of 94.30 ± 1.77 g were fed on four experimental diets including the control (C), 108 CFU g−1P. pentosaceus (PP), 100 mg kg−1 ferulic acid (FA) and combination of both elements (108 CFU g−1 + 100 mg kg−1, PP + FA) for 8 weeks. The final weight, weight gain (WG), and specific growth rate (SGR) were found to be markedly affected by PP and FA, with the most substantial growth being observed in the PP + FA treatment (P < 0.05). The experimental diets had a significant impact on the feed conversion ratio (FCR), with the PP + FA treatment resulting in lowest FCR (P < 0.05). The hematological indices, namely RBC, WBC, Hct, and Hb, were not observed to be significantly impacted by the experimental diets (P > 0.05). The levels of total immunoglobulin, ACH50, and lysozyme were observed to be enhanced by the dietary FA and PP, with statistical significance (P < 0.05). The activity of Malondialdehyde (MDA) was found to be declined by the experimental diets, and its lowest value was considerably observed in the PP + FA treatment when compared to the control group (P < 0.05). It was seen that the fish which were provided with diets supplemented with PP and/or FA exhibited noteworthy increase in the activity of catalase (CAT), superoxide dismutase (SOD), and glutathione peroxidase (GPx), as compared to the control group (P < 0.05). The treatments which were fed with diets supplemented with PP + FA showed marked up-regulation in the expression of immune-regulatory genes, specifically IL1β, IL6, IL-8, and TNF-α, in contrast to the control group (P < 0.05). Significant upregulations were observed in the relative expression levels of SOD, CAT, GPx, and Glutathione reductase (GSR) genes in the experimental treatments that were provided with PP and/or FA (P < 0.05). In conclusion, it was shown that these dietary supplements at the optimal dosage have the potential to enhance growth, innate immunity, and antioxidant factors, genes associated with immunity and antioxidant systems in rainbow trout. Both feed supplements showed synergetic effects and it is recommended to employ these compounds simultaneously for sustainable aquaculture.

Oleic Acid Activates Mitochondrial Energy Metabolism and Reduces Oxidative Stress Resistance in the Pancreatic β-Cell Line INS-1.

Elevated concentration of saturated fatty acids in plasma adversely affects pancreatic β-cells, but the effects of unsaturated fatty acids are controversial. In this study, we examined the effects of oleic acid (OA), a monounsaturated fatty acid, on mitochondrial function, which is important for insulin secretion, using INS-1 cells, a pancreatic β-cell line derived from rats. Observations of mitochondrial membrane potential and intracellular ATP concentration showed that the electron transport chain was enhanced and ATP production increased in cells treated with OA, indicating that the response that occurs from sensing an increase in glucose concentration to the production of ATP was accelerated. Measurements of intracellular reactive oxygen species (ROS) indicated that the rate of increase in ROS after glucose stimulation was significantly higher in OA-treated cells. The mRNA expression levels of superoxide dismutase 1 and 2, which are responsive to ROS and other substances, were significantly increased in OA 1-d treated cells, but decreased in OA 7-d treated cells. It can be inferred that continued exposure to high concentrations of OA reduced ROS processing capacity and increased intracellular ROS levels. The mRNA expression of apoptosis-inducing enzyme Caspase-3 was significantly increased in OA-treated cells, although its activity was not high. However, the apoptosis induction rate after H2O2 stimulation was significantly higher in OA-treated cells. The high OA environment was shown to promote mitochondrial energy metabolism, leading to an increase in glucose sensitivity and a decrease in oxidative stress resistance.

Cuttlefish ink nanoparticles against oxidative stress: Alleviation of TBHP-induced oxidative damage in Caco-2 cells and DSS-induced ulcerative colitis in C57BL/6

In this study, we investigated the protective effects of cuttlefish ink nanoparticles (CINP) against dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) and tert-butyl hydroperoxide (TBHP)-induced oxidative stress in human colon adenocarcinoma cells (Caco-2) and their underlying mechanisms. In vitro results showed that CINP reduced apoptosis, reactive oxygen species (ROS) production, and mitochondrial membrane potential. It also inhibited the expression levels of the oxidation indicators lactate dehydrogenase and malondialdehyde and increased the expression levels of superoxide dismutase (SOD) and catalase (CAT) in TBHP-induced Caco-2 cells. In vivo experiments showed that CINP ameliorated DSS-induced weight loss, colon shortening, and histopathological changes. In addition, CINP increased the enzymatic activities of SOD, CAT, and glutathione peroxidase and the levels of the anti-inflammatory factor interleukin-10. It also inhibited the expression of pro-inflammatory factors interleukin-6, tumor necrosis factor, and interleukin-1β. In summary, CINP attenuated TBHP-induced Caco-2 oxidative stress and DSS-induced UC through its antioxidant effects.

The effect of melatonin on physiological, biochemical, and enzymatic properties and the expression of antioxidant genes under different irrigation regimes in wheat

A split-plot experiment was conducted based on a randomized complete block design with four replications in two years to study the effect of melatonin on the biochemical properties and the gene expression related to antioxidant enzyme activity in bread wheat. Irrigation levels (normal (FC = 80%)), mild stress (FC = 60%), and severe stress (FC = 40%)) were assigned to the main plots, and melatonin foliar applications (zero, 50, 100, 150, and 200 μM) were assigned to Subplots. Results showed that, with the intensification of water stress, the 1000 kernel weight decreased, and the activity of ascorbate peroxidase enzyme and flavonoid content increased. Also, the level of 100 μM melatonin had the highest 1000 kernel weight, flavonoid content, and ascorbate peroxidase enzyme activity.  In this study, the highest number of grains per spike, biological yield, grain yield, chlorophyll a, chlorophyll b, carotenoids, and the lowest amount of malondialdehyde recorded for foliar treatment with 50 μM of melatonin under normal irrigation conditions and the highest proline content, total phenol, superoxide dismutase, and catalase were allocated to the 100 μM melatonin foliar treatment under severe water stress conditions. The synergistic effect of water deficit stress and melatonin foliar application increased the activity and expression level of genes related to antioxidant enzymes. So, the content of superoxide dismutase (21.30% and 65.16% respectively) and catalase (50.60% and 54.44% respectively) enzyme activity increased significantly under 100- and 150-mM melatonin foliar application in water severe water stress compared to the corresponding control treatment. Furthermore, mentioned melatonin levels increased the gene expression levels of superoxide dismutase (16.67% and 38.19% respectively), ascorbate peroxides (73.76% and 47.57% respectively), polyphenol oxidase (39.32 and 51.15%) and catalase (39.95% and 50.0% respectively) under an extreme water shortage compared with corresponding control treatment. In general, the application of 100- and 150-mM melatonin induced resistance to water deficit stress in wheat by increasing the expression of antioxidant genes.

Exogenous Phytosulfokine α (PSKα) Alleviates Chilling Injury of Kiwifruit by Regulating Ca2+ and Protein Kinase-Mediated Reactive Oxygen Species Metabolism.

Kiwifruit fruit stored at low temperatures are susceptible to chilling injury, leading to rapid softening, which therefore affects storage and marketing. The effect of 150 nM mL-1 of exogenous phytosulfokine α (PSKα) on reactive oxygen species (ROS) metabolism, Ca2+ signaling, and signal-transducing MAPK in kiwifruit, stored at 0 °C for 60 days, was investigated. The results demonstrated that PSKα treatment effectively alleviated chilling injury in kiwifruit, with a 15% reduction in damage compared to the control on day 60. In addition, PSKα enhanced the activities and gene expression levels of superoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), glutathione reductase (GR), Ca2+-ATPase, and mitogen-activated protein kinase (MAPK). In contrast, the activities and gene expression levels of NADPH oxidase (NOX) were inhibited, leading to a lower accumulation of O2- and H2O2, which were 47.2% and 42.2% lower than those in the control at the end of storage, respectively. Furthermore, PSKα treatment enhanced the calmodulin (CaM) content of kiwifruit, which was 1.41 times that of the control on day 50. These results indicate that PSKα can mitigate chilling injury and softening of kiwifruit by inhibiting the accumulation of ROS, increasing antioxidant capacity by inducing antioxidant enzymes, activating Ca2+ signaling, and responding to MAPK protein kinase. The present results provide evidence that exogenous PSKα may be taken for a hopeful treatment in alleviating chilling injury and maintaining the quality of kiwifruit.

Exosomes derived from human dermal fibroblasts protect against UVB‑induced skin photoaging.

Exosomes are used as innovative treatment options for repairing skin defects, such as aging, atopic dermatitis and wounds. However, the effects of exosomes obtained from human foreskin fibroblasts BJ‑5ta (BJ‑5ta Exo) on ultravioletB (UVB)‑mediated photoaging have not been previously reported, at least to the best of our knowledge. Therefore, the present study aimed to investigate the anti‑photoaging effects of BJ‑5ta Exo on UVB radiation in human skin fibroblasts and SKH‑1 hairless mice. The results revealed that BJ‑5ta Exo decreased the production of reactive oxygen species and inhibited the decrease in the expression levels of superoxide dismutase 1 and 2, glutathione peroxidase and catalase following UVB exposure. In addition, BJ‑5ta Exo attenuated the decrease in nuclear factor erythroid 2‑related factor 2 levels induced by UVB rays, indicating its scavenging activity against oxidative stress. Moreover, BJ‑5ta Exo inhibited the UVB‑induced increase in the levels of γH2AX, p53/21 and cleaved PARP, whereas it promoted DNA double‑strand break repair through radiation sensitive 52 and effectively activated the TGF‑β1/Smad pathway. BJ‑5ta Exo also protected against UVB‑induced senescence, as indicated by the downregulation in the levels of senescence‑associated β‑galactosidase and p16. In a mouse model of photoaging, BJ‑5ta Exo prevented the UVB‑induced increase in transepidermal water loss, wrinkle formation and MMP‑1 expression, while also suppressing the UVB‑mediated decrease in collagen typeI and elastin levels in the dorsal skin. Overall, the findings of the present study suggest that BJ‑5ta Exo represent an effective anti‑photoaging agent, which can be used as a component in cosmetic products.

Multilineage differentiation potential in the infant adipose- and umbilical cord-derived mesenchymal stem cells.

This study aims to compare the biological properties of infant adipose-derived mesenchymal stem cells (infant ADSCs) from excised polydactyly fat tissue and umbilical cord-derived mesenchymal stem cells (UCSCs) in terms of proliferation and differentiation capabilities. The proliferation of infant ADSCs and UCSCs was analyzed by determining the fold changes of cell numbers and doubling time periods. The state of senescence and replicative stress was compared by analyzing the expression of age-related genes, senescence-associated β-galactosidase (SA-β-gal) staining, and phosphorylated histone variant H2AX (γH2AX) immunofluorescence staining. The expression levels of superoxide dismutase ( SODs ) and genes related to multilineage differentiation were analyzed using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Differentiation levels were determined using histochemical staining, immunohistochemical staining, and immunofluorescence staining. Infant ADSCs exhibited higher proliferation rates and expression levels of SOD1 , SOD2 , and SOD3 at passages 3-5 compared with UCSCs. Senescence related genes ( p16 , p21 , and p53 ), SA-β-gal staining, and replicative stress analysis were reduced in infant ADSCs. The expression levels of chondrogenic genes ( COL2 and COL10 ), osteogenic genes ( RUNX2 and ALP ), adipogenic genes ( LPL ), and hepatogenic genes ( ALB and TAT ) in infant ADSC-differentiated cells were significantly higher than those in UCSCs. Histochemical and immunofluorescence staining confirmed these results. Only the expression levels of tenogenic genes ( MMP3 , DCN , and COL3 ) in infant ADSC-differentiated cells were lower than those in UCSCs. Infant ADSCs exhibit higher proliferation rates, reduced cellular senescence and replicative stress, better antioxidative activity, and higher differentiation potential toward chondrogenic, osteogenic, adipogenic and hepatogenic lineages than UCSCs.

Thymosin beta-4 participate in antibacterial immunity and wound healing in black tiger shrimp, Penaeus monodon

Thymosin beta-4 (Tβ4) is a ubiquitous protein with multiple and diverse intracellular and extracellular functions in vertebrates, which play fundamental roles in innate immune against pathogens and wound healing. In this study, the full-length cDNA of Tβ4 was cloned from Penaeus monodon (designated as PmTβ4), using the technology of rapid amplification of cDNA ends (RACE). The cDNA of PmTβ4 was 1361 bp with an open reading frame (ORF) of 501 bp, which encoding a polypeptide of 166 amino acid. The Quantitative Real-time PCR (qRT-PCR) analysis results showed that PmTβ4 was ubiquitously expressed in all the tested shrimp tissues, with the highest expression level was detected in the hemolymph, while the lowest expression level in the muscle. The expression level of PmTβ4 was significantly up-regulated in hepatopancreas after challenged by Vibrio parahaemolyticus, Vibrio harveyi and Staphylococcus aureus. In vitro antimicrobial test showed that the recombinant protein of PmTβ4 (rPmTβ4) had broad-spectrum of antimicrobial activity, which could inhibit both the growth of gram-negative bacteria and gram-positive bacteria, including Vibrio vulnificus, V. parahaemolyticus, Streptococcus agalactiae, S. aureus and Aeromonas hydrophila. Moreover, rPmTβ4 had a certain binding ability to different bacteria, and this binding ability exhibits a strong dose-dependent effect. In vivo, PmTβ4 could facilitate external bacterial clearance in shrimp, and have beneficial to shrimp survival post V. parahaemolyticus infection. Furthermore, wound-healing assay was carried out to study the role of PmTβ4 in the process of wound healing. The results showed that the PmTβ4 expression was significantly up-regulated by injury treatment, and exerted positive effects to promote wound healing. In addition, PmTβ4 can significantly increase the expression level of superoxide dismutase (SOD) and Catalase (CAT) after injury treatment in shrimp, which would involve in scavenging reactive oxygen species (ROS) caused by the wound. In conclusion, these results indicated that PmTβ4 may play important roles in antibacterial immunity and wound healing in Penaeus monodon.

Lysine-Specific Demethylase 4D Is Critical for the Regulation of the Cell Cycle and Antioxidant Capacity in Goat Fibroblast Cells.

Oxidative damage to skin fibroblast cells is a causative factor in many skin diseases. Previous studies have reported that lysine-specific demethylase 4D (Kdm4d) is involved in DNA replication, but its role on antioxidant capacity remains unclear. In the present study, we used goat fibroblast cells (GFCs) as the research model and identified 504 up-regulated and 1013 down-regulated genes following the knockdown of Kdm4d, respectively. The down-regulated genes of this enzyme were found to be enriched in the cell cycle, DNA replication, mitotic processes, and the oxidative phosphorylation pathway, as previously revealed from gene ontology (GO), Kyoto encyclopedia of genes and genomes (KEGG), and gene set enrichment analysis (GSEA), suggesting vital roles of the Kdm4d enzyme in the cell cycle and in antioxidant regulation. To this end, we found the cell proliferation rate was significantly decreased after the knockdown of Kdm4d. Moreover, both the mRNA and protein expression levels of superoxide dismutase 2 (SOD2), one of the major antioxidant enzymes, was decreased, while the reactive oxygen species (ROS) level was significantly increased in Kdm4d knocked-down cells. In addition, the expression of γH2A histone family member X (γH2AX) increased significantly, indicating the presence of DNA double-strand breaks after the knockdown of the Kdm4d enzyme. In conclusion, the knockdown of Kdm4d inhibited DNA replication and the cell cycle, repressed the expression of SOD2, and increased the generation of ROS, which led to the production of DNA damage in GFCs. Our data will be helpful for understanding the mechanism underlying antioxidant capacity regulation in fibroblast cells.

Antioxidant Activity, Phenolic Content, and Antioxidant Gene Expression in Genetic Resources of Sorghum Collected from Australia, Former Soviet Union, USA, Sudan and Guadeloupe

Functionality based on the biological activity of sorghum such as antioxidant activity is known worldwide for its excellence. In this study, we investigated the reactive oxygen species (ROS) scavenging activity, total phenolic and flavonoid contents, phenol compounds, and changes in antioxidant gene expression in sorghum seed cells collected from five countries (Australia, former Soviet Union, USA, Sudan, and Guadeloupe). Sorghum seeds were obtained from 12 genetic resources (K159041, K159042, K159078, K159081, K159088, K159089, K159093, K159097, K159100, K159096, K159048, and K159077). ROS scavenging activity was analyzed using 1,1-diphenyl-2-picrylhydrazyl (DPPH) and 2,20-azinobis 3-ethylbenzothiazoline-6-sulfonate (ABTS). K159097 showed high antioxidant activity values of 33.52 ± 0.70 μg/mL (DPPH) and 271.06 ± 13.41 μg/mL (ABTS), respectively. The reducing power of the resources improved in a concentration-dependent manner, and 10 sorghum resources, except K159078 and K159048, showed high reducing power. K159042 had the highest total phenol content (231 ± 2.17 mg·GAE/g), and K159081 had the highest total flavonoid content (67.71 ± 5.38 mg·QE/g). Among the six phenolic compounds (protocatechuic acid, caffeic acid, p-coumaric acid, ferulic acid, taxifolin, and naringenin) analyzed, the compound with the highest content was taxifolin (203.67 ± 4.99 mg/L in K159093). K159041, K159042, and K159048 had the highest expression levels of superoxide dismutase (SOD), ascorbate peroxidase 1 (APX1), and catalase (CAT), which are indicators of antioxidant activity. An evaluation of the diversity of sorghum provided useful information on antioxidant activity, physicochemical content, and antioxidant gene expression in seed cells, suggesting that sorghum can be used as a biomaterial from natural resources.

Protective effect of mussel polysaccharide on cyclophosphamide-induced intestinal oxidative stress injury via Nrf2-Keap1 signaling pathway.

The hard-shelled mussel (Mytilus coruscus) has been used as a traditional Chinese medicine and health food in China for centuries. Polysaccharides from mussel has been reported to have multiple biological functions, however, it remains unclear whether mussel polysaccharide (MP) exerts protective effects in intestinal functions, and the underlying mechanisms of action remain unclear. The aim of this study was to investigate the protective effects and mechanism of MP on intestinal oxidative injury in mice. In this study, 40 male BALB/C mice were used, with 30 utilized to produce an animal model of intestinal oxidative injury with intraperitoneal injection of cyclophosphamide (Cy) for four consecutive days. The protective effects of two different doses of MP (300 and 600 mg/kg) were assessed by investigating the change in body weight, visceral index, and observing colon histomorphology. Moreover, the underlying molecular mechanisms were investigated by measuring the antioxidant enzymes and related signaling molecules through ELISA, real-time PCR, and western blot methods. The results showed that MP pretreatment effectively protected the intestinal from Cy-induced injury: improved the colon tissue morphology and villus structure, increased superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-Px) activities, and reduced malondialdehyde (MDA) content in serum and colon tissues. Meanwhile, MP also significantly increased the expression levels of SOD, GSH-Px, heme oxygenase-1 (HO-1), and nuclear factor E2-related factor 2 (Nrf2) mRNA in colon tissues. Further, western blot results showed that the expression of Nrf2 protein was significantly upregulated while kelch-like ECH-associated protein 1 (Keap1) was significantly downregulated by MP in the colonic tissues. This study indicates that MP can ameliorate Cy-induced oxidative stress injury in mice, and Nrf2-Keap1 signaling pathway may mediate these protective effects.

Effects of Exogenous Nitric Oxide Treatment on Grape Berries Against Botrytis cinerea and Alternaria alternata Related Enzymes and Metabolites.

Postharvest losses of grape berries caused by the pathogenic fungi Botrytis cinerea and Alternaria alternata have been widely reported, and nitric oxide (NO) as a plant signaling molecule to control postharvest diseases has recently become an active research topic. This study aimed to investigate the regulatory effect of NO on the interaction between grape berries and fungi. During interactions between grape berries and pathogenic fungi, treatment with 10 mM sodium nitroprusside (SNP, an NO donor) delayed the decline of the physiological quality of the grape berries and had positive effects on the weight loss rate, firmness, and respiration intensity. SNP treatment increased the activities of superoxide dismutase (SOD) and polyphenol oxidase (PPO) and inhibited the activities of peroxidase (POD) and catalase (CAT) of grape berries during the resistance to fungal pathogen infection. In addition, the increase in browning degree and the accumulation of hydrogen peroxide were inhibited by SNP treatment. In the phenylpropane metabolic pathway, the activities of phenylalanine ammonia-lyase (PAL), cinnamate 4-hydroxylase (C4H), and 4-coumaric acid coenzyme A ligase (4CL) were increased during the activation of grape berries during the resistance to pathogen infection by SNP, and the intermediate metabolites lignin, flavonoids, and total phenols were accumulated. In addition, SNP treatment had a regulatory effect on the gene expression levels of SOD, POD, PPO, PAL, and 4CL. These results suggested that SNP treatment was effective for the preservation and disease reduction of grape berries.

Effects of dietary Humulus scandens extract on growth performance, antioxidant responses and intestinal microflora of Yellow River carp (Cyprinus carpio)

In the present study, the effects of Humulus scandens extract on growth performance, antioxidant responses and intestinal microflora of Yellow River carp (Cyprinus carpio) were investigated. 240 carps were assigned to four treatments received diets supplemented with 0 (control), 0.5, 1 and 2 g/kg Humulus scandens extract for 8 weeks. At end of the trial, the final body weight, specific growth rate (SGR) and intestinal body index of the 1.0 g/kg and 2.0 g/kg group were significantly higher than those of the control group (P < 0.05). No significantly differences were observed for food conversation ratio (FCR) or protein efficacy ratio (PER). The SOD (superoxide dismutase) content in gills of the treatment groups was significantly increased compared to the control group (P < 0.05), GPX (glutathione peroxidase) content in gills of 2.0 g/kg group was significantly higher than that of other groups (P < 0.05). Compared with the control group, the contents of SOD, CAT (catalase) and GPX in the liver of the treatment groups were significantly higher (P < 0.05). The gene expression levels of SOD, CAT and GPX in gill and liver of 1.0 g/kg and 2.0 g/kg group were significantly higher than those of control group (P < 0.05), and the gene expression level of HSP70 in gill and liver was decreased compared to the control group. The trypsin activity, fold height, crypt depth and goblet cell number in 1.0 g/kg and 2.0 g/kg group were significantly higher than those in control group and 0.5 g/kg group (P < 0.05). At the phyla level, the abundance of Actinobacteria in 0.5 g/kg group, Verrucomicrobia in 1.0 g/kg group and Fusobacteria in 2.0 g/kg group were significantly increased compare to the control group. At the genus level, compared with the control group, the abundances of Luteolibacter in 1.0 g/kg group, Gemmobacter and Cetobacterium in 0.5 g/kg and 2.0 g/kg groups were significantly higher. In conclusion, the present study demonstrated that 1.0 g/kg-2.0 g/kg level Humulus scandens extract is beneficial to growth performance, antioxidant activity and intestinal health of Yellow River carp.