Superoxide Dismutase mRNA Expression Research Articles

The effects of replacing corn with low-tannin sorghum in broiler’s diet on growth performance, nutrient digestibilities, lipid peroxidation and gene expressions related to growth and antioxidative properties

ABSTRACT The purpose of this study was to examine the effects of substituting yellow corn with low-tannin sorghum on growth performance, nutrient digestibilities, and some gene expressions. Chickens were divided into the four groups: 1) the basal diet containing 100% yellow corn, 2) 25% yellow corn was replaced with low-tannin sorghum, 3) 50% yellow corn was replaced with low-tannin sorghum, and 4) 100% yellow corn was replaced with low-tannin sorghum. Body weight significantly improved (P < 0.05) by low-tannin sorghum substitutions, however, feed conversion ratio (FCR) was improved numerically (P = 0.672). Moreover, a significant decrease in the thiobarbituric acid reactive substance in muscle was found in all sorghum groups (P < 0.05). The mRNAs of genes related to growth and antioxidative properties insulin growth factor (IGF), β-Actin, fatty acids synthesis (FAS), glutathione peroxidase (GPX) and superoxide dismutase (SOD) were significantly increased due to low-tannin sorghum substitutions. Interestingly, feeding the 50% yellow corn + 50% low-tannin sorghum diet led to a significant increase in mRNA expressions of IGF, β-Actin, FAS, GPX and SOD in broilers. It could be concluded that substitution of 50% yellow corn with low-tannin sorghum might improve performance, modify plasma lipids and enhance the antioxidative status in broilers.

Modulation of antioxidant enzyme gene expression by extremely low frequency electromagnetic field in post-stroke patients

Oxidative stress plays the most important role in the pathogenesis of stroke. Extremely low frequency electromagnetic field (ELF-EMF) therapy may be complementary in post-stroke therapy, as it modulates oxidative stress. The aim of this study was to evaluate the messenger ribonucleic acid (mRNA) levels of certain antioxidant genes in post-stroke patients given ELF-EMF therapy. Forty-eight post-stroke patients were divided into two groups: an ELF-EMF group and a non-ELF-EMF group. All patients underwent the same program of physical therapy, but the ELF-EMF group was additionally given ELF-EMF treatment. In order to determine the level of gene expression, we evaluated the level of mRNA expression of catalase, superoxide dismutase, and glutathione peroxidase. We observed that after ELF-EMF therapy, the mRNA expression of the studied genes (CAT, SOD1, SOD2, GPx1, and GPx4) significantly increased, which enhanced the antioxidant defence of the body. ELF-EMF therapy intensifies the endogenous antioxidant system by increasing the mRNA expression of genes encoding antioxidant enzymes and enhances the effectiveness of post-stroke patient therapy.

Fenofibrate ameliorates insulin resistance in lipoprotein lipase heterozygous knockout mice and its possible mechanisms

Objective To investigate the effect of fenofibrate on glucolipid metabolism and insulin sensitivity in lipoprotein lipase heterozygous knockout(LPL+ /-)mice, and to explore its mechanism. Methods LPL+ /- mice and wild type(WT)C57 mice were selected and divided into 3 groups(n=6 each group): LPL+ /-(FB)group, LPL+ /-(W)group, and WT group. Mice in LPL+ /-(FB)group were gavaged with fenofibrate(50 mg·kg-1·d-1)for 8 weeks. Mice in LPL+ /-(W)and WT groups were orally fed with the same volume water as that in LPL+ /-(FB)group for 8 weeks. Body weight was observed. Plasma triglyceride(TG)and free fatty acid(FFA)were measured. Intraperitoneal glucose tolerance test in 3 groups of mice were performed. The glucose area under the curve(AUCG)and homeostasis model assessment for insulin resistance index(HOMA-IR)were calculated. Insulin-stimulated Ser473 Akt phosphorylation in liver and skeletal muscle was measured by Western blot. Reactive oxygen species(ROS)levels in liver and skeletal muscle were determined by dihydroethidium staining method and superoxide dismutase(SOD)and catalase(CAT)mRNA expression levels were detected by real-time PCR. Results Compared with LPL+ /-(W)mice, body weight of LPL+ /-(FB)mice was lowered, plasma TG and FFA levels were decreased by about 46.0% and 76.5% respectively, and fasting insulin level and HOMA-IR were decreased while there were no significant differences in fasting glucose level and AUCG between two groups. Insulin-stimulated Ser473 Akt phosphorylation levels in liver and skeletal muscle of LPL+ /-mice were enhanced by fenofibrate. ROS level in skeletal muscle of LPL+ /-(FB)mice was lower than that in LPL+ /-(W)mice while there was no significant difference in ROS of liver between two groups. Fenofibrate significantly increased SOD and CAT mRNA expressions in skeletal muscle of LPL+ /-mice, but not in liver. Conclusion Fenofibrate reduces body weight, ameliorates lipid metabolism, and improves insulin sensitivity in LPL+ /- mice, with reduced oxidative stress. (Chin J Endocrinol Metab, 2018, 34: 867-871) Key words: Lipoprotein lipase; Insulin resistance; Fenofibrate; Oxidative stress

Tempeh attenuates cognitive deficit, antioxidant imbalance, and amyloid β of senescence-accelerated mice by modulating Nrf2 expression via MAPK pathway

This study investigated the effects of tempeh on the cognitive and antioxidant status in senescence-accelerated mouse prone 8 (SAMP8) mice. Six-month-old SAMP8 mice were divided into one control and three tempeh groups; the tempeh groups were administered 300, 600, and 900 mg/kg body weight (BW) tempeh. The results revealed tempeh groups had stronger cognitive ability, lower malondialdehyde and carbonyl protein levels, and higher superoxide dismutase (SOD) and catalase (CAT) activities in the hippocampus, striatum, and cortex. Of them, the 900 mg/kg BW tempeh showed the most superior results on increasing SOD and CAT mRNA expressions and reducing amyloid β (Aβ) and β-site amyloid precursor protein cleaving enzyme 1 levels; it also increased nuclear factor erythroid 2-related factor 2 (Nrf2) levels by reducing p-p38 and p-JNK expression. In conclusion, tempeh may protect neurons against oxidative stress and Aβ-induced damage and reduce memory impairment by modulating Nrf2 via mitogen-activated protein kinase pathway.

Dietary fructooligosaccharide can mitigate the negative effects of immunity on Chinese mitten crab fed a high level of plant protein diet

An 8-week feeding trial was carried out under controlled condition to evaluate the effect of dietary fructooligosaccharide (FOS) on growth performance, whole body composition, antioxidant status and immunity of crabs fed high levels of plant protein diets. Thus, six experimental diets were formulated (designated as F0P50, F0P60, F0P70, F0.2P50, F0.2P60 and F0.2P70), which contain two FOS levels (0 or 0.2%) and three plant protein levels (50, 60, or 70%) according to a 2 × 3 factorial design. The results showed that weight gain increased significantly as dietary plant protein level decreased from 70% to 50%. At 50% plant protein level, the addition of 0.2% FOS can significantly elevate weight gain (WG) (P < 0.05). The highest value in survival rate was observed in crabs fed F0.2P50 and F0.2P60 diet. Crabs fed F0.2P50 diet showed significantly higher crude protein content (P < 0.05) compared with those in other groups, but there were no significant differences in the contents of moisture, crude lipid and ash among all groups (P > 0.05). Catalase (CAT) activity in crabs fed F0.2P50 increased significantly (P < 0.05) compared with crabs fed F0P60, F0P70, F0.2P60 and F0.2P70, but malondialdehyde (MDA) concentrations decreased significantly (P < 0.05). Meanwhile, nitric oxide (NO) concentration, acid phosphatase (ACP) and alkaline phosphatase (AKP) activities of crabs fed 0.2% FOS diets increased significantly (P < 0.05) compared with crabs fed 0% FOS diets. The expressions of prophenoloxidase (propo) was significantly (P < 0.05) affected only by dietary plant protein levels with the highest values observed in 50% plant protein diet, whereas the opposite was true for Myeloid differentiation factor 88 (myd88). The mRNA expressions of mitochondrial manganese superoxide dismutase (mtmnsod), lipopolysaccharide-induced TNF-α factor (litaf) and toll like receptors (tlrs) were significantly affected (P < 0.05) by both FOS and plant protein levels. The cytosolic manganese superoxide dismutase (cytmnsod) mRNA expressions in F0.2P50 and F0.2P60 groups were significantly higher than those in F0P70 and F0.2P70 groups. The results in this study indicated that supplementation with 0.2% FOS can enhance growth performance in crabs fed lower plant protein diets and as well improve immunity in those fed with higher plant protein diets.

Effects of Ginkgo biloba leaf extract, shenmai and matrine on a human embryonic lung fibroblast fibrosis model.

The aim of the present study was to investigate the effects of Ginkgo biloba leaf extract (GBLE), shenmai (S), and matrine (M) on human embryonic lung fibroblasts (HELFs). HELFs were allocated into the following groups: Group A (control group), group B [transforming growth factor β1 (TGF-β1) model group], groups C1-3 (TGF-β1 + low-, moderate- and high-dose GBLE), groups D1-3 (TGF-β1 + low-, moderate- and high-dose S) and groups E1-3 (TGF-β1 + low-, moderate- and high-dose oM). Cell proliferation was assessed with an MTT assay and apoptosis was measured by annexin V/propidium iodide double staining and flow cytometry analysis. Collagen type I (COL-I), collagen type III (COL-III), α-smooth muscle actin (α-SMA) and extracellular superoxide dismutase (ECSOD) mRNA expression levels were measured using semi-quantitative reverse transcription-polymerase chain reaction, and protein content was measured using ELISA. The cell growth inhibition rates of the S groups were significantly higher than those of the other treatment groups (P<0.05). The rate of apoptosis was significantly increased in the treatment groups compared with the model group (P<0.05), and S induced a significant increase in HELF apoptosis compared with the other treatment groups (P<0.05). The mRNA and protein expressions of COL-III, COL-I and α-SMA in the GBLE, S and M groups were significantly decreased, while the expression of ECSOD was significantly increased when compared with the model group (P<0.05). In conclusion, GBLE, S and M inhibited the pro-fibrotic role of TGF-β1 by targeting different steps in TGF-β1-mediated fibrosis.

L-arginine protects against oxidative damage induced by T-2 toxin in mouse Leydig cells.

To explore the protective mechanism of l-arginine against T-2 toxin-induced oxidative damage in mouse Leydig cells, Leydig cells were isolated and cultured with control, T-2 toxin (10 nM), l-arginine (0.25, 0.5, and 1.0 mM), and T-2 toxin (10 nM T-2 toxin) with l-arginine (0.25, 0.5, or 1.0 mM) for 24 hours. Cells and supernatants were harvested to examine cell viability, activities, and messenger RNA (mRNA) expression of glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), and catalase (CAT), malondialdehyde (MDA) content, and DNA damage. Results showed that T-2 toxin significantly reduced cell viability, improved MDA content and DNA damage, and decreased activities and mRNA expression of GSH-Px, SOD, and CAT. However, l-argininereduced T-2 toxin-induced oxidative damage and tended to maintain normal levels. Furthermore, l-arginine upregulated mRNA expressions of GSH-Px, SOD, and CAT. Collectively, l-arginine, due to its antioxidative ability, could ameliorate T-2 toxin-induced cytotoxicities in mouse Leydig cells by regulating oxidative stress.

Oxidative stress modulates the expression of toll‑like receptor 3 during respiratory syncytial virus infection in human lung epithelial A549 cells.

Toll‑like receptor 3 (TLR3) can react with double stranded RNA and is involved in the inflammatory response to respiratory syncytial virus(RSV) infection. Also, oxidative stress has been reported to be involved in RSV infection. However, the correlation between oxidative stress and TLR3 activation during RSV infection is unclear. Therefore, the present study investigated the association between TLR3 expression and oxidative stress modulation during RSV infection in A549 cells. For comparison, seven treatment groups were established, including RSV‑treated cells, N‑acetyl‑L‑cysteine (NAC)+RSV‑treated cells, oxidant hydrogen peroxide (H2O2)+RSV‑treated cells, normal cell control, inactivatedRSV control, NAC control and H2O2 control. The mRNA expression changes of TLR3, interferon regulatory factor‑3 (IRF3), nuclear factor‑κB(NF‑κB) and superoxide dismutase 1(SOD1) were measured using semi‑quantitative reverse transcription‑polymerase chain reaction, and the protein changes of TLR3 and phospho‑NF‑κB p65 were determined using western blot in A549 cells from the different treatment groups. The present study also evaluated the differences in hydroxyl free radical (·OH), nitric oxide (NO) and total SOD activity in the different treatment groups. The results demonstrated that RSV infection of A549 cells increased the levels of ·OH and NO, while decreasing the activity of total SOD. Pretreatment of A549 cells with H2O2 prior to RSV infection upregulated the mRNA and protein expression of TLR3 and NF‑κB, and downregulated the mRNA expression of IRF3 and SOD1, as well as the total SOD activity. When the infected cells were pretreated with NAC, the mRNA and protein expression of these genes were reversed. These variations in the TLR3‑mediated signaling pathway molecules suggested that oxidative stress may be a key regulator for TLR3 activation during RSV infection. RSV‑induced oxidative stress may potentially activate TLR3 and enhance TLR3‑mediated inflammation. These results may provide better understanding of the RSV‑induced inflammatory and immune pathways, and may also contribute to the drug development and prevention of human RSV diseases.

A Cardioprotective Role of Nerium oleander with the Expression of Hypoxia Inducible Factor 2A mRNA by Increasing Antioxidant Enzymes in Rat Heart Tissue

Background: Nerium oleander (NO) distillate is used to either protect heart cells against oxidative stress or reduce the risk of cardiovascular disease by regulating the production of reactive oxygen species. Hypoxia-inducible factors (HIFs) regulate cellular antioxidant defense mechanisms under hypoxic conditions in which heart cells survive; however, the key responsible mechanism of NO distillate for cardioprotection remains elusive. The objective of this study was to evaluate the effects on heart tissue at different time intervals after administering NO distillateintraperitoneally (IP) while considering the transcriptional regulation of HIFs and representative antioxidant enzymes.Materials, Methods &amp;amp; Results: The NO plant was chopped, and distillated water was added. The mixture was distilled, and the distillate separated and collected into tubes, after which it was lyophilized to obtain dry material. Twenty male Wistar albino rats (2-3 month-old, 250-300 geach) were used in the study. The rats were randomly divided into four groups. The control group (n = 5) received IP injections of saline; the remaining 15 rats received IP injections of a single dose of 7.5 mL NO distillate. The NO distillate injected rats were divided into three groupsaccording to the time from injection to harvest the heart tissue samples. The tissues were collected at 0 h (control; n = 5), 2 h (group 2; n = 5), 4 h (group 3; n = 5), and 8 h (group 4; n = 5) after injection and under general anesthesia (60 mg/kg ketamine, IP + 10 mg/kg xylazine, IP).Quantitative polymerase chain reaction (qPCR) was used to assess the expression profiles of the genes of interest in the heart tissues. Hypoxanthine phosphoribosyltransferase was used as the reference gene. The expression of manganese superoxide dismutase (MnSOD) mRNA was in a steady state level between the control group and group 2 (P &amp;gt; 0.05); however, it significantly increased in group 3 and 4 compared with that in the control (P &amp;lt; 0.05). Expression of catalase (CAT) mRNA was significantly higher in group 2 than in the control group (P &amp;lt; 0.05) although it was lower in group 3 and 4 than in group 2 (P &amp;lt; 0.05); however, it appeared to be similar among the control group, group 3, and group 4 (P &amp;gt; 0.05). Copper (Cu) SOD mRNA was equallyexpressed in both the control group and group 2 (P &amp;gt; 0.05) but was lower in group 3 and 4 than in group 2 (P &amp;lt; 0.05). Expressions of HIF1A, HIF2A, and HIF3A mRNA were detected in the rat heart tissues in the control and 2, 4, and 8 h after administration of NO distillate. Expression ofHIF1A mRNA was in a steady state and did not differ among groups 2, 3, and 4 (P &amp;gt; 0.05). Similarly, the expression of HIF2A mRNA did not change between the control group and group 2 (P &amp;gt; 0.05); however, it was higher in group 3 than in the control (P &amp;lt; 0.05) and tended to behigher in group 3 than in group 2 (P = 0.063). HIF3A mRNA expression did not change significantly in the heart tissue of any of the groups (P &amp;gt; 0.05).Discussion: The present study using rats determined that MnSOD, CAT, CuSOD, HIF1A, HIF2A, and HIF3A mRNA are expressed in the heart tissues after administration of NO distillate. The increased expression of HIF2A mRNA after 4 h in accordance with a rise in CAT mRNA after 2 h, and MnSOD mRNA after 4 and 8 h might confirm the role of HIF2A mRNA in oxidative stress defense by regulating antioxidant enzymes; consequently, this study may expand our understanding of uses of NO distillate with respect to molecular pathways.

Neuroprotective effects of cryptotanshinone and 1,2-dihydrotanshinone I against MPTP induced mouse model of Parkinson’s disease

1,2-dihydrotanshinone I (DTs1) and cryptotanshinone (CTs) are the two major compounds isolated from Salvia castanea f. tomentosa Stib., which possess diverse biological effects such as antioxidant, anticancer, anti-​atherogenic, anti-​hepatic fibrosis. However, there is no any report available on the neuroprotective effects of DTs1 and CTs against MPTP induced dopaminergic neurons damage. Therefore, in this study, we investigated the neuroprotective effects of DTs1 and CTs in the MPTP-induced PD model of C57BL/6 mice. Locomotor behavior, tyrosine hydroxylase (TH) protein expression indicated that DTs1 and CTs significantly improved TH expression, inhibited dopaminergic cell loss induced by MPTP. Biochemical parameters determination and real-time polymerase chain reaction (PCR) analysis revealed that DTs1 and CTs increased the activities and mRNA expressions of Superoxide Dismutase (SOD), Glutathione peroxidase (GSH-Px), Catalase (CAT), deceased the level of Malondialdehyde (MDA). These findings indicated that DTs1 and CTs showed neuroprotective effects in MPTP-induced PD mice, which might be associated with their antioxidant properties.

Effect of pterostilbene on the expression and activity of antioxidant enzymes in ultraviolet B-radiated HaCaT Cells

Objective To evaluate the protective effect of pterostilbene against ultraviolet B (UVB) -induced acute damage in HaCaT cells, and to explore related mechanisms. Methods The 3- (4, 5-dimethylthiazol-2-yl) -5- (3-carboxymethoxyphenyl) -2- (4-sulfophenyl) -2H-tetrazolium (MTS) assay and flow cytometry were performed to estimate the proliferative activity and the apoptosis and necrosis rate of HaCaT cells treated with different concentrations of pterostilbene respectively, so as to screen the non-toxic concentration of pterostilbene. HaCaT cells were randomly divided into several groups: normal control group receiving no treatment, UVB group irradiated with 57 mJ/cm2 UVB, 3 pterostilbene groups treated with 2.44, 4.88 and 9.75 μmol/L pterostilbene respectively for 24 hours, 3 pterostilbene+ UVB groups treated with 2.44, 4.88 and 9.75 μmol/L pterostilbene respectively for 24 hours followed by UVB radiation. Western blot analysis was conducted to detect changes of the transcription factor NF-E2-related factor 2 (Nrf2) expression in cell nuclei and cytoplasm before and after the treatment with pterostilbene and UVB,quantitative PCR to determine the mRNA expression of catalase and superoxide dismutase in the HaCaT cells, and enzyme-linked immunosorbent assay (ELISA) to evaluate the activity of catalase and superoxide dismutase. Results MTS assay and flow cytometry showed that 2.44, 4.88 and 9.75 μmol/L pterostilbene had non-toxic effect on HaCaT cells. The protein expression of Nrf2 in the nuclei and cytoplasm in the normal control group was 1.03 ± 0.08 and 1.04 ± 0.11 respectively. Compared with the normal control group, the protein expression of Nrf2 in the nuclei and cytoplasm experienced no significant changes in the 2.44-, 4.88- and 9.75-μmol/L pterostilbene groups, and the UVB group showed similar protein expression of Nrf2 in the cytoplasm, but significantly increased protein expression of Nrf2 in the nuclei (1.77 ± 0.08, q= 17.24, P 0.05) . The mRNA expression of CAT and SOD experienced no significant changes in the 2.44-, 4.88- and 9.75-μmol/L pterostilbene groups compared with the normal control group (P > 0.05) . However, 2.44, 4.88 and 9.75 μmol/L pterostilbene could significantly reduce the inhibitory effect of UVB radiation on the mRNA expression of CAT (P < 0.05) and up-regulate the mRNA expression of SOD in the pterostilbene+ UVB groups (P < 0.05) . ELISA revealed that UVB radiation could inhibit the activity of CAT and SOD in the HaCaT cells (both P < 0.001) , while 2.44, 4.88 and 9.75 μmol/L pterostilbene could reduce the inhibitory effect of UVB radiation on the activity of CAT and SOD (all P < 0.05) . However, the activity of CAT and SOD were still lower in the 2.44-, 4.88- and 9.75-μmol/L pterostilbene + UVB groups than in the normal control group (P < 0.05) . Conclusion Pterostilbene can prevent UVB-induced acute damage in HaCaT cells by activating the Nrf2 pathway and up-regulating the expression of the downstream antioxidant enzymes CAT and SOD. Key words: Veratrum; NF-E2-related factor 2; Catalase; Superoxide dismutase; Ultraviolet rays; Pterostilbene; HaCaT cells

MTORC1 signaling regulates vascular endothelial function via reactive oxygen species and NFκB signaling

The mechanistic target of rapamycin complex 1 (mTORC1) is an important intracellular energy sensor that regulates protein synthesis through its downstream signaling components the S6‐kinase and the ribosomal S6 protein. Recently, our laboratory has demonstrated a critical role of the mTORC1 signaling pathway in the cardiovascular regulation with implications for obesity and hypertension. In this study, we tested the hypothesis that the mTORC1 signaling pathway in the vasculature is involved in the regulation of vascular endothelial function and that dysregulation of this pathway contributes to the cardiovascular disorders associated with obesity. We began by assessing the effects of activating mTORC1 signaling in cultured mouse lung endothelial cells (MLECs) using leucine (10 mM; 16hrs) or a constitutively active S6‐kinase adenoviral construct (Ad‐S6KCA; 48hrs). Activated mTORC1 signaling was confirmed by the increased phosphorylated levels of the ribosomal S6 protein (2–4 fold; p&lt;0.05). Increasing mTORC1 signaling elevated mRNA expression of oxidative stress markers (NOX1 and NOX2; 1.5–5 fold; p&lt;0.05), decreased mRNA expression of superoxide dismutase 2 (0.5 fold; p&lt;0.05) and increased reactive oxygen species (ROS) generation (via dihydroethidium staining; 1.5 fold; p&lt;0.05) in MLECs demonstrating a pro‐oxidant gene environment evoked by activation of mTORC1 signaling. Blockade of the IKKβ subunit of the NFκB transcriptional complex (BMS‐345541; 300nM) prevented mTORC1 signaling induced ROS generation in MLECs demonstrating a critical role of NFκB signaling in the cellular response to mTORC1 activation. Next, we used leucine and Ad‐S6KCA to test the consequence of enhancing mTORC1 signaling on endothelial function in aortic rings isolated from wildtype mice. Both leucine and an adenovirus expressing a constitutively active form of S6K (Ad‐S6KCA) impaired endothelial dependent acetylcholine‐induced relaxation (~10–15%; p&lt;0.05) without changing endothelial‐independent relaxation responses evoked by sodium nitroprusside indicating endothelial but not smooth muscle dysfunction in response to increased mTORC1 signaling. Blockade of mTORC1 signaling using a dominant negative S6K adenoviral construct (Ad‐S6KDN) as well as inhibition of ROS signaling by Tempol (non‐specific free radical scavenger) ameliorated mTORC1‐induced endothelial dysfunction (p&lt;0.05). To determine the involvement of mTORC1 signaling in the endothelial dysfunction associated with obesity, we utilized diet‐induced obese mice that display vascular endothelial dysfunction as compared to lean controls. Obese mice exhibited increased mTORC1 signaling in the aorta and mesenteric artery indicated by the elevated phosphorylated levels of the ribosomal S6 protein (1.5–3 fold; p&lt;0.05) and this was associated with increases in NOX2 expression (1.5 fold; p&lt;0.05) further indicating ROS signaling in the vascular pathology associated with obesity. We conclude that mTORC1 signaling is a novel regulator of vascular endothelial function through its effects on the NFκB complex and ROS signaling. Our data also indicate that dysregulation of the mTORC1 signaling pathway may be involved in the endothelial dysfunction associated with obesity.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Vitamin E and vitamin C supplementation improves antioxidant status and immune function in oxidative-stressed breeder roosters by up-regulating expression of GSH-Px gene

This study aimed to evaluate the effects of vitamin C and vitamin E on antioxidant capacity and immune function in oxidative-stressed breeder roosters. One hundred twenty 45-week-old Lveyang black-boned breeder roosters were randomly assigned to 5 dietary treatments, including negative control group (NC), positive control group (PC), and 3 trial groups, which were fed the diets containing 300mg/kg VC, 200mg/kg VE, or 300mg/kg VC and 200mg/kg VE (VC+VE). At 47 wk of age, the positive control and trial groups were subcutaneously injected 3 times every other d with dexamethasone (DEX) 4mg/kg of body weight, the negative control group was injected with saline. The experiment lasted for 35 d. The results showed that at 50 wk of age, average daily feed intake of birds challenged with DEX significantly increased (P < 0.05). During post-stress recovery period (52 wk of age), dietary supplemental VE or VC+VE notably increased body weight under oxidative stress (P < 0.01). Oxidative stress induced by DEX could significantly decrease superoxide dismutase (SOD), IgM, antibody titer of ND and mRNA expression of SOD or glutathion peroxidase activity (GSH-Px), increase serous malondialdehyde (MDA) (P < 0.05). Supplementation of VC or VE significantly decreased serous MDA, and increased SOD under oxidative stress (P < 0.05). Supplementation of VC or VE, or their combination significantly increased the relative expression of GSH-Px mRNA when compared to the oxidative-stressed control treatment (P < 0.05), whereas did not alleviate the relative expression of SOD mRNA (P > 0.05). Therefore, the results suggest that addition of 300mg/kg VC, 200mg/kg VE or their combination could improve antioxidant ability and immune performance in oxidative-stressed breeder roosters through up-regulating the expression of GSH-Px gene.

Research of the heart protective effect of exercise precondition mediated by calcitonin gene related peptide on acute exhaustion rats

To observe the heart protective effect of exercise preconditioning (EP) in the acute exhaustion exercise (EE) rats, and explore its action mechanism further. Eighty healthy male Sprague-Dawley (SD) rats were divided into control group (C group), EP group, EE group, and EP+EE group randomly, with 20 rats in each group. The rats in EP and EP+EE groups were trained for 3 weeks according to the daily swimming for 60 minutes (swimming 15 minutes, resting 5 minutes, repeating 3 times) with 6 days each week. The rats in EE and EP+EE groups on the last 1 day after 3 weeks, 3% weight heavy weight was carried once for swimming EE. Two hours after the last EE, abdominal aortic blood and heart was harvested, the levels of serum MB isoenzyme of creatine kinase (CK-MB) and calcitonin gene related peptide (CGRP) were determined by enzyme linked immunosorbent assay (ELISA); the ultrastructure of myocardium was observed by optical microscopy; the levels of myocardial malondialdehyde (MDA) and superoxide dismutase (SOD) were determined by ELISA, the mRNA expression of myocardial CGRP was assayed by reverse transcription-polymerase chain reaction (RT-PCR), and the protein expression of myocardial CGRP was assayed by Western Blot. Compared with C group, the levels of serum CK-MB and myocardial MDA were significantly increased, serum CGRP content, myocardial SOD activity, and mRNA and protein expressions of myocardial CGRP were significantly decreased in EE group and EP+EE group. Compared with EE group, the levels of serum CK-MB and myocardial MDA in EP+EE group were decreased [CK-MB (U/L): 13.11±0.77 vs. 15.55±0.90, MDA (μmol/L): 389.57±49.60 vs. 709.08±160.49], the level of serum CGRP, and mRNA and protein expressions of myocardium CGRP were increased [serum CGRP (ng/L): 120.41±9.07 vs. 97.97±9.05, CGRP mRNA (2 -ΔΔCT): 0.45±0.09 vs. 0.14±0.02, CGRP protein (gray value): 0.78±0.08 vs. 0.41±0.04, all P < 0.05], the degree of myocardial injury was obviously alleviated. There was no significant difference in the indexes between the EP group and C group. EP has the heart protective effect for the acute EE rats, and the mechanism is closely related to the endogenous protective substance CGRP.

Mercaptoethanol Protects the Aorta from Dissection by Inhibiting Oxidative Stress, Inflammation, and Extracellular Matrix Degeneration in a Mouse Model.

BackgroundThe aims of this study were to investigate the effects of mercaptoethanol treatment on the expression of mediators of oxidative stress, inflammation, and extracellular matrix (ECM) degeneration in a mouse aortic dissection (AD) model.Material/MethodsTwenty-four 8-month-old C57BL/6J mice were divided into three groups and studied for two weeks: 1) the aortic dissection (AD) Model group (N=8) underwent intraperitoneal injection of angiotensin II (Ang II) (5 ml/kg) three times every 24 h; 2) the mercaptoethanol Treated group (N=8) were given oral mercaptoethanol (2.5 mM); the Normal group (N=8) underwent intraperitoneal injection of noradrenaline (5 mg/kg) three times every 24 h. Sections of mouse aorta were prepared for histology with hematoxylin and eosin (H&E) staining; immunohistochemistry was performed to detect levels of: nuclear factor (erythroid-derived 2)-like 2 (NFE2L2), nuclear factor κB (NF-κB), p65, superoxide dismutase-1 (SOD1), glutamate cysteine ligase catalytic subunit (GCLC), tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and matrix metalloproteinase-9 (MMP9). Quantitative reverse transcription-polymerase chain reaction (RT-PCR) evaluated mRNA expression of SOD1, GCLC, TNF-α, IL-1β, and MMP9.ResultsMercaptoethanol treatment inhibited Ang II-induced aortic dissection in AD mice, as shown histologically. Mercaptoethanol treatment reduced the expression levels of NFE2L2, NF-κB, p65, TNF-α, IL-1β and increased the expression levels of SOD1, MMP9, and GCLC.ConclusionsIn an AD mouse model, mercaptoethanol treatment inhibited thoracic and abdominal aortic dissection and reduced aortic tissue expression of mediators of oxidative stress and inflammation and increased the activation of ECM signaling pathways.

Is 8% O2 more normoxic than 21% O2 for long-term in vitro cultures of human primary term cytotrophoblasts?

Is 8% O2 a better percentage of atmospheric oxygen for long-term cultures of human primary term cytotrophoblasts than the conventional 21% O2 traditionally used in cell culture? Human primary term cytotrophoblasts are able to differentiate into syncytiotrophoblasts under both atmospheric oxygen levels. Cell culture is traditionally done under 21% O2, which is equal to a pO2 of ~160 mm Hg. Based on the pO2 measured after instauration of the blood circulation within the placenta, it has been proposed that cytotrophoblasts culture should be under 8% O2, which is equivalent to 60 mm Hg, and that this percentage should be considered as the physiological normoxia for cytotrophoblasts. Cytotrophoblasts were isolated and purified from human term placentas (n > 4). Cells were cultured under 21% O2 and 8% O2 for 12 days. Several cellular parameters were assessed on Days 2, 4, 8 and 12. Placentas were obtained after vaginal or elective cesarean delivery from uncomplicated pregnancies at term (n ≥ 4). Cell viability was measured by a luminescent assay based on quantitation of the ATP content of living cells. Cell fusion was assessed by quantification of syncytin and e-cadherin mRNA expression by real-time PCR and determination of the fusion index by immunofluorescent microscopy. Trophoblast differentiation was assessed by measuring the expression levels of hCGβ, inhibin α subunit (InhA) and placental growth factor (PlGF) by real-time PCR and ELISA. Finally, the effect of the two oxygen levels on apoptosis and cellular oxidative stress was also investigated by quantifying caspase 3/7 activation, superoxide dismutase 1 (SOD-1) mRNA expression and H2O2 generation. There was no difference between 21% O2 and 8% O2 on cell viability. Cell fusion seemed to be enhanced during the first 4 days when the cells were cultured under 21% O2 compared to 8% O2. The expression level of hCGβ was equivalent in both oxygen conditions, indicating that there was no difference in trophoblast differentiation. Interestingly, InhA expression was higher under 8% O2, while PlGF expression was inhibited compared to 21% O2. This latter result indicates that 8% O2 may be more hypoxic than normoxic for in vitro culture of primary term cytotrophoblast. This is further corroborated by the fact that 21% O2 did not significantly increase caspase 3/7 activities and the oxidative stress (SOD-1 mRNA expression and H2O2 generation) in our cell cultures. Not applicable. The in vitro culture of cytotrophoblasts is artificial and does not reflect the in vivo situation. The cell population is nearly 100% pure, cultured as a monolayer, and the cells bath in a chemically defined culture medium deprived of any oxygen carrier. The oxygen molecules available to the cells are passively dissolved in the medium. The gas dissolution properties of the medium and the cellular consumption rate of oxygen may allow the cells to sustain a wide range of oxygen percentages from 8% to 21%. It is possible to culture human primary term cytotrophoblasts for at least 12 days. The O2 percentage of the air does not negatively affect in vitro cytotrophoblast differentiation. For in vitro culture of cytotrophoblasts, it is not necessary to lower the percentage of atmospheric oxygen to 8%. This work was fully supported by 'Fetus for Life' charity. The authors state that there is no conflict of interest to declare regarding the publication of this paper.

Roles of dietary supplementation with arginine or N-carbamylglutamate in modulating the inflammation, antioxidant property, and mRNA expression of antioxidant-relative signaling molecules in the spleen of rats under oxidative stress

This study evaluated the effects of arginine (Arg) or N-carbamylglutamate (NCG) on inflammation, antioxidant property, and antioxidant-related gene expression in rat spleen under oxidative stress. A total of 52 rats were randomly distributed into 4 treatment groups with 13 replicates per group. Rats were fed a basal diet (BD) or BD supplemented with Arg or NCG for 30 days. On day 28, half of the BD-fed rats were intraperitoneally injected with sterile saline (control group), and the other half with 12 mg/kg body weight of diquat (DT; DT group). The other 2 diet groups were intraperitoneally injected with 12 mg/kg body weight of DT with either Arg (1%) (DT + Arg) or NCG (0.1%) (DT + NCG). Rat spleen samples were collected for analysis at 48 h after DT injection. Results showed that DT damaged the antioxidant defense in rats compared with the control group (P < 0.05). Compared with the DT group, the DT + Arg and DT + NCG groups manifested improved anti-hydroxyl radical, catalase, and total superoxide dismutase (T-SOD) activities, increased glutathione content (P < 0.05), and decreased malondialdehyde content (P < 0.05). Moreover, compared with the DT group, the DT + Arg and DT + NCG groups enhanced mRNA expression of superoxide dismutase (SOD), glutathione peroxidase 1 (GPx1), glutathione reductase (GR), nuclear factor erythroid 2-related factor 2 (Nrf2), Kelch-like ECH-associated protein 1(Keap-1), and mammalian target of rapamycin (mTOR) (P < 0.05). Both NCG and Arg significantly increased anti-inflammatory cytokine mRNA level but suppressed the pro-inflammatory cytokine mRNA expression under oxidative stress (P < 0.05). In summary, NCG and Arg effectively alleviated oxidative stress, improved the antioxidant capacity and regulated the antioxidant-related signaling molecular expression in rat spleen. N-carbamylglutamate and Arg reduced the inflammation in the spleen by mediating the gene expression of anti-inflammatory and pro-inflammatory cytokines and transforming growth factor-β (TGF-β).

In ovo injection of ascorbic acid modulates antioxidant defense system and immune gene expression in newly hatched local Chinese yellow broiler chicks

The present study was conducted to investigate the effects of in ovo injection of ascorbic acid on antioxidant capacity and immune-related gene expression in the newly hatched local Chinese yellow broiler chicks. Fertile Chinese yellow broiler eggs (n = 90) were assigned to three equal groups. The first group was a non-injected control group. The second group was another control group where the eggs were injected with saline in the air sac after 18 days of incubation. The third group was injected with 3 mg/egg AA in the air sac after 18 days of incubation. In ovo injection of 3 mg/egg AA significantly (P < 0.0001) increased plasma glutathione peroxidase (GSH-PX), total antioxidant capacity (T-AOC), and significantly reduced plasma malondialdehyde (MDA) at 1 d old. Moreover, in ovo injection of 3 mg/egg AA significantly increased mRNA expression of glutathione peroxidase (GSH-PX) and superoxide Dismutase (SOD) in the chick’s spleen.Additionally, the mRNA level of interleukin 1 β (IL-1β), interleukin 6 (IL-6), and tumour necrosis factor α (TNF-α) in the spleen were significantly decreased (P < 0.0001), which indicates an improvement in chick’s immunity. In conclusion, our data suggest that in ovo injection of AA at 3 mg/egg enhance antioxidant defense system and immune system for newly hatched chicks.

Mesenchymal Stem Cell-Conditioned Medium Modulates Apoptotic and Stress-Related Gene Expression, Ameliorates Maturation and Allows for the Development of Immature Human Oocytes after Artificial Activation.

The aim of the present study was to determine whether mesenchymal stem cell-conditioned medium (MSC-CM) modulates apoptotic and stress-related gene expression, and ameliorates maturation and developmental potential of immature human oocytes after artificial activation. A total of 247 surplus immature germinal vesicle (GV) oocytes obtained from infertile women were allocated into two in vitro maturation (IVM) groups: 1: GV oocytes (n = 116) matured in vitro (fIVM), and 2: GV oocytes (n = 131) that were vitrified, then in vitro matured (vIVM). Also, two maturation media were used: Alpha-minimum essential medium (α-MEM) and human umbilical cord-derived MSCs (hUCM). After 36 h of incubation, the IVM oocytes were examined for nuclear maturation. In IVM-matured oocytes, cytoplasmic maturation was evaluated after artificial activation through Ionomycin. Moreover, the quantitative expressions of B-cell CLL/lymphoma 2 (BCL2), BCL2-associated X protein (BAX), superoxide dismutase (SOD), and Heat shock proteins (HSP70) in matured oocytes were assessed by quantitative Real-time polymerase chain reaction (qRT-PCR) and compared with fresh and vitrified in vivo matured oocytes, which were used as fIVM and vIVM controls, respectively. The highest maturation rate was found in hUCM in fIVM, and the lowest maturation rate was found using α-MEM in vIVM (85.18% and 71.42%, respectively). The cleavage rate in fIVM was higher than that in vIVM (83.4% vs. 72.0%). In addition, the cleavage rate in α-MEM was lower than that in the hUCM (66.0% vs. 89.4%). Furthermore, the difference between parthenote embryo arrested in 4–8 cells (p < 0.04) and the quality of embryo arrested in 8-cell (p < 0.007) were significant. The developmental stages of parthenote embryos in hUCM versus α-MEM were as follows: 2–4 cell (89.45% vs. 66.00%, respectively), 4–8 cell (44.31% vs. 29.11%, respectively), morula (12.27% vs. 2.63%, respectively), and blastocysts (2.5% vs. 0%, respectively). The messenger RNA (mRNA) expression levels of BCL2, BAX and SOD were significantly different (p < 0.05) between the matured IVM oocytes. Overall, hUCM showed potential efficacy in terms of ameliorating oocyte maturation and in promoting the development and mRNA expression of BAX, BCL2, and SOD.

Intestinal parameters of oxidative imbalance in celiac adults with extraintestinal manifestations.

AIMTo evaluate selected intestinal parameters of oxidative stress, and antioxidant capacity in adult celiac disease patients with extraintestinal manifestations.METHODSThe study involved 85 adult patients divided into the following subgroups: (1) patients with newly diagnosed celiac disease (CD) (n = 7); (2) celiac patients not adhering to a gluten-free diet (GFD) (n = 22); (3) patients with CD on the GFD (n = 31); and (4) patients with functional disorders of the gastrointestinal tract, serving as controls (n = 25). Celiac patients presented with non-classic symptoms or extraintestinal manifestations. Standard blood tests including serum antioxidant levels (uric acid, bilirubin, and vitamin D), celiac antibody levels, and histopathological status of duodenal biopsy specimens have been determined. The expression of mRNA for tumor necrosis factor α (TNF-α), interleukin 1β (IL-1β), interleukin 10 (IL-10), superoxide dismutase (SOD), heat-shock protein 70 (HSP-70), hypoxia-inducible factor 1 (HIF-1α), and BAX in the duodenal mucosa of patients was analyzed by reverse transcriptase-polymerase chain reaction.RESULTSThe mean plasma uric acid level in patients with active CD (newly diagnosed and nonadherent patients) and treated celiac patients was significantly higher than in controls (260.17 ± 53.65 vs 190.8 ± 22.98, P < 0.001, and 261.7 ± 51.79 vs 190.8 ± 22.98, P < 0.001, respectively). The mean bilirubin concentration in active and treated celiac patients was significantly lower than in controls (8.23 ± 5.04 vs 10.48 ± 4.08, P < 0.05 and 8.06 ± 3.31 vs 10.48 ± 4.08, P < 0.05, respectively). The mean plasma vitamin D level was significantly lower in active celiac patients than in treated celiac patients and controls (19.37 ± 9.03 vs 25.15 ± 11.2, P < 0.05 and 19.37 ± 9.03 vs 29.67 ± 5.12, P < 0.001, respectively). The expression of TNF-α, IL-10, and HSP-70 mRNAs was significantly elevated in the celiac groups regardless of the diet when compared with controls. Patients on the GFD presented a significantly lower mRNA expression of TNF-α and IL-10 than in newly diagnosed and nonadherent patients (P < 0.05). The expression of SOD mRNA was significantly elevated in celiac patients compared with controls (P < 0.05), with a significant difference between treated and untreated patients (P < 0.05). The expression of HIF-1α mRNA and BAX mRNA was significantly higher in patients with active CD compared with controls and patients on GFD, while no difference was observed between the latter two groups.CONCLUSIONIncreased intestinal expression of HSP-70 despite GFD indicates that GFD only partially reduced oxidative stress. CD patients exhibited an oxidative imbalance and inflammatory response despite GFD. Uric acid may act as an important antioxidant in CD.