Superoxide Dismutase Protein Levels Research Articles

Targeting SOD1 via RNAi with PEGylated graphene oxide nanoparticles in platinum-resistant ovarian cancer

Acquired platinum resistance poses a significant therapeutic impediment to ovarian cancer patient care, accounting for more than 200,000 deaths annually worldwide. We previously identified that overexpression of the antioxidant superoxide dismutase 1 (SOD1) in ovarian cancer is associated with a platinum-resistant phenotype via conferring oxidative stress resistance against platinum compounds. We further demonstrated that enzymatic inhibition using small-molecule inhibitors or silencing of SOD1 via RNA interference (RNAi) increased cisplatin sensitivity and potency in vitro. We launched this study to explore the potential therapeutic applications of SOD1 silencing in vivo in order to reverse cisplatin resistance using a graphene-based siRNA delivery platform. PEGylated graphene oxide (GO) polyethyleneimine (GOPEI-mPEG) nanoparticle was complexed with SOD1 siRNA. GOPEI-mPEG-siSOD1 exhibited high biocompatibility, siRNA loading capacity, and serum stability, and showed potent downregulation of SOD1 mRNA and protein levels. We further observed that cisplatin and PEI elicited mitochondrial dysfunction and transcriptionally activated the mitochondrial unfolded protein response (UPRmt) used as a reporter for their respective cytotoxicities. SOD1 silencing was found to augment cisplatin-induced cytotoxicity resulting in considerable tumour growth inhibition in cisplatin-sensitive A2780 and cisplatin-resistant A2780DDP subcutaneous mouse xenografts. Our study highlights the potential therapeutic applicability of RNAi-mediated targeting of SOD1 as a chemosensitizer for platinum-resistant ovarian cancers.

Melatonin alleviates ovarian function damage and oxidative stress induced by dexamethasone in the laying hens through FOXO1 signaling pathway

Oxidative stress can trigger follicular atresia, and decrease follicles quantity in each development stage, thereby alleviating reproductive activity. The induction of oxidative stress in chickens through intraperitoneal injection of dexamethasone is a reliable and stable method. Melatonin has been shown to mitigate oxidative stress in this model, but the underlying mechanism remains unclear. Therefore, this study aimed to investigate whether melatonin can recover aberrant antioxidant status induced by dexamethasone and the specific mechanism behind melatonin-dependent protection. A total of 150 healthy 40-wk-old Dawu Jinfeng laying hens with similar body weights and laying rates were randomly divided into three groups, with five replicates per group and 10 hens per replicate. The hens in the control group (NS) received intraperitoneal injections of normal saline for 30 d, the dexamethasone group (Dex+NS) received 20 mg/kg dose of dexamethasone for the first 15 d, followed by the 15 d of normal saline treatment. While in the melatonin group (Dex+Mel), dexamethasone (20 mg/kg dose) was injected intraperitoneally in the first 15 d, and melatonin (20 mg/kg/d) was injected in the last 15 d. The results showed that dexamethasone treatment significantly enhanced oxidative stress (P < 0.05), while melatonin not only inhibited the oxidative stress but also notably enhanced the antioxidant enzymes superoxide dismutase (SOD), catalase activity (CAT), glutathione peroxidase (GSH-Px), and antioxidant genes CAT, superoxide dismutase 1 (SOD1), glutathione peroxidase 3 (GPX3), and recombinant peroxiredoxin 3(PRDX3) expression (P < 0.05). Melatonin treatment also markedly reduced 8-hydroxy deoxyguanosine(8-OHdG), malondialdehyde (MDA), and reactive oxygen species (ROS) levels (P < 0.05) and apoptotic genes Caspase-3, Bim, and Bax in the follicle. In the Dex+Mel group, the Bcl-2 and SOD1 protein levels were also increased (P < 0.05). Melatonin inhibited the forkhead Box Protein O1(FOXO1) gene and its protein expression (P < 0.05). In general, this investigation revealed that melatonin might decrease oxidative stress and ROS by enhancing antioxidant enzymes and genes, activating the antiapoptotic genes, and inhibiting the FOXO1 pathway in laying hens.

Gum Arabic protects the rat heart from ischemia/reperfusion injury through anti-inflammatory and antioxidant pathways

Gum Arabic (GA) is a plant exudate with antioxidant and anti-inflammatory effects. GA has shown promise in protection from and treatment of kidney failure, however, its role in the protection of the heart from ischemia and reperfusion (I/R) has not been investigated. This study investigated the antioxidant and anti-inflammatory effects of Gum Arabic (GA) in the protection of the heart against ischemia/reperfusion (I/R) injury. Langendorff-perfused Wistar rat hearts were divided into seven groups. One group which was subjected to I/R with no other treatment served as the control group. The second group was subjected to buffer perfusion with no ischemia (sham group). The third group was perfused with GA in the absence of ischemia (sham + GA). The rest of the hearts were isolated from rats that had been treated with GA for 4 or 2 weeks in the drinking water, or GA that had been infused intravenously 2 h before sacrifice or added to perfusion buffer at reperfusion. Hemodynamics data were digitally computed; infarct size was measured using 2,3,5-triphenyltetrazolium chloride (TTC) staining and cardiomyocyte injury was assessed by quantifying creatine kinase (CK) and lactate dehydrogenase (LDH) enzymes. The total oxidants (TOS) and antioxidants (TAS), superoxide dismutase (SOD) and pro- and anti-inflammatory cytokines levels were estimated by ELISA. GA treatment for 2 weeks, 4 weeks or 2 hours before sacrifice resulted in a significant (P < 0.05) improvement in cardiac hemodynamics and reduction in infarct size and cardiac enzyme levels compared to respective controls. However, GA administration at the time of reperfusion did not protect the hearts against I/R injury. Furthermore, GA treatment decreased the pro-inflammatory and anti-inflammatory cytokines levels. The levels of TOS in the effluent were significantly decreased (P < 0.05) and SOD levels were significantly (P < 0.05) increased by GA administration. GA protected the heart against I/R injury when administered for 2 or 4 weeks or when infused 2 hours before sacrifice. GA treatment decreased the total oxidants levels, the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 protein levels and increases SOD and anti-inflammatory cytokine IL-10 protein levels.

Exposure to automobile exhaust-derived PM2.5 induces spermatogenesis dysfunction by damaging UPRmt of prepubertal rats

Automobile exhaust-derived particulate matter 2.5 (PM2.5) can cause spermatogenic cell damage, potentially resulting in male infertility. This study uses male prepubertal Sprague Dawley (SD) rats to explore the molecular mechanisms by which automobile exhaust-derived PM2.5 causes spermatogenic cell damage and induces spermatogenesis dysfunction during sexual maturity by disrupting the mitochondrial unfolded protein response (UPRmt) in spermatogenic cells. Male prepubertal SD rats were randomly divided into four groups: control (intratracheal instillation of normal saline), low-dose PM2.5 (5 mg/kg), high-dose PM2.5 (10 mg/kg), and PM2.5 10 mg/kg +Vit (100 mg/kg of vitamin C and 50 mg/kg of vitamin E). The rats were treated for four weeks, with five consecutive treatment days and two non-treatment days, followed by cohabitation. Testicular and epididymal tissues were harvested for analysis. The mitochondria in spermatogenic cells were observed under an electron microscope. UPRmt-, oxidative stress-, and apoptosis-related markers in spermatogenic cells were examined. Spermatogenic cell numbers and conception rate declined significantly with increasing PM2.5 dose, with their mitochondria becoming vacuolated, swollen, and degenerated to varying degrees. The apoptosis of spermatogenic cells was abnormally enhanced in PM2.5 exposed groups compared to the control group. Spermatogenic cell numbers of conception rate gradually recovered, mitochondrial damage in spermatogenic cells was alleviated, and spermatogenic cell apoptosis was significantly reduced after vitamin intervention. In addition, protein levels of superoxide dismutase 1 (Sod1), nuclear factor erythroid 2-related factor 2 (Nrf2), and B-cell lymphoma 2 (Bcl-2) were significantly lower, while those of Bcl2-associated X apoptosis regulator (Bax), cleaved caspase 3 (Casp3), and cytochrome c (Cyt-c) and malondialdehyde (MDA) levels were significantly higher in the high-dose PM2.5 group than in the control group. The levels of UPRmt-related proteins C/EBP homologous protein (Chop), heat shock protein 60 (Hsp60), and activating transcription factors 4 (Atf4) and 5 (Atf5) were higher in the low-dose PM2.5 group, lower in the high-dose PM2.5 group, and gradually recovered in PM2.5 10 mg/kg +Vit group. Our results show that exposure to automobile exhaust-derived PM2.5 induces oxidative stress responses, leads to post-sexual maturation UPRmt dysfunction and mitochondrial impairment, and abnormally enhances spermatogenic cell apoptosis in prepubertal rats, resulting in male infertility.

Modulation of aqueous humor melatonin levels by yellow-filter and its protective effect on lens.

Melatonin is mainly secreted by the pineal gland, and it is also produced by various ocular structures such as the lens. It has been recently demonstrated that melatonin ocular synthesis can be induced by blocking the blue component of white light by means of filters. Melatonin exhibits antioxidant properties that can be useful to face light-induced oxidative stress as well as oxidative events associated to ocular pathologies like cataracts. Moreover, as oxidative stress is a main event in cataract development, changes in melatonin levels could happen and be relevant in the progression of this pathology, a subject that remains uncertain. The goal of this work was to analyze the ability of a short wavelength light blocking (yellow) filter to modulate endogenous melatonin concentration and the antioxidant and cytoprotective actions induced by yellow filter's use in lens. Furthermore, we evaluated the potential changes in aqueous humor melatonin concentration from patients with cataracts. In human lens epithelial cells, white light-emitting diode (LED) light challenge reduced melatonin secretion, protein levels of the enzymes involved in melatonin synthesis (hydroxyindole-O-methyltransferase and unphosphorylated and phosphorylated forms of arylalkylamine N-acetyltransferase) and cell viability whereas increased reactive oxygen species production. Yellow filter exposure precluded melatonin secretion reduction and protected cells from oxidative damage. Consistent with cataract patient's results, significantly lower levels of melatonin were observed in aqueous humor of alloxan-induced diabetic cataract rabbits as compared to those of control rabbits. In contrast, aqueous humor melatonin levels of diabetic cataract animals maintaining in cages covered with a yellow filter resembled control values. This recovery seems to be mediated by the induction of melatonin biosynthetic enzymes protein expression. Yellow filter also preserved Nrf2 lens protein expression and superoxide dismutase protein levels and activity in diabetic animals. Modulation of endogenous ocular melatonin concentration using blocking filters might be a promising approach to prevent premature lens opacification.

Bortezomib alleviates myocardial ischemia reperfusion injury via enhancing of Nrf2/HO-1 signaling pathway

Bortezomib is a classical proteasome inhibitor and previous researches have reported its roles of anti-oxidation and anti-inflammatory functions in various diseases. However, the role of Bortezomib in myocardial ischemia reperfusion injury (MIRI) is unclear. Thus, our research seeks to reveal the protective effects of Bortezomib pretreatment in the mice model of MIRI. First, by the optimization of Bortezomib concentration and pretreatment timepoints, we found that 0.5 mg/kg Bortezomib pretreatment 2 h before MIRI significantly attenuated pathological damage and neutrophil infiltration. Then we found that pretreatment with Bortezomib obviously increased myocardial systolic function ((left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS)) and decreased infarct size, as well as serum Troponin T levels. Meanwhile, Bortezomib pretreatment also remarkably augmented oxidative stress related protein levels of Superoxide dismutase [Cu–Zn] (SOD1), Catalase (CAT) and Glutathione (GSH), while reactive oxygen species (ROS) contents and Malonaldehyde (MDA) protein level were significantly reduced. Mechanistically, Bortezomib pretreatment significantly promoted nuclear translocation of transcriptional factor nuclear factor erythroid 2-related factor 2(Nrf2) and Heme Oxygenase 1(HO-1) expression. Interestingly, co-treatment with ML-385, a new type and selective Nrf2 inhibitor, counteracted antioxidative effects induced by Bortezomib pretreatment. In conclusion, Bortezomib pretreatment mitigates MIRI by inhibiting oxidative damage which is regulated by Nrf2/HO-1 signaling pathway.

Treatment with Number 2 Feibi Recipe, a Compound Traditional Chinese Medicine Reduces Bleomycin-induced Pulmonary Fibrosis in Mice

Objective: Oxidative stress with reactive oxygen species (ROS) generated from exogenous oxidants and pollutants exposure is involved in the pathogenesis of Idiopathic pulmonary fibrosis (IPF). Number 2 Feibi Recipe (Number 2 FBR) is a traditional Chinese herbal formula which can attenuate the lung injury induced by PM2.5. The present study is to explore the effect and mechanism of Number 2 FBR on bleomycin (BLM)-induced pulmonary fibrosis in C57BL/6Cnc mice. Method: Bleomycin-induced C57BL/6Cnc mice were treated with Number 2 FBR and Sulforphane for two weeks. HE and Masson trichrome staining were performed to evaluate pathological changes in lung tissues. The extent of lung fibrosis was evaluated with fibrosis scores, collagen volume fraction, and hydroxyproline concentration. Levels of SOD and 8-iso-PGF2α in lung tissues were measured by using commercial assay kits. The levels of Nrf2, SOD, GSH-Px, and TGF-β1 relative protein and mRNA in lung tissues were measured by real time PCR and Western blot respectively. Results: The results showed that Number 2 FBR ameliorated bleomycin-induced pathological changes, collagen deposition and significantly decreased fibrosis scores, collagen volume fraction, and hydroxyproline concentration in the mice lungs. Additionally, Number 2 FBR inhibited the expression of 8-iso-prostaglandin F2α (8-iso-PGF2α) and transforming growth factor beta1 (TGF-β1), and increased the expression of Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2), superoxide dismutase (SOD) and Glutathione peroxidase (GSH-Px) in lung tissues. Conclusion: Number 2 FBR has an effect of anti-fibrosis by regulating the lung oxidants and antioxidants balance.

Discordant Monozygotic Parkinson Disease Twins: Role of Mitochondrial Integrity.

Even though genetic predisposition has proven to be an important element in Parkinson's disease (PD) etiology, monozygotic (MZ) twins with PD displayed a concordance rate of only about 20% despite their shared identical genetic background. We recruited 5 pairs of MZ twins discordant for idiopathic PD and established skin fibroblast cultures to investigate mitochondrial phenotypes in these cellular models against the background of a presumably identical genome. To test for genetic differences, we performed whole genome sequencing, deep mitochondrial DNA (mtDNA) sequencing, and tested for mitochondrial deletions by multiplex real-time polymerase chain reaction (PCR) in the fibroblast cultures. Further, the fibroblast cultures were tested for mitochondrial integrity by immunocytochemistry, immunoblotting, flow cytometry, and real-time PCR to quantify gene expression. Genome sequencing did not identify any genetic difference. We found decreased mitochondrial functionality with reduced cellular adenosine triphosphate (ATP) levels, altered mitochondrial morphology, elevated protein levels of superoxide dismutase 2 (SOD2), and increased levels of peroxisome proliferator-activated receptor-gamma coactivator-α (PPARGC1A) messenger RNA (mRNA) in skin fibroblast cultures from the affected compared to the unaffected twins. Further, there was a tendency for a higher number of somatic mtDNA variants among the affected twins. We demonstrate disease-related differences in mitochondrial integrity in the genetically identical twins. Of note, the clinical expression matches functional alterations of the mitochondria. ANN NEUROL 2021;89:158-164.

Sexually dimorphic and brain region-specific transporter adaptations in system xc− null mice

System xc− is a heterodimeric amino acid antiporter that, in the central nervous system, is best known for linking the import of L-cystine (CySS) with the export of L-glutamate for the production and maintenance of cellular glutathione (GSH) and extracellular glutamate levels, respectively. Yet, mice that are null for system xc− are healthy, fertile, and, morphologically, their brains are grossly normal. This suggests other glutamate and/or cyst(e)ine transport mechanisms may be upregulated in compensation. To test this, we measured the plasma membrane expression of Excitatory Amino Acid Transporters (EAATs) 1–3, the Alanine-Serine-Cysteine-Transporter (ASCT) 1, the sodium-coupled neutral amino acid transporter (SNAT) 3 and the L Amino Acid Transporter (LAT) 2 in striatum, hippocampus and cortex of male and female mice using Western Blot analysis. Present results demonstrate brain region and transporter-specific changes occurs in female system xc− null mice with increased expression of EAAT1 and ASCT1 occurring in the striatum and cortex, respectively, and decreased SNAT 3 expression in cortex. In male system xc− null brain, only SNAT3 was altered significantly - increasing in the cortex, but decreasing in the striatum. Total levels of GSH and CyS were similar to that found in age and sex-matched littermate control mice, however, reductions in the ratio of reduced to oxidized GSH (GSH/GSSG) — a hallmark of oxidative stress — were found in all three brain regions in female system xc− null mice, whereas this occurred exclusively in the striatum of males. Protein levels of Superoxide dismutase (SOD) 1 were reduced, whereas SOD2 was enhanced in the hippocampus of male xc− null mice only. Finally, striatal vulnerability to 3-nitropropionic acid (3-NP)-mediated oxidative stress in either sex showed no genotype difference, although 3-NP was more toxic to female mice of either genotype, as evidenced by an increase in moribundity as compared to males.

Impact of oxidative stress induced by chlorpyrifos in male rats: The ameliorating effect of rutin and vitamin E

The present study was designed to assess some toxicological effects in male rats following exposure to technical and formulated chlorpyrifos (CPF T. and CPF F.; at a dose of 10 mg a.i. kg-1 b.wt.) and to evaluate the possible ameliorative effect of Rutin (RT, 50 mg/kg) or Vitamin E (Vit.E, 100 mg/kg) administration. Nine groups of rats were designated for: control; [Vit.E]; [RT]; [CPF.T]; [CPF T.+Vit.E]; [CPF T.+RT]; [CPF F.]; [CPF F.+Vit.E] and [CPF.F+RT] treatments. Administration of CPF T. and CPF F. caused a significant increase in (LPO) level and decrease in the activities of antioxidant enzymes including catalase (CAT), superoxide dismutase (SOD), glutathione-s-transferase (GST), Glutathione peroxidase (GPx), and glutathione reductase (GR). Also, it caused a significant increase in serum aminotransferases, alanine transaminase (ALT) and aspartate transaminase (AST), alkaline phosphatase (ALP), lactate dehydrogenase (LDH), creatinine and uric acid levels, whereas albumin, total protein and cholinesterase (ChE) were decreased. In contrast, the present results demonstrated that co-administration of Vit.E or Rutin minimized oxidative damage included by CPF as the levels of LPO, CAT, SOD, GST, GPx, GR, AST, ALT, ALP, LDH, creatinine, uric acid, ChE, albumin and total protein were maintained to levels not significantly different from that of the normal control. These results suggest the protective role of Vit.E and Rutin in alleviating the oxidative stress and the toxicity induced by CPF in male adult rats.

Impact of Prolonged Mechanical Ventilation on Ferroptosis in Renal Ischemia/Reperfusion Injury in Rats.

We here investigated the impact of mechanical ventilation (MV) time on ferroptosis in a rat renal ischemia/reperfusion injury (IRI) model. Thirty-two male adult Sprague Dawley rats were divided into four groups (n = 8/group): the sham group, IRI group, IRI+MV-4 h group, and IRI+MV-12 h group. Rats in the IRI group were subjected to 45 min bilateral renal ischemia. Rats in the IRI+MV groups were additionally mechanically ventilated with tracheal intubation after 45 min bilateral renal ischemia. Morphological changes associated with kidney injury and ferroptosis were assessed by hematoxylin and eosin staining and electron microscopy. Levels of the central regulator of ferroptosis, glutathione peroxidase 4 (GPX4), and lipid peroxidation markers 4-hydroxynonenal (4HNE) and superoxide dismutase 2 (SOD2) were determined in the kidney tissue by western blotting. Glutathione (GSH) levels were assessed in the serum and kidney homogenate. Scr levels in the IRI+MV-12 h group were significantly higher than those in the sham, IRI, and IRI+MV-4 h groups (all P < 0.001). Electron microscopy revealed the most pronouncedly abnormal mitochondrial morphology, suggestive of ferroptosis, in the IRI+MV-12 h group. The GPX4 and SOD2 protein levels progressively decreased in the following order: sham group > IRI group > IRI+MV-4 h group > IRI+MV-12 h group (P < 0.05 for all comparisons). By contrast, the 4HNE levels progressively increased in the kidney, with the highest values in the IRI+MV-12 h group (P < 0.05, vs. the IRI group and vs. the IRI+MV-4 h group). Further, the GSH levels in the serum and kidney homogenates were significantly reduced in the IRI+MV-12 h group (P < 0.01, vs. IRI group and vs. the IRI+MV-4 h group). A significant positive correlation was observed between the serum and kidney GSH levels (r2 = 0.542, P = 0.03). These observations suggested that prolonged MV may exacerbate renal function failure, already initiated by IRI, by ferroptosis. Depletion of GSH may contribute to this effect, which requires further investigation.

Epigallocatechin Gallate Mitigates the Methamphetamine-Induced Striatal Dopamine Terminal Toxicity by Preventing Oxidative Stress in the Mouse Brain.

Methamphetamine (METH) is a popular psychostimulant due to its long-lasting effects and inexpensive production. METH intoxication is known to increase oxidative stress leading to neuronal damage. Thus, preventing the METH-induced oxidative stress can potentially mitigate neuronal damage. Previously, our laboratory found that epigallocatechin gallate (EGCG), a strong antioxidant found in green tea, can protect against the METH-induced apoptosis and dopamine terminal toxicity in the striatum of mice. In the present study, we evaluated the anti-oxidative properties of EGCG on the METH-induced oxidative stress using CD-1 mice. First, we demonstrated that mice pretreated with EGCG 30min prior to the METH injection (30mg/kg, ip) showed protection against the striatal METH-induced reduction of tyrosine hydroxylase without mitigating hyperthermia. In addition, injecting a single high dose of METH caused the reduction of striatal glutathione peroxidase activity at 24h after the METH injection. Interestingly, pretreatment with EGCG 30min prior to the METH injection prevented the METH-induced reduction of glutathione peroxidase activity. Moreover, we utilized Western blots to quantify the glutathione peroxidase 4 protein level in the striatum. The results showed that METH decreased striatal glutathione peroxidase 4 protein level, and the reduction was prevented by EGCG pretreatment. Finally, we observed that the METH-induced increase of striatal catalase and copper/zinc superoxide dismutase protein levels were also attenuated by pretreatment with EGCG. Taken together, our data indicate that EGCG is an effective agent that can be used to mitigate the METH-induced striatal toxicity in the mouse brain.

Hyperbaric oxygenation improves redox control and reduces mortality in the acute phase of myocardial infarction in a rat model.

Among the mechanisms of action of hyperbaric oxygenation (HBO), the chance of reducing injury by interfering with the mechanisms of redox homeostasis in the heart leads to the possibility of extending the period of viability of the myocardium at risk. This would benefit late interventions for reperfusion to the ischemic area. The objective of the present study was to investigate the changes in the redox system associated with HBO therapy maintained during the first hour after coronary occlusion in an acute myocardial infarction (MI) rat model. Surviving male rats (n=105) were randomly assigned to one of three groups: Sham (SH=26), myocardial infarction (MI=45) and infarction+hyperbaric therapy (HBO=34, 1 h at 2.5 atm). After 90 min of coronary occlusion, a sample of the heart was collected for western blot analysis of total protein levels of superoxide dismutase, catalase, peroxiredoxin and 3-nitrotyrosine. Glutathione was measured by enzyme-linked immunosorbent assay (ELISA). The detection of the superoxide radical anion was carried out by oxidation of dihydroethidium analyzed with confocal microscopy. The mortality rate of the MI group was significantly higher than that of the HBO group. No difference was noted in the myocardial infarction size. The oxidized/reduced glutathione ratio and peroxiredoxin were significantly higher in the SH and MI when compared to the HBO group. Superoxide dismutase enzymes and catalase were significantly higher in the HBO group compared to the MI and SH groups. 3-Nitrotyrosine and the superoxide radical were significantly lower in the HBO group compared to these in the MI and SH groups. These data demonstrated that hyperbaric oxygenation therapy decreased mortality by improving redox control in the hearts of rats in the acute phase of myocardial infarction.

Thymoquinone and curcumin modify inducible nitric oxide synthase, caspase 3, and thioredoxin immunohistochemical expression in acetaminophen hepatotoxicity.

Acetaminophen (APAP) hepatotoxicity is characterised by an extensive oxidative stress due to depletion of glutathione (GSH), which results in massive lipid peroxidation and subsequent liver injury. The current paradigm suggests that mitochondria are the main source of reactive oxygen species (ROS), which impair mitochondrial function and are responsible for cell signalling resulting in cell death. This study was designed to compare the potential impact of thymoquinone (THQ), and/or curcumin (CURC) on liver injury induced by APAP toxicity in rats. Serum levels of alanine transaminase, aspartate transaminase, total bilirubin, and total protein were measured. In addition, liver nitric oxide (NO), malondialdehyde, reduced glutathione (GSH), and superoxide dismutase (SOD) were estimated. Moreover, these biochemical parameters were confirmed by histopathological and immunohistochemical investigations for the expression of thioredoxin, iNOS and caspase 3. Acetaminophen toxicity elevated most of the above-mentioned parameters but decreased GSH, SOD, and total protein levels. Histologically, liver sections demonstrated liver injury characterised by hepatocellular necrosis with nuclear pyknosis, karyorrhexis and karyolysis. Immunohistochemical study revealed increased expression of iNOS and caspase 3 proteins, while the thioredoxin protein expression was decreased. Treatment with the THQ and CURC regulated the biochemical and histopathological alterations induced by APAP toxicity. It was concluded that the combination strategy of THQ and CURC might be considered as a potential antidote in combating liver injury induced by APAP with minimal side effects.

P6283Downregulation of Tim44 exacerbates oxidative stress-induced ROS production and cardiomyocytes death by reducing mitochondrial SOD2

Abstract Background Mitochondrial dysfunction has been highlighted as a critical driver of cardiac remodeling and failure. Mitochondria contains about 1500 proteins, 99% of which are encoded in the nuclear genome. Therefore, protein import into mitochondria is essential to maintain mitochondrial function. Previous reports suggest that nuclear-encoded mitochondrial precursor proteins import into mitochondria by multiple complex; translocase of outer membrane (TOM), translocase of inner membrane (TIM), and protein associated motor (PAM). However, the role of these protein import machineries of mitochondria in cardiac remodeling remains to be elucidated. Objective The purpose of this study was to elucidate the role of TOM, TIM, and PAM complex in cardiac remodeling and cardiomyocyte death. Methods and results C57BL/6J mice were subjected to myocardial infarction (MI) by permanent ligation of left anterior descending artery. Four weeks after operation, MI-mice demonstrated left ventricular (LV) dilation (LV end-diastolic dimension: 3.91 vs. 5.54 mm, n=8–11, p&lt;0.05) and dysfunction (LV fractional shortening: 33.3 vs. 7.7%, n=8–11, p&lt;0.05). Tim44 protein levels, a component of PAM complex, in mitochondrial fraction from non-infarcted left ventricle were significantly decreased compared with those in the heart from sham-operated mice by 39% (p&lt;0.05), whereas other proteins related to TOM, TIM and PAM complex such as Tom20, Tom22, Tom40, Tom70, Tim22, Tim23 and mtHSP70 were not altered between MI-mice and sham-mice. In addition, blue-native polyacrylamide gel electrophoresis revealed that a protein complex associated to Tim44 was significantly decreased in non-infarcted LV by 40% (p&lt;0.05). Superoxide dismutase 2 (SOD2), a mitochondrial matrix protein, was decreased in mitochondrial fraction from non-infarcted LV by 20% (p&lt;0.05), accompanied by enhancing protein carbonylation, a marker of oxidative stress, by 40% (p&lt;0.05). To assess the role of Tim44, it was downregulated by small interfering RNA in cultured neonatal rat ventricular myocytes (NRVMs). Knockdown of Tim44 significantly decreased SOD2 protein levels in mitochondrial fractionation (22%, p&lt;0.05), with no significant changes in its mRNA levels. Furthermore, knockdown of Tim44 significantly increased protein carbonylation (20%, p&lt;0.05) and cleaved caspase 3 (47%, p&lt;0.05) and decreased cell viability (69%, p&lt;0.05), assessed by cell titer assay, in H2O2-treatred NRVMs. Conclusions Downregulation of Tim44 exacerbates oxidative stress-induced ROS production and cardiomyocytes death, which is associated with a decrease in mitochondrial SOD2. Endogenous Tim44 might play a protective role in cardiac remodeling by attenuating oxidative stress and cardiomyocyte death via SOD2 import into mitochondria.

The Effects of Resistance Training and Berberine Chloride Supplementation on Oxidative Stress Markers in the Cerebellum Tissue of Diazinon-Poisoned Rats

Background: Diazinon is an insecticide from the organophosphorus group, which can increase the oxidative stress in the body. It has been established that training and antioxidant supplementation can improve oxidative stress system by decreasing malondialdehyde (MDA); meanwhile, increasing the activities of antioxidant enzymes, including superoxide dismutase (SOD), glutathione peroxidase (GPX), and catalase (CAT). Objectives: The aim of this study was to investigate the effect of resistance training (RT) and berberine chloride (BC) supplementation on oxidative stress indices in the cerebellum tissue of diazinon-poisoned rats. Methods: Fifty-six Wistar rats were divided into seven groups (n = 8), including resistance training (RT) (each session 2 sets of 6 repetitions, each one required 8 to 12 active movements with intensity of 10% - 50% of body weight, 3 sessions per week), BC2 (BC: 2 mg/kg), BC15 (BC: 15 mg/kg), RT + BC2, RT + BC15, diazinon, and control. MDA, SOD, and CAT were measured in the cerebellum tissue. Results: RT and BC significantly reduced MDA protein levels (P < 0.01), while increased SOD protein levels (P < 0.01) and CAT protein levels (P < 0.01) in the cerebellum tissue of diazinon-poisoned rats; RT with BC had interactive effects on decreasing MDA (P < 0.01), while increasing SOD (P < 0.01) and CAT protein levels (P < 0.01). Also, the consumption of 15 mg/kg BC rather than 2 mg/kg BC significantly increased SOD protein levels (P = 0.02). Conclusions: It seems that RT combination with BC has Interactive effects on the improvement of oxidative stress markers in the cerebellum tissue of diazinon-poisoned rats.

Cordycepin ameliorates cardiac hypertrophy via activating the AMPKα pathway.

Increase of myocardial oxidative stress is closely related to the occurrence and development of cardiac hypertrophy. Cordycepin, also known as 3'‐deoxyadenosine, is a natural bioactive substance extracted from Cordyceps militaris (which is widely cultivated for commercial use in functional foods and medicine). Since cordycepin suppresses oxidative stress both in vitro and in vivo, we hypothesized that cordycepin would inhibit cardiac hypertrophy by blocking oxidative stress‐dependent related signalling. In our study, a mouse model of cardiac hypertrophy was induced by aortic banding (AB) surgery. Mice were intraperitoneally injected with cordycepin (20 mg/kg/d) or the same volume of vehicle 3 days after‐surgery for 4 weeks. Our data demonstrated that cordycepin prevented cardiac hypertrophy induced by AB, as assessed by haemodynamic parameters analysis and echocardiographic, histological and molecular analyses. Oxidative stress was estimated by detecting superoxide generation, superoxide dismutase (SOD) activity and malondialdehyde levels, and by detecting the protein levels of gp91phox and SOD. Mechanistically, we found that cordycepin activated activated protein kinase α (AMPKα) signalling and attenuated oxidative stress both in vivo in cordycepin‐treated mice and in vitro in cordycepin treated cardiomyocytes. Taken together, the results suggest that cordycepin protects against post‐AB cardiac hypertrophy through activation of the AMPKα pathway, which subsequently attenuates oxidative stress.

Antioxidant effects of ginkgolides and bilobalide against cerebral ischemia injury by activating the Akt/Nrf2 pathway in vitro and in vivo.

Ginkgolide terpenoid lactones, including ginkgolides and bilobalide, are two crucial bioactive constituents of extract of Ginkgo biloba (EGb) which was used in the treatment of cardiovascular and cerebrovascular diseases. The aims of this study were to investigate the antioxidant effects and mechanism of ginkgolides (ginkgolide A (GA), ginkgolide B (GB), ginkgolide K (GK)) and bilobalide (BB) against oxidative stress induced by transient focal cerebral ischemia. In vitro, SH-SY5Y cells were exposed to oxygen-glucose deprivation (OGD) for 4h followed by reoxygenation with ginkgolides and BB treatments for 6h, and then cell viability, superoxide dismutase (SOD), and ROS were respectively detected using kit. Western blot was used to confirm the protein levels of hemeoxygenase-1 (HO-1), quinone oxidoreductase l (Nqo1), Akt, phosphorylated Akt (p-Akt), nuclear factor-E2-related factor2 (Nrf2), and phosphorylated Nrf2 (p-Nrf2). GB combined with different concentrations of LY294002 (PI3K inhibitor) were administrated to SH-SY5Y cells for 1h after OGD, and then p-Akt and p-Nrf2 levels were detected by western blot. In vivo, 2h of middle cerebral artery occlusion (MCAO) model was established, followed with reperfusion and GB treatments for 24 and 72h. The infarct volume ratios were confirmed by TTC staining. The protein levels of HO-1, Nqo1, SOD1, Akt, p-Akt, Nrf2, and p-Nrf2 were detected using western blot and immunohistochemistry (IHC). Experimental data in vitro confirm that GA, GB, GK, and BB resulted in significant decrease of ROS and increase of SOD activities and protein levels of HO-1 and Nqo1; however, GB group had a significant advantage in comparison with the GA and GK groups. Moreover, after ginkgolides and BB treatments, p-Akt and p-Nrf2 were significantly upregulated, which could be inhibited by LY294002 in a dose-dependent manner, meanwhile, GB exhibited more effective than GA and GK. In vivo, TTC staining indicated that the infarct volume ratios in MCAO rats were dramatically decreased by GB in a dose-dependent manner. Furthermore, GB significantly upregulated the protein levels of HO-1, Nqo1, SOD, p-Akt, p-Nrf2, and Nrf2. In conclusion, GA, GB, GK, and BB significantly inhibited oxidative stress damage caused by cerebral ischemia reperfusion. Compared with GA, GK, and BB, GB exerts the strongest antioxidant stress effects against ischemic stroke. Moreover, ginkgolides and BB upregulated the levels of antioxidant proteins through mediating the Akt/Nrf2 signaling pathway to protect neurons from oxidative stress injury.

Natural compound methyl protodioscin protects rat brain from ischemia/reperfusion injury through regulation of Mul1/SOD2 pathway

Methyl protodioscin (MPD) is reported to relieve angina pectoris and myocardial ischemia, and mitochondrial E3 ubiquitin ligase 1 (Mul1) plays a key role in maintaining mitochondrial functions. Bioinformatic analysis shows potential interactions between MPD and Mul1. This study aims to explore whether MPD could protect rat brain against ischemia/reperfusion (I/R) injury through regulation of Mul1/ superoxide dismutase 2 (SOD2) pathway. The SD rat brains were subjected to 2 h of ischemia following by 24 h of reperfusion, which showed I/R injury (increase in neurological deficit score and infarct volume), up-regulation of Mul1 and down regulation of SOD2, these phenomena were attenuated by MPD treatment (3 or 10 mg/kg, i.g.). Consistently, in cultured HT22 cells, hypoxia-reoxygenation (H/R) treatment induced cellular injury (apoptosis and LDH release) concomitant with up-regulation of Mul1 and down regulation of SOD2, these phenomena were blocked in the presence of MPD (5 μM). Knockdown of Mul1 could also decrease SOD2 protein levels in HT22 cells accompanied by alleviation of H/R injury (reduction of apoptosis and LDH release). In agreement with the change of SOD2, reactive oxygen species generation was increased in H/R-treated HT22 cells while decreased in the presence of MPD. Based on these observations, we conclude that upregulation of Mul1 in rat brain contributes to cerebral I/R injury via suppression of SOD2 and that MPD protects rat brain from I/R injury through a mechanism involving regulation of Mul1/SOD2 pathway.

The Effect of Swimming Training with Curcumin Consumption on Superoxide Dismutase and Glutathione Peroxidase Levels in Alcoholic Rats’ Heart Tissue

Background: Regarding the effects of excessive alcohol use on the heart tissue, the present study aimed at investigating the effect of swimming training and curcumin consumption on the levels of superoxide dismutase (SOD) and glutathione peroxidase (GPX) proteins in the heart tissue of rats after withdrawal of binge alcohol consumption. Methods: Forty rats were selected to receive alcohol (25 w/v% ethanol) every eight hours for four days through intragastric gavage. Then, they left the alcohol consumption for seven days to be divided into five groups of eight including (1) control (C), (2) curcumin (CU), (3) swimming training (ST), (4) swimming training with curcumin (STC), and (5) sham (S). Groups 3 and 4 performed swimming exercises five sessions per week for two weeks and groups 2 and 4 received 50 mg/kg intraperitoneal curcumin five times per week for two weeks. Results: CU, ST, and STCU had no significant effects on the increase of SOD protein levels in rats after excessive consumption of alcohol (P ≥ 0.05). However, CU, ST, and STCU significantly increased the GPX protein levels of rats after excessive consumption of alcohol (P ≤ 0.05). Conclusions: It seems that swimming training and curcumin consumption can be simultaneously used during withdrawal of binge alcohol abuse in order to improve GPX protein levels.