Glutathione Peroxidase Protein Expression Research Articles

Cytoprotective effects of iridoids from Gaultheria spp. berries in gastric and intestinal epithelial cells after in vitro digestion

Reactive oxygen species may induce oxidation of macromolecules in foods and during digestion in the gastrointestinal tract, negatively impacting human health. Antioxidants present in plant foods, including carotenoids, tocochromanols, ascorbic acid, as well as (poly)phenols and iridoids, may counteract oxidation of macromolecules. The aims of the present study were to determine the contents of carotenoids, tocochromanols, ascorbic acid from Gaultheria spp. berries and to investigate the cytoprotective and antioxidant activities of the fruit extracts and monotropein esters present in the berries. Moreover, the underlying mechanisms of action in gastrointestinal epithelial cells after in vitro digestion was also investigated. The content of ascorbic acid ranged from 3 to 24 mg/100 g fw, carotenoids from 9 to 56 μg/100 g fw, and total tocochromanols from 0.29 to 0.45 mg/100 g fw. The pre-incubation of gastric (AGS) and intestinal (Caco-2) cells with Gaultheria fruit extracts significantly increased viability of cells stressed with 10 mM H2O2 and 100 mM AAPH, respectively, compared to control cells. Furthermore, digested Gaultheria fruit extracts and monotropein esters increased the relative mRNA of the Nrf2/Keap1/ARE antioxidant signaling pathway and enzymes such as superoxide dismutase (SOD), glutathione peroxidase (GPX) and reductase, and catalase (CAT). The pre-incubation of Caco-2 cells with digested monotropein and monotropein esters increased the protein expression of GPX, CAT, Keap1, and Nrf2, and incremented the SOD activity in Caco-2 cells. In conclusion, Gaultheria spp. fruit extracts and monotropein esters may protect gastrointestinal epithelial cells against oxidative stress during digestive processes.

Curcumin exerts anti-inflammatory, antioxidant and anti-ferroptotic effects through the Nrf2/HO-1 pathway to protect cardiomyocytes against sepsis

This study explores the therapeutic effect of curcumin on H9c2 rat cardiac myoblasts in vitro sepsis model and its potential mechanisms. At first, Cell viability was measured using Cell Counting Kit 8 (CCK-8) and Cell-Light 5-ethynyl-2-deoxyuridine (EdU) staining, and inflammatory factors tumor necrosis factor-α (TNF-α), interleukin 6 (IL-6), interleukin 1β (IL-1β), oxygen species (ROS), malondialdehyde (MDA), superoxide dismutase (SOD), glutathione (GSH) and Fe2+ were calculated by ELISA and kits. Western blotting was used to quantitatively analyze nuclear factor erythroid 2-related factor 2 (Nrf2), heme oxygenase-1 (HO-1), glutathione peroxidase 4 (GPX4) and Acyl-CoA synthetase long-chain family (ACSL4) expression differences. Knocking down Nrf2 to study whether curcumin acts through the Nrf2/HO-1 pathway. The results show that curcumin significantly improved cell viability in lipopolysaccharide (LPS)-induced H9c2 cells (p < 0.01). Curcumin also significantly reduced inflammatory factor levels in LPS-induced cardiomyocytes (p < 0.001). Curcumin down-regulated ROS and MDA levels (p < 0.001), and up-regulated SOD and GSH levels (p < 0.001). A decrease in both Fe2+ content and protein expression of ACSL4 (p < 0.001), and increased protein expression of glutathione peroxidase 4 (GPX4) (p < 0.001) were observed with curcumin. By knocking down Nrf2 curcumin’s therapeutic effect against LPS was eliminated. So curcumin can inhibit LPS-induced oxidative stress, inflammation and ferroptosis in cardiomyocytes by regulating Nrf-2/HO-1 signaling.

Everolimus inhibits hepatoblastoma by inducing autophagy-dependent ferroptosis.

Everolimus, a known inhibitor of the mammalian target of rapamycin (mTOR), has shown uncertain efficacy in treating hepatoblastoma. This study delves into the potential anti-hepatoblastoma properties of everolimus and its intricate relationship with autophagy and ferroptosis, both in vitro and in vivo. In vivo, tumor tissue from hepatoblastoma patient and human hepatoblastoma cell line HuH-6 were xenografted into nude mice to establish xenograft models for observing the effect of everolimus on tumor growth. In vitro, HuH-6 cells were cultured to evaluate the anti-hepatoblastoma activity of everolimus. Transmission electron microscopy and microtubule-associated proteins 1 light chain 3 (LC3), beclin 1, and p62 protein expressions were employed to investigate autophagy. Additionally, indicators of cell apoptosis, reactive oxygen species (ROS) and proteins associated with ferroptosis were measured to evaluate ferroptosis. The results demonstrate that everolimus treatment effectively induced the formation of autophagosomes in hepatoblastoma cells, upregulated the LC3II/I ratio and beclin 1 expression, and downregulated p62 expression, indicating an enhanced autophagy level both in vitro and in vivo. Furthermore, everolimus treatment induced cell apoptosis, increased ROS level, elevated concentrations of malondialdehyde, 4-hydroxynonenal, and iron content, while reducing the ratio of glutathione/oxidized glutathione, and downregulating the protein expression of glutathione peroxidase 4 and solute carrier family 7 member 11, suggesting its ability to induce ferroptosis in hepatoblastoma cells. Importantly, the induction of ferroptosis by everolimus was significantly reversed in the presence of autophinib, an autophagy inhibitor, indicating the autophagy-dependent of everolimus-induced ferroptosis. Taken together, these findings suggest that everolimus holds promise as an effective anti-hepatoblastoma drug, with its mechanism of action potentially involving the induction of autophagy-dependent ferroptosis in hepatoblastoma cells.

Qi-Shen-Tang alleviates retinitis pigmentosa by inhibiting ferroptotic features via the NRF2/GPX4 signaling pathway

Ferroptosis has been observed during retinal photoreceptor cell death, suggesting that it plays a role in retinitis pigmentosa (RP) pathogenesis. Qi-Shen-Tang (QST) is a combination of two traditional Chinese medicines used for the treatment of ophthalmic diseases; however, its mechanism of action in RP and ferroptosis remains unclear. Therefore, this study aimed to explore the effect and potential molecular mechanisms of QST on RP. QST significantly improved tissue morphology and function of the retina in the RP model mice. A significant increase in retinal blood flow and normalization of the fundus structure were observed in mice in the treatment group. After QST treatment, the level of iron and the production of malondialdehyde decreased significantly; the levels of superoxide dismutase and glutathione increased significantly; and the protein expression of glutathione peroxidase 4 (GPX4), glutathione synthetase, solute carrier family 7 member 11, and nuclear factor erythroid 2-related factor 2 (NRF2) increased significantly. The molecular docking results demonstrated potential interactions between the small molecules of QST and the key proteins of NRF2/GPX4 signaling pathway. Our results indicate that QST may inhibit ferroptosis by inhibiting the NRF2/GPX4 signaling pathway, thereby reducing RP-induced damage to retinal tissue.

Wedelolactone alleviates hyperoxia-induced acute lung injury by regulating ferroptosis

To study whether wedelolactone can reduce hyperoxia-induced acute lung injury (HALI) by regulating ferroptosis, and provide a basic theoretical basis for the drug treatment of HALI. A total of 24 C57BL/6J mice were randomly divided into normal oxygen control group, HALI model group and wedelolactone pretreatment group, with 8 mice in each group. Mice in wedelolactone pretreatment group were treated with wedelolactone 50 mg/kg intraperitoneally for 6 hours, while the other two groups were not given with wedelolactone. After that, the HALI model was established by maintaining the content of carbon dioxide < 0.5% and oxygen > 90% in the molding chamber for 48 hours, and the normal oxygen control group was placed in indoor air. After modeling, the mice were sacrificed and lung tissues were collected. The lung histopathological changes were observed under light microscope and pathological scores were performed to calculate the ratio of lung wet/dry mass (W/D). The levels of tumor necrosis factor-α (TNF-α), interleukins (IL-6, IL-1β), superoxide dismutase (SOD), malondialdehyde (MDA) and glutathione (GSH) in lung tissues of mice in each group were determined. The protein expression of glutathione peroxidase 4 (GPX4) in lung tissue was detected by Western blotting. Under light microscope, the alveolar structure of HALI model group was destroyed, and a large number of neutrophils infiltrated the alveolar and interstitial lung, and the interstitial lung was thickened. The pathological score of lung injury (score: 0.75±0.02 vs. 0.11±0.01) and the ratio of lung W/D (6.23±0.34 vs. 3.68±0.23) were significantly higher than those in the normal oxygen control group (both P < 0.05). Wedelolactone pretreated mice had clear alveolar cavity and lower neutrophil infiltration and interstitial thickness than HALI group. Pathological scores (score: 0.43±0.02 vs. 0.75±0.02) and W/D ratio (4.56±0.12 vs. 6.23±0.34) were significantly lower than HALI group (both P < 0.05). Compared with the normal oxygen control group, the levels of SOD (kU/g: 26.41±4.25 vs. 78.64±3.95) and GSH (mol/g: 4.51±0.33 vs. 12.53±1.25) in HALI group were significantly decreased, while the levels of MDA (mmol/g: 54.23±4.58 vs. 9.65±1.96), TNF-α (μg/L: 96.32±3.67 vs. 11.65±2.03), IL-6 (ng/L: 163.35±5.89 vs. 20.56±3.63) and IL-1β (μg/L: 72.34±4.64 vs. 15.64±2.47) were significantly increased, and the protein expression of GPX4 (GPX4/β-actin: 0.44±0.02 vs. 1.00±0.09) was significantly decreased (all P < 0.05). Compared with the HALI group, the levels of SOD (kU/g: 53.28±3.69 vs. 26.41±4.25) and GSH (mol/g: 6.73±0.97 vs. 12.53±1.25) were significantly higher in the wedelolactone pretreatment group, and the levels of MDA (mmol/g: 25.36±1.98 vs. 54.23±4.58), TNF-α (μg/L: 40.25±4.13 vs. 96.32±3.67), IL-6 (ng/L: 78.32±4.65 vs. 163.35±5.89), and IL-1β (μg/L: 30.65±3.65 vs. 72.34±4.64) were significantly lower (all P < 0.05), and protein expression of GPX4 was significantly higher (GPX4/β-actin: 0.68±0.04 vs. 0.44±0.02, P < 0.05). Wedelolactone attenuates HALI injury by regulating ferroptosis.

Curcumin and Andrographis Exhibit Anti-Tumor Effects in Colorectal Cancer via Activation of Ferroptosis and Dual Suppression of Glutathione Peroxidase-4 and Ferroptosis Suppressor Protein-1.

Colorectal cancer (CRC) is the leading cause of cancer-related deaths worldwide. The limitations of current chemotherapeutic drugs in CRC include their toxicity, side effects, and exorbitant costs. To assess these unmet needs in CRC treatment, several naturally occurring compounds, including curcumin and andrographis, have gained increasing attention due to their multi-targeted functionality and safety vs. conventional drugs. In the current study, we revealed that a combination of curcumin and andrographis exhibited superior anti-tumor effects by inhibiting cell proliferation, invasion, colony formation, and inducing apoptosis. Genome-wide transcriptomic expression profiling analysis revealed that curcumin and andrographis activated the ferroptosis pathway. Moreover, we confirmed the gene and protein expression of glutathione peroxidase 4 (GPX-4) and ferroptosis suppressor protein 1 (FSP-1), the two major negative regulators of ferroptosis, were downregulated by this combined treatment. With this regimen, we also observed that intracellular accumulation of reactive oxygen species and lipid peroxides were induced in CRC cells. These cell line findings were validated in patient-derived organoids. In conclusion, our study revealed that combined treatment with curcumin and andrographis exhibited anti-tumorigenic effects in CRC cells through activation of ferroptosis and by dual suppression of GPX-4 and FSP-1, which have significant potential implications for the adjunctive treatment of CRC patients.

Chitosan Nanoparticles Alleviated the Adverse Effects of Sildenafil on the Oxidative Stress Markers and Antioxidant Enzyme Activities in Rats.

Sildenafil (SF) is widely used for erectile dysfunction and other conditions, though with limitations regarding oral absorption and adverse effects. Despite nanotechnological improvements, the effect of nanocarriers on SF hepatotoxicity has not been documented to date. This study aimed at assessing the impact of chitosan nanoparticles either uncoated (CS NPs) or Tween 80-coated (T-CS NPs) on the effects of SF on oxidative stress markers and antioxidant enzyme activities in rats. Test SF-CS NPs prepared by ionic gelation were uniform positively charged nanospheres (diameter 178-215 nm). SF was administered intraperitoneally to male rats (1.5 mg/kg body weight) in free or nanoencapsulated forms as SF-CS NPs and T-SF-CS NPs for 3 weeks. Free SF significantly suppressed the activity of the antioxidant enzymes glutathione S-transferase (GST), glutathione peroxidase (GPx), glutathione reductase (GR), catalase (CAT), and superoxide dismutase (SOD), as well as the levels of glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) as in an indirect measure of free radicals. Interestingly, SF-CS NPs and T-SF-CS-NPs treatments significantly attenuated the inhibitory effects of SF on the activity of these enzymes whereas, GST activity was inhibited. Moreover, the protein expression of GST was downregulated upon treatment of rats with free SF, SF-CS-NPs, and T-SF CS-NPs. In contrast, the activity and protein expression of GPx was induced by SF-CS NPs and T-SF-CS-NPs treatments. The histopathological study showed that SF induced multiple adverse effects on the rat liver architecture which were markedly suppressed particularly by T-SF-CS NPs. In conclusion, chitosan nanoencapsulation of SF counteracted the adverse effects of SF on the activity of antioxidant enzymes and liver architecture. Findings might have significant implications in improving the safety and efficacy of SF treatment of the widely expanding disease conditions.

Quercetin alleviates subarachnoid hemorrhage-induced early brain injury via inhibiting ferroptosis in the rat model.

Early brain injury (EBI) refers to a series of pathophysiological brain lesions that occur within 72 hr after subarachnoid hemorrhage (SAH), which is an extremely crucial factor in the poor prognosis of patients. In EBI, ferroptosis has been proven to cause neuronal death. Quercetin (QCT) is effective in deactivating reactive oxygen species (ROS), inhibiting lipid peroxidation, and even chelating iron, but its role in SAH remains unclear. In this study, the mortality rate, severity grade of SAH, brain water content (BWC), blood-brain barrier permeability, and neurological function of the rats were detected. Moreover, mitochondrial morphology in cortical neurons were observed and their sizes were subsequently quantified. The levels of lipid peroxidation on glutathione and malondialdehyde (MDA) and glutathione peroxidase (GSH-Px) were determined, whereas the protein expressions of glutathione peroxidase 4 (GPX4), SLC7A11 (xCT), transferrin receptor 1 (TfR1), and ferroportin-1 (FPN1) were analyzed by western immunoblotting. The neurodegeneration involved in EBI was investigated by fluoro-Jade C staining, while iron staining was utilized to measure iron content. Our results showed that inhibition of ferroptosis by QCT could suppress EBI and improve neurological function in SAH rats. QCT increased the expression levels of GPX4, xCT, and FPN1, while downregulated TfR1, and exerted protective effects on neurons as well as alleviated iron accumulation and lipid peroxidation in the cortex of SAH rats. In conclusion, our study revealed that QCT might alleviate the EBI by inhibiting ferroptosis in SAH rats.

Glutathione peroxidase 4 expression predicts poor overall survival in patients with resected lung adenocarcinoma

This study aimed to evaluate the protein expression of glutathione peroxidase 4 (GPX4) in resected non-small cell lung cancer (NSCLC). The clinical relevance and prognostic significance of GPX4 expression were analyzed. We reviewed patients with resected NSCLCs at Taipei Veterans General Hospital between September 2002 and January 2018. Available paraffin-embedded specimens were retrieved for immunohistochemistry (IHC) staining to detect GPX4 expression. The cutoff value for defining GPX4 positivity was determined according to the percentage of tumor stained in the microscopic field. The correlation between immune expression, clinicopathologic data, overall survival (OS), and disease-free survival (DFS) were analyzed. A total of 265 NSCLC specimens were retrieved for IHC staining. GPX4 expression positive was in 192 (72.5%) according to a cutoff value of 5%. GPX4 was a significant prognostic factor for OS and DFS on multivariate analysis at both 5% and 25% cutoff values. GPX4 expression was associated with poor OS and DFS, especially in lung adenocarcinoma (p = 0.008, and 0.027, respectively). In conclusions, IHC analysis revealed that GPX4 expression was associated with poor survival outcomes in patients with resected lung adenocarcinoma. Further research is needed to understand the role of GPX4 in tumorigenesis and the underlying mechanism responsible for survival outcomes in patients with resected lung adenocarcinoma.

Copper Depletion Strongly Enhances Ferroptosis via Mitochondrial Perturbation and Reduction in Antioxidative Mechanisms.

Copper serves as a co-factor for a host of metalloenzymes, particularly cytochrome c oxidase (COX). Although it is known that impaired COX function can lead to the excessive accumulation of reactive oxygen species (ROS), the mechanisms underlying how copper depletion leads to cell damage are poorly understood. Here, we have investigated the role of copper depletion during ferroptosis. The bathocuproinedisulfonic (BCS) treatment depolarized the mitochondrial membrane potential, increased the total cellular ROS levels, stimulated oxidative stress, and reduced the glutathione levels. Moreover, the depletion of copper limited the protein expression of glutathione peroxidase 4 (GPX4), which is the only enzyme that is known to prevent lipid peroxidation. Furthermore, we found that copper depletion decreased the sensitivity of the dermal papilla cells (DPCs) to erastin (an inducer of ferroptosis), and the ferroptosis inhibitor ferrostatin-1 (Fer-1) partially prevented BCS-mediated cell death. Overall, these findings establish a direct link between copper and ferroptosis; BCS-mediated copper depletion strongly enhances ferroptosis via mitochondrial perturbation and a reduction in antioxidative mechanisms.

5/6 nephrectomy affects enteric glial cells and promotes impaired antioxidant defense in the colonic neuromuscular layer

AimsChronic kidney disease (CKD) produces multiple repercussions in the gastrointestinal tract (GIT), such as alterations in motility, gut microbiota, intestinal permeability, and increased oxidative stress. However, despite enteric glial cells (EGC) having important neural and immune features in GIT physiology, their function in CKD remains unknown. The present study investigates colonic glial markers, inflammation, and antioxidant parameters in a CKD model. Main methodsA 5/6 nephrectomized rat model was used to induce CKD in rats and Sham-operated animals as a control to suppress. Biochemical measures in plasma and neuromuscular layer such as glutathione peroxidase (GPx) and superoxide dismutase (SOD) activity were carried out. Kidney histopathology was evaluated. Colon morphology analysis and glial fibrillary acid protein (GFAP), connexin-43 (Cx43), nuclear factor-kappa B (NF-κB) p65, and GPx protein expression were performed. Key findingsThe CKD group exhibited dilated tubules and tubulointerstitial fibrosis in the reminiscent kidney (p = 0.0002). CKD rats showed higher SOD activity (p = 0.004) in plasma, with no differences in neuromuscular layer (p = 0.9833). However, GPx activity was decreased in the CKD group in plasma (p = 0.013) and neuromuscular layer (p = 0.0338). Morphological analysis revealed alterations in colonic morphometry with inflammatory foci in the submucosal layer and neuromuscular layer straightness in CKD rats (p = 0.0291). In addition, GFAP, Cx43, NF-κBp65 protein expression were increased, and GPx decreased in the neuromuscular layer of the CKD group (p < 0.05). SignificanceCKD animals present alterations in colonic cytoarchitecture and decreased layer thickness. Moreover, CKD affects the enteric glial network of the neuromuscular layer, associated with decreased antioxidant activity and inflammation.

Endothelial cell ferroptosis mediates monocrotaline-induced pulmonary hypertension in rats by modulating NLRP3 inflammasome activation

Inflammation triggers pulmonary vascular remodelling. Ferroptosis, a nonapoptotic form of cell death that is triggered by iron-dependent lipid peroxidation and contributes to the pathogenesis of several inflammation-related diseases, but its role in pulmonary hypertension (PH) has not been studied. We examined endothelial cell ferroptosis in PH and the potential mechanisms. Pulmonary artery endothelial cells (PAECs) and lung tissues from monocrotaline (MCT)-induced PH rats were analysed for ferroptosis markers, including lipid peroxidation, the labile iron pool (LIP) and the protein expression of glutathione peroxidase 4 (GPX4), ferritin heavy chain 1 (FTH1) and NADPH oxidase-4 (NOX4). The effects of the ferroptosis inhibitor ferrostatin-1 (Fer-1) on endothelial cell ferroptosis and pulmonary vascular remodelling in MCT-induced rats were studied in vitro and in vivo. Ferroptosis was observed in PAECs from MCT-induced PH rats in vitro and in vivo and was characterized by a decline in cell viability accompanied by increases in the LIP and lipid peroxidation, the downregulation of GPX4 and FTH1 expression and the upregulation of NOX4 expression. High-mobility group box 1 (HMGB1)/Toll-like receptor 4 (TLR4)/NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome signalling was measured by western blotting. These changes were significantly blocked by Fer-1 administration in vitro and in vivo. These results suggest that Fer-1 plays a role in inhibiting ferroptosis-mediated PAEC loss during the progression of PH. The ferroptosis-induced inflammatory response depended on the activation of HMGB1/TLR4 signalling, which activated the NLRP3 inflammasome in vivo. We are the first to suggest that pulmonary artery endothelial ferroptosis triggers inflammatory responses via the HMGB1/TLR4/NLRP3 inflammasome signalling pathway in MCT-induced rats. Treating PH with a ferroptosis inhibitor and exploring new treatments based on ferroptosis regulation might be promising therapeutic strategies for PH.

Вплив мексидолу на глутатіонову систему мозку щурів при моделюванні хвороби Паркінсона

We studied the effects of mexidol (3-oxy-6-methyl-2-ethylpiridine succinate) on the antioxidant glutathione system in rat brain mitochondria in experimental Parkinson’s disease induced by rotenone administration. Wistar rats were divided into the following groups of 6 in each: I - intact rats (control); II - rotenone (3 mg/kg per day) was injected subcutaneously for 2 weeks; III - after rotenone intoxication, mexidol (50 mg/kg per day) was injected intraperitoneally for 2 weeks. In the suspension of brain mitochondria, the activity of NADH dehydrogenase (complex I of the mitochondrial respiratory chain), content of the active products of 2-thiobarbituric acid (TBA-AP), the reduced (GSH) and oxidized (GSSG) glutathione amounts, the activity of glutathione-dependent enzymes: glutathione peroxidase (GP) and glutathione reductase (GR) as well as NADH+-isocitrate-dehydrogenase activity (NADPH+- ICDH) were measured. The activity and protein expression of MnSOD and GP in rat brain mitochondria were estimated. Treatment of rats with mexidol led to a weakening of oxidative processes in brain mitochondria in comparison with rats exposed to rotenone intoxication. It was shown that intraperitoneal injections of mexidol led to a decrease in the TBA-AP and in the GSSG content and to an increase in GSH/GSSG ratio in comparison with rotenone intoxication. It was also registered an increase in the activity of NADH-dehydrogenase. Such changes indicated a weakening of the mitochondrial oxidative processes intensity. Treatment of rats with mexidol promoted an increase in GSH content, GR and NADPH+-ICDH activities in brain mitochondria in comparison with rotenone administration. Treatment with mexidol resulted to an increased activity and protein expression of GP and MnSOD. We conclude that mexidol reduced the rotenone-induced damage of rat brain mitochondria increasing the action of glutathione-dependent and NADPH+-generating enzymes.

Herceptin induces ferroptosis and mitochondrial dysfunction in H9c2cells.

Ferroptosis has been previously implicated in the pathological progression of cardiomyopathy. Herceptin (trastuzumab), which targets HER2, is commonly applied for the treatment of HER2+ breast cancer. However, its clinical use is limited by its cardiotoxicity. Therefore, the present study aimed to investigate if targeting ferroptosis could protect against Herceptin-induced heart failure in an in vitro model of H9c2 cells after treatment of Herceptin, Herceptin + ferroptosis inhibitor ferrostatin-1 (Fer-1) or Herceptin + Deferoxamine. H9c2 cell viability was measured by MTT assay. Reactive oxygen species (ROS) levels were detected by measuring the fluorescence of DCFH-DA-A and MitoSOX™ Red. Glutathione (GSH)/oxidized glutathione (GSSG) ratio was measured using the GSH/GSSG Ratio Detection Assay kit. Mitochondrial membrane potential and ATP content were evaluated by JC-1 staining and bioluminescent assay kits, respectively. Protein expressions of glutathione peroxidase 4, recombinant solute carrier family 7 member 11, mitochondrial optic atrophy1-1/2, mitofusin, Acyl-CoA synthetase long chain family member 4, cytochrome c, voltage-dependent anion-selective channel, dynamin-related protein, mitochondrial fission 1 protein and mitochondrial ferritin were evaluated by western blotting. It was found that Herceptin reduced H9c2 cell viability whilst increasing intracellular and mitochondrial ROS levels in a dose- and time-dependent manner. Furthermore, Herceptin decreased glutathione peroxidase (GPX) protein expression and the GSH/GSSG ratio in H9c2 cells in a dose- and time-dependent manner. The Fer-1 abolished this Herceptin-induced reduction in cell viability, GSH/GSSG ratio, mitochondrial membrane potential and ATP content. Fer-1 also reversed the suppressive effects of Herceptin on the protein expression levels of GPX4, recombinant solute carrier family 7 member 11, mitochondrial optic atrophy1-1/2 and mitofusin in H9c2 cells. Subsequently, Fer-1 was found to reverse the Herceptin-induced increase in mitochondrial ROS and iron levels in H9c2 cells, as well as the increased protein expression levels of Acyl-CoA synthetase long chain family member 4, cytochrome c, voltage-dependent anion-selective channel, dynamin-related protein, mitochondrial fission 1 protein and mitochondrial ferritin in H9c2 cells. However, compared with deferoxamine, an iron chelator, the effects of Fer-1 were less effective. Collectively, these findings provided insights into the pathogenic mechanism that underlie Herceptin-induced cardiomyopathy, which potentially provides a novel therapeutic target for the prevention of cardiotoxicity in HER2+ breast cancer treatment.

Вплив Капікору на зміни білкових маркерів розвитку окисного стресу в мітохондріях мозку щурів за умов моделювання хвороби Паркінсона

Досліджували вплив препарату Капікор (комбінація гамма-бутиробетаїн дигідрату і мельдонію дигідрату) на білкові маркери про- та антиоксидантної системи мітохондрій мозку щурів за умов ротенон-токсичної моделі хвороби Паркінсона (ХП).Мета дослідження – встановити ефективність використання препарату Капікор для корекції змін білкових компонентів про- та антиоксидантної системи мітохондрій мозку щурів за моделювання ХП ротеноном.Було показано, що внутрішньоочеревинне введення Капікору (50 мг/кг) щодня впродовж 14 днів підвищувало активність ферменту НАДН-дегідрогенази (комплекс І дихального ланцюга мітохондрій) і мітохондріальної аконітази, зменшувало вміст окисно модифікованих протеїнів, що свідчить про послаблення окисних процесів. Це відбувалося на тлі зростання вмісту відновленого глутатіону, відновлення активності та експресії білка Mn-супероксиддисмутази до контрольних показників. Про підвищення рівня антиперекисного захисту мітохондрій свідчить зростання активності та експресії білка глутатіонпероксидази.Таким чином, застосування Капікору за умов моделювання ХП призводить до зниження ротеноніндукованого пошкодження мітохондрій мозку щурів за рахунок посилення дії протективних білків ендогенного антиоксидантного захисту та послаблення процесу окисної модифікації мітохондріальних протеїнів.

Activation of p62-Keap1-Nrf2 Pathway Protects 6-Hydroxydopamine-Induced Ferroptosis in Dopaminergic Cells.

Parkinson's disease (PD) is a common neurodegenerative disorder primarily caused by the death of dopaminergic neurons in the substantia nigra pars compacta (SNpc). However, the manner of death of dopaminergic neurons remains indistinct. Ferroptosis is a form of cell death involving in the iron-dependent accumulation of glutathione depletion and lipid peroxide. Besides, previous studies indicated that ferroptosis might be involved in the death of dopaminergic neurons. In this study, we aim to explore the protective effect of the p62-Keap1-Nrf2 pathway against 6-hydroxydopamine (6-OHDA)-induced ferroptosis in dopaminergic cells. Firstly, our results demonstrated that 6-OHDA-induced ferroptosis could be observed in vivo zebrafish and in vitro human dopaminergic cell line (SH-SY5Y cells) model. Moreover, ferroptosis induced by 6-OHDA mitigates in SH-SY5Y cells upon ferrostatin-1 (Fer, an inhibitor of ferroptosis) treatment via upregulating the protein expression of glutathione peroxidase 4 (GPX4). Then, we found that high p62/SQSTM1 (p62) expression could protect SH-SY5Y cells against ferroptosis through promoting Nrf2 nuclear transfer and upregulating the expression of the antioxidant protein heme oxygenase-1 (HO-1). Ultimately, high p62 expression activates the Nrf2/HO-1 signaling pathway through binding to Kelch-like ECH-associated protein 1 (Keap1). Collectively, the activation of the p62-Keap1-Nrf2 pathway prevents 6-OHDA-induced ferroptosis in SH-SY5Y cells, targeting this pathway in combination with a pharmacological inhibitor of ferroptosis can be a potential approach for PD therapy.

NF-κB pathway took part in the development of apoptosis mediated by miR-15a and oxidative stress via mitochondrial pathway in ammonia-treated chicken splenic lymphocytes

Ammonia, a kind of gas with pungent smell, is harmful to livestock and people, and has bad influence on the atmosphere. However, the mechanism of splenic toxicity caused by ammonia is still poorly understood. The aim of present study was to investigate the effect of ammonia on chicken splenic lymphocytes from the perspective of apoptosis. Chicken splenic lymphocytes were divided into the control group and the two ammonium treatment groups (1 mmol/L and 5 mmol/L ammonia), and were cultured for 24 h. CCK-8, flow cytometry (FC), fluorescence microscope, quantitative real-time PCR (qRT-PCR), and Western blot were used to study the differences between different groups. The results showed that ammonia exposure increased the release of calcium (Ca)2+ and reactive oxygen species (ROS) from mitochondrion. Besides, we found an increase in mRNA levels of glutathione peroxidase (GPx), inflammation-related genes (nuclear factor-κB (NF-κB), cyclooxygenase-2 (COX-2), inducible nitric (iNOS), tumor necrosis factor-α (TNF-α), and transforming growth factor-β (TGF-β)), apoptosis-related genes (B-cell lymphoma-2 (BCL-2), Bcl-2 associated X protein (BAX), Cytochrome c (Cytc), apoptotic protease activating factor 1 (APAF1), Caspase-9, and Caspase-3), and an increase in protein levels of NF-κB, iNOS, BAX, Cytc, Caspase-9, and Caspase-3. At the same time, we found a decrease level of GPx protein expression, and a decrease level of glutathione S-transferase (GST) mRNA expression, and a decrease level of heme oxygenase-1 (HO-1) and BCL-2 mRNA and protein expression in splenic lymphocytes exposed to ammonia. Meanwhile, miR-15a expression increased under ammonia exposure. In summary, these results indicated that ammonia induced oxidative stress, promoted the release of Ca2+, Cytc, and ROS from mitochondria, and then induced mitochondria-mediated inflammatory response, finally triggered apoptosis in chicken splenic lymphocytes.

Ferrostatin-1 protects HT-22 cells from oxidative toxicity.

Ferroptosis is a type of programmed cell death dependent on iron. It is different from other forms of cell death such as apoptosis, classic necrosis and autophagy. Ferroptosis is involved in many neurodegenerative diseases. The role of ferroptosis in glutamate-induced neuronal toxicity is not fully understood. To test its toxicity, glutamate (1.25-20 mM) was applied to HT-22 cells for 12 to 48 hours. The optimal experimental conditions occurred at 12 hours after incubation with 5 mM glutamate. Cells were cultured with 3-12 μM ferrostatin-1, an inhibitor of ferroptosis, for 12 hours before exposure to glutamate. The cell viability was detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Autophagy was determined by monodansylcadaverine staining and apoptosis by caspase 3 activity. Damage to cell structures was observed under light and by transmission electron microscopy. The release of lactate dehydrogenase was detected by the commercial kit. Reactive oxygen species were measured by flow cytometry. Glutathione peroxidase activity, superoxide dismutase activity and malondialdehyde level were detected by the appropriate commercial kit. Prostaglandin peroxidase synthase 2 and glutathione peroxidase 4 gene expression was detected by real-time quantitative polymerase chain reaction. Glutathione peroxidase 4 and nuclear factor erythroid-derived-like 2 protein expression was detected by western blot analysis. Results showed that ferrostatin-1 can significantly counter the effects of glutamate on HT-22 cells, improving the survival rate, reducing the release of lactate dehydrogenase and reducing the damage to mitochondrial ultrastructure. However, it did not affect the caspase-3 expression and monodansylcadaverine-positive staining in glutamate-injured HT-22 cells. Ferrostatin-1 reduced the levels of reactive oxygen species and malondialdehyde and enhanced superoxide dismutase activity. It decreased gene expression of prostaglandin peroxidase synthase 2 and increased gene expression of glutathione peroxidase 4 and protein expressions of glutathione peroxidase 4 and nuclear factor (erythroid-derived)-like 2 in glutamate-injured HT-22 cells. Treatment of cultured cells with the apoptosis inhibitor Z-Val-Ala-Asp (OMe)-fluoromethyl ketone (2-8 μM), autophagy inhibitor 3-methyladenine (100-400 μM) or necrosis inhibitor necrostatin-1 (10-40 μM) had no effect on glutamate induced cell damage. However, the iron chelator deferoxamine mesylate salt inhibited glutamate induced cell death. Thus, the results suggested that ferroptosis is caused by glutamate-induced toxicity and that ferrostatin-1 protects HT-22 cells from glutamate-induced oxidative toxicity by inhibiting the oxidative stress.

Alteration of oxidative stress and expression of antioxidases in diaphragm of severely burned rats

Objective: To explore the occurrence of oxidative stress and antioxidases expression in diaphragm of severely burned rats, so that the mechanism of respiratory muscle atrophy and dysfunction post-burn injury will be further clarified. Methods: Eighty male Wistar rats (aged 7 to 8 weeks) were divided into sham injury group and burn injury group according to the random number table, with 40 rats in each group. Rats in burn injury group were inflicted with 50% total body surface area full-thickness scald (hereinafter referred to as burn) on the back and abdomen by immersing into 80 ℃ water for 15 s and 8 s respectively. Immediately after injury, 40 mL/kg normal saline was injected through abdomen for resuscitation, and the wounds were treated with iodine. Except for immersing into 37 ℃ warm water and no resuscitation, the other treatments of rats in sham injury group were the same as those of burn injury group. Whole diaphragms of 8 rats per time point per group were collected after anesthesia at post injury hour (PIH) 2 and on post injury day (PID) 1, 3, 7, and 14, and muscle mass was determined. The protein carbonyl content was determined by microplate reader. The protein expressions of catalase, superoxide dismutase 2 (SOD2), and glutathione peroxidase 1 were determined by Western blotting. Data were processed with analysis of variance of factorial design, t test, and Bonferroni correction. Results: (1) There were no statistically significant differences in the diaphragm mass of rats between the 2 groups at PIH 2 and on PID 1 (t=0.453, 0.755, P>0.05). The diaphragm mass of rats in burn injury group started to decrease from PID 3, which was significantly lower than that of sham injury group (t=3.321, P<0.01). The diaphragm mass of rats in burn injury group started to increase from PID 7 to PID 14, which was significantly lower than that of sham injury group (t=4.622, 4.380, P<0.01). (2) Protein carbonyl content in diaphragm of rats in burn injury group at PIH 2, and on PID 1, 3, 7, and 14 [(2.7±0.3), (2.5±0.5), (2.4±0.4), (2.5±0.4), (3.2±0.6) pg/mL] was significantly higher than that of sham injury group respectively [(1.2±0.4), (1.6±0.3), (1.5±0.7), (1.7±0.3), (1.8±0.4) pg/mL, t=5.994, 3.263, 3.666, 3.158, 5.763, P<0.05 or P<0.01]. (3) Protein expressions of catalase in diaphragm of rats in burn injury group on PID 1 and 3 were close to those of sham injury group (t=0.339, 0.324, P>0.05). There were no statistically significant differences in protein expressions of SOD2 in diaphragm of rats between the 2 groups at PIH 2 and on PID 1, 3, 7, and 14 (t=1.446, 1.385, 0.757, 1.561, 0.531, P>0.05). There were no statistically significant differences in protein expressions of glutathione peroxidase 1 in diaphragm of rats in the 2 groups at PIH 2 and on PID 1, 3, and 7 (t=0.200, 0.729, 0.385, 1.559, P>0.05). Conclusions: Continuous oxidative stress and relatively insufficient expression of antioxidases in diaphragm induced by burn injury could be a contributor to diaphragm atrophy.

L-Arginine Protects Ovine Intestinal Epithelial Cells from Lipopolysaccharide-Induced Apoptosis through Alleviating Oxidative Stress.

This research aims to explore the effect of l-arginine (Arg) upon lipopolysaccharide (LPS)-induced induction of the oxidative stress as well as subsequent apoptosis within ovine intestinal epithelial cells (IOECs). Through a 16 h incubation, cells were divided into four groups and the medium was replaced with different medium as follows: (1) control (Con), Arg-free Dulbecco's modified Eagle's F12 Ham medium (DMEM); (2) Arg treatment, Arg-free DMEM supplemented with 100 μM Arg; (3) LPS treatment, Arg-free DMEM supplemented with 10 μg/mL LPS; (4) LPS with Arg treatment, Arg-free DMEM supplemented with both 10 μg/mL LPS and 100 μM Arg. After culturing for 24 h in different mediums, some characteristics of cells in the four groups were measured. Addition of Arg increased cell viability induced with LPS compared with the LPS group ( p < 0.05). Arg significantly decreased the release of dehydrogenase (LDH) and the production of malonaldehyde (MDA) ( p < 0.05) within IOECs challenged by the LPS. Compared with the LPS group, cells treated with Arg and Arg + LPS increased ( p < 0.05) mRNA as well as protein expression of glutathione peroxidase 1 (GPx1), catalase (CAT), superoxide dismutase 2 (SOD2), B-cell lymphoma 2 (Bcl2), quinone oxidoreductase 1 (NQO1), heme oxygenase (HO-1), and nuclear factor erythroid 2-related factor 2 (Nrf2). IOEC treatment with Arg reduced significantly ( p < 0.05) apoptosis induced by the LPS (12.58 ± 0.79%). The results showed that Arg promoted the protein expression of Nrf2, up-regulated expression of the phase II metabolizing enzymes (NQO1 and HO-1), as well as antioxidative enzymes (GPx1, CAT, and SOD2) for alleviating oxidative injury and protected IOECs from LPS-induced apoptosis.