Attenuated Reactive Oxygen Species Production Research Articles

Connexin 43 modulates reverse electron transfer in cardiac mitochondria from inducible knock-out Cx43Cre-ER(T)/fl mice by altering the coenzyme Q pool.

Succinate accumulates during myocardial ischemia and is rapidly oxidized during reperfusion, leading to reactive oxygen species (ROS) production through reverse electron transfer (RET) from mitochondrial complex II to complex I, and favoring cell death. Given that connexin 43 (Cx43) modulates mitochondrial ROS production, we investigated whether Cx43 influences RET using inducible knock-out Cx43Cre-ER(T)/fl mice. Oxygen consumption, ROS production, membrane potential and coenzyme Q (CoQ) pool were analyzed in subsarcolemmal (SSM, expressing Cx43) and interfibrillar (IFM) cardiac mitochondria isolated from wild-type Cx43fl/fl mice and Cx43Cre-ER(T)/fl knock-out animals treated with 4-hydroxytamoxifen (4OHT). In addition, infarct size was assessed in isolated hearts from these animals submitted to ischemia-reperfusion (IR), and treated or not with malonate, a complex II inhibitor attenuating RET. Succinate-dependent ROS production and RET were significantly lower in SSM, but not IFM, from Cx43-deficient animals. Mitochondrial membrane potential, a RET driver, was similar between groups, whereas CoQ pool (2.165±0.338 vs. 4.18±0.55 nmol/mg protein, p<0.05) and its reduction state were significantly lower in Cx43-deficient animals. Isolated hearts from Cx43Cre-ER(T)/fl mice treated with 4OHT had a smaller infarct size after IR compared to Cx43fl/fl, despite similar concentration of succinate at the end of ischemia, and no additional protection by malonate. Cx43 deficiency attenuates ROS production by RET in SSM, but not IFM, and was associated with a decrease in CoQ levels and a change in its redox state. These results may partially explain the reduced infarct size observed in these animals and their lack of protection by malonate.

Sulforaphane Inhibits IL-1β-Induced IL-6 by Suppressing ROS Production, AP-1, and STAT3 in Colorectal Cancer HT-29 Cells.

Colorectal cancer (CRC) stands as a major cause of cancer-related mortality globally, accounting for approximately 881,000 deaths each year. Traditional approaches such as chemotherapy and surgery have been the primary treatment modalities, yet the outcomes for patients with metastatic CRC are often unsatisfactory. Recent research has focused on targeting the pathways involved in oxidative stress, inflammation, and metastasis to enhance the survival of CRC patients. Within this context, sulforaphane (SFN), a notable phytochemical found predominantly in cruciferous vegetables, has been recognized as a potential anticancer agent. However, the specific mechanisms through which SFN may exert its chemopreventive effects in CRC remain unclear. This study explores the impact of SFN on IL-1β-induced IL-6 activation and MAPK and AP-1 signaling in HT-29 cells. Our findings reveal that SFN treatment not only diminishes IL-1β-stimulated IL-6 expression but also reduces oxidative stress by curtailing reactive oxygen species (ROS) production. Furthermore, it hinders the proliferation and invasiveness of HT-29 cells through the modulation of MAPK/AP-1 and STAT3 signaling pathways. These results indicate that SFN mitigates IL-1β-induced IL-6 expression in CRC cells by attenuating ROS production and disrupting MAPK/AP-1 signaling. This suggests that SFN holds significant potential as a chemotherapeutic agent for both treating and preventing CRC.

Knockdown of CXCL1 improves ACLF by reducing neutrophil recruitment to attenuate ROS production and hepatocyte apoptosis.

Acute-on-chronic liver failure (ACLF) is an acute decompensated syndrome based on chronic liver disease, while neutrophil recruitment is the most critical early step. C-X-C motif chemokine ligand 1 (CXCL1), a cytokine that recruits neutrophils, was significantly upregulated in both ACLF mice and patients with ACLF. This present study aims to explore the role of CXCL1 in the pathogenesis of ACLF. We established an ACLF mouse model induced by carbon tetrachloride, lipopolysaccharide, and D-galactosamine, and used adeno-associated virus to achieve overexpression and knockdown of Cxcl1. We employed mass cytometry, flow cytometry, multiplex cytokine and chemokine analysis, Western blot, and reactive oxygen species (ROS) detection in mice blood and liver. ACLF patients (n = 10) and healthy controls (n = 5) were included, and their liver samples were stained using multiplex immunohistochemistry techniques. CXCL1 was significantly elevated in both ACLF mice and patients. CXCL1 recruits neutrophils by binding to the C-X-C motif chemokine receptor 2 on the surface of neutrophils, affects ACLF prognosis by generating ROS and mitochondrial depolarization and modulating caspase3-related apoptotic pathways. We found that the knockdown of CXCL1 attenuated the infiltration of neutrophils in the mouse liver, reduced the expression of inflammatory cytokines, and also significantly downregulated ROS production and caspase3-related hepatocyte apoptosis, thereby ameliorating the liver injury of ACLF. CXCL1 is a core player in the mobilization of neutrophils in ACLF, and the knockdown of Cxcl1 improves neutrophil infiltration, reduces ROS levels, and reduces hepatocyte apoptosis, thereby attenuating inflammation and liver injury in ACLF. Our results revealed a previously unknown link between CXCL1-induced neutrophil recruitment and ACLF, providing evidencing for potential therapies targeting ACLF.

Hyperbaric Oxygen Therapy Counters Oxidative Stress/Inflammation-Driven Symptoms in Long COVID-19 Patients: Preliminary Outcomes.

Long COVID-19 patients show systemic inflammation and persistent symptoms such as fatigue and malaise, profoundly affecting their quality of life. Since improving oxygenation can oppose inflammation at multiple tissue levels, we hypothesized that hyperbaric oxygen therapy (HBOT) could arrest inflammation progression and thus relieve symptoms of COVID-19. We evaluated oxy-inflammation biomarkers in long COVID-19 subjects treated with HBOT and monitored with non-invasive methods. Five subjects (two athletes and three patients with other comorbidities) were assigned to receive HBOT: 100% inspired O2 at 2.4 ATA in a multiplace hyperbaric chamber for 90 min (three athletes: 15 HBOT × 5 days/wk for 3 weeks; two patients affected by Idiopathic Sudden Sensorineural Hearing Loss: 30 HBOT × 5 days/wk for 6 weeks; and one patient with osteomyelitis: 30 HBOT × 5 days/wk for week for 6 weeks and, after a 30-day break, followed by a second cycle of 20 HBOT). Using saliva and/or urine samples, reactive oxygen species (ROS), antioxidant capacity, cytokines, lipids peroxidation, DNA damage, and renal status were assessed at T1_pre (basal level) and at T2_pre (basal level after treatment), and the results showed attenuated ROS production, lipid peroxidation, DNA damage, NO metabolites, and inflammation biomarker levels, especially in the athletes post-treatment. Thus, HBOT may represent an alternative non-invasive method for treating long COVID-19-induced long-lasting manifestations of oxy-inflammation.

Alternative oxidase causes cell type- and tissue-specific responses in mutator mice.

Energetic insufficiency, excess production of reactive oxygen species (ROS), and aberrant signaling partially account for the diverse pathology of mitochondrial diseases. Whether interventions affecting ROS, a regulator of stem cell pools, could modify somatic stem cell homeostasis remains unknown. Previous data from mitochondrial DNA mutator mice showed that increased ROS leads to oxidative damage in erythroid progenitors, causing lifespan-limiting anemia. Also unclear is how ROS-targeted interventions affect terminally differentiated tissues. Here, we set out to test in mitochondrial DNA mutator mice how ubiquitous expression of the Ciona intestinalis alternative oxidase (AOX), which attenuates ROS production, affects murine stem cell pools. We found that AOX does not affect neural stem cells but delays the progression of mutator-driven anemia. Furthermore, when combined with the mutator, AOX potentiates mitochondrial stress and inflammatory responses in skeletal muscle. These differential cell type-specific findings demonstrate that AOX expression is not a global panacea for curing mitochondrial dysfunction. ROS attenuation must be carefully studied regarding specific underlying defects before AOX can be safely used in therapy.

Pimobendan prevents cardiac dysfunction, mitigates cardiac mitochondrial dysfunction, and preserves myocyte ultrastructure in a rat model of mitral regurgitation

BackgroundPimobendan has been proven to delay the onset of congestive heart failure (CHF) in dogs with mitral regurgitation (MR); however, molecular underlying mechanisms have not been fully elucidated. This study aimed to investigate (1) the effects of pimobendan on cardiac function, cardiac mitochondrial quality and morphology, and cardiac ultrastructure in a rat model of chronic MR and (2) the direct effect of pimobendan on intracellular reactive oxygen species (ROS) production in cardiac cells. MR was surgically induced in 20 Sprague-Dawley rats, and sham procedures were performed on 10 rats. Eight weeks post-surgery, the MR rats were randomly divided into two groups: the MR group and the MR + pimobendan group. Pimobendan (0.15 mg/kg) was administered twice a day via oral gavage for 4 weeks, whereas the sham and MR groups received equivalent volumes of drinking water. Echocardiography was performed at baseline (8 weeks post-surgery) and at the end of the study (4 weeks after treatment). At the end of the study protocol, all rats were euthanized, and their hearts were immediately collected, weighed, and used for transmission electron microscopy and mitochondrial quality assessments. To evaluate the role of pimobendan on intracellular ROS production, preventive or scavenging properties were tested with H2O2-induced ROS generation in rat cardiac myoblasts (H9c2).ResultsPimobendan preserved cardiac functions and structure in MR rats. In addition, pimobendan significantly improved mitochondrial quality by attenuating ROS production and depolarization (P < 0.05). The cardiac ultrastructure and mitochondrial morphology were significantly preserved in the MR + pimobendan group. In addition, pimobendan appeared to play as a ROS scavenger, but not as a ROS preventer, in H2O2-induced ROS production in H9c2 cells.ConclusionsPimobendan demonstrated cardioprotective effects on cardiac function and ultrastructure by preserving mitochondrial quality and acted as an ROS scavenger in a rat model of MR.

Stigmasterol isolated from Azadirachta indica flowers attenuated glutamate-induced neurotoxicity via downregulation of the Cdk5/p35/p25 signaling pathway in the HT-22 cells

BackgroundGlutamate, an excitatory neurotransmitter, was elevated in the brain of neurodegenerative disease (ND) patients. The excessive glutamate induces Ca2+ influx and reactive oxygen species (ROS) production which exacerbates mitochondrial function, leading to mitophagy aberration, and hyperactivates Cdk5/p35/p25 signaling leading to neurotoxicity in ND. Stigmasterol, a phytosterol, has been reported for its neuroprotective effects; however, the underlying mechanism of stigmasterol on restoring glutamate-induced neurotoxicity is not fully investigated. PurposeWe investigated the effect of stigmasterol, a compound isolated from Azadirachta indica (AI) flowers, on ameliorating glutamate-induced neuronal apoptosis in the HT-22 cells. Study designTo further understand the underlying molecular mechanisms of stigmasterol, we investigated the effect of stigmasterol on Cdk5 expression, which was aberrantly expressed in glutamate-treated cells. Cell viability, Western blot analysis, and immunofluorescence are employed. ResultsStigmasterol significantly inhibited glutamate-induced neuronal cell death via attenuating ROS production, recovering mitochondrial membrane depolarization, and ameliorating mitophagy aberration by decreasing mitochondria/lysosome fusion and the ratio of LC3-II/LC3-I. In addition, stigmasterol treatment downregulated glutamate-induced Cdk5, p35, and p25 expression via enhancement of Cdk5 degradation and Akt phosphorylation. Although stigmasterol demonstrated neuroprotective effects on inhibiting glutamate-induced neurotoxicity, the efficiency of stigmasterol is limited due to its poor water solubility. We conjugated stigmasterol to soluble soybean polysaccharides with chitosan nanoparticles to overcome the limitations. We found that the encapsulated stigmasterol increased water solubility and enhanced the protective effect on attenuating the Cdk5/p35/p25 signaling pathway compared with free stigmasterol. ConclusionOur findings illustrate the neuroprotective effect and the improved utility of stigmasterol in inhibiting glutamate-induced neurotoxicity.

Sodium acetate ameliorates cisplatin-induced kidney injury in vitro and in vivo

Cisplatin is an effective chemotherapeutic drug against tumors. Studies often report on the improvement of kidney injury by probiotics or short-chain fatty acids (SCFAs); however, the effects of SCFAs on cisplatin-induced kidney injury are rarely studied. The aim of this study is to evaluate the function of sodium acetate on preventing cisplatin-induced kidney injury. Cell viability was detected by MTT assay. SA-β-gal staining was performed to investigate premature senescence. Reactive oxygen species (ROS) production was analyzed by H2DCFDA staining. Propidium iodide (PI) staining was analyzed by cell cycle. Protein expression was determined by Western blot assay. Annexin Ⅴ/PI staining was used to investigate cisplatin-induced apoptosis. Tumor growth and kidney injury were evaluated in C57BL/6 mice. Sodium acetate ameliorated cisplatin-induced premature senescence and ROS production in SV40 MES-13 glomerular cells, NRK-52E renal tubular cells, and NRK-49F renal fibroblast cells. Cisplatin-induced cell cycle arrest was inhibited by sodium acetate in SV40 MES-13 and NRK-49F cells. Sodium acetate alleviated cisplatin-induced apoptosis in vivo and in vitro but not cisplatin-induced fibrosis. Our study demonstrated that sodium acetate inhibited cisplatin-induced premature senescence, cell cycle arrest, and apoptosis by attenuating ROS production. This strategy may be useful in the treatment of cisplatin-induced kidney injury.

N-Acetylcysteine Enhances the Recovery of Ischemic Limb in Type-2 Diabetic Mice

Critical limb ischemia (CLI) is a severe complication of diabetes mellitus that occurs without effective therapy. Excessive reactive oxygen species (ROS) production and oxidative stress play critical roles in the development of diabetic cardiovascular complications. N-acetylcysteine (NAC) reduces ischemia-induced ROS production. The present study aimed to investigate the effect of NAC on the recovery of ischemic limb in an experimental model of type-2 diabetes. TALLYHO/JngJ diabetic and SWR/J non-diabetic mice were used for developing a CLI model. For NAC treatment, mice received NAC (1 mg/mL) in their drinking water for 24 h before initiating CLI, and continuously for the duration of the experiment. Blood flow, mechanical function, histology, expression of antioxidant enzymes including superoxide dismutase (SOD)-1, SOD-3, glutathione peroxidase (Gpx)-1, catalase, and phosphorylated insulin receptor substrate (IRS)-1, Akt, and eNOS in ischemic limb were evaluated in vivo or ex vivo. Body weight, blood glucose, plasma advanced glycation end-products (AGEs), plasma insulin, insulin resistance index, and plasma TNF-a were also evaluated during the experiment. NAC treatment effectively attenuated ROS production with preserved expressions of SOD-1, Gpx-1, catalase, phosphorylated Akt, and eNOS, and enhanced the recovery of blood flow and function of the diabetic ischemic limb. NAC treatment also significantly decreased the levels of phosphorylated IRS-1 (Ser307) expression and plasma TNF-α in diabetic mice without significant changes in blood glucose and AGEs levels. In conclusion, NAC treatment enhanced the recovery of blood flow and mechanical function in ischemic limbs in T2D mice in association with improved tissue redox/inflammatory status and insulin resistance.

Ergosterol isolated from cloud ear mushroom (Auricularia polytricha) attenuates bisphenol A-induced BV2 microglial cell inflammation

Bisphenol A (BPA) has been reported to have neurotoxic properties that may increase the risk of neurodegenerative diseases by inducing neuroinflammation. Auricularia polytricha (AP) is an edible mushroom with several medicinal properties. Herein, the anti-neuroinflammatory effects of AP extracts against BPA-induced inflammation of BV2 microglial cells were investigated. Hexane (APH) and ethanol (APE) extracts of AP inhibited BPA-induced neuroinflammation in BV2 microglia by reducing microglial activation and the expression of pro-inflammatory cytokines. These anti-inflammatory effects were regulated by the NF-κB signaling pathway. In addition, APH and APE exhibited antioxidative effects by increasing the activity of the SOD-1 enzyme and restoring the accumulation of reactive oxygen species (ROS) in BPA-induced BV2 cells. Moreover, the conditioned medium prepared using BPA-induced BV2 cells demonstrated that the presence of APH or APE could attenuate ROS production in HT-22 cells. Further, ergosterol was isolated from APE and also showed anti-inflammatory and antioxidative activities. In conclusion, AP extracts and ergosterol attenuated neuroinflammation against BPA induction in BV2 microglial cells through the NF-κB signaling pathway.

Targeting p21‐highly‐expressing Senescent Cells Enhances Skeletal Muscle Function through Mitochondrial Function and Reactive Oxygen Species

BACKGROUNDThe accumulation of senescent cells is a hallmark of aging in the skeletal muscle which causes reactive oxygen species (ROS) and mitochondrial dysfunction. p21 is one of the major regulators and most recognized cellular markers for senescent cells. Recent evidence demonstrated that clearance of p21high cells enhances muscle function including grip strength, hanging endurance, and maximal walking speed in mice. However, it is still unclear how the muscle performance enhances through the clearance of p21high cells. One of the potential mechanisms is mitochondrial function and/or mitochondria‐derived ROS. Therefore, this study investigated the linkage between mitochondria and p21high cells in aging or high fat diet‐induced muscle dysfunction.METHODSFive p21‐Cre/+; +/+ (P) and p21‐Cre/+; DTA/+ (PD) obese mice fed by high fat diet were administrated with tamoxifen for twice. Five P lean mice fed by normal chow were adopted as normal control. We previously demonstrated that p21high cells accumulate in obese P mice, and can be eliminated in obese PD mice by tamoxifen treatment. Mitochondrial respiration was measured, by high‐resolution respirometry, in permeabilized muscle fibers from the soleus muscle. Mitochondria‐derive ROS production was determined using Amplex Red assays. One‐way ANOVAs with Bonferroni post‐hoc were used to determine differences between groups (P &lt; 0.05).RESULTSReductions in complex I + II state 3 respiration were observed in p21‐Cre mice with high fat diet but the clearance of p21high cells using tamoxifen enhanced complex I + II state 3 respiration (Lean P: 35.2 ± 3.13 pmol·s‐1·mg‐1; Obese P: 17.45 ± 2.85 pmol·s‐1·mg‐1; Obese PD: 30.04 ± 3.19 pmol·s‐1·mg‐1; P &lt; 0.05). State 4 respiration did not differ between groups (P &gt; 0.05). Respiratory Control Ratio (RCR), defined as respiration in state 3 divided by respiration in state 4, significantly decreased in high fat diet group but clearing p21high cells restored respiratory function (Lean P: 4.20 ± 1.18; Obese P: 2.21 ± 1.11; Obese PD: 3.85 ± 1.60; P&lt;0.05). Also, high fat diet increased mitochondrial ROS production, but tamoxifen treatment attenuated ROS production (Lean P: 10.16 ± 0.41 pmol·s‐1·mg‐1; Obese P: 26.83 ± 0.54 pmol·s‐1·mg‐1; Obese PD: 12.23 ± 0.54 pmol·s‐1·mg‐1; P&lt;0.05).CONCLUSIONThese results demonstrated that p21high cells may play a causal role in mitochondrial dysfunction and ROS emission in the skeletal muscle with obesity and other chronic diseases. Therefore, targeting p21high cells may enhance muscle performance through decreasing mitochondria‐derived ROS and enhancing mitochondrial function.

Eriodictyol Suppresses Porphyromonas gingivalis-Induced Reactive Oxygen Species Production by Gingival Keratinocytes and the Inflammatory Response of Macrophages.

Porphyromonas gingivalis is a key pathogen of periodontitis, an inflammatory disease that affects the tooth-supporting tissues. The aim of the present study was to investigate the effects of the flavanone eriodictyol on P. gingivalis-induced reactive oxygen species (ROS) production by gingival keratinocytes and the inflammatory response of macrophages. Porphyromonas gingivalis and H2O2 acted synergistically to induce ROS production by keratinocytes. The presence of eriodictyol significantly attenuated ROS production in a dose-dependent manner. We used a macrophage model to show that eriodictyol decreases the secretion of IL-1β, IL-6, IL-8, and TNF-α induced by P. gingivalis. Evidence has been brought that this anti-inflammatory property of eriodictyol may be related to its ability to prevent the activation of the NF-κB signaling pathway by P. gingivalis. This periodontal pathogen was also found to be a potent inducer of matrix metalloproteinase (MMP) production by macrophages, including MMP-2, MMP-8, and MMP-9. Eriodictyol dose-dependently inhibited the production of all three MMPs. Lastly, eriodictyol inhibited the catalytic activity of both MMP-9 and P. gingivalis collagenase. In conclusion, eriodictyol may be a potential therapeutic agent for preventing and/or treating periodontal disease due to its antioxidant, anti-inflammatory, and anti-proteinase properties.

Effect of expansion of human umbilical cord blood CD34\u2009+\u2009cells on neurotrophic and angiogenic factor expression and function

The use of CD34 + cell-based therapies has largely been focused on haematological conditions. However, there is increasing evidence that umbilical cord blood (UCB) CD34 + -derived cells have neuroregenerative properties. Due to low cell numbers of CD34 + cells present in UCB, expansion is required to produce sufficient cells for therapeutic purposes, especially in adults or when frequent applications are required. However, it is not known whether expansion of CD34 + cells has an impact on their function and neuroregenerative capacity. We addressed this knowledge gap in this study, via expansion of UCB-derived CD34 + cells using combinations of LDL, UM171 and SR-1 to yield large numbers of cells and then tested their functionality. CD34 + cells expanded for 14 days in media containing UM171 and SR-1 resulted in over 1000-fold expansion. The expanded cells showed an up-regulation of the neurotrophic factor genes BDNF, GDNF, NTF-3 and NTF-4, as well as the angiogenic factors VEGF and ANG. In vitro functionality testing showed that these expanded cells promoted angiogenesis and, in brain glial cells, promoted cell proliferation and reduced production of reactive oxygen species (ROS) during oxidative stress. Collectively, this study showed that our 14-day expansion protocol provided a robust expansion that could produce enough cells for therapeutic purposes. These expanded cells, when tested in in vitro, maintained functionality as demonstrated through promotion of cell proliferation, attenuation of ROS production caused by oxidative stress and promotion of angiogenesis.

Putative Prevention of XML Injection Against Myocardial Ischemia Is Mediated by PKC and PLA2 Proteins

Background: Xinmailong (XML) injection is a CFDA-approved traditional Chinese medicine with clinical value for heart failure treatment. The present investigation was aimed to evaluate the potential protective roles of this injection on myocardial ischemia and the underlying molecular mechanism. Methods: In our study, we selected two models of myocardial ischemia rats. Rats were randomly divided into six groups, with saline or XML administrated 4 days before ischemia model establishment. ECG of different time intervals and biochemical parameters of end point were measured. The potential mechanisms of the protective role of XML were explored using system pharmacology and molecular biology approaches. Results: Myocardial ischemia rats demonstrated abnormal ECG and serum levels of cTnT. Pretreatment with XML significantly attenuated these damages, especially the medium doses. GO and KEGG analysis revealed that the 90 putative target genes were associated with pathways of fatty acid absorption/metabolism, inflammation, RAAS, and vascular smooth muscle. Further network pharmacology method identified five main chemical ingredients and potential targets of XML injection for myocardial ischemia. Mechanically, the beneficial effect of XML injection was mediated by the reactive oxygen species (ROS) inhibition and inflammation attenuation via regulating the expression levels of targets of PKC and PLA2. Conclusion: These findings indicate that XML exerts protective effects against myocardial injury, with attenuated ROS production, apoptosis, and inflammation. Therefore, we speculate that XML may be an alternative supplementary therapeutic agent for myocardial ischemia prevention.

(-)-Epicatechin Alters Reactive Oxygen and Nitrogen Species Production Independent of Mitochondrial Respiration in Human Vascular Endothelial Cells.

Introduction Vascular endothelial dysfunction is characterised by lowered nitric oxide (NO) bioavailability, which may be explained by increased production of reactive oxygen species (ROS), mitochondrial dysfunction, and altered cell signalling. (-)-Epicatechin (EPI) has proven effective in the context of vascular endothelial dysfunction, but the underlying mechanisms associated with EPI's effects remain unclear. Objective(s). Our aim was to investigate whether EPI impacts reactive oxygen and nitrogen species (RONS) production and mitochondrial function of human vascular endothelial cells (HUVECs). We hypothesised that EPI would attenuate ROS production, increase NO bioavailability, and enhance indices of mitochondrial function. Methods HUVECs were treated with EPI (0-20 μM) for up to 48 h. Mitochondrial and cellular ROS were measured in the absence and presence of antimycin A (AA), an inhibitor of the mitochondrial electron transport protein complex III, favouring ROS production. Genes associated with mitochondrial remodelling and the antioxidant response were quantified by RT-qPCR. Mitochondrial bioenergetics were assessed by respirometry and signalling responses determined by western blotting. Results Mitochondrial superoxide production without AA was increased 32% and decreased 53% after 5 and 10 μM EPI treatment vs. CTRL (P < 0.001). With AA, only 10 μM EPI increased mitochondrial superoxide production vs. CTRL (25%, P < 0.001). NO bioavailability was increased by 45% with 10 μM EPI vs. CTRL (P = 0.010). However, EPI did not impact mitochondrial respiration. NRF2 mRNA expression was increased 1.5- and 1.6-fold with 5 and 10 μM EPI over 48 h vs. CTRL (P = 0.015 and P = 0.001, respectively). Finally, EPI transiently enhanced ERK1/2 phosphorylation (2.9 and 3.2-fold over 15 min and 1 h vs. 0 h, respectively; P = 0.035 and P = 0.011). Conclusion(s). EPI dose-dependently alters RONS production of HUVECs but does not impact mitochondrial respiration. The induction of NRF2 mRNA expression with EPI might relate to enhanced ERK1/2 signalling, rather than RONS production. In humans, EPI may improve vascular endothelial dysfunction via alteration of RONS and activation of cell signalling.

Methylglyoxal Scavengers Attenuate Angiogenesis Dysfunction Induced by Methylglyoxal and Oxygen-Glucose Deprivation.

Cerebral endothelial cells play an essential role in brain angiogenesis, and their function has been found to be impaired in diabetes. Methylglyoxal (MG) is a highly reactive dicarbonyl metabolite of glucose formed mainly during glycolysis, and its levels can be elevated in hyperglycemic conditions. MG is a potent precursor of AGEs (advanced glycation end-products). In this study, we investigated if MG can induce angiogenesis dysfunction and whether MG scavengers can ameliorate angiogenesis dysfunction induced by MG. Here, we used cultured human brain microvascular endothelial cells (HBMECs) treated with MG and oxygen-glucose deprivation (OGD) to mimic diabetic stroke in vitro. We also used the MG challenged chicken embryo chorioallantoic membrane (CAM) to study angiogenesis in vivo. Interestingly, administration of MG significantly impaired cell proliferation, cell migration, and tube formation and decreased protein expression of angiogenesis-related factors, which was rescued by three different MG scavengers, glyoxalase 1 (GLO1), aminoguanidine (AG), and N-acetyl cysteine (NAC). In cultured CAM, MG exposure significantly reduced angiogenesis and the angiogenesis-related dysfunction could be attenuated by pretreatment with AG or NAC. Treatment of cultured HBMECs with MG plus OGD increased cellular apoptosis significantly, which could be prevented by exposure to GLO1, AG, or NAC. We also noted that administration of MG increased cellular oxidative stress as measured by reactive oxygen species (ROS) generation, enhanced AGE accumulation, and receptor for advanced glycation end-product (RAGE) expression in the cultured HBMECs, which were partially reversed by GLO1, AG, or NAC. Taken together, our findings demonstrated that GLO1, AG, or NAC administration can ameliorate MG-induced angiogenesis dysfunction, and this can be mainly attributed to attenuated ROS production, reduced cellular apoptosis, and increased levels of angiogenic factors. Overall, this study suggested that GLO1, AG, or NAC may be promising candidate compounds for the treatment of angiogenesis dysfunction caused by hyperglycemia in diabetic ischemic stroke.

The effects of agomelatine on endoplasmic reticulum stress related to mitochondrial dysfunction in hippocampus of aging rat model

BackgroundAgomelatine, a novel antidepressant, is a melatonin MT receptor agonist and serotonin 5HT2C receptor antagonist. In this study, agomelatine was used to investigate the molecular mechanisms of hippocampal aging associated with endoplasmic reticulum (ER) stress, mitochondrial dysfunction, and apoptosis, all of which led to short-term memory impairment. MethodHippocampal aging was induced in male Wistar rats by d-galactose (D-gal) intraperitoneal injection (100 mg/kg) for 14 weeks. During the last 4 weeks of D-gal treatment, rats were treated with agomelatine (40 mg/kg) or melatonin (10 mg/kg). At the end of the experiment, all rats were assessed for short-term memory by using the Morris water maze test. Subsequently, rats were sacrified and the hippocampus was removed from each rat for determination of reactive oxygen species (ROS), malondialdehyde (MDA), and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assays; and immunohistochemistry related to ER stress, mitochondrial dysfunction, and apoptosis. ResultsAgomelatine suppressed the expression of the aging-related proteins P16 and receptor for advanced glycation endproducts (RAGE), the expression of NADPH oxidase (NOX) 2 and 4, and ROS production. This treatment also shifted the morphology of astrocytes and microglia toward homeostasis. Furthermore, agomelatine decreased inositol-requiring enzyme 1 (pIRE1), protein kinase R-like endoplasmic reticulum kinase (pPERK), and chaperone binding immunoglobulin protein (BiP), leading to suppression of ER stress markers C/EBP homologous protein (CHOP) and caspase-12. Agomelatine reduced Ca2+ from the ER and stabilized the mitochondrial membrane stability, which was denoted by the BCL2 Associated X (Bax)/B-cell lymphoma 2 (Bcl2) balance. Agomelatine decreased cleaved caspase-3 production and the Terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL)-positive area, and glutamate excitotoxicity was prevented via suppression of N-methyl-d-aspartate (NMDA) receptor subunit expression. Agomelatine exhibited effects that were similar to melatonin. ConclusionAgomelatine improved neurodegeneration in a rat model of hippocampal aging by attenuating ROS production, ER stress, mitochondrial dysfunction, excitotoxicity, and apoptosis.

LncDACH1 promotes mitochondrial oxidative stress of cardiomyocytes by interacting with sirtuin3 and aggravates diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is a common complication in diabetic patients. The molecular mechanisms of DCM remain to be fully elucidated. The intronic long noncoding RNA of DACH1 (lncDACH1) has been demonstrated to be closely associated with heart failure and cardiac regeneration. In this study, we investigated the role of lncDACH1 in DCM and the underlying molecular mechanisms. The expression of lncDACH1 was increased in DCM hearts and in high glucose-treated cardiomyocytes. Knockout of lncDACH1 reduced mitochondrial oxidative stress, cell apoptosis, cardiac fibrosis and hypertrophy, and improved cardiac function in DCM mice. Overexpression of lncDACH1 exacerbated mitochondria-derived reactive oxygen species (ROS) level and apoptosis, decreased activity of manganese superoxide dismutase (Mn-SOD); while silencing of lncDACH1 attenuated ROS production, mitochondrial dysfunction, cell apoptosis, and increased the activity of Mn-SOD in cardiomyocytes treated with high glucose. LncDACH1 directly bound to sirtuin3 (SIRT3) and facilitated its degradation by ubiquitination, therefore promoting mitochondrial oxidative injury and cell apoptosis in mouse hearts. In addition, SIRT3 silencing abrogated the protective effects of lncDACH1 deficiency in cardiomyocytes. In summary, lncDACH1 aggravates DCM by promoting mitochondrial oxidative stress and cell apoptosis via increasing ubiquitination-mediated SIRT3 degradation in mouse hearts. Inhibition of lncDACH1 represents a novel therapeutic strategy for the intervention of diabetic cardiomyopathy.

Dual Regulation of Tank Binding Kinase 1 by BRG1 in Hepatocytes Contributes to Reactive Oxygen Species Production.

Excessive accumulation of reactive oxygen species (ROS) is considered a major culprit for the pathogenesis of non-alcoholic fatty liver disease (NAFLD). We have previously shown that deletion of Brahma related gene 1 (BRG1) mitigated NAFLD in mice in part by attenuating ROS production in hepatocyte. Here we report that BRG1 deletion led to simultaneous down-regulation in expression and phosphorylation of tank binding kinase 1 (TBK1) in vivo and in vitro. On the one hand, BRG1 interacted with AP-1 to bind to the TBK1 promoter and directly activated TBK1 transcription in hepatocytes. On the other hand, BRG1 interacted with Sp1 to activate the transcription of c-SRC, a tyrosine kinase essential for TBK1 phosphorylation. Over-expression of c-SRC and TBK1 corrected the deficiency in ROS production in BRG1-null hepatocytes whereas depletion of TBK1 or c-SRC attenuated ROS production. In conclusion, our data suggest that dual regulation of TBK1 activity, at the transcription level and the post-transcriptional level, by BRG1 may constitute an important mechanism underlying excessive ROS production in hepatocytes.

Prussian Blue Nanozymes Prevent Anthracycline-Induced Liver Injury by Attenuating Oxidative Stress and Regulating Inflammation.

Anthracycline-induced liver injury (AILI) is becoming an increasingly serious and potential clinical complication and is linked to reactive oxygen species (ROS) production and subsequent inflammatory response. Herein, we demonstrated that artificial Prussian blue nanozymes (PBZs) prevented daunorubicin-induced liver injury, a prototype of AILI, by attenuating ROS production and regulating inflammation. PBZs exhibited multienzyme activity and could scavenge ROS and free radicals. At the cellular level, PBZs could effectively eliminate ROS, suppress hepatocyte apoptosis, reduce deoxyribonucleic acid damage, and decrease the levels of inflammatory cytokines and chemokines. According to the results of the in vivo study, pretreatment with PBZs also resulted in a desirable protective effect against AILI, as indicated by both a decrease in biochemical indicator levels and hepatocyte necrosis. PBZs upregulated antioxidative genes by activating the Nrf2 pathway to reduce oxidative stress. Meanwhile, PBZs counteracted the inflammatory response based on the decreased expression levels of myeloperoxidase and F4/80 in the liver. Collectively, our findings indicate that PBZ-based nanotherapy is a novel strategy for protecting against AILI.