Reduced Reactive Oxygen Species Generation Research Articles

Protective effects of extracts from six local strains of <italic>Pyropia yezoensis</italic> against oxidative damage <italic>in vitro</italic> and in zebrafish model

Pyropia yezoensis has been used as functional food in East Asia, especially in Korea and Japan, for more than five hundred years. This study aims to evaluate the antioxidant effect of polyphenols and proteins-rich extracts from P. yezoensis (PPPs) against 2,2′-azobis (2-amidinopropane) dihydrochloride (AAPH)-induced oxidative cell damage. Among six Korean local strains obtained from Jinhae (JiH), Haenam (HN), Jangheung (JaH), Jindo (JD), Wando (WD), and Sinan (SA) areas, the extracts of P. yezoensis from SA and JD are relatively higher in polyphenols and proteins contents. SA showed the lowest IC50 scavenging activities against 1,1-diphenyl-2-picryl-hydrazyl and alkyl radicals and displayed protective effects against reactive oxygen species (ROS) in AAPH-induced Vero cells. Especially, the PPPs extracts from SA and JD showed protective activities against AAPH-induced apoptosis, as observed by nuclear staining with Hoechst 33342. Furthermore, in vivo studies of the SA extract in zebrafish showed significantly reduced ROS generation, lipid peroxidation, and cell damage. This is the first study, to our knowledge, to evaluate the antioxidant bioactivity of PPP in the Korean Peninsula using a zebrafish model. Due to SA and JD both located in the west coast of Korea, we deduced that the chemical content of the different PPP extracts was mildly influenced by their geographic location, and this alga has potential of protective activity against AAPH-induced ROS both in vitro and in vivo. Key words: antioxidant effect; Gim; polyphenols; proteins; Pyropia yezoensis; zebrafish

Adiponectin ameliorates lung injury induced by intermittent hypoxia through inhibition of ROS-associated pulmonary cell apoptosis.

Obstructive sleep apnea hypopnea syndrome has been reported to be associated with pulmonary hypertension (PH). Adiponectin (Ad) has many protective roles in the human body, including its function as an anti-inflammatory and an anti-oxidant, as well as its role in preventing insulin resistance and atherosclerosis. This study aimed to investigate the molecular mechanism of chronic intermittent hypoxia (CIH)-induced pulmonary injury and the protective role of Ad in experimental rats. Thirty male Sprague-Dawley rats were randomly divided into three groups with 10 rats in each group: normal control (NC) group, CIH group, and CIH + Ad group. Rats in the NC group were kept breathing room air for 12weeks. Rats in the CIH group were intermittently exposed to a hypoxic environment for 8h/day for 12weeks. Rats in the CIH + Ad group received 10μg Ad twice weekly via intravenous injection. After 12weeks of CIH exposure, we detected the pulmonary function, pulmonary artery pressure, lung histology, pulmonary cell apoptosis, pulmonary artery endothelial cell apoptosis, mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) level. We also analyzed expression proteins involved in the mitochondria-, endoplasmic reticulum (ER) stress-, and Fas receptor-associated pulmonary apoptosis pathways, as well as the SIRT3/SOD2 pathway. CIH exposure for 12weeks did not lead to abnormal pulmonary function, PH, or pulmonary artery endothelial cell apoptosis. However, we observed a significant increase in the rate of pulmonary cell apoptosis, the expression of proteins involved in mitochondria-, ER stress-, and Fas receptor-associated pulmonary apoptosis pathways, and the generation of ROS in the CIH group compared with the NC group. In contrast, the MMP and protein expressions of SIRT3/SOD2 pathway were significantly decreased in the CIH group compared with the NC group. Ad supplementation in the CIH + Ad group partially improved these changes induced by CIH. Even though CIH did not cause abnormal pulmonary function or PH, early lung injury was detected at the molecular level in rats exposed to CIH. Treatment with Ad ameliorated the pulmonary injury by activating the SIRT3/SOD2 pathway, reducing ROS generation, and inhibiting ROS-associated lung cell apoptosis.

P47phox deficiency impairs platelet function and protects mice against arterial and venous thrombosis

NADPH oxidase-derived reactive oxygen species (ROS) regulates platelet function and thrombosis. It remains controversial regarding NOX2’s role in platelet function. As a regulatory subunit for NOX2, whether p47phox regulates platelet function remains unclear. Our study intends to evaluate p47phox’s role in platelet function. Platelets were isolated from wild-type or p47phox-/- mice followed by analysis of platelet aggregation, granule secretion, surface receptors expression, spreading, clot retraction and ROS generation. Additionally, in vivo hemostasis, arterial and venous thrombosis was assessed. Moreover, human platelets were treated with PR-39 to inhibit p47phox activity followed by analysis of platelet function. p47phox deficiency significantly prolonged tail-bleeding time, delayed arterial and venous thrombus formation in vivo as well as reduced platelet aggregation, ATP release and αIIbβ3 activation. In addition, p47phox-/- platelets presented impaired spreading on fibrinogen or collagen and defective clot retraction concomitant with decreased phosphorylation of Syk and PLCγ2. Moreover, CRP or thrombin-stimulated p47phox-/- platelets displayed reduced intracellular ROS generation which was further decreased after inhibition of NOX1. Meanwhile, p47phox deficiency increased VASP phosphorylation and decreased phosphorylation of ERK1/2, p38, ERK5 and JNK without affecting AKT and c-PLA2 phosphorylation. Furthermore, p47phox translocates to membrane to interact with both NOX1 and NOX2 after stimulation with CRP or thrombin. Finally, inhibition of p47phox activity by PR-39 reduced ROS generation, platelet aggregation and clot retraction in human platelets. In conclusion, p47phox regulates platelet function, arterial and venous thrombus formation and ROS generation, indicating that p47phox might be a novel therapeutic target for treating thrombotic or cardiovascular diseases.

Calcitonin gene-related peptide inhibits angiotensin II-induced NADPH oxidase-dependent ROS via the Src/STAT3 signalling pathway.

We had previously demonstrated that the calcitonin gene‐related peptide (CGRP) suppresses the oxidative stress and vascular smooth muscle cell (VSMC) proliferation induced by vascular injury. A recent study also indicated that CGRP protects against the onset and development of angiotensin II (Ang II)‐induced hypertension, vascular hypertrophy and oxidative stress. However, the mechanism behind the effects of CGRP on Ang II‐induced oxidative stress is unclear. CGRP significantly suppressed the level of reactive oxygen species (ROS) generated by NADPH oxidase in Ang II‐induced VSMCs. The Ang II‐stimulated activation of both Src and the downstream transcription factor, STAT3, was abrogated by CGRP. However, the antioxidative effect of CGRP was lost following the expression of constitutively activated Src or STAT3. Pre‐treatment with H‐89 or CGRP8–37 also blocked the CGRP inhibitory effects against Ang II‐induced oxidative stress. Additionally, both in vitro and in vivo analyses show that CGRP treatment inhibited Ang II‐induced VSMC proliferation and hypertrophy, accompanied by a reduction in ROS generation. Collectively, these results demonstrate that CGRP exhibits its antioxidative effect by blocking the Src/STAT3 signalling pathway that is associated with Ang II‐induced VSMC hypertrophy and hyperplasia.

Inhibition of soluble epoxide hydrolase attenuates renal tubular mitochondrial dysfunction and ER stress by restoring autophagic flux in diabetic nephropathy

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease (ESRD), and renal tubular cell dysfunction contributes to the pathogenesis of DN. Soluble epoxide hydrolase (sEH) is an enzyme that can hydrolyze epoxyeicosatrienoic acids (EETs) and other epoxy fatty acids (EpFAs) into the less biologically active metabolites. Inhibition of sEH has multiple beneficial effects on renal function, however, the exact role of sEH in hyperglycemia-induced dysfunction of tubular cells is still not fully elucidated. In the present study, we showed that human proximal tubular epithelial (HK-2) cells revealed an upregulation of sEH expression accompanied by the impairment of autophagic flux, mitochondrial dysfunction, ubiquitinated protein accumulation and enhanced endoplasmic reticulum (ER) stress after high glucose (HG) treatment. Furthermore, dysfunctional mitochondria accumulated in the cytoplasm, which resulted in excessive reactive oxygen species (ROS) generation, Bax translocation, cytochrome c release, and apoptosis. However, t-AUCB, an inhibitor of sEH, partially reversed these negative outcomes. Moreover, we also observed increased sEH expression, impaired autophagy flux, mitochondrial dysfunction and enhanced ER stress in the renal proximal tubular cells of db/db diabetic mice. Notably, inhibition of sEH by treatment with t-AUCB attenuated renal injury and partially restored autophagic flux, improved mitochondrial function, and reduced ROS generation and ER stress in the kidneys of db/db mice. Taken together, these results suggest that inhibition of sEH by t-AUCB plays a protective role in hyperglycemia-induced proximal tubular injury and that the potential mechanism of t-AUCB-mediated protective autophagy is involved in modulating mitochondrial function and ER stress. Thus, we provide new evidence linking sEH to the autophagic response during proximal tubular injury in the pathogenesis of DN and suggest that inhibition of sEH can be considered a potential therapeutic strategy for the amelioration of DN.

Short Exposure to Ethanol Diminishes Caspase-1 and ASC Activation in Human HepG2 Cells In Vitro.

This paper discusses how the assembly of pro-caspase-1 and apoptosis-associated speck-like protein containing a caspase-recruitment domain (ASC) in macromolecular protein complexes, inflammasomes, activates caspase-1. The present study investigates the molecular mechanisms of inflammasome activation in HepG2 cells and examines how short exposures to ethanol (EtOH) affect inflammasome activation. HepG2 cells were treated with lipopolysaccharide (LPS), ATP or nigericin (NIG) in a two-step model. After LPS priming, ATP or NIG were added. As inhibitors, sodium orthovanadate (general inhibitor of tyrosine phosphatases), AC-YVAD-CMK (caspase-1 inhibitor) or AZ10606120 (purinergic receptor P2X7R inhibitor) were applied after LPS priming. To monitor the inflammasome activation, the caspase-1 activity, ASC speck formation, reactive oxygen species (ROS) production and cell death were analyzed. To elucidate the mechanistical approach of EtOH to the inflammasome assembly, the cells were treated with EtOH either under simultaneous LPS administration or concurrently with ATP or NIG application. The co-stimulation with LPS and ATP induced a significant ASC speck formation, caspase-1 activation, cell death and ROS generation. The inhibition of the ATP-dependent purinoreceptor P2X7 decreased the caspase-1 activation, whereas sodium orthovanadate significantly induced caspase-1. Additional treatment with EtOH reversed the LPS and ATP-induced caspase-1 activation, ASC speck formation and ROS production. The ASC speck formation and caspase-1 induction require a two-step signaling with LPS and ATP in HepG2 cells. Inflammasome activation may depend on P2X7. The molecular pathway of an acute effect of EtOH on inflammasomes may involve a reduction in ROS generation, which in turn may increase the activity of tyrosine phosphatases.

Neuroprotective Effects of Emodin against Ischemia/Reperfusion Injury through Activating ERK-1/2 Signaling Pathway.

Background: Stroke is one of the leading causes of death and disability worldwide and places a heavy burden on the economy in our society. Current treatments, such as the use of thrombolytic agents, are often limited by a narrow therapeutic time window. However, the regeneration of the brain after damage is still active days, even weeks, after stroke occurs, which might provide a second window for treatment. Emodin, a traditional Chinese medicinal herb widely used to treat acute hepatitis, has been reported to possess antioxidative capabilities and protective effects against myocardial ischemia/reperfusion injury. However, the underlying mechanisms and neuroprotective functions of Emodin in a rat middle cerebral artery occlusion (MCAO) model of ischemic stroke remain unknown. This study investigates neuroprotective effects of Emodin in ischemia both in vitro and in vivo. Methods: PC12 cells were exposed to oxygen-glucose deprivation to simulate hypoxic injury, and the involved signaling pathways and results of Emodin treatment were evaluated. The therapeutic effects of Emodin in ischemia animals were further investigated. Results: Emodin reduced infarct volume and cell death following focal cerebral ischemia injury. Emodin treatment restored PC12 cell viability and reduced reactive oxygen species (ROS) production and glutamate release under conditions of ischemia/hypoxia. Emodin increased Bcl-2 and glutamate transporter-1 (GLT-l) expression but suppressed activated-caspase 3 levels through activating the extracellular signal-regulated kinase (ERK)-1/2 signaling pathway. Conclusion: Emodin induced Bcl-2 and GLT-1 expression to inhibit neuronal apoptosis and ROS generation while reducing glutamate toxicity via the ERK-1/2 signaling pathway. Furthermore, Emodin alleviated nerve cell injury following ischemia/reperfusion in a rat MCAO model. Emodin has neuroprotective effects against ischemia/reperfusion injury both in vitro and in vivo, which may be through activating the ERK-1/2 signaling pathway.

Inhibition of LPS-induced Nox2 activation by VAS2870 protects alveolar epithelial cells through eliminating ROS and restoring tight junctions

Inhibiting the production of reactive oxygen species (ROS) in alveolar epithelial cells (AECs) under oxidative distress becomes a new therapeutic strategy for acute respiratory distress syndrome (ARDS). Herein in the present study, we investigated effects of Nox2, the catalytic subunit of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase type 2, on LPS-induced epithelium injury in ARDS mice and in human alveolar epithelial A549 cells. Severe lung injury, disruption of alveolar-capillary barrier with the loss of zonula occluden (ZO)-1 and up-regulated expression of Nox2 in AECs were exhibited in ARDS mice. In vitro, LPS induced decreased cell viability coupled with activated Nox2, high level of ROS, and destroyed ZO-1 distribution. Moreover, VAS2870 proved to inhibit Nox2 expression, reduce ROS generation, restore epithelium barrier integrity, and preserve cell viability in LPS-induced A549 cells. These data demonstrate that Nox2/ROS/ZO-1 axis is of great importance in AECs damage induced by LPS, and the utilization of VAS2870 to inhibit this pathway might lighten LPS-induced ARDS.

Nicotinamide N-methyltransferase expression in SH-SY5Y human neuroblastoma cells decreases oxidative stress.

Nicotinamide N-methyltransferase (NNMT) plays a central role in cellular metabolism, regulating pathways including epigenetic regulation, cell signalling, and energy production. Our previous studies have shown that the expression of NNMT in the human neuroblastoma cell line SH-SY5Y increased complex I activity and subsequent ATP synthesis. This increase in ATP synthesis was lower than the increase in complex I activity, suggesting uncoupling of the mitochondrial respiratory chain. We, therefore, hypothesised that pathways that reduce oxidative stress are also increased in NNMT-expressing SH-Y5Y cells. The expression of uncoupling protein-2 messenger RNA and protein were significantly increased in NNMT-expressing cells (57% ± 5.2% and 20.1% ± 1.5%, respectively; P = .001 for both). Total GSH (22 ± 0.3 vs 35.6 ± 1.1 nmol/mg protein), free GSH (21.9 ± 0.2 vs 33.5 ± 1 nmol/mg protein), and GSSG (0.6 ± 0.02 vs 1 ± 0.05 nmol/mg protein; P = .001 for all) concentrations were significantly increased in NNMT-expressing cells, whereas the GSH:GSSG ratio was decreased (39.4 ± 1.8 vs 32.3 ± 2.5; P = .02). Finally, reactive oxygen species (ROS) content was decreased in NNMT-expressing cells (0.3 ± 0.08 vs 0.12 ± 0.03; P = .039), as was the concentration of 8-isoprostane F2α (200 ± 11.5 vs 45 ± 2.6 pg/mg protein; P = .0012). Taken together, these results suggest that NNMT expression reduced ROS generation and subsequent lipid peroxidation by uncoupling the mitochondrial membrane potential and increasing GSH buffering capacity, most likely to compensate for increased complex I activity and ATP production.

Astragalus Polysaccharide Attenuates Cisplatin-Induced Acute Kidney Injury by Suppressing Oxidative Damage and Mitochondrial Dysfunction.

Cisplatin is a widely used chemotherapeutic drug in the treatment of various solid tumors. However, the cisplatin-induced acute kidney injury remains a disturbing complication, which still lacks effective prevention. Cisplatin-induced oxidative damage and mitochondrial dysfunction are anticipated to be crucial in the occurrence of kidney injury. Astragalus polysaccharide (APS) has been reported to possess multiple biological activities including anti-inflammatory, antioxidant, and mitochondria protection. In this study, we investigated the potentially protective effect of APS against cisplatin-induced kidney injury both in vivo and in vitro. We found that APS pretreatment attenuated the cisplatin-induced renal dysfunction and histopathological damage in mice; in addition, it also protected the viability of HK-2 cells upon cisplatin exposure. APS attenuated the cisplatin-induced oxidative damage by reducing reactive oxygen species (ROS) generation and recovering the activities of total superoxide dismutase and glutathione peroxidase in mice kidney. In addition, electron microscope analysis indicated that cisplatin induced extensive mitochondrial vacuolization in mice kidney. However, APS administration reversed these mitochondrial morphology changes. In HK-2 cells, APS reduced the cisplatin-induced mitochondrial and intracellular ROS generation. Furthermore, APS protected the normal morphology of mitochondria, blocked the cisplatin-induced mitochondrial permeability transition pore opening, and reduced the cytochrome c leakage. Subsequently, APS reduced the cisplatin-induced apoptosis in mice renal and HK-2 cells. In conclusion, our data suggested that APS pretreatment might prevent cisplatin-induced kidney injury through attenuating oxidative damage, protecting mitochondria, and ameliorating mitochondrial-mediated apoptosis.

Amifostine ameliorates cerebral ischaemia-reperfusion injury via p38-mediated oxidative stress and mitochondrial dysfunction.

Amifostine is acytoprotective compound that is beneficial in ischaemic stroke cases. However, the neuroprotective effect of amifostine on ischaemia/reperfusion (I/R)-induced brain injury and its underlying mechanism are still poorly understood. Herein, we constructed an animal model of middle cerebral artery occlusion and reperfusion (MCAO/R) injury and an in vitro model of oxygen and glucose deprivation and reperfusion (OGD/R) injury. After administration of amifostine, we found significant improvements in neurological deficits, infarct size, and cerebral oedema. Moreover, amifostine alleviated histopathological alteration and increased the number of surviving neurons. Biochemical analysis showed that treatment with amifostine obviously improved the brain damage of MCAO/R mice, as manifested by adecrease in reactive oxygen species (ROS) and malondialdehyde (MDA) generation, and an increase in superoxide dismutase (SOD) activity. Moreover, amifostine decreased the mitochondrial membrane potential (m) loss, and cytochrome c escaping to cytoplasm, but increased the ATP level. In vitro, amifostine also showed an antioxidant effect, which was reflected by the reduced ROS generation, decreased mitochondrial superoxide generation, increased total SOD, SOD1 (Cu/Zn SOD, cytoplasmic SOD), and SOD2 (mitochondrial SOD) activities, and decreased m loss. Furthermore, amifostine suppressed neuronal apoptosis, accompanied by the reduction of Bax, cleaved caspase-9, cleaved caspase-3, and Bcl-2 upregulation. Amifostine also reduced the expression of p-p38 (Thr 180/Tyr 182) in vivo and in vitro. In short, amifostine exhibits aprotective effect on cerebral I/R damage through modulating p38-related oxidative stress, mitochondrial dysfunction, and apoptosis.

TGF-β3 Induces Autophagic Activity by Increasing ROS Generation in a NOX4-Dependent Pathway.

Higher concentrations of reactive oxygen species (ROS) have been associated with epithelial cell damage, cell shedding, and airway hyperresponsiveness. Previous studies have indicated that transforming growth factor-beta (TGF-β) mediates ROS production and NADPH oxidase (NOX) activity. In our previous study, we also observed that TGF-β3 increases mucus secretion in airway epithelial cells in an autophagy-dependent fashion. Although it is well known that the relationship between ROS and autophagy is cell context-dependent, the exact mechanism of action remains unclear. The following study examined whether ROS act as upstream of autophagy activation in response to TGF-β3 induction. Using an allergic inflammation mouse model induced by house dust mite (HDM), we observed elevated lung amounts of TGF-β3 accompanied by increased ROS levels. And we found that ROS levels were elevated and NOX4 expression was increased in TGF-β3-induced epithelial cells, while the lack of NOX4 in the epithelial cells could reduce ROS generation and autophagy-dependent MUC5AC expression treated with TGF-β3. Furthermore, our studies demonstrated that the Smad2/3 pathway was involved in TGF-β3-induced ROS generation by promoting NOX4 expression. The inhibition of ROS generation by N-Acetyl-L-cysteine (NAC) resulted in a decrease in mucus expression and autophagy activity in vivo as well as in vitro. Finally, TGF-β3-neutralizing antibody significantly reduced the ROS generation, mucus expression, and autophagy activity and also decreased the phosphorylation of Smad2 and Smad3. Taken together, the obtained results revealed that persistent TGF-β3 activation increased ROS levels in a NOX4-dependent pathway and subsequently induced autophagy as well as MUC5AC expression in the epithelial cells.

Honokiol ameliorates oxidative stress-induced DNA damage and apoptosis of c2c12 myoblasts by ROS generation and mitochondrial pathway

ABSTRACT Honokiol is one of the main active components of Magnolia officinalis, and has been demonstrated to have multiple pharmacological activities against a variety of diseases. Recently, this phenolic compound is known to have antioxidant activity, but its mechanism of action remains unclear. The purpose of the current study was to evaluate the preventive effects of honokiol against oxidative stress-induced DNA damage and apoptosis in C2C12 myoblasts. The present study found that honokiol inhibited hydrogen peroxide (H2O2)-induced DNA damage and mitochondrial dysfunction, while reducing reactive oxygen species (ROS) formation. The inhibitory effect of honokiol on H2O2-induced apoptosis was associated with the up-regulation of Bcl-2 and down-regulation of Bax, thus reducing the Bax/Bcl-2 ratio that in turn protected the activation of caspase-9 and -3, and inhibition of poly (ADP-ribose) polymerase cleavage, which was associated with the blocking of cytochrome c release to the cytoplasm. Collectively, these results demonstrate that honokiol defends C2C12 myoblasts against H2O2-induced DNA damage and apoptosis, at least in part, by preventing mitochondrial-dependent pathway through scavenging excessive ROS.

Design and Biological Evaluation of Colchicine-CD44-Targeted Peptide Conjugate in an In Vitro Model of Crystal Induced Inflammation.

Gout is an inflammatory arthritis due to the joint deposition of monosodium urate (MSU) crystals. Phagocytosis of MSU crystals by tissue macrophages results in the generation of reactive oxygen species (ROS) and production of inflammatory cytokines and chemokines. Colchicine use in gout is limited by severe toxicity. CD44 is a transmembrane glycoprotein that is highly expressed in tissue macrophages and may be involved in gout pathogenesis. The P6 peptide is a 20-amino acid residue peptide that binds to CD44. We hypothesized that the conjugation of colchicine to the P6 peptide would reduce its off-target cytotoxicity while preserving its anti-inflammatory effect. A modified version of P6 peptide and colchicine-P6 peptide conjugate were synthesized using Fmoc/tBu solid-phase and solution-phase chemistry, respectively. A glutaryl amide was used as a linker. The P6 peptide was evaluated for its binding to CD44, association, and internalization by macrophages. Cytotoxic effects of P6 peptide, colchicine, and colchicine-P6 peptide on macrophages were compared and the inhibition of ROS generation and interleukin-8 (IL-8) secretion in MSU-stimulated macrophages treated with P6 peptide, colchicine, or colchicine-P6 peptide was studied. We confirmed that the P6 peptide binds to CD44 and its association and internalization by macrophages were CD44-dependent. Colchicine (1, 10, and 25 μM) demonstrated a significant cytotoxic effect on macrophages while the P6 peptide and colchicine-P6 peptide conjugate (1, 10 and 25 μM) did not alter the viability of the macrophages. The P6 peptide (10 and 25 μM) reduced ROS generation and IL-8 secretion mediated by a reduction in MSU phagocytosis by macrophages. The colchicine-P6 peptide significantly reduced ROS generation and IL-8 secretion compared to the P6 peptide alone at 1 and 10 μM concentrations. Conjugation of colchicine to the P6 peptide reduced the cytotoxic effect of colchicine while preserving its anti-inflammatory activity.

Celastrol Alleviates Aortic Valve Calcification Via Inhibition of NADPH Oxidase 2 in Valvular Interstitial Cells

This study sought to investigate whether reactive oxygen species (ROS)-generating reduced nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) contributes to calcific aortic valve disease (CAVD) or whether celastrol, a natural Nox2 inhibitor, may provide potential therapeutic target for CAVD. CAVD is an active and cellular-driven fibrocalcific process characterized by differentiation of aortic valvular interstitial cells (AVICs) toward an osteogenic-like phenotype. ROS levels increase in calcified aortic valves, while the sources of ROS and their roles in the pathogenesis of CAVD are elusive. The roles of Nox2 and the effects of celastrol were studied using cultured porcine AVICs invitro and a rabbit CAVD model invivo. Nox2 proteins were significantly upregulated in human aortic valves with CAVD. Invitro, Nox2 was markedly induced upon stimulation of AVICs with osteogenic medium, along with the increases in ROS production and calcium nodule formation. Celastrol significantly decreased calcium deposition of AVICs by 35%, with a reduction of ROS generation. Knockdown of endogenous Nox2 substantially suppressed AVIC calcification by 39%, the inhibitory effect being similar to celastrol treatment. Mechanistically, either celastrol treatment or knockdown of Nox2 significantly inhibited glycogen synthase kinase 3 beta/β-catenin signaling, leading to attenuation of fibrogenic and osteogenic responses of AVICs. In a rabbit CAVD model, administration of celastrol significantly reduced aortic valve ROS production, fibrosis, calcification, and severity of aortic stenosis, with less left ventricular dilatation and better preserved contractile function. Upregulation of Nox2 is critically involved in CAVD. Celastrol is effective to alleviate CAVD, likely through the inhibition of Nox2-mediated glycogen synthase kinase 3 beta/β-catenin pathway in AVICs.

Microglial Hv1 proton channels promote white matter injuries after chronic hypoperfusion in mice.

Microglia are critical in damage/repair processes during ischemic white matter injury (WMI). Voltage-gated proton channel (Hv1) is expressed in microglia and contributes to nicotinamide adenine dinucleotide phosphate oxidase complex-dependent production of reactive oxygen species (ROS). Recent findings have shown that Hv1 is involved in regulating luminal pH of M1-polarized microglial phagosomes and inhibits endocytosis in microglia. We previously reported that Hv1 facilitated production of ROS and pro-inflammatory cytokines in microglia and enhanced damage to oligodendrocyte progenitor cells from oxygen and glucose deprivation. To investigate the role of Hv1 in hypoperfusion-induced WMI, we employed mice that were genetically devoid of Hv1 (Hv1-/- ), as well as a model of subcortical vascular dementia via bilateral common carotid artery stenosis. Integrity of myelin was assessed using immunofluorescent staining and transmission electron microscopy, while cognitive impairment was assessed using an eight-arm radial maze test. Hv1 deficiency was found to attenuate bilateral common carotid artery stenosis-induced disruption of white matter integrity and impairment of working memory. Immunofluorescent staining and western blotting were used to assay changes in oligodendrocytes, OPCs, and microglial polarization. Compared with that in wild-type (WT) mice, Hv1-/- mice exhibited reduced ROS generation, decreased pro-inflammatory cytokines production, and an M2-dominant rather than M1-dominant microglial polarization. Furthermore, Hv1-/- mice exhibited enhanced OPC proliferation and differentiation into oligodendrocytes. Results of mouse-derived microglia-OPC co-cultures suggested that PI3K/Akt signaling was involved in Hv1-deficiency-induced M2-type microglial polarization and concomitant OPC differentiation. These results suggest that microglial Hv1 is a promising therapeutic target for reducing ischemic WMI and cognitive impairment.

Betulin Protects HT-22 Hippocampal Cells against ER Stress through Induction of Heme Oxygenase-1 and Inhibition of ROS Production

A key pathologic event in neurodegenerative diseases, such as Alzheimer’s disease and Parkinson’s disease, is endoplasmic reticulum (ER) stress-induced neuronal cell death. ER stress-induced generation of reactive oxygen species (ROS) has been implicated in neurological disease processes. Betulin is one of the major triterpenoids found in Betula platyphylla that possesses several biological properties, including cytoprotective and antioxidative effects. Therefore, we investigated whether betulin could prevent ER stress-induced neurotoxicity in HT-22 hippocampal neuronal cells. We observed that betulin reduced the thapsigargin (TG, an ER stress inducer)-induced apoptosis of HT-22 cells. Moreover, the cytoprotective effects of betulin were comparable to those of tauroursodeoxycholic acid, a potent ER stress-reducing agent. In our study, we confirmed that the ER stress-induced accumulation of ROS plays an important role in HT-22 cell death. Betulin also displayed cytoprotective effects in TG-injured HT-22 cells by reducing ROS generation; these results were comparable to those for N-acetyl-L-cysteine, a known ROS inhibitor. In addition, SnPP, a heme oxygenase-1 (HO-1) inhibitor significantly blocked the cytoprotective effects and ROS scavenging activity of betulin. Based on these results, we believe that betulin-mediated induction of HO-1 may contribute to the neuroprotective effects against ER stress in HT-22 hippocampal cells. We also found that betulin significantly inhibited the TG-induced expression of CHOP and caspase-12. These results demonstrated that betulin could serve as a potential therapeutic agent against ER stress-induced neurodegenerative diseases.

Cerium Oxide Nanoparticles Protect against Oxidant Injury and Interfere with Oxidative Mediated Kinase Signaling in Human-Derived Hepatocytes.

Cerium oxide nanoparticles (CeO2NPs) possess powerful antioxidant properties, thus emerging as a potential therapeutic tool in non-alcoholic fatty liver disease (NAFLD) progression, which is characterized by a high presence of reactive oxygen species (ROS). The aim of this study was to elucidate whether CeO2NPs can prevent or attenuate oxidant injury in the hepatic human cell line HepG2 and to investigate the mechanisms involved in this phenomenon. The effect of CeO2NPs on cell viability and ROS scavenging was determined, the differential expression of pro-inflammatory and oxidative stress-related genes was analyzed, and a proteomic analysis was performed to assess the impact of CeO2NPs on cell phosphorylation in human hepatic cells under oxidative stress conditions. CeO2NPs did not modify HepG2 cell viability in basal conditions but reduced H2O2- and lipopolysaccharide (LPS)-induced cell death and prevented H2O2-induced overexpression of MPO, PTGS1 and iNOS. Phosphoproteomic analysis showed that CeO2NPs reverted the H2O2-mediated increase in the phosphorylation of peptides related to cellular proliferation, stress response, and gene transcription regulation, and interfered with H2O2 effects on mTOR, MAPK/ERK, CK2A1 and PKACA signaling pathways. In conclusion, CeO2NPs protect HepG2 cells from cell-induced oxidative damage, reducing ROS generation and inflammatory gene expression as well as regulation of kinase-driven cell survival pathways.

BRD4 blockage alleviates pathological cardiac hypertrophy through the suppression of fibrosis and inflammation via reducing ROS generation

Hypertension is an essential regulator of cardiac injury and remodeling. However, the pathogenesis that contributes to cardiac hypertrophy remains to be fully explored. BRD4, as a bromodomain and extra-terminal (BET) family member, plays an important role in critical biological processes. In the study, our results showed that BRD4 expression was up-regulated in human and mouse hypertrophied hearts, and importantly these effects were modulated by reactive oxygen species (ROS) generation. In angiotensin II (Ang II)-treated cardiomyocytes, BRD4 decrease markedly blunted the prohypertrophic effect, which was further promoted by the combinational treatment of ROS scavenger (N-acetyl-cysteine, NAC). In addition, NAC pre-treatment markedly elevated the anti-fibrotic role of BRD4 suppression in Ang II-incubated cardiomyocytes by repressing transforming growth factor β1 (TGF-β1)/SMADs signaling pathway. NAC combined with BRD4 reduction further alleviated inflammation and oxidative stress in Ang II-exposed cardiomyocytes, which was partly through inhibiting nuclear factor-κB (NF-κB) signaling and improving nuclear erythroid factor 2-related factor 2 (Nrf-2)/heme oxygenase-1 (HO-1) pathway, respectively. Furthermore, the in vivo results confirmed the protective effects of BRD4 suppression on mice against aortic banding (AB)-induced cardiac hypertrophy, as evidenced by the reduced cross sectional area and fibrotic area using H&E and Masson trichrome staining. What’s more, the degree of cardiac hypertrophy (ANP and BNP), the expression of pro-fibrotic genes (TGF-β1, Collagen I, Collagen III and CTGF), the levels of inflammation and oxidative stress were all significantly attenuated by the blockage of BRD4 in AB-operated mice. Taken together, repressing BRD4 expression was found to confer a protective effect against experimental cardiac hypertrophy in mice, demonstrating its potential as an effective therapeutic target for pathological cardiac hypertrophy.

Oxidised low-density lipoprotein (OXLDL) causes calcium influx and activates mitochondrial permeability transition (MPT) in human macrophages

Oxidized low-density lipoprotein (oxLDL) plays an important role in atherosclerosis development. Studies have implicated the involvement of macrophages and the presence of oxLDL in atherosclerotic plaque. OxLDL is cytotoxic to human macrophages, causing a rapid and concentration and time-dependent cell death. This study aimed to investigate the mechanism of cell death induced by oxLDL in human macrophages. Human macrophages were treated with different concentrations of tetramethylrhodamine methyl ester (TMRM), ruthenium red (RuR) or cyclosporin A (CsA) for 30 minutes followed by addition of median lethal concentration (LC50) of oxLDL and were incubated at 37oC. Fluorescence levels were measured using flow cytometry for TMRM and dihydroethidium (DHE) after a designated incubation time. Cell viability was determined using propidium iodide (PI) via flow cytometry after 24 hours. TMRM results showed that oxLDL caused a time-dependent mitochondrial membrane potential (∆Ψm) loss. Cells treated with RuR and CsA were protected from oxLDL toxicity, possibly by reducing reactive oxygen species (ROS) production and preventing MPT pores opening, respectively. Our findings showed that blocking calcium uptake into the mitochondria may reduce ROS generation and prevents MPT activation. This indicates that oxLDL insult to the mitochondria might play a vital role in oxLDL-induced cell death.