H2O2-induced Oxidative Injury Research Articles

Hydroxysafflor Yellow A Attenuates Hydrogen Peroxide-Induced Oxidative Damage on Human Umbilical Vein Endothelial Cells.

Oxidative stress of endothelial cells is thought to be a principal cause that induces many cardiovascular diseases. Hydroxysafflor yellow A (HSYA) is a major active component in traditional Chinese medicine safflower and has been used to cure ischemic cardiovascular diseases in China for many years. This study aims to investigate whether HSYA has a repairing effect on oxidative damage of human umbilical vein endothelial cells (HUVECs) induced by H2O2 and to provide a theoretical basis for the clinical treatment of cardiovascular diseases related to traditional Chinese medicine. Based on the establishment of an H2O2-induced HUVEC oxidative injury model, the cell viability and proliferation rate were measured by the MTT assay and EdU staining. The intracellular GSH/GSSG ratio and SOD activity were determined by kits. The ROS level was detected by flow cytometry. And the BAX, Bcl-2, PTEN, and AKT expressions were evaluated with western blotting methods. The results showed that HSYA treatment significantly attenuated the H2O2-induced HUVEC cell damage, increased the intracellular GSH/GSSG ratio and unit SOD activity also, and decreased the intracellular ROS levels. Furthermore, HSYA increased the expressions of AKT and Bcl-2 proteins and inhibited the expressions of BAX and PTEN proteins. These suggest that HSYA exerts repair effects on H2O2-induced oxidative damage in HUVECs, and the mechanisms may be related to the influence of BAX/Bcl-2 expression and AKT/PTEN signal pathway expression.

Rhizoma Paridis total saponins alleviate H2O2‑induced oxidative stress injury by upregulating the Nrf2 pathway.

Rhizoma Paridis total saponins (RPTS) is an active substance isolated from the traditional Chinese medicine Rhizoma Paridis, which possesses multiple biological activities. The aim of the present study was to explore the roles and mechanisms of RPTS in oxidative stress injury of ARPE-19 human retinal pigment epithelial cells. Cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP) and apoptosis were determined by Cell Counting kit-8 assay and flow cytometry, respectively. Enzyme-linked immunosorbent assay was performed to detect the expression of oxidative stress markers. Western blotting and reverse transcription-quantitative polymerase chain reaction were used to determine the expression levels of related genes and proteins. The results revealed that RPTS enhanced cell viability and reduced H2O2-induced oxidative stress of ARPE-19 human retinal pigment epithelial cells. RPTS increased the MMP of ARPE-19 cells compared with in H2O2-treated ARPE-19 cells. In addition, RPTS suppressed ROS production and apoptosis of H2O2-treated ARPE-19 cells. Additionally, RPTS modulated the expression levels of apoptosis-associated proteins and the nuclear factor 2-related factor 2 (Nrf2) pathway. In conclusion, RPTS alleviated H2O2-induced oxidative stress injury by upregulating the Nrf2 pathway. The potential effects of RPTS on protection against H2O2-induced apoptosis of ARPE-19 cells suggested that RPTS may be a potential therapeutic target for preventing age-related macular degeneration.

Paeoniflorin Alleviates H2O2-Induced Oxidative Injury Through Down-Regulation of MicroRNA-135a in HT-22 Cells.

Paeoniflorin (PF) has been reported to possess neuroprotective influences on cognitive dysfunction illness. In current research, we attempted to probe into the protective influences of PF against H2O2-induced damage and the underlying regulating mechanismson hippocampal HT-22 cells. HT-22 cells were pretreated with PF, and then induced by H2O2. Afterwards, the influences of PF pretreatment were examined using CCK-8 assay, apoptosis assay, western blot andROS assay, respectively. In addition, the expression of microRNA-135a (miR-135a) was analyzedand altered by qRT-PCRandcell transfection, respectively. After overexpression of miR-135a, the effects of miR-135a mimic on cell functions were detected again. Moreover, influences of H2O2, PFand miR-135a overexpression on JAK2/STAT3 and ERK1/2 signal pathways were further investigated. Further experiments verified that PF pretreatment alleviated H2O2-induced oxidative stress through increasing cell viability, inhibiting cell apoptosis, reducing ROS generation and activating JAK2/STAT3 and ERK1/2 pathways. Besides, expression of miR-135a was declined by PF pretreatment. Whereas, miR-135a mimic abrogated the protective effects triggered by PF pretreatment. These results indicated that PF can alleviate H2O2-induced oxidative stress by down-regulation of miR-135a via activation of JAK2/STAT3 and ERK1/2 pathways.

Discovery of coumarin-derived imino sulfonates as a novel class of potential cardioprotective agents

The burst of reactive oxygen species (ROS) contributes to and exacerbates cardiac injury. Exogenous supplementation of antioxidants or upregulation of endogenous antioxidant defense genes should be the potential therapies for cardiovascular disease. Sixteen coumarin-derived imino sulfonates compounds were synthesized with the ability of attenuating oxidative stress directly by reducing intracellular ROS level via promoting Nrf2 pathway. The cell-based assays showed that most of the compounds had significant protective activity against H2O2-induced oxidative injury in H9c2 cells. Compound 5h with the highest activity and low cytotoxicity was demonstrated to remarkably remove the intracellular ROS accumulation by activating expressions of Nrf2 and its downstream antioxidant proteins (ie. HO-1 and NQO1), indicating a novel promising antioxidant and Nrf2 activator. Overall, these findings demonstrated that compound 5h could serve as a potential cardioprotective agent. Moreover, our study features developing new antioxidants and Nrf2 activators by introducing a sulfonyl group and nitrogen atom to the α,β-unsaturated carbonyl entity in coumarin, rather than adding new functional groups or active fragments to coumarin itself.

CH(II), a cerebroprotein hydrolysate, exhibits potential neuro-protective effect on Alzheimer's disease.

Alzheimer’s disease (AD) is a progressive neurodegenerative disorder, and is the most common type of cognitive impairment and dementia. There is a pressing need to improve the clinical efficacy and quality of life for AD patients, as limited treatments options for AD patients have been developed until now. In this study, we aim to investigate the protective effect of CH(II), a cerebroprotein hydrolysate consisted of abundant biological peptides, on preclinical model of AD. We found that CH(II) treatment effectively protects oxygen glucose deprivation (OGD)-induced N2A cell viability impairment and cell apoptosis. In addition, CH(II) significantly reduces H2O2-induced ROS accumulation and exhibits the protective activities against H2O2-induced oxidative injury. Intriguingly, we found that CH(II) treatment can effectively promote neurite outgrowth of N2A cells. Moreover, CH(II) obviously improve the cognitive and memorial function in scopolamine-induced amnesia mice model. Taken together, this study provides evidences of the neuroprotective activities of CH(II) and offers a potential therapeutic strategy for AD patients.

MiR-208a aggravates H2O2-induced cardiomyocyte injury by targeting APC

Oxidative stress injury, inducing cardiomyocyte injury, is the major denominator of many cardiovascular diseases. In present study, we aimed to explore the molecular mechanism of microRNA-208a (miR-208a) in oxidative stress-induced cardiomyocyte injury. In this study, hydrogen peroxide (H2O2)-induced injury in H9c2 and AC16 cardiomyocytes was used as a model of myocardial injury. The pro-apoptosis potential and mechanism of miR-208a for oxidative injury were evaluated by MTT, flow cytometry, qRT-PCR and Western blot assays. Intracellular reactive oxygen species and detection of lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD) were performed to analyze the effect of miR-208a on H2O2-induced injury in H9c2 cardiomyocytes. The association between miR-208a and activated protein C (APC) was confirmed by luciferase reporter and RIP assays. We foundthatmiR-208a mimic aggravated H2O2-induced apoptosis and oxidative injury in cardiomyocytes, while miR-208a inhibitor hadan inverse effect. APC was a target gene of miR-208a and miR-208a negatively regulated the expression of APC. APC reduced H2O2-induced injury in H9c2 cardiomyocytes. Knockdown of APC attenuated the inhibitiveeffect of miR-208a inhibitor on H2O2-induced injuryin H9c2 cardiomyocytes. We concluded thatmiR-208a could aggravate H2O2-induced injury in H9c2 cardiomyocytes by targeting APC. A new signaling pathway miR-208a/APC was first observed in myocardial injury.

SGK1 Attenuates Oxidative Stress-Induced Renal Tubular Epithelial Cell Injury by Regulating Mitochondrial Function.

Mitochondrial dysfunction has been implicated in the early stages or progression of many renal diseases. Improving mitochondrial function and homeostasis has the potential to protect renal function. Serum- and glucocorticoid-induced kinase 1 (SGK1) is known to regulate various cellular processes, including cell survival. In this study, we intend to demonstrate the effect and molecular mechanisms of SGK1 in renal tubular cells upon oxidative stress injury and to determine whether regulation of mitochondrial function is implicated in this process. HK-2 cells were exposed to H2O2, and cell viability and apoptosis were dynamically detected by the CCK-8 assay and annexin-V/PI staining. The concentrations of cellular reactive oxygen species (ROS) and adenosine triphosphate (ATP) and the expression of the SGK1/GSK3β/PGC-1α signaling pathway were analyzed by flow cytometry or western blot. In addition, shRNA targeting SGK1 and SB216763 were added into the culture medium before H2O2 exposure to downregulate SGK1 and GSK3β, respectively. Cell viability and mitochondrial functions, including mitochondrial membrane potential (Δψm), Cytochrome C release, mtDNA copy number, and mitochondrial biogenesis, were examined. Protein levels and SGK1 activation were significantly stimulated by H2O2 exposure. HK-2 cells with SGK1 inhibition were much more sensitive to H2O2-induced oxidative stress injury than control group cells, as they exhibited increased apoptotic cell death and mitochondrial dysfunction involving the deterioration of cellular ATP production, ROS accumulation, mitochondrial membrane potential reduction, and release of Cytochrome C into the cytoplasm. Studies on SGK1 knockdown also indicated that SGK1 is required for the induction of proteins associated with mitochondrial biogenesis, including PGC-1α, NRF-1, and TFAM. Moreover, the deleterious effects of SGK1 suppression on cell apoptosis and mitochondrial function, including mitochondrial biogenesis, were related to the phosphorylation of GSK3β and partially reversed by SB216763 treatment. H2O2 leads to SGK1 overexpression in HK-2 cells, which protects human renal tubule cells from oxidative stress injury by improving mitochondrial function and inactivating GSK3β.

364 Adenosine triphosphate-P2X7 axis mediates melanocyte death and skin trafficking of CD8+ T cells by CXCL9 through inflammasome activation in vitiligo

Emerging evidence demonstrates a critical role for inflammasome activation in vitiligo pathogenesis. However, the specific molecular mechanism of inflammasome-dependent melanocyte degeneration in vitiligo is still not clear. Extracellular adenosine triphosphate (ATP) is a versatile mediator of inflammatory processes, which may be involved in vitiligo pathogenesis. This study presents how extracellular ATP, released from keratinocytes by oxidative stress, affects melanocyte survival in vitiligo skins. H2O2-induced oxidative injury increased ATP release from keratinocytes and skin tissues. High concentration of extracellular ATP induced both ROS production and cell death in melanocytes. Treatment with ATP in melanocytes led to caspase-1 activation as well as production of active forms of IL-1β and IL-18 via P2X7 receptor (P2X7R). Lesional and perilesional skins of vitiligo showed higher levels of ATP as well as upregulation of active caspase-1 compared to non-lesional skins, suggesting its possible role in inflammasome activation in vitiligo. Moreover, the expression of CXCL9 in keratinocytes, mediated through ATP-P2X7R-dependent inflammasome activation, was responsible for CLA+ CD8+ T-cell chemotaxis into the skin. In conclusion, extracellular ATP as a danger signal not only activates inflammasome pathway as well as increases cutaneous chemotaxis of CD8+ T cells via CXCL9 in vitiligo.

Sesamin Enhances Nrf2-Mediated Protective Defense against Oxidative Stress and Inflammation in Colitis via AKT and ERK Activation.

Ulcerative colitis (UC) is a major form of inflammatory bowel disease (IBD) with high incidence and prevalence in many countries. Patients with UC usually suffer from a lifetime of debilitating physical symptoms. Therefore, developing effective therapeutic strategy that can manage this disease better and improve patients' life quality is in urgent need. Sesamin (SSM) is a lignan derived from sesame seeds. In this study, the protective effect of SSM against UC and the underlying mechanism were investigated in vitro and in vivo. Our data showed that SSM protected Caco-2 cells from H2O2-induced oxidative stress injury via GSH-mediated scavenging of reactive oxygen species (ROS). Dual luciferase reporter assay showed that the transcriptional activity of nuclear factor erythroid-related factor 2 (Nrf2) was significantly increased by SSM, and the ability of SSM to activate Nrf2-targeted genes was further confirmed in Caco-2 cells using western blot and quantitative real-time PCR (qRT-PCR). In contrast, Nrf2 knockdown abolished the protective effect of SSM. Additionally, we found that SSM also activated advanced protein kinase B (AKT) and extracellular signal-regulated kinase (ERK) in Caco-2 cells, while either AKT or ERK inhibition can prevent SSM-mediated nuclear translocation of Nrf2. Furthermore, SSM displayed a better protective effect against dextran sulfate sodium- (DSS-) induced UC compared with 5-aminosalicylic acid (5-ASA) in C57BL/6 mice. The enhanced Nrf2 signaling and activated AKT/ERK were also observed in the colon of mice after SSM administration. These results first demonstrate the protective effect of SSM against UC and indicate that the effect is associated with AKT/ERK activation and subsequent Nrf2 signaling enhancement. This study provides a new insight into the medicinal value of SSM and proposes it as a new natural nutrition for better managing the symptoms of UC.

Cyclosporin A protects trophoblasts from H2O2-induced oxidative injury via FAK-Src pathway

Oxidative stress is associated with functional disorder of trophoblast cells. Our previous studies have demonstrated that cyclosporin A (CsA) promotes the activity of normal human trophoblast cells. We further investigated the role and mechanism of CsA on oxidative stress in trophoblast cells. JEG-3 cells were co-cultured with H2O2 and CsA. Cell viability and morphology were measured by MTT assay and inverted microscope. Reactive oxygen species (ROS) was analyzed by fluorescence microscopy. Cell mitochondrial membrane potential (MMP) was determined by flow cytometric analysis. Malondialdehyde (MDA) production, superoxide dismutase (SOD) and catalase (CAT) activities were examined using colorimetric assays. The expression and phosphorylation of FAK and Src kinase proteins were examined by western blotting. CsA increased JEG-3 cell viability and reduced the morphologic injury induced by H2O2 treatment. CsA decreased ROS and MDA production, increased SOD and CAT activities, and restored the MMP of H2O2 treated JEG-3 cells. CsA administration suppressed H2O2-induced reduction of FAK and Src phosphorylation. Blocking the activation of FAK or Src attenuated the protective effect of CsA on JEG-3 cells in H2O2-induced oxidative injury. CsA protects JEG-3 cells from H2O2-induced oxidative injury, and the FAK/Src signaling pathway plays an important role in this process.

Protective effect of polysaccharides of sea cucumber Acaudina leucoprocta on hydrogen peroxide-induced oxidative injury in RAW264.7 cells

The aim of this experiment was to investigate the protective effects of polysaccharides of sea cucumber Acaudina leucoprocta (ALP) against hydrogen peroxide (H2O2) induced oxidative injury in RAW264.7 cells. Analysis of monosaccharide composition and structure of one fraction from ALP (ALPN) were analyzed by High Performance Liquid Chromatography (HPLC) and Fourier Transform Infrared Spectoscopy (FT-IR). The results showed that ALPN contain sulfate groups, which is sulfated polysaccharides. The results from MTT assay indicated that ALPN could markedly increase viability of cells compared with RAW264.7 cells exposed to H2O2. Moreover, ALPN significantly increased the levels of catalase (CAT), glutathione peroxidase (GSH-PX) and superoxide dismutase (SOD), decreased the production of malondialdehyde (MDA) and lactate dehydrogenase (LDH) in RAW264.7 cells. The data from RT-PCR showed that ALPN (300 μg/mL) could increase the gene expression levels of SOD1 and GPX1. ALPN could also observably increase the protein expression level of Nrf2 and decrease the protein expression level of Keap1 with western blot. Collectively, this study suggested that polysaccharides of sea cucumber Acaudina leucoprocta (ALP) could effectively protect RAW264.7 cells against H2O2-induced oxidative injury. This protection mechanism may be related to activation of the Nrf2/Keap1 signaling pathway.

Brazilin Treatment Produces Antidepressant- and Anxiolytic-Like Effects in Mice.

Increasing evidence shows depression relevant to oxidative stress and inflammation. Anti-inflammatory strategies or antioxidants have led to the development of new antidepressants. Brazilin is a natural product from the Chinese traditional medicine Caesalpinia sappan L., exerting anti-inflammatory, antioxidant, anti-platelet concentration, and anti-cancer effects. While the antidepressant effect of brazilin is largely unknown. In present study, we investigated the effects of brazilin on H2O2-induced oxidative injury in PC12 cells and on depression- and anxiety-like behaviors of chronically mild stressed (CMS)-induced depression mice. It was found that brazilin pre-treatment (both 10 and 20 µM) significantly increased cell viability and decreased cell apoptosis in H2O2-treated PC12 cells. Furthermore, repetitive administration of brazilin to CMS-induced depression mice by intraperitoneal injection (10 mg/kg) made the mice significantly lose their latency of feeding in novelty-suppressed feeding test (NSF), have more the sucrose preference in sucrose preference test (SPT), and more time spent in the central zone without affecting their crossing activity in open field test (OFT). These results suggested that brazilin can play a role in antidepressant and anxiolytic-like behaviors for CMS-induced depression mice probably through inhibiting the oxidative stress. Therefore, brazilin is worth to be further explored for treating depressive and anxiety disorders.

Targeting cullin 3 by miR-601 activates Nrf2 signaling to protect retinal pigment epithelium cells from hydrogen peroxide

Activation of Nrf2 cascade can protect retinal pigment epithelium (RPE) cells from hydrogen peroxide (H2O2) and other oxidative injury. The current study identified microRNA-601 (miR-601) as a novel cullin 3 (Cul3)-targeting miRNA that activates Nrf2 cascade. In ARPE-19 cells and primary human RPE cells, forced overexpression of miR-601 significantly inhibited Cul3 3′-UTR activity and downregulated Cul3 mRNA/protein expression, leading to Nrf2 protein stabilization and its nuclear translocation as well as expression of anti-oxidant response elements (ARE)-dependent genes (HO1, NQO1 and GCLC). H2O2 treatment increased miR-601 levels in RPE cells. Significantly, ectopic miR-601 overexpression attenuated H2O2-induced oxidative injury and apoptosis in RPE cells. In contrast, miR-601 inhibition promoted Cul3 expression, lowered basal Nrf2 activation, and enhanced H2O2-induced oxidative stress and apoptosis in RPE cells. In ARPE-19 cells, CRISPC/Cas9-mediated knockout (KO) of Cul3 or Keap1 not only mimicked, but also nullified, miR-601-inudced anti-H2O2 actions. Furthermore, Nrf2 silencing by targeted shRNAs abolished miR-601-inudced cytoprotection in H2O2-treated ARPE-19 cells. Taken together, we show that miR-601 activates Nrf2 signaling to protect RPE cells from H2O2 by targeting Cul3.

Nrf2/Keap1/ARE Signaling Mediated an Antioxidative Protection of Human Placental Mesenchymal Stem Cells of Fetal Origin in Alveolar Epithelial Cells.

The oxidative stresses are a major insult in pulmonary injury such as acute lung injury (ALI) and acute respiratory distress syndrome (ARDS), two clinical manifestations of acute respiratory failure with substantially high morbidity and mortality. Mesenchymal stem cells (MSCs) hold a promise in treatments of many human diseases, mainly owing to their capacities of immunoregulation and antioxidative activity. The strong immunoregulatory role of human placental MSCs of fetal origin (hfPMSCs) has been previously demonstrated; their antioxidant activity, however, has yet been interrogated. In this report, we examined the antioxidative activity of hfPMSCs by accessing the ability to scavenge oxidants and radicals and to protect alveolar epithelial cells from antioxidative injury using both a cell coculture model and a conditioned culture medium (CM) of hfPMSCs. Results showed a comparable antioxidative capacity of the CM with 100 μM of vitamin C (VC) in terms of the total antioxidant capacity (T-AOC), scavenging abilities of free radicals DPPH, hydroxyl radical (·OH), and superoxide anion radical (O2 −), as well as activities of antioxidant enzymes of SOD and GSH-PX. Importantly, both of the CM alone and cocultures of hfPMSCs displayed a protection of A549 alveolar epithelial cells from oxidative injury of 600 μM hydrogen peroxide (H2O2) exposure, as determined in monolayer and transwell coculture models, respectively. Mechanistically, hfPMSCs and their CM could significantly reduce the apoptotic cell fraction of alveolar epithelial A549 cells exposed to H2O2, accompanied with an increased expression of antiapoptotic proteins Bcl-2, Mcl-1, Nrf-2, and HO-1 and decreased proapoptotic proteins Bax, caspase 3, and Keap1, in comparison with naïve controls. Furthermore, hfPMSCs-CM (passage 3) collected from cultures exposed an inhibition of the Nrf2/Keap1/ARE signaling pathway which led to a significant reduction in caspase 3 expression in A549 cells, although the addition of Nrf2 inhibitor ML385 had no effect on the antioxidative activity of hfPMSCs-CM. These data clearly suggested that hfPMSCs protected the H2O2-induced cell oxidative injury at least in part by regulating the Nrf2-Keap1-ARE signaling-mediated cell apoptosis. Our study thus provided a new insight into the antioxidative mechanism and novel functions of hfPMSCs as antioxidants in disease treatments, which is warranted for further investigations.

Thymosin beta 4 attenuates oxidative stress-induced injury of spinal cord-derived neural stem/progenitor cells through the TLR4/MyD88 pathway

Neural stem/progenitor cells (NSPCs) can enhance regeneration after spinal cord injury (SCI), but survival of transplanted cells remains poor. Understanding how NSPCs respond to the chemical mediators of secondary injury thus is essential for treating SCI. Thymosin β4 (Tβ4) has physiological functions that are highly relevant to SCI. We exposed NSPCs to oxidative stress and found reduced expression of Tβ4 in H2O2-injured NSPCs. Using an MTT assay, we found that Tβ4 dose dependently increased viability of the injured NSPCs. Tβ4 also reversed the decreases of intracellular Ca2+ concentration and increases of lactate dehydrogenase in NSPCs induced by H2O2 treatment. H2O2 exposure increased NSPC apoptosis, which Tβ4 decreased. In H2O2-induced NSPCs, ROS production and pro-inflammatory cytokines increased, and again, Tβ4 reversed these effects. We investigated the toll-like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) signaling pathway as an underlying mechanism in Tβ4's protective effect on H2O2-exposed NSPCs. Our results showed that Tβ4 reduced expression of TLR4 and MyD88. Moreover, H2O2-exposed NSPCs that were treated with the TLR4/MyD88 pathway inhibitor showed a reversal of all the effects caused by H2O2, similar to Tβ4's effects. In conclusion, our study determined that Tβ4 attenuated H2O2-induced oxidative stress injury in NSPCs via the TLR4/MyD88 pathway.

Luteolin protects PC-12 cells from H2O2-induced injury by up-regulation of microRNA-21

BackgroundIschemic cerebrovascular disease (ICVD) is the third leading cause of death worldwide. Luteolin is a naturally flavonoid widely distributed in many plant leaves. This study aimed to explore the effects of luteolin on H2O2-induced ICVD cell oxidative injury model, as well as underlying molecular mechanisms. MethodsViability and apoptosis of PC-12 cells and rat brain microvascular endothelial cells (rBMECs) were detected using CCK-8 assay and FITC-Annexin V/PI staining, respectively. The levels of ROS and MDA were measured using DCFH-DA staining and MDA assay kit, respectively. Cell transfection was conducted to change the expression level of miR-21. Expression levels of key factors involved in cell proliferation, oxidative stress, apoptosis, PI3K/AKT and PDCD4/p21 pathways were evaluated using western blotting. ResultsLow concentration of luteolin had no significant effect on PC-12 cell viability and presented protective effects on H2O2-induced PC-12 cell viability loss, proliferation inhibition, ROS generation, oxidative stress increase and apoptosis. Moreover, luteolin up-regulated the expression level of miR-21 in H2O2-treated PC-12 cells. Overexpression of miR-21 strengthened the protective effects of luteolin on H2O2-induced PC-12 cell oxidative injury. Suppression of miR-21 had opposite effects. Furthermore, luteolin alleviated H2O2-induced inactivation of PI3K/AKT pathway and activation of PDCD4/p21 pathway in PC-12 cells by up-regulating miR-21. Besides, luteolin also protected rBMECs from H2O2-induced oxidative injury. ConclusionOur research revealed the protective effects of luteolin on H2O2-induced ICVD cell oxidative injury. Luteolin protected PC-12 cells from H2O2-induced oxidative injury by up-regulating miR-21, activating PI3K/AKT pathway and inactivating PDCD4/p21 pathway.

Resveratrol alleviates chemotherapy-induced oogonial stem cell apoptosis and ovarian aging in mice.

Chemotherapy-induced ovarian aging not only increases the risk for early menopause-related complications but also results in infertility in young female cancer survivors. Oogonial stem cells have the ability to generate new oocytes and thus provide new opportunities for treating ovarian aging and female infertility. Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a natural phenol derived from plants, that has been shown to have positive effects on longevity and redox flow in lipid metabolism and a preventive function against certain tumors. To evaluate whether resveratrol could promote the repair of oogonial stem cells damage in a busulfan/cyclophosphamide (Bu/Cy)-induced accelerated ovarian aging model, female mice were administered 30 and 100 mg/kg/d resveratrol through a gavage for 2 weeks. We demonstrated that resveratrol (30 mg/kg/d) relieved oogonial stem cells loss and showed an attenuating effect on Bu/Cy-induced oxidative apoptosis in mouse ovaries, which may be attributed to the attenuation of oxidative levels in ovaries. Additionally, we also showed that Res exerted a dose-dependent effect on oogonial stem cells and attenuated H2O2-induced cytotoxicity and oxidative stress injury by activating Nrf2 in vitro. Therefore, resveratrol could be of a potential therapeutic drug used to prevent chemotherapy-induced ovarian aging.

Protective effects of ginsenoside Rb1 on H2O2-induced oxidative injury in human endothelial cell line (EA.hy926) via miR-210.

Ginsenoside Rb1 (Rb1) possesses a cardioprotective effect via mediating microRNAs (miRs), while it is unexplored whether miR-210 is regulated by Rb1 in response to oxidative stress. Human endothelial EA.hy926 cells were stimulated with H2O2 before Rb1 treatment. After transfection, cell viability, apoptosis, migration, and invasion assays were conducted. Western blot was applied to quantify protein. BCL2/adenovirus E1B 19-kDa interacting protein 3 (BNIP3) and miR-210 were analyzed with quantitative reverse transcription polymerase chain reaction. Dual luciferase activity assay was performed. Rb1 elevated viability, migration, and invasion of H2O2-treated cells. H2O2-induced apoptosis was moderated by Rb1. miR-210 was augmented in H2O2-treated cells after Rb1 stimulation. miR-210 inhibitor abolished the positive effects of Rb1. BNIP3 was negatively modulated by miR-210 and implicated in modulating viability, apoptosis, and migration and invasion. In addition, BNIP3 modulated phosphorylation of regulators. Rb1 repressed oxidative injury via elevating miR-210. miR-210 negatively mediated BNIP3, which participated in oxidative damage via regulating mammalian targets of rapamycin (mTOR) and nuclear factor-κB (NF-κB).

Protective effect of WSY6, a caffeic acid derivative, on oxidative stress injury in melanocytes and its mechanism

Objective To evaluate the protective effect of WSY6 (a caffeic acid derivative) on hydrogen peroxide (H2O2) -induced oxidative stress injury in melanocytes, and to explore its potential molecular mechanism. Methods In vitro cultured human primary melanocytes were divided into 5 groups: control group receiving no treatment, H2O2 group treated with 1 mmol/L H2O2, 6.25, 12.5, 25 μmol/L WSY6 groups pretreated with 6.25, 12.5, 25 μmol/L WSY6 respectively followed by 1-hour treatment with 1 mmol/L H2O2. After 24-hour treatment, MTS assay was performed to determine the survival rate of melanocytes, and the lactate dehydrogenase (LDH) kit was used to detect the LDH leakage level. Some melanocytes were divided into 2 groups: inhibitor group pretreated with the p38 inhibitor for 1 hour followed by 1-hour treatment with 1 mmol/L H2O2, and H2O2 group treated with 1 mmol/L H2O2 for 1 hour. After 24-hour treatment, the LDH kit was used to detect the LDH leakage level. Some other melanocytes were pretreated with 25 μmol/L WSY6 for 1, 2, 4 hours separately, followed by 1-hour treatment with H2O2. Then, flow cytometry was conducted to detect the level of intracellular reactive oxygen species (ROS) . Some melanocytes were treated with 6.25, 12.5, 25 μmol/L WSY6 separately for 1 hour, followed by 1-hour treatment with H2O2. Then, Western bolt analysis was performed to determine the protein expression of cytochrome c (cyto-c) , caspase-3, caspase-9, phosphorylated (p) -p38 MAPK, p-ERK and p-JNK. Results Compared with the control group, the H2O2 group showed significantly decreased survival rate of melanocytes (29.22% ± 1.31%, P < 0.05) , but significantly increased intracellular LDH leakage level (47.19% ± 4.85%, P < 0.05) , elevated intracellular ROS level (18.37 ± 1.59, P < 0.05) , and increased expression of caspase-3 and caspase-9. Compared with the H2O2 group, the 6.25, 12.5, 25 μmol/L WSY6 groups showed significantly increased cell survival rate (52.48% ± 1.17%, 60.21% ± 0.25%, 78.32% ± 1.73%, P < 0.05) , but significantly decreased LDH leakage level (21.99% ± 0.22%, 15.38% ± 0.45%, 13.18% ± 0.38%, P < 0.05) , and the intracellular ROS level was significantly decreased in the 25 μmol/L WSY6 group after 1, 2, 4 hours of treatment (7.59 ± 1.00, 6.22 ± 0.52, 5.1 ± 0.48, P < 0.05) . The LDH leakage level in melanocytes was significantly lower in the p38 inhibitor group than in the H2O2 group (P < 0.05) . Western blot analysis revealed that after the pretreatment with 6.25, 12.5, 25 μmol/L WSY6 separately, the WSY6 groups all showed obviously decreased expression of caspase-3, caspase-9 and p-p38 compared with the H2O2 group. However, there was no obvious difference in the expression of p-ERK and p-JNK between the WSY6 groups and the H2O2 group. Besides, the WSY6 groups showed decreased expression of p-p53 (a downstream product of p38 MAPK) , which decreased along with the increase in the concentration of WSY6. Conclusion WSY6 shows a markedly protective effect on H2O2-induced oxidative stress injury in melanocytes, likely through the p38 MAPK pathway. Key words: Vitiligo; Melanocytes; Oxidative stress; Caffeic acid; Lactate dehydrogenases; Extracellular signal-regulated MAP kinases

A novel mechanism of Gamma-aminobutyric acid (GABA) protecting human umbilical vein endothelial cells (HUVECs) against H2O2-induced oxidative injury

Vascular endothelial cell damage is related to many vascular diseases, including cardiovascular disease (CVD). Reactive oxygen species (ROS) play a vital role in the pathogenesis of many cardiovascular diseases. Herein, H2O2-induced human umbilical vein endothelial cell (HUVEC) injury model was used to explore the mechanisms involved in the pathogenesis of ROS-induced oxidative stress and cell dysfunction. Gamma-aminobutyric acid (GABA), a naturally occurring four-carbon non-protein amino acid, has antioxidant activity and anti-inflammatory action. In the present study, we demonstrated that GABA could scavenge free radicals including DPPH and ABTS, reverse H2O2-induced suppression on HUVEC proliferation, HUVEC apoptosis and ROS formation via p65 signaling. Interestingly, GABA treatment alone did not cause significant changes in p65 phosphorylation, suggesting that GABA will not cause imbalance in NF-κB signaling and ROS formation without oxidative stress. Moreover, GABA also modulated Keap1-Nrf2 and Notch signaling pathways upon H2O2 stimulation, suggesting that GABA may exert its effect via multi mechanisms. In conclusion, the present study demonstrated that GABA inhibits H2O2-induced oxidative stress in HUVECs via inhibiting ROS-induced NF-κB and Caspase 3 pathway activation. GABA may, therefore, have potential as a pharmacological agent in the prevention or treatment of oxidative injury-related cardiovascular disease.