H2O2-induced Stress Research Articles

Protection of Bovine Mammary Epithelial Cells from Hydrogen Peroxide-Induced Oxidative Cell Damage by Resveratrol.

The mammary epithelial cells (MECs) of high-producing dairy cows are likely to be subject to oxidative stress (OS) due to the intensive cell metabolism. The objectives of this study were to investigate the cytoprotective effects of resveratrol against hydrogen peroxide- (H2O2-) induced OS in cultured bovine MECs (MAC-T). Pretreatment of MAC-T cells with resveratrol could rescue the decrease in cell viability and resulted in lower intracellular reactive oxygen species (ROS) accumulation after H2O2 exposure. Resveratrol helped MAC-T cells to prevent H2O2-induced endoplasmic reticulum stress and mitochondria-related cell apoptosis. Moreover, resveratrol induced mRNA expression of multiple antioxidant defense genes in MAC-T cells under normal/oxidative conditions. Nuclear factor erythroid 2-related factor 2 (Nrf2) was required for the cytoprotective effects on MAC-T cells by resveratrol, as knockdown of Nrf2 significantly abolished resveratrol-induced cytoprotective effects against OS. In addition, by using selective inhibitors, we further confirmed that the induction of Nrf2 by resveratrol was mediated through the prolonged activation of PI3K/Akt and ERK/MAPK pathways but negatively regulated by p38/MAPK pathway. Overall, resveratrol has beneficial effects on bovine MECs redox balance and may be potentially used as a therapeutic medicine against oxidative insult in lactating animals.

Gensenoside Rb1 protects rat PC12 cells from oxidative stress-induced endoplasmic reticulum stress: the involvement of thioredoxin-1.

Oxidative stress plays an important role in many diseases and hydrogen peroxide (H2O2) plays a central role in the stress. Gensenoside Rb1 is the one of active ingredients in the traditional Chinese medicine Panax notoginseng. It has been reported that gensenoside Rb1 possesses various pharmacological activities. Here we report that gensenoside Rb1 exhibits potent protective effects against oxidative injury induced by H2O2 through inhibiting endoplasmic reticulum stress in PC12 cells. Cell viability assay demonstrated that incubation with H2O2 for 24 h led to a significant loss of cultured rat PC12 cells, and the cell viability was pronouncedly increased by pretreatment of gensenoside Rb1 for 24 h. H2O2-induced endoplasmic reticulum stress pathway was also suppressed after gensenoside Rb1 pretreatment, which was related with thioredoxin-1 (Trx-1) induction. Trx-1 siRNA abolished the protective effects of gensenoside Rb1. Our results of the present study demonstrate that gensenoside Rb1 shows a potent anti-oxidative effect on cultured PC12 cells by inducing Trx-1 expression.

Sulfated polysaccharides from Cyclocarya paliurus reduce H2O2-induced oxidative stress in RAW264.7 cells

In this study, two sulfated polysaccharides (S-CP1–4 and S-CP1–8) from Cyclocarya paliurus were produced by chlorosulfonic acid–pyridine method. Hydrogen peroxide (H2O2) was used to develop an oxidative stress model in the mouse macrophage cell line RAW264.7. Effects of the two sulfated polysaccharides on H2O2-induced oxidative stress were investigated. The results showed that S-CP1–8 improved the viability of the H2O2-induced stressed RAW264.7 cells, as well as inhibited the lipid oxidation as determined by the level of malondialdehyde (MDA). Meanwhile, treatment with S-CP1–4 increased superoxide dismutase (SOD) activity in these cells. The sulfated polysaccharides were found to have a better protective effect against H2O2-induced oxidative stress as compared to the native polysaccharide. Scanning electron microscopy also showed a significant change in the surface morphology of sulfated polysaccharides, but the degradation of main chain of polysaccharides was unconspicuous according to the results of monosaccharide composition. In addition, the sulfated polysaccharides had noticeable DPPH radical scavenging activity. In summary, our results demonstrated that H2O2 was able to induce oxidative stress in RAW264.7 cells, and sulfated group might play an important role in resistance to H2O2-induced oxidative damage.

CKA2 functions in H2O2-induced apoptosis and high-temperature stress tolerance by regulating NO accumulation in yeast.

Nitric oxide (NO) plays key roles in yeast responses to various environmental factors, such as H2O2 and high temperature. However, the gene encoding NO synthase (NOS) in yeast has not yet been identified, and the mechanism underlying the regulation of NOS-like activity is poorly understood. Here, we report on the involvement of CKA2 in H2O2-induced yeast apoptosis and yeast high-temperature stress tolerance. Our results showed that although Δcka2 mutant had reduced NO accumulation with decreased apoptosis after H2O2 exposure, treatment with a NO donor, sodium nitroprusside, resulted in similar survival rate of Δcka2 mutant compared to that of wild-type yeast when subjected to H2O2 stress. This finding occurred because H2O2-enhanced NOS-like activity in wild-type yeast was significantly repressed in Δcka2. Our additional experiments indicated that both high-temperature-enhanced NO accumulation and NOS-like activity were also suppressed in Δcka2, leading to the hypersensitivity of the mutant to high temperature in terms of changes in survival rate. Thus, our results showed that CKA2 functioned in H2O2-induced apoptosis and high-temperature stress tolerance by regulating NOS-like-dependent NO accumulation in yeast.

Involvement of Catalase in Saccharomyces Cerevisiae Hormetic Response to Hydrogen Peroxide

In this study, we investigated the relationship between catalase activity and H2O2-induced hormetic response in budding yeast S. cerevisiae. In general, our data suggest that: (i)hydrogen peroxide induces hormesis in a concentration- and time-dependent manner; and (ii) theeffect of hydrogen peroxide on yeast colony growth positively correlates with the activity ofcatalase that suggests the enzyme involvement in overall H2O2-induced stress response andhormetic response in yeast.

Aging-related changes in oxidative stress response of human endothelial cells.

Oxidative stress is strongly associated with aging and age-related diseases and plays a crucial role in endothelial dysfunction development. To better understand the molecular mechanisms of aging and stress response in humans, we examined changes to young and older human endothelial cells over time (72, 96 and 120h), before and after H2O2-induced stress. We measured the expression of the deacetylase Sirtuin 1 (Sirt1) and its transcriptional target Forkhead box O3a (Foxo3a); TBARS, a well-known marker of overall oxidative stress, and catalase activity as index of antioxidation. Moreover, we quantified levels of cellular senescence by senescence-associated β galactosidase (SA-βgal) assay. Under oxidative stress induction older cells showed a progressive decrease of Sirt1 and Foxo3a expression, persistently high TBARS levels with high, but ineffective Cat activity to counteract such levels. In addition cellular senescence drastically increased in older cells compared with Young cells both in presence and in the absence of oxidative stress. By following the cell behavior during the time course, we can hypothesize that while in young cells an oxidative stress induction stimulated an adequate response through activation of molecular factor crucial to counteract oxidative stress, the older cells are not able to adequately adapt themselves to external stress stimuli. During their life, endothelial cells impair the ability to defend themselves from oxidative stress stimuli. This dysfunction involves the pathway of Sirt1 a critical regulator of oxidative stress response and cellular lifespan, underlining its crucial role in endothelial homeostasis control during aging and age-associated diseases.

Structural characterization of an Indian squid antioxidant peptide and its protective effect against cellular reactive oxygen species

Antioxidant peptides were derived from the edible part of Indian squid, Loligo duvauceli, via the use of gastrointestinal enzymes for up to 12 hours. The active α-chymotrypsin hydrolysate exhibited the maximum activity compared to the trypsin and pepsin hydrolysates, as assessed by various radical scavenging and metal chelating assays. Moreover, essential and non-essential amino acids were present in the active hydrolysate. Furthermore, the hydrolysate was purified by ion exchange and gel filtration chromatography using fast protein liquid chromatography (FPLC). The purified peptide exhibited strong free radical scavenging, metal chelation and reducing power abilities that were confirmed with a UV-visible and electron spin resonance (ESR) spectrometer. The peptide also prevented DNA damage and inhibited lipid peroxidation due to its small size (682.5 Da) and content of the high redox potential amino acid sequence Trp-Cys-Thr-Ser-Val-Ser, which was confirmed by ESI-MS/MS. This peptide exhibited no cytotoxic effects on breast cancer cells (MCF7) and scavenged reactive oxygen species at the cellular level under H2O2-induced stress. These results suggest that the peptide derived from squid mantle protein acts as a potent antioxidant against oxidative stress and could be used as an efficient and safer adjunct in food processing.

Functional Significance of Point Mutations in Stress Chaperone Mortalin and Their Relevance to Parkinson Disease

Mortalin/mtHsp70/Grp75 (mot-2), a heat shock protein 70 family member, is an essential chaperone, enriched in cancers, and has been shown to possess pro-proliferative and anti-apoptosis functions. An allelic form of mouse mortalin (mot-1) that differs by two amino acids, M618V and G624R, in the C terminus substrate-binding domain has been reported. Furthermore, genome sequencing of mortalin from Parkinson disease patients identified two missense mutants, R126W and P509S. In the present study, we investigated the significance of these mutations in survival, proliferation, and oxidative stress tolerance in human cells. Using mot-1 and mot-2 recombinant proteins and specific antibodies, we performed screening to find their binding proteins and then identified ribosomal protein L-7 (RPL-7) and elongation factor-1 α (EF-1α), which differentially bind to mot-1 and mot-2, respectively. We demonstrate that mot-1, R126W, or P509S mutant (i) lacks mot-2 functions involved in carcinogenesis, such as p53 inactivation and hTERT/hnRNP-K (heterogeneous nuclear ribonucleoprotein K) activation; (ii) causes increased level of endogenous oxidative stress; (iii) results in decreased tolerance of cells to exogenous oxidative stress; and (iv) shows differential binding and impact on the RPL-7 and EF-1α proteins. These factors may mediate the transformation of longevity/pro-proliferative function of mot-2 to the premature aging/anti-proliferative effect of mutants, and hence may have significance in cellular aging, Parkinson disease pathology, and prognosis.

Antioxidant effect of a fermented powder of Lady Joy bean in primary rat hepatocytes.

The role and beneficial effects of plant and food extracts against various diseases induced by oxidative stress have received much attention in recent years. Legumes are rich in bioactive compounds, and some studies suggest a correlation between their consumption and a reduced incidence of diseases. Primary cultures of rat hepatocytes were used to investigate whether and how an extract obtained from a fermented powder of bean named Lady Joy (Phaseolus vulgaris L.) is able to regulate antioxidant and detoxifying enzymes through the NRF2 pathway, inhibit NF-kB activation, and reduce H2O2-induced endoplasmic reticulum (ER) stress. All of the antioxidant and detoxifying enzymes studied were significantly up-regulated by Lady Joy treatment. Western blot showed that Nrf2 was activated by Lady Joy treatment. Also, cells treated with this fermented bean were partially protected against NF-kB activation resulting from H2O2 stress. As a link between oxidative stress and ER dysfunction is hypothesized, we verified whether Lady Joy was able to protect cells from H2O2-induced ER stress, by studying the response of the proteins CHOP, BiP and caspase 12. The results of this study show that Lady Joy can induce the Nrf2 pathway, inhibit NF-kB, and protect ER from stress induced by H2O2.

Bacterial fucose-rich polysaccharide stabilizes MAPK-mediated Nrf2/Keap1 signaling by directly scavenging reactive oxygen species during hydrogen peroxide-induced apoptosis of human lung fibroblast cells.

Continuous free radical assault upsets cellular homeostasis and dysregulates associated signaling pathways to promote stress-induced cell death. In spite of the continuous development and implementation of effective therapeutic strategies, limitations in treatments for stress-induced toxicities remain. The purpose of the present study was to determine the potential therapeutic efficacy of bacterial fucose polysaccharides against hydrogen peroxide (H2O2)-induced stress in human lung fibroblast (WI38) cells and to understand the associated molecular mechanisms. In two different fermentation processes, Bacillus megaterium RB-05 biosynthesized two non-identical fucose polysaccharides; of these, the polysaccharide having a high-fucose content (∼42%) conferred the maximum free radical scavenging efficiency in vitro. Structural characterizations of the purified polysaccharides were performed using HPLC, GC-MS, and 1H/13C/2D-COSY NMR. H2O2 (300 µM) insult to WI38 cells showed anti-proliferative effects by inducing intracellular reactive oxygen species (ROS) and by disrupting mitochondrial membrane permeability, followed by apoptosis. The polysaccharide (250 µg/mL) attenuated the cell death process by directly scavenging intracellular ROS rather than activating endogenous antioxidant enzymes. This process encompasses inhibition of caspase-9/3/7, a decrease in the ratio of Bax/Bcl2, relocalization of translocated Bax and cytochrome c, upregulation of anti-apoptotic members of the Bcl2 family and a decrease in the phosphorylation of MAPKs (mitogen activated protein kinases). Furthermore, cellular homeostasis was re-established via stabilization of MAPK-mediated Nrf2/Keap1 signaling and transcription of downstream cytoprotective genes. This molecular study uniquely introduces a fucose-rich bacterial polysaccharide as a potential inhibitor of H2O2-induced stress and toxicities.

Involvement of PG2212 zinc finger protein in the regulation of oxidative stress resistance in Porphyromonas gingivalis W83.

The adaptation of Porphyromonas gingivalis to H2O2-induced stress while inducible is modulated by an unknown OxyR-independent mechanism. Previously, we reported that the PG_2212 gene was highly upregulated in P. gingivalis under conditions of prolonged oxidative stress. Because this gene may have regulatory properties, its function in response to H2O2 was further characterized. PG2212, annotated as a hypothetical protein of unknown function, is a 10.3-kDa protein with a cysteine 2-histidine 2 (Cys2His2) zinc finger domain. The isogenic mutant P. gingivalis FLL366 (ΔPG_2212) showed increased sensitivity to H2O2 and decreased gingipain activity compared to the parent strain. Transcriptome analysis of P. gingivalis FLL366 revealed that approximately 11% of the genome displayed altered expression (130 downregulated genes and 120 upregulated genes) in response to prolonged H2O2-induced stress. The majority of the modulated genes were hypothetical or of unknown function, although some are known to participate in oxidative stress resistance. The promoter region of several of the most highly modulated genes contained conserved motifs. In electrophoretic mobility shift assays, the purified rPG2212 protein did not bind its own promoter region but bound a similar region in several of the genes modulated in the PG_2212-deficient mutant. A metabolome analysis revealed that PG2212 can regulate a number of genes coding for proteins involved in metabolic pathways critical for its survival under the conditions of oxidative stress. Collectively, our data suggest that PG2212 is a transcriptional regulator that plays an important role in oxidative stress resistance and virulence regulation in P. gingivalis.

Cardiovascular effects of resveratrol and atorvastatin treatments in an H2O2-induced stress model.

Oxidative stress has been implicated in the pathophysiology of several types of cardiovascular disease (CVD). Statins are widely used to inhibit the progression of atherosclerosis and reduce the incidence of CVD. Certain over-the-counter products, including resveratrol, show similar effects to statins and may thus be used in conjunction with statins for the treatment of the majority of patients with CVD. The aim of the present study was to evaluate the effects of atorvastatin, resveratrol and resveratrol + atorvastatin (R+A) pretreatment on myocardial contractions and vascular endothelial functions in the presence of H2O2 as an experimental model of oxidative stress in rats. Four groups were established and referred to as the control, atorvastatin, resveratrol and R+A groups. Atorvastatin (40 mg/kg, per oral) and/or resveratrol (30 mg/kg, intraperitoneal) treatments were administered for 14 days. On the 15th day, the thoracic aortas and hearts of the rats were dissected and placed into isolated organ baths. Vascular responses to cumulative doses of H2O2 (1×10−8–1×10−4 M H2O2) with and without N (G)-nitro-L-arginine methyl ester (L-NAME) incubation were measured. In addition, myocardial electrical stimulation (ES) responses to various H2O2 concentrations (1×10−7–1×10−5 M H2O2) were evaluated. In the control and atorvastatin groups, H2O2 application caused a significant dose-dependent decrease in the ES-induced contractions in the myocardial tissue of rats. In the resveratrol and R+A groups, H2O2 application did not significantly affect myocardial contraction at any dose. In all groups, incubation with L-NAME caused a significant augmentation in the H2O2 response, revealing that this effect was mediated via the vascular endothelium. In conclusion, pretreatment with R+A for CVD appears to be superior to pretreatment with either agent alone.

Protein polymer nanoparticles engineered as chaperones protect against apoptosis in human retinal pigment epithelial cells

αB-Crystallin is a protein chaperone with anti-apoptotic and anti-inflammatory activity that is apically secreted in exosomes by polarized human retinal pigment epithelium. A 20 amino acid mini-peptide derived from residues 73–92 of αB-crystallin protects human retinal pigment epithelial (RPE) cells from oxidative stress, a process involved in the progression of age-related macular degeneration (AMD). Unfortunately, due to its small size, its development as a therapeutic requires a robust controlled release system. To achieve this goal, the αB-crystallin peptide was re-engineered into a protein polymer nanoparticle/macromolecule with the purpose of increasing the hydrodynamic radius/molecular weight and enhancing potency via multivalency or an extended retention time. The peptide was recombinantly fused with two high molecular weight (~40kDa) protein polymers inspired by human tropoelastin. These elastin-like polypeptides (ELPs) include the following: (i) a soluble peptide called S96 and (ii) a diblock copolymer called SI that assembles multivalent nanoparticles at physiological temperature. Fusion proteins, cryS96 and crySI, were found to reduce aggregation of alcohol dehydrogenase and insulin, which demonstrates that ELP fusion did not diminish chaperone activity. Next their interaction with RPE cells was evaluated under oxidative stress. Unexpectedly, H2O2-induced stress dramatically enhanced cellular uptake and nuclear localization of both cryS96 and crySI ELPs. Accompanying uptake, both fusion proteins protected RPE cells from apoptosis, as indicated by reduced caspase 3 activation and TUNEL staining. This study demonstrates the in vitro feasibility of modulating the hydrodynamic radius for small peptide chaperones by seamless fusion with protein polymers; furthermore, they may have therapeutic applications in diseases associated with oxidative stress, such as AMD.

Protective effect of polysaccharides from Sargassum horneri against oxidative stress in RAW264.7 cells

This study was designed to investigate chemical composition and the protective effects of polysaccharides isolated from Sargassum horneri against hydrogen peroxide (H2O2)-induced oxidative injury in RAW264.7 cells. Results showed that isolated polysaccharides (SHSc) and the major fractions (SHS1, SHS0.5) contained sulfate ester, and SHS1 was high fucose-containing sulfated polysaccharide. After preincubation with three isolated polysaccharides, RAW264.7 cells viability were significantly restored and decreased in cellular LDH release (P<0.05). SHS1 and SHS0.5 decreased intracellular ROS level, intracellular NO and malonic dialdehyde (MDA) level (P<0.05), restoring activities of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) (P<0.05), decreasing inducible nitric oxide synthase (iNOS) (P<0.05). Moreover, preincubation of SHS1 with RAW264.7 cells resulted in the increase of the gene expression level of endogenous antioxidant enzymes such as MnSOD and GSH-Px (P<0.05). These results clearly showed that SHSc and its fractions could attenuate H2O2-induced stress injury in RAW264.7 cells, and a similar efficiency in protecting RAW264.7 cells against H2O2-induced oxidative injury between SHS1 and Vitamin C. Taken together, our findings suggested that SHS1 can effectively protect RAW264.7 cells against oxidative stress by H2O2, which might be used as a potential natural antioxidant in the functional food and pharmaceutical industries.

DPP4 Deficiency Preserved Cardiac Function in Abdominal Aortic Banding Rats

Dipeptidyl peptidase-4 (DPP4) enzyme inhibition has been reported to increase plasma glucagon-like peptide-1 (GLP-1) level for controlling postprandial glucose concentration. A prominent GLP-1 level in DPP4-deficient rats contributed to the resistance of endotoxemia and myocardial infarction. DPP4 deficiency also increased the capability against H2O2-induced stress in cardiomyocyte. However, long term effect of loss DPP4 activity on cardiac performance remained unclear. We used abdominal aortic banding (AAB) to induce pressure overload in wild-type and DPP4-deficient rats, and investigated the progression of heart failure. Cardiac histology and function were determined. Blood sample was collected for the plasma biochemical marker measurement. Heart weight to body weight ratio increased 1.2-fold after 6 weeks of AAB surgery. Cardiac function was compensated against pressure overload after 6 weeks of AAB surgery, but progressed to deterioration after 10 weeks of AAB surgery. AAB induced cardiac dysfunction was alleviated in DPP4-deficient rats. DPP4 activity increased significantly in wild-type rats after 10 weeks of AAB surgery, but remained unchanged in DPP4-deficient rats. In contrast, GLP-1 concentration was elevated by AAB after 6 weeks of surgery in DPP4-deficient rats, and remained high after 10 weeks of surgery. Ang II level markedly increased after 6 weeks of AAB surgery, but were less in DPP4-deficient rats. Massive collagen deposits in wild-type rat hearts appeared after 10 weeks of AAB surgery, which were alleviated in DPP4-deficient rats. Long term deficiency of DPP4 activity improved cardiac performance against pressure overload in rat, which may be attributed to a great quantity of GLP-1 accumulation during AAB.

Antiapoptotic Actions of Methyl Gallate on Neonatal Rat Cardiac Myocytes Exposed to H2O2

Reactive oxygen species trigger cardiomyocyte cell death via increased oxidative stress and have been implicated in the pathogenesis of cardiovascular diseases. The prevention of cardiomyocyte apoptosis is a putative therapeutic target in cardioprotection. Polyphenol intake has been associated with reduced incidences of cardiovascular disease and better overall health. Polyphenols like epigallocatechin gallate (EGCG) can reduce apoptosis of cardiomyocytes, resulting in better health outcomes in animal models of cardiac disorders. Here, we analyzed whether the antioxidant N-acetyl cysteine (NAC) or polyphenols EGCG, gallic acid (GA) or methyl gallate (MG) can protect cardiomyocytes from cobalt or H2O2-induced stress. We demonstrate that MG can uphold viability of neonatal rat cardiomyocytes exposed to H2O2 by diminishing intracellular ROS, maintaining mitochondrial membrane potential, augmenting endogenous glutathione, and reducing apoptosis as evidenced by impaired Annexin V/PI staining, prevention of DNA fragmentation, and cleaved caspase-9 accumulation. These findings suggest a therapeutic value for MG in cardioprotection.

Hydrogen peroxide leads to cell damage and apoptosis in the gill of the freshwater crab Sinopotamon henanense (Crustacea, Decapoda)

Hydrogen peroxide (H2O2), a major reactive oxygen species, has been shown to be a critical mediator of apoptosis induced by several toxic metals such as cadmium. In this study, we used the freshwater crab Sinopotamon henanense to study whether H2O2 can cause apoptosis in gill cells. The crabs were incubated in H2O2 and the DNA fragmentation, ultrastructural changes and caspase-3/8/9 activities were measured. The results showed that in freshwater crab, H2O2 was found to induce apoptosis, as confirmed by DNA fragmentation analysis and morphological observation of transmission electron microscopy. This apoptosis occurs in a concentration-dependent pattern. During the apoptotic process, caspase-3, caspase-8 and caspase-9 were activated by H2O2. In addition, multiple physiological and pathological changes of gill cells were discovered after 24 h exposure to 5 mM H2O2, including aggregation and condensation of nuclear chromatin, appearance of extremely irregular nuclei with finger-like buds, disappearance of the organelles around the nuclei, swollen and dissolved cristae of mitochondria. We propose that H2O2-induced stress leads to mitochondria lesions oxidative injury and triggers apoptotic response through the caspase pathway in freshwater crab.

Abstract 19186: Role of Exosomes and Their microRNA Constituents in Mediating the Therapeutic Benefits of Human Cardiosphere-Derived Cells in vitro and in Mice With Myocardial Infarction

Background: Exosomes are nano-sized bilayer vesicles that are secreted by most cell types. Exosomes are rich in microRNAs (mirs) which may function in a paracrine fashion. Cardiosphere-derived cells (CDCs) have been shown to regenerate heart after myocardial infarction (MI) in animal models and in the CADUCEUS clinical trial. However, most of the regenerated muscle is of innate origin, which suggests that CDCs function mainly through indirect pathways. Methods and Results: In vitro and in vivo, we compared three treatment groups: vehicle only (control), CDC-derived exosomes and normal human dermal fibroblast (NHDF)-derived exosomes. Neonatal rat cardiomyocytes (NRCMs) incubated with CDC exosomes were more frequently positive for Ki67 (n=4, p&lt;0.001), and less frequently tunel-positive, compared to NHDF exosomes or control (n=4, p&lt;0.01). CDC exosomes also stimulated tube formation in HUVEC cells compared to NHDF exosomes or control (p&lt;0.05). SCID mice injected with exosomes from CDCs during acute MI showed higher LVEF at two weeks (n=6, p&lt;0.05) and four weeks (n=6, p&lt;0.05) post MI, as well as increased viable mass. The therapeutic benefits of exosomes were equivalent to those of 105 injected human CDCs (p=ns). Inhibition of exosome secretion by ceramide synthesis inhibition abrogated the beneficial effects by CDCs in vivo and in vitro. Mir microarray analysis identified mirs146a as the most highly-up regulated mirs in CDC-exosomes compared to NHDF (262-fold, n=4). Mir-146a-treated NRCMs were more resistant to H2O2-induced stress compared to a mimic control. Array analysis of these NRCMs treated also showed suppression of IRAK1 and TRAF6 transcripts. SCID mice injected with 80 ng of mir-146a during acute MI showed higher LVEF at two weeks (n=6, p&lt;0.05) and four weeks (n=6, p&lt;0.05) post MI, as well as increased viable mass compared to mimic control. Conclusions: Mir-containing exosomes secreted by CDCs exhibit multiple beneficial effects on injured myocardium, suggesting that exosomes may mediate some of the therapeutic effects of CDCs. Most notably mir-146a provides cardioprotection in an acute model of MI.

MiR-146a regulates SOD2 expression in H2O2 stimulated PC12 cells.

SOD2 (superoxide dismutase 2) is one of the endogenous antioxidant enzymes that protect against reactive oxygen species. While explorations of SOD2 expression regulation are mainly focused on transcriptional and post-translational activation, there are few reports about the post-transcriptional regulation of SOD2. MicroRNAs (miRNAs) are 21nt-25nt (nucleotide) small noncoding RNAs that have emerged as indispensable regulators of gene expression. Here we show that miR-146a, a widely expressed miRNA, is up-regulated by H2O2-induced stress. By sequence analysis we found a binding site for miR-146a in the sod2 mRNA 3′UTR, and a luciferase reporter assay confirmed that miR-146a can interact with this sod2 regulatory region. Our results further show that miR-146a could down-regulate the SOD2 protein expression, and antisense-miR-146a could reverse the decrease of both the SOD2 level and cell viability in H2O2 treated PC12 cells. In conclusion, here we have identified a novel function of miR-146a in the post-transcriptional regulation of SOD2 expression.

Superoxide radicals have a protective role during H2O2stress

Reactive oxygen species (ROS) consist of potentially toxic, partly reduced oxygen species and free radicals. After H(2)O(2) treatment, yeast cells significantly increase superoxide radical production. Respiratory chain complex III and possibly cytochrome b function are essential for this increase. Disruption of complex III renders cells sensitive to H(2)O(2) but not to the superoxide radical generator menadione. Of interest, the same H(2)O(2)-sensitive mutant strains have the lowest superoxide radical levels, and strains with the highest resistance to H(2)O(2) have the highest levels of superoxide radicals. Consistent with this correlation, overexpression of superoxide dismutase increases sensitivity to H(2)O(2), and this phenotype is partially rescued by addition of small concentrations of menadione. Small increases in levels of mitochondrially produced superoxide radicals have a protective effect during H(2)O(2)-induced stress, and in response to H(2)O(2), the wild-type strain increases superoxide radical production to activate this defense mechanism. This provides a direct link between complex III as the main source of ROS and its role in defense against ROS. High levels of the superoxide radical are still toxic. These opposing, concentration-dependent roles of the superoxide radical comprise a form of hormesis and show one ROS having a hormetic effect on the toxicity of another.