Cytoprotective Capacity Research Articles

Neuroprotection of Kolaviron by Regulation of Nuclear Factor Erythroid 2-related Factor 2 in 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine Mice Model of Parkinson Disease

Objectives: Parkinson’s disease (PD) is the most prevalent movement disorder. Available therapies are palliative with no effect on disease progression. We have previously demonstrated that kolaviron (KV), a natural anti-inflammatory and antioxidant agent, suppressed behavioral defect, redo-inflammation, and nigrostriatal pathology in rotenone PD model. The present study investigates the neuroprotective effect of KV focusing on DJ-1/nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. Material and Methods: All-trans retinoic acid (ATRA, 10 mg/kg/day) was used to inhibit Nrf2. PD was established with four doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (20 mg/kg) at 2 h interval. MPTP mice were pre-treated with either KV (200 mg/kg/day), ATRA or both for 7 days before MPTP. Mice were evaluated for locomotor defects and indices of oxidative stress, neuroinflammation and neurotransmission as well as pathological tyrosine hydroxylase expression PD were evaluated in the striatum. Results: ATRA alone in mice did not exhibit neurobehavioral defect but caused striatal toxicity, mild nigrostriatal pathology, significant nitrosative stress, and Nrf2 cascade inhibition. KV+ATRA mice were slow in movement with frequent short-lived interruptions and oxidative striatal pathology. ATRA aggravated MPTP-associated locomotor incompetence and could not prevent nigrostriatal toxicity with evident vacuolated striosome and pyknotic/degenerating dopaminergic neurons. MPTP induced acute locomotor, exploratory, and motor incompetence, which was prevented by KV treatment. In addition, KV treatment restored MPTP-mediated depletion of endogenous antioxidant, striatal nitrosative stress, and oxidative damage with elevated DJ-1 level, potentiated Nrf2/NAD(P)H; quinone oxidoreductase-1 cytoprotective capacity, reduced Kelch-like ECH-associated protein 1 expression, and limited striatal pathology. However, ATRA treatment attenuated all the protective effects of KV on MPTP-challenged mice. Meanwhile, other ATRA-combinations elicited significant DJ-1 and Nrf2 induction but are associated striatal toxicity/pathology. Conclusion: This suggests that KV may be conferring protection through a yet-undetermined DJ-1 downstream cytoprotective effect dependent on the KV-mediated attenuation of oxidative environment.

Effects of Deoxynivalenol and Fumonisins on Broiler Gut Cytoprotective Capacity.

Mycotoxins are a crucial problem for poultry production worldwide. Two of the most frequently found mycotoxins in feedstuffs are deoxynivalenol (DON) and fumonisins (FUM) which adversely affect gut health and poultry performance. The current knowledge on DON and FUM effects on broiler responses relevant for gut detoxification, antioxidant capacity, and health is still unclear. The aim of this study was to assess a range of selected molecular intestinal biomarkers for their responsiveness to the maximum allowable European Union dietary levels for DON (5 mg/kg) and FUM (20 mg/kg) in broilers. For the experimental purpose, a challenge diet was formulated, and biomarkers relevant for detoxification, antioxidant response, stress, inflammation, and integrity were profiled across the broiler intestine. The results reveal that DON significantly (p < 0.05) induced aryl hydrocarbon receptor (AhR) and cytochrome P450 enzyme (CYP) expression mainly at the duodenum. Moreover, DON and FUM had specific significant (p < 0.05) effects on the antioxidant response, stress, inflammation, and integrity depending on the intestinal segment. Consequently, broiler molecular responses to DON and FUM assessed via a powerful palette of biomarkers were shown to be mycotoxin and intestinal site specific. The study findings could be highly relevant for assessing various dietary bioactive components for protection against mycotoxins.

Efficient production of lycopene from CO2 via microbial electrosynthesis

Valorization of CO2 is key to establishing a carbon–neutral society. However, CO2 possesses a strong C = O with bond energy of 750 kJ/mol, which makes it difficult for electrochemical conversion. As a result, the conversion of CO2 to complex molecules remains a grand challenge and product profiles based on inorganic catalysis are only limited to a few simple chemicals, such as formate, acetate, and ethanol. Here, we employed microbial electrosynthesis to diversify the product spectrum. First, an engineered Cupriavidus necator strain was constructed to produce lycopene from H2 and CO2. Then, by coupling de novo microbial catalyzed lycopene synthesis with inorganic catalyzed water electrolysis, a microbial electrosynthesis (MES) system was developed. Lycopene, representing the most complex nonnative molecules in the MES system, was produced with CO2 as the sole carbon source and H2 derived from electrochemical water splitting as the reducing power. Moreover, the produced lycopene provided long-term cytoprotective capacity against reactive oxygen species, thereby addressing the incompatibility between microbial and inorganic catalysis. Finally, 1.73 mg/L lycopene was produced from real exhaust gas from a coal-fired power plant in the MES system. The present study opens a possible route to turn trash to cash for industrial exhaust gas.

Dry-Cured Ham-Derived Peptide (Asp-Leu-Glu-Glu) Exerts Cytoprotective Capacity in Human Intestinal Epithelial Caco-2 Cells.

Dry-cured hams are well-known and highly appreciated products in the Mediterranean and China. The long-term fermentation endows dry-cured hams with a unique flavor and quality. Our previous study has identified Asp–Leu–Glu–Glu (DLEE) from dry-cured Xuanwei ham with remarkable antioxidant capacity. In the current study, the Caco-2 cells were cultured in vitro and treated with different doses of DLEE. The cellular reactive oxygen species (ROS) level and antioxidant enzyme activities were then determined to investigate the intracellular protection effect of DLEE. According to the results, the cellular ROS level was reduced, whereas the antioxidant enzyme activities of glutathione reductase, catalase, and glutathione peroxidase were improved following DLEE treatment. The DLEE treatment also increased the Nrf2 expression, along with downregulating the Keap1 expression. Thus, the dry-cured ham-derived peptide DLEE exhibited excellent bioactive capacity by reducing the ROS level and regulating the antioxidant enzyme activities. In addition, Nrf2/Keap1 was shown to be the main signaling pathway underlying DLEE-induced antioxidant activities in Caco-2 cells.

Antioxidant and Cytoprotective Effects of New Diarylheptanoids from Rhynchanthus beesianus.

Rhynchanthus beesianus (Zingiberaceae) has been an important food spice and vegetable in southern China. Fifteen phenolic compounds (1-15) including three new diarylheptanoids, rhynchanines A-C (1-3) and one new phenylpropanoid, 4-O-methylstroside B (9), were isolated from R. beesianus rhizomes. The structures of new compounds were elucidated by comprehensive analyses through NMR, HRMS technique, acid hydrolysis, and Mosher's reaction. Among them, compound 5 is the first isolated natural product and its NMR data are reported. Most of the isolated compounds, especially 3-6 and 8, showed significant antioxidant activities on DPPH, ABTS+ radical scavenging, and FRAP assays. Furthermore, the antioxidant phenolic compounds were evaluated for their cytoprotective capacity against H2O2-induced oxidative stress in HepG-2 cells. Compounds 3 and 5 could significantly inhibit reactive oxygen species production, and compounds 3, 5, and 6 could remarkably prevent the cell apoptosis. Then, the R. beesianus rhizome, which contained phenolic compounds, might serve as a functional food for potential application on preventing oxidative stress-connected diseases.

Graphene-Based Sensors for the Detection of Bioactive Compounds: A Review

Over the last years, different nanomaterials have been investigated to design highly selective and sensitive sensors, reaching nano/picomolar concentrations of biomolecules, which is crucial for medical sciences and the healthcare industry in order to assess physiological and metabolic parameters. The discovery of graphene (G) has unexpectedly impulsed research on developing cost-effective electrode materials owed to its unique physical and chemical properties, including high specific surface area, elevated carrier mobility, exceptional electrical and thermal conductivity, strong stiffness and strength combined with flexibility and optical transparency. G and its derivatives, including graphene oxide (GO) and reduced graphene oxide (rGO), are becoming an important class of nanomaterials in the area of optical and electrochemical sensors. The presence of oxygenated functional groups makes GO nanosheets amphiphilic, facilitating chemical functionalization. G-based nanomaterials can be easily combined with different types of inorganic nanoparticles, including metals and metal oxides, quantum dots, organic polymers, and biomolecules, to yield a wide range of nanocomposites with enhanced sensitivity for sensor applications. This review provides an overview of recent research on G-based nanocomposites for the detection of bioactive compounds, providing insights on the unique advantages offered by G and its derivatives. Their synthesis process, functionalization routes, and main properties are summarized, and the main challenges are also discussed. The antioxidants selected for this review are melatonin, gallic acid, tannic acid, resveratrol, oleuropein, hydroxytyrosol, tocopherol, ascorbic acid, and curcumin. They were chosen owed to their beneficial properties for human health, including antibiotic, antiviral, cardiovascular protector, anticancer, anti-inflammatory, cytoprotective, neuroprotective, antiageing, antidegenerative, and antiallergic capacity. The sensitivity and selectivity of G-based electrochemical and fluorescent sensors are also examined. Finally, the future outlook for the development of G-based sensors for this type of biocompounds is outlined.

In vitro cytoprotective effect and antioxidant capacity of salmon, mackerel and herring hydrolysates in Caco-2 clone (TC7) cells

In vitro cytoprotective effect and antioxidant capacity of salmon, mackerel and herring hydrolysates in Caco-2 clone (TC7) cells

Cytoprotective organoselenium compounds for oligodendrocytes

Herein we report the synthesis of peptide-like and tetrazole-based organoselenium compounds via Ugi and Ugi-azide reactions, respectively. The organoselenium compounds' intrinsic cytoprotective and antioxidant capacities were evaluated in 158 N and 158JP murine oligodendrocytes. Furthermore, their redox properties were theoretically evaluated using Molecular Operating Environment-docking studies. Most of the compounds did not exhibit any cytotoxicity against the 158JP and 158 N cells. Among the tested compounds, the tetrazole- (e.g., 6, 7, and 9) and the pseudopeptide-based organoselenium compounds (e.g., 11, 15, and 17) displayed antioxidant properties. On the other hand, the quinones- (e.g., 4c and 18) and the pseudopeptides-based (e.g., 12, 14, and 17) organoselenium compounds exhibited prooxidant activities. Furthermore, the tetrazole-based organoselenium compounds 5 and 9 and the selenopeptide 11 and 15 showed good GPx-like activity. Some of the newly synthesized organoselenium compounds presented interesting antioxidant and cytoprotective activities and are therefore considered potential myelin diseases drug candidates.

Octopus vulgaris ink extracts exhibit antioxidant, antimutagenic, cytoprotective, antiproliferative, and proapoptotic effects in selected human cancer cell lines.

Cancer is a noncommunicable disease of rising worldwide concern. Marine food products such as Octopus vulgaris ink (OI) could be sources of compounds addressing these concerns. This study aimed to evaluate the antimutagenic, cytoprotective, antiproliferative, proapoptotic, and antioxidant capacity of OI extracts on human cancer cell lines (22Rv1, HeLa, A549). The ARPE-19 cell line was used as a reference human cell line to evaluate the ink's cytotoxicity. The water extract exhibited the highest antimutagenic and cytoprotective effect, but the dichloromethane extract (DM) showed the lowest half lethal concentration against 22Rv1 cells. Structural elucidation of purified DM fractions (F1, F2, F3) identified an unreported compound, N-(2-ozoazepan-3-yl)-pyrrolidine-2-carboxamide (OPC). DM-F2 showed high antiproliferative effect (LC50 = 27.6 µg/mL), reactive species modulation, early-apoptosis induction (42.9%), and nuclei disruption in 22Rv1 cells. In silico analysis predicted high OPC affinity with Cyclin D1 (-6.70kcal/mol), suggesting its potential impact on cell cycle arrest. These results highlight the antimutagenic, cytoprotective, and antiproliferative potential health benefits derived from underutilized marine food products such as OI. Further investigations at in vitro or in vivo levels are required to elucidate mechanisms and health benefits from OI. PRACTICAL APPLICATION: O. vulgaris ink is an underutilized marine natural product that could be a source of biological compounds with potential health benefits such as antioxidant activity and cancer prevention.

In-Vitro Effect of Kalanchoe daigremontiana and Its Main Component, Quercetin against Entamoeba histolytica and Trichomonas vaginalis.

Background:Parasitic infections represent one of the main public health problems in humans according to the WHO. Therefore, the need has arisen to find new treatments that can be used as an alternative cure to parasitosis. We aimed to investigate the in-vitro effects of the methanolic extract of Kalanchoe daigremontiana as well as its main component, quercetin against Entamoeba histolytica and Trichomonas vaginalis.Methods:For this purpose, the in-vitro activity of the methanol extract of K. daigremontiana also its main component, quercetin, against trophozoites of E. histolytica and T. vaginalis was evaluated, using the microassay technique. Furthermore, the antioxidant activity was determined. Finally, the cytotoxic and cytoprotective capacity was determined using the hemolysis technique.Results:The IC50 indicated that quercetin significantly (P < 0.05) inhibited the growth rate of the trophozoite stage of E. histolytica and T. vaginalis in comparison to the methanolic extract of K. daigremontiana (KalL). Also, quercetin significantly (P < 0.05) was a better antioxidant as compared with the positive control. In the evaluation of cytotoxicity effects, it could be observed that KalL as compared with quercetin exhibited more cytotoxicity against human erythrocytes. Quercetin significantly (P < 0.001) exhibited better cytoprotective activity compared to KalL.Conclusion:Both K. daigremontiana methanolic extract and quercetin alone demonstrated high antiparasitic activity against E. histolytica and T. vaginalis. However, the in-vivo efficacy of K. daigremontiana and quercetin also requires to be evaluated using an animal model.

Biochemical and Molecular Investigation of In Vitro Antioxidant and Anticancer Activity Spectrum of Crude Extracts of Willow Leaves Salix safsaf.

Organic fractions and extracts of willow (Salix safsaf) leaves, produced by sequential solvent extraction as well as infusion and decoction, exhibited anticancer potencies in four cancerous cell lines, including breast (MCF-7), colorectal (HCT-116), cervical (HeLa) and liver (HepG2). Results of the MTT assay revealed that chloroform (CHCl3) and ethyl acetate (EtOAc)-soluble fractions exhibited specific anticancer activities as marginal toxicities were observed against two non-cancerous control cell lines (BJ-1 and MCF-12). Ultra-high-resolution mass spectrometry Q-Exactive™ HF Hybrid Quadrupole-Orbitrap™ coupled with liquid chromatography (UHPLC) indicated that both extracts are enriched in features belonging to major phenolic and purine derivatives. Fluorescence-activated cell sorter analysis (FACS), employing annexin V-FITC/PI double staining indicated that the observed cytotoxic potency was mediated via apoptosis. FACS analysis, monitoring the increase in fluorescence signal, associated with oxidation of DCFH to DCF, indicated that the mechanism of apoptosis is independent of reactive oxygen species (ROS). Results of immunoblotting and RT-qPCR assays showed that treatment with organic fractions under investigation resulted in significant up-regulation of pro-apoptotic protein and mRNA markers for Caspase-3, p53 and Bax, whereas it resulted in a significant reduction in amounts of both protein and mRNA of the anti-apoptotic marker Bcl-2. FACS analysis also indicated that pre-treatment and co-treatment of human amniotic epithelial (WISH) cells exposed to the ROS H2O2 with EtOAc fraction provide a cytoprotective and antioxidant capacity against generated oxidative stress. In conclusion, our findings highlight the importance of natural phenolic and flavonoid compounds with unparalleled and unique antioxidant and anticancer properties.

Priming of intestinal cytoprotective genes and antioxidant capacity by dietary phytogenic inclusion in broilers.

The potential of a phytogenic premix (PP) based on ginger, lemon balm, oregano and thyme to stimulate the expression of cytoprotective genes at the broiler gut level was evaluated in this study. In particular, the effects of PP inclusion levels on a selection of genes related to host protection against oxidation (catalase [CAT], superoxide dismutase 1 [SOD1], glutathione peroxidase 2 [GPX2], heme oxygenase 1 [HMOX1], NAD(P)H quinone dehydrogenase 1 [NQO1], nuclear factor (erythroid-derived 2)-like 2 [Nrf2] and kelch like ECH associated protein 1 [Keap1]), stress (heat shock 70 kDa protein 2 [HSP70] and heat shock protein 90 alpha family class A member 1 [HSP90]) and inflammation (nuclear factor kappa B subunit 1 [NF-κB1], Toll-like receptor 2 family member B (TLR2B) and Toll-like receptor 4 [TLR4]) were profiled along the broiler intestine. In addition, broiler intestinal segments were assayed for their total antioxidant capacity (TAC). Depending on PP inclusion level (i.e. 0, 750, 1,000 and 2,000 mg/kg diet) in the basal diets, 1-d-old Cobb broiler chickens (n = 500) were assigned into the following 4 treatments: CON, PP-750, PP-1000 and PP-2000. Each treatment had 5 replicates of 25 chickens with ad libitum access to feed and water. Data were analyzed by ANOVA and means compared using Tukey's honest significant difference (HSD) test. Polynomial contrasts tested the linear and quadratic effect of PP inclusion levels. Inclusion of PP increased (P ≤ 0.05) the expression of cytoprotective genes against oxidation, except CAT. In particular, the cytoprotective against oxidation genes were up-regulated primarily in the duodenum and the ceca and secondarily in the jejunum. Most of the genes were up-regulated in a quadratic manner with increasing PP inclusion level with the highest expression levels noted in treatments PP-750 and PP-1000 compared to CON. Similarly, intestinal TAC was higher in PP-1000 in the duodenum (P = 0.011) and the ceca (P = 0.050) compared to CON. Finally, increasing PP inclusion level resulted in linearly reduced (P ≤ 0.05) expression of NF-κB1, TLR4 and HSP70, the former in the duodenum and the latter 2 in the ceca. Overall, PP inclusion consistently up-regulated cytoprotective genes and down-regulated stress and inflammation related ones. The effect is dependent on PP inclusion level and the intestinal site. The potential of PP to beneficially prime bird cytoprotective responses merit further investigation under stress-challenge conditions.

Protective Effect of Graviola (Annona muricata) Leaves Extract Against Cadmium-Induced Testicular Toxicity In Male Albino Rats

Graviola (Annona muricata), an edible plant belonging to the Annonaceae family, has a long history in traditional medicine due to its wide range of therapeutic effects on many ailments. This study was planned to assess the protective potentials of Graviola leaves extract (GLE) on cadmium-induced testicular damage in male albino rats. The study was conducted on 32 male adult Wistar rats randomly divided into four groups (n=8/each). A (control group); B (GLE, 100 mg/kg); C (cadmium chloride (CdCl2), 5 mg/kg) and D (GLE, 100 mg/kg + CdCl2 5 mg/kg). All administrations were performed by daily gavage for 8 weeks. Blood, testes, and epididymis were harvested for biochemical and histological investigations. In addition to sperm analysis, sperm DNA comet assay, and apoptotic indices. Results revealed that GLE significantly reversed (p < 0.05) testicular oxidative stress induced by CdCl2, maintained male sex hormones levels, and restored testis architecture via increasing the testicular antioxidant status. GLE co-treatment significantly increased (p < 0.05) sperm count, motility, viability, and DNA integrity along with significantly reducing sperm DNA damage and sperm head abnormality in comparison with CdCl2 group. Additionally, GLE protected germ cells from Cd-induced apoptosis. In conclusion, results of the present study confirmed that GLE has potent antioxidant potentials and cytoprotective capacity against the deleterious effects of CdCl2 on testicular tissue by reducing cellular oxidative stress prompted by the generation of the reactive oxygen species.

The Metabolite Urolithin-A Ameliorates Oxidative Stress in Neuro-2a Cells, Becoming a Potential Neuroprotective Agent.

Urolithin A is a metabolite generated from ellagic acid and ellagitannins by the intestinal microbiota after consumption of fruits such as pomegranates or strawberries. The objective of this study was to determine the cytoprotective capacity of this polyphenol in Neuro-2a cells subjected to oxidative stress, as well as its direct radical scavenging activity and properties as an inhibitor of oxidases. Cells treated with this compound and H2O2 showed a greater response to oxidative stress than cells only treated with H2O2, as mitochondrial activity (MTT assay), redox state (ROS formation, lipid peroxidation), and the activity of antioxidant enzymes (CAT: catalase, SOD: superoxide dismutase, GR: glutathione reductase, GPx: glutathione peroxidase) were significantly ameliorated; additionally, urolithin A enhanced the expression of cytoprotective peroxiredoxins 1 and 3. Urolithin A also acted as a direct radical scavenger, showing values of 13.2 μM Trolox Equivalents for Oxygen Radical Absorbance Capacity (ORAC) and 5.01 µM and 152.66 µM IC50 values for superoxide and 2,2-diphenyss1-picrylhydrazyl (DPPH) radicals, respectively. Finally, inhibition of oxidizing enzymes, such as monoamine oxidase A and tyrosinase, was also detected in a dose-dependent manner. The cytoprotective effects of urolithin A could be attributed to the improvement of the cellular antioxidant battery, but also to its role as a direct radical scavenger and enzyme inhibitor of oxidases.

Piceatannol Increases Antioxidant Defense and Reduces Cell Death in Human Periodontal Ligament Fibroblast under Oxidative Stress.

Piceatannol is a resveratrol metabolite that is considered a potent antioxidant and cytoprotector because of its high capacity to chelate/sequester reactive oxygen species. In pathogenesis of periodontal diseases, the imbalance of reactive oxygen species is closely related to the disorder in the cells and may cause changes in cellular metabolism and mitochondrial activity, which is implicated in oxidative stress status or even in cell death. In this way, this study aimed to evaluate piceatannol as cytoprotector in culture of human periodontal ligament fibroblasts through in vitro analyses of cell viability and oxidative stress parameters after oxidative stress induced as an injury simulator. Fibroblasts were seeded and divided into the following study groups: control, vehicle, control piceatannol, H2O2 exposure, and H2O2 exposure combined with the maintenance in piceatannol ranging from 0.1 to 20 μM. The parameters analyzed following exposure were cell viability by trypan blue exclusion test, general metabolism status by the 3-[4,5-dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method, mitochondrial activity through the ATP production, total antioxidant capacity, and reduced gluthatione. Piceatannol was shown to be cytoprotective due the maintenance of cell viability between 1 and 10 μM even in the presence of H2O2. In a concentration of 0.1 μM piceatannol decreased significantly cell viability but increased cellular metabolism and antioxidant capacity of the fibroblasts. On the other hand, the fibroblasts treated with piceatannol at 1 μM presented low metabolism and antioxidant capacity. However, piceatannol did not protect cells from mitochondrial damage as measured by ATP production. In summary, piceatannol is a potent antioxidant in low concentrations with cytoprotective capacity, but it does not prevent all damage caused by hydrogen peroxide.

Antioxidant, Antimutagenic and Cytoprotective Properties of Hydrosos Pistachio Nuts.

Pistachio nuts are included among the foods with the highest antioxidant capacity. Stressed cultivating conditions, such as the use of regulated deficit irrigation (RDI), are expected to create a plant response that might increase the production of secondary metabolites. Fruits that are obtained under RDI treatments are commonly called hydroSOS products. The aim of this work was to study the influence of using different rootstocks (P. atlantica, P. integerrima, and P. terebinthus) and two RDI treatments on the antioxidant (ABTS, ferric reducing antioxidant power (FRAP), and DPPH), antimutagenic (Ames test), and cytotoxicity (MTT assay in five human cell lines) activities of pistachios. P. terebinthus showed the best antioxidant activity, and the RDI treatments maintained and improved the antioxidant properties of pistachios. Neither the rootstock nor the RDI had significant impact on the antimutagenic potential of pistachios. The nut extracts had no toxic effect on non-cancerous cells and the application of RDI did not reduce their cytoprotective capacity. Furthermore, neither rootstock nor RDI treatments affected the ability of the pistachio extracts of preventing the oxidative damage by H2O2. The application of RDI strategies, in addition to allowing irrigation water saving, led to obtaining pistachios with the same or even better biofunctional characteristics as compared to fully irrigated pistachios.

Genistein protects against acute pancreatitis via activation of an apoptotic pathway mediated through endoplasmic reticulum stress in rats

Acute pancreatitis (AP) is a severe and frequently lethal disorder, but the precise mechanisms are not well understood and there is lack of effective drugs. Therefore, our study examined the in vivo intervention effects of genistein and elucidated its mechanism in acute experimental pancreatitis models. We used cerulein or taurocholate to induce acute pancreatitis (AP) in Sprague-Dawley rats with prior genistein treatment. Histological examination of the pancreas was performed and the expression of unfolded protein response (UPR) components and apoptotic mediators like caspase 12 and c-Jun N-terminal protein kinase (JNK) were measured. The amount of apoptosis in pancreatic acinar cells was also determined. Our studies found that the severity of cerulein- or taurocholate-induced AP was rescued by prior genistein treatment. Genistein stimulated the activation of multiple endoplasmic reticulum (ER) stress-related regulators like GRP78, PERK, eIF2α, and upregulated the expression of the apoptotic genes, caspase 12 and CHOP. Moreover, TUNEL assays showed that genistein treatment promoted acinar cell apoptosis. Taken together, we speculated that ER stress-associated apoptotic pathways in AP are induced by genistein, which showed cytoprotective capacity in the exocrine pancreas. These data suggest novel therapeutic strategies that employ genistein in the prevention of AP.

111 - Application of resonance raman spectroscopy on manganese porphyrin detection and redox state in endothelial cells: implications on cytoprotection towards peroxynitrite

Manganese(III) ortho N-substituted pyridylporphyrins (MnP) act as efficient antioxidants catalyzing superoxide dismutation and accelerating peroxynitrite reduction. Importantly, MnP can reach mitochondria offering protection against reactive species in different animal models of disease. In this scenario, we applied Resonance Raman (RR) spectroscopy as a direct method to detect both the uptake and the redox state of MnP in living cells. Using RR we were able to analyze the intracellular redox state changes of two potent MnP (MnTnBuOE-2-PyP5+ and MnTnHex-2-PyP5+) within endothelial cells exposed to SIN-1. RR experiments with cells and isolated mitochondria revealed that the reduction of Mn(III)P was affected by inhibitors of the electron transport chain, supporting the action of MnP as efficient redox active compounds in mitochondria. Indeed, RR spectra confirmed that MnP added in the Mn(III) state can be incorporated into the cells, readily reduced by intracellular components to the Mn(II) state and oxidized by peroxynitrite. We also evaluated the cytoprotective capacity of MnP by exposing the endothelial cells to nitro-oxidative stress induced by peroxynitrite. We observed a preservation of normal mitochondrial function, attenuation of cell damage and prevention of apoptotic cell death. These data introduce a novel application of RR spectroscopy for the direct detection of MnP and their redox states inside living cells, and helps to rationalize their antioxidant capacity in biological systems.

Manganese porphyrin redox state in endothelial cells: Resonance Raman studies and implications for antioxidant protection towards peroxynitrite

Cationic manganese(III) ortho N-substituted pyridylporphyrins (MnP) act as efficient antioxidants catalyzing superoxide dismutation and accelerating peroxynitrite reduction. Importantly, MnP can reach mitochondria offering protection against reactive species in different animal models of disease. Although an LC-MS/MS-based method for MnP quantitation and subcellular distribution has been reported, a direct method capable of evaluating both the uptake and the redox state of MnP in living cells has not yet been developed. In the present work we applied resonance Raman (RR) spectroscopy to analyze the intracellular accumulation of two potent MnP-based lipophilic SOD mimics, MnTnBuOE-2-PyP5+ and MnTnHex-2-PyP5+ within endothelial cells. RR experiments with isolated mitochondria revealed that the reduction of Mn(III)P was affected by inhibitors of the electron transport chain, supporting the action of MnP as efficient redox active compounds in mitochondria. Indeed, RR spectra confirmed that MnP added in the Mn(III) state can be incorporated into the cells, readily reduced by intracellular components to the Mn(II) state and oxidized by peroxynitrite. To assess the combined impact of reactivity and bioavailability, we studied the kinetics of Mn(III)TnBuOE-2-PyP5+ with peroxynitrite and evaluated the cytoprotective capacity of MnP by exposing the endothelial cells to nitro-oxidative stress induced by peroxynitrite. We observed a preservation of normal mitochondrial function, attenuation of cell damage and prevention of apoptotic cell death. These data introduce a novel application of RR spectroscopy for the direct detection of MnP and their redox states inside living cells, and helps to rationalize their antioxidant capacity in biological systems.

E-Configuration Improves Antioxidant and Cytoprotective Capacities of Resveratrols

The antioxidant and cytoprotective capacities of E-resveratrol and Z-resveratrol were compared using chemical and cellular assays. Chemical assays revealed that the two isomers were dose-dependently active in •O2−-scavenging, ferric reducing antioxidant power (FRAP), Cu2+-reducing antioxidant capacity (CUPRAC), 2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide radical (PTIO•)-scavenging (pH 7.4 and pH 4.5), and 1,1-diphenyl-2-picryl-hydrazyl (DPPH•)-scavenging assays. The cellular assay indicated that the two isomers could also increase cell viabilities. However, quantitative analyses suggested that E-resveratrol exhibited stronger effects than Z-resveratrol in all chemical and cellular assays. Finally, the conformations of E-resveratrol and Z-resveratrol were analyzed. It can be concluded that both E-resveratrol and Z-resveratrol can promote redox-related pathways to exhibit antioxidant action and consequently protect bone marrow-derived mesenchymal stem cells (bmMSCs) from oxidative damage. These pathways include electron transfer (ET) and H+-transfer, and likely include hydrogen atom transfer (HAT). The E-configuration, however, improves antioxidant and cytoprotective capacities of resveratrols. The detrimental effect of the Z-configuration may be attributed to the non-planar preferential conformation, where two dihedral angles block the extension of the conjugative system.