Identification of Major Antioxidant Compounds from the Edible Mushroom Basidiomycetes-X (Echigoshirayukidake).

Basidiomycetes-X, of which Japanese vernacular name is Echigoshirayukidake, is a local speciality mushroom found and cultivated in Japan that has been distributed as a precious cuisine material or as a functional food with medicinal properties. Antioxidant activity-guided isolation of major ingredients in Basidiomycetes-X revealed the presence of ergosterol, trans-10,cis-12-octadecadienoic acid (a conjugated linolenic acid, 10(E),12(Z)-CLA) and 2,3-dihydro-3,5-dihydroxy-6-methyl­4H­pyran-4-one (DDMP). Approximately 21% of the 2,2-diphenyl-1-(2,4,6-trinitrophenyl)hydrazino radical (DPPH) scavenging activities in the methanolic extract were related to 10(E),12(Z)-CLA, while approximately 6.2% of the activity was related to ergosterol. DDMP was present in both methanolic and water extracts, and the activity related to DDMP was conspicuously detected in water extracts. Moreover, uridine and adenosine were identified as major components of Basidiomycetes-X. The ingredients identified in Basidiomycetes-X are expected to be involved in biological functions observed in this mushroom, which is an attractive functional food resource.

Identification of a Novel Pyrrole Alkaloid from the Edible Mushroom Basidiomycetes-X (Echigoshirayukidake).

Three pyrrole alkaloid derivatives were isolated from the edible mushroom Basidiomycetes-X (Echigoshirayukidake) by water extraction followed by ethyl acetate fractionation. The chemical structures determined by MS and NMR were 4-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl] butanoic acid (compound I), 4-[2-formyl-5-(hydroxymethyl)-1H-pyrrol-1-yl] butanamide (compound II), and 5-(hydroxymethyl)-1H-pyrrole-2-carboxaldehyde (compound III). Compound I was found to be the major component, followed by compound II, and compound III was the minor component. The dry powder of Basidiomycetes-X contained approximately 825 µg g-1 compound I and 484 µg g-1 compound II. Compound II was found to be a novel pyrrole aldehyde homologue not previously reported and thus is a specific component of this mushroom.

Obesity preventive function of novel edible mushroom, Basidiomycetes-X (Echigoshirayukidake): Manipulations of insulin resistance and lipid metabolism.

Echigoshirayukidake is an edible mushroom found in Uonuma, Japan in 1994. It was assigned to a new species of Basidiomycetes (BDM-X) but is uniquely defect of forming bashidium. The high antioxidant activity and ß-glucan content of BDM-X suggest possible functions preventing type 2 diabetes. In the present study, anti-obesity and insulin resistance preventive functions of BDM-X were examined using genetically defined obese model rat, OLETF (Otsuka Long Evans Tokushima Fatty) by feeding regular diet with and without supplementation of 5% dried BDM-X powder (BDMP) for 15 weeks. BDMP supplementation to the diet significantly (p < 0.01) suppressed the body weight gain and also visceral fat accumulation during the feeding period compared to control diet. Simultaneously, the insulin resistance and the plasma levels of adiponectin and triglycerides were significantly (p = 0.003) ameliorated in the BDMP supplemented diet group. A statistical multivariate analysis showed the weight of three types of adipose tissue (epididymal, retroperirenal, and mesenteric fat) positively correlated with HOMA-IR (Homeostasis Model Assessment of Insulin Resistance), and negatively correlated with plasma adiponectin. These results indicate BDM-X is a new resource applicable to the functional foods or the complementary biomedicines to prevent metabolic syndromes leading to type 2 diabetes.

3,4-Dihydroxybenzalacetone (DBL) Prevents Aging-Induced Myocardial Changes in Senescence-Accelerated Mouse-Prone 8 (SAMP8) Mice.

Aging is a predominant risk factor for the development and progression of cardiovascular complications. Physiologically and anatomically, the heart undergoes numerous changes that result in poor cardiac function in the elderly population. Recently, several studies have provided promising results, confirming the ability of the senescence-accelerated mouse-prone 8 (SAMP8) model to accurately model age-related cardiovascular alterations. In this study, using a murine model of senescence, SAMP8, we aimed to investigate the effect of 3,4-dihydroxybenzalacetone (DBL), a catechol-containing phenylpropanoid derivative isolated from Inonotusobliquus (Chaga), on cardiac aging. DBL was administered at the doses of 10 mg/kg and 20 mg/kg by oral gavage to SAMP8 mice to examine aging-mediated cardiac changes, such as oxidative DNA damage, oxygen radical antioxidant capacity (ORAC) value, fibrosis, inflammation, and apoptosis. The treatment with DBL at both doses significantly reduced aging-mediated oxidative DNA damage, and simultaneously increased the ORAC value in the SAMP8 assay. Cardiac fibrosis was assessed with Azan-Mallory staining, and the number of cardiac remodeling markers was found to be significantly reduced after the treatment with DBL. We also observed a decrease in cardiomyocyte apoptosis as measured by the terminal transferase-mediated dUTP nick end labeling (TUNEL) staining method and the caspase-3 levels in SAMP8 mice compared with senescence-resistant control (SAMR1) mice. The findings from this study suggest that DBL has a potentially beneficial effect on aging-mediated myocardial alterations. Further studies are warranted to confirm the promising potential of this catechol compound against aging-associated myocardial dysfunction.

The Adjuvant Effect of Squalene, an Active Ingredient of Functional Foods, on Doxorubicin-Treated Allograft Mice.

Many functional foods or physiologically active ingredients derived from plants and animals are actively being investigated for their role in chronic disease prevention. Squalene (SQ) is found as active ingredient in the functional foods predominantly present in olive oil and shark liver oil. It is known that during chemotherapy anticancer drugs induce inflammation. SQ has been thought to prevent and suppress inflammation; however, there is little direct evidence available. We examined the adjuvant effect of SQ on tumor-transplanted mice along with anticancer drug doxorubicin (DOX). SQ significantly suppressed the DOX-induced increase in prostaglandin E2 (PGE2) concentration (P < 0.05) in plasma of tumor-bearing mice. SQ inhibited the numbers of writhing response (P < 0.05), formalin-induced pain and decreased COX-2 and substance P expression in the tumor tissue compared to control mice and also enhanced the antitumor efficacy of DOX in allograft mice. Thus, SQ reduces inflammation through modulation of PGE2 production indicating its potential as an adjuvant during chemotherapy in tumor-bearing mice.

Inhibition of nuclear factor-κB p65 phosphorylation by 3,4-dihydroxybenzalacetone and caffeic acid phenethyl ester.

3,4-Dihydroxybenzalacetone (DBL) and caffeic acid phenethyl ester (CAPE) are both catechol-containing phenylpropanoid derivatives with various bioactivities. In the present study, we compared the effects of these compounds and other phenylpropanoid derivatives on the activation of nuclear factor-κB (NF-κB) signaling, a major pathway in the inflammatory response, using RAW 264.7 cells. Lipopolysaccharide (LPS)- and interferon γ-induced production of nitrite was strongly suppressed by CAPE and, to a lesser extent, by DBL and caffeic acid ethyl ester. Consistent with these results, induction of NF-κB downstream genes, such as Nitric oxide synthase, interleukin 1 beta, and interleukin 6, and translocation of NF-κB p65 to the nucleus were reduced after LPS stimulation, to a greater extent with CAPE than with DBL. Interestingly, the phosphorylation of p65 was reduced by both compounds, especially by CAPE, even when the level of IκB was not altered. Furthermore, the thiol groups of p65 were modified by CAPE, and the inhibitory effects of CAPE and DBL on the p65 phosphorylation and nitrite production were reversed by pretreatment with thiol-containing reagents. These results suggest that CAPE has strong inhibitory effects on the NF-κB activation that are associated with the modification of thiol groups and phosphorylation of p65.

3,4-dihydroxybenzalacetone and caffeic acid phenethyl ester induce preconditioning ER stress and autophagy in SH-SY5Y cells.

3,4-dihydroxybenzalacetone (DBL) and Caffeic acid phenethyl ester (CAPE) are both catechol-containing phenylpropanoid derivatives with diverse bioactivities. In the present study, we analyzed the ability of these compounds to activate the unfolded protein response (UPR) and the oxidative stress response. When human SH-SY5Y neuroblastoma cells were treated with DBL or CAPE, the expression of endoplasmic reticulum (ER) stress-related genes such as HSPA5, HYOU1, DDIT3, and SEC61b increased to a larger extent in response to CAPE treatment, while that of antioxidant genes such as HMOX1, GCLM, and NQO1 increased to a larger extent in response to DBL treatment. DNA microarray analysis confirmed the strong link of these compounds to ER stress. Regarding the mechanism, activation of the UPR by these compounds was associated with enhanced levels of oxidized proteins in the ER, and N-acetyl cysteine (NAC), which provides anti-oxidative effects, suppressed the induction of the UPR-target genes. Furthermore, both compounds enhanced the expression of LC3-II, a marker of autophagy, and 4-Phenylbutyric acid (4-PBA), a chemical chaperone that reduces ER stress, suppressed it. Finally, pretreatment of cells with DBL, CAPE or low doses of ER stressors protected cells against a neurotoxin 6-hydroxydopamine (6-OHDA) in an autophagy-dependent manner. These results suggest that DBL and CAPE induce oxidized protein-mediated ER stress and autophagy that may have a preconditioning effect in SH-SY5Y cells.

Tiny molecule, big power: Multi-target approach for curcumin in diabetic cardiomyopathy.

Diabetic cardiomyopathy (DCM) is described as impaired cardiac diastolic and systolic functions. Diabetes mellitus (DM), a related cardiovascular disease, has become one of the major causes of death in DM patients. Mortality in these diseases is 2 to 3 times higher than in non-DM patients with cardiovascular disease. The progression of DCM and the cellular and molecular perturbations associated with the pathogenesis are complex and multifactorial. Although considerable progress has been achieved, the molecular etiologies of DCM remain poorly understood. There is an expanding need for natural antidiabetic medicines that do not cause the side effects of modern drugs. Curcumin, a pleiotropic molecule, from Curcuma longa, is known to possess numerous impacts such as scavenging free radical, antioxidant, antitumor, and antiinflammatory activities. The reports from preclinical and clinical findings revealed that curcumin can reverse insulin resistance, hyperglycemia, obesity, and obesity-related metabolic diseases. The current review provides an updated overview of the possible molecular mechanism of DCM and multitarget approach of curcumin in alleviating DCM and diabetic complication. Additionally, we mentioned the approaches that are currently being implemented to improve the bioavailability of this promising natural product in diabetes therapeutics.

Pork Loin Treated with High Hydrostatic Pressure as a Food Processing Technology: Subacute Toxicity of the Freeze-Dried Powder and Cytotoxicity of the Methanol Extracts.

High hydrostatic pressure (HP) treatment is used in food processing owing to its sterilization effect. Meat or meat products are sterilized and become tender by HP processing. Therefore, the variety of HP-processed meat products has increased worldwide. However, little is known about the safety of HP-processed meat products. The aim of this study was to determine the effects of HP processing and HP combined with 0.4 M sodium carbonate treatment (HP-Na) on pork loins and to evaluate the subacute toxicity and cytotoxicity of these processing methods. In an in vivo study, we performed 90- and 180-day feeding tests in mice and did not detect any adverse effects in HP-processed and HP-Na-processed pork loins. In addition, we evaluated the cytotoxicity of HP-processed meats, and did not observe any obvious toxicity associated with pork loin extracts in vitro. These results suggest that HP is not associated with risk factors during processing.

Modulation of Macrophage Polarization and HMGB1-TLR2/TLR4 Cascade Plays a Crucial Role for Cardiac Remodeling in Senescence-Accelerated Prone Mice.

The aim of this study was to investigate the role of macrophage polarization in aging heart. Macrophage differentiation is pathogenically linked to many inflammatory and immune disorders. It is often preceded by myocardial inflammation, which is characterized by increased cardiac damage and pro-inflammatory cytokine levels. Therefore, we investigated the hypothesis that senescence accelerated-prone (SAMP8) mice cardiac tissue would develop macrophage polarization compared with senescence-resistant control (SAMR1) mice. Both SAMP8 and SAMR1 mice were sacrificed when they became six month old. We evaluated, histo-pathological changes and modifications in protein expression by Western blotting and immuno-histochemical staining for M1 and M2 macrophage markers, high mobility group protein (HMG)B1 and its cascade proteins, pro-inflammatory factors and inflammatory cytokines in cardiac tissue. We observed significant upregulation of HMGB1, toll-like receptor (TLR)2, TLR4, nuclear factor (NF)κB p65, tumor necrosis factor (TNF)α, cyclooxygenase (COX)2, interferon (IFN)γ, interleukin (IL)-1ß, IL-6 and M1 like macrophage specific marker cluster of differentiation (CD)68 expressions in SAMP8 heart. In contrast, M2 macrophage specific marker CD36, and IL-10 expressions were down-regulated in SAMP8 mice. The results from the study demonstrated that, HMGB1-TLR2/TLR4 signaling cascade and induction of phenotypic switching to M1 macrophage polarization in SAMP8 mice heart would be one of the possible reasons behind the cardiac dysfunction and thus it could become an important therapeutic target to improve the age related cardiac dysfunction.

Role of MAPK-mediated endoplasmic reticulum stress signaling in the heart during aging in senescence-accelerated prone mice.

Heart failure is typically related to aging as there is a definite relationship between age-related changes in the heart and the pathogenesis of heart failure. We have previously reported the involvement of p38 mitogen-activated protein kinase protein in cardiac function using animal models of heart failure. To further understand its relationship with aging-induced heart failure, we have compared its expression in the hearts of senescence accelerated-prone (SAMP8) mice and their control (SAMR1) with normal aging behavior. We have identified its activation along with reduced expression of 14-3-3η protein in SAMP8 mice hearts than in SAMR1 mice. To reveal the downstream signaling, we have measured the endoplasmic reticulum stress marker proteins along with some inflammatory and apoptosis markers and identified a significant increase in SAMP8 mice hearts than that of SAMR1. In addition, we have performed comet assay and revealed a significant DNA damage in the cardiomyocytes of SAMP8 mice when compared with SAMR1 mice. All these results demonstrate the role of 14-3-3η protein and the downstream mitogen-activated protein kinase-mediated endoplasmic reticulum stress, and apoptosis and DNA damage in aging-induced cardiac malfunction in SAMP8 mice. Thus targeting this signaling might be effective in treating age-related cardiac dysfunction. © 2016 BioFactors, 42(4):368-375, 2016.

Squalene Inhibits ATM-Dependent Signaling in γIR-Induced DNA Damage Response through Induction of Wip1 Phosphatase.

Ataxia telangiectasia mutated (ATM) kinase plays a crucial role as a master controller in the cellular DNA damage response. Inhibition of ATM leads to inhibition of the checkpoint signaling pathway. Hence, addition of checkpoint inhibitors to anticancer therapies may be an effective targeting strategy. A recent study reported that Wip1, a protein phosphatase, de-phosphorylates serine 1981 of ATM during the DNA damage response. Squalene has been proposed to complement anticancer therapies such as chemotherapy and radiotherapy; however, there is little mechanistic information supporting this idea. Here, we report the inhibitory effect of squalene on ATM-dependent DNA damage signals. Squalene itself did not affect cell viability and the cell cycle of A549 cells, but it enhanced the cytotoxicity of gamma-irradiation (γIR). The in vitro kinase activity of ATM was not altered by squalene. However, squalene increased Wip1 expression in cells and suppressed ATM activation in γIR-treated cells. Consistent with the potential inhibition of ATM by squalene, IR-induced phosphorylation of ATM effectors such as p53 (Ser15) and Chk1 (Ser317) was inhibited by cell treatment with squalene. Thus, squalene inhibits the ATM-dependent signaling pathway following DNA damage through intracellular induction of Wip1 expression.

Antihyperlipidemic effect of Acanthopanax senticosus (Rupr. et Maxim) Harms leaves in high-fat-diet fed mice.

BACKGROUND: Metabolic syndrome is a major risk factor for a variety of obesity-related diseases. Recently, the effects of functional foods have been investigated on lipid metabolism as a means to reduce lipid content in the blood, liver and adipose tissues associated with carnitine O-palmitoyltransferase (CPT) activity. Acanthopanax senticosus (Rupr. et Maxim) Harms (AS) is a medicinal herb possessing a wide spectra of functions including antioxidant, anti-inflammatory and anti-fatigue actions. Despite much research being focused on the cortical roots of AS, little information is available regarding its leaves, which are also expected to promote human health, for example by improving abnormal lipid metabolism. Here, we explored whether AS leaves affect lipid metabolism in mice fed a high-fat diet. RESULTS: The administration of AS to BALB/c mice fed a high-fat diet significantly decreased plasma triglycerides (TG). CPT activity in the liver of these mice was significantly enhanced by AS treatment. CONCLUSION: These findings indicate that AS leaves have the potential to alleviate increase in plasma TG levels due to high-fat diet intake in mice, possibly by increasing mitochondrial fatty acid ß-oxidation, especially via CPT activation. Consequently, daily intake of AS leaves could promote beneficial health effects including the prevention of metabolic syndrome. © 2015 Society of Chemical Industry.

Schisandrin B Ameliorates ICV-Infused Amyloid ß Induced Oxidative Stress and Neuronal Dysfunction through Inhibiting RAGE/NF-κB/MAPK and Up-Regulating HSP/Beclin Expression.

Amyloid ß (Aß)-induced neurotoxicity is a major pathological mechanism of Alzheimer's disease (AD). Our previous studies have demonstrated that schisandrin B (Sch B), an antioxidant lignan from Schisandra chinensis, could protect mouse brain against scopolamine- and cisplatin-induced neuronal dysfunction. In the present study, we examined the protective effect of Sch B against intracerebroventricular (ICV)-infused Aß-induced neuronal dysfunction in rat cortex and explored the potential mechanism of its action. Our results showed that 26 days co-administration of Sch B significantly improved the behavioral performance of Aß (1-40)-infused rats in step-through test. At the same time, Sch B attenuated Aß-induced increases in oxidative and nitrosative stresses, inflammatory markers such as inducible nitric oxide syntheses, cyclooxygenase-2, interleukin-1ß (IL-1ß), IL-6, and tumor necrosis factor-α, and DNA damage. Several proteins such as receptor for advanced glycation end products (RAGE), nuclear factor-κB, mitogen-activated protein kinases, and apoptosis markers were over expressed in Aß-infused rats but were significantly inhibited by Sch B treatment. Furthermore, Sch B negatively modulated the Aß level with simultaneous up-regulation of HSP70 and beclin, autophagy markers in Aß-infused rats. The aforementioned effects of Sch B suggest its protective role against Aß-induced neurotoxicity through intervention in the negative cycle of RAGE-mediated Aß accumulation during AD patho-physiology.

Antioxidant Formulae, Shengmai San, and LingGuiZhuGanTang, Prevent MPTP Induced Brain Dysfunction and Oxidative Damage in Mice.

The present study was designed to evaluate the preventive effect of antioxidative traditional oriental medicine formulae, Shengmai San (SMS) and LingGuiZhuGanTang (LGZGT), against 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (i.p 30 mg·kg(-1) for 5 consecutive days) induced neurotoxicity. In in vitro antioxidant assays measured with Trolox and butyl hydroxyl toluene as reference antioxidant revealed that SMS has higher scavenging potential against hydroxyl radical than superoxide anion radical, but LGZGT was the reverse. The neuroprotective effect of SMS and LGZGT against MPTP was evaluated in mice by behavioral, biochemical, and immunohistochemical studies. In the behavioral study, both SMS and LGZGT significantly reversed the locomotive impairment induced by MPTP. Simultaneously, both formulae significantly prevented the MPTP induced dopaminergic neuron loss assessed by tyrosine hydroxylase in the midbrain. Both SMS and LGZGT significantly attenuated the elevated lipid peroxidation and protein carbonyls levels by MPTP. The DNA damage induced by MPTP was also prevented by both formulae. Although a little difference in the protective functions was observed between the two formulae, such as in DNA damage and behavioral studies, the results indicate that both SMS and LGZGT with antioxidant property act as a good candidate applicable for the antioxidant based complementary therapies of neurodegenerative diseases.

Schisandrin B prevents doxorubicin induced cardiac dysfunction by modulation of DNA damage, oxidative stress and inflammation through inhibition of MAPK/p53 signaling.

Doxorubicin (Dox) is a highly effective antineoplastic drug. However, Dox-induced apoptosis in cardiomyocytes leads to irreversible degenerative cardiomyopathy, which limits Dox clinical application. Schisandrin B (Sch B), a dibenzocyclooctadiene derivative isolated from the fruit of Schisandra chinensis, has been shown to protect against oxidative damage in liver, heart and brain tissues in rodents. In current study, we investigated possible protective effects of Sch B against Dox-induced cardiomyopathy in mice. Mice received a single injection of Dox (20 mg/kg IP). Five days after Dox administration, left ventricular (LV) performance was significantly depressed and was improved by Sch B treatment. Sch B prevented the Dox-induced increase in lipid peroxidation, nitrotyrosine formation, and metalloproteinase activation in the heart. In addition, the increased expression of phospho-p38 MAPK and phospho-MAPK activated mitogen kinase 2 levels by Dox were significantly suppressed by Sch B treatment. Sch B also attenuated Dox-induced higher expression of LV proinflammatory cytokines, cardiomyocyte DNA damage, myocardial apoptosis, caspase-3 positive cells and phopho-p53 levels in mice. Moreover, LV expression of NADPH oxidase subunits and reactive oxygen species were significantly less in Sch B treatment mice after Dox injection. These findings suggest that Sch B attenuates Dox-induced cardiotoxicity via antioxidative and anti-inflammatory effects.

Anti-Gastric Ulcer Activity of Polysaccharide Fraction Isolated from Mycelium Culture of Lion's Mane Medicinal Mushroom, Hericium erinaceus (Higher Basidiomycetes).

Hericium erinaceus is a culinary-medicinal mushroom that is used in traditional medicine, in folk medicine, and as medicinal cuisine in Asian countries such as China, Japan, and Korea. H. erinaceus exhibits various pharmacological properties, such as anti-cancer, immunomodulation, anti-dementia, and anti-gastric ulcer effects. The extracts of the fruiting body of H. erinaceus demonstrate anti-gastritis activity. However, the active principle in the extract, as well as the mechanism to treat gastric ulcers, remains uncertain. The current study aims to identify the active component, with anti-gastric ulcer function, from the extracts of the H. erinaceus mycelium culture. In the experiment, anti-gastric ulcer activity was evaluated using an ethanol-induced ulcer model in mice and with an 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide assay using MC cell lines. The results suggest that the polysaccharide fraction can significantly decrease the ulcerated area compared with the control group and the effect is fairly dose dependent, irrespective of animal or cell experiments. These results indicate that the polysaccharide fraction is the active component of the H. erinaceus mycelium culture, which protects against gastric ulcers.

Hericium erinaceus (Yamabushitake): a unique resource for developing functional foods and medicines.

Hericium erinaceus (HE) is a fungus inhabiting the mountainous areas of the northeast territories in Asia. HE has been used in traditional folk medicine and medicinal cuisine in China, Korea and Japan. Evidence has been adduced for a variety of physiological effects, including anti-aging, anti-cancer, anti-gastritis, and anti-metabolic disease properties. Hence, HE is an attractive target resource for developing not only medicines, but also functional foods. Basic studies on the physiological functions of HE and on the chemical identification of its active ingredients have progressed in recent decades. In this article, we provide an overview of the biochemical and pharmacological studies on HE, especially of its antitumor and neuroprotective functions, together with a survey of recent developments in the chemical analysis of its polysaccharides, which comprise its major active components.

3,4-dihydroxybenzalacetone protects against Parkinson's disease-related neurotoxin 6-OHDA through Akt/Nrf2/glutathione pathway.

Oxidative stress is implicated in the pathogenesis of various neurodegenerative diseases including Parkinson's disease (PD). 3,4-Dihydroxybenzalacetone (DBL) is a small catechol-containing compound isolated from Chaga (Inonotus obliquus [persoon] Pilat), and has been reported to have beneficial bioactivities, including antioxidative, anti-inflammatory, and anti-tumorigenic activities, with a relatively low toxicity to normal cells. We, therefore, investigated the neuroprotective activity of DBL against the PD-related neurotoxin 6-hydroxydopamine (6-OHDA). Pretreatment of human neuroblastoma SH-SY5Y cells with DBL, but not with another Chaga-derived catechol-containing compound, caffeic acid, dose-dependently improved the survival of 6-OHDA-treated cells. Although DBL did not reduce 6-OHDA-induced reactive oxygen species in the cell-free system, it promoted the translocation of Nrf2 to the nucleus, activated the transcription of Nrf2-dependent antioxidative genes, and increased glutathione synthesis in the cells. Buthionine sulfoximine, an inhibitor of glutathione synthesis, but not Sn-mesoporphyrin IX, a heme oxygenase-1 inhibitor, or dicoumarol, an NAD(P)H: quinone oxidoreductase 1 inhibitor, abolished the protective effect of DBL against 6-OHDA. Furthermore, DBL activated stress-associated kinases such as Akt, ERK, and p38 MAPK, and PI3K or Akt inhibitors, but not ERK, p38, or JNK inhibitors, diminished DBL-induced glutathione synthesis and protection against 6-OHDA. These results suggest that DBL activates the Nrf2/glutathione pathway through PI3K/Akt, and improves survival of SH-SY5Y cells against 6-OHDA toxicity.

Differential effect of schisandrin B stereoisomers on ATR-mediated DNA damage checkpoint signaling.

We have previously reported that schisandrin B (SchB) is a specific inhibitor of ATR (ataxia telangiectasia and Rad-3-related) protein kinase. Since SchB consists of a mixture of its diastereomers gomisin N (GN) and γ-schisandrin (γ-Sch), the inhibitory action of SchB might result from a stereospecific interaction between one of the stereoisomers of SchB and ATR. Therefore, we investigated the effect of GN and γ-Sch on UV (UVC at 254 nm)-induced activation of DNA damage checkpoint signaling in A549 cells. UV-induced cell death (25 - 75 J/m(2)) was amplified by the presence of the diastereomers, especially GN. At the same time, GN, but not γ-Sch, inhibited the phosphorylation of checkpoint proteins such as p53, structural maintenance of chromosomes 1, and checkpoint kinase 1 in UV-irradiated cells. Moreover, GN inhibited the G2/M checkpoint during UV-induced DNA damage. The in vitro kinase activity of immunoaffinity-purified ATR was dose-dependently inhibited by GN (IC50: 7.28 µM) but not by γ-Sch. These results indicate that GN is the active component of SchB and suggest that GN inhibits the DNA damage checkpoint signaling by stereospecifically interacting with ATR.