Neuroprotective Effects of Chemical Constituents of Leaves of Euonymus hamiltonianus Wall.

Euonymus hamiltonianus Wall. is considered a medicinal plant and is used to treat pain, cough, dysuria, and cancer, but a clear phytochemical investigation of its biological activities has yet to be performed. Investigation of chemical constituents of the leaves of Euonymus hamiltonianus Wall. led to the isolation of three new compounds by chromatography techniques, euonymusins A-C (1, 10, and 11), and the acquisition of new spectroscopic data for euonymusin D (2), along with the identification of ten known compounds. The chemical structures of the compounds were established using extensive spectroscopic techniques, including NMR, MS, and hydrolysis, and compared with the published data. These compounds were tested in vitro for their inhibitory effects on beta amyloid production (Aß42). Compounds 13 and 14 displayed weak inhibition, with IC50 values ranging from 53.15 to 65.43 µM. Moreover, these compounds were also assessed for their inhibitory effects on nitric oxide production. Of these compounds, 3, 4, and 14 displayed inhibitory effects on NO production, with IC50 values ranging from 14.38 to 17.44 µM. Compounds 3, 4, and 14 also suppressed LPS-induced expression of nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) protein.

Anti-inflammatory effect and metabolic mechanism of BS012, a mixture of Asarum sieboldii, Platycodon grandiflorum, and Cinnamomum cassia extracts, on atopic dermatitis in vivo and in vitro.

BACKGROUND: Atopic dermatitis (AD) is a chronic, relapsing skin disease accompanied by itchy and dry skin. AD is caused by complex interactions between innate and adaptive immune response. AD treatment include glucocorticoids and immunosuppressants. However, long-term treatment can have serious side effects. Thus, an effective AD treatment with fewer side effects is required. Natural materials, including herbal medicines, have potential applications. PURPOSE: This study evaluated the in vivo and in vitro therapeutic effects of BS012, a mixture of Asarum sieboldii, Platycodon grandiflorum, and Cinnamomum cassia extracts, on AD and investigated the underlying metabolic mechanisms. METHODS: The anti-inflammatory effects of BS012 were assessed using a mouse model of AD induced by 1­chloro-2,4-dinitrobenzene (DNCB) and in tumor necrosis factor-alpha/interferon-gamma (TNF-α/IFN-γ) stimulated normal human epidermal keratinocytes (NHEKs). In DNCB-induced mice, total dermatitis score, histopathological analysis, and immune cell factors were assessed to evaluate the anti-atopic activity. In TNF-α/IFN-γ-stimulated NHEKs, pro-inflammatory cytokines, chemokines, and related signaling pathways were investigated. Serum and intracellular metabolomics were performed to identify the metabolic mechanism underlying the therapeutic effects of BS012 treatment. RESULTS: In DNCB-induced mice, BS012 showed potent anti-atopic activity, including reducing AD-like skin lesions and inhibiting the expression of Th2 cytokines and thymic stromal lymphopoietin. In TNF-α/IFN-γ-stimulated keratinocytes, BS012 dose-dependently inhibited the expression of pro-inflammatory cytokines and chemokines by blocking nuclear factor-kappa B and signal transducer and activator of transcription signaling pathways. Serum metabolic profiles of mice revealed significant changes in lipid metabolism related to inflammation in AD. Intracellular metabolome analysis revealed that BS012 treatment affected the metabolism associated with inflammation, skin barrier function, and lipid organization of the stratum corneum. CONCLUSION: BS012 exerts anti-atopic activity by reducing the Th2-specific inflammatory response and improving skin barrier function in AD in vivo and in vitro. These effects are mainly related to the inhibition of inflammation and recovery of metabolic imbalance in lipid organization. BS012, a novel combination with strong activity in suppressing the Th2-immune response, could be a potential alternative for AD treatment. Furthermore, the metabolic mechanism in vivo and in vitro using a metabolomics approach will provide crucial information for the development of natural products for AD treatment.

Sinapic Acid Attenuates the Neuroinflammatory Response by Targeting AKT and MAPK in LPS-Activated Microglial Models.

Sinapic acid (SA) is a phenolic acid that is widely distributed in fruits and vegetables, which has various bioactivities, such as antidiabetic, anticancer and anti-inflammatory functions. Over-activated microglial is involved in the development progress of neurodegenerative diseases, such as Parkinson's disease and Alzheimer's disease. The objective of this study was to investigate the effect of SA in microglia neuroinflammation models. Our results demonstrated that SA inhibited secretion of the nitric oxide (NO) and interleukin (IL)-6, reduced the expression of inducible nitric oxide synthase (iNOS) and enhanced the release of IL-10 in a dose-dependent manner. Besides, our further investigation revealed that SA attenuated the phosphorylation of AKT and MAPK cascades in LPS-induced microglia. Consistently, oral administration of SA in mouse regulated the production of inflammation-related cytokines and also suppressed the phosphorylation of MAPK cascades and AKT in the mouse cerebral cortex. These results suggested that SA may be a possible therapy candidate for anti-inflammatory activity by targeting the AKT/MAPK signaling pathway.

Narciclasine inhibits LPS-induced neuroinflammation by modulating the Akt/IKK/NF-κB and JNK signaling pathways.

BACKGROUND: Neuroinflammation is defined as innate immune system activation in the central nervous system, and is a complex response involved in removing pathogens, toxic components, and dead cells by activating microglial cells. However, over-activated microglia have been implicated in the pathogenesis of neurodegenerative diseases, because they release large amounts of neurotoxic factors. Thus, inhibiting microglial activation may represent an attractive approach for preventing neuroinflammatory disorders. The objective of this study was to investigate the effect of narciclasine (NA) on lipopolysaccharide (LPS)-induced neuroinflammation by evaluating related markers and neurotoxic factors. METHODS: BV-2 cells were pre-incubated with NA at 0.1, 0.2, and 0.3 µM for 1h, and then co-treated with LPS for 12 h. Cellular medium and lysates were measured using a nitric oxide assay, enzyme-link immunosorbent assay (ELISA), western blotting, kinase activity assay, luciferase assay, and immunofluorescence assay. C57BL/6N mice were orally administered NA and intraperitoneally injected with LPS, and the cerebral cortex was examined using western blotting and immunofluorescence assays. RESULTS: NA showed novel pharmacological activity, inhibiting pro-inflammatory factors, including TNF-α, IL-6, IL-18, NO, and PGE2, but increasing the anti-inflammatory cytokines IL-10 and TGF-ß1 in LPS-induced microglial cells. Moreover, NA also attenuated the LPS-induced mRNA and proteins of iNOS and COX-2. The mechanistic study indicated that NA attenuates the secretion of pro-inflammatory factor by down-regulating the Akt/IKK/NF-κB and JNK signaling pathways, and directly inhibits the catalytic activity of IKKα/ß. Furthermore, we found that NA also reduced the expression of the microglial markers Iba-1, COX-2, and TNF-α in the mouse brain. CONCLUSION: NA inhibits the over-expression of pro-inflammatory factors but it promotes anti-inflammatory cytokines by down-regulating the Akt/IKK/NF-κB and JNK signaling pathways in experimental models. Thus, NA may be a potential candidate for relieving neuroinflammation.

Extract of Polygala tenuifolia, Angelica tenuissima, and Dimocarpus longan Reduces Behavioral Defect and Enhances Autophagy in Experimental Models of Parkinson's Disease.

The 20% ethanol extract of Polygala tenuifolia, Angelica tenuissima, and Dimocarpus longan (WIN-1001X) was derived from a modified version of Korean traditional herbal formula 'Chungsimyeolda-tang' which has been used for the treatment of cerebrovascular disorders. The Parkinson's disease presents with impaired motor functions and loss of dopaminergic neurons. However, the treatment for Parkinson's disease is not established until now. This study aims to elucidate the therapeutic advantages of WIN-1001X on animal models of Parkinson's disease. WIN-1001X administration successfully relieved the Parkinsonism symptoms in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson's disease mice tested by rota-rod and pole tests. The loss of tyrosine hydroxylase activities in substantia nigra and striatum was also attenuated by administration of WIN-1001X. In mice with sub-chronical MPTP injections, autophagy-related proteins, such as LC3, beclin-1, mTOR, and p62, were measured using the immunoblot assay. The results were favorable to induction of autophagy after the WIN-1001X administration. WIN-1001X treatment on 6-hydroxydopamine-injected rats also exhibited protective effects against striatal neuronal damage and loss of dopaminergic cells. Such protection is expected to be due to the positive regulation of autophagy by administration of WIN-1001X with confirmation both in vivo and in vitro. In addition, an active compound, onjisaponin B was isolated and identified from WIN-1001X. Onjisaponin B also showed significant autophagosome-inducing effect in human neuroblastoma cell line. Our study suggests that relief of Parkinsonism symptoms and rescue of tyrosine hydroxylase activity in dopaminergic neurons are affected by autophagy enhancing effect of WIN-1001X which the onjisaponin B is one of the major components of activity.

Substrate-Specific Activation of α-Secretase by 7-Deoxy-Trans-Dihydronarciclasine Increases Non-Amyloidogenic Processing of ß-Amyloid Protein Precursor.

Accumulation of ß-amyloid (Aß) in the brain has been implicated in the pathology of Alzheimer's disease (AD). Aß is produced from the Aß precursor protein (APP) through the amyloidogenic pathway by ß-, and γ-secretase. Alternatively, APP can be cleaved by α-, and γ-secretase, precluding the production of Aß. Thus, stimulating α-secretase mediated APP processing is considered a therapeutic option not only for decreasing Aß production but for increasing neuroprotective sAPPα. We have previously reported that 7-deoxy-trans-dihydronarciclasine (E144), the active component of Lycoris chejuensis, decreases Aß production by attenuating APP level, and retarding APP maturation. It can also improve cognitive function in the AD model mouse. In this study, we further analyzed the activating effect of E144 on α-secretase. Treatment of E144 increased sAPPα, but decreased ß-secretase products from HeLa cells stably transfected with APP. E144 directly activated ADAM10 and ADAM17 in a substrate-specific manner both in cell-based and in cell-free assays. The Lineweaver-Burk plot analysis revealed that E144 enhanced the affinities of A Disintegrin and Metalloproteinases (ADAMs) towards the substrate. Consistent with this result, immunoprecipitation analysis showed that interactions of APP with ADAM10 and ADAM17 were increased by E144. Our results indicate that E144 might be a novel agent for AD treatment as a substrate-specific activator of α-secretase.

An Autophagy Inducing Triterpene Saponin Derived from Aster koraiensis.

Autophagy is an important self-degradative mechanism that plays a key role in treating neurodegeneration diseases. This research aimed at discovering bioactive compounds from Aster koraiensis. A new triterpene saponin, astersaponin I (1), was isolated from the EtOH extract of A. koraiensis. The structure of 1 was characterized by spectroscopic methods, ECD calculation, and acid hydrolysis. The biochemical analysis showed that compound 1 significantly increased the expression of microtubule-associated protein 1A/1B light chain 3B (LC3-II) expression in SH-SY5Y cells, which indicates the induction of autophagy. Thus, further study may be needed to clarify whether compound 1 exerts beneficial effects on neurodegeneration diseases like Parkinson's disease through autophagy induction.

7-Deoxy-trans-dihydronarciclasine Isolated from Lycoris chejuensis Inhibits Neuroinflammation in Experimental Models.

Overactivated microglia and persistent neuroinflammation hold an important role in the pathophysiology of neurodegenerative diseases. The extract of Lycoris chejuensis (CJ) and its active compound, 7-deoxy-trans-dihydronarciclasine (named E144), attenuated expressions of pro-inflammatory factors, including nitric oxide, prostaglandin E2, inducible nitric oxide synthase, cyclooxygenase-2 (COX-2), tumor necrosis factor α (TNF-α), and interleukin 6, secreted by lipopolysaccharide-activated BV-2 microglial cells, as measured by an enzyme-linked immunosorbent assay or western blotting. In contrast, CJ extract and E144 promoted the secretion of the anti-inflammatory cytokine, interleukin 10. Moreover, we found that E144 attenuated the expression of TNF-α and COX-2 in the cerebral cortex of lipopolysaccharide-treated mice and/or T2576 transgenic mice as well as reduced the reactive immune cells visualized by ionized calcium-binding adaptor molecule 1. Our results suggest the possibility of E144 to serve as a potential anti-neuroinflammatory agent by preventing excess production of pro-inflammatory factors.

Neuroprotective effect of anodal transcranial direct current stimulation on 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity in mice through modulating mitochondrial dynamics.

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the accumulation of protein inclusions and the loss of dopaminergic neurons. Abnormal mitochondrial homeostasis is thought to be important for the pathogenesis of PD. Transcranial direct current stimulation (tDCS), a noninvasive brain stimulation technique, constitutes a promising approach for promoting recovery of various neurological conditions. However, little is known about its mechanism of action. The present study elucidated the neuroprotective effects of tDCS on the mitochondrial quality control pathway in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced PD mouse model. We used the MPTP-induced neurotoxicity in vivo model. Mice were stimulated for 5 consecutive days with MPTP treatment. After observation of behavioral alteration using the rotarod test, mice were sacrificed for the measurement of the PD- and mitochondrial quality control-related protein levels in the substantia nigra. tDCS improved the behavioral alterations and changes in tyrosine hydroxylase levels in MPTP-treated mice. Furthermore, tDCS attenuated mitochondrial damage, as indicated by diminished mitochondrial swelling and mitochondrial glutamate dehydrogenase activity in the MPTP-induced PD mouse model. MPTP significantly increased mitophagy and decreased mitochondrial biogenesis-related proteins. These changes were attenuated by tDCS. Furthermore, MPTP significantly increased fission-related protein dynamin-related protein 1 with no effect on fusion-related protein mitofusin-2, and tDCS attenuated these changes. Our findings demonstrated the neuroprotective effect of anodal tDCS on the MPTP-induced neurotoxic mouse model through suppressing excessive mitophagy and balancing mitochondrial dynamics. The neuroprotective effect of anodal tDCS with modulation of mitochondrial dynamics provides a new therapeutic strategy for the treatment of PD.

Anti-neuroinflammatory Effects of 12-Dehydrogingerdione in LPS-Activated Microglia through Inhibiting Akt/IKK/NF-κB Pathway and Activating Nrf-2/HO-1 Pathway.

Ginger, one of worldwide consumed dietary spice, is not only famous as food supplements, but also believed to exert a variety of remarkable pharmacological activity as herbal remedies. In this study, a ginger constituent, 12-dehydrogingerdione (DHGD) was proven that has comparable anti-inflammatory activity with positive control 6-shogaol in inhibiting LPS-induced interleukin (IL)-6, tumor necrosis factor (TNF)-α, prostaglandin (PG) E2, nitric oxide (NO), inducible NO synthase (iNOS) and cyclooxygenase (COX)-2, without interfering with COX-1 in cultured microglial cells. Subsequent mechanistic studies indicate that 12-DHGD may inhibit neuro-inflammation through suppressing the LPS-activated Akt/IKK/NF-κB pathway. Furthermore, 12-DHGD markedly promoted the activation of NF-E2-related factor (Nrf)-2 and heme oxygenase (HO)-1, and we demonstrated that the involvement of HO-1 on the production of pro-inflammatory mediators such as NO and TNF-α by using a HO-1 inhibitor, Zinc protoporphyrin (Znpp). These results indicate that 12-DHGD may protect against neuro-inflammation by inhibiting Akt/IKK/IκB/NF-κB pathway and promoting Nrf-2/HO-1 pathway.

Anti-Neuroinflammatory Effects of Fucoxanthin via Inhibition of Akt/NF-κB and MAPKs/AP-1 Pathways and Activation of PKA/CREB Pathway in Lipopolysaccharide-Activated BV-2 Microglial Cells.

Microglia play a critical role in controlling the homeostasis of the brain, but over-activated microglia secrete pro-inflammatory mediators and cytokines, which induce neuronal cell death. Fucoxanthin (Fx), a marine carotenoid, has demonstrated a variety of beneficial health effects. Despite accumulating evidence supporting the immune-modulating effects of Fx in vitro, the underlying signaling pathways remain unknown. In the present study, Fx dose-dependently inhibited the secretion of lipopolysaccharide (LPS)-induced pro-inflammatory mediators including interleukin (IL)-6, tumor necrosis factor (TNF)-α, reactive oxygen species (ROS), prostaglandin (PG) E2, and nitric oxide (NO) productions, and also suppressed the expression of inducible NO synthase (iNOS) and cyclooxygenase (COX)-2 enzymes. Further, the reverse transcription-polymerase chain reaction (RT-PCR) analysis indicated IL-6, TNF-α, iNOS, and COX-2 mRNA expression were suppressed by treatment with Fx in a dose-dependently manner. The mechanism studies indicated that Fx blocks protein kinase B (Akt)/nuclear factor-kappaB (NF-κB) and mitogen-activated protein kinase (MAPKs)/transcription factor (AP)-1 pathways. In addition, we demonstrated that Fx increases nuclear factor erythroid 2-related factor (Nrf)-2 activation and heme oxygenase (HO)-1 expression in LPS-activated BV-2 microglia. Subsequently, we found that Fx also mediates the reactive oxygen species (ROS) by activating protein kinase A (PKA)/cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) pathway, and promotes the production of brain-derived neurotrophic factor (BDNF). These results indicate that Fx may be more effective and potential than other candidates via either decreasing the pro-inflammatory factors production or increasing the neuroprotective molecules expression for therapy of neurodegenerative diseases.

The Neuroprotective Effects of Justicidin A on Amyloid Beta25-35-Induced Neuronal Cell Death Through Inhibition of Tau Hyperphosphorylation and Induction of Autophagy in SH-SY5Y Cells.

Justicidin A is a structurally defined arylnaphthalide lignan, which has been shown anti-cancer activity; however, the neuroprotective effect of justicidin A is still untested. In this study, we investigated the action of justicidin A on amyloid beta (Aß)25-35-induced neuronal cell death via inhibition of the hyperphosphorylation of tau and induction of autophagy in SH-SY5Y cells. Pretreatment with justicidin A significantly elevated cell viability in cells treated with Aß25-35. Western blot data demonstrated that justicidin A inhibited the Aß25-35-induced up-regulation the levels of hyperphosphorylation of tau in SH-SY5Y cells. In addition, treatment with justicidin A significantly induced autophagy as measured by the increasing LC3 II/I ratio, an important autophagy marker. These studies showed that justicidin A inhibited activity of glycogen synthase kinase-3beta (GSK-3ß), which is an important kinase in up-stream signaling pathways; inhibited hyperphosphorylation of tau in AD; and enhanced activity of AMP-activated protein kinase (AMPK), which is the key molecule for both hyperphosphorylation of tau and induction of autophagy. These data provide the first evidence that justicidin A protects SH-SY5Y cells from Aß25-35-induced neuronal cell death through inhibition of hyperphosphorylation of tau and induction of autophagy via regulation the activity of GSK-3ß and AMPK, and they also provide some insights into the relationship between tau protein hyperphosphorylation and autophagy. Thus, we conclude that justicidin A may have a potential role for neuroprotection and, therefore, may be used as a therapeutic agent for AD.

The Neuroprotective Effect of Erythropoietin on Rotenone-Induced Neurotoxicity in SH-SY5Y Cells Through the Induction of Autophagy.

Currently, the autophagy pathway is thought to be important for the pathogenesis of Parkinson's disease (PD), and the modulation of autophagy may be a novel strategy for the treatment of this disease. Erythropoietin (EPO) has been reported to have neuroprotective effects through anti-oxidative, anti-apoptotic, and anti-inflammatory mechanisms, and it has also been shown to modulate autophagy signaling in an oxygen toxicity model. Therefore, we investigated the effects of EPO on autophagy markers and evaluated its neuroprotective effect on rotenone-induced neurotoxicity. We adapted the rotenone-induced neurotoxicity model to SH-SY5Y cells as an in vitro model of PD. We measured cell viability using MTT and annexin V/propidium iodide assays and measured intracellular levels of reactive oxygen species. Immunofluorescence analysis was performed to measure the expression of LC3 and α-synuclein. Intracellular signaling proteins associated with autophagy were examined by immunoblot analysis. EPO mono-treatment increased the levels of mammalian target of rapamycin (mTOR)-independent/upstream autophagy markers, including Beclin-1, AMPK, and ULK-1. Rotenone treatment of SH-SY5Y cells reduced their viability, increased reactive oxygen species levels, and induced apoptosis and α-synuclein expression, and simultaneous exposure to EPO significantly reduced these effects. Rotenone enhanced mTOR expression and suppressed Beclin-1 expression, indicating suppression of the autophagy system. However, combined treatment with EPO restored Beclin-1 expression and decreased mTOR expression. EPO protects against rotenone-induced neurotoxicity in SH-SY5Y cells by enhancing autophagy-related signaling pathways. The experimental evidence for the EPO-induced neuroprotection against rotenone-induced dopaminergic neurotoxicity may significantly impact the development of future PD treatment strategies.

Anti-apoptotic effect of modified Chunsimyeolda-tang, a traditional Korean herbal formula, on MPTP-induced neuronal cell death in a Parkinson's disease mouse model.

ETHNOPHARMACOLOGICAL RELEVANCE: The modified-Chungsimyeolda-tang (DG) is an important traditional Korean herbal formula used in traditional oriental medicine for treatment of cerebrovascular disorders, including stroke. The formula is based on the book "Dongui Sasang Shinpyun". AIM OF THE STUDY: In the previous studies, the neuroprotective effect of DG is demonstrated in an in vitro Parkinson's disease (PD) model, and in this study, the 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) animal model of PD is used to evaluate the behavioral effect of DG and possible mechanism through anti-apoptosis of DG. 6-Hydroxydopamine (6-OHDA) also is used to evaluate the anti-apoptosis effect of DG in SH-SY5Y cells. MATERIALS AND METHODS: MPTP was used to evaluate the behavioral damage and neurotoxicity in mice. The bradykinesia symptom was measured by a Pole test and a Rota-rod test in mice. Also the loss of tyrosine hydroxylase (TH)-positive neurons induced by MPTP was examined by an immunohistochemical assay. The DG-mediated anti-apoptosis effect was measured using an immunoblotting assay with apoptosis-related markers such as Bax and cleaved caspase-3. DG and 1-methyl-4-phenylpyridinium (MPP(+)) were co-treated with primary dopaminergic neurons to evaluate the protective effect of DG. The expression of caspase-3 and PARP was measured to detect the protective effect of DG from the damage by 6-OHDA. RESULTS AND CONCLUSIONS: The treatment with DG resulted in prophylactic effects on MPTP-induced Parkinsonian bradykinesia and the immunohistochemical analysis showed that DG provided the neuroprotection against the MPP(+)-induced dopaminergic neurons loss through the anti-apoptosis effect. The present results suggested that it might be possible to use DG for the prevention of substantia nigra pars compacta (SNpc) degeneration induced by exposure to the toxic substances, such as MPTP/MPP(+), in PD mouse model.

Neuroprotective effect of modified Chungsimyeolda-tang, a traditional Korean herbal formula, via autophagy induction in models of Parkinson's disease.

AIM OF THE STUDY: Previous studies in our laboratory revealed the neuroprotective effect of modified Yeoldahanso-tang (MYH) in models of Parkinson׳s disease (PD). In this study, we investigated another traditional Korean herbal formula, modified Chungsimyeolda-tang (termed DG), as a potential treatment for PD. Chungsimyeolda-tang has been used in Korea to treat cerebrovascular diseases, such as stroke. Here, we verify the neuroprotective and autophagy-inducing effects of DG to evaluate any potential anti-parkinsonian properties. MATERIALS AND METHODS: 1-Methyl-4-phenylpyridinium (MPP(+)) and rotenone were used to induce cytotoxicity in nerve growth factor (NGF)-differentiated rat pheochromocytoma (PC12) cells. Cell viability was measured using an MTT assay. Induction of autophagy by DG in NGF-differentiated PC12 cells was measured using an immunoblotting assay with an LC3 antibody. The proteasomal inhibitor lactacystin was used to induce ubiquitin-proteasome system (UPS) dysfunction in NGF-differentiated PC12 cells. DG-mediated clearance of aggregated proteins was measured using an immunoblotting assay with a ubiquitin antibody. RESULTS AND CONCLUSIONS: Our findings indicate that DG robustly protects NGF-differentiated PC12 cells against the neurotoxic effects of MPP(+) and rotenone in an in vitro model. Furthermore, DG protects NGF-differentiated PC12 cells against lactacystin-induced cell death. This effect is partially mediated by an increased autophagy associated with the enhanced degradation of aggregated proteins. This study suggests that DG is an attractive candidate drug for inducing autophagy and, therefore, may represent a promising strategy to prevent diseases associated with misfolded/aggregated proteins in various neurodegenerative disorders, including Parkinson׳s disease.

Neuroprotective Effect of Chebulagic Acid via Autophagy Induction in SH-SY5Y Cells.

Autophagy is a series of catabolic process mediating the bulk degradation of intracellular proteins and organelles through formation of a double-membrane vesicle, known as an autophagosome, and fusing with lysosome. Autophagy plays an important role of death-survival decisions in neuronal cells, which may influence to several neurodegenerative disorders including Parkinson's disease. Chebulagic acid, the major constituent of Terminalia chebula and Phyllanthus emblica, is a benzopyran tannin compound with various kinds of beneficial effects. This study was performed to investigate the autophagy enhancing effect of chebulagic acid on human neuroblastoma SH-SY5Y cell lines. We determined the effect of chebulagic acid on expression levels of autophago-some marker proteins such as, DOR/TP53INP2, Golgi-associated ATPase Enhancer of 16 kDa (GATE 16) and Light chain 3 II (LC3 II), as well as those of its upstream pathway proteins, AMP-activated protein kinase (AMPK), mammalian target of rapamycin (mTOR) and Beclin-1. All of those proteins were modulated by chebulagic acid treatment in a way of enhancing the autophagy. Additionally in our study, chebulagic acid also showed a protective effect against 1-methyl-4-phenylpyridinium (MPP(+)) - induced cytotoxicity which mimics the pathological symptom of Parkinson's disease. This effect seems partially mediated by enhanced autophagy which increased the degradation of aggregated or misfolded proteins from cells. This study suggests that chebulagic acid is an attractive candidate as an autophagy-enhancing agent and therefore, it may provide a promising strategy to prevent or cure the diseases caused by accumulation of abnormal proteins including Parkinson's disease.

Glionitrin A, a new diketopiperazine disulfide, activates ATM-ATR-Chk1/2 via 53BP1 phosphorylation in DU145 cells and shows antitumor effect in xenograft model.

In a recent study, we isolated the diketopiperazine disulfide glionitrin A from the co-culture broth of a mine drainage-derived fungus (Aspergillus fumigatus KMC901) and bacterium (Sphingomonas KMK001). Here, we investigated the antitumor activity of glionitrin A and its underlying molecular mechanisms in human prostate cancer DU145 cells. Glionitrin A showed significant cytotoxicity, promoting cell cycle arrest and apoptosis. Glionitrin A-treated cells exhibited elevated levels of phospho-histone 2AX (Ser139), a marker of DNA damage, and accumulated in both S phase and G2/M phase due to the activation of checkpoints associated with the ataxia-telangiectasia-mutated and ataxia-telangiectasia-mutated-Rad3-related Chk1/2 pathway downstream of p53-binding protein 1 phosphorylation at Ser1778. In addition, glionitrin A induced apoptosis through both caspase-dependent and -independent pathways. Glionitrin A activated caspase-8, -9 and -3 and also released endonuclease G from the mitochondria to the nucleus in a dose-dependent manner. Our in vivo study performed in nude mice bearing xenografts of DU145 cells showed that glionitrin A dramatically reduced the tumor volume by an average of 38.2% (5 mg/kg, per os (p.o.)) and 71.3% (10 mg/kg, p.o.) at 27 d after the beginning of treatment. Taken together, these findings provide a detailed description of the mechanism underlying the biological activity of the new natural product glionitrin A, which has the potential to be developed as an anti-prostate cancer agent.

Ropinirole protects against 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced neurotoxicity in mice via anti-apoptotic mechanism.

Ropinirole is a pharmacologically active agent used in the treatment of Parkinson's disease (PD) that directly acts on dopamine receptors. Recent studies reported that ropinirole has neuroprotective potential. However, there have been no detailed studies on apoptosis. Here we demonstrated the protective effects of ropinirole against 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine (MPTP)-induced neurotoxicity in a mouse model of PD. Unlike previous studies, we focused on apoptotic pathways. To confirm the protective effect of ropinirole, we conducted behavioral tests and tyrosine hydroxylase (TH)-immunohistochemistry. In the pole test and the rotarod test, commonly used behavioral tests in the mouse model of PD, ropinirole treatment (0.5, 1, or 2 mg/kg) maintained movement ability against MPTP-induced changes in motor coordination and postural balance, and bradykinesia. Our histological analyses illustrated that ropinirole significantly reduced MPTP-induced dopaminergic neuron damage in both the substantia nigra pars compacta (SNpc) and the striatum. In addition, we performed Western blot or kit analysis to measure apoptosis-related protein levels in the SNpc. Ropinirole increased the Bcl-2/Bax ratio, transcription factor A, and nuclear respiratory factor 1 and inhibited cytosolic cytochrome c release and caspase-3 activity, indicating that ropinirole inhibited the apoptotic cascade. These results suggest that ropinirole has neuroprotective effects in a mouse model of PD by inhibiting apoptosis.

Identification of the urinary metabolites of glionitrin A in rats using ultra-performance liquid chromatography combined with quadrupole time-of-flight mass spectrometry.

Glionitrin A (GN A) is a new diketopiperazine disulfide with an aromatic nitro group, which is isolated from the coculture of an Aspergillus fumigatus fungal strain and a Sphingomonas bacterial strain. After intravenous administration of GN A in rats, 13 urinary metabolites of GN A were identified using ultra-performance liquid chromatography/quadrupole time-of-flight mass spectroscopy (UPLC-QTOP-MS) analysis in conjunction with data processing programs such as MetaboLynx™ and MassFragnent™. Reduction, nitro-reduction and hydration were the primary metabolic processes affecting GN A in vivo, followed by demethylation or oxidative deamination to alcohol, as well as cysteine, glycine, glucuronide or sulfate conjugation. The metabolite resulting from reduction was found to be a molecule with a dithiol group, and the metabolite made by nitro reduction was found to be an aromatic amine corresponding to GN A. Both of these products may have pharmacological or toxicological activity, which is valuable information in terms of using GN A as a lead compound. In addition, this work showed that UPLC-QTOP-MS analysis coupled with efficient data processing programs is useful for rapid and reliable characterization of GN A metabolites in vivo.

9-Cis-retinoic acid induces growth inhibition in retinoid-sensitive breast cancer and sea urchin embryonic cells via retinoid X receptor α and replication factor C3.

There is widespread interest in defining factors and mechanisms that suppress the proliferation of cancer cells. Retinoic acid (RA) is a potent suppressor of mammary cancer and developmental embryonic cell proliferation. However, the molecular mechanisms by which 9-cis-RA signaling induces growth inhibition in RA-sensitive breast cancer and embryonic cells are not apparent. Here, we provide evidence that the inhibitory effect of 9-cis-RA on cell proliferation depends on 9-cis-RA-dependent interaction of retinoid X receptor α (RXRα) with replication factor C3 (RFC3), which is a subunit of the RFC heteropentamer that opens and closes the circular proliferating cell nuclear antigen (PCNA) clamp on DNA. An RFC3 ortholog in a sea urchin cDNA library was isolated by using the ligand-binding domain of RXRα as bait in a yeast two-hybrid screening. The interaction of RFC3 with RXRα depends on 9-cis-RA and bexarotene, but not on all-trans-RA or an RA receptor (RAR)-selective ligand. Truncation and mutagenesis experiments demonstrated that the C-terminal LXXLL motifs in both human and sea urchin RFC3 are critical for the interaction with RXRα. The transient interaction between 9-cis-RA-activated RXRα and RFC3 resulted in reconfiguration of the PCNA-RFC complex. Furthermore, we found that knockdown of RXRα or overexpression of RFC3 impairs the ability of 9-cis-RA to inhibit proliferation of MCF-7 breast cancer cells and sea urchin embryogenesis. Our results indicate that 9-cis-RA-activated RXRα suppresses the growth of RA-sensitive breast cancer and embryonic cells through RFC3.