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.

Protective Roles of Monsonia angustifolia and Its Active Compounds in Experimental Models of Alzheimer's Disease.

Alzheimer's disease (AD), a progressive neurodegenerative disorder, is characterized by the accumulation of neurotoxic ß-amyloid (Aß) peptides, which consequently affects cognitive decline and memory impairment. Current research on AD treatment is actively focusing on the prevention of neurotoxic Aß peptide accumulation. Monsonia angustifolia is reported to be consumed as an indigenous vegetable in Tanzania. In this study, we investigated the effect of the ethanol (EtOH) extract of M. angustifolia dried ground material on Aß production and spatial learning ability as protection against AD. The formation of Aß peptides was significantly reduced in HeLa cells stably transfected with the Swedish mutant form of ß-amyloid precursor protein (APPsw) after treatment with a 60% EtOH extract of M. angustifolia. We next examined the cognitive-improving effects of the EtOH extract in vivo. Tg2576 mice were treated with extract for 6 months and subjected to Morris water maze and novel object recognition tests. The results showed that the 60% EtOH extract of M. angustifolia significantly ameliorated behavioral deficits of the AD transgenic mice and reduced the level of insoluble Aß42 in the cerebral cortex and hippocampus. We further found that the 60% EtOH extract was effective for memory function recovery after shorter treatment (4 months). In addition, we isolated and identified several single compounds, justicidin A, 5-methoxyjusticidin A, chinensinaphthol, retrochinensinaphthol methyl ether, and suchilactone, from M. angustifolia and tested these compounds. Among them, justicidin A potently decreased the formation of Aß in APPsw-transfected cells. These data suggest that the 60% EtOH extract of M. angustifolia has the potential to be developed as a treatment of AD. Furthermore, justicidin A may contribute, at least partially, to the Aß alteration observed with the extract treatment.

Phytochemicals in Ischemic Stroke.

Stroke is the second foremost cause of mortality worldwide and a major cause of long-term disability. Due to changes in lifestyle and an aging population, the incidence of stroke continues to increase and stroke mortality predicted to exceed 12 % by the year 2030. However, the development of pharmacological treatments for stroke has failed to progress much in over 20 years since the introduction of the thrombolytic drug, recombinant tissue plasminogen activator. These alarming circumstances caused many research groups to search for alternative treatments in the form of neuroprotectants. Here, we consider the potential use of phytochemicals in the treatment of stroke. Their historical use in traditional medicine and their excellent safety profile make phytochemicals attractive for the development of therapeutics in human diseases. Emerging findings suggest that some phytochemicals have the ability to target multiple pathophysiological processes involved in stroke including oxidative stress, inflammation and apoptotic cell death. Furthermore, epidemiological studies suggest that the consumption of plant sources rich in phytochemicals may reduce stroke risk, and so reinforce the possibility of developing preventative or neuroprotectant therapies for stroke. In this review, we describe results of preclinical studies that demonstrate beneficial effects of phytochemicals in experimental models relevant to stroke pathogenesis, and we consider their possible mechanisms of action.

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.

Effect of Lycoris chejuensis and Its Active Components on Experimental Models of Alzheimer's Disease.

We found that an extract of Lycoris chejuensis and its three isolated active components, narciclasine, 7-deoxynarciclasine, and 7-deoxy-trans-dihydronarciclasine, each significantly reduced the formation of amyloid-ß peptides in HeLa cells transfected with an amyloid precursor protein carrying the Swedish mutation up to 45 ± 3.6%. The extract down-regulated amyloid precursor protein, especially the mature form by up to 88%, and reduced the ability of secretases to generate toxic amyloid-ß. Double-transgenic mice treated with the extract for 4 months also showed significantly reduced levels of amyloid-ß and plaques while exhibiting improved memory functions in the Morris water maze and novel object recognition tests. In conclusion, the extract and isolated active components of L. chejuensis decreased the production of amyloid-ß by attenuating amyloid precursor protein levels. Furthermore, the extract improved the disrupted memory functions in animals while inhibiting amyloid plaque formation. Thus, this extract, as well as its active components, could prove beneficial in the treatment of Alzheimer's disease.

Effect of GCSB-5, a Herbal Formulation, on Monosodium Iodoacetate-Induced Osteoarthritis in Rats.

Therapeutic effects of GCSB-5 on osteoarthritis were measured by the amount of glycosaminoglycan in rabbit articular cartilage explants in vitro, in experimental osteoarthritis induced by intra-articular injection of monoiodoacetate in rats in vivo. GCSB-5 was orally administered for 28 days. In vitro, GCSB-5 inhibited proteoglycan degradation. GCSB-5 significantly suppressed the histological changes in monoiodoacetate-induced osteoarthritis. Matrix metalloproteinase (MMP) activity, as well as, the levels of serum tumor necrosis factor-α, cyclooxygenase-2, inducible nitric oxide synthase protein, and mRNA expressions were attenuated by GCSB-5, whereas the level of interleukin-10 was potentiated. By GCSB-5, the level of nuclear factor-κB p65 protein expression was significantly attenuated but, on the other hand, the level of inhibitor of κB-α protein expression was increased. These results indicate that GCSB-5 is a potential therapeutic agent for the protection of articular cartilage against progression of osteoarthritis through inhibition of MMPs activity, inflammatory mediators, and NF-κB activation.

Protective effect of GCSB-5, an herbal preparation, against peripheral nerve injury in rats.

AIM OF THE STUDY: GCSB-5 (traditional name: Chungpa-Juhn), an herbal medicine composed of 6 crude herbs (Saposhnikovia divaricata Schiskin, Achyranthis bidentata Blume, Acanthopanax sessiliflorum Seem, Cibotium baromets J. Smith, Glycine max Meriill, and Eucommia ulmoides Oliver), has been widely used in Asia for treatment of neuropathic and inflammatory diseases. This study investigated the protective effect of GCSB-5 against peripheral nerve injury in vitro and in vivo. MATERIALS AND METHODS: After left sciatic nerve transection, rats received oral administration of GCSB-5 (30, 100, 300, and 600 mg/kg), or saline (vehicle), respectively, once daily for 8 weeks. Motor functional recovery and axonal nerve regeneration were evaluated by measurement of sciatic functional index (SFI), sensory regeneration distance, and gastrocnemius muscle mass ratio. The myelinated axon number was counted by morphometric analysis. In the in vitro study, the effects of GCSB-5 on H(2)O(2)-induced oxidative damage in SH-SY5Y cells were investigated by measurement of cell viability, production of reactive oxygen species (ROS), lipid peroxidation, release of lactate dehydrogenease (LDH), and cellular glutathione contents. Neurite outgrowth was also determined. RESULTS: After 8 weeks of nerve transection, SFI, regeneration distance, and gastrocnemius muscle mass ratio and myelinated axon number showed a significant decrease and these decreases were attenuated by GCSB-5. GCSB-5 significantly inhibited H(2)O(2)-induced cell death and oxidative stress, as evidenced by decreases in production of ROS and lipid peroxidation and release of LDH, and by increase in total GSH content. CONCLUSIONS: The neuroprotective effect afforded by GCSB-5 is due in part to reduced oxidative stress.

Hepatoprotective effect of pinoresinol on carbon tetrachloride-induced hepatic damage in mice.

Forsythiae Fructus is known to have diuretic, anti-bacterial, and anti-inflammatory activities. This study examined the hepatoprotective effects of pinoresinol, a lignan isolated from Forsythiae Fructus, against carbon tetrachloride (CCl(4))-induced liver injury. Mice were treated intraperitoneally with vehicle or pinoresinol (25, 50, 100, and 200 mg/kg) 30 min before and 2 h after CCl4 (20 microl/kg) injection. In the vehicle-treated CCl(4 )group, serum aminotransferase activities were significantly increased 24 h after CCl4 injection, and these increases were attenuated by pinoresinol at all doses. Hepatic glutathione contents were significantly decreased and lipid peroxidation was increased after CCl4 treatment. These changes were attenuated by 50 and 100 mg/kg of pinoresinol. The levels of protein and mRNA expression of inflammatory mediators, including tumor necrosis factor-alpha, inducible nitric oxide synthase, and cyclooxygenase-2, were significantly increased after CCl4 injection; and these increases were attenuated by pinoresinol. Nuclear translocation of nuclear factor-kappaB (NF-kappaB) and phosphorylation of c-Jun, one of the components of activating protein 1 (AP-1), were inhibited by pinoresinol. Our results suggest that pinoresinol ameliorates CCl4)-induced acute liver injury, and this protection is likely due to anti-oxidative activity and down-regulation of inflammatory mediators through inhibition of NF-kappaB and AP-1.

Palmatine attenuates D-galactosamine/lipopolysaccharide-induced fulminant hepatic failure in mice.

Palmatine is an isoquinoline alkaloid from Coptis chinensis, an herbal medicine used to treat various inflammatory diseases such as gastritis, edema and dermatitis. The present study examined the cytoprotective properties of palmatine on d(+)-galactosamine (GalN)/lipopolysaccharide (LPS)-induced fulminant hepatic failure. Mice were intraperitoneally given GalN (700 mg/kg)/LPS (10 microg/kg). Palmatine (25, 50, 100, and 200mg/kg) was administered 1h before GalN/LPS. GalN/LPS increased the mortality and serum aminotransferase activities. These increases were attenuated by palmatine. GalN/LPS increased hepatic lipid peroxidation and decreased the contents of reduced glutathione. Palmatine did not affect the lipid peroxidation and glutathione content. GalN/LPS increased the circulating levels of tumor necrosis factor (TNF)-alpha, interleukin-6 (IL-6) and IL-10. Palmatine prevented the increase of serum TNF-alpha and augmented that of serum IL-10. GalN/LPS treatment also increased the levels of TNF-alpha, IL-6 and IL-10 mRNA expression in liver tissue. Palmatine decreased the TNF-alpha mRNA expression and increased the IL-10 mRNA expression. Palmatine attenuated the apoptosis of hepatocytes, as evidenced by the TUNEL method and capase-3 analysis. Our data suggest that palmatine alleviates GalN/LPS-induced liver injury by modulating the cytokine response and inhibiting apoptosis.

Identification of new microsatellite markers in Panax ginseng.

Microsatellites, also called simple sequence repeats (SSR), are very useful molecular genetic markers commonly used in crop breeding, species identification and linkage analysis. In the present study, we constructed a microsatellite-enriched genomic library of Panax ginseng, and identified 251 novel microsatellite sequences. Tri-nt repeat units were the most abundant (46.6%), followed by di-nt repeats (35.5%). The (AG)n motif was most common (23.1%), followed by the (AAC)n motif (22.3%). From the genotyping of 94 microsatellites using marker-specific primer sets, we identified 11 intraspecific polymorphic markers as well as 14 possible interspecific polymorphic markers differing between P. ginseng and P. quinquefolius. The exact allele structures of the polymorphic markers were determined and the alleles were named. This study represents the first report of the bulk isolation of microsatellites by screening a microsatellite-enriched genomic library in P. ginseng. The microsatellite markers could be useful for linkage analysis, genetic breeding and authentication of Panax species.

Effect of intratesticular injection of xylazine/ketamine combination on canine castration.

This study was performed to compare the effect of intratesticular (IT) injection of xylazine/ketamine combination for canine castration with those of intramuscular (IM) or intravenous (IV) injection. Xylazine and ketamine was administered simultaneously via intratesticularly (IT group), intramuscularly (IM group) or intravenously (IV group) at doses of 2 and 10 mg/kg, respectively. Pain response at the time of injection, mean induction time, mean arousal time, mean walking time and cardiopulmonary function during anesthesia were monitored after the xylazine and ketamine administration. In IV and IM groups, heart rates were significantly decreased 30 and 45 min after xylazine and ketamine administration, respectively (p < 0.05). Respiratory rates were significantly decreased in the IV group (p < 0.05). In the IT group, there was no significant changes in heart and respiratory rates. The occurrence of cardiac arrhythmias was less severe in IT group compared with those in IM and IV groups. The route of administration did not affect rectal temperature. Mean induction time was significantly (p < 0.05) longer in IT group than in IM and IV groups. On the contrary, mean arousal time and mean walking time were shortened in IT group. Clinical signs related to pain response at the time of injection and vomiting were less observed in IT group than in IM group, and head shaking was less shown in IT group than in IM and IV groups during recovery period. These results indicated that intratesticular injection of xylazine/ketamine for castration has several advantages such as less inhibition of cardiopulmonary function and fast recovery from anesthesia without severe complications, and would be an effective anesthetic method for castration in small animal practice.