Medicarpin isolated from Radix Hedysari ameliorates brain injury in a murine model of cerebral ischemia.

The development of effective post-stroke therapy is highly demanded. Medicarpin is a key active component of a famous Chinese herbal prescription used for post-stroke treatment in Taiwan; however, little is known about its biological effects and mechanisms of action. Herein, we implemented a murine model of cerebral ischemic/reperfusional injury-related stroke to elucidate medicarpin's neuroprotective effect. In male ICR mice 24 h after stroke induction, treatment with medicarpin (0.5 and 1.0 mg/kg, i.v.) markedly enhanced the survival rates, improved moving distance and walking area coverage, reduced brain infarction, and preserved the blood-brain barrier, supporting medicarpin's protective effect on stroke-induced injury. Immunohistochemistry analysis further revealed that medicarpin treatment decreased the expression/activation of p65NF-κB and caspase 3, especially near the infarct cortex, while promoting the expression of neurogenesis-associated proteins, including doublecortin (DCX), brain-derived neurotrophic factor (BDNF), and tyrosine receptor kinase B (TrkB). These changes of expression levels were accompanied by GSK-3 inactivation and ß-catenin upregulation. Notably, pretreatment with LY294002, a PI3K inhibitor, abolished the aforementioned beneficial effects of medicarpin, illustrating an essential role of PI3K/Akt activation in medicarpin's neuroprotective and reparative activities. In vitro studies revealed that medicarpin displayed strong anti-inflammatory activity by reducing nitric oxide (NO) production in lipopolysaccharide-stimulated microglial cells (BV2) with an IC50 around 5 ±1 (µM) and anti-apoptotic activity in neuronal cells (N2A) subjected to oxygen-glucose deprivation with an IC50 around 13 ± 2 (µM). Collectively, this is the first report to demonstrate that medicarpin, isolated from Radix Hedysari, ameliorates ischemic brain injury through its anti-inflammatory microglia/NO), anti-apoptotic (neuronal cells/OGD) and neuroprotective effects by activating the PI3K/Akt-dependent GSK-3 inactivation for upregulating ß-catenin, which in turn decreases the expression/activation of p65NF-κB and caspase 3 and promotes the expression of neurogenic (DCX, BDNF, TrkB) and neuroprotective (Bcl2) factors in the brain.

Suppression of Inflammatory and Fibrotic Signals by Cinnamon (Cinnamomum cassia) and Cinnamaldehyde in Cyclophosphamide-Induced Overactive Bladder in Mice.

Cinnamon (Cinnamomum cassia) is a well-known traditional Chinese medicine used to treat nocturia by tonifying and warming the kidney. Our recent clinical study found that overactive bladder (OAB) patients treated with cinnamon powder (CNP) patches exhibited significantly ameliorated OAB symptoms without significant side effects, but the mechanism of action is unclear. To explore the beneficial effects and action mechanisms of CNP and its major active component cinnamaldehyde (CNA) in an OAB-related murine model, cyclophosphamide- (CYP-) induced OAB injury was performed on male ICR mice in the presence or absence of CNP and CNA, as well as solifenacin, a clinical drug for OAB as a reference. Twenty-four-hour micturition patterns (frequency of urination and volume of urine per time), as well as histopathological examination, immunohistochemistry (IHC), and Western blotting of the bladder, were analyzed for mechanism elucidation. Administration of CYP (300 mg/kg, i.p.) induced typical OAB pathophysiological changes, including increased frequency of urination and reduced volume of urine. CYP-induced mice displayed strong edema of the bladder and hemorrhagic cystitis, accompanied by loss of normal corrugated folds and decreased muscarinic receptors (M2/M3) in the urothelium, and disordered/broken structures of the lamina propria and detrusor. These changes were correlated with increased leukocyte (CD11b) infiltration colocalized with inflammatory (pp65 NFκB, macrophage migration inhibitory factor (MIF)/Toll-like receptor 4 (TLR4)) and fibrotic (stem cell factor (SCF)/c-Kit, α-smooth muscle actin (α-SMA)/ß-catenin) signals. Treatment with CNP (600 mg/kg, p.o.) and CNA (10-50 mg/kg, p.o.), but not solifenacin (50 mg/kg), 30 min after CYP induction significantly ameliorated CYP-induced dysfunction in micturition patterns and pathophysiological changes. CNP and CNA further suppressed MIF/TLR4-associated inflammatory and SCF/c-Kit-related fibrotic signaling pathways. Our findings indicate that suppression of inflammatory and fibrotic signals contributes to the crucial mechanism in the improvement of CYP-induced OAB by CNP and CNA.

Osthole ameliorates cartilage degradation by downregulation of NF-κB and HIF-2α pathways in an osteoarthritis murine model.

Osteoarthritis (OA) is a common and disabling joint disease mainly characterized by cartilage degradation, with the knees most commonly affected. No effective treatment for the cartilage degradation of OA exists. Preliminary studies have revealed the protective and osteogenic effects of osthole, a natural coumarin first isolated from Cnidium monnieri (Fructus Cnidii); however, no evidence of osthole in an OA-related model has been published to date. This study further explored the effects of osthole in a monoiodoacetate (MIA)-induced OA-related animal model and focused on the molecular mechanism(s) behind the anti-inflammatory and cartilage protective effects of osthole. This study revealed that the cartilage protective effect of osthole in a MIA-induced osteoarthritis (OA) murine model can be explained by downregulation of COX-2 and RUNX2 by inhibition of NF-κB and HIF-2α up-regulated by OA induction, resulting in downregulation of MMP-13, Syndecan IV and ADAMTS-5. In addition, osthole might have anti-inflammatory and analgesic effects due to COX-2 inhibition. Osthole can be considered as a potential component of the treatment of OA, for it possesses a cartilage protective effect, as well as anti-inflammation, analgesic, and movement improving effects. Further preclinical and human clinical studies are needed to examine the efficacy and safety profile of long-term therapy.

Ergostatrien-7,9(11),22-trien-3ß-ol from Antrodia camphorata ameliorates ischemic stroke brain injury via downregulation of p65NF-κ-B and caspase 3, and activation of Akt/GSK3/catenin-associated neurogenesis.

Antrodia camphorata is a well-known traditional Chinese mushroom used as a functional food and nutraceutical in Taiwan and China. The aim of this study was to explore the protective effects and mechanism(s) of the ethyl acetate crude extract of A. camphorata (EtOAc-AC) and its active constituent ergostatrien-7,9(11),22-trien-3ß-ol (EK100) in an acute ischemic stroke (AIS) murine model. Treating mice with induced AIS injury by using EtOAc-AC (0.3-0.6 g kg-1, p.o.) and EK100 (60 and 120 mg kg-1, p.o.) 2 h after AIS induction significantly increased the tracking distance and reduced brain infarction. Both EtOAc-AC and EK-100 reduced the expression levels of p65NF-κB and caspase 3 near the peri-infarct cortex and promoted the expression of neurogenesis-associated protein doublecortin (DCX) near the hippocampus, accompanied by glycogen synthase kinase 3 (GSK-3) inhibition and ß-catenin upregulation. Signaling pathway analysis revealed that the advantageous effects of EtOAc-AC and EK-100 involved triggering the activation of PI3K/Akt and inhibition of GSK-3. Our findings suggest that EtOAc-AC and its active constituent EK100 display anti-inflammatory and anti-apoptotic activities. Both EtOAc-AC and EK100 reduce ischemic brain injury by decreasing p65NF-κB and caspase 3 expression, and they promote neurogenesis (DCX) and neuroprotection (Bcl2) by activating the PI3k/Akt-associated GSK3 inhibition and ß-catenin activation.

GSK-3 inhibition through GLP-1R allosteric activation mediates the neurogenesis promoting effect of P7C3 after cerebral ischemic/reperfusional injury in mice.

An aminopropyl carbazole compound, P7C3, has been shown to be a potent neurogenesis promoting agent; however, its fundamental signaling action has yet to be elucidated. A cerebral ischemic/reperfusional (CI/R) injury model in mice was implemented to elucidate the neuronal protective mechanism(s) of P7C3. Treating CI/R mice using P7C3 (50-100 µg/kg, i.v.) significantly improved tracking distance and walking behavior, and reduced brain damage. Specifically, P7C3 promoted the expression of neurogenesis-associated proteins, including doublecortin, beta tubulin III (ß-tub3), adam11 and adamts20, near the peri-infarct cortex, accompanied by glycogen synthase kinase 3 (GSK-3) inhibition and ß-catenin upregulation. The application of a specific inhibitor against glucagon-like peptide 1 receptor (GLP-1R), exendin(9-39), revealed that the beneficial effects of P7C3 involved triggering the activation of GLP-1R-associated PKA/Akt signaling. P7C3 elicited the GLP-1R-dependent intracellular cAMP increment and the insulin secretion in cellular models. Surface plasmon resonance assay of P7C3 showed a Kd value of 0.53 µM for GLP-1R binding, and the docking of P7C3 to the putative active site on GLP-1R was successfully predicted by molecular modeling. Our findings indicate that P7C3 promotes the expression of neurogenesis proteins by activation of the cAMP/PKA-dependent and Akt/GSK3-associated ß-catenin through positive allosteric stimulation of GLP-1R. Within the P7C3 class of neuroprotective molecules, this mechanism appears to be unique to the prototypical P7C3 molecule, as other active derivatives such as P7C2-A20 and P7C3-S243 they do not engage this same pathway and have been shown to work by nicotinamide phosphoribosyltransferase (NAMPT) stimulation.

1,6-Bis[4-(4-amino-3-hydroxyphenoxy)phenyl] diamantane potentiates in vitro and in vivo antitumor effects of irinotecan on human colorectal cancer cells.

1,6-Bis[4-(4-amino-3-hydroxyphenoxy)phenyl] diamantane (DPD), a diamantane derivative, was previously noted as an anticancer compound through anticancer drug screening with NCI-60 human tumor cells. Irinotecan (CPT-11), a semisynthetic derivative of camptothecin, is clinically active in the treatment of colorectal cancer, with no cross-resistance. The current study conducted a pharmacokinetic evaluation of DPD, an essential component of drug discovery. Subsequent pathway analysis of microarray gene expression data indicated that the anticancer mechanisms of DPD were associated with cell cycle progression and apoptosis. The combined effect of DPD and CPT-11 with regard to the mechanisms of apoptosis-related pathways in COLO 205 cells, and the antitumor effects in colon cancer xenograft mice, were investigated. The plasma concentration and pharmacokinetic parameters of DPD in male albino rats were analyzed following a single dose of DPD by injection. The protein expression of active caspase-3, procaspase-3 and poly ADP-ribose polymerase (PARP) in COLO 205 cells treated with DPD and CPT-11, alone or combined, was evaluated by western blotting. A trypan blue dye exclusion assay revealed that, whilst DPD alone demonstrated good antitumor effects, this effect was potentiated when combined with CPT-11. Combined treatment with DPD and CPT-11 upregulated the expression of cleaved PARP, procaspase-3, caspase-3 and active caspase-3 in COLO 205 cells. In the colon cancer xenograft model, compared with the control (vehicle-treated) mice, the sizes of the tumors were significantly lower in mice treated with DPD and CPT-11, alone or in combination. Thus, DPD may be a potential therapeutic agent for the treatment of colorectal cancer via upregulating apoptosis-related pathways.

Anticolorectal cancer effects and pharmacokinetic application of 2, 2-Bis [4-(4-amino-3-hydroxyphenoxy) phenyl] adamantane.

2, 2-Bis (4-(4-amino-3-hydroxyphenoxy) phenyl) adamantane (DPA) induced growth inhibition in human cancer cells using the national cancer institute (NCI) anticancer drug screen. In our previous study, we demonstrated that DPA exerted growth inhibitory activities in the three human colon cancer cell lines (Colo 205, HT-29, and HCT-15). To identify the detailed mechanism, we examined the functional importance of p21 and p53 in DPA-induced anticancer effect. We used three isogenic colon cancer cell lines, HCT-116, HCT-116 p53(-/-), and HCT-116 p21(-/-), to evaluate the roles of p21 and p53 in the in vitro anticancer effects of DPA. DPA dose-dependently inhibited cell growth, cell migration and increased cell cycle at the G0/G1 phase in HCT116 cells but not in p21(-/-) and p53(-/-) isogenic HCT-116 cells. Additionally, Western blot showed that DPA treatment induced the p21, p53, and cyclin-E protein expressions in HCT-116 cells. The p21 associated cell cycle regulatory protein such as cyclin D, CDK4, and pRb were decreased after DPA treatment in HCT-116 cells. DPA decreased cell migration in HCT-116 and HCT-116 p53(-/-) but not in HCT-116 p21(-/-) cells. We observed the up-regulation of E-cadherin, p-p38, and p-Erk in DPA-treated HCT-116 group but not in HCT-116 p21(-/-) and HCT-116 p53(-/-) groups. We assumed that p21 was required for DPA-induced anti-colon cancer effect through the Erk and p38 pathway leading to cell cycle arrest and inhibition of cell motility. Mean (± SE) pharmacokinetic parameters of the DPA were as follows: AUC = 64.44 ± 8.41, Cmax = 1.56 ± 0.48 and t1/2 = 113.92 ± 58.19. The pharmacokinetic data suggest DPA can be applied to further clinical study. This is the first pharmacokinetic study of DPA, and indicated that anti-proliferation and the cell mobility inhibition effects of DPA in HCT116 WT cells may result from the induction of p21 through activation of ERK and p38 pathway.

Common and unique mechanisms of Chinese herbal remedies on ischemic stroke mice revealed by transcriptome analyses.

ETHNOPHARMACOLOGICAL RELEVANCE: Four traditional Chinese herbal remedies (CHR) including Buyang Huanwu decoction (BHD), Xuefu Zhuyu decoction (XZD), Tianma Gouteng decoction (TGD) and Shengyu decoction (SYD) are popular used in treating brain-related dysfunction clinically with different syndrome/pattern based on traditional Chinese medicine (TCM) principles, yet their neuroprotective mechanisms are still unclear. MATERIALS AND METHODS: Mice were subjected to an acute ischemic stroke to examine the efficacy and molecular mechanisms of action underlying these CHR. RESULTS: CHR treatment significantly enhanced the survival rate of stroke mice, with BHD being the most effective CHR. All CHR were superior to recombinant tissue-type plasminogen activator (rt-PA) treatment in successfully ameliorating brain function, infarction, and neurological deficits in stroke mice that also paralleled to improvements in blood-brain barrier damage, inflammation, apoptosis, and neurogenesis. Transcriptome analyses reveals that a total of 774 ischemia-induced probe sets were significantly modulated by four CHR, including 52 commonly upregulated genes and 54 commonly downregulated ones. Among them, activation of neurogenesis-associated signaling pathways and down-regulating inflammation and apoptosis pathways are key common mechanisms in ischemic stroke protection by all CHR. Besides, levels of plasma CX3CL1 and S100a9 in patients could be used as biomarkers for therapeutic evaluation before functional recovery could be observed. CONCLUSION: Our results suggest that using CHR, a combinatory cocktail therapy, is a better way than rt-PA for treating cerebral ischemic-associated diseases through modulating a common as well as a specific group of genes/pathways that may partially explain the syndrome differentiation and treatment principle in TCM.

Preventive effect of baicalein on methamphetamine-induced amnesia in the passive avoidance test in mice.

BACKGROUND/AIMS: Methamphetamine abuse may produce cognitive impairment. Baicalein, a bioactive flavonoid, has antioxidative, anti-inflammatory and neuroprotective effects. This study examined the effects of baicalein pretreatment on memory performance in the passive avoidance test after either one dose or an acute binge of methamphetamine in Institute of Cancer Research (ICR) mice. METHODS: Methamphetamine was administered by intraperitoneal (i.p.) injection of either one dose (3 mg/kg) or an acute binge (3 mg/kg, 4 i.p. injections at 2-hour intervals). The effects of baicalein pretreatment (1 mg/kg, i.p.) on methamphetamine-induced changes of locomotor activity and memory performance were compared with those of eticlopride, a selective dopamine D2 receptor antagonist. The effects of baicalein on acute binge methamphetamine-induced oxidative stress (malondialdehyde- and nitrotyrosine-modified protein production) in the mouse hippocampus were also examined. RESULTS: One-dose methamphetamine treatment (i.p., 30 min before or immediately after the training trial) induced hyperlocomotion and amnesia in mice, which were blocked by eticlopride but not by baicalein pretreatment. The memory performance in mice was impaired 5 days after acute binge methamphetamine, which was significantly attenuated by baicalein but not by eticlopride pretreatment. Baicalein pretreatment also attenuated acute binge methamphetamine-induced oxidative stress in the mouse hippocampus. CONCLUSIONS: Baicalein exhibits antioxidative and neuroprotective effects in attenuating acute binge methamphetamine-induced memory deficits and oxidative hippocampal damage.

2-Methoxystypandrone ameliorates brain function through preserving BBB integrity and promoting neurogenesis in mice with acute ischemic stroke.

2-Methoxystypandrone (2-MS), a naphthoquinone, has been shown to display an immunomodulatory effect in a cellular model. To explore whether 2-MS could protect mice against cerebral ischemic/reperfusion (I/R)-induced brain injury, we evaluated 2-MS's protective effects on an acute ischemic stroke by inducing a middle cerebral artery occlusion/reperfusion (MCAO) injury in murine model. Treatment of mice that have undergone I/R injury with 2-MS (10-100 µg/kg, i.v.) at 2 h after MCAO enhanced survival rate and ameliorated neurological deficits, brain infarction, neural dysfunction and massive oxidative stress, due to an enormous production of free radicals and breakdown of blood-brain barrier (BBB) by I/R injury; this primarily occurred with extensive infiltration of CD11b-positive inflammatory cells and upexpression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 and p65 nuclear factor-kappa B (NF-κB). All of these pathological changes were diminished by 2-MS; 2-MS also intensively limited cortical infarction and promoted upexpression of neurodevelopmental genes near peri-infarct cortex and endogenous neurogenesis near subgranular zone of hippocampal dentate gyrus and the subventricular zone, most possibly by inactivation of GSK3ß which in turn upregulating ß-catenin, Bcl-2 adam11 and adamts20. We conclude that 2-MS blocks inflammatory responses by impairing NF-κB signaling to limit the inflammation and oxidative stress for preservation of BBB integrity; 2-MS also concomitantly promotes neurodevelopmental protein expression and endogenous neurogenesis through inactivation of GSK3ß to enhance ß-catenin signaling for upexpression of neuroprotective genes and proteins.

Unique mechanisms of sheng yu decoction ( shèng yù tang) on ischemic stroke mice revealed by an integrated neurofunctional and transcriptome analysis.

Sheng Yu Decoction ( Shèng Yù Tang; SYD) is a popular traditional Chinese medicine (TCM) remedy used in treating cardiovascular and brain-related dysfunction clinically; yet, its neuroprotective mechanisms are still unclear. Here, mice were subjected to an acute ischemic stroke to examine the efficacy and mechanisms of action of SYD by an integrated neurofunctional and transcriptome analysis. More than 80% of the mice died within 2 days after ischemic stroke with vehicle treatment. Treatments with SYD (1.0 g/kg, twice daily, orally or p.o.) and recombinant thrombolytic tissue plasminogen activator (rt-PA; 10 mg/kg, once daily, intravenously or i.v.) both significantly extended the lifespan as compared to that of the vehicle-treated stroke group. SYD successfully restored brain function, ameliorated cerebral infarction and oxidative stress, and significantly improved neurological deficits in mice with stroke. Molecular impact of SYD by a genome-wide transcriptome analysis using brains from stroke mice showed a total of 162 out of 2081 ischemia-induced probe sets were significantly influenced by SYD. Mining the functional modules and genetic networks of these 162 genes revealed a significant upregulation of neuroprotective genes in Wnt receptor signaling pathway (3 genes) and regulation of cell communication (7 genes) and downregulation of destructive genes in response to stress (13 genes) and in the induction of inflammation (5 genes), cytokine production (4 genes), angiogenesis (3 genes), vasculature (6 genes) and blood vessel (5 genes) development, wound healing (7 genes), defense response (7 genes), chemotaxis (4 genes), immune response (7 genes), antigen processing and presenting (3 genes), and leukocyte-mediated cytotoxicity (2 genes) by SYD. Our results suggest that SYD could protect mice against ischemic stroke primarily through significantly downregulating the damaging genes involved in stress, inflammation, angiogenesis, blood vessel formation, immune responses, and wound healing, as well as upregulating the genes mediating neurogenesis and cell communication, which make SYD beneficial for treating ischemic stroke.

Melatonin ameliorates neural function by promoting endogenous neurogenesis through the MT2 melatonin receptor in ischemic-stroke mice.

Melatonin has many protective effects against ischemic stroke, but the underlying neuroprotective mechanisms are not fully understood. Our aim was to explore the relationship between melatonin's neuroprotective effects and activation of the MT2 melatonin receptor in a murine ischemic-stroke model. Male ICR mice were subjected to a transient middle cerebral ischemic/reperfusional injury, and melatonin (5 and 10 mg/kg, ip) was administrated once daily starting 2 h after ischemia. More than 80% of the mice died within 5 days after stroke without treatment. Melatonin treatment significantly improved the survival rates and neural functioning with modestly prolonged life span of the stroke mice by preserving blood-brain barrier (BBB) integrity via a reduction in the enormous amount of stroke-induced free radical production and significant gp91(phox) cell infiltration. These protective effects of melatonin were reversed by pretreatment with MT2 melatonin receptor antagonists (4-phenyl-2-propionamidotetralin (4P-PDOT) and luzindole). Moreover, treatment with melatonin after stroke dramatically enhanced endogenous neurogenesis (doublecortin positive) and cell proliferation (ki67 positive) in the peri-infarct regions. Most ki67-positive cells were nestin-positive and NG2-positive neural stem/progenitor cells that coexpressed two neurodevelopmental proteins (adam11 and adamts20) and the MT2 melatonin receptor. RT-PCR revealed that the gene expression levels of doublecortin, ki67, adamts20, and adam11 are markedly reduced by stroke, but are restored by melatonin treatment; furthermore, pretreatment with 4P-PDOT and luzindole antagonized melatonin's restorative effect. Our results support the hypothesis that melatonin is able to protect mice against stroke by activating MT2 melatonin receptors, which reduces oxidative/inflammatory stress. This results in the preservation of BBB integrity and enhances endogenous neurogenesis by upregulating neurodevelopmental gene/protein expression.

Prodigiosin inhibits gp91(phox) and iNOS expression to protect mice against the oxidative/nitrosative brain injury induced by hypoxia-ischemia.

This study aimed to explore the mechanisms by which prodigiosin protects against hypoxia-induced oxidative/nitrosative brain injury induced by middle cerebral artery occlusion/reperfusion (MCAo/r) injury in mice. Hypoxia in vitro was modeled using oxygen-glucose deprivation (OGD) followed by reoxygenation of BV-2 microglial cells. Our results showed that treatment of mice that have undergone MCAo/r injury with prodigiosin (10 and 100µg/kg, i.v.) at 1h after hypoxia ameliorated MCAo/r-induced oxidative/nitrosative stress, brain infarction, and neurological deficits in the mice, and enhanced their survival rate. MCAo/r induced a remarkable production in the mouse brains of reactive oxygen species (ROS) and a significant increase in protein nitrosylation; this primarily resulted from enhanced expression of NADPH oxidase 2 (gp91(phox)), inducible nitric oxide synthase (iNOS), and the infiltration of CD11b leukocytes due to breakdown of blood-brain barrier (BBB) by activation of nuclear factor-kappa B (NF-κB). All these changes were significantly diminished by prodigiosin. In BV-2 cells, OGD induced ROS and nitric oxide production by up-regulating gp91(phox) and iNOS via activation of the NF-κB pathway, and these changes were suppressed by prodigiosin. In conclusion, our results indicate that prodigiosin reduces gp91(phox) and iNOS expression possibly by impairing NF-κB activation. This compromises the activation of microglial and/or inflammatory cells, which then, in turn, mediates prodigiosin's protective effect in the MCAo/r mice.

Deciphering the neuroprotective mechanisms of Bu-yang Huan-wu decoction by an integrative neurofunctional and genomic approach in ischemic stroke mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Bu-yang Huan-wu decoction (BHD) is a famous traditional Chinese medicine formula that has been used clinically in Asia to treat stroke-induced disability for centuries, but the underlying neuroprotective mechanisms are not fully understood. AIM OF THE STUDY: In this study, we aim to investigate the mechanisms of action using an integrative neurofunctional and broad genomics approach. MATERIALS AND METHODS: Male ICR mice were subjected to an acute ischemic stroke by inducing a middle cerebral ischemic/reperfusion (CI/R) injury. To examine whether BHD could extend the lifespan of mice with a stroke, we used oral administration of BHD (0.5 and 1.0g/kg) twice daily starting from 2h after ischemia and compared this with vehicle control treatments, recombinant tissue-type plasminogen activator (rt-PA, 10mg/kg, i.v.), and MK-801 (0.2mg/kg, i.p.). An integrative neurofunctional and genomic approach was performed to elucidate the underlying molecular mechanisms of BHD. RESULTS: More than 80% of the mice died within 2 days after stroke induction in the vehicle control treatment group. However, the survival rates and life-spans of mice treated with BHD, rt-PA and MK-801 were significantly enhanced as compared to the vehicle-treated CI/R group in all three cases. Mice treated with BHD (1.0g/kg) showed the greatest protective effect across all groups. BHD successfully restored brain function, ameliorated the cerebral infarction, and significantly improved the neurological deficits of the mice with a stroke. BHD also reduced inflammation, oxidative stress, and apoptosis, as well as improved neurogenesis. The molecular impacts of BHD were assessed by genome-wide transcriptome analysis using brains from the CI/R mice. The results showed a total of 377 ischemia-induced probe-sets that were significantly influenced by BHD including 93 probe-sets that were commonly more abundant in BHD-treated and sham mice, and another 284 ischemia-induced probe sets that were suppressed by BHD. Mining the functional modules and genetic networks of these 377 genes revealed a significant upregulation of neuroprotective genes associated with neurogenesis (6 genes) and nervous system development (9 genes), and a significant down-regulation of destructive genes associated with the induction of inflammation (14 genes), apoptosis (15 genes), angiogenesis (11 genes) and blood coagulation (7 genes) by BHD. CONCLUSIONS: Our results suggested that BHD is able to protect mice against stroke and extend lifespan primarily through a significant down-regulation of genes involved in inflammation, apoptosis, angiogenesis and blood coagulation, as well as an up-regulation of genes mediating neurogenesis and nervous system development. The changes in expression after treatment with BHD are beneficial after ischemic stroke.

Andrographolide inhibits PI3K/AKT-dependent NOX2 and iNOS expression protecting mice against hypoxia/ischemia-induced oxidative brain injury.

This study aimed to explore the mechanisms by which andrographolide protects against hypoxia-induced oxidative/nitrosative brain injury provoked by cerebral ischemic/reperfusion (CI/R) injury in mice. Hypoxia IN VITRO was modeled using oxygen-glucose deprivation (OGD) followed by reoxygenation of BV-2 microglial cells. Our results showed that treatment of mice that have undergone CI/R injury with andrographolide (10-100 µg/kg, i. v.) at 1 h after hypoxia ameliorated CI/R-induced oxidative/nitrosative stress, brain infarction, and neurological deficits in the mice, and enhanced their survival rate. CI/R induced a remarkable production in the mouse brains of reactive oxygen species (ROS) and a significant increase in protein nitrosylation; this primarily resulted from enhanced expression of NADPH oxidase 2 (NOX2), inducible nitric oxide synthase (iNOS), and the infiltration of CD11b cells due to activation of nuclear factor-kappa B (NF- κB) and hypoxia-inducible factor 1-alpha (HIF-1 α). All these changes were significantly diminished by andrographolide. In BV-2 cells, OGD induced ROS and nitric oxide production by upregulating NOX2 and iNOS via the phosphatidylinositol-3-kinase (PI3K)/AKT-dependent NF- κB and HIF-1 α pathways, and these changes were suppressed by andrographolide and LY294002. Our results indicate that andrographolide reduces NOX2 and iNOS expression possibly by impairing PI3K/AKT-dependent NF- κB and HIF-1 α activation. This compromises microglial activation, which then, in turn, mediates andrographolide's protective effect in the CI/R mice.

Camphoratins A-J, potent cytotoxic and anti-inflammatory triterpenoids from the fruiting body of Taiwanofungus camphoratus.

Ten new triterpenoids, camphoratins A-J (1-10), along with 12 known compounds were isolated from the fruiting body of Taiwanofungus camphoratus. Their structures were established by spectroscopic analysis and chemical methods. Compound 10 is the first example of a naturally occurring ergosteroid with an unusual cis-C/D ring junction. Compounds 2-6 and 11 showed moderate to potent cytotoxicity, with EC(50) values ranging from 0.3 to 3 µM against KB and KB-VIN human cancer cell lines. Compounds 6, 10, 11, 14-16, 18, and 21 exhibited anti-inflammatory NO-production inhibition activity with IC(50) values of less than 5 µM, and were more potent than the nonspecific NOS inhibitor N(ω)-nitro-L-arginine methyl ester.

Preventive effect of silymarin in cerebral ischemia-reperfusion-induced brain injury in rats possibly through impairing NF-κB and STAT-1 activation.

Silymarin and silibinin are bioactive components isolated from Silybum marianum. They have been reported to exhibit anti-oxidative and anti-inflammatory effects. Many studies revealed that drugs with potent anti-inflammatory potential can protect animals against inflammation-associated neurodegenerative disease, e.g., stroke. In this current work we established an animal model of acute ischemic stroke injury by inducing cerebral ischemic/reperfusion (CI/R) in rats to elucidate whether silymarin or silibinin can protect animals from CI/R injury. Pretreatment with silymarin, but not silibinin, dose-dependently (1-10µg/kg, i.v.) reduced CI/R-induced brain infarction by 16-40% and improved neurological deficits in rats with a stroke. Elevated pathophysiological biomarkers for CI/R-induced brain injury, including lipid peroxidation, protein nitrosylation, and oxidative stress, were all reduced by silymarin. In addition, expression of inflammation-associated proteins (e.g., inducible nitric oxide synthase, cyclooxygenase-2 and myeloperoxidase), and transcriptional factors (e.g., nuclear factor (NF)-kappa B and signal transducer and activator of transcription (STAT)-1), as well as production of proinflammatory cytokine (e.g., interleukin-1ß and tumor necrosis factor-α) was all significantly prevented by silymarin. Furthermore, an in vitro study on microglial BV2 cells showed that silymarin could inhibit nitric oxide and superoxide anion production, possibly by interfering with NF-κB nuclear translocation/activation. Likewise, silymarin pretreatment also inhibited IκB-α degradation and NF-κB nuclear translocation in brain tissues of ischemic rats. Our results reveal that silymarin, but not its active component silibinin, protected rats against CI/R-induced stroke injury by amelioration of the oxidative and nitrosative stresses and inflammation-mediated tissue injury through impeding the activation of proinflammatory transcription factors (e.g., NF-κB and STAT-1) in the upregulation of proinflammatory proteins and cytokines in stroke-damaged sites. In conclusion, silymarin displays beneficial effects of preventing inflammation-related neurodegenerative disease, e.g., stroke, which needs further investigation and clinical evidences.

Honokiol protects rats against eccentric exercise-induced skeletal muscle damage by inhibiting NF-kappaB induced oxidative stress and inflammation.

Honokiol, a bioactive component isolated from the Chinese herb Magnolia officinalis, is known for its potent antioxidative and anti-inflammatory effects. To study whether honokiol can protect skeletal muscle from sports injuries, we set up an eccentric exercise bout protocol for rats consisting of downhill running on a treadmill and examined the effect of oral administration of honokiol at 1 h before eccentric exercise at a dose of 5 mg/kg on day 1 (HK5 x 1) or 1 mg/kg/day for 5 consecutive days (HK1 x 5). Eccentric exercise was implemented for 3-5 consecutive days, and induced remarkable tissue damage. This damage was associated with an increase in serum creatine levels, increase in protein nitrotyrosylation, poly-ADP-ribose-polymerase (PARP) upregulation, lipid peroxidation, and leukocyte infiltration. The degree of muscle damage also paralleled dramatic gene expression for cyclooxygenase-2 (COX-2), inducible nitric oxide synthase (iNOS), and inflammation-associated cytokines (interleukin (IL)-1beta, IL-6, tumor necrosis factor-alpha, and monocyte chemoattractant protein-1), possibly through activation of nuclear factor kappa-B (NF-kappaB), a crucial proinflammatory transcription factor. Both honokiol treatments (HK5 x 1 and HK1 x 5) significantly ameliorated eccentric exercise-induced muscle damage as revealed by suppression of cell fragmentation, protein nitrotyrosylation and PARP upregulation, as well as reductions in lipid peroxidation and leukocyte infiltration, possibly through downregulating gene expression for COX-2, iNOS, and the proinflammatory cytokines by modulation of NF-kappaB activation. In conclusion, the present study demonstrates for the first time that honokiol exhibits protective effects against eccentric exercise-induced skeletal muscle damage in rats, probably by modulating inflammation-mediated damage to muscle cells.

Dimemorfan protects rats against ischemic stroke through activation of sigma-1 receptor-mediated mechanisms by decreasing glutamate accumulation.

Dimemorfan, an antitussive and a sigma-1 (sigma(1)) receptor agonist, has been reported to display neuroprotective properties. We set up an animal model of ischemic stroke injury by inducing cerebral ischemia (for 1 h) followed by reperfusion (for 24 h) (CI/R) in rats to examine the protective effects and action mechanisms of dimemorfan against stroke-induced damage. Treatment with dimemorfan (1.0 microg/kg and 10 microg/kg, i.v.) either 15 min before ischemia or at the time of reperfusion, like the putative sigma(1) receptor agonist, PRE084 (10 microg/kg, i.v.), ameliorated the size of the infarct zone by 67-72% or 51-52%, respectively, which was reversed by pre-treatment with the selective sigma(1) receptor antagonist, BD1047 (20 microg/kg, i.v.). Major pathological mechanisms leading to CI/R injury including excitotoxicity, oxidative/nitrosative stress, inflammation, and apoptosis are all downstream events initiated by excessive accumulation of extracellular glutamate. Dimemorfan treatment (10 microg/kg, i.v., at the time of reperfusion) inhibited the expressions of monocyte chemoattractant protein-1 and interleukin-1beta, which occurred in parallel with decreases in neutrophil infiltration, activation of inflammation-related signals (p38 mitogen-activated protein kinase, nuclear factor-kappaB, and signal transducer and activator of transcription-1), expression of neuronal and inducible nitric oxide synthase, oxidative/nitrosative tissue damage (lipid peroxidation, protein nitrosylation, and 8-hydroxy-guanine formation), and apoptosis in the ipsilateral cortex after CI/R injury. Dimemorfan treatment at the time of reperfusion, although did not prevent an early rise of glutamate level, significantly prevented subsequent glutamate accumulation after reperfusion. This inhibitory effect was lasted for more than 4 h and was reversed by pre-treatment with BD1047. These results suggest that dimemorfan activates the sigma(1) receptor to reduce glutamate accumulation and then suppresses initiation of inflammation-related events and signals as well as induction of oxidative and nitrosative stresses, leading to reductions in tissue damage and cell death. In conclusion, our results demonstrate for the first time that dimemorfan exhibits protective effects against ischemic stroke in CI/R rats probably through modulation of sigma(1) receptor-dependent signals to prevent subsequent glutamate accumulation and its downstream pathologic events.

Anti-inflammatory ergostanes from the basidiomata of Antrodia salmonea.

Three new anti-oxidative ergostanes, methyl antcinate L (1), antcin M (2), and methyl antcinate K (3), together with nine additional known compounds, 3-ketodehydrosulphurenic acid, sulphurenic acid, dehydrosulphurenic acid, 3beta,15alpha-dihydroxylanosta-7,9(11),24-trien-21-oic acid, zhankuic acid A, zhankuic acid B, zhankuic acid C, antcin C, and antcin K were isolated from the basidiomata of Antrodia salmonea, a newly identified species of Antrodia (Polyporaceae) in Taiwan. These three new compounds were identified as methyl 3alpha,7alpha,12alpha-trihydroxy-4alpha-methylergosta-8,24(29)-dien-11-on-26-oate (1), 3alpha,12alpha-dihydroxy-4alpha-methylergosta-8,24(29)-dien-11-on-26-oic acid (2), and methyl 3alpha,4beta,7beta-trihydroxy-4alpha-methylergosta-8,24(29)-dien-11-on-26-oate (3) by spectroscopic analysis. We studied their antioxidative potential on the production of reactive oxygen species and nitric oxide (NO) in neutrophils and microglial cells, respectively. Compounds 1-3 displayed potent antioxidative activity with IC50 values of around 2.0-8.8 microM that was partially due to inhibition (6-67%) of NADPH oxidase activity but not through direct radical-scavenging properties. Compounds 1-3 also inhibited NO production with IC50 values of around 1.7-16.5 microM and were more potent than a non-specific NOS inhibitor. We conclude that these three new compounds 1, 2, and 3 exhibit anti-inflammatory activities in activated inflammatory cells.