GW4064 attenuates lipopolysaccharide­induced hepatic inflammation and apoptosis through inhibition of the Toll­like receptor 4­mediated p38 mitogen­activated protein kinase signaling pathway in mice.

Liver injury is associated with devastating consequences caused by inflammation and apoptosis. The farnesoid X receptor (FXR) is a nuclear receptor that has an essential role in hepatoprotection by maintaining the homeostasis of liver metabolism. The present study investigated the capacity of the FXR agonist GW4064 to protect the livers of mice from lipopolysaccharide (LPS)­induced inflammation and apoptosis. Male C57BL/6J [wild­type (WT)] and FXR knockout (KO) mice were intraperitoneally injected with LPS or saline. LPS­treated mice were intraperitoneally injected with vehicle or GW4064 (20 mg/kg) twice and then sacrificed. Activation of FXR by GW4064 alleviated hepatic inflammation in the LPS­induced murine liver injury model as reflected by reduced serum levels of aspartate aminotransferase and pro­inflammatory cytokine mRNA expression, including tumor necrosis factor­α, as well as interleukin­6 and ­1ß in WT mice. In addition, Toll­like receptor 4 (TLR4), p38 mitogen­activated protein kinase (MAPK), B­cell lymphoma­2­associated X protein and cytochrome c protein levels were decreased in WT mice receiving LPS with simultaneous GW4064 administration compared with those receiving LPS alone, while this was not observed in FXR KO mice. These results indicated that in WT mice, administration of GW4064 ameliorated LPS­mediated liver injury by upregulation of FXR expression, which was in part mediated by the TLR4/p38 MAPK pathway.

Farnesoid X receptor agonist GW4064 ameliorates lipopolysaccharide-induced ileocolitis through TLR4/MyD88 pathway related mitochondrial dysfunction in mice.

Inflammatory bowel disease (IBD) is a complex and relapsing inflammatory condition of the gastro intestinal tract characterized by diarrhoea and abdominal pain. Farnesoid X receptor (FXR) plays an important role in enteroprotection and mucosal injury by regulating inflammatory responses and barrier function in the intestinal tract. Here we show the mechanisms of FXR agonist, GW4064, inhibits mucosal injury in ileum caused by lipopolysaccharides (LPS). Ileum injury was induced by intraperitoneal injection of LPS in Wild-type (WT) and FXR knockout (KO) mice. GW4064 alleviates LPS-mediated tight junction dysfunction as well as macrophage infiltration in WT mice, but not in FXR KO mice. Interesting, GW4064 suppresses NACHT, LRR and PYD domains-containing protein 3 (NALP3) inflammasome mediates tumor necrosis factor-alpha (TNF-α), interleukin (IL)-6 and IL-1ß, as well as mitochondrial respiratory complexes mRNA expression in WT and FXR KO mice treated with LPS. This results demonstrated that central roles of FXR in coordinating regulation of both inflammation and mitochondrial dysfunction. We propose that GW4064 is promising therapeutic agent for treatment of ileocolitis.

Dicentrine Analogue-Induced G2/M Arrest and Apoptosis through Inhibition of Topoisomerase II Activity in Human Cancer Cells.

Lindera megaphylla has been traditionally used as an antineoplastic and wound healing remedy. We previously demonstrated the antitumor effects of D-dicentrine, a natural aporphine alkaloid from the root of L. megaphylla. To generate analogues, series of phenanthrene alkaloids from D-dicentrine were synthesized by degradation with ethyl chloroformate in pyridine, base hydrolysis, and N-alkylation. In this study, we demonstrated that one of the synthesized D-dicentrine analogues (here after designated as analogue 1) exhibited more potent cytotoxic effects than D-dicentrine in colon adenocarcinoma, hepatoma, leukemia, and epidermoid carcinoma cells. We performed cell cycle and apoptotic analysis by flow cytometry, an apoptotic DNA detection ELISA assay, and topoisomerase II activity by the kinetoplast DNA concatenation assay for studying their cytotoxic mechanisms. We found that both D-dicentrine and analogue 1 induced apoptosis and G2/M arrest in HL-60 leukemia cells. The percentage of apoptotic cells induced by analogue 1 was 4.5-fold higher than that induced by D-dicentrine as evident from measuring the amount of histone-bound DNA fragments. Moreover, we found that analogue 1 was 28-fold more potent than D-dicentrine for inhibition of topoisomerase II activity by the kinetoplast DNA concatenation assay. Our findings indicate that D-dicentrine analogue 1 is very promising as a potential antitumor agent for future study.

P2X7 is involved in the anti-inflammation effects of levobupivacaine.

BACKGROUND: We sough to elucidate whether purinergic P2X7 receptor is actively involved in the effects of levobupivacaine on inhibiting microglia activation. MATERIALS AND METHODS: Microglia were treated with lipopolysaccharide (LPS, 50 ng/mL), LPS plus levobupivacaine (50 µM), or LPS plus levobupivacaine plus the P2X7 receptor agonist Bz-ATP (100 µM) and denoted as the LPS, LPS + Levo, and LPS + Levo + Bz-ATP group, respectively. Microglia activation was measured by assaying inflammatory molecules expression. Microglia activation was also measured by assaying neuronal cell viability using coculture of microglia and neurons, as activated microglia may cause neuron injury. We also measured the levels of P2X7 receptor activation in microglia using ethidium uptake assay. RESULTS: Our data confirmed the effects of levobupivacaine on inhibiting inflammatory molecules upregulation in activated microglia, as the concentrations of interleukin (IL)-1ß, tumor necrosis factor α, IL-6, and macrophage inflammatory protein 2, of the LPS + Levo group were significantly lower than those of the LPS group (all P < 0.05). Moreover, Bz-ATP significantly abrogated the inhibitory effects of levobupivacaine, as concentrations of IL-1ß, tumor necrosis factor α, IL-6, and macrophage inflammatory protein 2 of the LPS + Levo + Bz-ATP group were significantly higher than those of the LPS + Levo group (all P < 0.05). In contrast, neuronal cell viability of the LPS + Levo group was significantly higher than those of the LPS and LPS + Levo + Bz-ATP groups (P = 0.012 and 0.002). Moreover, levels of P2X7 receptor activation of the LPS and LPS + Levo + Bz-ATP groups were significantly higher than that of the LPS + Levo group (P = 0.003 and 0.006). CONCLUSIONS: P2X7 receptor is involved in the effects of levobupivacaine on inhibiting microglial activation.

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.

Dual effects of silymarin on nasopharyngeal carcinoma cells (NPC-TW01).

BACKGROUND: Silymarin is an active component from the seeds of Silybum marianum and is widely used as a hepatic protection agent. Apoptosis induced by silymarin has been mentioned in cervical cancer cells. However, silymarin shows dual effects on tumor cells: cytostatic action at a low dose and cytotoxic action at higher dose. Thus, in the present study, we focused on low-dosing of silymarin in nasopharyngeal carcinoma cells, NPC-TW01 (TW01). METHODS: Cell viability assay was used to screen the effect of silymarin in TW01 cells. Western blot analysis was used to identify the expressions of antioxidant enzymes and anti-/proapoptotic proteins. Fluorescent dyes are employed to detect the content of reactive oxygen species (ROS) and cell apoptosis. RESULTS: Treatment of TW01 cells with silymarin at a low dose (80 µmol/l) resulted in a significant increase of antioxidant enzymes. Silymarin increased the expressions of superoxide dismutase 1, catalase, and glutathione peroxidase. Consequently, the cell apoptosis was reduced markedly. An increase of Bcl-2 expression and a decrease of activated caspase-3 or apoptosis-inducing factor (AIF) were observed in TW01 cells at a low dose (80 µmol/l) treatment. CONCLUSION: Silymarin at a low dose can induce cytostatic effect on TW01 cells mainly through an increase of antioxidant-like action. Thus, silymarin should be applied carefully to patients with nasopharyngeal carcinoma.

Increase of phosphatase and tensin homolog by silymarin to inhibit human pharynx squamous cancer.

Silymarin is an active principle from the seeds of the milk thistle plant and is widely used as a hepatoprotective gent due to its antioxidant-like activity. In the present study, we evaluated the potential efficacy of silymarin against oral cancer and investigated its possible mechanism of action. Cell viability assay and western blotting analyses were used to identify silymarin-induced apoptotic cell death in human pharynx squamous cell carcinoma (FaDu) cells. The short interfering RNA (siRNA) is used to confirm the role of phosphatase and tensin homolog (PTEN) in silymarin-induced apoptosis. Treatment of FaDu cells with silymarin resulted in a significant decrease in cell viability (up to 70%). Silymarin inhibited the phosphorylation of Akt (over 10-fold) with an increase in expression of PTEN (five to sixfold). Consequently, the level of Bcl-2 expression was decreased five to sixfold and caspase 3 activated to induce apoptosis. Treatment with siRNA specific to PTEN gene diminished the action of silymarin. The results suggest that silymarin inhibits the Akt signaling pathway by increasing PTEN expression in FaDu cells and directly affects Bcl-2 family members. Also, we demonstrated the inhibitory activity of silymarin for oral cancer is related to cell survival. These mechanisms may in part explain the actions of silymarin and provide a rationale for the development of silymarin as an anticancer agent.

Suppressive effects of levobupivacaine on endotoxin-induced microglial activation.

BACKGROUND: We sought to elucidate the effects of levobupivacaine on modulating endotoxin-induced upregulation of inflammatory mediators and activation of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) signaling pathways in activated microglia. MATERIALS AND METHODS: Confluent murine microglia (BV-2) were treated with endotoxin (lipopolysaccharide, 50 ng/mL) or endotoxin plus levobupivacaine (5, 25, or 50 µM) and denoted as the LPS, LPS + L(5), LPS + L(25), and LPS + L(50) groups, respectively. Levobupivacaine was administered immediately after endotoxin. Control groups were run simultaneously. RESULTS: The concentrations of inflammatory mediators, including macrophage inflammatory protein-2 (P = 0.023 and 0.016), tumor necrosis factor-α (P = 0.025 and 0.020), interleukin (IL)-1ß (P = 0.018 and 0.014), IL-6 (P = 0.029 and 0.023), nitric oxide (P = 0.025 and 0.026), and prostaglandin E2 (P = 0.028 and 0.016) of the LPS + L(25) and LPS + L(50) groups were significantly lower than those of the LPS group. The concentrations of macrophage inflammatory protein-2 (P = 0.035), IL-1ß (P = 0.024), nitric oxide (P = 0.031), and prostaglandin E2 (P = 0.036) but not tumor necrosis factor-α and interleukin-6 of the LPS + L(5) group were also significantly lower than those of the LPS group. These data revealed that effects of endotoxin on upregulating inflammatory mediators were inhibited by levobupivacaine. Moreover, effects of endotoxin on activating NF-κB, including inhibitor-κB degradation, NF-κB nuclear translocation, and NF-κB-DNA binding, were also inhibited by levobupivacaine. Similarly, effects of endotoxin on activating MAPKs, including extracellular signal-regulated kinase, c-jun N-terminal kinase, and p38 MAPK, were also significantly inhibited by levobupivacaine. CONCLUSIONS: Levobupivacaine significantly inhibited endotoxin-induced upregulation of inflammatory mediators and activation of NF-κB and MAPKs signaling pathways in activated microglia.

Activation of Akt by advanced glycation end products (AGEs): involvement of IGF-1 receptor and caveolin-1.

Diabetes is characterized by chronic hyperglycemia, which in turn facilitates the formation of advanced glycation end products (AGEs). AGEs activate signaling proteins such as Src, Akt and ERK1/2. However, the mechanisms by which AGEs activate these kinases remain unclear. We examined the effect of AGEs on Akt activation in 3T3-L1 preadipocytes. Addition of AGEs to 3T3-L1 cells activated Akt in a dose- and time-dependent manner. The AGEs-stimulated Akt activation was blocked by a PI3-kinase inhibitor LY 294002, Src inhibitor PP2, an antioxidant NAC, superoxide scavenger Tiron, or nicotinamide adenine dinucleotide phosphate (NAD(P)H) oxidase inhibitor DPI, suggesting the involvement of Src and NAD(P)H oxidase in the activation of PI3-kinase-Akt pathway by AGEs. AGEs-stimulated Src tyrosine phosphorylation was inhibited by NAC, suggesting that Src is downstream of NAD(P)H oxidase. The AGEs-stimulated Akt activity was sensitive to Insulin-like growth factor 1 receptor (IGF-1R) kinase inhibitor AG1024. Furthermore, AGEs induced phosphorylation of IGF-1 receptorßsubunit (IGF-1Rß) on Tyr1135/1136, which was sensitive to PP2, indicating that AGEs stimulate Akt activity by transactivating IGF-1 receptor. In addition, the AGEs-stimulated Akt activation was attenuated by ß-methylcyclodextrin that abolishes the structure of caveolae, and by lowering caveolin-1 (Cav-1) levels with siRNAs. Furthermore, addition of AGEs enhanced the interaction of phospho-Cav-1 with IGF-1Rß and transfection of 3T3-L1 cells with Cav-1 Y14F mutants inhibited the activation of Akt by AGEs. These results suggest that AGEs activate NAD(P)H oxidase and Src which in turn phosphorylates IGF-1 receptor and Cav-1 leading to activation of IGF-1 receptor and the downstream Akt in 3T3-L1 cells. AGEs treatment promoted the differentiation of 3T3-L1 preadipocytes and addition of AG1024, LY 294002 or Akt inhibitor attenuated the promoting effect of AGEs on adipogenesis, suggesting that IGF-1 receptor, PI3-Kinase and Akt are involved in the facilitation of adipogenesis by AGEs.

Norcantharidin suppresses cell growth and migration with enhanced anticancer activity of gefitinib and cisplatin in human non-small cell lung cancer cells.

Norcantharidin is the demethylated analog of cantharidin isolated from blister beetles (Mylabris phalerata Pall.). In this study, we evaluated whether norcantharidin exhibits anticancer effects against the human non-small cell lung cancer cell lines A549 (epidermal growth factor receptor (EGFR) mutation-negative) and PC9 (EGFR mutation-positive). Our results revealed that norcantharidin dose-dependently retards cell growth, arrests cell cycle at G2/M phase, reduces cell migration, and even induces apoptosis at the concentration of 100 µM. Moreover, we found that norcantharidin enhances the anticancer effects of gefitinib and cisplatin. Norcantharidin exhibited similar potency of anticancer effects against the two cell lines with different EGFR mutation status and did not affect EGF-induced EGFR phosphorylation, suggesting that the EGFR signaling may not be the target of norcantharidin. In conclusion, our results suggest that norcantharidin exhibits anticancer effects against non-small cell lung cancer cells in vitro and support its potential as a chemotherapeutic agent for treating non-small cell lung cancer.

Mirtazapine inhibits tumor growth via immune response and serotonergic system.

To study the tumor inhibition effect of mirtazapine, a drug for patients with depression, CT26/luc colon carcinoma-bearing animal model was used. BALB/c mice were randomly divided into six groups: two groups without tumors, i.e. wild-type (no drug) and drug (mirtazapine), and four groups with tumors, i.e. never (no drug), always (pre-drug, i.e. drug treatment before tumor inoculation and throughout the experiment), concurrent (simultaneously tumor inoculation and drug treatment throughout the experiment), and after (post-drug, i.e. drug treatment after tumor inoculation and throughout the experiment). The "psychiatric" conditions of mice were observed from the immobility time with tail suspension and spontaneous motor activity post tumor inoculation. Significant increase of serum interleukin-12 (sIL-12) and the inhibition of tumor growth were found in mirtazapine-treated mice (always, concurrent, and after) as compared with that of never. In addition, interferon-γ level and immunocompetent infiltrating CD4+/CD8+ T cells in the tumors of mirtazapine-treated, tumor-bearing mice were significantly higher as compared with that of never. Tumor necrosis factor-α (TNF-α) expressions, on the contrary, are decreased in the mirtazapine-treated, tumor-bearing mice as compared with that of never. Ex vivo autoradiography with [(123)I]ADAM, a radiopharmaceutical for serotonin transporter, also confirms the similar results. Notably, better survival rates and intervals were also found in mirtazapine-treated mice. These findings, however, were not observed in the immunodeficient mice. Our results suggest that tumor growth inhibition by mirtazapine in CT26/luc colon carcinoma-bearing mice may be due to the alteration of the tumor microenvironment, which involves the activation of the immune response and the recovery of serotonin level.

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.

Ugonin K-stimulated osteogenesis involves estrogen receptor-dependent activation of non-classical Src signaling pathway and classical pathway.

We have reported previously that ugonin K, a flavonoid isolated from Helminthostachys zeylanica (L.) Hook, potently induces cell differentiation and mineralization of MC3T3-E1 mouse osteoblast-like cells. Here we aimed to elucidate whether ugonin K evoked osteogenesis required interaction with estrogen receptor. Results showed that ugonin K induced increases in alkaline phosphatase (ALP) activity, expressions of bone sialoprotein (BSP) and osteocalcin (OCN), and subsequent bone nodule formation were concentration-dependently inhibited by estrogen receptor antagonist ICI 182,780, suggesting that an estrogen receptor-dependent pathway was involved. In the presence of ICI 182,780, ugonin K induced up-regulation of the expressions of runt-related transcription factor 2 (Runx2) and osterix was also significantly repressed. Numerous studies have demonstrated that estrogens induced rapid and transient activation of the c-Src phosphorylation cascade. We found that ugonin K indeed raised the phosphorylated level of c-Src and such phosphorylation was significantly attenuated by ICI 182,780 treatment. Application of c-Src specific inhibitor PP2 concentration-dependently repressed ugonin K-induced osteogenesis. In the nuclear translocation assay, results showed that ugonin K increased the nuclear level of estrogen receptor-α protein, suggesting that an enhanced transcriptional activity might be observed. Excepting MC3T3-E1 cells, results obtained from ALP activity assay revealed that ugonin K also stimulated osteoblastic differentiation of human MG-63 osteosarcoma cells and rat primary osteoblasts isolated from femora. Our results demonstrate that ugonin K stimulated osteogenesis might act through an estrogen receptor-dependent activation of a non-classical signaling pathway mediated by phosphorylation of c-Src. Moreover, a transactivation potential toward estrogen receptor-α through a classical pathway might not be precluded.

Ugonin K promotes osteoblastic differentiation and mineralization by activation of p38 MAPK- and ERK-mediated expression of Runx2 and osterix.

Ugonin K is a flavonoid isolated from the roots of Helminthostachys zeylanica, a folk medicine used to strengthen bone mass and cure bone fracture. It is of interest to determine whether ugonin K has beneficial effect on osteoblast maturation. In this study, MC3T3-E1 osteoblasts were treated with ugonin K. Cell differentiation and mineralization were identified by alkaline phosphatase (ALP) activity and Alizarin red S staining, respectively. RT-PCR and Western blot were used to analyze osteoblast-associated gene expression and signaling pathways. Our results showed that ugonin K significantly induced the increase of ALP activity, expressions of bone sialoprotein (BSP) and osteocalcin (OCN), and mineralization. The mRNA expressions of the transcription factors Runx2 and osterix were also up-regulated by ugonin K. Ugonin K increased the phosphorylated level of p38 and ERK, respectively. In the presence of SB203580, ugonin K induced expressions of Runx2 and osterix, ALP activity, BSP level and bone nodule formation were all completely inhibited, but ugonin K induced OCN expression was not affected. On the other hand, ugonin K-induced ALP activity and mineralization were mildly attenuated by PD98059, but the over-expressed Runx2, osterix, BSP and OCN also were significantly repressed by PD98059. These suggested that both p38 and ERK participate in regulating ugonin K evoked osteogenesis but p38 seemed to play a more important role. Take together, the potential anabolic effect of ugonin K on bone might act through activations of p38- and ERK-mediated Runx2 and osterix expressions to induce the synthesis of osteoids and formation of bone nodule.

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.

Anti-inflammatory and anti-infectious effects of Evodia rutaecarpa (Wuzhuyu) and its major bioactive components.

This article reviews the anti-inflammatory relative and anti-infectious effects of Evodia rutaecarpa and its major bioactive components and the involvement of the nitric oxide synthases, cyclooxygenase, NADPH oxidase, nuclear factor kappa B, hypoxia-inducible factor 1 alpha, reactive oxygen species, prostaglandins, tumor necrosis factor, LIGHT, amyloid protein and orexigenic neuropeptides. Their potential applications for the treatment of endotoxaemia, obesity, diabetes, Alzheimer's disease and their uses as cardiovascular and gastrointestinal protective agents, analgesics, anti-oxidant, anti-atherosclerosis agents, dermatological agents and anti-infectious agents are highlighted. Stimulation of calcitonin gene-related peptide release may partially explain the analgesic, cardiovascular and gastrointestinal protective, anti-obese activities of Evodia rutaecarpa and its major bioactive components.

8-Prenylkaempferol accelerates osteoblast maturation through bone morphogenetic protein-2/p38 pathway to activate Runx2 transcription.

AIMS: In this study, we investigated the effect of 8-prenylkaempferol (8-PK), a prenyl-flavonoid isolated from Sophora flavescens, on osteoblast differentiation and maturation. MAIN METHODS: MC3T3-E1 cells were exposed to 8-PK and the cytotoxicity was assayed. Osteoblast differentiation and maturation were evaluated by analyzing alkaline phosphatase (ALP) activity and cell mineralization, respectively. RT-PCR and Western blot were executed to determine the effects of 8-PK on osteoblast differentiation-related gene expression and signaling pathway. KEY FINDINGS: 8-PK significantly promoted ALP activity, up-regulated mRNA expressions of osteocalcin, osteopontin, and type I collagen, and induced bone nodules formation. Induction of differentiation by 8-PK was associated with increased bone morphogenetic protein (BMP)-2 expression, and sequentially up-regulated the phosphorylations of Smad1/5/8 and p38, and increased the nuclear translocation of runt-related transcription factor 2 (Runx2). Addition of BMP-2 antagonist noggin blocked 8-PK and recombinant mouse BMP-2-induced ALP activity, reconfirming that BMP-2 production is required in 8-PK-mediated osteoblast differentiation. Noggin also abrogated 8-PK evoked phosphorylations of Smad1/5/8 and p38, suggesting that BMP-2 signaling is required for p38 activation in 8-PK-treated cells. Application of p38 inhibitor SB203580 repressed not only 8-PK-mediated activation of ALP, but also the nuclear translocation of Runx2 and bone nodules formation. SIGNIFICANCE: The present results suggested that BMP-2/p38/Runx2 pathways were involved in 8-PK-induced differentiation/maturation of MC3T3-E1 osteoblasts and firstly demonstrated that 8-PK might be a promising agent for inducing osteogenesis.

Characterized polysaccharides from black soybean induce granulocyte colony-stimulated factor gene expression in a phosphoinositide 3-kinase-dependent manner.

Black soybean (Glycine max L. merr.) is an edible Chinese medicine for nourishment spleen. In the present study, effects of characterized polysaccharides from black soybean (PGM) on granulocyte colony-stimulated factor (G-CSF) production in human peripheral blood mononuclear cells (PBMC) were determined and their action mechanisms were examined. The results indicated that PGM concentration-dependently enhanced G-CSF production in PBMC through modulation of mRNA expression. Data from Western blotting showed that PGM significantly induced the extracellular signal-regulated protein kinase (ERK) activation in PBMC. The nuclear factor (NF)-κB activation in PBMC was increased with PGM by modulation of IκB degradation and PKC θ activation. The levels of G-CSF mRNA in PGM-treated PBMC could be reduced by ERK inhibitor U0126 and NF-κB inhibitor pyrrolidine dithiocarbamate, respectively. Furthermore, the data showed that PGM stimulated phosphoinositide 3-kinase (PI3K)-regulated Akt phosphorylation. The PI3K inhibitor, Ly294002, blocked ERK, NF-κB, and PKC θ activation and G-CSF mRNA expression in PBMC induced by PGM. Thus, we first proved that the enhancement mechanisms of PGM on G-CSF production, appeared to be mediated, at least in part, through activation of PI3K, ERK, PKC θ, and NF-κB signaling pathways in PBMC. We suggest that PGM from black soybean is a potential G-CSF stimulator.

Psoralidin inhibits LPS-induced iNOS expression via repressing Syk-mediated activation of PI3K-IKK-IκB signaling pathways.

Psoralidin has been reported to inhibit lipopolysaccharide (LPS)-induced nitric oxide (NO) production, but the mechanisms of the action remain unclear. Thus, the impact of psoralidin on signaling pathways known to be implicated in NO synthesis was explored in LPS-activated RAW264.7 macrophages by using RT-PCR and Western blotting. Consistent with NO inhibition, psoralidin suppressed LPS-induced expression of inducible NO synthase (iNOS) by abolishing IκB kinase (IKK) phosphorylation, IκB degradation and nuclear factor κB (NF-κB) nuclear translocation without effecting mitogen-activated protein kinases (MAPKs) phosphorylation. Exposure to wortmannin abrogated IKK/IκB/NF-κB-mediated iNOS expression, suggesting activation of such a signal pathway might also be phosphoinositide-3-kinase (PI3K) dependent. By using Src inhibitor PP2, Janus kinase 2 (JAK-2) inhibitor AG490, Bruton's tyrosine kinase (Btk) inhibitor LFM-A13 and spleen tyrosine kinase (Syk) inhibitor piceatannol, the results showed that piceatannol clearly repressed NO production more potently than the other inhibitors. Furthermore, piceatannol significantly repressed LPS-induced PI3K/Akt phosphorylation and the downstream IKK/IκB activation, suggesting that Syk is an upstream key regulator in the activation of PI3K/Akt-mediated signaling. In fact, transfection with siRNA targeting Syk obviously reduced iNOS expression. Interestingly, LPS-induced phosphorylations of Syk and PI3K-p85 were both significantly blunted by psoralidin treatment. The present results show that interfering with Syk-mediated PI3K phosphorylation might contribute to the NO inhibitory effect of psoralidin via blocking IKK/IκB signaling propagation in LPS-stimulated RAW 264.7 macrophages.

Silymarin protects spinal cord and cortical cells against oxidative stress and lipopolysaccharide stimulation.

Contusive spinal cord injury (SCI) is a devastating event which leads to a loss of neurological function below the level of injury. A secondary degenerative process is initiated following acute SCI. This secondary cascade provides opportunities for the delivery of therapeutic interventions. Silymarin, a widely used "liver herb", is frequently used for the protection against various hepatobiliary problems. However, the effectiveness of silymarin in central nervous system (CNS), especially in spinal cord, is not firmly established. The present work evaluates the effects of silymarin and its major constituent, silybin, on oxidative stress and lipopolysaccharide (LPS) stimulation in primary neuronal/glial cell cultures and in vivo. Silymarin or silybin inhibited glial cell proliferation in a concentration-dependent manner. Furthermore, it protected glial cells against peroxide-induced reactive oxygen species (ROS) formation, ATP depletion, and cell damage. Interestingly, the inhibition of peroxide-induced ROS by silybin could be partially attenuated by inhibitors of NFκB or protein kinase C (PKC), suggesting an involvement of NFκB and PKC signaling pathways. In mixed neuronal/glial cell cultures from cerebral cortex or spinal cord, silymarin or silybin effectively attenuated peroxide-induced ROS formation, with silymarin being more effective than silybin, implicating other constituents of silymarin that may be involved. Consistently, silymarin reduced LPS-induced injures in spinal neuronal/glial cell cultures. In vivo, intrathecal administration of silymarin immediately after eliciting contusive SCI effectively improved hindlimb locomotor behavior in the rats. Taken together, silymarin or silybin shows promise in protecting the CNS cells from toxin- or injury-induced damages and might be used to treat head- or spinal cord-injuries related to free radical assault.