Hyperoside exerts anti-inflammatory and anti-arthritic effects in LPS-stimulated human fibroblast-like synoviocytes in vitro and in mice with collagen-induced arthritis.

AIM: Hyperoside is a flavonol glycoside mainly found in plants of the genera Hypericum and Crataegus, which has shown anti-oxidant, anti-cancer and anti-inflammatory activities. In this study, we investigated the effects of hyperoside on human rheumatoid fibroblast-like synoviocytes (FLSs) in vitro and on mouse collagen-induced arthritis (CIA) in vivo. METHODS: FLSs were isolated from primary synovial tissues obtained from rheumatoid arthritis (RA) patients and exposed to LPS (1 µg/mL). Cell viability and proliferation were measured with MTT and BrdU assay. Cell migration was assessed using wound-healing assay and Transwell assay. DNA binding of NF-κB was measured using a TransAM-NFkappaB kit. The localization of p65 subunit was detected with immunocytochemistry. CIA was induced in mice by primary immunization with Bovine Type II collagen (CII) emulsified in CFA, followed by a booster injection 3 weeks later. The arthritic mice were treated with hyperoside (25, 50 mg·kg(-1)·d(-1), ip) for 3 weeks, and the joint tissues were harvested for histological analysis. RESULTS: Hyperoside (10, 50, 100 µmol/L) dose-dependently inhibited LPS-induced proliferation and migration of human RA FLSs in vitro. Furthermore, hyperoside decreased LPS-stimulated production of TNF-α, IL-6, IL-1 and MMP-9 in the cells. Moreover, hyperoside inhibited LPS-induced phosphorylation of p65 and IκBα, and suppressed LPS-induced nuclear translocation of p65 and DNA biding of NF-κB in the cells. Three-week administration of hyperoside significantly decreased the clinical scores, and alleviated synovial hyperplasia, inflammatory cell infiltration and cartilage damage in mice with CIA. CONCLUSION: Hyperoside inhibits LPS-induced proliferation, migration and inflammatory responses in human RA FLSs in vitro by suppressing activation of the NF-κB signaling pathway, which contributes to the therapeutic effects observed in mice with CIA.

Chronic treatment with anti-bipolar drugs suppresses glutamate release from astroglial cultures.

Astroglial cells are fundamental elements of most neurological diseases, including bipolar disorders in which astrocytes show morphological and functional deficiency. Here we report the suppression of astroglial glutamate release by chronic treatment with three anti-bipolar drugs, lithium salt (Li(+)), carbamazepine (CBZ) and valproic acid (VPA). Release of glutamate was triggered by transient exposure of astrocytes to ATP (which activated purinoceptors) and 45 mM K(+) (which depolarised cell membrane to ~-30 mV). In both types of stimulation glutamate release was regulated by Ca(2+) entry through plasmalemmal channels and by Ca(2+) release from the endoplasmic reticulum (ER) intracellular stores. Exposure of astroglial cultures to Li(+), CBZ and VPA for 2 weeks led to a significant (more than 2 times) inhibition of glutamate release, which may alleviate the hyperactivity of the glutamatergic transmission in the brain of patients with bipolar disorders and thus contribute the underlying mechanism of drug action.

Astrocytic glycogenolysis: mechanisms and functions.

Until the demonstration little more than 20 years ago that glycogenolysis occurs during normal whisker stimulation glycogenolysis was regarded as a relatively uninteresting emergency procedure. Since then, a series of important astrocytic functions has been shown to be critically dependent on glycogenolytic activity to support the signaling mechanisms necessary for these functions to operate. This applies to glutamate formation and uptake and to release of ATP as a transmitter, stimulated by other transmitters or elevated K(+) concentrations and affecting not only other astrocytes but also most other brain cells. It is also relevant for astrocytic K(+) uptake both during the period when the extracellular K(+) concentration is still elevated after neuronal excitation, and capable of stimulating glycogenolytic activity, and during the subsequent undershoot after intense neuronal activity, when glycogenolysis may be stimulated by noradrenaline. Both elevated K(+) concentrations and several transmitters, including the ß-adrenergic agonist isoproterenol and vasopressin increase free cytosolic Ca(2+) concentration in astrocytes, which stimulates phosphorylase kinase so that it activates the transformation of the inactive glycogen phosphorylase a to the active phosphorylase b. Contrary to common belief cyclic AMP plays at most a facilitatory role, and only when free cytosolic Ca(2+) concentration is also increased. Cyclic AMP is not increased during activation of glycogenolysis by either elevated K(+) concentrations or the stimulation of the serotonergic 5-HT(2B) receptor. Not all agents that stimulate glycogenolysis do so by directly activating phophorylase kinase--some do so by activating processes requiring glycogenolysis, e.g. for synthesis of glutamate.

Inhibition of brain swelling after ischemia-reperfusion by ß-adrenergic antagonists: correlation with increased K+ and decreased Ca2+ concentrations in extracellular fluid.

Infarct size and brain edema following ischemia/reperfusion are reduced by inhibitors of the Na+, K+, 2Cl-, and water cotransporter NKCC1 and by ß1-adrenoceptor antagonists. NKCC1 is a secondary active transporter, mainly localized in astrocytes, driven by transmembrane Na+/K+ gradients generated by the Na+,K+-ATPase. The astrocytic Na+,K+-ATPase is stimulated by small increases in extracellular K+ concentration and by the ß-adrenergic agonist isoproterenol. Larger K+ increases, as occurring during ischemia, also stimulate NKCC1, creating cell swelling. This study showed no edema after 3 hr medial cerebral artery occlusion but pronounced edema after 8 hr reperfusion. The edema was abolished by inhibitors of specifically ß1-adrenergic pathways, indicating failure of K+-mediated, but not ß1-adrenoceptor-mediated, stimulation of Na+,K+-ATPase/NKCC1 transport during reoxygenation. Ninety percent reduction of extracellular Ca2+ concentration occurs in ischemia. Ca2+ omission abolished K+ uptake in normoxic cultures of astrocytes after addition of 5 mM KCl. A large decrease in ouabain potency on K+ uptake in cultured astrocytes was also demonstrated in Ca2+-depleted media, and endogenous ouabains are needed for astrocytic K+ uptake. Thus, among the ionic changes induced by ischemia, the decrease in extracellular Ca2+ causes failure of the high-K+-stimulated Na+,K+-ATPase/NKCC1 ion/water uptake, making ß1-adrenergic activation the only stimulus and its inhibition effective against edema.

Mechanisms for L-channel-mediated increase in [Ca(2+)]i and its reduction by anti-bipolar drugs in cultured astrocytes combined with its mRNA expression in freshly isolated cells support the importance of astrocytic L-channels.

The importance of Ca(2+) signaling in astrocytes is undisputed but a potential role of Ca(2+) influx via L-channels in the brain in vivo is disputed, although expression of these channels in cultured astrocytes is recognized. This study shows that an increase in free cytosolic Ca(2+) concentration ([Ca(2+)]i) in astrocytes in primary cultures in response to an increased extracellular K(+) concentration (45mM) is inhibited not only by nifedipine (confirming previous observations) but also to a very large extent by ryanodine, inhibiting ryanodine receptor-mediated release of Ca(2+), known to occur in response to an elevation in [Ca(2+)]i. This means that the actual influx of Ca(2+) is modest, which may contribute to the difficulty in demonstrating L-channel-mediated Ca(2+) currents in astrocytes in intact brain tissue. Chronic treatment with any of the 3 conventional anti-bipolar drugs lithium, carbamazepine or valproic acid similarly causes a pronounced inhibition of K(+)-mediated increase in [Ca(2+)]i. This is shown to be due to an inhibition of capacitative Ca(2+) influx, reflected by decreased mRNA and protein expression of the 'transient receptor potential channel' (TRPC1), a constituent of store-operated channels (SOCEs). Literature data are cited (i) showing that depolarization-mediated Ca(2+) influx in response to an elevated extracellular K(+) concentration is important for generation of Ca(2+) oscillations and for the stimulatory effect of elevated K(+) concentrations in intact, non-cultured brain tissue, and (ii) that Ca(2+) channel activity is dependent upon availability of metabolic substrates, including glycogen. Finally, expression of mRNA for Cav1.3 is demonstrated in freshly separated astrocytes from normal brain.

Astrocytic and neuronal accumulation of elevated extracellular K(+) with a 2/3 K(+)/Na(+) flux ratio-consequences for energy metabolism, osmolarity and higher brain function.

Brain excitation increases neuronal Na(+) concentration by 2 major mechanisms: (i) Na(+) influx caused by glutamatergic synaptic activity; and (ii) action-potential-mediated depolarization by Na(+) influx followed by repolarizating K(+) efflux, increasing extracellular K(+) concentration. This review deals mainly with the latter and it concludes that clearance of extracellular K(+) is initially mainly effectuated by Na(+),K(+)-ATPase-mediated K(+) uptake into astrocytes, at K(+) concentrations above ~10 mM aided by uptake of Na(+),K(+) and 2 Cl(-) by the cotransporter NKCC1. Since operation of the astrocytic Na(+),K(+)-ATPase requires K(+)-dependent glycogenolysis for stimulation of the intracellular ATPase site, it ceases after normalization of extracellular K(+) concentration. This allows K(+) release via the inward rectifying K(+) channel Kir4.1, perhaps after trans-astrocytic connexin- and/or pannexin-mediated K(+) transfer, which would be a key candidate for determination by synchronization-based computational analysis and may have signaling effects. Spatially dispersed K(+) release would have little effect on extracellular K(+) concentration and allow K(+) accumulation by the less powerful neuronal Na(+),K(+)-ATPase, which is not stimulated by increases in extracellular K(+). Since the Na(+),K(+)-ATPase exchanges 3 Na(+) with 2 K(+), it creates extracellular hypertonicity and cell shrinkage. Hypertonicity stimulates NKCC1, which, aided by ß-adrenergic stimulation of the Na(+),K(+)-ATPase, causes regulatory volume increase, furosemide-inhibited undershoot in [K(+)]e and perhaps facilitation of the termination of slow neuronal hyperpolarization (sAHP), with behavioral consequences. The ion transport processes involved minimize ionic disequilibria caused by the asymmetric Na(+),K(+)-ATPase fluxes.

Brain glycogenolysis, adrenoceptors, pyruvate carboxylase, Na(+),K(+)-ATPase and Marie E. Gibbs' pioneering learning studies.

The involvement of glycogenolysis, occurring in astrocytes but not in neurons, in learning is undisputed (Duran et al., 2013). According to one school of thought the role of astrocytes for learning is restricted to supply of substrate for neuronal oxidative metabolism. The present "perspective" suggests a more comprehensive and complex role, made possible by lack of glycogen degradation, unless specifically induced by either (1) activation of astrocytic receptors, perhaps especially ß-adrenergic or (2) even small increases in extracellular K(+) concentration above its normal resting level. It discusses (1) the known importance of glycogenolysis for glutamate formation, requiring pyruvate carboxylation; (2) the established role of K(+)-stimulated glycogenolysis for K(+) uptake in cultured astrocytes, which probably indicates that astrocytes are an integral part of cellular K(+) homeostasis in the brain in vivo; and (3) the plausible role of transmitter-induced glycogenolysis, stimulating Na(+),K(+)-ATPase/NKCC1 activity and thereby contributing both to the post-excitatory undershoot in extracellular K(+) concentration and the memory-enhancing effect of transmitter-mediated reduction of slow neuronal afterhyperpolarization (sAHP).

Chronic treatment with anti-bipolar drugs causes intracellular alkalinization in astrocytes, altering their functions.

Bipolar disorder I and II are affective disorders with mood changes between depressive and manic (bipolar I) or hypomanic (bipolar II) periods. Current therapy of these conditions is chronic treatment with one or more of the anti-bipolar drugs, Li(+) ('lithium'), carbamazepine and valproic acid. The pathophysiology of bipolar disorder is multifactorial and far from clear. Recent data on the dependence of normal brain function on neuronal-astrocytic interactions raise the possibility of astrocytic involvement. We will discuss our previously published and new results on effects of chronic treatment of primary cultures of normal mouse astrocytes with any of three conventional anti-bipolar drugs. The focus will be on several drug-induced events in relation to therapeutic effects of the drugs, such as myo-inositol uptake, intracellular pH and alkalinization, drug-induced modulation of glutamatergic activity in astrocytes and release of astrocytic 'gliotransmitters'. Finally, we will discuss the importance of phospholipase A2 (PLA(2)) and arachidonic acid cascade in drug-treated astrocytes, partly based on Dr. Barneda Cuirana's published thesis. All three drugs cause gradual intracellular alkalinization through different mechanisms. Alkalinization inhibit myo-inositol uptake, resulting in reduced inositolphosphate/phospholipid signaling. Accordingly, transmitter-induced increase in free intracellular Ca(2+) ([Ca(2+)](i)) becomes inhibited, aborting release of astrocytic 'gliotransmitters'. The reduction of "gliotransmitter" effects on neurons may have therapeutic effects in mania. Alkalinization also up-regulates expression of cPLA(2), an enzyme releasing arachidonic acid, and triggered arachidonic acid cascade and production, but perhaps not release, of prostaglandins. Whenever tested, identical effects were observed in freshly isolated astrocytes, but not neurons, from carbamazepine-treated healthy animals.

Neuroprotective effect of sodium ferulate and signal transduction mechanisms in the aged rat hippocampus.

AIM: To investigate whether the age-related increase in interleukin-1beta (IL-1beta) and c-Jun N-terminal kinases (JNK) pathway was coupled with a decrease in cell survival signaling pathways and whether sodium ferulate (SF) treatment was effective in preventing these age-associated changes. METHODS: Groups of young and aged rats were fed for 4 weeks on a diet enriched in SF (100 mg/kg and 200 mg/kg per day). At the end of the period of dietary manipulation, Western blotting analysis was used to determine the expressions of IL-1beta, phosphorylated mitogen-activated protein kinase kinase (MKK)4, phospho-JNK, phospho-c-Jun, phosphorylated extracellular signal-regulated kinase (ERK1/2), phospho-MEK, phospho-Akt, phosphorylated ribosomal protein S6 protein kinase (p70S6K), and activated caspase-3 and caspase-7. Nissl staining was used to observe the morphological change in hippocampal CA1 regions. Immunohistochemical techniques for glial fibrillary acidic protein (GFAP) and integrin alphaM (OX-42) were used to determine the astrocyte and microglia activation. RESULTS: IL-1beta protein levels, and phospho-MKK4, phospho-JNK1/2, and phospho-c-Jun were significantly enhanced in hippocampus prepared from age-matched control rats. Increased IL-1beta production and JNK1/2 activation was accompanied by downregulation of MEK/ERK1/2 pathway and Akt/p70S6K pathway, leading to cell apoptosis assessed by activation of caspase-3. Significantly, treatment of aged rats with SF (100 mg/kg and 200 mg/kg per day) for 4 weeks prevented the agerelated increase in IL-1beta and IL-1beta-induced JNK signaling pathway and also the age-related changes in ERK and Akt kinase. CONCLUSION: SF plays neuroprotective roles through suppression of IL-1beta and IL-1beta-induced JNK signaling and upregulation of MEK/ERK1/2 and Akt/p70S6K survival pathways.

Neuroprotection by sodium ferulate against glutamate-induced apoptosis is mediated by ERK and PI3 kinase pathways.

AIM: To investigate whether sodium ferulate (SF) can protect cortical neurons from glutamate-induced neurotoxicity and the mechanisms responsible for this protection. METHODS: Cultured cortical neurons were incubated with 50 micromol/L glutamate for either 30 min or 24 h, with or without pre-incubation with SF (100, 200, and 500 micromol/L, respectively). LY294002, wortmannin, PD98059, and U0126 were added respectively to the cells 1 h prior to SF treatment. After incubation with glutamate for 24 h, neuronal apoptosis was quantified by scoring the percentage of cells with apoptotic nuclear morphology after Hoechst 33258 staining. After incubation with glutamate for either 30 min or 24 h, cellular extracts were prepared for Western blotting of active caspase-3, poly (ADP-ribose) polymerase (PARP), mu-calpain, Bcl-2, phospho-Akt, phosphorylated ribosomal protein S6 protein kinase (p70S6K), phospho-mitogen-activated protein kinase kinase (MEK1/2) and phosphorylated extracellular signal-regulated kinase (ERK) 1/2. RESULTS: SF reduced glutamate-evoked apoptotic morphology, active caspase-3 protein expression, and PARP cleavage and inhibited the glutamate-induced upregulation of the mu-calpain protein level. The inhibition of the phosphatidylinositol 3-kinase (PI3K) and the MEK/ERK1/2 pathways partly abrogated the protective effect of SF against glutamate-induced neuronal apoptosis. SF prevented the glutamate-induced decrease in the activity of the PI3K/Akt/p70S6K and the MEK/ERK1/2 pathways. Moreover, incubation of cortical neurons with SF for 30 min inhibited the reduction of the Bcl-2 expression induced by glutamate. CONCLUSION: The results indicate that PI3K/Akt/p70S6K and the MEK/ERK signaling pathways play important roles in the protective effect of SF against glutamate toxicity in cortical neurons.

Effects of sodium ferulate on amyloid-beta-induced MKK3/MKK6-p38 MAPK-Hsp27 signal pathway and apoptosis in rat hippocampus.

AIM: To observe the effects of sodium ferulate (SF) on amyloid beta (Abeta)1-40-induced p38 mitogen-activated protein kinase (MAPK) signal transduction pathway and the neuroprotective effects of SF. METHODS: Rats were injected intracerebroventricularly with Abeta1-40. Six hours after injection, Western blotting was used to determine the expressions of phosphorylated mitogen-activated protein kinase kinase (MKK) 3/MKK6, phospho-p38 MAPK, interleukin (IL)-1beta, phospho-MAPK activating protein kinase 2 (MAPKAPK-2), the 27 kDa heat shock protein (Hsp27), procaspase-9, -3, and -7 cleavage, and poly (ADP-ribose) polymerase (PARP) cleavage. Seven days after injection, Nissl staining was used to observe the morphological change in hippocampal CA1 regions. RESULTS: Intracerebroventricular injection of Abeta1-40 induced an increase in phosphorylated MKK3/MKK6 and p38 MAPK expressions in hippocampal tissue. These increases, in combination with enhanced interleukin (IL)-1beta protein expression and reduced phospho-MAPKAPK2 and phospho-Hsp27 expression, mediate the Abeta-induced activation of cell death events as assessed by cleavage of procaspase-9, -3, and -7 and caspase-3 substrate PARP cleavage. Pretreatment with SF (100 mg/kg and 200 mg/kg daily, 3 weeks) significantly prevented Abeta1-40-induced increases in phosphorylated MKK3/MKK6 and p38 MAPK expression. The Abeta1-40-induced increase in IL-1beta protein level was attenuated by pretreatment with SF. In addition, Abeta1-40-induced decreases in phosphorylated MAPKAPK2 and Hsp27 expression were abrogated by administration of SF. In parallel with these findings, Abeta1-40-induced changes in activation of caspase-9, caspase-7, and caspase-3 were inhibited by pretreatment with SF. CONCLUSION: SF prevents Abeta1-40-induced neurotoxicity through suppression of MKK3/MKK6-p38 MAPK activity and IL-1beta expression and upregulation of phospho-Hsp27 expression.

Sodium ferulate prevents amyloid-beta-induced neurotoxicity through suppression of p38 MAPK and upregulation of ERK-1/2 and Akt/protein kinase B in rat hippocampus.

AIM: To observe whether an amyloid beta (Abeta)-induced increase in interleukin (IL)-1beta was accompanied by an increase in the p38 mitogen-activated protein kinase (MAPK) pathway and a decrease in the cell survival pathway, and whether sodium ferulate (SF) treatment was effective in preventing these Abeta-induced changes. METHODS: Rats were injected intracerebroventricularly with Abeta25-35. Seven days after injection, immunohistochemical techniques for glial fibrillary acidic protein (GFAP) were used to determine the astrocyte infiltration and activation in hippocampal CA1 areas. The expression of IL-1beta, extracellular signal-regulated kinase (ERK), p38 MAPK, Akt/protein kinase B (PKB), Fas ligand and caspase-3 were determined by Western blotting. The caspase-3 activity was measured by cleavage of the caspase-3 substrate (Ac-DEVD-pNA). Reverse transcription-polymerase chain reaction was used to analyze the changes in IL-1beta mRNA levels. RESULTS: Intracerebroventricular injection of Abeta25-35 elicited astrocyte activation and infiltration and caused a strong inflammatory reaction characterized by increased IL-1beta production and elevated levels of IL-1beta mRNA. Increased IL-1beta synthesis was accompanied by increased activation of p38 MAPK and downregulation of phospho-ERK and phospho-Akt/PKB in hippocampal CA regions prepared from Abeta-treated rats, leading to cell death as assessed by activation of caspase-3. SF significantly prevented Abeta-induced increases in IL-1beta and p38 MAPK activation and also Abeta-induced changes in phospho-ERK and phospho-Akt/PKB expression levels. CONCLUSION: SF prevents Abeta-induced neurotoxicity through suppression of p38 MAPK activation and upregulation of phospho-ERK and phospho-Akt/PKB expression.