Synthesis and Analgesic Effects of μ-TRTX-Hhn1b on Models of Inflammatory and Neuropathic Pain

μ-TRTX-Hhn1b (HNTX-IV) is a 35-amino acid peptide isolated from the venom of the spider, Ornithoctonus hainana. It inhibits voltage-gated sodium channel Nav1.7, which has been considered as a therapeutic target for pain. The goal of the present study is to elucidate the analgesic effects of synthetic μ-TRTX-Hhn1b on animal models of pain. The peptide was first synthesized and then successfully refolded/oxidized. The synthetic peptide had the same inhibitory effect on human Nav1.7 current transiently expressed in HEK 293 cells as the native toxin. Furthermore, the analgesic potentials of the synthetic peptide were examined on models of inflammatory pain and neuropathic pain. μ-TRTX-Hhn1b produced an efficient reversal of acute nociceptive pain in the abdominal constriction model, and significantly reduced the pain scores over the 40-min period in the formalin model. The efficiency of μ-TRTX-Hhn1b on both models was equivalent to that of morphine. In the spinal nerve model, the reversal effect of μ-TRTX-Hhn1b on allodynia was longer and higher than mexiletine. These results demonstrated that μ-TRTX-Hhn1b efficiently alleviated acute inflammatory pain and chronic neuropathic pain in animals and provided an attractive template for further clinical analgesic drug design.     

Synapse Loss Induced by Interleukin-1β Requires Pre- and Post-synaptic Mechanisms

Interleukin-1β (IL-1β) is an inflammatory cytokine that exerts marked effects on neuronal function and survival. Here we examined the effects of IL-1β on synapses between rat hippocampal neurons in culture using an imaging-based assay to quantify clusters of the scaffolding protein postsynaptic density 95 fused to green fluorescent protein. Treatment with IL-1β for 24?h induced a 23?±?3% loss in the number of synaptic sites. Pharmacological studies indicated that synapse loss was mediated by the IL-1 receptor with subsequent activation of two pathways. COX2-mediated prostaglandin production and postsynaptic activation of a Src family tyrosine kinase were required. Presynaptic release of glutamate with subsequent activation of NMDA receptors was necessary for IL-1β-induced synapse loss. Neither Src activation nor prostaglandin E2 (PGE2) application alone was sufficient to reduce the number of synapses. However, in cells expressing constitutively active or pharmacologically activated Src, PGE2 induced synapse loss. Thus, IL-1β reduces the number of synaptic connections by simultaneously activating multiple pathways that require both pre- and post-synaptic activity. These results highlight targets that may prove important for pharmacotherapy of neuroinflammatory disease.     

Effects of a Corticosteroid,Budesonide, on Alveolar Macrophage and Blood Monocyte Secretion of Cytokines: Differential Sensitivity of GM-CSF,IL-1β, and IL-6

Summary: Down-regulation of cytokine production in activated human blood monocytes (BMs) and alveolar macrophages (AMs) can be achieved in vitro by treatment with corticosteroids. The inhibition of cytokine secretion by corticosteroids may have important therapeutic consequences in e.g. asthma. However, relatively little is known about possible differences in the sensitivity of different cytokines to corticosteroid treatment. Homologous BMs and AMs were obtained from six healthy volunteers. Secretion of interleukin-1β (IL-1β), interleukin-6 (IL-6) and granulocyte-macrophage colony-stimulating factor (GM-CSF) in the cultures of lipopolysaccharide (LPS) stimulated adherent BMs and AMs was analysed using specific immunoassays. Sensitivity of the IL-1β, IL-6, and GM-CSF secretion to the in vitro treatment with a synthetic corticosteroid, budesonide, was compared. BMs and AMs displayed significant differences in both cytokine secretion and susceptibility to regulation by budesonide. When added to the BM cultures concomitantly with LPS, budesonide suppressed IL-1β and IL-6 only partially (to 30% of the control level). In contrast, GM-CSF release in these cultures was almost totally inhibited by budesonide (⩾10^-8 M). The IC₅₀ for inhibition of the GM-CSF secretion was as low as 2 × 10^-10 M. In the AM cultures, budesonide had very little effect on IL-1β and IL-6 secretion (inhibition to 80% and 60% of control levels, respectively), while GM-CSF secretion was suppressed to 20% of control by budesonide concentrations ⩾10^-7 M. These in vitro studies suggest that GM-CSF secretion from BMs and AMs is more sensitive to corticosteroid treatment than IL-1β and IL-6 secretion. Moreover, these data support the view that human monocytes derived from different compartments or at different stages of differentiation exhibit different responsiveness to corticosteroids.     

Differential Effects of Anionic,Cationic, Nonionic,and Physiologic Surfactants on the Dissociation, α-Chymotryptic Degradation,and Enteral Absorption of Insulin Hexamers

Various surfactants were investigated to compare their effects on insulin dissociation, -chymotryptic degradation, and rat enteral absorption. With a circular dichroism technique, sodium dodecyl sulfate (SDS) at a 5 mM concentration was found to completely dissociate procine-zinc insulin hexamers (0.5 mg/ml) into monomers. The catalytic activity of -chymotrypsin (0.5 µM) was also abolished by 5 mM SDS. When insulin was injected into the distal jejunum/ proximal ileum segment of the rat, 5 mM SDS greatly enhanced its pharmacological availability, from a negligible value to 2.8%. Being a cationic surfactant, hexadecyl trimethylammonium bromide (CTAB) also efficiently dissociated insulin hexamers at concentrations of 1–5 mM. However, extensive charge–charge interaction was observed below a CTAB concentration of 0.6 mM, leading to insulin precipitation at a molar CTAB:insulin ratio of 1:1 to 2:1. An -chymotryptic degradation study also revealed near-complete dissociation of insulin hexamers at 1 mM CTAB. Above 1 mM, however, CTAB acted as an enzyme inhibitor, most likely by means of charge repulsion. Enteral absorption studies showed a much lower pharmacological availability, only 0.29%. Nonionic surfactants such as Tween 80 and polyoxyethylene 9 lauryl ether were ineffective in dissociating insulin hexamers. Tween 80, at 5 mM, neither significantly altered the -chymotryptic degradation pattern nor enhanced the enteral absorption of insulin. The relative effectiveness of different species of bile salts on insulin hexamer dissociation appeared to be similar. Sodium glycocholate at a 30 mM concentration also significantly increased insulin pharmacological availability, to 2.3%. A morphological study did not reveal any significant alteration of the rat intestinal mucosal integrity after exposure to 5 mM SDS for 30 min. The results further emphasize the importance of the degree of insulin aggregation on its enteral transport.     

Effects of corticotrophin-releasing factor receptor 1 antagonists on amyloid-β and behavior in Tg2576 mice

Rationale

Previous studies indicate that psychosocial stressors could accelerate amyloid-β (Aβ) levels and accelerate plaque deposition in mouse models of Alzheimer disease (AD). Stressors enhanced the release of corticotrophin-releasing factor (CRF), and exogenous CRF administration mimicked the effects of stress on Aβ levels in mouse models of AD. However, whether CRF receptor 1 (CRF1) antagonists could influence the stress-induced acceleration of an AD-like process in mouse models has not been well studied.

Objective

We sought to examine whether CRF1 antagonists inhibit the effects of isolation stress on tissue Aβ levels, Aβ plaque deposition, and behaviors related to anxiety and memory in Tg2576 mice, and to investigate the molecular mechanism underlying such effects.

Methods

Cohorts of Tg2576 mouse pups were isolated or group-housed at 21 days of age, and then the subgroups of these cohorts received daily intraperitoneal injections of the CRF1 antagonists, antalarmin or R121919 (5, 10, and 20 mg/kg), or vehicle for 1 week. Other cohorts of Tg2576 mouse pups were isolated or group-housed at 21 days of age, and then at 4 months of age, subgroups of these mice were administered antalarmin (20 mg/kg) or vehicle in their drinking water for 6 months. Finally, cultured primary hippocampal neurons from regular Tg2576 pups (P0) were incubated with CRF (0.1, 1, and 10 nM), antalarmin (100 nM) or H-89 (1 μM) for 48 h. Brain tissues or cultured neurons were collected for histological and biochemical analyses, and behavioral measures were collected in the cohorts of mice that were chronically stressed.

Results

Administration of antalarmin at 20 mg/kg dose for 1 week significantly reduced Aβ_1-42 levels in isolation stressed mice. Administration of antalarmin for 6 months significantly decreased plasma corticosterone levels, tissue Aβ_1-42 levels, and Aβ plaque deposition in the brain and blocked the effects of isolation stress on behaviors related to anxiety and memory. Finally, incubation of neurons with 100 nM antalarmin inhibited the ability of 10 nM CRF to increase Aβ_1-42 levels and protein kinase A IIβ expression. The effect of CRF1 on Aβ_1-42 levels was also diminished by treatment with H-89, a c-AMP/PKA inhibitor.

Conclusions

These results suggest that CRF1 antagonists can slow an AD-like process in Tg2576 mice and that the c-AMP/PKA signaling pathway may be involved in this effect.     

Effects of Aspirin and Mefenamic Acid on Soman Poisoning–Induced Neuropathology in Mice

The efficacy of aspirin and mefenamic acid to counteract soman-induced brain damage was investigated in mice. Neuronal damage was evaluated in the hippocampus and amygdala by performing ω3 receptor density measurements and hemalun–phloxin staining. The effect of both drugs on the proliferation of neural progenitors after soman exposure was also assessed. Mefenamic acid aggravated the soman-induced hippocampal neuropathology. On the other hand, aspirin recorded a weak neuroprotective effect in the amygdala. However, this drug also diminished the proliferation of neural precursor cells. The possible neurochemical mechanisms underlying such differences in the efficacy of the two drugs are also reviewed.     

Effects of Breviscapine on the Changes in Antioxidant Enzyme Activity Induced by Cerebral Ischemia-reperfusion in Rats

本文研究了灯盏花素对大鼠脑缺血再灌注引起抗氧化酶活性改变的影响，结果表明，灯盏花素明显提高脑缺血再灌注引起的脑组织超氧歧化酶（ＳＯＤ）、谷胱甘肽过氧化物酶（ＧＳＨｐｅｒｏｘｉｄａｓｅ）和过氧化氢酶（Ｃａｔａｌａｓｅ）的活性，减少脑组织丙二醛（ＭＤＡ）含量。这些作用有利于减轻脑缺血再灌注损伤。     

Effects of deferoxamine and sympathectomy on endothelin-1-induced contraction and acetylcholine—Induced relaxation following subarachnoid hemorrhage in carotid artery

The role of endothelium-related factors in the pathogenesis of cerebral vasospasm after subarachnoid hemorrhage (SAH) has gained interest since the discovery of EDRF and of endothelin-1 (ET-1). The effect of SAH and both treatment of deferoxamine (DFO) and sympathectomy on endothelium-dependent vasodilation and ET-1-induced vasoconstriction of isolated rabbit carotid artery was examined using an isometric tension recording method. Thirty-five rabbits were divided into four groups: control animals, 7 days after SAH, treatment with DFO after SAH for 7 days and sympathectomy after SAH. Acetylcholine (10⁻⁸ to 10⁻⁵ M) was used to evoke concentration-dependent vasodilation of isolated arterial rings previously contracted by 10⁻⁶ M phenylephrine. In the animals killed 7 days after SAH, acetylcholine-induced relaxation was suppressed and the degree of relaxation of this group was 50% of the initial contractile tone in response to the 10⁻⁵ M acetylcholine. These relaxant responses did not return to control values in carotid arteries obtained from animals treated with DFO and subjected to sympathectomy. In isolated carotid arteries, ET-1 (10⁻¹⁰ to 10⁻⁸ M) produced concentration-dependent contractions. These contractile responses were significantly enhanced in animals 7 days after SAH compared with controls and did not return to control values in carotid arteries obtained from animals both treated with DFO and sympathectomized for 7 days after SAH. The present experiments suggest that impairment of endothelium-dependent vasodilation and the hyperreactivity of ET-1 of the carotid artery as well as cerebral arteries may be involved in the pathogenesis of cerebral vasospasm. Both treatment with DFO and sympathectomy during the chronic stage for vasospasm after SAH did not affect these vascular responses of the extradural part of the carotid artery to ET-1 and acetylcholine.     

Effects of intracellular application of CsCL on action potential and contractio,

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Effect of 17-β-Estradiol and Ginsenoside Rg1 on Reactive Microglia Induced by β-Amyloid Peptides

Abstract

The reactive microglias induced by 25 μmol of β-amyloid peptides (Aβ25–35) and/or IFN-γ can initiate the microglial respiratory burst and release NO, respectively. Oxidative stress and inflammatory function have been implicated in Alzheimer's disease (AD). We showed that 10 μmol 17-β-estradiol (E₂) and 1–10 μmol ginsenoside Rg₁ (Rg₁) could prevent the toxicity of Aβ25–35 and/or IFN-γ to microglias, inhibit the microglial respiratory burst activity and decrease the accumulation of NO. These results demonstrated the protectional effect of E₂ or Rg₁ on neuron from damaging by reactive microglias in AD.     

Effects of naloxone, β-endorphin and ACTH on acquisition of schedule-induced polydipsia

A series of three experiments examined the possible involvement of endogenous opioid peptides in the development of schedule-induced polydipsia in rats. Repeated pretraining treatment with 2 mg/kg naloxone impaired acquisition of schedule-induced polydipsia, whereas the same treatment injected after training increased drinking. This later effect was time dependent, since a 30-min delay in the injection of naloxone resulted in a disappearance of its effect. Post-training injections of 10 g/kg -endorphin or ACTH delayed the development of drinking. These findings are consistent with the hypothesis that endogenous opioid peptides modulate the development of schedule-induced polydipsia.     

Effects of dexamethasone and ibuprofen on LPS-induced gene expresion of TNF_ α,IL-1β,and MIP-1α in rat lung

研究地塞米松（Ｄｅｘ）和布洛芬（Ｉｂｕ）对脂多糖（ＬＰＳ）诱导的大鼠肺ＴＮＦα、ＩＬ-１β和ＭＩＰ-１α基因表达的影响．方法：荧光法测定肺中伊文斯蓝含量，Ｓｌｏｔｂｌｏｔ对细胞因子ｍＲＮＡ表达相对定量．结果：腹腔注射ＬＰＳ导致大鼠肺中ＴＮＦα、ＩＬ-１β和ＭＩＰ-１αｍＲＮＡ表达明显增加，与ＬＰＳ剂量有依赖关系，峰值分别在２，６，１２小时．在ＬＰＳ前１小时给药，Ｄｅｘ５０ｍｇ·ｋｇ－１和Ｉｂｕ９０ｍｇ·ｋｇ－１均明显降低肺中伊文斯蓝含量，同时ＴＮＦα、ＩＬ-１β和ＭＩＰ-１αｍＲＮＡ表达量亦明显减少．结论：ＬＰＳ诱导大鼠肺中ＴＮＦα、ＩＬ-１β和ＭＩＰ-１α基因表达，Ｄｅｘ和Ｉｂｕ通过抑制细胞因子表达而减轻肺损伤．     

Reversal of Signs of Induced Cotton Effects of Dicumarol-α1-Acid Glycoprotein Systems by Phenothiazine Neuroleptics Through Ternary Complexation

The interaction of dicumarol and phenothiazine neuroleptics binding to ₁-acid glycoprotein (AGP) was investigated by circular dichroism (CD) and equilibrium dialysis. The induced CD spectra of the dicumarol–AGP complex were affected differently by the different substituents of the phenothiazine molecule. The sign of the induced Cotton effect of dicumarol bound to AGP was reversibly changed with the introduction of the propyldimethylamine substituent at position 10 or chloride group at position 2 of the phenothiazine molecule. Chlorpromazine, which contains both of these substituents reversed the sign of the induced Cotton effect with the highest intensity. The addition of trifluoperazine, fluphenazine, and promethazine containing neither of the two substituents generated a new negative CD band. However, the addition of opromazine, which contains sulfoxide at position 5, decreased the CD intensity of the dicumarol–AGP complex without changing the shape of the CD spectra. Equilibrium dialysis studies revealed that the interaction of dicumarol–AGP with phenothiazine derivatives occurred simultaneously, and the interaction followed a cooperative and anticooperative binding model. Further, among the six phenothiazine derivatives that reversed the signs of the induced Cotton effects of the dicumarol–AGP complex, a linear relationship was observed between coupling constants and the difference in the induced optical ellipticity. The opromazine and dicumarol interaction was competitive for a common binding site on the AGP molecule. Removal of sialic acid did not have any effect on this interaction. These data support the hypothesis that the acidic and the basic drug binding sites overlap each other.     

Differential effects of harmaline and ouabain on intestinal sodium,phenylalanine and β-methyl-glucoside transport

Summary Harmaline inhibits both the Na⁺-K⁺-ATPase activity and the uptake of l-phenylalanine in guinea-pig intestinal mucosa. The latter effect is not a direct consequence of the former, since higher concentrations are needed to inhibit the enzyme than the influx into the mucosa. Furthermore the uptake is still sensitive to harmaline when the Na⁺-K⁺-ATPase has been fully inhibited by ouabain.Harmaline can inhibit l-phenylalanine influx at a concentration at which it does not affect intracellular ion concentrations. Ouabain, however, inhibits the uptake of l-phenylalanine only after a 30 min preincubation period, when the intracellular sodium concentration reached the extracellular level.Harmaline also interferes with the influx of -methyl-d-glucoside in the mucosa of the dog colon. Addition of harmaline at the mucosal face of the tissue suppresses all net transport of sodium and chloride ions and l-phenylalanine across the mucosa. Thus the same mode of action appears to apply in both the guinea-pig ileum and the dog colon.     

Effects of complete Freund＇s adjuvant on immunohistochemical distribution of IL-1β and IL-1R I in neurons and glia cells of dorsal root ganglion

AIM: To investigate the effects of complete Freund adjuvant (CFA) on inflammatory hyperalgesia and morphological change of the coexistence of interleukin-1 beta (IL-1beta) and type I IL-1 receptor (IL-1RI) in neurons and glia cells of rat dorsal root ganglion (DRG). METHODS: The pain-related parameters and the expression of IL-1RI and IL-1beta positive neurons and glia cells of DRG in normal saline (NS) and adjuvant-induced arthritic (AA) group were examined with pain behavior assessment methods and immunohistochemical assay, respectively. RESULTS: Five hours, 1 d, and 2 d after intra-articular injection of 50 microL CFA, tactile hyperalgesia induced by CFA was observed in the foot flexion and extension scores of the ipsilateral hindpaw of AA group. Three days after injection, the distribution of IL-1RI/IL-1beta double-stained coexisted neurons and glia cells were observed in ipsilateral DRG of both groups. The number of IL-1beta positive neurons, IL-1RI positive neurons, IL-1beta/IL-1RI double-stained neurons, and IL-1RI positive glia cells in ipsilateral DRG of the AA group were higher than that of NS group (P<0.05 or P<0.01). CONCLUSION: The coexistence of IL-1beta and IL-1RI in neurons and nonneuronal cells suggests an as yet unknown autocrine and/or paracrine function of IL-1beta in the DRG. The function was enhanced in articular arthritis induced by CFA and could play an important role in hyperalgesia under inflammatory conditions.     

Effects of saffron and its constituents, crocin-1, crocin-2, and crocetin on α-synuclein fibrils

Journal of Natural Medicines - Saffron, the stigma of Crocus sativus Linné (Iridaceae family), has been known to inhibit aggregation of β-amyloid, a nerve tissue protein. α-Synuclein...     

Effects of GM-CSF, IL-3, and GM-CSF／IL-3 fusion protein on apoptosis of human myeloid leukemic cell line Tf-1 induced by irradiation

AIM: To observe the effects of three cytokines on the apoptosis of Tf-1 cells induced by γ irradiation and investigate the relationship between apoptosis and caspase-3 activity. METHODS: Different cytokines GM-CSF, IL-3 and GM-CS/IL-3 fusion protein were added into the irradiated Tf-1 cells. MTT assay, morphology, flow cytometry,and DNA fragmentation assay were used to observe the effects of cytokines on apoptosis. The caspase-3 activity was determined with a fluorocytometer. RESULTS: Irradiated Tf-1 cells showed typical morphological characteristic of apoptosis demonstrated by transmission electron microscopy and were accumulated in G0/G1 phase. In the groups treated with growth factors after irradiation, three cytokines significantly increased the viability rate, distinctly decreased the apoptosis rate and the proportion of DNA fragmentation. When Tf-1 cells were irradiated by γ irradiation, caspase-3 activity was increased at different time points. In comparison with the control group in which no growth factor was added after the cells were irradiated, the caspase-3 activity of irradiated Tf-1 cells was significantly inhibited by addition of the above cytokines. Thirty-six hours after irradiation, in the control group,GM-CSF, IL-3, GM-CSF and IL-3 in combination, and two GM-CSF/IL-3 fusion protein groups, the apoptosis ratewas 73 %, 11%, 15 %, 13 %, 12 %, and 13 %. The percent of fragmented DNA was 36 %, 19 %, 18 %, 14 %,13 %, and 14 %. The fluorescence intensity was 16923, 5529, 6581, 5322, 5426, and 5485. CONCLUSION:GM-CSF, IL-3, and GM-CSF/IL-3 fusion protein could protect Tf-1 cells from apoptosis induced by γ irradiation.After Tf-1 cells were irradiated, the caspase-3 activity was significantly increased but was dramatically decreased by the above cytokines. The remarkable inhibition of caspase-3 activity may be one of the mechanisms of these hematopoietic growth factors exerting their anti-apoptotic effects.     

Effects of GM-CSF, IL-3, and GM-CSG/IL-3 fusion protein on apoptosis of human myeloid leukemic cell line Tf-1 induced by irradiation

AIM: To observe the effects of three cytokines on the apoptosis of Tf-1 cells induced by y irradiation and investigate the relationship between apoptosis and caspase-3 activity. METHODS: Different cytokines GM-CSF, IL-3 and GM-CS/IL-3 fusion protein were added into the irradiated Tf-1 cells. MTT assay, morphology, flow cytometry, and DNA fragmentation assay were used to observe the effects of cytokines on apoptosis. The caspase-3 activity was determined with a fluorocytometer. RESULTS: Irradiated Tf-1 cells showed typical morphological characteristic of apoptosis demonstrated by transmission electron microscopy and were accumulated in G0/G1 phase. In the groups treated with growth factors after irradiation, three cytokines significantly increased the viability rate, distinctly decreased the apoptosis rate and the proportion of DNA fragmentation. When Tf-1 cells were irradiated by y     

Heme oxygenase-1 mediates protective effects on inflammatory, catabolic and senescence responses induced by interleukin-1β in osteoarthritic osteoblasts

Osteoarthritis (OA) is a chronic degenerative joint disease showing altered bone metabolism. Osteoblasts contribute to the regulation of cartilage metabolism and bone remodeling. We have shown previously that induction of heme oxygenase-1 (HO-1) protects OA cartilage against inflammatory and degradative responses. In this study, we investigated the effects of HO-1 induction on OA osteoblast metabolism. HO-1 was induced with cobalt protoporphyrin IX (CoPP) and by transduction with LV-HO-1. In osteoblasts stimulated with interleukin (IL)-1β, CoPP enhanced mineralization, the expression of a number of markers of osteoblast differentiation such as Runx2, bone morphogenetic protein-2, osteocalcin, and collagen 1A1 and 1A2, as well as the ratio osteoprotegerin/receptor activator of nuclear factor-κB ligand. HO-1 induction significantly reduced the expression of matrix metalloproteinase (MMP)-1, MMP-2 and MMP-3, and the production of pro-inflammatory cytokines such as tumor necrosis factor-α and IL-6 whereas IL-10 levels increased. HO-1 also exerted inhibitory effects on prostaglandin (PG)E(2) production which could be dependent on cyclooxygenase-2 and microsomal PGE synthase-1 down-regulation. The activity of senescence-associated β-galactosidase and the expression of the senescence marker caveolin-1 were significantly decreased after HO-1 induction. The inhibition of nuclear factor-κB activation induced by IL-1β in OA osteoblasts may contribute to some HO-1 effects. Our results have shown that HO-1 decreases the production of relevant inflammatory and catabolic mediators that participate in OA pathophysiology thus eliciting protective effects in OA osteoblasts.