Activation of spinal glucagon-like peptide-1 receptors specifically suppresses pain hypersensitivity.

This study aims to identify the inhibitory role of the spinal glucagon like peptide-1 receptor (GLP-1R) signaling in pain hypersensitivity and its mechanism of action in rats and mice. First, GLP-1Rs were identified to be specifically expressed on microglial cells in the spinal dorsal horn, and profoundly upregulated after peripheral nerve injury. In addition, intrathecal GLP-1R agonists GLP-1(7-36) and exenatide potently alleviated formalin-, peripheral nerve injury-, bone cancer-, and diabetes-induced hypersensitivity states by 60-90%, without affecting acute nociceptive responses. The antihypersensitive effects of exenatide and GLP-1 were completely prevented by GLP-1R antagonism and GLP-1R gene knockdown. Furthermore, exenatide evoked ß-endorphin release from both the spinal cord and cultured microglia. Exenatide antiallodynia was completely prevented by the microglial inhibitor minocycline, ß-endorphin antiserum, and opioid receptor antagonist naloxone. Our results illustrate a novel spinal dorsal horn microglial GLP-1R/ß-endorphin inhibitory pathway in a variety of pain hypersensitivity states.

Local protective effects of oral 45S5 bioactive glass on gastric ulcers in experimental animals.

Bioactive glass has been shown to stimulate bone regeneration and soft tissue healing. In this study, we evaluated the local protective effects of bioactive glass on experimental gastric ulcers, in comparison with omeprazole and hydrotalcite. Single and multiple gavage of 45S5 bioactive glass dose-dependently protected stress ulcers in mice and chronic ulcers in rats. Multi-daily gavage of bioactive glass for 7 days prevented chronic ulcer recurrence by 50 %. Bioactive glass ionic dissolution produced marked proliferation of ethanol-injured GES-1 human gastric mucosa epithelial cells 48 and 72 h after exposure. Bioactive glass was shown to be hardly absorbed after single or multi-daily gavage. This study, for the first time, demonstrates that bioactive glass is effective in protecting against gastric ulcers, with its high efficacy comparable to omeprazole and superior to hydrotalcite. The lack of oral absorption makes bioactive glass a potential for treatment of peptic ulcers omitting systemic toxicity or side-effects.

Histone deacetylation directs DNA methylation in survivin gene silencing.

DNA methylation and histone acetylation are major epigenetic modifications in gene silencing. In our previous research, we found that the methylated oligonucleotide (SurKex) complementary to a region of promoter of survivin could induce DNA methylation in a site-specific manner leading to survivin silencing. Here, we further studied the role of histone acetylation in survivin silencing and the relationship between histone acetylation and DNA methylation. First we observed the levels of histone H4 and H4K16 acetylation that were decreased after SurKex treatment by using the chromatin immunoprecipitation (ChIP) assay. Next, we investigated the roles of histone acetylation and DNA methylation in survivin silencing after blockade of histone deacetylation with Trichostatin A (TSA). We assessed survivin mRNA expression by RT-PCR, measured survivin promoter methylation by bisulfite sequencing and examined the level of histone acetylation by the ChIP assay. The results showed that histone deacetylation blocked by TSA reversed the effects of SurKex on inhibiting the expression of survivin mRNA, inducing a site-specific methylation on survivin promoter and decreasing the level of histone acetylation. Finally, we examined the role of histone acetylation in the expression of DNA methyltransferase 1 (DNMT1) mRNA. The results showed that histone deacetylation blocked by TSA reversed the increasing effect of histone deacetylation on the expression of survivin mRNA. This study suggests that histone deacetylation guides SurKex-induced DNA methylation in survivin silencing possibly through increasing the expression of DNMT1 mRNA.

Induced epigenetic modifications of the promoter chromatin silence survivin and inhibit tumor growth.

Anti-apoptotic survivin is over-expressed in a variety of human carcinomas and is considered as a therapeutic target in cancers. Suppression of survivin mRNA by RNAi and anti-sense nucleotides has proved to be a powerful anti-tumor therapy in both animal models and human investigations. In this communication, we tested an alternative approach to silence survivin by knocking down its gene transcription through a short methylated oligonucleotide (SurKex) that is complementary to the survivin gene promoter. Treatment of NCI-H460 cells with SurKex significantly suppressed the production of survivin mRNA and its oncoprotein. DNA bisulfite sequencing showed that SurKex induced site-specific de novo CpG methylation in the complementary region of survivin promoter. Chromatin immunoprecipitation assay also demonstrated that SurKex induced histone hypermethylation at histone H3K9 and H3K27 as well as deacetylation of histone H4 in the same regulatory region. SurKex remarkably inhibited tumor growth in nude mice bearing xenograft tumors. This study demonstrates that the synthetic methylated oligonucleotide SurKex inhibits tumor growth by silencing the survivin gene using a mechanism of altering the epigenotype in the survivin promoter. Thus, targeted epigenetic modification in the gene promoter may offer a new general strategy to silence tumor-related genes in tumor therapy.

Potential role of spinal TRPA1 channels in antinociceptive tolerance to spinally administered morphine.

BACKGROUND: Prolonged morphine treatment leads to antinociceptive tolerance. Suppression of spinal astrocytes or d-amino acid oxidase (DAAO), an astroglial enzyme catalyzing oxidation of d-amino acids, has reversed morphine antinociceptive tolerance. Since the astrocyte-DAAO pathway generates hydrogen peroxide, an agonist of the TRPA1 channel expressed spinally on nociceptive nerve terminals and astrocytes, we tested a hypothesis that the spinal TRPA1 contributes to antinociceptive tolerance to prolonged spinal morphine treatment. METHODS: Nociception was assessed using hot-plate test in rats with an intrathecal (it) catheter. Drugs were administered it twice daily from day one to seven in five treatment groups: (i) Saline, (ii) Chembridge-5861528 (a TRPA1 antagonist; 10µg), (iii) morphine (10µg), (iv) Chembridge-5861528 (10µg)+morphine (10µg), (v) DMSO. Antinociceptive action of morphine was assessed at day one and eight. Additionally, mRNA for DAAO and TRPA1 in the spinal cord was determined on day 8. RESULTS: Morphine treatment produced antinociceptive tolerance, which was attenuated by co-administration of Chembridge-5861528 that alone had no effect on hot-plate latencies. In animals treated with morphine only, spinal mRNA for DAAO but not TRPA1 was increased. DAAO increase was prevented by co-administration of Chembridge-5861528. CONCLUSIONS: Antinociceptive morphine tolerance and up-regulation of spinal DAAO were attenuated in morphine-treated animals by blocking the spinal TRPA1. This finding suggests that spinal TRPA1 may contribute, at least partly, to facilitation of morphine antinociceptive tolerance through mechanisms that possibly involve TRPA1-mediated up-regulation of the astroglial DAAO, a generator of hydrogen peroxide, a pronociceptive compound acting also on TRPA1.

Mechanical antihypersensitivity effect induced by repeated spinal administrations of a TRPA1 antagonist or a gap junction decoupler in peripheral neuropathy.

Spinal transient receptor potential ankyrin 1 (TRPA1) channel is associated with various pain hypersensitivity conditions. Spinally, TRPA1 is expressed by central terminals of nociceptive nerve fibers and astrocytes. Among potential endogenous agonists of TRPA1 is H2O2 generated by d-amino acid oxidase (DAAO) in astrocytes. Here we studied whether prolonged block of the spinal TRPA1 or astrocytes starting at time of injury attenuates development and/or maintenance of neuropathic hypersensitivity. Additionally, TRPA1 and DAAO mRNA were determined in the dorsal root ganglion (DRG) and spinal dorsal horn (SDH). Experiments were performed in rats with spared nerve injury (SNI) and chronic intrathecal catheter. Drugs were administered twice daily for the first seven injury days or only once seven days after injury. Mechanical hypersensitivity was assessed with monofilaments. Acute and prolonged treatment with Chembridge-5861528 (a TRPA1 antagonist), carbenoxolone (an inhibitor of activated astrocytes), or gabapentin (a comparison drug) attenuated tactile allodynia-like responses evoked by low (2g) stimulus. However, antihypersensitivity effect of these compounds was short of significance at a high (15g) stimulus intensity. No preemptive effects were observed. In healthy controls, carbenoxolone failed to prevent hypersensitivity induced by spinal cinnamaldehyde, a TRPA1 agonist. TRPA1 and DAAO mRNA in the DRG but not SDH were slightly increased in SNI, independent of drug treatment. The results indicate that prolonged peri-injury block of spinal TRPA1 or inhibition of spinal astrocyte activation attenuates maintenance but not development of mechanical (tactile allodynia-like) hypersensitivity after nerve injury.

The non-peptide GLP-1 receptor agonist WB4-24 blocks inflammatory nociception by stimulating ß-endorphin release from spinal microglia.

BACKGROUND AND PURPOSE: Two peptide agonists of the glucagon-like peptide-1 (GLP-1) receptor, exenatide and GLP-1 itself, exert anti-hypersensitive effects in neuropathic, cancer and diabetic pain. In this study, we have assessed the anti-allodynic and anti-hyperalgesic effects of the non-peptide agonist WB4-24 in inflammatory nociception and the possible involvement of microglial ß-endorphin and pro-inflammatory cytokines. EXPERIMENTAL APPROACH: We used rat models of inflammatory nociception induced by formalin, carrageenan or complete Freund's adjuvant (CFA), to test mechanical allodynia and thermal hyperalgesia. Expression of ß-endorphin and pro-inflammatory cytokines was measured using real-time quantitative PCR and fluorescent immunoassays. KEY RESULTS: WB4-24 displaced the specific binding of exendin (9-39) in microglia. Single intrathecal injection of WB4-24 (0.3, 1, 3, 10, 30 and 100 µg) exerted dose-dependent, specific, anti-hypersensitive effects in acute and chronic inflammatory nociception induced by formalin, carrageenan and CFA, with a maximal inhibition of 60-80%. Spinal WB4-24 was not effective in altering nociceptive pain. Subcutaneous injection of WB4-24 was also antinociceptive in CFA-treated rats. WB4-24 evoked ß-endorphin release but did not inhibit expression of pro-inflammatory cytokines in either the spinal cord of CFA-treated rats or cultured microglia stimulated by LPS. WB4-24 anti-allodynia was prevented by a microglial inhibitor, ß-endorphin antiserum and a µ-opioid receptor antagonist. CONCLUSIONS AND IMPLICATIONS: Our results suggest that WB4-24 inhibits inflammatory nociception by releasing analgesic ß-endorphin rather than inhibiting the expression of proalgesic pro-inflammatory cytokines in spinal microglia, and that the spinal GLP-1 receptor is a potential target molecule for the treatment of pain hypersensitivity including inflammatory nociception.

Geniposide and its iridoid analogs exhibit antinociception by acting at the spinal GLP-1 receptors.

We recently discovered that the activation of the spinal glucagon-like peptide-1 receptors (GLP-1Rs) by the peptidic agonist exenatide produced antinociception in chronic pain. We suggested that the spinal GLP-1Rs are a potential target molecule for the management of chronic pain. This study evaluated the antinociceptive activities of geniposide, a presumed small molecule GLP-1R agonist. Geniposide produced concentration-dependent, complete protection against hydrogen peroxide-induced oxidative damage in PC12 and HEK293 cells expressing rat and human GLP-1Rs, but not in HEK293T cells that do not express GLP-1Rs. The orthosteric GLP-1R antagonist exendin(9-39) right-shifted the concentration-response curve of geniposide without changing the maximal protection, with identical pA2 values in both cell lines. Subcutaneous and oral geniposide dose-dependently blocked the formalin-induced tonic response but not the acute flinching response. Subcutaneous and oral geniposide had maximum inhibition of 72% and 68%, and ED50s of 13.1 and 52.7 mg/kg, respectively. Seven days of multidaily subcutaneous geniposide and exenatide injections did not induce antinociceptive tolerance. Intrathecal geniposide induced dose-dependent antinociception, which was completely prevented by spinal exendin(9-39), siRNA/GLP-1R and cyclic AMP/PKA pathway inhibitors. The geniposide iridoid analogs geniposidic acid, genipin methyl ether, 1,10-anhydrogenipin, loganin and catalpol effectively inhibited hydrogen peroxide-induced oxidative damage and formalin pain in an exendin(9-39)-reversible manner. Our results suggest that geniposide and its iridoid analogs produce antinociception during persistent pain by activating the spinal GLP-1Rs and that the iridoids represented by geniposide are orthosteric agonists of GLP-1Rs that function similarly in humans and rats and presumably act at the same binding site as exendin(9-39).

Targeted gene suppression by inducing de novo DNA methylation in the gene promoter.

BACKGROUND: Targeted gene silencing is an important approach in both drug development and basic research. However, the selection of a potent suppressor has become a significant hurdle to implementing maximal gene inhibition for this approach. We attempted to construct a 'super suppressor' by combining the activities of two suppressors that function through distinct epigenetic mechanisms. RESULTS: Gene targeting vectors were constructed by fusing a GAL4 DNA-binding domain with a epigenetic suppressor, including CpG DNA methylase Sss1, histone H3 lysine 27 methylase vSET domain, and Kruppel-associated suppression box (KRAB). We found that both Sss1 and KRAB suppressors significantly inhibited the expression of luciferase and copGFP reporter genes. However, the histone H3 lysine 27 methylase vSET did not show significant suppression in this system. Constructs containing both Sss1 and KRAB showed better inhibition than either one alone. In addition, we show that KRAB suppressed gene expression by altering the histone code, but not DNA methylation in the gene promoter. Sss1, on the other hand, not only induced de novo DNA methylation and recruited Heterochromatin Protein 1 (HP1a), but also increased H3K27 and H3K9 methylation in the promoter. CONCLUSIONS: Epigenetic studies can provide useful data for the selection of suppressors in constructing therapeutic vectors for targeted gene silencing.

Beneficial effects of natural Jeju groundwaters on lipid metabolism in high-fat diet-induced hyperlipidemic rats.

BACKGROUND: Groundwater is believed to possess many beneficial effects due to its natural source of various minerals. In this study, we examined the effects of natural Jeju groundwater S1 (Samdasoo™), S2 and S3 pumped up from different locations of Jeju Island, Korea, along with local tap water, on body weight gain, serum lipids and lipoproteins, and liver histopathology in high-fat diet-induced hyperlipidemic rats. MATERIALS/METHODS: Rats were randomly and equally divided into 6 groups. Different water samples were supplied to the hyperlipidemic rats as their daily drinking water and the widely-used anti-hyperlipidemic drug simvastatin was used as a positive control. Body weight, serum lipids and lipoproteins were measured weekly. Liver weight, liver index and liver histopathology were examined after the execution of the rats. RESULTS: After drinking Jeju groundwaters for two months, S2 but not S3 significantly reduced weight growth and serum triglycerides levels and increased high density lipoprotein-C (HDL-C) without affecting total cholesterol or LDL-C. S1 and particularly S2 significantly reduced the severity of liver hypertrophy and steatosis. All Groundwaters had much higher contents of vanadium (S3>S2>S1>>tap water) whereas S1 and S2 but not S3 markedly blocked autoxidation of ferrous ions. CONCLUSION: Jeju Groundwater S1 and particularly S2 exhibit protective effects against hyperlipidemia and fatty liver and hypothesize that the beneficial effect of Jeju Groundwaters may be contributed from blockade of autoxidation of ferrous ions rather than their high contents of vanadium.

Spinal D-amino acid oxidase contributes to mechanical pain hypersensitivity induced by sleep deprivation in the rat.

We studied the hypothesis that spinal d-amino acid oxidase (DAAO) that is expressed in astrocytes and that has been reported to promote tonic pain in various pathophysiological conditions plays a role in 'physiological' pain hypersensitivity induced by rapid eye movement sleep deprivation (REMSD). The experiments were performed in healthy rats with a chronic intrathecal (i.t.) catheter. Pain behavior was assessed by determining limb withdrawal response to repetitive stimulation of the hind paw with a calibrated series of monofilaments. REMSD of 48 h duration produced a significant mechanical hypersensitivity. At 48 h of REMSD, the animals were treated i.t. with a DAAO inhibitor or vehicle. Three structurally different DAAO inhibitors were tested in this study: 6-chlorobenzo[d]isoxazol-3-ol (CBIO), sodium benzoate, and 5-methylpyrazole-3-carboxylic acid (AS-057278). CBIO (1-3 µg), sodium benzoate (30-100 µg) and AS-057278 (3-10 µg) produced dose-related antihypersensitivity effects in sleep-deprived animals. In control animals (with no sleep deprivation), the currently used doses of DAAO inhibitors failed to produce significant changes in mechanically evoked pain behavior. The results indicate that among spinal pain facilitatory mechanisms that contribute to the sleep deprivation-induced mechanical pain hypersensitivity is DAAO, presumably due to production of reactive oxygen species, such as hydrogen peroxide, an endogenous agonist of the pronociceptive TRPA1 ion channel.

Interactions of the potent D-amino acid oxidase inhibitor CBIO with morphine in pain and tolerance to analgesia.

A series of experiments using technologies of gene mutation and silencing as well as chemical biology have demonstrated that spinal D-amino acid oxidase (DAAO) contributes to the development of central sensitization-mediated chronic pain and might be a potential molecular target for the treatment of chronic pain. DAAO inhibitors are now under clinical investigations for the management of chronic neuropathic pain. This study examined the interactions between morphine and the DAAO inhibitor CBIO (5-chloro-benzo[d]isoxazol-3-ol) in pain and analgesia tolerance mainly in the formalin test. Given subcutaneously CBIO acutely interacted with morphine in analgesia in an additive manner both in the acute nociception settings (the formalin acute phase nociception, hot-plate test and tail immersion test) and in formalin-induced tonic pain. Bi-daily exposure of CBIO given subcutaneously for 7 days did not produce self-tolerance to analgesia or cross-tolerance to morphine whereas 7-day subcutaneous morphine induced self-tolerance to analgesia but not cross-tolerance to CBIO. More importantly, subcutaneous co-administrations or even single dose of CBIO completely prevented or reversed morphine tolerance to analgesia (exhibited by a single dose or a dose-response curve of morphine) in both formalin-induced acute phase nociception and tonic phase pain. These results, for the first time, identified DAAO as an efficacious molecule mediating morphine tolerance, in addition to clarifying the complex interactions between morphine and DAAO inhibitors probed by CBIO, and provided a pharmacological basis for DAAO inhibitors in combination with morphine to clinically manage pain.

Acute oral toxicity of 3-MCPD mono- and di-palmitic esters in Swiss mice and their cytotoxicity in NRK-52E rat kidney cells.

The acute oral toxicity of 1-palmitoyl-3-chloropropanediol (3-MCPD 1-monopalmitate) and 1,2-bis-palmitoyl-3-chloropropanediol (3-MCPD dipalmitate) in Swiss mice were examined, along with their cytotoxicity in NRK-52E rat kidney cells. LD50 (median lethal dose) value of 3-MCPD 1-monopalmitate was determined 2676.81 mg/kg body weight (BW). The results showed that 3-MCPD 1-monopalmitate dose-dependently decreased the mean body weight, and caused significant increase of serum urea nitrogen and creatinine in dead mice compared to the control and survived mice. Major histopathological changes in mice fed 3-MCPD 1-monopalmitate were renal tubular necrosis, protein casts and spermatids decrease in the seminiferous tubules. According to the limit test for 3-MCPD dipalmitate, LD50 value of 3-MCPD dipalmitate was presumed to be greater than 5000 mg/kg BW. Obvious changes were not observed on mean body weight, absolute and relative organ weight or serum urea nitrogen and creatinine levels in mice fed 3-MCPD dipalmitate. However, renal tubular necrosis, protein casts and spermatids decrease were also observed in the dead mice. In addition, MTT and LDH assay results only showed the cytotoxicity of 3-MCPD 1-monopalmitate in NRK-52E rat kidney cells in a dose-dependent manner. Together, the results indicated a greater toxicity of 3-MCPD 1-monopalmitate compared to 3-MCPD dipalmitate.

Site-specific PEGylation of exenatide analogues markedly improved their glucoregulatory activity.

BACKGROUND AND PURPOSE: Exenatide is a 39-amino-acid peptide widely used to manage type 2 diabetes mellitus. However, it has a short plasma half-life and requires a twice daily injection regime. To overcome these drawbacks we used maleimide-polyethylene glycol to induce site-specific PEGylation. EXPERIMENTAL APPROACH: The analogue PB-105 (ExC39) was produced by replacing cysteine at position 39 of exenatide to provide a free thiol group. PB-105 showed the same glucoregulatory activity as exenatide in mice. Site-specific PEGylation of PB-105 was performed to produce PB-110 (ExC39PEG5kDa), PB-106 (ExC39PEG20kDa), PB-107 (ExC39PEG30kDa) and PB-108 (ExC39PEG40kDa). Their effects on intracellular cAMP, acute glucoregulatory activity and pharmacokinetic profile were compared in mice and rats. KEY RESULTS: PEGylation shifted the concentration-response curve of PB-105 to the right in a parallel, polyethylene glycol mass-dependent manner but with an inflexion point of at least 20 kDa. The activities of PB-107 and PB-108 but not PB-106 were reduced by 90% and 99%. PEGylation affected in vivo glucoregulatory activity in the same 'Inflexion-Shift' fashion at least at 20 kDa, but linearly increased plasma duration and systemic exposure without inflexion. PB-106 had a plasma t(1/2) approximately 10-fold that of PB-105, and exhibited superior glucoregulatory activity compared with PB-105 in normal and diabetic mice. CONCLUSIONS AND IMPLICATIONS: Site-specific PEGylation of exenatide with a permanent amide linkage affects its activity in a new type of 'Inflexion-Shift' fashion. PB-106 is a putative new analogue for treating diabetes; it possesses no loss of in vitro activity, prolonged plasma duration and superior, improved in vivo glucoregulatory activity compared with exenatide.

Intrathecal administration of antioxidants attenuates mechanical pain hypersensitivity induced by REM sleep deprivation in the rat.

Background Sleep deprivation as well as peripheral neuropathy and cutaneous neurogenic inflammation has a facilitatory effect on pain perception. Here we studied whether oxidative stress-related mechanisms in the spinal cord that have been shown to contribute to pain facilitation in peripheral neuropathy and cutaneous neurogenic inflammation play a role in sleep deprivation-induced pain hypersensitivity Methods Flower pot method was used to induce rapid eye movement sleep deprivation (REMSD) of 48 h duration in the rat that had a chronic intrathecal (i.t.) catheter for spinal administration of drugs. Pain behavior was assessed by determining the monofilament-induced limb withdrawal response. Results REMSD of 48 h produced mechanical hypersensitivity that was attenuated in a dose-related fashion by i.t. administration of two different antioxidants, phenyl-N-tert-butylnitrone (PBN) or 4-hydroxy-2,2,6,6-tetramethylpiperidine-1 oxyl (TEMPOL). While both antioxidants attenuated mechanical pain behavior also in control animals, their effects were significantly stronger after REMSD than in control conditions. Conversely, i.t. administration of a reactive oxygen species (ROS) donor, tert-butylhydroperoxide (t-BOOH), in control animals produced pain hypersensitivity that was prevented by i.t. pretreatment with an antioxidant, TEMPOL. I.t. treatment with PBN or TEMPOL at the currently used doses failed to influence motor behavior in the Rotarod test. Conclusions The results indicate that among common mechanisms contributing to mechanical pain hypersensitivity following sleep deprivation as well as nerve injury or neurogenic inflammation is oxidative stress in the spinal cord. Implications Compounds with antioxidant properties might prove useful in suppressing the vicious pronociceptive interaction between chronic pain and sleep-deprivation.

Intrathecal administration of a gap junction decoupler, an inhibitor of Na(+)-K(+)-2Cl(-) cotransporter 1, or a GABA(A) receptor agonist attenuates mechanical pain hypersensitivity induced by REM sleep deprivation in the rat.

We studied the hypothesis that some of the spinal mechanisms that are involved in neuropathic hypersensitivity play a role in hypersensitivity induced by REM sleep deprivation (REMSD). Rats with a chronic intrathecal (i.t.) catheter had REMSD of 48h duration that induced hypersensitivity to mechanical stimulation. After REMSD, the animals were treated i.t. with carbenoxolone (a gap junction decoupler), bumetanide (a blocker of Na(+)-K(+)-2Cl(-) cotransporter 1 or NKCC1), muscimol (a GABA(A) receptor agonist), or pretreated intraperitoneally with minocycline (an inhibitor of microglia activation). Previously, all these treatments attenuated neuropathic hypersensitivity. Following REMSD, carbenoxolone, bumetanide and muscimol had a strong antihypersensitivity effect, whereas pretreatment with minocycline failed to prevent development of hypersensitivity. The results suggest that among spinal pain facilitatory mechanisms that are common to REMSD and neuropathy are NKCC1 blocker- and gap junction decoupler-reversible mechanisms. Moreover, there is a net pain inhibitory effect by spinal administration of an exogenous GABA(A) receptor agonist following REMSD as shown earlier in neuropathy. In contrast, activation of spinal microglia may not be as important for the development of hypersensitivity induced by REMSD as following nerve injury.

Sensitive quantitative determination of oxymatrine and matrine in rat plasma by capillary electrophoresis with stacking induced by moving reaction boundary.

A quantitative method of capillary electrophoresis with sample stacking induced by moving reaction boundary (MRB) was developed for sensitive determination of oxymatrine (OMT) and matrine (MT) in rat plasma. The experimental conditions were optimized firstly. Below are the optimized experimental conditions: 20 mM sodium formate solution (HCOONa, adjusted to pH 10.70 by ammonia) as sample solution, 3 min 14 mbar sample injection, 40 mM formic buffer (HCOOH-HCOONa, pH 2.60) as stacking buffer, 7 min 14 mbar injection of stacking buffer, 100 mM HCOOH-HCOONa (pH 4.80) as separation buffer, 73 cm capillary (effective length 64 cm), 21 kV voltage, 210 nm wavelength. Under the optimized conditions, higher than 60-fold sensitivity improvement of the stacking was simply achieved as compared with capillary zone electrophoresis, and the detectable limits obtained for OMT and MT were 0.26 and 0.19 microg mL(-1), respectively. Then, numerous demonstrations were carefully performed for the methodological validations of OMT and MT in rate plasma, including high specificity of method, good linearity (r=0.9993 for OMT, r=0.9991 for MT), fair wide linear concentration range (1.30-65.00 microg mL(-1) for OMT, 0.84-42.00 microg mL(-1) for MT), low limit of detection (1.03 microg mL(-1) for OMT, 0.38 microg mL(-1) for MT), less than 5% intra- and inter-day variance value, and higher than 96% recovery of OMT and MT in plasma. The developed method could be used for the trace analyses of OMT and MT in plasma and was finally used for the investigation on pharmacokinetic study of OMT in rat plasma.