The Preventive Effect of Urinary Trypsin Inhibitor on Postoperative Cognitive Dysfunction, on the Aspect of Behavior, Evaluated by Y-Maze Test, via Modulation of Microglial Activity.

This experimental study was designed to evaluate the effect of ulinastatin, a urinary trypsin inhibitor, on postoperative cognitive dysfunction (POCD) in rats under general anesthesia with isoflurane, on the aspect of behavior, as evaluated using a Y-maze test and focusing on microglial activity. Ulinastatin (50,000 U/mL) and normal saline (1 mL) were randomly (1:1) administered intraperitoneally to the ulinastatin and control groups, respectively, before general anesthesia. Anesthesia with isoflurane 1.5 volume% was maintained for 2 h. The Y-maze test was used to evaluate cognitive function. Neuronal damage using caspase-1 expression, the degree of inflammation through cytokine detection, and microglial activation with differentiation of the phenotypic expression were evaluated. Twelve rats were enrolled in the study and evenly allocated into the two groups, with no dropouts from the study. The Y-maze test showed similar results in the two groups before general anesthesia (63 ± 12% in the control group vs. 64 ± 12% in the ulinastatin group, p = 0.81). However, a significant difference was observed between the two groups after general anesthesia (17 ± 24% in the control group vs. 60 ± 12% in the ulinastatin group, p = 0.006). The ulinastatin group showed significantly lower expression of caspase-1. Pro-inflammatory cytokine levels were significantly lower in the ulinastatin group than in the control group. The ulinastatin group had a significantly lower microglial activation (41.74 ± 10.56% in the control group vs. 4.77 ± 0.56% in the ulinastatin, p < 0.001), with a significantly lower activation of M1 phenotypes (52.19 ± 7.83% in the control group vs. 5.58 ± 0.76% in the ulinastatin group, p < 0.001). Administering ulinastatin before general anesthesia prevented neuronal damage and cognitive decline after general anesthesia, in terms of the aspect of behavior, as evaluated by the Y-maze test. The protective effect of ulinastatin was associated with the inhibition of microglial activation, especially the M1 phenotype.

Employing the sustained-release properties of poly(lactic-co-glycolic acid) nanoparticles to reveal a novel mechanism of sodium-hydrogen exchanger 1 in neuropathic pain.

Neuropathic pain is caused by injury or disease of the somatosensory system, and its course is usually chronic. Several studies have been dedicated to investigating neuropathic pain-related targets; however, little attention has been paid to the persistent alterations that these targets, some of which may be crucial to the pathophysiology of neuropathic pain. The present study aimed to identify potential targets that may play a crucial role in neuropathic pain and validate their long-term impact. Through bioinformatics analysis of RNA sequencing results, we identified Slc9a1 and validated the reduced expression of sodium-hydrogen exchanger 1 (NHE1), the protein that Slc9a1 encodes, in the spinal nerve ligation (SNL) model. Colocalization analysis revealed that NHE1 is primarily co-localized with vesicular glutamate transporter 2-positive neurons. In vitro experiments confirmed that poly(lactic-co-glycolic acid) nanoparticles loaded with siRNA successfully inhibited NHE1 in SH-SY5Y cells, lowered intracellular pH, and increased intracellular calcium concentrations. In vivo experiments showed that sustained suppression of spinal NHE1 expression by siRNA-loaded nanoparticles resulted in delayed hyperalgesia in naïve and SNL model rats, whereas amiloride-induced transient suppression of NHE1 expression yielded no significant changes in pain sensitivity. We identified Slc9a1, which encodes NHE1, as a key gene in neuropathic pain. Utilizing the sustained release properties of nanoparticles enabled us to elucidate the chronic role of decreased NHE1 expression, establishing its significance in the mechanisms of neuropathic pain.

LPC20K modified from krill oil ameliorates the scopolamine-induced cognitive impairment.

Alzheimer's disease (AD) is characterized by cognitive impairment. It is common in the elderly. Etiologically, dysfunction of cholinergic neurotransmitter system is prominent in AD. However, disease modifying drug for AD is still unavailable. We hypothesized that krill oil and modified krill oil containing 20 % lysophosphatidylcholine-docosahexaenoic acid (LPC-DHA, LPC20K) could play a crucial role in AD by improving cognitive functions measured by several behavioral tests. We found that LPC20K could ameliorate short-term, long-term, spatial, and object recognition memory under cholinergic hypofunction states. To find the underlying mechanism involved in the effect of LPC20K on cognitive function, we investigated changes of signaling molecules using Western blotting. Expression levels of protein kinase C zeta (PKCζ) and postsynaptic density protein 95 (PSD-95), and phosphorylation levels of extracellular signal-regulated kinase (ERK), Ca2+/calmodulin-dependent protein kinase Ⅱ (CaMKⅡ), and cAMP response element-binding protein (CREB) were significantly increased in LPC20K-administered group compared to those in the memory impairment group. Moreover, the expression levels of BDNF were temporally increased especially 6 or 9 h after administration of LPC20K compared with the control group. These results suggest that LPC20K could ameliorate memory impairment caused by hypocholinergic state by enhancing the expression levels of PKCζ and PSD-95, and phosphorylation levels of ERK, CaMKⅡ and CREB and increasing BDNF expression levels. Therefore, LPC20K could be used as a dietary supplement against cognitive impairment observed in diseases such as AD with a hypocholinergic state.

Effects of oleanolic acid and ursolic acid on depression-like behaviors induced by maternal separation in mice.

Oleanolic acid (OA) and ursolic acid (UA) are structural isomeric triterpenoids. Both triterpenoids have been reported to be able to improve depression. However, no studies have compared their effects in the same system. Whether OA or UA could ameliorate depression-like behaviors in maternal separation (MS)-induced depression-like model was investigated. MS model is a well-accepted mouse model that can reflect the phenotype and pathogenesis of depression. Depression is a mental illness caused by neuroinflammation or changes in neuroplasticity in certain brain regions, such as the prefrontal cortex and hippocampus. Depression-like behaviors were measured using splash test or forced swimming test. In addition, anxiety-like behaviors were also measured using the open field test or elevated plus-maze test. MS-treated female mice showed greater depression-like behaviors than male mice, and that OA improved several depression-like behaviors, whereas UA only relieved anxiety-like behavior of MS-treated mice. Microglial activation, expression levels of TNF-α, and mRNA levels of IDO1 were increased in the hippocampi of MS-treated female mice. However, OA and UA treatments attenuated such increases. In addition, expression levels of synaptophysin and PSD-95 were decreased in the hippocampi of MS-treated female mice. These decreased expression levels of synaptophysin were reversed by both OA and UA treatments, although decreased PSD-95 expression levels were only reversed by OA treatment. Our findings suggest that MS cause depression-like behaviors through female-specific neuroinflammation, changes of tryptophan metabolism, and alterations of synaptic plasticity. Our findings also suggest that OA could reverse MS-induced depression-like behaviors more effectively than UA.

N-Cyano sulfilimine functional group as a nonclassical amide bond bioisostere in the design of a potent analogue to anthranilic diamide insecticide.

To explore the potential of the N-cyano sulfilimine group as an amide bond isostere, a derivative of the blockbuster anthranilic diamide, chlorantramiliprole, was synthesized and evaluated with regard to its physicochemical properties, permeability, and biological activity. Given the combination of N-cyano sulfilimine chlorantraniliprole 1 and its strong hydrogen bond acceptor character, high permeability, and excellent insecticidal activity, the N-cyano sulfilimine functional group could be considered as an amide bond isostere.

Ionic Diffusion-Driven Ionovoltaic Transducer for Probing Ion-Molecular Interactions at Solid-Liquid Interface.

Ion-solid surface interactions are one of the fundamental principles in liquid-interfacing devices ranging from various electrochemical systems to electrolyte-driven energy conversion devices. The interplays between these two phases, especially containing charge carriers in the solid layer, work as a pivotal role in the operation of these devices, but corresponding details of those effects remain as unrevealed issues in academic fields. Herein, an ion-charge carrier interaction at an electrolyte-semiconductor interface is interrogated with an ion-dynamics-induced (ionovoltaic) energy transducer, controlled by interfacial self-assembled molecules. An electricity generating mechanism from interfacial ionic diffusion is elucidated in terms of the ion-charge carrier interaction, originated from a dipole potential effect of the self-assembled molecular layer (SAM). In addition, this effect is found to be modulated via chemical functionalization of the interfacial molecular layer and transition metal ion complexation therein. With the aiding of surface analytic techniques and a liquid-interfacing Hall measurement, electrical behaviors of the device depending on the magnitude of the ion-ligand complexation are interrogated, thereby demonstrating the ion-charge carrier interplays spanning at electrolyte-SAM-semiconductor interface. Hence, this system can be applied to study molecular interactions, including chemical and physical influences, occurring at the solid-liquid interfacial region.

Probing an Interfacial Ionic Pairing-Induced Molecular Dipole Effect in Ionovoltaic System.

A surficial molecular dipole effect depending on ion-molecular interactions has been crucial issues regarding to an interfacial potential, which can modulate solid electronic and electrochemical systems. Their properties near the interfacial region can be dictated by specific interactions between surface and adsorbates, but understandings of the corresponding details remain at interesting issues. Here, intuitive observations of an ionic pair formation-induced interfacial potential shifts are presented with an ionovoltaic system, and corresponding output signal variations are analyzed in terms of the surficial dipole changes on self-assembled monolayer. With aiding of photoelectron spectroscopies and density function theory simulation, the ionic pair formation-induced potential shifts are revealed to strongly rely on a paired molecular structure and a binding affinity of the paired ionic moieties. Chemical contributions to the binding event are interrogated in terms of polarizability in each ionic group and consistent with chaotropic/kosmotropic character of the ionic groups. Based on these findings, the ionovoltaic output changes are theoretically correlated with an adsorption isotherm reflecting the molecular dipole effect, thereby demonstrating as an efficient interfacial molecular probing method under electrolyte interfacing conditions.

Verification of Carrier Concentration-Dependent Behavior in Water-Infiltration-Induced Electricity Generation by Ionovoltaic Effect.

Water-infiltration-induced power generation has the renewable characteristic of generating electrical energy from ambient water. Importantly, it is found that the carrier concentration in semiconductor constituting the energy generator seriously affect the electricity generation. Nevertheless, few studies are conducted on the influence of semiconductor carrier concentration, a crucial factor on electricity generation. Due to this, understanding of the energy harvesting mechanism is still insufficient. Herein, the semiconductor carrier concentration-dependent behavior in water-infiltration-induced electricity generation and the energy harvesting mechanism by ionovoltaic effect are comprehensively verified. A clue to enhance the electric power generation efficiency is also proposed. When 20 µL of water (NaCl, 0.1 m) infiltrates into a porous CuO nanowires film (PCNF), electric power of ≈0.5 V and ≈1 µA are produced for 25 min. Moreover, the PCNF shows good practicability by generating electricity using various ambient water, turning on LEDs, and being fabricated as a curved one.

Application of PLGA nanoparticles to enhance the action of duloxetine on microglia in neuropathic pain.

Duloxetine (DLX) is a selective serotonin and noradrenaline reuptake inhibitor (SNRI) used for the treatment of pain, but it has been reported to show side effects in 10-20% of patients. Its analgesic efficacy in central pain is putatively related to its influence on descending inhibitory neuronal pathways. However, DLX can also affect the activation of microglia. This study was performed to investigate whether PLGA nanoparticles (NPs), which are expected to enhance targeting to microglia, can improve the analgesic efficacy and limit the side effects of DLX. PLGA NPs encapsulating a low dose of DLX (DLX NPs) were synthesized and characterized and their localization was determined. The analgesic and anti-inflammatory effects of DLX NPs were evaluated in a spinal nerve ligation (SNL)-induced neuropathic pain model. The analgesic effect of DLX lasted for only a few hours and disappeared within 1 day. However, DLX NPs alleviated mechanical allodynia, and the effect was maintained for 1 week. DLX NPs were localized to the spinal microglia and suppressed microglial activation, phosphorylation of p38/NF-κB-mediated pathways and the production of inflammatory cytokines in the spinal dorsal horn of SNL rats. We demonstrated that DLX NPs can provide a prolonged analgesic effect by enhanced targeting of microglia. Our observations imply that DLX delivery through nanoparticle encapsulation allows drug repositioning with a prolonged analgesic effect, and reduces the potential side effects of abuse and overdose.

IKBKB siRNA-Encapsulated Poly (Lactic-co-Glycolic Acid) Nanoparticles Diminish Neuropathic Pain by Inhibiting Microglial Activation.

Activation of nuclear factor-kappa B (NF-κB) in microglia plays a decisive role in the progress of neuropathic pain, and the inhibitor of kappa B (IκB) is a protein that blocks the activation of NF-κB and is degraded by the inhibitor of NF-κB kinase subunit beta (IKBKB). The role of IKBKB is to break down IκB, which blocks the activity of NF-kB. Therefore, it prevents the activity of NK-kB. This study investigated whether neuropathic pain can be reduced in spinal nerve ligation (SNL) rats by reducing the activity of microglia by delivering IKBKB small interfering RNA (siRNA)-encapsulated poly (lactic-co-glycolic acid) (PLGA) nanoparticles. PLGA nanoparticles, as a carrier for the delivery of IKBKB genes silencer, were used because they have shown potential to enhance microglial targeting. SNL rats were injected with IKBKB siRNA-encapsulated PLGA nanoparticles intrathecally for behavioral tests on pain response. IKBKB siRNA was delivered for suppressing the expression of IKBKB. In rats injected with IKBKB siRNA-encapsulated PLGA nanoparticles, allodynia caused by mechanical stimulation was reduced, and the secretion of pro-inflammatory mediators due to NF-κB was reduced. Delivering IKBKB siRNA through PLGA nanoparticles can effectively control the inflammatory response and is worth studying as a treatment for neuropathic pain.

NFAT5 Deficiency Alleviates Formalin-Induced Inflammatory Pain Through mTOR.

Nuclear factor of activated T cells (NFAT5) is a well-known transcription factor that regulates the expression of genes involved in osmotic stress. However, the role of NFAT5 in inflammatory pain remains unknown. Here, we studied the function of NFAT5 in inflammatory pain using NFAT5-heterozygous (Het) mice. To study inflammatory pain, we injected 10 µL of 2% formalin into the right hind paws of mice and monitored pain behaviors, such as licking, lifting, and flinching, for 60 min. After the first 15 min (phase I), there were no significant differences in pain behaviors between wild-type (WT) and NFAT5-Het mice. However, from 15-60 min (phase II), NFAT5-Het mice displayed significantly fewer pain behaviors compared to WT mice. Further, the expression levels of inflammatory-pain-related factors, including c-Fos, phosphorylated extracellular signal-regulated kinase (p-ERK), and phosphorylated n-methyl-D-aspartate receptor subunit 2B (p-NR2B), were significantly elevated in the spinal dorsal neurons of formalin-treated WT mice but was not elevated in NFAT5-Het mice. Similarly, c-Fos, p-ERK, and p-NR2B levels were significantly higher in glutamate-treated PC12 neuronal cells but were not affected by Nfat5 silencing in glutamate-treated PC12 cells. Altogether, our findings suggest that NFAT5 deficiency may mitigate formalin-induced inflammatory pain by upregulating mammalian target of rapamycin (mTOR) expression and downregulating its downstream factors in spinal dorsal neurons. Therefore, NFAT5 is a potential therapeutic target for the treatment of inflammatory pain.

Electron Density-Change in Semiconductor by Ion-Adsorption at Solid-Liquid Interface.

The change in electrical properties of electrodes by adsorption or desorption at interfaces is a well-known phenomenon required for signal production in electrically transduced sensing technologies. Furthermore, in terms of electrolyte-insulator-semiconductor (EIS) structure, several studies of energy conversion techniques focused on ion-adsorption at the solid-liquid interface have suggested that the electric signal is generated by ionovoltaic phenomena. However, finding substantial clues for the ion-adsorption phenomena in the EIS structure is still a difficult task because direct evidence for carrier accumulation in semiconductors by Coulomb interactions is insufficient. Here, a sophisticated Hall measurement system is demonstrated to quantitatively analyze accumulated electron density-change inside the semiconductor depending on the ion-adsorption at the solid-liquid interface. Also, an enhanced EIS-structured device is designed in an aqueous-soaked system that works with the ionovoltaic principle to monitor the ion-dynamics in liquid electrolyte media, interestingly confirming ion-concentration dependence and ion-specificity by generated peak voltages. This newly introduced peculiar method contributes to an in-depth understanding of the ionovoltaic phenomena in terms of carrier actions in the semiconductors and ionic behaviors in the aqueous-bulk phases, providing informative analysis about interfacial adsorptions that can expand the scope of ion-sensing platforms.

Design and synthesis of acrylate and acrylamide substituted pyrimidinediones as potential PPO herbicides.

PPO herbicides emerge to be widely use in the agricultural field and a focus of research to many scientists due to its environmentally-friendly properties. In lieu with this, this study presents acrylate and acrylamide substituted pyrimidinediones as PPO herbicide candidates. Most synthesized compounds exhibits herbicidal activities against both monocot and dicot weeds, especially, compound 5a which showed non-selective superior activity against the commercialized, Saflufenacil. Compound 5a was further tested for residual effect and showed promising results as shorter period is needed to cultivate the next crops. The synthesized acrylate and acrylamide substituted pyrimidinediones, especially, 5a could potentially be utilized in the development of commercial protoporphyrinogen oxidase inhibitors with further tests and studies.

Synthesis and Properties of Pentafluorosulfanyl Group (SF5)-Containing Meta-Diamide Insecticides.

Herein, we describe novel pentafluorosulfanyl (SF5) group-containing meta-diamide insecticides. For the facile preparation of the SF5-based compounds 4a-d, practical synthetic methods were applied. Among newly synthesized compounds, 3-benzamido-N-(2,6-dimethyl-4-(pentafluoro-λ6-sulfanyl)phenyl)-2-fluorobenzamide 4d showed (i) a high insecticidal activity, (ii) an excellent selectivity to insects, and (iii) good levels of water solubility and log P values. In this study, we demonstrated that the pentafluorosulfanyl moiety could serve as an attractive functionality for the discovery of a new scope of crop-protecting agents.

CX3CR1-Targeted PLGA Nanoparticles Reduce Microglia Activation and Pain Behavior in Rats with Spinal Nerve Ligation.

Activation of CX3CR1 in microglia plays an important role in the development of neuropathic pain. Here, we investigated whether neuropathic pain could be attenuated in spinal nerve ligation (SNL)-induced rats by reducing microglial activation through the use of poly(D,L-lactic-co-glycolic acid) (PLGA)-encapsulated CX3CR1 small-interfering RNA (siRNA) nanoparticles. After confirming the efficacy and specificity of CX3CR1 siRNA, as evidenced by its anti-inflammatory effects in lipopolysaccharide-stimulated BV2 cells in vitro, PLGA-encapsulated CX3CR1 siRNA nanoparticles were synthesized by sonication using the conventional double emulsion (W/O/W) method and administered intrathecally into SNL rats. CX3CR1 siRNA-treated rats exhibited significant reductions in the activation of microglia in the spinal dorsal horn and a downregulation of proinflammatory mediators, as well as a significant attenuation of mechanical allodynia. These data indicate that the PLGA-encapsulated CX3CR1 siRNA nanoparticles effectively reduce neuropathic pain in SNL-induced rats by reducing microglial activity and the expression of proinflammatory mediators. Therefore, we believe that PLGA-encapsulated CX3CR1 siRNA nanoparticles represent a valuable new treatment option for neuropathic pain.

BMAL1 Suppresses Proliferation, Migration, and Invasion of U87MG Cells by Downregulating Cyclin B1, Phospho-AKT, and Metalloproteinase-9.

Several studies have shown that brain and muscle aryl hydrocarbon receptor nuclear translocator-like 1 (BMAL1), an important molecule for maintaining circadian rhythms, inhibits the growth and metastasis of tumor cells in several types of cancer, including lung, colon, and breast cancer. However, its role in glioblastoma has not yet been established. Here, we addressed the function of BMAL1 in U87MG glioblastoma cells with two approaches-loss and gain of function. In the loss of function experiments, cell proliferation in U87MG cells transfected with small interfering RNA (siRNA) targeting BMAL1 was increased by approximately 24% (small interfering (si)-NC 0.91 ± 0.00 vs. si-BMAL1 1.129 ± 0.08) via upregulation of cyclin B1. In addition, cell migration and invasion of BMAL1 siRNA-treated glioblastoma cells were elevated by approximately 20% (si-NC 51.00 ± 1.53 vs. si-BMAL161.33 ± 0.88) and 209% (si-NC 21.28 ± 1.37 vs. si-BMAL1 44.47 ± 3.48), respectively, through the accumulation of phosphorylated-AKT (p-AKT) and matrix metalloproteinase (MMP)-9. Gain of function experiments revealed that adenovirus-mediated ectopic expression of BMAL1 in U87MG cells resulted in a 19% (Adenovirus (Ad)-vector 0.94± 0.03 vs. Ad-BMAL1 0.76 ± 0.03) decrease in cell proliferation compared with the control via downregulation of cyclin B1 and increased early and late apoptosis due to changes in the levels of BCL2-associated X protein (BAX), B-cell lymphoma 2 (BCL-2), and cleaved caspase-3. Likewise, cell migration and invasion were attenuated by approximately 24% (Ad-vector 55.00 ± 0.00 vs. Ad-BMAL1 41.83 ± 2.90) and 49% (Ad-vector 70.01 ± 1.24 vs. Ad-BMAL1 35.55 ± 1.78), respectively, in BMAL1-overexpressing U87MG cells following downregulation of p-AKT and MMP-9. Taken together, our results suggest that BMAL1 acts as an anti-cancer gene by altering the proliferation, migration, and invasion of glioblastoma cells. Therefore, the BMAL1 gene could be a potential therapeutic target in the treatment of glioblastoma.

The mTOR Inhibitor Rapamycin Prevents General Anesthesia-Induced Changes in Synaptic Transmission and Mitochondrial Respiration in Late Postnatal Mice.

Preclinical animal studies have continuously reported the possibility of long-lasting neurotoxic effects after general anesthesia in young animals. Such studies also show that the neurological changes induced by anesthesia in young animals differ by their neurodevelopmental stage. Exposure to anesthetic agents increase dendritic spines and induce sex-dependent changes of excitatory/inhibitory synaptic transmission in late postnatal mice, a critical synaptogenic period. However, the mechanisms underlying these changes remain unclear. Abnormal activation of the mammalian target of rapamycin (mTOR) signaling pathway, an important regulator of neurodevelopment, has also been shown to induce similar changes during neurodevelopment. Interestingly, previous studies show that exposure to general anesthetics during neurodevelopment can activate the mTOR signaling pathway. This study, therefore, evaluated the role of mTOR signaling after exposing postnatal day (PND) 16/17 mice to sevoflurane, a widely used inhalation agent in pediatric patients. We first confirmed that a 2-h exposure of 2.5% sevoflurane could induce widespread mTOR phosphorylation in both male and female mice. Pretreatment with the mTOR inhibitor rapamycin not only prevented anesthesia-induced mTOR phosphorylation, but also the increase in mitochondrial respiration and male-dependent enhancement of excitatory synaptic transmission. However, the changes in inhibitory synaptic transmission that appear after anesthesia in female mice were not affected by rapamycin pretreatment. Our results suggest that mTOR inhibitors may act as potential therapeutic agents for anesthesia-induced changes in the developing brain.

Synthesis, Physicochemical Properties, and Biological Activities of 4-(S-Methyl-N-(2,2,2-Trifluoroacetyl)Sulfilimidoyl) Anthranilic Diamide.

Novel anthranilic diamides with sulfilimidoyl and sulfoximidoyl functionalities were successfully prepared. Among newly-prepared organosulfur compounds, 3-bromo-1-(3-chloropyridin-2-yl)-N-(2-methyl-6-(methylcarbamoyl)-4-(methylthio)phenyl)-1H-pyrazole-5-carboxamide and (S,E)-3-bromo-1-(3-chloropyridin-2-yl)-N-(2-methyl-4-(S-methyl-N-(2,2,2-trifluoroacetyl)sulfinimidoyl)-6-(methylcarbamoyl)phenyl)-1H-pyrazole-5-carboxamide showed good levels of efficacy and a strong correlation between insecticidal activities and physical properties, respectively. In particular, available data indicated that the N-trifluoroacetyl sulfilimine moiety could be an appealing structural scaffold for the discovery of a new crop-protecting agent.

Evans Blue Reduces Neuropathic Pain Behavior by Inhibiting Spinal ATP Release.

Upon peripheral nerve injury, vesicular ATP is released from damaged primary afferent neurons. This extracellular ATP subsequently activates purinergic receptors of the spinal cord, which play a critical role in neuropathic pain. As an inhibitor of the vesicular nucleotide transporter (VNUT), Evans blue (EB) inhibits the vesicular storage and release of ATP in neurons. Thus, we tested whether EB could attenuate neuropathic pain behavior induced by spinal nerve ligation (SNL) in rats by targeting VNUT. An intrathecal injection of EB efficiently attenuated mechanical allodynia for five days in a dose-dependent manner and enhanced locomotive activity in an SNL rat model. Immunohistochemical analysis showed that EB was found in VNUT immunoreactivity on neurons in the dorsal root ganglion and the spinal dorsal horn. The level of ATP in cerebrospinal fluid in rats with SNL-induced neuropathic pain decreased upon administration of EB. Interestingly, EB blocked ATP release from neurons, but not glial cells in vitro. Eventually, the loss of ATP decreased microglial activity in the ipsilateral dorsal horn of the spinal cord, followed by a reduction in reactive oxygen species and proinflammatory mediators, such as interleukin (IL)-1ß and IL-6. Finally, a similar analgesic effect of EB was demonstrated in rats with monoiodoacetate-induced osteoarthritis (OA) pain. Taken together, these data demonstrate that EB prevents ATP release in the spinal dorsal horn and reduces the ATP/purinergic receptor-induced activation of spinal microglia followed by a decline in algogenic substances, thereby relieving neuropathic pain in rats with SNL.

Analgesic Effect of Toll-like Receptor 4 Antagonistic Peptide 2 on Mechanical Allodynia Induced with Spinal Nerve Ligation in Rats.

Neuroinflammation is one of the key mechanisms of neuropathic pain, which is primarily mediated by the Toll-like receptor 4 (TLR4) signaling pathways in microglia. Therefore, TLR4 may be a reasonable target for treatment of neuropathic pain. Here, we examined the analgesic effect of TLR4 antagonistic peptide 2 (TAP2) on neuropathic pain induced by spinal nerve ligation in rats. When lipopolysaccharide (LPS)-stimulated BV2 microglia cells were treated with TAP2 (10 µM), the mRNA levels of proinflammatory mediators, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, cyclooxygenase (COX)-2, and inducible nitric oxide synthase (iNOS), were markedly decreased by 54-83% as determined by quantitative PCR (qPCR) analysis. Furthermore, when TAP2 (25 nmol in 20 µL PBS) was intrathecally administered to the spinal nerve ligation-induced rats on day 3 after surgery, the mechanical allodynia was markedly decreased for approximately 2 weeks in von Frey filament tests, with a reduction in microglial activation. On immunohistochemical and qPCR analyses, both the level of reactive oxygen species and the gene expression of the proinflammatory mediators, such as TNF-α, IL-1ß, IL-6, COX-2, and iNOS, were significantly decreased in the ipsilateral spinal dorsal horn. Finally, the analgesic effect of TAP2 was reproduced in rats with monoiodoacetate-induced osteoarthritic pain. The findings of the present study suggest that TAP2 efficiently mitigates neuropathic pain behavior by suppressing microglial activation, followed by downregulation of neuropathic pain-related factors, such as reactive oxygen species and proinflammatory molecules. Therefore, it may be useful as a new analgesic for treatment of neuropathic pain.