APT1-Mediated Depalmitoylation Regulates Hippocampal Synaptic Plasticity.

Palmitoylation may be relevant to the processes of learning and memory, and even disorders, such as post-traumatic stress disorder and aging-related cognitive decline. However, underlying mechanisms of palmitoylation in these processes remain unclear. Herein, we used acyl-biotin exchange, coimmunoprecipitation and biotinylation assays, and behavioral and electrophysiological methods, to explore whether palmitoylation is required for hippocampal synaptic transmission and fear memory formation, and involved in functional modification of synaptic proteins, such as postsynapse density-95 (PSD-95) and glutamate receptors, and detected if depalmitoylation by specific enzymes has influence on glutamatergic synaptic plasticity. Our results showed that global palmitoylation level, palmitoylation of PSD-95 and glutamate receptors, postsynapse density localization of PSD-95, surface expression of AMPARs, and synaptic strength of cultured hippocampal neurons were all enhanced by TTX pretreatment, and these can be reversed by inhibition of palmitoylation with palmitoyl acyl transferases inhibitors, 2-bromopalmitate and N-(tert-butyl) hydroxylamine hydrochloride. Importantly, we also found that acyl-protein thioesterase 1 (APT1)-mediated depalmitoylation is involved in palmitoylation of PSD-95 and glutamatergic synaptic transmission. Knockdown of APT1, not protein palmitoyl thioesterase 1, with shRNA, or selective inhibition, significantly increased AMPAR-mediated synaptic strength, palmitoylation levels, and synaptic or surface expression of PSD-95 and AMPARs. Results from hippocampal tissues and fear-conditioned rats showed that palmitoylation is required for synaptic strengthening and fear memory formation. These results suggest that palmitoylation and APT1-mediated depalmitoylation have critical effects on the regulation of glutamatergic synaptic plasticity, and it may serve as a potential target for learning and memory-associated disorders.SIGNIFICANCE STATEMENT Fear-related anxiety disorders, including post-traumatic stress disorder, are prevalent psychiatric conditions, and fear memory is associated with hyperexcitability in the hippocampal CA1 region. Palmitoylation is involved in learning and memory, but mechanisms coupling palmitoylation with fear memory acquisition remain poorly understood. This study demonstrated that palmitoylation is essential for postsynapse density-95 clustering and hippocampal glutamatergic synaptic transmission, and APT1-mediated depalmitoylation plays critical roles in the regulation of synaptic plasticity. Our study revealed that molecular mechanism about downregulation of APT1 leads to enhancement of AMPAR-mediated synaptic transmission, and that palmitoylation cycling is implicated in fear conditioning-induced synaptic strengthening and fear memory formation.

Upregulation of dynorphin/kappa opioid receptor system in the dorsal hippocampus contributes to morphine withdrawal-induced place aversion.

Aversive emotion of opioid withdrawal generates motivational state leading to compulsive drug seeking and taking. Kappa opioid receptor (KOR) and its endogenous ligand dynorphin have been shown to participate in the regulation of aversive emotion. In the present study, we investigated the role of dynorphin/KOR system in the aversive emotion following opioid withdrawal in acute morphine-dependent mice. We found that blockade of KORs before pairing by intracerebroventricular injection of KOR antagonist norBNI (20, 40 µg) attenuated the development of morphine withdrawal-induced conditioned place aversion (CPA) behavior. We further found that morphine withdrawal increased dynorphin A expression in the dorsal hippocampus, but not in the amygdala, prefrontal cortex, nucleus accumbens, and thalamus. Microinjection of norBNI (20 µg) into the dorsal hippocampus significantly decreased morphine withdrawal-induced CPA behavior. We further found that p38 MAPK was significantly activated in the dorsal hippocampus after morphine withdrawal, and the activation of p38 MAPK was blocked by pretreatment with norBNI. Accordingly, microinjection of p38 MAPK inhibitor SB203580 (5 µg) into the dorsal hippocampus significantly decreased morphine withdrawal-produced CPA behavior. This study demonstrates that upregulation of dynorphin/KOR system in the dorsal hippocampus plays a critical role in the formation of aversive emotion associated with morphine withdrawal, suggesting that KOR antagonists may have therapeutic value for the treatment of opioid withdrawal-induced mood-related disorders.

Koumine Attenuates Neuroglia Activation and Inflammatory Response to Neuropathic Pain.

Despite decades of studies, the currently available drugs largely fail to control neuropathic pain. Koumine-an alkaloidal constituent derived from the medicinal plant Gelsemium elegans Benth.-has been shown to possess analgesic and anti-inflammatory properties; however, the underlying mechanisms remain unclear. In this study, we aimed to investigate the analgesic and anti-inflammatory effects and the possible underlying mechanisms of koumine. The analgesic and anti-inflammatory effects of koumine were explored by using chronic constriction injury of the sciatic nerve (CCI) neuropathic pain model in vivo and LPS-induced injury in microglia BV2 cells in vitro. Immunofluorescence staining and Western blot analysis were used to assess the modulator effect of koumine on microglia and astrocyte activation after CCI surgery. Enzyme-linked immunosorbent assay (ELISA) was used to evaluate the levels of proinflammatory cytokines. Western blot analysis and quantitative real-time polymerase chain reaction (qPCR) were used to examine the modulator effect of koumine on microglial M1 polarization. We found that single or repeated treatment of koumine can significantly reduce neuropathic pain after nerve injury. Moreover, koumine showed inhibitory effects on CCI-evoked microglia and astrocyte activation and reduced proinflammatory cytokine production in the spinal cord in rat CCI models. In BV2 cells, koumine significantly inhibited microglia M1 polarization. Furthermore, the analgesic effect of koumine was inhibited by a TSPO antagonist PK11195. These findings suggest that the analgesic effects of koumine on CCI-induced neuropathic pain may result from the inhibition of microglia activation and M1 polarization as well as the activation of astrocytes while sparing the anti-inflammatory responses to neuropathic pain.

Effects of Danggui Sini decoction on neuropathic pain: experimental studies and clinical pharmacological significance of inhibiting glial activation and proinflammatory cytokines in the spinal cord
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AIM: Neuropathic pain responds poorly to drug treatments. Partial relief is achieved in only about half of the patients. Danggui Sini decoction (DSD), an aqueous extract of Angelica sinensis, Ramulus Cinnamomi, and Radix Puerariae, has been used extensively in China to treat inflammatory and ischemic diseases. The current study examined the putative effects of DSD on neuropathic pain. METHOD: We used two commonly-used animal models: chronic constriction injury (CCI) and diabetic neuropathy for the study. And we examined effects of DSD on pain response, activation of microglia and astroglia in spinal dorsal horn, and expression of proinflammatory cytokines in the spinal cord. RESULTS: Consecutive intragastric administration of DSD (25 - 100 mg/kg) for 10 days inhibited the mechanical and thermal nociceptive response induced by CCI and diabetes without interfering with the normal pain response. Meanwhile, in both models, DSD inhibited the over-expression of specific markers for microglia (Iba-1) and astroglia (GFAP) activation in the spinal dorsal horn. DSD also reduced the elevated nuclear NF-κB level and inhibited the up-regulation of proinflammatory cytokines, such as IL-6, IL-1ß, and TNF-α, in the spinal cord. CONCLUSION: DSD can alleviate CCI and diabetes-induced neuropathic pain, and its effectiveness might be due to the inhibition of neuroinflammation in the spinal dorsal horn. The anti-inflammation effect of DSD may be related to the suppression of spinal NF-κB activation and/or cytokines expression.
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Effects of Koumine on Adjuvant- and Collagen-Induced Arthritis in Rats.

To examine the effect of koumine, a Gelsemium alkaloid, on two experimental models of rheumatoid arthritis (RA), rats with adjuvant-induced arthritis (AIA) and collagen-induced arthritis (CIA) were administered koumine (0.6, 3, or 15 mg/kg/day) or vehicle through gastric gavage (i.g.). Clinical evaluation was performed via measurements of hind paw volume, arthritis index (AI) score, mechanical withdrawal threshold, organ weight, and by radiographic and histological examinations. Levels of interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, and antitype II collagen (CII) antibody were also examined. In rats with AIA, koumine reduced the AI score and mechanical allodynia of the injected hind paw in a dose-dependent manner and significantly inhibited increase in thymus and liver weights. In rats with CIA, koumine inhibited increase in hind paw volume, AI score, and mechanical allodynia in a dose-dependent manner and reduced joint space narrowing. Furthermore, koumine also attenuated the increase in the expression of IL-1ß and TNF-α, as well as the robust increase of serum anti-CII antibodies in response to immunization. These results suggested that koumine effectively attenuated arthritis progression in two rat models of RA and that this therapeutic effect may be associated with its immunoregulatory action.

Koumine enhances spinal cord 3α-hydroxysteroid oxidoreductase expression and activity in a rat model of neuropathic pain.

BACKGROUND: Koumine is an alkaloid monomer found abundantly in Gelsemium plants. It has been shown to reverse thermal hyperalgesia and mechanical allodynia induced by sciatic nerve chronic constriction injury (CCI) in rats in a dose-dependent manner. Interestingly, this effect is mediated by elevated allopregnanolone levels in the spinal cord (SC). Since 3α-hydroxysteroid oxidoreductase (3α-HSOR), the key synthetase of allopregnanolone, is responsible for allopregnanolone upregulation in the SC, the objective of the present study was to investigate the role of its expression in the SC in koumine-induced analgesia using a rat model of neuropathic pain following peripheral nerve injury. RESULTS: Time-course investigations of immunohistochemistry and real-time polymerase chain reaction revealed that the immunoreactivity and mRNA expression of 3α-HSOR markedly increased in a time-dependent manner in the SC of koumine-treated CCI rats. Furthermore, 3α-HSOR activity in the SC of koumine-treated CCI rats increased by 15.8% compared to the activity in untreated CCI rats. Intrathecal injection of medroxyprogesterone acetate, a selective 3α-HSOR inhibitor, reversed the analgesic effect of koumine on CCI-induced mechanical pain perception. Our results confirm that koumine alleviates neuropathic pain in rats with CCI by enhancing 3α-HSOR mRNA expression and bioactivity in the SC. CONCLUSION: This study demonstrates that 3α-HSOR is an important molecular target of koumine for alleviating neuropathic pain. Koumine may prove a promising compound for the development of novel analgesic agents effective against intractable neuropathic pain.

Anti-allodynic and neuroprotective effects of koumine, a Benth alkaloid, in a rat model of diabetic neuropathy.

Diabetic neuropathy is characterized by progressive degeneration of nerve fibers associated with diabetes mellitus. Antidepressants and anticonvulsants are the mainstay of pharmacological treatment, but are often limited in effectiveness against the core clinical feature of pain. In the current study, we examined the potential effects of koumine, a Gelsemium elegans Benth alkaloid, using a rat model of diabetic neuropathy. Rats were administered intraperitoneally a single dose of streptozocin (60 mg/kg) to induce type 1 diabetes. Koumine was given at a dose range of 0.056-7 mg/kg subcutaneously for one week starting 3 weeks after streptozocin adminstration. Behavioral responses to mechanical stimuli were evaluated every day after streptozocin injection. At 4 weeks after streptozocin injection, sensory nerve conduction velocity (SNCV) and morphological alternation of sciatic nerves were assessed by electron microscopy. Diabetic rats developed mechanical hyperalgesia within 3 weeks after streptozocin injection and exhibited reduced SNCV and impaired myelin/axonal structure. Koumine treatment of diabetic rats decreased neuropathic pain behavior as early as after the first administration. At a dose of 7 mg/kg, koumine was more effective than gabapentin (100 mg/kg), and decreased mechanical sensitivity threshold to a level comparable to healthy control. Repeated treatment of koumine significantly reduced the damage to axon and myelin sheath of the sciatic nerve and increased SNCV, without affecting body weight and blood glucose. These findings encourage the use of koumine in the treatment of diabetic neuropathy.

Glycogen synthase kinase-3ß in the ventral tegmental area mediates diurnal variations in cocaine-induced conditioned place preference in rats.

Cocaine sensitization and reward are reported to be under the influence of diurnal rhythm. However, no previous studies have reported brain areas that play a role as modulators and underlie the mechanism of diurnal variations in cocaine reward. We examined (1) the diurnal rhythm of glycogen synthase kinase-3ß (GSK-3ß) activity in the suprachiasmatic nucleus (SCN) and reward-related brain areas in naive rats; (2) the effect of day and night on the acquisition of cocaine-induced conditioned place preference (CPP); (3) the influence of cocaine-induced CPP on GSK-3ß activity in the SCN and reward-related brain areas; and (4) the effect of the GSK-3ß inhibitor SB216763 microinjected bilaterally into the ventral tegmental area (VTA) on cocaine-induced CPP. A significant diurnal rhythm of GSK-3ß activity was found in the SCN and reward-related brain areas, with diurnal variations in cocaine-induced CPP. GSK-3ß activity in the SCN and reward-related brain areas exhibited marked diurnal variations in rats treated with saline. GSK-3ß activity in rats treated with cocaine exhibited distinct diurnal variations only in the prefrontal cortex and VTA. Cocaine decreased the expression of phosphorylated GSK-3ß (i.e. increased GSK-3ß activity) only in the VTA in rats trained and tested at ZT4 and ZT16. SB216763 microinjected into the VTA bilaterally eliminated the diurnal variations in cocaine-induced CPP, but did not affect the acquisition of cocaine-induced CPP. These findings suggest that the VTA may be a critical area involved in the diurnal variations in cocaine-induced CPP, and GSK-3ß may be a regulator of diurnal variations in cocaine-induced CPP.

The alleviatory effects of koumine on MSU-induced gouty arthritis via the TLR4/NF-κB/NLRP3 pathway.

The aim of this study was to validate the preventive effects of koumine (KM), a monoterpene indole alkaloid, on gouty arthritis (GA) and to explore its possible mechanisms. C57BL/6 mice were intraperitoneally administered KM (0.8, 2.4 or 7.2 mg/kg), colchicine (3.0 mg/kg) or sterile saline. One hour later, a monosodium urate (MSU) suspension was injected into the right hind paws of the mice to establish an acute gout model. Inflammation symptoms were evaluated at 0, 3, 6, 12 and 24 h, and the mechanical withdrawal threshold was evaluated at 0, 6 and 24 h. After 24 h, the mice were euthanized, and the joint tissue, kidney and blood were collected for subsequent experiments. Histological examination and antioxidant enzyme, kidney index and serum uric acid (UA) measurements were taken. The expression levels of the signalling pathway components were determined. KM effectively alleviated the symptoms of redness, swelling and pain; counteracted inflammatory cell infiltration; and increased antioxidant enzyme levels, reduced kidney index and serum UA levels through regulating UA excretion in MSU-induced mice. The expression of toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NF-κB)/nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain-containing 3 (NLRP3) signalling pathway proteins and mRNA were reduced in the KM group. These results suggest that KM may be effective in alleviating GA through the TLR4/NF-κB/NLRP3 pathway.

Koumine alleviates rheumatoid arthritis by regulating macrophage polarization.

ETHNOPHARMACOLOGICAL RELEVANCE: The imbalance between M1-and M2-polarized macrophages is one of the major pathophysiological changes in RA. Therefore, targeted macrophage polarization may be an effective therapy for RA. Koumine, an alkaloid monomer with the highest content and low toxicity in Gelsemium elegans Benth., has the effect of treating RA by playing an immunomodulatory role by influencing various immune cells. However, whether koumine affects macrophage polarization in RA and the associated molecular mechanisms remain unknown. AIM OF THE STUDY: To investigate the mechanism of the anti-RA effect of koumine on macrophage polarization. MATERIALS AND METHODS: The effect of koumine on macrophage polarization was investigated in vivo and in vitro. We first explored the effects of koumine on AIA rats and detected the levels of M1/M2 macrophage polarization markers in the spleen by western blotting. Then, we explored the regulatory effect of koumine on M1/M2 macrophage polarization and the effect on the PI3K/AKT signaling pathway in vitro. Finally, we verified the effects of koumine on macrophage polarization in CIA mice. RESULTS: We found that koumine alleviated symptoms, including relieving pain, reducing joint redness and swelling in AIA rats and restoring the M1/M2 macrophage balance in vivo. Interestingly, koumine had an inhibitory effect on both M1 and M2 macrophage polarization in vitro, but it had a stronger inhibitory effect on M1 macrophage. In a mixed polarization experiment, koumine mainly inhibited M1 macrophage polarization and had an inhibitory effect on the PI3K/AKT signaling pathway. Finally, we found that koumine had therapeutic effects on CIA mice, regulated macrophage polarization and inhibited the PI3K/AKT signaling pathway. CONCLUSIONS: Our results reveal that koumine regulates macrophage polarization through the PI3K/AKT signaling pathway. This may be one of the important mechanisms of its anti-RA effect, which provides a theoretical and scientific basis for the possible clinical application of koumine.

D-Optimal Design and Development of a Koumine-Loaded Microemulsion for Rheumatoid Arthritis Treatment: In vivo and in vitro Evaluation.

Introduction: Koumine (KME) is the most abundant active ingredient separated from Gelsemium elegans Benth and exhibits a significant therapeutic effect on rheumatoid arthritis (RA). It is a lipophilic compound with poor aqueous solubility, and there is an urgent need to develop novel dosage forms of KME and promote its clinical application for the treatment of RA. The aim of this study was to design and develop KME-loaded microemulsions (KME-MEs) for the effective management of RA. Methods: The composition of the microemulsion was selected by carrying out a solubility study and generating pseudoternary phase diagrams, and further optimized by D-Optimal design. The optimized KME-MEs was evaluated for particle size, viscosity, drug release, storage stability, cytotoxicity, cellular uptake, Caco-2 cell transport and everted gut sac investigations. In vivo fluorescence imaging and the therapeutic effects of KME and KME-MEs on collagen-induced arthritis (CIA) rats were also evaluated. Results: The optimized microemulsion contained 8% oil, 32% Smix (surfactant/cosurfactant) and 60% water and was used for in vivo and in vitro studies. The optimal KME-MEs exhibited a small globule size of 18.5 ± 0.14 nm and good stability over 3 months, and the release kinetics followed a first-order model. These KME-MEs had no toxic effect on Caco-2 cells but were efficiently internalized into the cytoplasm. Compared to KME, the KME-MEs displayed significantly increased permeability and absorption in Caco-2 cell monolayer assay and ex vivo everted gut sac experiment. As expected, the KME-MEs attenuated the progression of RA in CIA rats and were more effective than free KME with a reduced frequency of administration. Conclusion: The KME-MEs improved the solubility and therapeutic efficacy of KME by employing formulation technology. These results provide a promising vehicle for the oral delivery of KME to treat RA and have attractive potential for clinical translation.

Basolateral amygdala astrocytes modulate diabetic neuropathic pain and may be a potential therapeutic target for koumine.

BACKGROUND AND PURPOSE: New remedies are required for the treatment of diabetic neuropathic pain (DNP) due to insufficient efficacy of available therapies. Here, we used chemogenetic approaches combined with in vivo pharmacology to elucidate the role of basolateral amygdala (BLA) astrocytes in DNP pathogenesis and provide new insights into therapeutic strategies for DNP. EXPERIMENTAL APPROACH: A streptozotocin-induced DNP model was established. Designer receptors exclusively activated by designer drugs (DREADDs) were used to regulate astrocyte activity. Mechanical hyperalgesia was assessed using the electronic von Frey test. Anxiety-like behaviours were detected using open field and elevated plus maze tests. Astrocytic activity was detected by immunofluorescence, and cytokine content was determined by ELISA. KEY RESULTS: BLA astrocytes were regulated by DREADDs, and inhibition of BLA astrocytes attenuated mechanical allodynia and pain-related negative emotions in DNP rats. In contrast, temporary activation of BLA astrocytes induced allodynia without anxious behaviours in naive rats. In addition, koumine (KM) alleviated mechanical allodynia and anxiety-like behaviours in DNP rats, inhibited the activation of BLA astrocytes and suppressed the inflammatory response. Furthermore, persistent activation of BLA astrocytes through chemogenetics mimicked chronic pain, and KM alleviated the pain hypersensitivity and anxiety-like behaviours. CONCLUSION AND IMPLICATIONS: DREADDs bidirectionally regulate the activity of BLA astrocytes, which proves for the first time the role of BLA astrocyte activation in the pathogenesis of DNP and represents a novel therapeutic strategy for DNP. KM ameliorates DNP, perhaps by inhibiting the activation of BLA astrocytes and reveal KM as a potential candidate for treating DNP.

Regulation effect of koumine on T-helper cell polarization in rheumatoid arthritis.

Koumine, an alkaloid, exerts therapeutic effects against rheumatoid arthritis (RA), and thus may have a potential application in novel treatment strategies against this disease. Herein, we investigated the regulatory effect of koumine on Th cell polarization using a "pyramid" structure model to elucidate the mechanism underlying its therapeutic effect on RA. The third layer of the model comprises the cytokine secretion layer, in which the effects of koumine on the balance of Th-related cytokines were investigated in mice with collagen-induced arthritis (CIA). Koumine showed significant therapeutic effects and reversed the imbalance of Th1/Th2 and Th17/Treg cytokines. In the Th cell polarization layer, the effects of koumine on the relative numbers of Th cell subsets in splenocytes of rats with CIA were examined. Koumine attenuated both of the increased Th1/Th2 and Th17/Treg subset ratios accompanied with its therapeutic effects. Finally, the primary cultured splenocytes from BALB/c mice were used to further investigate the effect of koumine on Th cell activation by evaluating cell proliferation induced by concanavalin A (Con A), lipopolysaccharides (LPS) and phytohemagglutinin (PHA). Koumine inhibited the cell proliferation responses and its effects on proliferation induced by Con A and PHA were greater than those by LPS, showing the relatively selective inhibition on the proliferation of Th cells. Our results suggest that koumine might restore the homeostasis of the network system with Th subsets and cytokines by inhibiting the activation of T cells, subsequently regulating the polarization of Th subsets and the downstream imbalance of pro/anti-inflammatory cytokines in RA.

Koumine regulates macrophage M1/M2 polarization via TSPO, alleviating sepsis-associated liver injury in mice.

BACKGROUND: Translocator protein (TSPO) is an 18-kDa transmembrane protein found primarily in the mitochondrial outer membrane, and it is implicated in inflammatory responses, such as cytokine release. Koumine (KM) is an indole alkaloid extracted from Gelsemium elegans Benth. It has been reported to be a high-affinity ligand of TSPO and to exert anti-inflammatory and immunomodulatory effects in our recent studies. However, the protective effect of KM on sepsis-associated liver injury (SALI) and its mechanisms are unknown. PURPOSE: To explore the role of TSPO in SALI and then further explore the protective effect and mechanism of KM on SALI. METHODS: The effect of KM on the survival rate of septic mice was confirmed in mouse models of caecal ligation and puncture (CLP)-induced and lipopolysaccharide (LPS)-induced sepsis. The protective effect of KM on CLP-induced SALI was comprehensively evaluated by observing the morphology of the mouse liver and measuring liver injury markers. The serum cytokine content was detected in mice by flow cytometry. Macrophage polarization in the liver was examined using western blotting. TSPO knockout mice were used to explore the role of TSPO in sepsis liver injury and verify the protective effect of KM on sepsis liver injury through TSPO. RESULTS: KM significantly improved the survival rate of both LPS- and CLP-induced sepsis in mice. KM has a significant liver protective effect on CLP-induced sepsis in mice. KM treatment ameliorated liver ischaemia, improved liver pathological injuries, and decreased the levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH) and proinflammatory cytokines in serum. Western blotting results showed that KM inhibited M1 polarization of macrophages and promoted M2 polarization. In TSPO knockout mice, we found that TSPO knockout can improve the survival rate of septic mice, ameliorate liver ischaemia, improve liver pathological injuries, and decrease the levels of ALT, AST, and LDH. In addition, TSPO knockout inhibits the M1 polarization of macrophages in the liver of septic mice and promotes M2 polarization and the serum levels of proinflammatory cytokines. Interestingly, in TSPO knockout septic mice, these protective effects of KM were no longer effective. CONCLUSIONS: We report for the first time that TSPO plays a critical role in sepsis-associated liver injury by regulating the polarization of liver macrophages and reducing the inflammatory response. KM, a TSPO ligand, is a potentially desirable candidate for the treatment of SALI that may regulate macrophage M1/M2 polarization through TSPO in the liver.

Gelsenicine from Gelsemium elegans attenuates neuropathic and inflammatory pain in mice.

Gelsemium elegans BENTH and its crude extract are widely used to treat pain in China despite its apparent toxicity. The analgesic effects of gelsenicine, an active component of G. elegans, however, have not been reported. The current study examined potential analgesic effects of subcutaneously injected gelsenicine using acetic acid-induced writhing, formalin-induced nociceptive behavior, and thermal hyperalgesia caused by chronic constriction injury (CCI) in mice. Gelsenicine produced dose-dependent analgesic effects in both inflammatory and neuropathic pain models. The ED(50), for either the inflammatory pain (10.4 µg/kg for writhing test, 7.4 µg/kg for formalin test) or neuropathic pain (9.8 µg/kg for thermal hyperalgesia caused by CCI model), was far below the LD(50) (95% confidence interval at 100-200 µg/kg). Repeated subcutaneous injections of gelsenicine in CCI mice led to sustained attenuation of neuropathic pain after drug discontinuation. These results revealed that gelsenicine could be used safely to attenuate both inflammatory and neuropathic pain.

Streptozotocin-Induced Hyperglycemia Affects the Pharmacokinetics of Koumine and its Anti-Allodynic Action in a Rat Model of Diabetic Neuropathic Pain.

Koumine (KM), the most abundant alkaloid in Gelsemium elegans, has anti-neuropathic, anti-inflammatory, and analgesic activities; thus, it has the potential to be developed as a broad-spectrum analgesic drug. However, factors determining the relationship between analgesic efficacy and the corresponding plasma KM concentration are largely unclear. The pharmacokinetics and pharmacodynamics of KM and their optimization in the context of neuropathic pain have not been reported. We investigated the pharmacokinetics and pharmacodynamics of KM after oral administration in a streptozotocin-induced rat model of diabetic neuropathic pain (DNP) using a population approach. A first-order absorption and elimination pharmacokinetics model best described the plasma KM concentration. This pharmacokinetic model was then linked to a linear pharmacodynamic model with an effect compartment based on the measurement of the mechanical withdrawal threshold. KM was rapidly absorbed (time to maximum plasma concentration: 0.14-0.36 h) with similar values in both DNP and naïve rats, suggesting that DNP did not influence the KM absorption rate. However, the area under the curve (AUC0-∞) of KM in DNP rats was over 3-fold higher than that in naïve rats. The systemic clearance rate and volume of KM distribution were significantly lower in DNP rats than in naïve rats. Blood glucose value prior to KM treatment was a significant covariate for the systemic clearance rate of KM and baseline value of the threshold. Our results suggest that streptozotocin-induced hyperglycemia is an independent factor for decreased KM elimination and its anti-allodynic effects in a DNP rat model. To the best of our knowledge, this is the first study to investigate the role of DNP in the pharmacokinetics and pharmacokinetics-pharmacodynamics of KM in streptozotocin-induced diabetic rats.

Koumine modulates spinal microglial M1 polarization and the inflammatory response through the Notch-RBP-Jκ signaling pathway, ameliorating diabetic neuropathic pain in rats.

BACKGROUND: Diabetic neuropathic pain (DNP), a complication of diabetes, has serious impacts on human health. As the pathogenesis of DNP is very complex, clinical treatments for DNP is limited. Koumine (KM) is an active ingredient extracted from Gelsemium elegans Benth. that exerts an inhibitory effect on neuropathic pain (NP) in several animal models. PURPOSE: To clarify the anti-NP effect of KM on rats with DNP and the molecular mechanisms involving the Notch- Jκ recombination signal binding protein (RBP-Jκ) signaling pathway. METHODS: Male Sprague-Dawley rats were administered streptozocin (STZ) by intraperitoneal injection to induce DNP. The effect of KM on mechanical hyperalgesia in rats with DNP was evaluated using the Von Frey test. Microglial polarization in the spinal cord was examined using western blotting and quantitative real-time PCR. The Notch-RBP-Jκ signaling pathway was analysed using western blotting. RESULTS: KM attenuated DNP during the observation period. In addition, KM alleviated M1 microglial polarization in STZ-induced rats. Subsequent experiments revealed that Notch-RBP-Jκ signaling pathway was activated in the spinal cord of rats with DNP, and the activation of this pathways was decreased by KM. Additionally, KM-mediated analgesia and deactivation of the Notch-RBP-Jκ signaling pathway were inhibited by the Notch signaling agonist jagged 1, indicating that the anti-DNP effect of KM may be regulated by the Notch-RBP-Jκ signaling pathway. CONCLUSIONS: KM is a potentially desirable candidate treatment for DNP that may inhibit microglial M1 polarization through the Notch-RBP-Jκ signaling pathway.

Orally Administered Koumine Persists Longer in the Plasma of Aged Rats Than That of Adult Rats as Assessed by Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry.

Aging leads to changes in nearly all pharmacokinetic phases. Koumine (KM), an alkaloid derived from Gelsemium elegans Benth., is effective against age-associated chronic diseases, but its dose proportionality following oral administration in aged individuals remains unknown. Herein, we established and validated a simple method that requires low sample volumes to determine KM concentration in rats using ultra-performance liquid chromatography-tandem mass spectrometry. The maximum plasma concentration (Cmax) of 7 mg·kg-1 KM was ~12-fold and ~24-fold higher than that of 0.28 mg·kg-1 KM in adult and aged rats, respectively (P < 0.01). Time to reach Cmax (Tmax) for 7 mg·kg-1 KM was 4-fold longer in aged rats (P < 0.05). The area under the curve (AUC) of 7 mg·kg-1 KM was >17-fold and >43-fold higher than those of 0.28 mg·kg-1 KM in adult and aged rats, respectively (P < 0.01). The half-life (t1/2) of 7 mg·kg-1 KM was over 4-fold longer than that of 0.28 mg·kg-1 KM in adult rats (P < 0.01). The t1/2 of 1.4 and 7 mg·kg-1 KM were 1.5~2-fold longer, than that of 0.28 mg·kg-1 KM in aged rats (P < 0.05). The clearance rate of 7 mg·kg-1 KM was significantly lower in aged than in adult rats (P < 0.05). For 7.0 mg·kg-1 KM, the Cmax in aged rats was higher than in adult rats during the Tmax period (P < 0.05). In aged rats, the AUC for KM was >2.5-fold higher (P < 0.05) and the t1/2 was >60% longer than in adult rats (P < 0.05). These results help interpret the pharmacokinetics of KM in aging-associated diseases.

Ventral tegmental area GABAergic neurons induce anxiety-like behaviors and promote palatable food intake.

The obesity epidemic is a global problem and a great challenge for public health. Overconsumption of food, especially palatable food, is the leading cause of obesity. The precise neural circuits underlying food overconsumption remain unclear and require further characterization. In the present study, we showed that Ca2+ signals of GABAergic neurons within the ventral tegmental area (VTA) increased after the onset of food intake, especially high-fat or high-sugar chow. Optogenetic activation of VTA GABAergic neurons evoked immediate eating of palatable food and significantly increased palatable food intake in satiated mice. Photoinhibition of VTA GABAergic neurons suppressed palatable food intake. Surprisingly, photoactivation of VTA GABAergic neurons suppressed the intake of standard chow in fasted mice, but did not reduce the duration of eating of standard chow. Moreover, we found that photoactivation of these neurons drove a series of anxiety-like behaviors in the open field, elevated plus maze, and marble-burying test. Additionally, we found that VTA GABAergic neurons sent abundant projections to the lateral hypothalamus and photoactivation of GABAergic VTA terminals in the lateral hypothalamus induced overconsumption of palatable food, but not anxiety-like behaviors. Taken together, our results illustrate that GABAergic VTA neurons are a key node in the neural circuitry underlying anxiety-like behavior and over-feeding of palatable food, and that over-excitation of GABAergic VTA neurons may underlie clinical diseases related to anxiety and obesity.

Basal forebrain GABAergic neurons promote arousal and predatory hunting.

Predatory hunting is an important approach for animals to obtain valuable nutrition and energy, which critically depends on heightened arousal. Yet the neural substrates underlying predatory hunting remain largely undefined. Here, we report that basal forebrain (BF) GABAergic neurons play an important role in regulating predatory hunting. Our results showed that BF GABAergic neurons were activated during the prey (cricket)-hunting and food feeding in mice. Optogenetic activation of BF GABAergic neurons evoked immediate predatory-like actions to both artificial and natural preys, significantly reducing the attack latency while increasing the attack probability and the number of killed natural prey (crickets). Similar to the effect of activating the soma of BF GABAergic neurons, photoactivation of their terminals in the ventral tegmental area (VTA) also strongly promotes predatory hunting. Moreover, photoactivation of GABAergic BF - VTA pathway significantly increases the intake of various food in mice. By synchronous recording of electroencephalogram and electromyogram, we showed that photoactivation of GABAergic BF - VTA pathway induces instant arousal and maintains long-term wakefulness. In summary, our results clearly demonstrated that the GABAergic BF is a key neural substrate for predatory hunting, and promotes this behavior through GABAergic BF - VTA pathway.