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.

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.

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.

The analgesic effect and possible mechanisms by which koumine alters type II collagen-induced arthritis in rats.

Gelsemium elegans Benth. is a toxic plant that has been used as an ancient Chinese herbal remedy for rheumatoid arthritis (RA) and nervous pain, spasticity, skin ulcers, and cancers. Koumine, one of its representative alkaloids, shows numerous promising pharmacological activities, including anti-inflammatory and analgesic activities. Here, we investigated the analgesic effect of koumine on the collagen-induced arthritis (CIA) rat model of RA and explored the potential pharmacological mechanisms underlying the analgesia. In the CIA rats, repeated koumine treatments significantly reduced pain compared to controls and attenuated the collagen-induced increase in levels of glial fibrillary acidic protein (GFAP) and the pro-inflammatory cytokines tumour necrosis factor α (TNF-α) and interleukin 1ß (IL-1ß). Cultured astrocytes showed reduced astrocyte reactivation and decreased production of both tested cytokines. Based on our results, koumine exerted both analgesic and anti-inflammatory effects on the CIA rat model that were apparently mediated by inhibiting astrocyte reactivation and pro-inflammatory cytokine production.

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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Koumine exhibits anxiolytic properties without inducing adverse neurological effects on functional observation battery, open-field and Vogel conflict tests in rodents.

Koumine, an active alkaloid of neurotoxic plant Gelsemium, has been focused on its therapeutic uses, especially in central nervous system. Nevertheless, less is known about the neurological effects of koumine, which hampers its potential therapeutic exploitation. Moreover, as the anxiolytic potential of Gelsemium has raised many critical issues, its active principles on the anxiolytic and other neurological effects need to be further investigated. Here, we used functional observation battery (FOB) of mice to systematically measure the neurological effects of koumine at the effective doses, and then further confirmed its anxiolytic properties in open-field test (OFT) of mice and Vogel conflict test (VCT) of rats. Koumine exhibited anxiolytic-like activities but did not affect other autonomic, neurological and physical functions in FOB. Furthermore, koumine released anxiolytic responses and anti-punishment action in a manner similar to diazepam in OFT and VCT, respectively. The results constitutes solid set of fundamental data further demonstrating anxiolytic properties of koumine at the therapeutic doses without inducing adverse neurological effects, which supports the perspectives for the development of safe and effective koumine medicine against pathological anxiety.

Antidepressant-like effects of albiflorin extracted from Radix paeoniae Alba.

ETHNOPHARMACOLOGY RELEVANCE: Albiflorin, a monoterpene glycoside, is a main component of Radix paeoniae Alba, which could be a Chinese herbal medicine used in the treatment of psychiatric disorders. However, the exact role of albiflorin in depression is poorly understood. AIM OF THE STUDY: The current study aimed to evaluate the antidepressant effect of albiflorin in mice and rats, and the possible mechanism was also determined. MATERIALS AND METHODS: The antidepressant-like effects of albiflorin was determined by using animal models of depression including forced swim and tail suspension tests in mice and chronic unpredictable stress (CUS) in rats. The acting mechanism was explored by determining the effect of albiflorin on the expression of brain-derived neurotrophic factor (BDNF) in the hippocampus by western blot and the levels of monoamine in the hippocampus by HPLC. RESULTS: Our results showed that 7 days treatment with albiflorin significantly decreased immobility time in the forced swimming test (FST) and the tail suspension test (TST) at doses of 3.5, 7.0 and 14.0mg/kg without alter the locomotor activity in mice. Moreover, western blot analysis showed that albiflorin could increase the expression of BDNF in the hippocampus. We further exposed rats to a chronic unpredictable stress (CUS) protocol for a period of 35d to induce depressive-like behaviors. We found that chronic treatment with albiflorin, at doses of 7.0 and 14.0mg (i.g., once daily for 35d), restored the sucrose preference in CUS rats. In the open-field test, albiflorin significantly increased the number of crossings and rearings in the CUS rats at three doses. Moreover, chronic treatment with albiflorin up-regulated the hippocampal BDNF expression levels and the hippocampal 5-HT, 5-HIAA, and NA levels. CONCLUSION: Albiflorin produced significant antidepressant-like effects, which were closely related to the hippocampal 5-HT/NE increase and BDNF expression. Our data indicated that albiflorin could be a potential anti-depressant drug.

Medicinal plants of the genus Gelsemium (Gelsemiaceae, Gentianales)--a review of their phytochemistry, pharmacology, toxicology and traditional use.

ETHNOPHARMACOLOGICAL RELEVANCE: In the genus Gelsemium, Gelsemium elegans (Gardn. & Champ.) Benth. has been recognized as a toxic plant that is widely distributed in Southeast Asia and has been used as traditional Chinese medicine for the treatment of rheumatoid pain, neuropathic pain, spasticity, skin ulcers and cancers for many years. Gelsemium sempervirens (L.) J.St.-Hil. has been used since the nineteenth century in homeopathy for treating anxiety, neuralgia, migraine and spasmodic disorders, such as asthma and whooping cough in North America. This review aims to provide comprehensive information on the botany, traditional uses, phytochemistry, pharmacological research and toxicology of medicinal plants in the genus Gelsemium. The overall objective is to explore the evidence supporting its ethnopharmacological effectiveness. MATERIALS AND METHODS: A literature survey was performed by searching the scientific databases Pubmed, Google Scholar, SciFinder, Scopus, Web of Science and the Chinese CNKI, in addition to traditional Chinese medicine and homeopathic texts for information on Gelsemium. RESULTS: Plants of the genus Gelsemium have been used in traditional medicine for the treatment of migraines, neuralgia, sciatica, cancer and various types of sores. Studies into the phytochemical composition of this genus have shown that all of the species are rich sources of monoterpene indole alkaloids and that they have attracted the attention of many researchers due to their markedly diverse and complex architecture. To date, a total of 121 alkaloids have been isolated and identified from the genus. The crude extracts, as well as the monomeric compounds, from the genus possess anti-tumor, analgesic, anxiolytic, anti-inflammatory and immunomodulating pharmacological activities. CONCLUSION: It is evident from the available literature that Gelsemium species possess potential for use as a beneficial therapeutic remedy. However, the analysis of previous pharmacological research suggests that a clear assignment of active molecules and mechanisms of action is remain lacking. Due to their high toxicity, the studies available on toxicity and safety are inadequate for providing information on clinical utilization.

The active alkaloids of Gelsemium elegans Benth. are potent anxiolytics.

RATIONALE: An increasing number of herbal products has been introduced to treat anxiety and depression. Gelsemium elegans Benth (G. elegans) is a well-known herbal plant in Asia. Four major alkaloids (gelsemine, koumine, gelsevirine, and gelsenicine) have been isolated from G. elegans. Recently, interest has arisen to investigate the pharmaceutical potential of G. elegans alkaloids in the context of neuropsychopharmacology. OBJECTIVES: We investigated whether G. elegans alkaloids are capable of producing anxiolytic and antidepressant effects in mouse models. In particular, we examined whether the anxiolytic action of G. elegans alkaloids is due to the agonist effects of glycine receptor in the brain. METHODS: Two mouse models (elevated plus-maze and light-dark transition model) were used to examine potential anxiolytic effects. Forced swim test and tail suspension test were used to test the antidepressive action of G. elegans alkaloids. Moreover, we also explored the anxiolytic mechanisms of G. elegans alkaloids by intracerebroventricular administration of strychnine, an antagonist of glycine receptor, in the elevated plus-maze. RESULTS: Gelsemine, koumine, and gelsevirine, but not gelsenicine, exhibited potent anxiolytic effects in the two anxiety models. None of the four G. elegans alkaloids exerted antidepressant effects in the two depression models. None of G. elegans alkaloids impaired spontaneous motor activities. The intracerebroventricular administration of strychnine significantly antagonized the anxiolytic effects of gelsemine, koumine, and gelsevirine administrated subcutaneously. CONCLUSIONS: Gelsemine, koumine, and gelsevirine could be developed as the treatment of anxiety-related disorders in human patients. Their anxiolytic mechanism may be involved in the agonist action of glycine receptor in the brain.

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.

Preparative separation of alkaloids from Gelsemium elegans Benth. using pH-zone-refining counter-current chromatography.

Alkaloids in Gelsemium elegans possess a variety of therapeutic properties, including tumor suppression, analgesic and anti-inflammatory effects. In China, G. elegans has been used for centuries to treat a variety of medical conditions, including chronic pain and skin ulcer. Methods currently used to separate the active components of G. elegans are time-consuming and have low recovery. In the present study, we used pH-zone-refining counter-current chromatography to separate major alkaloids from a crude extract of G. elegans. The two-phase solvent system was methyl tert-butyl ether (MtBE)/acetonitrile/water (3:1.5:4, v/v). Triethylamine (20 mM) was added to the upper organic stationary phase as a retainer. Hydrochloric acid (10 mM) was added to the lower aqueous phase as an eluter. From 1.5 g of crude extract, we obtained 312 mg gelsemine, 420 mg koumine and 195 mg gelsevirine, with purities at 94.8%, 95.9% and 96.7%, respectively, which were determined by HPLC at 256 nm. The chemical identity of the isolated compounds was verified by electrospray ionization-mass spectrometry (ESI-MS), ¹H NMR and ¹³C NMR. These results demonstrated that pH-zone-refining counter-current chromatography is an effective method to separate and purify major alkaloids from G. elegans.