Design, semi-synthesis and bioevaluation of koumine-like derivatives as potential antitumor agents in vitro and in vivo.

Aims: Five series of novel koumine-like compounds were designed, semi-synthesized and systematically evaluated for antitumor activities.Methods: All compounds were evaluated for antiproliferative activity against four human cancer cell lines, including HT-29, HCT-116, HCT-15 and Caco-2.Results: Most compounds exhibited much higher antiproliferation activities (IC50 <10 µM) than koumine. Two selected compounds A4 and C5 showed comparable antitumor effects to 5-FU in vivo, as well as better safety profiles. Further studies suggested that A4 and C5 could arrest HT-29 cell cycle in G2 phase and raise reactive oxygen species level, thus inducing cell apoptosis related to Erk MAPK and NF-κB signaling pathways inhibition.Conclusion: These results will greatly promote the druggability study of these koumine-like compounds.

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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.

Effect of cytochrome P450 3A4 on tissue distribution of humantenmine, koumine, and gelsemine, three alkaloids from the toxic plant Gelsemium.

Humantenmine, koumine, and gelsemine are three indole alkaloids found in the highly toxic plant Gelsemium. Humantenmine was the most toxic, followed by gelsemine and koumine. The aim of this study was to investigate and analyze the effects of these three substances on tissue distribution and toxicity in mice pretreated with the Cytochrome P450 3A4 (CYP3A4) inducer ketoconazole and the inhibitor rifampicin. The in vivo test results showed that the three alkaloids were absorbed rapidly and had the ability to penetrate the blood-brain barrier. At 5 min after intraperitoneal injection, the three alkaloids were widely distributed in various tissues and organs, the spleen and pancreas were the most distributed, and the content of all tissues decreased significantly at 20 min. Induction or inhibition of CYP3A4 in vivo can regulate the distribution and elimination effects of the three alkaloids in various tissues and organs. Additionally, induction of CYP3A4 can reduce the toxicity of humantenmine, and vice versa. Changes in CYP3A4 levels may account for the difference in toxicity of humantenmine. These findings provide a reliable and detailed dataset for drug interactions, tissue distribution, and toxicity studies of Gelsemium alkaloids.

An efficient method for the preparative isolation and purification of alkaloids from Gelsemium by using high speed counter-current chromatography and preparative HPLC.

We established an efficient method using high-speed countercurrent chromatography (HSCCC) combined with preparative high-performance liquid chromatography (prep-HPLC) for isolating and purifying Gelsemium elegans (G. elegans) alkaloids. First, the two-phase solvent system composed of 1% triethylamine aqueous solution/n-hexane/ethyl acetate/ethanol (volume ratio 4:2:3:2) was employed to separate the crude extract (350 mg) using HSCCC. Subsequently, the mixture that resulted from HSCCC was further separated by Prep-HPLC, resulting in seven pure compounds including: 14-hydroxygelsenicine (1, 12.1 mg), sempervirine (2, 20.8 mg), 19-(R)-hydroxydihydrogelelsevirine (3, 10.1 mg), koumine (4, 50.5 mg), gelsemine (5, 32.2 mg), gelselvirine (6, 50.5 mg), and 11-hydroxyhumanmantenine (7, 12.5 mg). The purity of these seven compounds were 97.4, 98.9, 98.5, 99, 99.5, 96.8, and 85.5%, as determined by HPLC. The chemical structures of the seven compounds were analyzed and confirmed by electrospray ionization mass spectrometry (ESI-MS), 1H-nuclear magnetic resonance (1H NMR), and 13 C-nuclear magnetic resonance (13 C NMR) spectra. The results indicate that the HSCCC-prep-HPLC method can effectively separate the major alkaloids from the purified G. elegans, holding promising prospects for potential applications in the separation and identification of other traditional Chinese medicines.

Koumine inhibits IL-1ß-induced chondrocyte inflammation and ameliorates extracellular matrix degradation in osteoarthritic cartilage through activation of PINK1/Parkin-mediated mitochondrial autophagy.

Osteoarthritis (OA) is a degenerative joint disease, Increasingly, mitochondrial autophagy has been found to play an important regulatory role in the prevention and treatment of osteoarthritis. Koumine is a bioactive alkaloid extracted from the plant Gelsemium elegans. In previous research, Koumine was found to have potential in improving the progression of OA in rats. However, the specific mechanism of its action has not been fully explained. Therefore, the aim of this study was to investigate whether Koumine can alleviate OA in rats by influencing mitochondrial autophagy. In the in vitro study, rat chondrocytes (RCCS-1) were induced with IL-1ß (10 ng/mL) to induce inflammation, and Koumine (50 µg/mL) was co-treated. In the in vivo study, a rat OA model was established by intra-articular injection of 2% papain, and Koumine was administered orally (1 mg/kg, once daily for two weeks). It was found that Koumine effectively reduced cartilage erosion in rats with osteoarthritis. Additionally, it decreased the levels of inflammatory factors such as IL-1ß, IL-6, and extracellular matrix (ECM) components MMP13 and ADAMTS5 in chondrocytes and articular cartilage tissue, while increasing the level of Collagen II.Koumine inhibited the production of reactive oxygen species (ROS) in cartilage tissue and increased the number of autophagosomes in chondrocytes and articular cartilage tissue. Additionally, it upregulated the expression of mitochondrial autophagy proteins LC3Ⅱ/Ⅰ, PINK1, Parkin, and Drp1. The administration of Mdivi-1 (50 µM) reversed the enhanced effect of Koumine on mitochondrial autophagy, as well as its anti-inflammatory and anti-ECM degradation effects in rats with OA. These findings suggest that Koumine can alleviate chondrocyte inflammation and improve the progression of OA in rats by activating PINK1/Parkin-mediated mitochondrial autophagy.

Koumine inhibits RANKL-induced ubiquitination and NF-κB activation to prevent ovariectomy and aging-induced bone loss.

Osteoporosis (OP) is a bone remodeling disease characterized by an imbalance between bone resorption and formation. Osteoclasts are the primary therapeutic targets for treating bone destruction. Koumine (KM), the most bioactive component in Gelsemium alkaloids, exhibits antitumor, immunosuppressive, anti-inflammatory, and analgesic properties. However, the effects of bone loss have not been well studied. This study conducted in vitro and in vivo verification experiments on KM. The results showed that KM inhibited bone resorption and tartrate-resistant acid phosphatase positive (TRAP+) osteoclasts development by mature osteoclasts in a dose-dependent manner. Moreover, KM prevented OVX-induced OP in vivo and potentially inhibited ubiquitination, a process closely related to various biological activities, including protein interaction, transcription, and transmembrane signal transduction regulation, especially within the nuclear factor-κB (NF-κB) pathway. Previous studies have demonstrated that several proteins ubiquitination promotes osteoclastogenesis, our study indicated that KM inhibits early NF-κB activation and receptor activator of NF-κB ligand induced ubiquitination, a critical factor in osteoclast differentiation. In conclusion, our research suggests that KM holds potential as an effective therapeutic agent for OP.

Effect of dietary koumine on the immune and antioxidant status of carp (Cyprinus carpio) after Aeromonas hydrophila infection.

Koumine (KM) has anxiolytic, anti-inflammatory and growth-promoting effects in pigs and sheep. Based on the growth-promoting and immunological effects of koumine, the present study was conducted on Cyprinus carpio (C. carpio) with four KM concentrations: 0 mg/kg, 0.2 mg/kg, 2 mg/kg, and 20 mg/kg for 10 weeks, followed by a 1-week Aeromonas hydrophila (A. hydrophila) infection experiment. The effect of KM on the immunity of A. hydrophila infected carp was analyzed by histopathology, biochemical assay, and qRT-PCR to assess the feasibility of KM in aquaculture. The results showed that the presence of KM alleviated pathogen damage to carp tissues. At 2 mg/kg and 20 mg/kg concentrations of KM successively and significantly elevated (p < 0.05) the SOD activities in the intestinal tract, hepatopancreas and kidney of carp. The expression levels of hepatopancreatic antioxidant genes Nrf2 and IGF-1 were significantly up-regulated in the same group (p < 0.05), while the expression levels of immune genes IL-8 and IL-10 were down-regulated. In summary, KM at concentrations of 2 mg/kg and 20 mg/kg could regulate the expression of antioxidant and immune genes in various tissues in an orderly and rapid manner, and significantly improve the antioxidant and immune abilities of carp, which is conducive to the improvement of the resilience of carp.

Toxic Effects of Koumine on the Early-Life Development Stage of Zebrafish.

Koumine is one of the most abundant alkaloids found in Gelsemium elegans, and it has a wide range of pharmacological effects including antitumor, anti-inflammatory, analgesic treatment effects, and antianxiety. However, its high toxicity and unclear mechanism of action have greatly limited the medicinal development and use of koumine. We investigated the toxic effects of koumine on the developmental toxicity and behavioral neurotoxicity of zebrafish embryos and larvae. Embryos at 6 h postfertilization (hpf) were exposed to 12.5, 25, 50, 75, and 100 mg/L of koumine until 120 hpf. Koumine affected the hatching and heartbeats of the embryos. The morphological analysis also revealed many abnormalities, such as shortened bodies, yolk sac edemas, tail malformations, and pericardial edemas. To identify the neurotoxicity of koumine, the behavior of the larvae was measured. Koumine at 50 and 100 mg/L affect the escape response. The embryos exhibited uncoordinated muscle contractions along the body axis in response to touch at 36 hpf. More importantly, we found that the neurotoxicity of koumine is mainly caused by influencing the ACh content and the activity of AChE without impairing motor neuron development. A comprehensive analysis shows that a high concentration of koumine has obvious toxic effects on zebrafish, and the safe concentration of koumine for zebrafish should be less than 25 mg/L. These results will be valuable for better understanding the toxicity of koumine and provide new insights into the application of koumine.

Koumine ameliorates neuroinflammation by regulating microglia polarization via activation of Nrf2/HO-1 pathway.

BACKGROUND: Gelsemium elegans (Gardner & Chapm.) Benth (G. elegans) has been widely used as a traditional folk medicine in China and Southeast Asia. As the most abundant alkaloid in G. elegans, Koumine (KM) has been revealed the effect of inflammatory attenuation modulating by macrophage activation and polarization. PURPOSE: This study aimed to explore the effect of KM on modulation of microglia polarization that led to the suppression of neuroinflammation and further improved neurodegenerative behavior. METHODS: Inflammatory mediators, microglia M1 and M2 phenotype markers and Nrf2/HO-1 pathway related protein were assessed in LPS-induced BV2 cells and LPS-treated mice by RT-PCR, immunohistochemistry, immunofluorescence and Western blotting. Moreover, the learning and memory abilities of mice were evaluated by Morris water maze test, and the neuronal damage was evaluated by the Nissl staining. RESULTS: KM attenuated LPS-induced viability and morphological changes in BV2 microglial cells. Our findings showed that KM activated the Nrf2/HO-1 signaling pathway to promote phenotypic switch from M1 to M2 phenotypes. This switch suppresses the release of inflammatory mediators in LPS-induced BV2 cells. Meanwhile, KM attenuated neuroinflammation through modulating microglia polarization and subsequently reversed the behavioral alterations in LPS-induced mice model of neuroinflammation. CONCLUSIONS: KM may alleviate neuroinflammation by regulating microglia polarization with the involvement of Nrf2/HO-1 pathway, resulting of the neuroprotective effect.

Antinociceptive effect of gelsenicine, principal toxic alkaloids of gelsemium, on prostaglandin E2-induced hyperalgesia in mice: Comparison with gelsemine and koumine.

Gelsemium elegans (G.elegans) is a plant of the Loganiaceae family, known for its indole alkaloids, including gelsemine, koumine, and gelsenicine. Gelsemine and koumine are well-studied active alkaloids with low toxicity, valued for their anti-anxiety and analgesic properties. However, gelsenicine, another important alkaloid, remains underexplored due to its high toxicity. This study focuses on evaluating the analgesic properties of gelsenicine and comparing them with gelsemine and koumine. The results indicate that all three alkaloids exhibit robust analgesic properties, with gelsemine, koumine, and gelsenicine showing ED50 values of 0.82 mg/kg, 0.60 mg/kg, and 8.43 µg/kg, respectively, as assessed by the hot plate method. Notably, the therapeutic dose of gelsenicine was significantly lower than its toxic dose (LD50 = 0.185 mg/kg). The study also investigated the mechanism of action by analyzing the expression levels of GlyRα3 and Gephyrin. The PGE2 model group showed decreased expression levels of GlyRα3 and Gephyrin, while groups treated with gelsemine, koumine, and gelsenicine were able to reverse this decrease. These results suggest that gelsenicine effectively alleviates PGE2-induced hyperalgesia by upregulating the expression of GlyRα3 and Gephyrin, which are key targets of the Gly receptor pathway.

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.

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.

PI3K/AKT/mTOR, NF-κB and ERS pathway participated in the attenuation of H2O2-induced IPEC-J2 cell injury by koumine.

ETHNOPHARMACOLOGICAL RELEVANCE: Koumine, an indole alkaloid extracted from Gelsemium elegans Benth, exerts anti-inflammation and antioxidant activities. However, the effects of koumine on intestinal injury induced by H2O2 and its potential molecular mechanisms need larger studies. AIM OF THE STUDY: We established an IPEC-J2 cell damage model induced by H2O2 to explore the protective mechanism of koumine on intestinal injury. MATERIALS AND METHODS: In the experiment, cell damage models were made with hydrogen peroxide. To assess the protective effect of koumine on H2O2-induced IPEC-J2 cell injury, CCK-8, the release of LDH and ROS, transmission electron microscopy and Annexin V-FITC/PI were employed. Western Blot and Quantitative Real-time PCR were used to determine the potential alleviated mechanism of koumine on H2O2-trigged IPEC-J2 cell damage. RESULTS: The results of CCK-8 and LDH implied that koumine has a mitigative effect on H2O2-induced cell damage via upregulating cell viability and suppressing cell membrane fragmentation. Simultaneously, koumine notably inhibited the level of pro-inflammatory factors (IL-1ß, IL-6, IL-8, TNF-α and TGF-ß), the over-production of ROS along with decreasing the injury of mitochondrion, endoplasmic reticulum and lysosome induced by H2O2. Moreover, koumine dramatically attenuated H2O2-triggered IPEC-J2 cell apoptosis and autophagy. Subsequently, Western blot analysis identified NF-ΚB, PI3K and ERS as possible pathway responsible for the protective effect of koumine on H2O2-stimulated IPEC-J2 cell inflammation. CONCLUSIONS: This in vitro experimental study suggests that koumine suppresses the H2O2-induced activation of inflammatory pathways, oxidative injury, ER stress, apoptosis and autophagy, which provide a rationale for therapeutically use in major intestinal diseases.

Koumine ameliorates concanavalin A-induced autoimmune hepatitis in mice: involvement of the Nrf2, NF-κB pathways, and gut microbiota.

Gelsemiumelegans(Gardner. & Chapm.) Benth. has long been considered a traditional Chinese medicine effective against rheumatoid pain, cancer, cirrhosis, and skin diseases. Koumine (KM), the most abundant alkaloid in G.elegans Benth., demonstrates a variety of biological effects, including antitumor, analgesic, anxiolytic, anti-inflammatory, antidepressant, antioxidant, immunoregulatory, and hepatoprotective effects. Furthermore, the relatively low toxicity of KM makes it a promising drug candidate. This study aimed to investigate the protective effects of KM and its possible mechanisms using a concanavalin A (Con A)-induced autoimmune hepatitis (AIH) model in mice. Mice were orally administered different doses of KM for 14 d before Con A tail vein injections. The effects of KM on serum biochemical markers and liver histopathology were then evaluated 12 h after Con A exposure. The Nrf2 and NF-κB signaling pathways and alterations in gut microbiota were determined using western blotting, immunohistochemistry, and 16S rRNA sequencing to explore the underlying mechanisms of KM exposure. KM pretreatment dose-dependently decreased serum liver injury markers (Alanine aminotransferase, and aspartate aminotransferase) and cytokine levels (Tumor necrosis factor-α and interleukin-6), as well as the liver pathological damage triggered by Con A. Furthermore, the results of the multi-technique analysis indicated that KM activated the Nrf2 pathway, upregulated the expression of anti-oxidation factors HO-1 and Nrf2, and downregulated the expression of Keap1. Moreover, the NF-κB signaling pathway was inhibited. Interestingly, pre-treatment with KM also significantly improved the composition of the gut microbiota probably because it increases the richness of probiotics. Our findings suggest that KM pretreatment could attenuate Con A-induced AIH, the Nrf2 and NF-κB signaling pathways, and that gut microbiota are involved in the process of the hepatoprotective effect. This study provides a theoretical basis for the development of KM as an effective agent against AIH.

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.

Koumine induces apoptosis in Cyprinus carpio liver cells by regulating JAK-STAT and p53 signaling pathways.

Koumine is an alkaloid with significant anti-anxiety, anticancer cell proliferation, and analgesic activities, and our previous studies have shown that koumine can be used as an immunostimulant in aquaculture, but the molecular mechanism of its effect remains unclear. We fed a basal diet with 0, 0.2, 2, and 20 mg/kg koumine to C. carpio for 10 weeks, and comprehensive studies of the histological and biochemical parameters and transcriptomes of the four groups were performed. Histological results indicated that the number of apoptotic cells in the liver increased with increasing koumine concentration. Compared with those of the control group, the malondialdehyde, superoxide dismutase, catalase, acid phosphatase, alkaline phosphatase, and lactate dehydrogenase levels of the treatment group increased to varying degrees. In total, 100.11 GB of clean data, 4774 DEGs, and 138 differentially expressed genes were obtained from the transcriptome data. Differentially expressed genes were classified into 187 signalling pathways, and the circadian rhythm signalling pathway, the JAK-STAT signalling pathway, the p53 signalling pathway and the PPAR signalling pathway were the top enriched pathways. The qRT-PCR results confirmed that the key genes ifnar1, socs3l, epoa, ghra, cMyc, mcl-1, shisa4, and gtse1 involved in balancing cell proliferation and apoptosis were enriched in these pathways. We discovered that the JAK-STAT and p53 pathways are important targets of koumine. Such information contributes to a better understanding of the potential mechanism by which koumine regulates hepatic immunity as well as to lays the theoretical foundation for its application.

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.

Two-Dimensional Liquid Chromatography Method for the Determination of Gelsemium Alkaloids in Honey.

Toxic Chinese medicine residues in honey pose a serious threat to consumer health. Gelsemium is one of the nine ancient poisons, making the whole plant virulent. The residue of Gelsemium alkaloid in honey causes poisoning from time to time. Therefore, it is very important to establish a method for the detection of Gelsemium alkaloids in honey. In this study, a method of solid phase extraction (SPE) with two-dimensional liquid chromatography (2D-LC) was developed for the first time for the simultaneous determination of Gelsemium alkaloids in honey, including gelsemine, koumine and humantenmine. First, the honey samples were purified by a PRS cation exchange column and extracted with 5% ammoniated methanol. Then, we verified the methodological indicators, which were in line with the Codex Guideline requirements. The verification results are as follows: matrix-matched calibrations indicated that the correlation coefficients were higher than 0.998. The recovery was in the range of 81%-94.2% with an intraday precision (RSD) of ≤5.0% and interday RSD of ≤3.8%. The limit of detection for the three alkaloids was 2 ng/g. The limits of quantification for gelsemine and koumine were 5 ng/g, and humantenmine was 20 ng/g. This method can be applied to the monitoring of Gelsemium alkaloids in honey.

The anxiolytic effect of koumine on a predatory sound stress-induced anxiety model and its associated molecular mechanisms.

BACKGROUND: Koumine is the most abundant alkaloid extracted from Gelsemium elegans Benth.. Preliminary studies by our research group have shown that koumine has significant anxiolytic effect, but this needs to be further confirmed. HYPOTHESIS/PURPOSE: To investigate the potential anxiolytic effect of koumine on predatory sound (PS) stress-induced anxiety models and preliminarily explore its therapeutic targets and molecular mechanisms. STUDY DESIGN AND METHODS: The anxiolytic effect of koumine in an animal model of acute PS stress-induced anxiety were determined. Then, neurosteroids levels in the main brain regions involved in anxiety disorders, as well as plasma adrenocorticotropic hormone (ACTH) and corticosterone (CORT) levels, were determinated. Finally, to clarify the effect of koumine on translocator protein 18 kDa (TSPO), the affinity between koumine and TSPO was evaluated by surface plasmon resonance (SPR) technology. RESULTS: Koumine treatment mitigated anxiety-like behavior following acute PS stress in the open field test and elevated plus maze test. PS exposure significantly decreased progesterone and allopregnanolone levels in the PFC, Hip, and Amy and increased ACTH and CORT levels in plasma, and koumine administration significantly reversed these effects. Finally, the reliable SPR results showed that the KD of koumine with TSPO was 155.33 ± 11.0 µM, indicating that koumine is a human TSPO high-affinity ligand that has an affinity comparable to typical TSPO ligands. CONCLUSION: Our results show that koumine has obvious anxiolytic effect in the PS-induced anxiety model. Targeting TSPO-neurosteroids-HPA axis may be an important mechanism by which koumine exerts its anxiolytic effect.