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.

CVB3 VP1 interacts with MAT1 to inhibit cell proliferation by interfering with Cdk-activating kinase complex activity in CVB3-induced acute pancreatitis.

Coxsackievirus B3 (CVB3) belongs to the genus Enterovirus of the family Picornaviridae and can cause acute acinar pancreatitis in adults. However, the molecular mechanisms of pathogenesis underlying CVB3-induced acute pancreatitis have remained unclear. In this study, we discovered that CVB3 capsid protein VP1 inhibited pancreatic cell proliferation and exerted strong cytopathic effects on HPAC cells. Through yeast two-hybrid, co-immunoprecipitation, and confocal microscopy, we show that Menage a trois 1 (MAT1), a subunit of the Cdk-Activating Kinase (CAK) complex involved in cell proliferation and transcription, is a novel interaction protein with CVB3 VP1. Moreover, CVB3 VP1 inhibited MAT1 accumulation and localization, thus interfering with its interaction with CDK7. Furthermore, CVB3 VP1 could suppress CAK complex enzymic phosphorylation activity towards RNA Pol II and CDK4/6, direct substrates of CAK. VP1 also suppresses phosphorylation of retinoblastoma protein (pRb), an indirect CAK substrate, especially at phospho-pRb Ser780 and phospho-pRb Ser807/811 residues, which are associated with cell proliferation. Finally, we present evidence using deletion mutants that the C-terminal domain (VP1-D8, 768-859aa) is the minimal VP1 region required for its interaction with MAT1, and furthermore, VP1-D8 alone was sufficient to arrest cells in G1/S phase as observed during CVB3 infection. Taken together, we demonstrate that CVB3 VP1 can inhibit CAK complex assembly and activity through direct interaction with MAT1, to block MAT1-mediated CAK-CDK4/6-Rb signaling, and ultimately suppress cell proliferation in pancreatic cells. These findings substantially extend our basic understanding of CVB3-mediated pancreatitis, providing strong candidates for strategic therapeutic targeting.

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.

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.

Gentiana macrophylla flavonoids from Tibetan medicine decreases circ_0059665 to alleviate the progression of non-small cell lung cancer under hypoxia.

BACKGROUND: Gentiana macrophylla Pall. is a traditional Tibetan medicinal herb possessing antinociceptive and anti-inflammatory activities. Circular RNAs (circRNAs) have been identified to be involved in the tumorigenesis of non-small cell lung cancer (NSCLC). Here, this study focused on investigating the function and mechanism of Gentiana macrophylla flavonoids (GF) and circ_0059665 in NSCLC progression. METHODS: The contents of mRNA and protein were detected using qRT-PCR and western blotting analysis. Cell proliferative and invasive abilities were evaluated by cell counting kit-8, EdU, colony formation and transwell assays, respectively. M2 macrophage polarization was analyzed by flow cytometry. RESULTS: GF treatment suppressed NSCLC cell proliferation, invasion and M2 macrophage polarization under hypoxic conditions. Circ_0059665 was highly expressed in NSCLC tissues and cells. Its expression was increased under hypoxic conditions but was reduced following GF treatment. Furthermore, circ_0059665 overexpression reversed the anticancer effects of GF on NSCLC cells under hypoxic conditions. Mechanistically, circ_0059665 acted as a sponge for miR-512-5p to regulate NOVA2 expression. Hypoxia decreased miR-512-5p levels, and increased NOVA2 levels in NSCLC cells, while these tendencies were abolished after GF treatment. Circ_0059665 silencing inhibited NSCLC cell proliferation, invasion and M2 macrophage polarization in hypoxic environments, which were counteracted by NOVA2 overexpression. Moreover, NOVA2 upregulation reversed the suppressive effects of GF on NSCLC cells with hypoxia treatment. In addition, GF impeded NSCLC tumor growth in vivo via suppressing circ_0059665. CONCLUSION: GF treatment in hypoxic environments suppressed NSCLC cell proliferation, invasion and M2 macrophage polarization via the circ_0059665/miR-512-5p/NOVA2 axis.

Gelsevirine ameliorates sepsis-associated encephalopathy by inhibiting the STING signalling-mediated pyroptosis pathway in microglia.

BACKGROUND: Sepsis-associated encephalopathy (SAE) is among the most prevalent and deadly complications associated with sepsis, but satisfactory treatments and therapeutic agents are lacking. Gelsevirine, an active ingredient derived from Gelsemium elegans Benth., has shown promising effects in animal models of anxiety, ischaemic stroke and osteoarthritis. However, its protective effect against SAE and its mechanism of action are still unknown. PURPOSE: To elucidate the efficacy of gelsevirine against SAE and the mechanism of its protective effect through the STING signalling-mediated pyroptosis pathway. METHODS: We constructed a mouse model of caecum ligation and puncture (CLP)-induced sepsis and explored the protective effects of gelsevirine in mice with SAE by assessing survival rates and behavioural alterations. To further explore its mechanism of action, we investigated the modulatory effects of gelsevirine on the levels of inflammatory factors, microglial activation and pyroptosis by Western blotting, immunohistochemistry staining and PCR. STING knockout mice were used to verify the protective effect of gelsevirine against SAE through the STING pathway. RESULTS: Gelsevirine increased the survival rate of mice with SAE. The Morris water maze and open field tests revealed that gelsevirine significantly alleviated cognitive dysfunction and increased exploratory behaviour in mice with SAE. Gelsevirine inhibited the activation of microglia and decreased inflammatory factor levels in the hippocampus of mice with SAE. In mice with SAE and in vitro BV2 microglia, gelsevirine reduced levels of inflammatory factors and inhibited STING protein phosphorylation and microglial pyroptosis. However, after STING knockout, the inhibitory effect of gelsevirine on microglial pyroptosis was significantly weakened, and gelsevirine-mediated protective effects were abolished. CONCLUSIONS: Gelsevirine increased the survival rate, ameliorated cognitive impairment, inhibited glial cell activation and reduced inflammation in the hippocampi of mice with SAE; the mechanism may be related to the inhibition of STING signalling pathway-mediated pyroptosis in microglia.

Salidroside suppresses the multiple oncogenic activates and immune escape of lung adenocarcinoma through the circ_0009624-mediated PD-L1 pathway.

BACKGROUND: Lung adenocarcinoma (LUAD) is a fatal malignancy all over the world. Salidroside (SAL) is an active component extracted from Rhodiola rosea that has been reported to exert antitumor activity against several human cancers, containing lung adenocarcinoma (LUAD). The purpose of this study was to explore the effect and underlying mechanism of SAL in LUAD. METHODS: Cell viability, proliferation, migration, and invasion were measured using cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), and transwell assays. Effects of LUAD cells on the cytotoxicity, percentage, and death of CD8+ cells were detected using lactate dehydrogenase (LDH) and flow cytometry assays. Programmed cell death ligand 1 (PD-L1) protein level was examined using western blot. Circ_0009624, enolase 1 (ENO1), and PD-L1 levels were determined using real-time quantitative polymerase chain reaction (RT-qPCR). The biological role of SAL on LUAD tumor growth was assessed using the xenograft tumor model in vivo. RESULTS: SAL restrained LUAD cell proliferation, migration, invasion, and immune escape in vitro via modulating PD-L1. Circ_0009624 expression was increased in LUAD. Applying SAL repressed circ_0009624 and PD-L1 expression in LUAD cells. SAL treatment hindered suppressed various oncogenic activates and immune escape of LUAD cells by regulating the circ_0009624/PD-L1 pathway. SAL blocked LUAD xenograft growth in vivo. CONCLUSION: Applying SAL might constrain malignant phenotypes and immune escape of LUAD cells partially through the circ_0009624-mediated PD-L1 pathway, providing a novel insight for LUAD treatment.

Neuroprotective strategies for neonatal hypoxic-ischemic brain damage: Current status and challenges.

Neonatal hypoxic-ischemic brain damage (HIBD) is a prominent contributor to both immediate mortality and long-term impairment in newborns. The elusive nature of the underlying mechanisms responsible for neonatal HIBD presents a significant obstacle in the effective clinical application of numerous pharmaceutical interventions. This comprehensive review aims to concentrate on the potential neuroprotective agents that have demonstrated efficacy in addressing various pathogenic factors associated with neonatal HIBD, encompassing oxidative stress, calcium overload, mitochondrial dysfunction, endoplasmic reticulum stress, inflammatory response, and apoptosis. In this review, we conducted an analysis of the precise molecular pathways by which these drugs elicit neuroprotective effects in animal models of neonatal hypoxic-ischemic brain injury (HIBD). Our objective was to provide a comprehensive overview of potential neuroprotective agents for the treatment of neonatal HIBD in animal experiments, with the ultimate goal of enhancing the feasibility of clinical translation and establishing a solid theoretical foundation for the clinical management of neonatal HIBD.

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 new xanthones from Hypericum sampsonii and biological activity of the isolated compounds.

Phytochemical investigation of the CH(2) Cl(2) extract of the aerial part of Hypericum sampsonii yielded two new prenylated xanthones, hypericumxanthone A and B, together with three known xanthones. Their structures were elucidated by analysis of physical and spectral (UV, IR, mass and NMR) data and comparison of spectroscopic data with those reported previously. All these compounds were evaluated for in vitro antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). Two new compounds were also tested for their cytotoxicity against human breast (MCF-7), hepatoma (HepG2), colon (HT-29) and lung (A549) tumour cell lines. Two new compounds showed moderate antibacterial activities at minimum inhibitory concentrations (MIC) of 16 and 32 µg/mL, respectively, whereas the positive standard antibacterial drug, vancomycin, showed an MIC of 8 µg/mL. The other compounds were inactive against MRSA. In addition, hypericumxanthone B showed weak inhibitory activities against four human tumour cell lines.

Investigation of the Possible Allostery of Koumine Extracted From Gelsemium elegans Benth. And Analgesic Mechanism Associated With Neurosteroids.

Translocator protein 18 kDa (TSPO) is an evolutionarily conserved 5-transmembrane domain protein, and has been considered as an important therapeutic target for the treatment of pain. We have recently reported the in vitro and in vivo pharmacological characterization of koumine as a TSPO positive allosteric modulator (PAM), more precisely ago-PAM. However, the probe dependence in the allostery of koumine is an important question to resolve, and the possible analgesic mechanism of koumine remains to be clarified. Here, we report the in vivo evaluation of the allostery of koumine when orthosteric ligand PK11195 was used and preliminarily explore the possible analgesic mechanism of koumine associated with neurosteroids. We find that koumine is an ago-PAM of the PK11195-mediated analgesic effect at TSPO, and the analgesic mechanism of this TSPO ago-PAM may be associated with neurosteroids as the analgesic effects of koumine in the formalin-induced inflammatory pain model and chronic constriction injury-induced neuropathic pain model can be antagonized by neurosteroid synthesis inhibitor aminoglutethimide. Although our results cannot fully clarify the allosteric modulatory effect of koumine, it further prove the allostery in TSPO and provide a solid foundation for koumine to be used as a new clinical candidate drug to treat pain.

Sempervirine Inhibits Proliferation and Promotes Apoptosis by Regulating Wnt/ß-Catenin Pathway in Human Hepatocellular Carcinoma.

Gelsemium elegans (G. elegans) Benth., recognized as a toxic plant, has been used as traditional Chinese medicine for the treatment of neuropathic pain and cancer for many years. In the present study, we aim to obtain the anti-tumor effects of alkaloids of G. elegans and their active components in hepatocellular carcinoma (HCC) and the potential mechanism was also further investigated. We demonstrated that sempervirine induced HCC cells apoptosis and the apoptosis was associated with cell cycle arrest during the G1 phase, up-regulation of p53 and down-regulation of cyclin D1, cyclin B1 and CDK2. Furthermore, sempervirine inhibited HCC tumor growth and enhances the anti-tumor effect of sorafenib in vivo. In addition, inactivation of Wnt/ß-catenin pathway was found to be involved in sempervirine-induced HCC proliferation. The present study demonstrated that alkaloids of G. elegans were a valuable source of active compounds with anti-tumor activity. Our findings justified that the active compound sempervirine inhibited proliferation and induced apoptosis in HCC by regulating Wnt/ß-catenin pathway.

Identification of Koumine as a Translocator Protein 18 kDa Positive Allosteric Modulator for the Treatment of Inflammatory and Neuropathic Pain.

Koumine is an alkaloid that displays notable activity against inflammatory and neuropathic pain, but its therapeutic target and molecular mechanism still need further study. Translocator protein 18 kDa (TSPO) is a vital therapeutic target for pain treatment, and recent research implies that there may be allostery in TSPO. Our previous competitive binding assay hint that koumine may function as a TSPO positive allosteric modulator (PAM). Here, for the first time, we report the pharmacological characterization of koumine as a TSPO PAM. The results imply that koumine might be a high-affinity ligand of TSPO and that it likely acts as a PAM since it could delay the dissociation of 3H-PK11195 from TSPO. Importantly, the allostery was retained in vivo, as koumine augmented Ro5-4864-mediated analgesic and anti-inflammatory effects in several acute and chronic inflammatory and neuropathic pain models. Moreover, the positive allosteric modulatory effect of koumine on TSPO was further demonstrated in cell proliferation assays in T98G human glioblastoma cells. In summary, we have identified and characterized koumine as a TSPO PAM for the treatment of inflammatory and neuropathic pain. Our data lay a solid foundation for the use of the clinical candidate koumine to treat inflammatory and neuropathic pain, further demonstrate the allostery in TSPO, and provide the first proof of principle that TSPO PAM may be a novel avenue for the discovery of analgesics.

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.

Efficacy and safety of oral traditional Chinese patent medicine for chronic cerebral circulation insufficiency patients: A protocol for a systematic review and network meta-analysis.

BACKGROUND: Chronic cerebral circulation insufficiency (CCCI) is a common clinical cerebrovascular disease, especially among middle-aged and elderly patients, which seriously endangers their quality of life and physical and mental health. At present, Oral traditional Chinese patent medicine (OTCPM) is widely used in the treatment of CCCI in China, but its actual efficacy and safety lack of evidence-based evidence. Therefore, we will screen out the most effective OTCPM through a systematic review and network meta-analysis to provide a reliable theoretical basis for clinical decisions. METHODS: We will search electronic databases to collect relevant RCT studies from inception to October 2019. Those electronic databases include PubMed, Cochrane Library, Web of Science, EMBASE, China Biomedical Literature Database (CBM), China National Knowledge Infrastructure (CNKI), Chinese Scientific Journal Database (VIP), and Wan-fang database. Only randomized clinical trials (RCTs) concerned any OTCPM treatments for CCCI will be collected. The included studies will no restrictions on language or publication year. There were no publication year or language for the included literature. Risk bias tools will assess the quality of the included literature. A Bayesian NMA will be performed to combine the direct and indirect comparisons of TCPMs interventions. The surface under the cumulative ranking curve (SUCRA) will be drawn to display the hierarchy of each TCPMs treatment. All statistical analyses will be implemented using R v3.5.2. and GeMTC v1.4.3.We will publish this systematic review in academic journals. Since this literature review will not involve directly contacting patients, ethical approval and informed consent are not required. TRIAL REGISTRATION NUMBER: CRD42019123878.

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.

Immunoregulatory Effect of Koumine on Nonalcoholic Fatty Liver Disease Rats.

Nonalcoholic fatty liver disease (NAFLD) is the most common and important chronic liver disease all over the world. In the present study, we found that koumine, the main and active ingredient isolated from Gelsemium elegans, has the potential therapeutic effect on NAFLD rats by immunomodulatory activity. Koumine could significantly reduce the level of TG, TC, LDL-C, ALT, and AST in the serum of NAFLD rats and increase the level of HDL-C, reduce the liver index, and improve the adipose-like lesions of liver cells in NAFLD rats. Furthermore, treatment with koumine inhibited the severity of NAFLD. In addition, koumine-treated rats significantly increased the proportion of CD4+/CD8+ T cells and also decreased the percentages of Th1 and Th17 cells and increased Th2 and Treg cells in the liver. Moreover, koumine reduced the production and mRNA expression of proinflammatory cytokines in vivo. This result showed that koumine could effectively modulate different subtypes of helper T cells and prevent NAFLD. The present study revealed the novel immunomodulatory activity of koumine and highlighted the importance to further investigate the effects of koumine on hepatic manifestation of the metabolic syndrome.

Koumine Decreases Astrocyte-Mediated Neuroinflammation and Enhances Autophagy, Contributing to Neuropathic Pain From Chronic Constriction Injury in Rats.

Koumine, an indole alkaloid, is a major bioactive component of Gelsemium elegans. Previous studies have demonstrated that koumine has noticeable anti-inflammatory and analgesic effects in inflammatory and neuropathic pain (NP) models, but the mechanisms involved are not well understood. This study was designed to explore the analgesic effect of koumine on chronic constriction injury (CCI)-induced NP in rats and the underlying mechanisms, including astrocyte autophagy and apoptosis in the spinal cord. Rats with CCI-induced NP were used to evaluate the analgesic and anti-inflammatory effects of koumine. Lipopolysaccharide (LPS)-induced inflammation in rat primary astrocytes was also used to evaluate the anti-inflammatory effect of koumine. We found that repeated treatment with koumine significantly reduced and inhibited CCI-evoked astrocyte activation as well as the levels of pro-inflammatory cytokines. Meanwhile, we found that koumine promoted autophagy in the spinal cord of CCI rats, as reflected by decreases in the LC3-II/I ratio and P62 expression. Double immunofluorescence staining showed a high level of colocalization between LC3 and GFAP-positive glia cells, which could be decreased by koumine. Intrathecal injection of an autophagy inhibitor (chloroquine) reversed the analgesic effect of koumine, as well as the inhibitory effect of koumine on astrocyte activation in the spinal cord. In addition, TUNEL staining suggested that CCI-induced apoptosis was inhibited by koumine, and this inhibition could be abolished by chloroquine. Western blot analysis revealed that koumine significantly increased the level of Bcl-xl while inhibiting Bax expression and decreasing cleaved caspase-3. In addition, we found that koumine could decrease astrocyte-mediated neuroinflammation and enhance autophagy in primary cultured astrocytes. These results suggest that the analgesic effects of koumine on CCI-induced NP may involve inhibition of astrocyte activation and pro-inflammatory cytokine release, which may relate to the promotion of astrocyte autophagy and the inhibition for apoptosis in the spinal cord.

Analgesic effects and pharmacologic mechanisms of the Gelsemium alkaloid koumine on a rat model of postoperative pain.

Postoperative pain (POP) of various durations is a common complication of surgical procedures. POP is caused by nerve damage and inflammatory responses that are difficult to treat. The neuroinflammation-glia-steroid network is known to be important in POP. It has been reported that the Gelsemium alkaloid koumine possesses analgesic, anti-inflammatory and neurosteroid modulating activities. This study was undertaken to test the analgesic effects of koumine against POP and explore the underlying pharmacologic mechanisms. Our results showed that microglia and astroglia were activated in the spinal dorsal horn post-incision, along with an increase of proinflammatory cytokines (interleukin 1ß, interleukin 6, and tumor necrosis factor α). Both subcutaneous and intrathecal (i.t.) koumine treatment after incision significantly prevented mechanical allodynia and thermal hyperalgesia, inhibited microglial and astroglial activation, and suppressed expression of proinflammatory cytokines. Moreover, the analgesic effects of koumine were antagonized by i.t. administration of translocator protein (18 kDa) (TSPO) antagonist PK11195 and GABAA receptor antagonist bicuculline. Together, koumine prevented mechanical allodynia and thermal hyperalgesia caused by POP. The pharmacologic mechanism of koumine-mediated analgesia might involve inhibition of spinal neuroinflammation and activation of TSPO. These data suggested that koumine might be a potential pharmacotherapy for the management of POP.