RESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Amyotrophic lateral sclerosis (ALS) is a fetal neuromuscular disorder characterized by the gradual deterioration of motor neurons. Semen Strychni pulveratum (SSP), a processed version of Semen Strychni (SS) powder, is widely used to treat ALS in China. Vomicine is one of the most primary components of SS. However, their pharmacological effects and mechanisms for ALS remain elusive. AIM OF THE STUDY: This study aimed to evaluate the neuroprotective and anti-neuroinflammatory effects of SSP and vomicine, as well as to explore their protective roles in ALS and the underlying mechanisms. MATERIALS AND METHODS: In vivo, 8-week-old hSOD1-WT mice and hSOD1-G93A mice were orally administered different concentrations of SSP (SSP-L = 5.46 mg/ml, SSP-M = 10.92 mg/ml or SSP-H = 16.38 mg/ml) once every other day for 8 weeks. A series of experiments, including body weight measurement, footprint tests, Hematoxylin & Eosin staining, and Nissl staining, were performed to evaluate the preventive effect of SSP. Immunofluorescence staining, western blotting, and RT-qPCR were subsequently performed to evaluate activation of the cGAS-STING-TBK1 pathway in the spinal cord. In vitro, hSOD1G93A NSC-34 cells were treated with vomicine to further explore the pharmacological mechanism of vomicine in the treatment of ALS via the cGAS-STING-TBK1 pathway. RESULTS: SSP improved motor function, body weight loss, gastrocnemius muscle atrophy, and motor neuron loss in the spine and cortex of hSOD1-G93A mice. Furthermore, the cGAS-STING-TBK1 pathway was activated in the spinal cord of hSOD1-G93A mice, with activation predominantly observed in neurons and microglia. However, the levels of cGAS, STING, and pTBK1 proteins and cGAS, IRF3, IL-6, and IL-1ß mRNA were reversed following intervention with SSP. Vomicine not only downregulated the levels of cGAS, TBK1, IL-6 and IFN-ß mRNA, but also the levels of cGAS and STING protein in hSOD1G93A NSC-34 cells. CONCLUSION: This study demonstrated that SSP and vomicine exert neuroprotective and anti-neuroinflammatory effects in the treatment of ALS. SSP and vomicine may reduce neuroinflammation by regulating the cGAS-STING-TBK1 pathway, and could thereby play a role in ALS treatment.
Assuntos
Esclerose Lateral Amiotrófica , Proteínas de Membrana , Fármacos Neuroprotetores , Nucleotidiltransferases , Proteínas Serina-Treonina Quinases , Animais , Proteínas Serina-Treonina Quinases/metabolismo , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Camundongos , Proteínas de Membrana/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Nucleotidiltransferases/metabolismo , Masculino , Transdução de Sinais/efeitos dos fármacos , Camundongos Transgênicos , Doenças Neuroinflamatórias/tratamento farmacológico , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Modelos Animais de DoençasRESUMO
Traumatic spinal cord injury is characterized by immediate and irreversible tissue loss at the lesion site and secondary tissue damage. Secondary injuries should, in principle, be preventable, although no effective treatment options currently exist for patients with acute spinal cord injury. Traumatized tissues release excessive amounts of adenosine triphosphate and activate the P2X purinoceptor 7/pannexin1 complex, which is associated with secondary injury. We investigated the neuroprotective effects of the blue dye Brilliant Blue FCF, a selective inhibitor of P2X purinoceptor 7/pannexin1 that is approved for use as a food coloring, by comparing it with Brilliant Blue G, a P2X7 purinoceptor antagonist, and carbenoxolone, which attenuates P2X purinoceptor 7/pannexin1 function, in a rat spinal cord injury model. Brilliant Blue FCF administered early after spinal cord injury reduced spinal cord anatomical damage and improved motor recovery without apparent toxicity. Brilliant Blue G had the highest effect on this neurological recovery, with Brilliant Blue FCF and carbenoxolone having comparable improvement. Furthermore, Brilliant Blue FCF administration reduced local astrocytic and microglial activation and neutrophil infiltration, and no differences in these histological effects were observed between compounds. Thus, Brilliant Blue FCF protects spinal cord neurons after spinal cord injury and suppresses local inflammatory responses as well as Brilliant Blue G and carbenoxolone.
Assuntos
Trifosfato de Adenosina , Carbenoxolona , Conexinas , Proteínas do Tecido Nervoso , Recuperação de Função Fisiológica , Corantes de Rosanilina , Traumatismos da Medula Espinal , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/metabolismo , Animais , Conexinas/metabolismo , Conexinas/antagonistas & inibidores , Trifosfato de Adenosina/metabolismo , Carbenoxolona/farmacologia , Carbenoxolona/uso terapêutico , Corantes de Rosanilina/farmacologia , Corantes de Rosanilina/uso terapêutico , Proteínas do Tecido Nervoso/metabolismo , Proteínas do Tecido Nervoso/antagonistas & inibidores , Recuperação de Função Fisiológica/efeitos dos fármacos , Ratos , Antagonistas do Receptor Purinérgico P2X/farmacologia , Antagonistas do Receptor Purinérgico P2X/uso terapêutico , Ratos Sprague-Dawley , Modelos Animais de Doenças , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Microglia/efeitos dos fármacos , Microglia/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2X7/efeitos dos fármacos , Feminino , Infiltração de Neutrófilos/efeitos dos fármacosRESUMO
An important goal in the opioid field is to discover effective analgesic drugs with minimal side effects. MCRT demonstrated potent antinociceptive effects with limited side effects, making it a promising candidate. However, its pharmacological properties and how it minimizes side effects remain unknown. Various mouse pain and opioid side effect models were used to evaluate the antinociceptive properties and safety at the spinal level. The targets of MCRT were identified through cAMP measurement, isolated tissue assays, and pharmacological experiments. Immunofluorescence was employed to visualize protein expression. MCRT displayed distinct antinociceptive effects between acute and chronic inflammatory pain models due to its multifunctional properties at the µ opioid receptor (MOR), µ-δ heterodimer (MDOR), and neuropeptide FF receptor 2 (NPFFR2). Activation of NPFFR2 reduced MOR-mediated antinociception, leading to bell-shaped response curves in acute pain models. However, activation of MDOR produced more effective antinociception in chronic inflammatory pain models. MCRT showed limited tolerance and opioid-induced hyperalgesia in both acute and chronic pain models and did not develop cross-tolerance to morphine. Additionally, MCRT did not exhibit addictive properties, gastrointestinal inhibition, and effects on motor coordination. Mechanistically, peripheral chronic inflammation or repeated administration of morphine and MCRT induced an increase in MDOR in the spinal cord. Chronic administration of MCRT had no apparent effect on microglial activation in the spinal cord. These findings suggest that MCRT is a versatile compound that provides potent antinociception with minimal opioid-related side effects. MDOR could be a promising target for managing chronic inflammatory pain and addressing the opioid crisis.
Assuntos
Analgésicos Opioides , Dor Crônica , Modelos Animais de Doenças , Inflamação , Injeções Espinhais , Receptores Opioides mu , Animais , Dor Crônica/tratamento farmacológico , Receptores Opioides mu/metabolismo , Camundongos , Masculino , Inflamação/tratamento farmacológico , Analgésicos Opioides/administração & dosagem , Analgésicos Opioides/efeitos adversos , Analgésicos Opioides/farmacologia , Receptores de Neuropeptídeos/metabolismo , Receptores de Neuropeptídeos/antagonistas & inibidores , Receptores Opioides delta/metabolismo , Camundongos Endogâmicos C57BL , Analgésicos/farmacologia , Analgésicos/administração & dosagem , Morfina/administração & dosagem , Morfina/farmacologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Hiperalgesia/tratamento farmacológico , Humanos , Oligopeptídeos/administração & dosagem , Oligopeptídeos/farmacologiaRESUMO
The aim of this study was to elucidate the therapeutic effect of simvastatin on experimental autoimmune encephalomyelitis (EAE) by regulating the balance between Th17 and Treg cells in mice. C57BL/6 mice were randomly divided into four groups: normal group, EAE group, simvastatin (2 and 10 mg/kg) group, and AG490 group (with AG490 serving as the positive control). Neurological function scores of mice were assessed daily. The four groups received treatments of normal saline, normal saline, and simvastatin (2 and 10 mg/kg), respectively. In the AG490 group, mice were injected intraperitoneally with AG490 (1 mg) every other day, and treatment was halted after 3 weeks. The spinal cord was stained with hematoxylin and eosin (H&E), and immunohistochemical staining for retinoic acid receptor-related orphan receptor γ(RORγ) and Foxp3 (Foxp3) was performed. Spleen samples were taken for Th17 and Treg analysis using flow cytometry. The levels of interleukin-17 and transforming growth factor-ß (TGF-ß) were detected using enzyme-linked immunosorbent assay (ELISA). In the simvastatin and AG490 groups, recovery from neurological impairment was earlier compared to the EAE group, and the symptoms were notably improved. Both simvastatin and AG490 reduced focal inflammation, decreased RORγ-positive cell infiltration, and significantly increased the number of FOXP3-positive cells. The number of Th17 cells and the level of IL-17 in the spleen were decreased in the simvastatin and AG490 treatment groups, while the number of Treg cells and TGF-ß levels were significantly increased across all treatment groups. Simvastatin exhibits anti-inflammatory and immunomodulatory effects, potentially alleviating symptoms of neurological dysfunction of EAE. Regulating the balance between Th17 and Treg may represent a therapeutic mechanism for simvastatin in treating EAE.
Assuntos
Encefalomielite Autoimune Experimental , Camundongos Endogâmicos C57BL , Sinvastatina , Linfócitos T Reguladores , Células Th17 , Animais , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Células Th17/imunologia , Células Th17/efeitos dos fármacos , Linfócitos T Reguladores/imunologia , Linfócitos T Reguladores/efeitos dos fármacos , Sinvastatina/farmacologia , Sinvastatina/administração & dosagem , Camundongos , Feminino , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Interleucina-17/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Medula Espinal/imunologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Humanos , Fator de Crescimento Transformador beta/metabolismo , Modelos Animais de DoençasRESUMO
Itch is a protective sensation that drives scratching. Although specific cell types have been proposed to underlie itch, the neural basis for itch remains unclear. Here, we used two-photon Ca2+ imaging of the dorsal horn to visualize neuronal populations that are activated by itch-inducing agents. We identify a convergent population of spinal interneurons recruited by diverse itch-causing stimuli that represents a subset of neurons that express the gastrin-releasing peptide receptor (GRPR). Moreover, we find that itch is conveyed to the brain via GRPR-expressing spinal output neurons that target the lateral parabrachial nuclei. We then show that the kappa opioid receptor agonist nalfurafine relieves itch by selectively inhibiting GRPR spinoparabrachial neurons. These experiments provide a population-level view of the spinal neurons that respond to pruritic stimuli, pinpoint the output neurons that convey itch to the brain, and identify the cellular target of kappa opioid receptor agonists for the inhibition of itch.
Assuntos
Morfinanos , Prurido , Receptores da Bombesina , Receptores Opioides kappa , Prurido/tratamento farmacológico , Prurido/metabolismo , Animais , Receptores Opioides kappa/metabolismo , Receptores Opioides kappa/agonistas , Receptores da Bombesina/metabolismo , Receptores da Bombesina/antagonistas & inibidores , Receptores da Bombesina/agonistas , Camundongos , Morfinanos/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Medula Espinal/metabolismo , Medula Espinal/efeitos dos fármacos , Compostos de Espiro/farmacologia , Interneurônios/metabolismo , Interneurônios/efeitos dos fármacos , MasculinoRESUMO
AIMS: Neuropathic pain remains a significant unmet medical challenge due to its elusive mechanisms. Recent clinical observations suggest that vitamin D (VitD) holds promise in pain relief, yet its precise mechanism of action is still unclear. This study explores the therapeutical role and potential mechanism of VitD3 in spared nerve injury (SNI)-induced neuropathic pain rat model. METHODS: The analgesic effects and underlying mechanisms of VitD3 were evaluated in SNI and naïve rat models. Mechanical allodynia was assessed using the Von Frey test. Western blotting, immunofluorescence, biochemical assay, and transmission electron microscope (TEM) were employed to investigate the molecular and cellular effects of VitD3. RESULTS: Ferroptosis was observed in the spinal cord following SNI. Intrathecal administration of VitD3, the active form of VitD, activated the vitamin D receptor (VDR), suppressed ferroptosis, and alleviated mechanical nociceptive behaviors. VitD3 treatment preserved spinal GABAergic interneurons, and its neuroprotective effects were eliminated by the ferroptosis inducer RSL3. Additionally, VitD3 mitigated aberrant mitochondrial morphology and oxidative metabolism in the spinal cord. Mechanistically, VitD3 inhibited SNI-induced activation of spinal PKCα/NOX4 signaling. Inhibition of PKCα/NOX4 signaling alleviated mechanical pain hypersensitivity, accompanied by reduced ferroptosis and mitochondrial dysfunction in SNI rats. Conversely, activation of PKCα/NOX4 signaling in naïve rats induced hyperalgesia, ferroptosis, loss of GABAergic interneurons, and mitochondrial dysfunction in the spinal cord, all of which were reversed by VitD3 treatment. CONCLUSIONS: Our findings provide evidence that VitD3 attenuates neuropathic pain by preserving spinal GABAergic interneurons through the suppression of mitochondria-associated ferroptosis mediated by PKCα/NOX4 signaling, probably via VDR activation. VitD, alone or in combination with existing analgesics, presents an innovative therapeutic avenue for neuropathic pain.
Assuntos
Colecalciferol , Ferroptose , Mitocôndrias , NADPH Oxidase 4 , Neuralgia , Proteína Quinase C-alfa , Ratos Sprague-Dawley , Transdução de Sinais , Animais , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , NADPH Oxidase 4/metabolismo , NADPH Oxidase 4/antagonistas & inibidores , Ferroptose/efeitos dos fármacos , Ferroptose/fisiologia , Masculino , Ratos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-alfa/antagonistas & inibidores , Colecalciferol/farmacologia , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologiaRESUMO
AIM: We aimed to explore whether the combination of CLP290 and bumetanide maximally improves neuropathic pain following spinal cord injury (SCI) and its possible molecular mechanism. METHODS: Rats were randomly divided into five groups: Sham, SCI + vehicle, SCI + CLP290, SCI + bumetanide, and SCI + combination (CLP290 + bumetanide). Drug administration commenced on the 7th day post-injury (7 dpi) and continued for 14 days. All rats underwent behavioral assessments for 56 days to comprehensively evaluate the effects of interventions on mechanical pain, thermal pain, cold pain, motor function, and other relevant parameters. Electrophysiological assessments, immunoblotting, and immunofluorescence detection were performed at different timepoints post-injury, with a specific focus on the expression and changes of KCC2 and NKCC1 proteins in the lumbar enlargement of the spinal cord. RESULTS: CLP290 and bumetanide alleviated SCI-associated hypersensitivity and locomotor function, with the combination providing enhanced recovery. The combined treatment group exhibited the most significant improvement in restoring Rate-Dependent Depression (RDD) levels. In the combined treatment group and the two individual drug administration groups, the upregulation of potassium chloride cotransporter 2 (K+-Cl-cotransporter 2, KCC2) expression and downregulation of sodium potassium chloride cotransporter 1 (Na+-K+-Cl-cotransporter 1, NKCC1) expression in the lumbar enlargement area resulted in a significant increase in the KCC2/NKCC1 ratio compared to the SCI + vehicle group, with the most pronounced improvement seen in the combined treatment group. Compared to the SCI + vehicle group, the SCI + bumetanide group showed no significant paw withdrawal thermal latency (PWTL) improvement at 21 and 35 dpi, but a notable enhancement at 56 dpi. In contrast, the SCI + CLP290 group significantly improved PWTL at 21 days, with non-significant changes at 35 and 56 days. At 21 dpi, KCC2 expression was marginally higher in monotherapy groups versus SCI + vehicle, but not significantly. At 56 dpi, only the SCI + bumetanide group showed a significant difference in KCC2 expression compared to the control group. CONCLUSION: Combined application of CLP290 and bumetanide effectively increases the ratio of KCC2/NKCC1, restores RDD levels, enhances GABAA receptor-mediated inhibitory function in the spinal cord, and relieves neuropathic pain in SCI; Bumetanide significantly improves neuropathic pain in the long term, whereas CLP290 demonstrates a notable short-term effect.
Assuntos
Bumetanida , Cotransportadores de K e Cl- , Neuralgia , Ratos Sprague-Dawley , Membro 2 da Família 12 de Carreador de Soluto , Traumatismos da Medula Espinal , Simportadores , Animais , Bumetanida/farmacologia , Bumetanida/uso terapêutico , Traumatismos da Medula Espinal/complicações , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/metabolismo , Neuralgia/tratamento farmacológico , Neuralgia/etiologia , Neuralgia/metabolismo , Ratos , Masculino , Simportadores/metabolismo , Membro 2 da Família 12 de Carreador de Soluto/metabolismo , Inibidores de Simportadores de Cloreto de Sódio e Potássio/farmacologia , Inibidores de Simportadores de Cloreto de Sódio e Potássio/uso terapêutico , Quimioterapia Combinada , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Acetatos , IndenosRESUMO
Experimental autoimmune encephalomyelitis is a demyelinating disease that causes paralysis in laboratory rats. This condition lacks treatment that reverses damage to the myelin sheaths of neuronal cells. Therefore, in this study, treatment with EPO as a neuroprotective effect was established to evaluate the ERK 1/2 signaling pathway and its participation in the EAE model. EPO was administered in 5000 U/Kg Sprague Dawley rats. U0126 was used as an inhibitor of the ERK 1/2 pathway to demonstrate the possible activation of this pathway in the model. Spinal cord and optic nerve tissues were evaluated using staining techniques such as H&E and the Luxol Fast Blue myelin-specific technique, as well as immunohistochemistry of the ERK 1/2 protein. The EPO-treated groups showed a decrease in cellular sampling in the spinal cord tissues but mainly in the optic nerve, as well as an increase in the expression of the ERK 1/2 protein in both tissues. The findings of this study suggest that EPO treatment reduces cellular death in EAE-induced rats by regulating the ERK pathway.
Assuntos
Encefalomielite Autoimune Experimental , Eritropoetina , Sistema de Sinalização das MAP Quinases , Fármacos Neuroprotetores , Nervo Óptico , Ratos Sprague-Dawley , Medula Espinal , Animais , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/metabolismo , Encefalomielite Autoimune Experimental/patologia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Eritropoetina/farmacologia , Nervo Óptico/efeitos dos fármacos , Nervo Óptico/patologia , Nervo Óptico/metabolismo , Ratos , Medula Espinal/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Feminino , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismoRESUMO
Traumatic spinal cord injury (tSCI) has complex pathophysiological events that begin after the initial trauma. One such event is fibroglial scar formation by fibroblasts and reactive astrocytes. A strong inhibition of axonal growth is caused by the activated astroglial cells as a component of fibroglial scarring through the production of inhibitory molecules, such as chondroitin sulfate proteoglycans or myelin-associated proteins. Here, we used neural precursor cells (aldynoglia) as promoters of axonal growth and a fibrin hydrogel gelled under alkaline conditions to support and guide neuronal cell growth, respectively. We added Tol-51 sulfoglycolipid as a synthetic inhibitor of astrocyte and microglia in order to test its effect on the axonal growth-promoting function of aldynoglia precursor cells. We obtained an increase in GFAP expression corresponding to the expected glial phenotype for aldynoglia cells cultured in alkaline fibrin. In co-cultures of dorsal root ganglia (DRG) and aldynoglia, the axonal growth promotion of DRG neurons by aldynoglia was not affected. We observed that the neural precursor cells first clustered together and then formed niches from which aldynoglia cells grew and connected to groups of adjacent cells. We conclude that the combination of alkaline fibrin with synthetic sulfoglycolipid Tol-51 increased cell adhesion, cell migration, fasciculation, and axonal growth capacity, promoted by aldynoglia cells. There was no negative effect on the behavior of aldynoglia cells after the addition of sulfoglycolipid Tol-51, suggesting that a combination of aldynoglia plus alkaline fibrin and Tol-51 compound could be useful as a therapeutic strategy for tSCI repair.
Assuntos
Axônios , Fibrina , Gânglios Espinais , Animais , Gânglios Espinais/efeitos dos fármacos , Gânglios Espinais/metabolismo , Gânglios Espinais/citologia , Axônios/metabolismo , Axônios/efeitos dos fármacos , Fibrina/metabolismo , Hidrogéis/química , Hidrogéis/farmacologia , Ratos , Glicolipídeos/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Células-Tronco Neurais/citologia , Neurônios/metabolismo , Neurônios/efeitos dos fármacos , Células Cultivadas , Técnicas de Cocultura , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/tratamento farmacológico , Traumatismos da Medula Espinal/patologia , Neuroglia/efeitos dos fármacos , Neuroglia/metabolismo , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Medula Espinal/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/citologia , Movimento Celular/efeitos dos fármacosRESUMO
Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease. Syncytin-1 (Syn), an envelope glycoprotein encoded by the env gene of the human endogenous retrovirus-W family, has been resorted to be highly expressed in biopsies from the muscles from ALS patients; however, the specific regulatory role of Syn during ALS progression remains uncovered. In this study, C57BL/6 mice were injected with adeno-associated virus-overexpressing Syn, with or without Fasudil administration. The Syn expression was assessed by quantitative real-time polymerase chain reaction and immunohistochemistry analysis. The histological change of anterior tibial muscles was determined by hematoxylin-eosin staining. Qualitative ultrastructural analysis of electron micrographs obtained from lumbar spinal cords was carried out. Serum inflammatory cytokines were assessed by enzyme linked immunosorbent assay (ELISA) assay and motor function was recorded using Basso, Beattie, and Bresnahan (BBB) scoring, climbing test and treadmill running test. Immunofluorescence and western blot assays were conducted to examine microglial- and motor neurons-related proteins. Syn overexpression significantly caused systemic inflammatory response, muscle tissue lesions, and motor dysfunction in mice. Meanwhile, Syn overexpression promoted the impairment of motor neuron, evidenced by the damaged structure of the neurons and reduced expression of microtubule-associated protein 2, HB9, neuronal nuclei and neuron-specific enolase in Syn-induced mice. In addition, Syn overexpression greatly promoted the expression of CD16/CD32 and inducible nitric oxide synthase (M1 phenotype markers), and reduced the expression of CD206 and arginase 1 (M2 phenotype markers). Importantly, the above changes caused by Syn overexpression were partly abolished by Fasudil administration. This study provides evidence that Syn-activated microglia plays a pivotal role during the progression of ALS.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina , Camundongos Endogâmicos C57BL , Microglia , Neurônios Motores , Animais , Microglia/efeitos dos fármacos , Microglia/metabolismo , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/metabolismo , Camundongos , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Produtos do Gene env , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Proteínas da Gravidez/metabolismo , Masculino , Citocinas/metabolismo , Modelos Animais de Doenças , Atividade Motora/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/efeitos dos fármacosRESUMO
Modifications to the small intestine and liver are known to occur during the symptomatic disease period of amyotrophic lateral sclerosis (ALS), a member of the motor neuron disease (MND) family of neurodegenerative disorders. How these modifications impact on oral absorption and pharmacokinetics of drugs remains unknown. In this study, model drugs representing different mechanisms of intestinal transport (caffeine for passive diffusion, digoxin for P-glycoprotein efflux, and sulfasalazine for breast cancer resistance protein efflux) were administered via oral gavage to postnatal day 114-120 male and female SOD1G93A mice (model of familial ALS) and wild-type (WT) littermates. Samples of blood, brain and spinal cord were taken at either 15, 30, 60 or 180 min after administration. In addition, the in vivo gastric emptying of 70 kDa fluorescein isothiocyanate-dextran (FITC-dextran) and the ex vivo intestinal permeability of caffeine were assessed. The area under the plasma concentration-time curves (AUCplasma) of digoxin and sulfasalazine were not significantly different between SOD1G93A and WT mice for both sexes. However, the AUCplasma of caffeine was significantly lower (female: 0.79-fold, male: 0.76-fold) in SOD1G93A compared to WT mice, which was associated with lower AUCbrain (female: 0.76-fold, male: 0.80-fold) and AUCspinal cord (female: 0.81-fold, male: 0.82-fold). The AUCstomach of caffeine was significantly higher (female: 1.5-fold, male: 1.9-fold) in SOD1G93A compared to WT mice, suggesting reduced gastric emptying in SOD1G93A mice. In addition, there was a significant reduction in gastric emptying of FITC-dextran (0.66-fold) and ex vivo intestinal permeability of caffeine (0.52-fold) in male SOD1G93A compared to WT mice. Reduced systemic and brain/spinal cord exposure of caffeine in SOD1G93A mice may therefore result from alterations to gastric emptying and small intestinal permeability. Specific dosing requirements may therefore be required for certain medicines in ALS to ensure that they remain in a safe and effective concentration range.
Assuntos
Esclerose Lateral Amiotrófica , Encéfalo , Cafeína , Modelos Animais de Doenças , Camundongos Transgênicos , Medula Espinal , Animais , Cafeína/administração & dosagem , Cafeína/farmacocinética , Esclerose Lateral Amiotrófica/tratamento farmacológico , Esclerose Lateral Amiotrófica/metabolismo , Medula Espinal/metabolismo , Medula Espinal/efeitos dos fármacos , Masculino , Feminino , Camundongos , Administração Oral , Encéfalo/metabolismo , Encéfalo/efeitos dos fármacos , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Digoxina/farmacocinética , Digoxina/administração & dosagem , Sulfassalazina/farmacocinética , Sulfassalazina/administração & dosagem , Absorção Intestinal/efeitos dos fármacos , Absorção Intestinal/fisiologiaRESUMO
Opioids are widely used, effective analgesics to manage severe acute and chronic pain, although they have recently come under scrutiny because of epidemic levels of abuse. While these compounds act on numerous central and peripheral pain pathways, the neuroanatomical substrate for opioid analgesia is not fully understood. By means of single-cell transcriptomics and manipulation of morphine-responsive neurons, we have identified an ensemble of neurons in the rostral ventromedial medulla (RVM) that regulates mechanical nociception in mice. Among these, forced activation or silencing of excitatory RVMBDNF projection neurons mimicked or completely reversed morphine-induced mechanical antinociception, respectively, via a brain-derived neurotrophic factor (BDNF)/tropomyosin receptor kinase B (TrkB)-dependent mechanism and activation of inhibitory spinal galanin-positive neurons. Our results reveal a specific RVM-spinal circuit that scales mechanical nociception whose function confers the antinociceptive properties of morphine.
Assuntos
Analgésicos Opioides , Fator Neurotrófico Derivado do Encéfalo , Bulbo , Morfina , Neurônios , Nociceptividade , Animais , Masculino , Camundongos , Analgésicos Opioides/farmacologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Fator Neurotrófico Derivado do Encéfalo/genética , Bulbo/efeitos dos fármacos , Bulbo/metabolismo , Camundongos Endogâmicos C57BL , Morfina/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/fisiologia , Nociceptividade/efeitos dos fármacos , Receptor trkB/metabolismo , Análise de Célula Única , Medula Espinal/efeitos dos fármacos , Transcriptoma , FemininoRESUMO
While Resolvin D1 (RvD1) shows promise in resolving inflammation in experimental autoimmune encephalomyelitis (EAE), its pro-resolving roles on dendritic cells (DCs) remain unknown, and the chemical instability of RvD1 poses significant challenges to its drug development. This study aims to investigate whether 4-(2'-methoxyphenyl)-1-[2'-[N-(2â³-pyridinyl)-p-fluorobenzamido]ethyl]piperazine (p-MPPF), a novel chemically stable analogue of RvD1, can play a pro-resolving role in EAE, particularly on DCs, and if p-MPPF could serve as a potential substitute for RvD1. We showed that both RvD1 and p-MPPF mediated the resolution of inflammation in EAE, as evidenced by ameliorated EAE progression, attenuated pathological changes in the spinal cord, altered cytokine expression profile in serum, and reduced proportion of pro-inflammatory immune cells in the spleen. Utilizing DCs derived from both the spleen and bone marrow of EAE, our investigation showed that RvD1 and p-MPPF prevented DC maturation, decreased pro-inflammatory cytokine secretion, shifted DCs away from a pro-inflammatory phenotype, increased the phagocytosis capacity of DCs, and suppressed their ability to induce differentiation of CD4+ T cells into Th1 and Th17 subsets. For underlying intracellular mechanisms, we found that RvD1 and p-MPPF down-regulated the lactate dehydrogenase A signaling pathways. Comparisons between RvD1 and p-MPPF showed that they exerted overlapped pro-resolving effects to a large extent. This study demonstrates that both RvD1 and p-MPPF exert therapeutic effects on EAE by mediating inflammation resolution, which is closely associated with modulating DC immune function towards a tolerogenic phenotype. SPM mimetics may serve as a more promising therapeutic drug.
Assuntos
Citocinas , Células Dendríticas , Ácidos Docosa-Hexaenoicos , Encefalomielite Autoimune Experimental , Camundongos Endogâmicos C57BL , Encefalomielite Autoimune Experimental/tratamento farmacológico , Encefalomielite Autoimune Experimental/imunologia , Animais , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Ácidos Docosa-Hexaenoicos/uso terapêutico , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Docosa-Hexaenoicos/química , Feminino , Camundongos , Citocinas/metabolismo , Anti-Inflamatórios/uso terapêutico , Anti-Inflamatórios/farmacologia , Medula Espinal/efeitos dos fármacos , Medula Espinal/imunologia , Medula Espinal/patologia , Medula Espinal/metabolismo , Inflamação/tratamento farmacológico , Inflamação/imunologia , Piperazinas/farmacologia , Piperazinas/uso terapêutico , Piperazinas/química , Células Cultivadas , Baço/efeitos dos fármacos , Baço/imunologia , Células Th17/imunologia , Células Th17/efeitos dos fármacosRESUMO
Immune cells are critical in promoting neuroinflammation and neuropathic pain and in facilitating pain resolution, depending on their inflammatory and immunoregulatory cytokine response. Interleukin (IL)-35, secreted by regulatory immune cells, is a member of the IL-12 family with a potent immunosuppressive function. In this study, we investigated the effects of IL-35 on pain behaviors, spinal microglia phenotype following peripheral nerve injury, and in vitro microglial cultures in male and female mice. Intrathecal recombinant IL-35 treatment alleviated mechanical pain hypersensitivity prominently in male mice, with only a modest effect in female mice after sciatic nerve chronic constriction injury (CCI). IL-35 treatment resulted in sex-specific microglial changes following CCI, reducing inflammatory microglial markers and upregulating anti-inflammatory markers in male mice. Spatial transcriptomic analysis revealed that IL-35 suppressed microglial complement activation in the superficial dorsal horn in male mice after CCI. Moreover, in vitro studies showed that IL-35 treatment of cultured inflammatory microglia mitigated their hypertrophied morphology, increased their cell motility, and decreased their phagocytic activity, indicating a phenotypic shift towards homeostatic microglia. Further, IL-35 altered microglial cytokines/chemokines in vitro, suppressing the release of IL-9 and monocyte-chemoattractant protein-1 and increasing IL-10 in the supernatant of male microglial cultures. Our findings indicate that treatment with IL-35 modulates spinal microglia and alleviates neuropathic pain in male mice, suggesting IL-35 as a potential sex-specific targeted immunomodulatory treatment for neuropathic pain.
Assuntos
Interleucinas , Microglia , Neuralgia , Traumatismos dos Nervos Periféricos , Animais , Masculino , Microglia/metabolismo , Microglia/efeitos dos fármacos , Camundongos , Neuralgia/metabolismo , Neuralgia/tratamento farmacológico , Interleucinas/metabolismo , Feminino , Traumatismos dos Nervos Periféricos/metabolismo , Traumatismos dos Nervos Periféricos/complicações , Camundongos Endogâmicos C57BL , Citocinas/metabolismo , Medula Espinal/metabolismo , Medula Espinal/efeitos dos fármacos , Nervo Isquiático/lesões , Nervo Isquiático/metabolismo , Hiperalgesia/metabolismo , Hiperalgesia/tratamento farmacológico , Anti-Inflamatórios/farmacologia , Modelos Animais de Doenças , Inflamação/metabolismoRESUMO
Providing antioxidants and targeting acetylcholinesterase (AChE) are key strategies in treating neurocognitive dysfunction. In this study, bioactive sturgeon (Acipenser schrenckii) spinal cord peptides (SSCPs) with antioxidant and AChE inhibitory potency were extracted and separated from sturgeon spinal cord by enzymatic hydrolysis and ultrafiltration, and targeted peptide PGGW was screened via computer simulated molecular docking. Further, the molecular dynamic interactions of the PGGW with superoxide dismutase (SOD) and AChE were analyzed, and the protective effect of PGGW on glutamate-induced PC12 cells in vitro was evaluated. The <3 kDa fraction of SSCPs displays the most potent antioxidative efficacy (1 mg/mL, DPPHâ¢: 89.07%, ABTS+: 76.35%). Molecular dynamics simulation showed that PGGW was stable within AChE and tightly bound to residues SER203, PHE295, ILE294 and TRP236. When combined with SOD, the indole group of PGGW was stuck inside SOD, but the tail chain PGG fluctuated greatly outside. Surface plasmon resonance demonstrated that PGGW has a high binding affinity for AChE (KD = 1.4 mM) and 0.01 mg/mL PGGW provided good protection against glutamate-induced apoptosis. The findings suggest a promising strategy for drug research on neurodegenerative diseases.
Assuntos
Acetilcolinesterase , Antioxidantes , Inibidores da Colinesterase , Proteínas de Peixes , Peixes , Simulação de Acoplamento Molecular , Peptídeos , Medula Espinal , Animais , Antioxidantes/química , Antioxidantes/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Acetilcolinesterase/química , Acetilcolinesterase/metabolismo , Inibidores da Colinesterase/farmacologia , Inibidores da Colinesterase/química , Proteínas de Peixes/química , Proteínas de Peixes/farmacologia , Ratos , Medula Espinal/química , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Células PC12 , Superóxido Dismutase/metabolismo , Superóxido Dismutase/químicaRESUMO
BACKGROUND: Spinal cord injury (SCI) is considered a central nervous system (CNS) disorder. Nuclear factor kappa B (NF-κB) regulates inflammatory responses in the CNS and is implicated in SCI pathogenesis. The mechanism(s) through which NF-κB contributes to the neuroinflammation observed during SCI however remains unclear. METHODS: SCI rat models were created using the weight drop method and separated into Sham, SCI and SCI+NF-κB inhibitor groups (n = 6 rats per-group). We used Hematoxylin-Eosin Staining (H&E) and Nissl staining for detecting histological changes in the spinal cord. Basso-Beattie-Bresnahan (BBB) behavioral scores were utilized for assessing functional locomotion recovery. Mouse BV2 microglia were exposed to lipopolysaccharide (LPS) to mimic SCI-induced microglial inflammation in vitro. RESULTS: Inhibition of NF-κB using JSH-23 alleviated inflammation and neuronal injury in SCI rats' spinal cords, leading to improved locomotion recovery (p < 0.05). NF-κB inhibition reduced expression levels of CD86, interleukin-6 (IL-6), IL-1ß, and inducible Nitric Oxide Synthase (iNOS), and improved expression levels of CD206, IL-4, and tissue growth factor-beta (TGF-ß) in both LPS-treated microglia and SCI rats' spinal cords (p < 0.05). Inhibition of NF-κB also effectively suppressed mitochondrial fission, evidenced by the reduced phosphorylation of dynamin-related protein 1 (DRP1) at Ser616 (p < 0.001). CONCLUSION: We show that inhibition of the NF-κB/DRP1 axis prevents mitochondrial fission and suppresses pro-inflammatory microglia polarization, promoting neurological recovery in SCI. Targeting the NF-κB/DRP1 axis therefore represents a novel approach for SCI.
Assuntos
Dinaminas , Microglia , NF-kappa B , Traumatismos da Medula Espinal , Animais , Masculino , Camundongos , Ratos , Linhagem Celular , Polaridade Celular/efeitos dos fármacos , Modelos Animais de Doenças , Dinaminas/metabolismo , Dinaminas/genética , Inflamação/metabolismo , Lipopolissacarídeos , Locomoção/efeitos dos fármacos , Microglia/metabolismo , Microglia/efeitos dos fármacos , Neuroproteção , NF-kappa B/metabolismo , Quinazolinonas , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/efeitos dos fármacos , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/patologiaRESUMO
INTRODUCTION: This study aimed to investigate whether the maternal administration of minocycline, a tetracycline antibiotic known to have anti-inflammatory and neuroprotective properties in models of neural injury, reduces inflammation and neural cell death in a fetal rat model of myelomeningocele (MMC). METHODS: E10 pregnant rats were gavaged with olive oil or olive oil + retinoic acid to induce fetal MMC. At E12, the dams were exposed to regular drinking water or water containing minocycline (range, 40-140 mg/kg/day). At E21, fetal lumbosacral spinal cords were isolated for immunohistochemistry and quantitative gene expression studies focused on microglia activity, inflammation, and apoptosis (P < 0.05). RESULTS: There was a trend toward decreased activated Iba1+ microglial cells within the dorsal spinal cord of MMC pups following minocycline exposure when compared to water (H2O) alone (P = 0.052). Prenatal minocycline exposure was correlated with significantly reduced expression of the proinflammatory cytokine, IL-6 (minocycline: 1.75 versus H2O: 3.52, P = 0.04) and apoptosis gene, Bax (minocycline: 0.71 versus H2O: 1.04, P < 0.001) among MMC pups. CONCLUSIONS: This study found evidence that the maternal administration of minocycline reduces selected markers of inflammation and apoptosis within the exposed dorsal spinal cords of fetal MMC rats. Further study of minocycline as a novel prenatal treatment strategy to mitigate spinal cord damage in MMC is warranted.
Assuntos
Modelos Animais de Doenças , Meningomielocele , Minociclina , Ratos Sprague-Dawley , Medula Espinal , Animais , Feminino , Minociclina/farmacologia , Minociclina/administração & dosagem , Gravidez , Meningomielocele/patologia , Ratos , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Medula Espinal/patologia , Apoptose/efeitos dos fármacos , Terapias Fetais/métodos , Antibacterianos , Microglia/efeitos dos fármacos , Microglia/patologia , Microglia/metabolismo , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/administração & dosagemRESUMO
Our previous in vitro studies showed that excitotoxicity evoked by glutamate analogue kainate (KA) significantly decreased the number of rat spinal neurons and triggered high release of glutamate leading to locomotor network block. Our current objective was to assess the role of CREB as a predictive marker of damage following chemically-induced spinal cord injury by using in vivo and in vitro models. Thus, in vivo excitotoxicity in Balb/c adult mice was induced by KA intraspinal injection, while in vitro spinal cord excitotoxicity was produced by bath-applied KA. KA application evoked significant neuronal loss, deterioration in hindlimb motor coordination and thermal allodynia. In addition, immunohistochemical analysis showed that KA application resulted in decreased number of CREB positive nuclei in the ventral horn and in dorsal layers III-IV. Our data suggests that excitotoxic-induced neuronal loss may be potentially predicted by altered CREB nuclear translocation.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico , Ácido Caínico , Camundongos Endogâmicos BALB C , Nociceptividade , Medula Espinal , Animais , Ácido Caínico/farmacologia , Camundongos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Nociceptividade/efeitos dos fármacos , Masculino , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Agonistas de Aminoácidos Excitatórios/farmacologia , Agonistas de Aminoácidos Excitatórios/toxicidade , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/induzido quimicamente , Locomoção/efeitos dos fármacos , Núcleo Celular/metabolismo , Núcleo Celular/efeitos dos fármacos , Hiperalgesia/induzido quimicamente , Hiperalgesia/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismoRESUMO
Morphine is an important pain reliever employed in pain management, its extended utilize is hindered by the onset of analgesic tolerance and oxidative stress. Long-term morphine administration causes elevated production of reactive oxygen species (ROS), disrupting mitochondrial function and inducing oxidation. Sirtuin 3 (SIRT3), a mitochondrial protein, is essential in modulating ROS levels by regulating mitochondrial antioxidant enzymes as manganese superoxide dismutase (MnSOD). Our investigation focused on the impact of SIRT3 on hyperalgesia and morphine tolerance in mice, as evaluating the antioxidant effect of the polyphenolic fraction of bergamot (BPF). Mice were administered morphine twice daily for four consecutive days (20 mg/kg). On the fifth day, mice received an acute dose of morphine (3 mg/kg), either alone or in conjunction with BPF or Mn (III)tetrakis (4-benzoic acid) porphyrin (MnTBAP). We evaluated levels of malondialdehyde (MDA), nitration, and the activity of SIRT3, MnSOD, glutamine synthetase (GS), and glutamate 1 transporter (GLT1) in the spinal cord. Our findings demonstrate that administering repeated doses of morphine led to the development of antinociceptive tolerance in mice, accompanied by increased superoxide production, nitration, and inactivation of mitochondrial SIRT3, MnSOD, GS, and GLT1. The combined administration of morphine with either BPF or MnTBAP prevented these effects.
Assuntos
Tolerância a Medicamentos , Hiperalgesia , Mitocôndrias , Morfina , Estresse Oxidativo , Polifenóis , Sirtuína 3 , Animais , Morfina/farmacologia , Camundongos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Masculino , Hiperalgesia/tratamento farmacológico , Hiperalgesia/induzido quimicamente , Polifenóis/farmacologia , Sirtuína 3/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Superóxido Dismutase/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Medula Espinal/efeitos dos fármacos , Medula Espinal/metabolismo , Antioxidantes/farmacologia , Analgésicos Opioides/farmacologia , Malondialdeído/metabolismo , Glutamato-Amônia Ligase/metabolismo , Metaloporfirinas/farmacologiaRESUMO
Homeostatic plasticity represents a set of mechanisms thought to stabilize some function of neural activity. Here, we identified the specific features of cellular or network activity that were maintained after the perturbation of GABAergic blockade in two different systems: mouse cortical neuronal cultures where GABA is inhibitory and motoneurons in the isolated embryonic chick spinal cord where GABA is excitatory (males and females combined in both systems). We conducted a comprehensive analysis of various spiking activity characteristics following GABAergic blockade. We observed significant variability in many features after blocking GABAA receptors (e.g., burst frequency, burst duration, overall spike frequency in culture). These results are consistent with the idea that neuronal networks achieve activity goals using different strategies (degeneracy). On the other hand, some features were consistently altered after receptor blockade in the spinal cord preparation (e.g., overall spike frequency). Regardless, these features did not express strong homeostatic recoveries when tracking individual preparations over time. One feature showed a consistent change and homeostatic recovery following GABAA receptor block. We found that spike rate within a burst (SRWB) increased after receptor block in both the spinal cord preparation and cortical cultures and then returned to baseline within hours. These changes in SRWB occurred at both single cell and population levels. Our findings indicate that the network prioritizes the burst spike rate, which appears to be a variable under tight homeostatic regulation. The result is consistent with the idea that networks can maintain an appropriate behavioral response in the face of challenges.