RESUMEN
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.
Asunto(s)
Esclerosis Amiotrófica Lateral , Proteínas de la Membrana , Fármacos Neuroprotectores , Nucleotidiltransferasas , Proteínas Serina-Treonina Quinasas , Animales , Proteínas Serina-Treonina Quinasas/metabolismo , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/metabolismo , Ratones , Proteínas de la Membrana/metabolismo , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Nucleotidiltransferasas/metabolismo , Masculino , Transducción de Señal/efectos de los fármacos , Ratones Transgénicos , Enfermedades Neuroinflamatorias/tratamiento farmacológico , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patología , Modelos Animales de EnfermedadRESUMEN
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.
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1-(5-Isoquinolinesulfonil)-2-Metilpiperazina , Ratones Endogámicos C57BL , Microglía , Neuronas Motoras , Animales , Microglía/efectos de los fármacos , Microglía/metabolismo , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Ratones , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Productos del Gen env , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/metabolismo , Proteínas Gestacionales/metabolismo , Masculino , Citocinas/metabolismo , Modelos Animales de Enfermedad , Actividad Motora/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacosRESUMEN
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.
Asunto(s)
Bumetanida , Cotransportadores de K Cl , Neuralgia , Ratas Sprague-Dawley , Miembro 2 de la Familia de Transportadores de Soluto 12 , Traumatismos de la Médula Espinal , Simportadores , Animales , Bumetanida/farmacología , Bumetanida/uso terapéutico , Traumatismos de la Médula Espinal/complicaciones , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/metabolismo , Neuralgia/tratamiento farmacológico , Neuralgia/etiología , Neuralgia/metabolismo , Ratas , Masculino , Simportadores/metabolismo , Miembro 2 de la Familia de Transportadores de Soluto 12/metabolismo , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/farmacología , Inhibidores del Simportador de Cloruro Sódico y Cloruro Potásico/uso terapéutico , Quimioterapia Combinada , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/etiología , Acetatos , IndenosRESUMEN
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.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Eritropoyetina , Sistema de Señalización de MAP Quinasas , Fármacos Neuroprotectores , Nervio Óptico , Ratas Sprague-Dawley , Médula Espinal , Animales , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/metabolismo , Encefalomielitis Autoinmune Experimental/patología , Fármacos Neuroprotectores/farmacología , Fármacos Neuroprotectores/uso terapéutico , Eritropoyetina/farmacología , Nervio Óptico/efectos de los fármacos , Nervio Óptico/patología , Nervio Óptico/metabolismo , Ratas , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Femenino , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismoRESUMEN
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.
Asunto(s)
Encefalomielitis Autoinmune Experimental , Ratones Endogámicos C57BL , Simvastatina , Linfocitos T Reguladores , Células Th17 , Animales , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Células Th17/inmunología , Células Th17/efectos de los fármacos , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/efectos de los fármacos , Simvastatina/farmacología , Simvastatina/administración & dosificación , Ratones , Femenino , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Interleucina-17/metabolismo , Factores de Transcripción Forkhead/metabolismo , Médula Espinal/inmunología , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Humanos , Factor de Crecimiento Transformador beta/metabolismo , Modelos Animales de EnfermedadRESUMEN
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.
Asunto(s)
Axones , Fibrina , Ganglios Espinales , Animales , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Ganglios Espinales/citología , Axones/metabolismo , Axones/efectos de los fármacos , Fibrina/metabolismo , Hidrogeles/química , Hidrogeles/farmacología , Ratas , Glucolípidos/farmacología , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Neuronas/metabolismo , Neuronas/efectos de los fármacos , Células Cultivadas , Técnicas de Cocultivo , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/patología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/citología , Movimiento Celular/efectos de los fármacosRESUMEN
AIMS: Poly (ADP-ribose) polymerase (PARP) has been extensively investigated in human cancers. Recent studies verified that current available PARP inhibitors (Olaparib or Veliparib) provided clinical palliation of clinical patients suffering from paclitaxel-induced neuropathic pain (PINP). However, the underlying mechanism of PARP overactivation in the development of PINP remains to be investigated. METHODS AND RESULTS: We reported induction of DNA oxidative damage, PARP-1 overactivation, and subsequent nicotinamide adenine dinucleotide (NAD+) depletion as crucial events in the pathogenesis of PINP. Therefore, we developed an Olaparib PROTAC to achieve the efficient degradation of PARP. Continuous intrathecal injection of Olaparib PROTAC protected against PINP by inhibiting the activity of PARP-1 in rats. PARP-1, but not PARP-2, was shown to be a crucial enzyme in the development of PINP. Specific inhibition of PARP-1 enhanced mitochondrial redox metabolism partly by upregulating the expression and deacetylase activity of sirtuin-3 (SIRT3) in the dorsal root ganglions and spinal cord in the PINP rats. Moreover, an increase in the NAD+ level was found to be a crucial mechanism by which PARP-1 inhibition enhanced SIRT3 activity. CONCLUSION: The findings provide a novel insight into the mechanism of DNA oxidative damage in the development of PINP and implicate PARP-1 as a possible therapeutic target for clinical PINP treatment.
Asunto(s)
Daño del ADN , Mitocondrias , Neuralgia , Paclitaxel , Poli(ADP-Ribosa) Polimerasa-1 , Animales , Masculino , Ratas , Modelos Animales de Enfermedad , Daño del ADN/efectos de los fármacos , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , NAD/metabolismo , Neuralgia/inducido químicamente , Neuralgia/metabolismo , Neuralgia/tratamiento farmacológico , Estrés Oxidativo/efectos de los fármacos , Paclitaxel/toxicidad , Ftalazinas/farmacología , Piperazinas/farmacología , Poli(ADP-Ribosa) Polimerasa-1/metabolismo , Poli(ADP-Ribosa) Polimerasa-1/antagonistas & inhibidores , Inhibidores de Poli(ADP-Ribosa) Polimerasas/farmacología , Ratas Sprague-Dawley , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismoRESUMEN
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.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico , Ácido Kaínico , Ratones Endogámicos BALB C , Nocicepción , Médula Espinal , Animales , Ácido Kaínico/farmacología , Ratones , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Nocicepción/efectos de los fármacos , Masculino , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Agonistas de Aminoácidos Excitadores/toxicidad , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/inducido químicamente , Locomoción/efectos de los fármacos , Núcleo Celular/metabolismo , Núcleo Celular/efectos de los fármacos , Hiperalgesia/inducido químicamente , Hiperalgesia/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismoRESUMEN
HIV associated neuropathic pain (HANP) is a common complication of AIDS. Intrathecal injection of recombinant HIV-1 gp120 in mice is a well-known model. Previous RNA sequencing revealed spinal TLR2 acts as a differentially expressed gene in HANP mice. The spinal TLR2 is involved in HANP, but its role and underlying mechanism remains unclear. In this study the transcription, expression and distribution characteristics of TLR2 in the spinal cord of HANP male mice have been analyzed by qRT-PCR, Western blotting, and immunofluorescent staining. We found that TLR2 expression was upregulated in the spinal dorsal horn and mainly distributed in microglial cells, and blocking TLR2 relieved pain of HANP mice. Following stimulation by gp120 microglial cells upregulate TLR2 expression and become activated. The activation stimulates their differentiation into the M1 type, increasing IL-1ß and TNF-α expression while inhibiting IL-10 expression. Silencing the Tlr2 gene slows down the activation, polarization, and secretion of pro-inflammatory factors in microglial cells induced by gp120, and enhances the expression of anti-inflammatory factors. Further analysis of the impact of gp120 on downstream signaling pathways of TLR2 in microglial cells, including NF-κB, MAPK (p38MAPK, ERK, and JNK) and PI3K/AKT, revealed that TLR2-NF-κB signaling plays a crucial role in the activation and polarization of microglial cells by gp120. Activation of NF-κB signaling aggravates pain in HANP mice, while blocking it lightens pain. This data indicates that gp120, through the TLR2-NF-κB signaling, activates spinal microglial cells, promotes the secretion of inflammatory cytokines, leading to HANP. This provides new targets to develop drugs for HANP.
Asunto(s)
Citocinas , Proteína gp120 de Envoltorio del VIH , Microglía , FN-kappa B , Neuralgia , Transducción de Señal , Receptor Toll-Like 2 , Regulación hacia Arriba , Animales , Receptor Toll-Like 2/metabolismo , Receptor Toll-Like 2/genética , Proteína gp120 de Envoltorio del VIH/toxicidad , Microglía/metabolismo , Microglía/efectos de los fármacos , Neuralgia/metabolismo , Ratones , FN-kappa B/metabolismo , Masculino , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiología , Citocinas/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Ratones Endogámicos C57BL , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacosRESUMEN
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.
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Tolerancia a Medicamentos , Hiperalgesia , Mitocondrias , Morfina , Estrés Oxidativo , Polifenoles , Sirtuina 3 , Animales , Morfina/farmacología , Ratones , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Masculino , Hiperalgesia/tratamiento farmacológico , Hiperalgesia/inducido químicamente , Polifenoles/farmacología , Sirtuina 3/metabolismo , Estrés Oxidativo/efectos de los fármacos , Superóxido Dismutasa/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Antioxidantes/farmacología , Analgésicos Opioides/farmacología , Malondialdehído/metabolismo , Glutamato-Amoníaco Ligasa/metabolismo , Metaloporfirinas/farmacologíaRESUMEN
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.
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Citocinas , Células Dendríticas , Ácidos Docosahexaenoicos , Encefalomielitis Autoinmune Experimental , Ratones Endogámicos C57BL , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Encefalomielitis Autoinmune Experimental/inmunología , Animales , Células Dendríticas/efectos de los fármacos , Células Dendríticas/inmunología , Ácidos Docosahexaenoicos/uso terapéutico , Ácidos Docosahexaenoicos/farmacología , Ácidos Docosahexaenoicos/química , Femenino , Ratones , Citocinas/metabolismo , Antiinflamatorios/uso terapéutico , Antiinflamatorios/farmacología , Médula Espinal/efectos de los fármacos , Médula Espinal/inmunología , Médula Espinal/patología , Médula Espinal/metabolismo , Inflamación/tratamiento farmacológico , Inflamación/inmunología , Piperazinas/farmacología , Piperazinas/uso terapéutico , Piperazinas/química , Células Cultivadas , Bazo/efectos de los fármacos , Bazo/inmunología , Células Th17/inmunología , Células Th17/efectos de los fármacosRESUMEN
Oxidative stress is a hallmark of secondary injury of spinal cord injuries. Controlling oxidative stress is crucial for mitigating secondary injury and promoting functional recovery after spinal cord injuries. Calycosin is an O-methylated isoflavone with antioxidant activity. To evaluate the effect of calycosin on spinal cord neurons under oxidative stress and clarify the molecular mechanism underlying the effect, we tested the neuroprotective activity of calycosin in a primary spinal cord neuron culture model. We found that calycosin protected neurons from H2O2-induced neuronal death in a dose-dependent manner. Further experiments revealed that calycosin decreased H2O2-induced mitochondrial fragmentation and mitochondrial membrane potential loss, and subsequently reduced H2O2-triggered release of mitochondrial cytochrome c into the cytoplasm. In addition, calycosin inhibited H2O2-induced reactive oxygen species generation and activation of NF-κB signaling in spinal cord neurons. Furthermore, the expression of several antioxidant enzymes such as HO-1, NQO1, GCLC, GCLM, TrxR1, and Trx1 was significantly promoted by calycosin. More importantly, we revealed that the Nrf2/Keap1 signal is crucial for the effect of calycosin, because calycosin increased the amount of nuclear Nrf2 while decreasing the amount of cytoplasmic Nrf2. Nrf2 knockdown with siRNA transfection abolished the neuroprotective effect of calycosin. Taken together, this study disclosed a novel mechanism by which calycosin combats oxidative stress. Our study thus sheds light on the potential clinical application of calycosin in SCI treatment.
Asunto(s)
Peróxido de Hidrógeno , Isoflavonas , Proteína 1 Asociada A ECH Tipo Kelch , Mitocondrias , Factor 2 Relacionado con NF-E2 , Neuronas , Transducción de Señal , Médula Espinal , Isoflavonas/farmacología , Factor 2 Relacionado con NF-E2/metabolismo , Proteína 1 Asociada A ECH Tipo Kelch/metabolismo , Animales , Peróxido de Hidrógeno/farmacología , Peróxido de Hidrógeno/toxicidad , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Transducción de Señal/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Muerte Celular/efectos de los fármacos , Ratas , Fármacos Neuroprotectores/farmacologíaRESUMEN
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.
Asunto(s)
Analgésicos Opioides , Factor Neurotrófico Derivado del Encéfalo , Bulbo Raquídeo , Morfina , Neuronas , Nocicepción , Animales , Masculino , Ratones , Analgésicos Opioides/farmacología , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Factor Neurotrófico Derivado del Encéfalo/genética , Bulbo Raquídeo/efectos de los fármacos , Bulbo Raquídeo/metabolismo , Ratones Endogámicos C57BL , Morfina/farmacología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Nocicepción/efectos de los fármacos , Receptor trkB/metabolismo , Análisis de la Célula Individual , Médula Espinal/efectos de los fármacos , Transcriptoma , FemeninoRESUMEN
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.
Asunto(s)
Acetilcolinesterasa , Antioxidantes , Inhibidores de la Colinesterasa , Proteínas de Peces , Peces , Simulación del Acoplamiento Molecular , Péptidos , Médula Espinal , Animales , Antioxidantes/química , Antioxidantes/farmacología , Péptidos/química , Péptidos/farmacología , Acetilcolinesterasa/química , Acetilcolinesterasa/metabolismo , Inhibidores de la Colinesterasa/farmacología , Inhibidores de la Colinesterasa/química , Proteínas de Peces/química , Proteínas de Peces/farmacología , Ratas , Médula Espinal/química , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Células PC12 , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa/químicaRESUMEN
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.
Asunto(s)
Esclerosis Amiotrófica Lateral , Encéfalo , Cafeína , Modelos Animales de Enfermedad , Ratones Transgénicos , Médula Espinal , Animales , Cafeína/administración & dosificación , Cafeína/farmacocinética , Esclerosis Amiotrófica Lateral/tratamiento farmacológico , Esclerosis Amiotrófica Lateral/metabolismo , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Masculino , Femenino , Ratones , Administración Oral , Encéfalo/metabolismo , Encéfalo/efectos de los fármacos , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Digoxina/farmacocinética , Digoxina/administración & dosificación , Sulfasalazina/farmacocinética , Sulfasalazina/administración & dosificación , Absorción Intestinal/efectos de los fármacos , Absorción Intestinal/fisiologíaRESUMEN
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.
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Interleucinas , Microglía , Neuralgia , Traumatismos de los Nervios Periféricos , Animales , Masculino , Microglía/metabolismo , Microglía/efectos de los fármacos , Ratones , Neuralgia/metabolismo , Neuralgia/tratamiento farmacológico , Interleucinas/metabolismo , Femenino , Traumatismos de los Nervios Periféricos/metabolismo , Traumatismos de los Nervios Periféricos/complicaciones , Ratones Endogámicos C57BL , Citocinas/metabolismo , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Nervio Ciático/lesiones , Nervio Ciático/metabolismo , Hiperalgesia/metabolismo , Hiperalgesia/tratamiento farmacológico , Antiinflamatorios/farmacología , Modelos Animales de Enfermedad , Inflamación/metabolismoRESUMEN
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.
Asunto(s)
Potenciales de Acción , Homeostasis , Neuronas Motoras , Médula Espinal , Animales , Homeostasis/fisiología , Homeostasis/efectos de los fármacos , Médula Espinal/fisiología , Médula Espinal/efectos de los fármacos , Potenciales de Acción/fisiología , Potenciales de Acción/efectos de los fármacos , Femenino , Neuronas Motoras/fisiología , Neuronas Motoras/efectos de los fármacos , Ratones , Masculino , Células Cultivadas , Embrión de Pollo , Corteza Cerebral/fisiología , Corteza Cerebral/efectos de los fármacos , Antagonistas de Receptores de GABA-A/farmacología , Neuronas/fisiología , Neuronas/efectos de los fármacos , Receptores de GABA-A/metabolismo , Plasticidad Neuronal/fisiología , Plasticidad Neuronal/efectos de los fármacos , Ratones Endogámicos C57BLRESUMEN
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.
Asunto(s)
Dinaminas , Microglía , FN-kappa B , Traumatismos de la Médula Espinal , Animales , Masculino , Ratones , Ratas , Línea Celular , Polaridad Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Dinaminas/metabolismo , Dinaminas/genética , Inflamación/metabolismo , Lipopolisacáridos , Locomoción/efectos de los fármacos , Microglía/metabolismo , Microglía/efectos de los fármacos , Neuroprotección , FN-kappa B/metabolismo , Quinazolinonas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/patologíaRESUMEN
Oxaliplatin (OXA) as the platinum-based agent induces the cumulative neuropathy including functional impairment and neuropathic pain. OXA treatment triggered oxidative stress and inflammatory reaction in the spinal cord. Puerarin as a natural product has the neuroprotective effect on neuropathic pain. Hence, the roles and mechanisms of Pue on OXA induced neuropathic pain were studied. In this study, OXA-induced neuropathic pain mouse model was constructed by oxaliplatin injection for 5 consecutive days and two cycles. Pue (10 mg/kg) was administered intraperitoneally for seven consecutive days. The changes of behavior, morphology and levels of related proteins were detected. As a result, OXA-induced mice exhibited as the increased pain hypersensitivity, the impaired motor coordination, the activated NLRP3 inflammasome mediated inflammation and the suppressed nuclear factor erythroid 2-related factor 2 (Nrf2) mediated antioxidative reaction in the spinal cord (P<0.05 vs Control). After Pue administration, the mechanical pain threshold, thermal pain latency, spontaneous pain number and motor latency were improved (P<0.05 vs OXA). In the spinal cord, Pue administration reduced the levels of inflammatory elements, increased the levels of antioxidative elements and decreased the levels of oxidative factors (P<0.05 vs OXA). Furthermore, Pue also bind with Nrf2 and increased the association of Nrf2 to glutathione peroxidase 4 (GPX4). In summary, Pue alleviates oxaliplatin induced neuropathic pain by enhancing Nrf2/GPX4-mediated antioxidant response and suppressing inflammatory reaction in the spinal cord.
Asunto(s)
Antioxidantes , Isoflavonas , Factor 2 Relacionado con NF-E2 , Neuralgia , Oxaliplatino , Fosfolípido Hidroperóxido Glutatión Peroxidasa , Animales , Oxaliplatino/efectos adversos , Neuralgia/tratamiento farmacológico , Neuralgia/inducido químicamente , Neuralgia/metabolismo , Ratones , Masculino , Factor 2 Relacionado con NF-E2/metabolismo , Antioxidantes/farmacología , Antioxidantes/metabolismo , Isoflavonas/farmacología , Isoflavonas/uso terapéutico , Fosfolípido Hidroperóxido Glutatión Peroxidasa/metabolismo , Médula Espinal/metabolismo , Médula Espinal/efectos de los fármacos , Médula Espinal/patología , Estrés Oxidativo/efectos de los fármacos , Glutatión Peroxidasa/metabolismo , Modelos Animales de Enfermedad , Ratones Endogámicos C57BLRESUMEN
So far, only a small number of medications are effective in progressive multiple sclerosis (MS). The sphingosine-1-phosphate-receptor (S1PR)-1,5 modulator siponimod, licensed for progressive MS, is acting both on peripheral immune cells and in the central nervous system (CNS). So far it remains elusive, whether those effects are related to the neurotrophin brain derived neurotrophic factor (BDNF). We hypothesized that BDNF in immune cells might be a prerequisite to reduce disease activity in experimental autoimmune encephalomyelitis (EAE) and prevent neurotoxicity. MOG35-55 immunized wild type (WT) and BDNF knock-out (BDNFko) mice were treated with siponimod or vehicle and scored daily in a blinded manner. Immune cell phenotyping was performed via flow cytometry. Immune cell infiltration and demyelination of spinal cord were assessed using immunohistochemistry. In vitro, effects on neurotoxicity and mRNA regulation were investigated using dorsal root ganglion cells incubated with EAE splenocyte supernatant. Siponimod led to a dose-dependent reduction of EAE scores in chronic WT EAE. Using a suboptimal dosage of 0.45 µg/day, siponimod reduced clinical signs of EAE independent of BDNF-expression in immune cells in accordance with reduced infiltration and demyelination. Th and Tc cells in secondary lymphoid organs were dose-dependently reduced, paralleled with an increase of regulatory T cells. In vitro, neuronal viability trended towards a deterioration after incubation with EAE supernatant; siponimod showed a slight rescue effect following treatment of WT splenocytes. Neuronal gene expression for CCL2 and CX3CL1 was elevated after incubation with EAE supernatant, which was reversed after siponimod treatment for WT, but not for BNDFko. Apoptosis markers and alternative death pathways were not affected. Siponimod exerts both anti-inflammatory and neuroprotective effects, partially related to BDNF-expression. This might in part explain effectiveness during progression in MS and could be a target for therapy.