RESUMEN
Individuals suffering from diabetic polyneuropathy (DPN) experience debilitating symptoms such as pain, paranesthesia, and sensory disturbances, prompting a quest for effective treatments. Dipeptidyl-peptidase (DPP)-4 inhibitors, recognized for their potential in ameliorating DPN, have sparked interest, yet the precise mechanism underlying their neurotrophic impact on the peripheral nerve system (PNS) remains elusive. Our study delves into the neurotrophic effects of DPP-4 inhibitors, including Diprotin A, linagliptin, and sitagliptin, alongside pituitary adenylate cyclase-activating polypeptide (PACAP), Neuropeptide Y (NPY), and Stromal cell-derived factor (SDF)-1a-known DPP-4 substrates with neurotrophic properties. Utilizing primary culture dorsal root ganglia (DRG) neurons, we meticulously evaluated neurite outgrowth in response to these agents. Remarkably, all DPP-4 inhibitors and PACAP demonstrated a significant elongation of neurite length in DRG neurons (PACAP 0.1 µM: 2221 ± 466 µm, control: 1379 ± 420, p < 0.0001), underscoring their potential in nerve regeneration. Conversely, NPY and SDF-1a failed to induce neurite elongation, accentuating the unique neurotrophic properties of DPP-4 inhibition and PACAP. Our findings suggest that the upregulation of PACAP, facilitated by DPP-4 inhibition, plays a pivotal role in promoting neurite elongation within the PNS, presenting a promising avenue for the development of novel DPN therapies with enhanced neurodegenerative capabilities.
Asunto(s)
Neuropatías Diabéticas , Inhibidores de la Dipeptidil-Peptidasa IV , Ganglios Espinales , Proyección Neuronal , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa , Animales , Ganglios Espinales/metabolismo , Ganglios Espinales/efectos de los fármacos , Inhibidores de la Dipeptidil-Peptidasa IV/farmacología , Polipéptido Hipofisario Activador de la Adenilato-Ciclasa/farmacología , Proyección Neuronal/efectos de los fármacos , Neuropatías Diabéticas/tratamiento farmacológico , Neuropatías Diabéticas/metabolismo , Ratones , Neuropéptido Y/metabolismo , Neuropéptido Y/farmacología , Quimiocina CXCL12/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Linagliptina/farmacología , Dipeptidil Peptidasa 4/metabolismo , Fosfato de Sitagliptina/farmacología , Células Cultivadas , Neuritas/efectos de los fármacos , Neuritas/metabolismo , OligopéptidosRESUMEN
We previously demonstrated that orally supplemented Bifidobacterium breve MCC1274 (B. breve MCC1274) mitigated Alzheimer's disease (AD) pathologies in both 7-month-old AppNL-G-F mice and wild-type mice; thus, B. breve MCC1274 supplementation might potentially prevent the progression of AD. However, the possibility of using this probiotic as a treatment for AD remains unclear. Thus, we investigated the potential therapeutic effects of this probiotic on AD using 17-month-old AppNL-G-F mice with memory deficits and amyloid beta saturation in the brain. B. breve MCC1274 supplementation ameliorated memory impairment via an amyloid-cascade-independent pathway. It reduced hippocampal and cortical levels of phosphorylated extracellular signal-regulated kinase and c-Jun N-terminal kinase as well as heat shock protein 90, which might have suppressed tau hyperphosphorylation and chronic stress. Moreover, B. breve MCC1274 supplementation increased hippocampal synaptic protein levels and upregulated neuronal activity. Thus, B. breve MCC1274 supplementation may alleviate cognitive dysfunction by reducing chronic stress and tau hyperphosphorylation, thereby enhancing both synaptic density and neuronal activity in 17-month-old AppNL-G-F mice. Overall, this study suggests that B. breve MCC1274 has anti-AD effects and can be used as a potential treatment for AD.
Asunto(s)
Enfermedad de Alzheimer , Bifidobacterium breve , Aplicaciones Móviles , Ratones , Animales , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Bifidobacterium breve/metabolismo , Ratones Transgénicos , Modelos Animales de Enfermedad , Trastornos de la Memoria/tratamiento farmacológico , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismoRESUMEN
Morphological analysis of peripheral nerves in mouse models can be used to characterize the pathophysiology of peripheral nerve disease, but obtaining high-quality electron micrographs can be challenging. Here, we present a protocol to obtain electron micrographs of mouse peripheral nerves. We detail the procedures of sampling, fixation, and embedding of peripheral nerves. We then outline the steps for ultrathin sectioning and transmission electron microscopy imaging. Finally, we describe morphological evaluation of nerve fibers in these images using ImageJ and AxonSeg. For complete details on the use and execution of this protocol, please refer to Nakai-Shimoda et al. (2021).
Asunto(s)
Técnicas Histológicas , Nervios Periféricos , Animales , Técnicas Histológicas/métodos , Ratones , Microscopía Electrónica de Transmisión , Nervios Periféricos/diagnóstico por imagen , Manejo de EspecímenesRESUMEN
Background: Electrical stimulation (ES) therapy is recommended for healing pressure injuries. Monophasic pulsed microcurrent stimulation promotes the migration of human dermal fibroblasts (HDFs) to the cathode, and ES potentially accelerates pressure injury healing. A reverse current is generated after ES in the human body; however, the effects of the electrical shunt in preventing the reverse current from migrating are unclear. Therefore, this study aimed to investigate the effects of an electrical shunt on the migration of HDFs. Methods: In the shunt groups, HDFs were electrically stimulated (0, 200, 400, and 600 µA) for 8 hours, and an electrical shunt was used to remove the electricity after ES. HDFs were observed under time-lapse microscopy for 24 hours. The migration ratio toward the cathode was calculated for each dish. Results: The migration ratio was significantly higher in the 200-µA group than in the other groups. HDFs migrated toward the anode after ES in the non-shunt groups with greater than 400 µA ES; however, HDFs did not migrate toward the anode with electrical shunting. Conclusions: A post-ES electrical shunt is important in preventing a decline in the migration effect of ES.
RESUMEN
Diabetic peripheral neuropathy (DPN) includes symptoms of thermosensory impairment, which are reported to involve changes in the expression or function, or both, of nociceptive TRPV1 and TRPA1 channels in rodents. In the present study, we did not find changes in the expression or function of TRPV1 or TRPA1 in DPN mice caused by STZ, although thermal hypoalgesia was observed in a murine model of DPN or TRPV1-/- mice with a Plantar test, which specifically detects temperature avoidance. With a Thermal Gradient Ring in which mice can move freely in a temperature gradient, temperature preference can be analyzed, and we clearly discriminated the temperature-dependent phenotype between DPN and TRPV1-/- mice. Accordingly, we propose approaches with multiple behavioral methods to analyze the progression of DPN by response to thermal stimuli. Attention to both thermal avoidance and preference may provide insight into the symptoms of DPN.
Asunto(s)
Diabetes Mellitus , Neuropatías Diabéticas , Animales , Ratones , Neuropatías Diabéticas/etiologíaRESUMEN
Glucose-responsive ATP-sensitive potassium channels (KATP) are expressed in a variety of tissues including nervous systems. The depolarization of the membrane potential induced by glucose may lead to hyperexcitability of neurons and induce excitotoxicity. However, the roles of KATP in the peripheral nervous system (PNS) are poorly understood. Here, we determine the roles of KATP in the PNS using KATP-deficient (Kir6.2-deficient) mice. We demonstrate that neurite outgrowth of dorsal root ganglion (DRG) neurons was reduced by channel closers sulfonylureas. However, a channel opener diazoxide elongated the neurite. KATP subunits were expressed in mouse DRG, and expression of certain subunits including Kir6.2 was increased in diabetic mice. In Kir6.2-deficient mice, the current perception threshold, thermal perception threshold, and sensory nerve conduction velocity were impaired. Electron microscopy revealed a reduction of unmyelinated and small myelinated fibers in the sural nerves. In conclusion, KATP may contribute to the development of peripheral neuropathy.
RESUMEN
BACKGROUND AND AIMS: Chronic HBV infection is a major health problem worldwide. Currently, the first-line treatment for HBV is nucleos(t)ide analogs or interferons; however, efficient therapeutic approaches that enable cure are lacking. Therefore, anti-HBV agents with mechanisms distinct from those of current drugs are needed. Sodium taurocholate cotransporting polypeptide (NTCP) was previously identified as an HBV receptor that is inhibited by several compounds. Farnesoid X receptor (FXR) activation also inhibits NTCP function. APPROACH AND RESULTS: In this study, we investigated the inhibitory effect of bile acid (BA) derivatives-namely obeticholic acid (OCA), 6α-ethyl-24-nor-5ß-cholane-3α,7α,23-triol-23 sulfate sodium salt (INT-767; a dual agonist of FXR and Takeda G protein-coupled receptor [TGR5]), and 6α-ethyl-23(S)-methyl-cholic acid (INT-777; a TGR5 agonist)-3-(2,6-dichlorophenyl)-4-(3'-carboxy-2-chlorostilben-4-yl)oxymethyl-5-isopropylisoxazole (GW4064; a FXR agonist), cyclosporin A, and irbesartan. OCA and INT-777 suppressed HBV infection in HepG2-human NTCP-C4 cells. Interestingly, INT-767 showed potent inhibition by attaching to HBV particles rather than binding to NTCP. As an entry inhibitor, INT-767 was stronger than various natural BAs. Furthermore, in chimeric mice with humanized liver, INT-767 markedly delayed the initial rise of HBsAg, HBeAg, and HBV DNA and reduced covalently closed circular DNA. The strong inhibitory effect of INT-767 may be due to the cumulative effect of its ability to inhibit the entry of HBV and to stimulate FXR downstream signaling, which affects the postentry step. CONCLUSIONS: Our results suggest that BA derivatives, particularly INT-767, are prospective candidate anti-HBV agents. Clarifying the underlying mechanisms of BA derivatives would facilitate the development of anti-HBV agents.
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Antivirales/farmacología , Hepatitis B Crónica/tratamiento farmacológico , Receptores Citoplasmáticos y Nucleares/agonistas , Receptores Acoplados a Proteínas G/agonistas , Internalización del Virus/efectos de los fármacos , Animales , Antivirales/uso terapéutico , Ácidos y Sales Biliares/farmacología , Ácidos y Sales Biliares/uso terapéutico , Ácido Quenodesoxicólico/análogos & derivados , Ácido Quenodesoxicólico/farmacología , Ácido Quenodesoxicólico/uso terapéutico , Ácidos Cólicos/farmacología , Ácidos Cólicos/uso terapéutico , Modelos Animales de Enfermedad , Células Hep G2 , Virus de la Hepatitis B/efectos de los fármacos , Virus de la Hepatitis B/metabolismo , Hepatitis B Crónica/virología , Humanos , Masculino , Ratones , Transportadores de Anión Orgánico Sodio-Dependiente/metabolismo , Estudios Prospectivos , Receptores Citoplasmáticos y Nucleares/metabolismo , Simportadores/metabolismo , Quimera por TrasplanteRESUMEN
Although diabetic polyneuropathy (DPN) is the commonest diabetic complication, its pathology remains to be clarified. As previous papers have suggested the neuroprotective effects of glucagon-like peptide-1 in DPN, the current study investigated the physiological indispensability of glucagon gene-derived peptides (GCGDPs) including glucagon-like peptide-1 in the peripheral nervous system (PNS). Neurological functions and neuropathological changes of GCGDP deficient (gcg-/-) mice were examined. The gcg-/- mice showed tactile allodynia and thermal hyperalgesia at 12-18 weeks old, followed by tactile and thermal hypoalgesia at 36 weeks old. Nerve conduction studies revealed a decrease in sensory nerve conduction velocity at 36 weeks old. Pathological findings showed a decrease in intraepidermal nerve fiber densities. Electron microscopy revealed a decrease in circularity and an increase in g-ratio of myelinated fibers and a decrease of unmyelinated fibers in the sural nerves of the gcg-/- mice. Effects of glucagon on neurite outgrowth were examined using an ex vivo culture of dorsal root ganglia. A supraphysiological concentration of glucagon promoted neurite outgrowth. In conclusion, the mice with deficiency of GCGDPs developed peripheral neuropathy with age. Furthermore, glucagon might have neuroprotective effects on the PNS of mice. GCGDPs might be involved in the pathology of DPN.
Asunto(s)
Neuropatías Diabéticas/etiología , Péptidos Similares al Glucagón/deficiencia , Animales , Neuropatías Diabéticas/genética , Neuropatías Diabéticas/patología , Modelos Animales de Enfermedad , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Glucagón/deficiencia , Glucagón/genética , Glucagón/metabolismo , Péptido 1 Similar al Glucagón/deficiencia , Péptido 1 Similar al Glucagón/genética , Péptido 1 Similar al Glucagón/metabolismo , Péptidos Similares al Glucagón/genética , Péptidos Similares al Glucagón/metabolismo , Hiperalgesia/etiología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Fibras Nerviosas Mielínicas/patología , Conducción Nerviosa , Proyección Neuronal , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Glucagón/genética , Receptores de Glucagón/metabolismoRESUMEN
AIM: Hepatitis B virus (HBV) relies on glycosylation for crucial functions, such as entry into host cells, proteolytic processing and protein trafficking. The aim of this study was to identify candidate molecules for the development of novel antiviral agents against HBV using an siRNA screening system targeting the host glycosylation pathway. METHODS: HepG2.2.15.7 cells that consistently produce HBV were employed for our in vitro study. We investigated the effects of siRNAs that target 88 different host glycogenes on hepatitis B surface antigen (HBsAg) and HBV DNA secretion using the siRNA screening system. RESULTS: We identified four glycogenes that reduced HBsAg and/or HBV DNA secretion; however, the observed results for two of them may be due to siRNA off-target effects. Knocking down ST8SIA3, a member of the sialyltransferase family, significantly reduced both HBsAg and HBV DNA secretion. Knocking down GALNT7, which transfers N-acetylgalactosamine to initiate O-linked glycosylation in the Golgi apparatus, also significantly reduced both HBsAg and HBV DNA levels. CONCLUSIONS: These results showed that knocking down the ST8SIA3 and GALNT7 glycogenes inhibited HBsAg and HBV DNA secretion in HepG2.2.15.7 cells, indicating that the host glycosylation pathway is important for the HBV life cycle and could be a potential target for the development of novel anti-HBV agents.
RESUMEN
Distal sensory-motor polyneuropathy is one of the most frequent diabetic complications. However, few therapies address the etiology of neurodegeneration in the peripheral nervous systems of diabetic patients. Several metabolic mechanisms have been proposed as etiologies of this polyneuropathy. In this study, we revisited one of those mechanisms, the polyol pathway, and investigated the curative effects of a novel strong aldose reductase inhibitor, ranirestat, in streptozotocin-induced diabetic rats with preexisting polyneuropathy. Twelve weeks after the onset of diabetes, rats which had an established polyneuropathy were treated once daily with a placebo, ranirestat, or epalrestat, over 6 weeks. Before and after the treatment, nerve conduction velocities and thermal perception threshold of hindlimbs were examined. After the treatment, intraepidermal fiber density was evaluated. As an ex vivo assay, murine dorsal root ganglion cells were dispersed and cultured with or without 1 µmol/l ranirestat for 48 hours. After the culture, neurite outgrowth was quantified using immunological staining. Sensory nerve conduction velocity increased in diabetic rats treated with ranirestat (43.3 ± 3.6 m/s) compared with rats treated with placebo (39.8 ± 2.3). Motor nerve conduction velocity also increased in the ranirestat group (45.6 ± 3.9) compared with the placebo group (38.9 ± 3.5). The foot withdrawal latency to noxious heating was improved in the ranirestat group (17.7 ± 0.6 seconds) compared with the placebo group (20.6 ± 0.6). The decrease in the intraepidermal fiber density was significant in the diabetic placebo group (21.6 ± 1.7/mm) but not significant in the diabetic ranirestat group (26.2 ± 1.2) compared with the nondiabetic placebo group (30.3 ± 1.5). Neurite outgrowth was promoted in the neurons supplemented with ranirestat (control 1446 ± 147 µm/neuron, ranirestat 2175 ± 149). Ranirestat improved the peripheral nervous dysfunctions in rats with advanced diabetic polyneuropathy. Ranirestat could have potential for regeneration in the peripheral nervous system of diabetic rats.
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Diabetes Mellitus Experimental/complicaciones , Neuropatías Diabéticas/fisiopatología , Inhibidores Enzimáticos/farmacología , Fibras Nerviosas/efectos de los fármacos , Conducción Nerviosa/efectos de los fármacos , Pirazinas/farmacología , Compuestos de Espiro/farmacología , Sensación Térmica/efectos de los fármacos , Aldehído Reductasa/antagonistas & inhibidores , Animales , Neuropatías Diabéticas/etiología , Epidermis/patología , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Técnicas In Vitro , Ratones , Fibras Nerviosas/patología , Conducción Nerviosa/fisiología , Proyección Neuronal/efectos de los fármacos , Proyección Neuronal/fisiología , Ratas , Ratas Wistar , Células Receptoras Sensoriales/efectos de los fármacos , Células Receptoras Sensoriales/fisiología , Sensación Térmica/fisiologíaRESUMEN
OBJECTIVE: Diabetic polyneuropathy (DPN) is one of the most prevalent diabetic complications. We previously demonstrated that exendin-4 (Ex4), a glucagon-like peptide-1 receptor agonist (GLP-1RA), has beneficial effects in animal models of DPN. We hypothesized that GLP-1 signaling would protect neurons of the peripheral nervous system from oxidative insult in DPN. Here, the therapeutic potential of GLP-1RAs on DPN was investigated in depth using the cellular oxidative insult model applied to the dorsal root ganglion (DRG) neuronal cell line. RESEARCH DESIGN AND METHODS: Immortalized DRG neuronal 50B11 cells were cultured with and without hydrogen peroxide in the presence or absence of Ex4 or GLP-1(7-37). Cytotoxicity and viability were determined using a lactate dehydrogenase assay and MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium inner salt), respectively. Antioxidant enzyme activity was evaluated using a superoxide dismutase assay. Alteration of neuronal characteristics of 50B11 cells induced by GLP-1RAs was evaluated with immunocytochemistry utilizing antibodies for transient receptor potential vanilloid subfamily member 1, substance P, and calcitonin gene-related peptide. Cell proliferation and apoptosis were also examined by ethynyl deoxyuridine incorporation assay and APOPercentage dye, respectively. The neurite projection ratio induced by treatment with GLP-1RAs was counted. Intracellular activation of adenylate cyclase/cyclic adenosine monophosphate (cAMP) signaling was also quantified after treatment with GLP-1RAs. RESULTS: Neither Ex4 nor GLP-1(7-37) demonstrated cytotoxicity in the cells. An MTS assay revealed that GLP-1RAs amended impaired cell viability induced by oxidative insult in 50B11 cells. GLP-1RAs activated superoxide dismutase. GLP-1RAs induced no alteration of the distribution pattern in neuronal markers. Ex4 rescued the cells from oxidative insult-induced apoptosis. GLP-1RAs suppressed proliferation and promoted neurite projections. No GLP-1RAs induced an accumulation of cAMP. CONCLUSIONS: Our findings indicate that GLP-1RAs have neuroprotective potential which is achieved by their direct actions on DRG neurons. Beneficial effects of GLP-1RAs on DPN could be related to these direct actions on DRG neurons.
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Apoptosis , Ganglios Espinales/citología , Ganglios Espinales/efectos de los fármacos , Receptor del Péptido 1 Similar al Glucagón/agonistas , Estrés Oxidativo , Animales , Antioxidantes/metabolismo , Calcitonina/metabolismo , Línea Celular , Proliferación Celular , Supervivencia Celular , AMP Cíclico/metabolismo , Neuropatías Diabéticas/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/uso terapéutico , Ratas , Sustancia P/metabolismo , Superóxido Dismutasa/metabolismoRESUMEN
GABA inhibits mature neurons and conversely excites immature neurons due to lower K(+)-Cl(-) cotransporter 2 (KCC2) expression. We observed that ectopically expressed KCC2 in embryonic cerebral cortices was not active; however, KCC2 functioned in newborns. In vitro studies revealed that taurine increased KCC2 inactivation in a phosphorylation-dependent manner. When Thr-906 and Thr-1007 residues in KCC2 were substituted with Ala (KCC2T906A/T1007A), KCC2 activity was facilitated, and the inhibitory effect of taurine was not observed. Exogenous taurine activated the with-no-lysine protein kinase 1 (WNK1) and downstream STE20/SPS1-related proline/alanine-rich kinase (SPAK)/oxidative stress response 1 (OSR1), and overexpression of active WNK1 resulted in KCC2 inhibition in the absence of taurine. Phosphorylation of SPAK was consistently higher in embryonic brains compared with that of neonatal brains and down-regulated by a taurine transporter inhibitor in vivo. Furthermore, cerebral radial migration was perturbed by a taurine-insensitive form of KCC2, KCC2T906A/T1007A, which may be regulated by WNK-SPAK/OSR1 signaling. Thus, taurine and WNK-SPAK/OSR1 signaling may contribute to embryonic neuronal Cl(-) homeostasis, which is required for normal brain development.