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1.
Mol Med Rep ; 27(1)2023 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-36367170

RESUMO

3,4­Dihydroxybenzaldehyde (DBD), one of the active components of Gastrodia elata, has a cerebral protective effect and can effectively combat cerebral ischemia/reperfusion (I/R) injury in rats. However, the metabolite profiles and underlying mechanisms associated with DBD remain unclear. To explore the level of energy metabolism and pharmacological targets in brain tissue following DBD treatment of stroke. The right middle cerebral artery of the rats was occluded for 2 h and reperfused for 24 h to simulate brain I/R injury. Pharmacological results showed that DBD reduced cerebral infarct volume, improved neurological function and increased adenosine triphosphate (ATP) content. Mitochondria are the primary sites for ATP generation and cellular energy supply and decreasing mitochondrial dysfunction can alleviate the energy expenditure of ischemic stroke. Through further experiments, it was found that mitochondrial damage was recovered following DBD treatment, which was manifested by the improvement of mitochondrial morphology under an electron microscope and the reduction of oxidative stress damage. The metabolomics of middle cerebral artery occlusion/reperfusion (MCAO/R) rat brain tissue was studied by the liquid chromatography­tandem mass spectrometry metabolomics method. Significantly different metabolites were screened and the pathways involved included amino sugar and nucleotide sugar metabolism and pentose phosphate pathway. Finally, the present study performed targeted metabolic profiling and validated potential therapeutic targets. Uridine diphosphate N­acetylglucosamine (UDP­GlcNAc) levels were decreased in the MCAO/R group but significantly increased in the DBD group. TdT­mediated dUTP nick end labeling (TUNEL) staining, hematoxylin and eosin staining and western blotting showed that brain cell apoptosis was inhibited and neuronal morphology improved. Among them, the regulatory enzyme O­GlcNAc transferase (OGT) of UDP­GlcNAc appeared to be significantly increased and neuronal apoptosis was inhibited following DBD treatment, which was verified by western blotting. Therefore, by analyzing mitochondrial dysfunction following I/R and the characterization of potential markers in mitochondrial energy metabolism, it was shown that OGT could inhibit neuronal apoptosis as a potential therapeutic target for brain I/R injury.


Assuntos
Isquemia Encefálica , Traumatismo por Reperfusão , Ratos , Animais , Isquemia Encefálica/metabolismo , Traumatismo por Reperfusão/metabolismo , Apoptose , Infarto da Artéria Cerebral Média/tratamento farmacológico , Trifosfato de Adenosina/farmacologia , Difosfato de Uridina
2.
Sci Rep ; 12(1): 18613, 2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36329102

RESUMO

Pseudoallergies caused by drugs make disease treatment difficult. Mas-relate G protein-coupled receptor X2 (MRGPRX2), which is specifically expressed in mast cells (MCs), has been implicated in pseudoallergies. High concentrations of therapeutic agents are typically required to stimulate MRGPRX2. Although regulatory mechanisms may enhance this response, the factors involved in this regulation are not well-understood. In this study, the effects of extracellular ATP on MC activation induced by MrgprB2, the mouse ortholog of human MRGPRX2, were examined in mouse peritoneal MCs (PMCs). ATP alone induced minimal PMC degranulation but markedly enhanced degranulation induced by the MrgprB2 agonist compound 48/80 (CP48/80), substance P, PAMP-12, and vancomycin. ATP promoted CP48/80-induced increase in intracellular Ca2+ in PMCs. This enhancement effect of ATP was absent in PMCs prepared from P2X4 receptor (P2X4R)-deficient mice and inhibited by the PI3K inhibitor wortmannin. In addition, P2X4R deficiency reduced the skin-specific and systemic anaphylactic responses to CP48/80 in vivo. In MC-deficient KitW-sh/W-sh mice, reconstitution with MCs obtained from wild-type mice led to a more severe anaphylactic response to CP48/80 compared to that from P2X4R-deficient mice. P2X4R-mediated effect may be involved in MrgprB2-mediated MC activation in vivo and is a potential target for alleviating pseudoallergic reactions.


Assuntos
Anafilaxia , Degranulação Celular , Camundongos , Humanos , Animais , Mastócitos/metabolismo , Receptores de Neuropeptídeos/genética , Receptores de Neuropeptídeos/metabolismo , Receptores Purinérgicos P2X4 , Fosfatidilinositol 3-Quinases , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Anafilaxia/induzido quimicamente , Trifosfato de Adenosina/farmacologia
3.
Nat Commun ; 13(1): 6536, 2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-36344520

RESUMO

Astrocytes can affect animal behavior by regulating tripartite synaptic transmission, yet their influence on affective behavior remains largely unclear. Here we showed that hippocampal astrocyte calcium activity reflects mouse affective state during virtual elevated plus maze test using two-photon calcium imaging in vivo. Furthermore, optogenetic hippocampal astrocyte activation elevating intracellular calcium induced anxiolytic behaviors in astrocyte-specific channelrhodopsin 2 (ChR2) transgenic mice (hGFAP-ChR2 mice). As underlying mechanisms, we found ATP released from the activated hippocampal astrocytes increased excitatory synaptic transmission in dentate gyrus (DG) granule cells, which exerted anxiolytic effects. Our data uncover a role of hippocampal astrocytes in modulating mice anxiety-like behaviors by regulating ATP-mediated synaptic homeostasis in hippocampal DG granule cells. Thus, manipulating hippocampal astrocytes activity can be a therapeutic strategy to treat anxiety.


Assuntos
Astrócitos , Cálcio , Animais , Camundongos , Astrócitos/metabolismo , Cálcio/metabolismo , Hipocampo/metabolismo , Channelrhodopsins/genética , Camundongos Transgênicos , Trifosfato de Adenosina/farmacologia , Ansiedade
4.
Sci Rep ; 12(1): 18856, 2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-36344587

RESUMO

This study investigated the mechanism of membrane damage by protocatechualdehyde (PCA) against Micrococcus luteus and assessed effects of PCA on the sensory and physicochemical properties of pork. The mechanism of PCA inhibition on M. luteus was studied by determining the minimum inhibitory concentration (MIC) based on membrane potential, intracellular ATP concentration, intracellular pH, confocal laser scanning microscopy (CLSM), and field emission gun scanning electron microscopy (FEG-SEM). The results showed that the MIC of PCA against M. luteus was 1.25 mg/mL. Hyperpolarization of the bacterial cell membrane, a decrease in the intracellular ATP concentration, and intracellular pH indicated that PCA damaged the cell membrane of M. luteus. FEG-SEM observation revealed that PCA could cause surface collapse, cell membrane rupture, and content outflow of M. luteus. Additionally, PCA was found to inhibit increases in the total number of colonies, the thiobarbituric acid reactive substances (TBARS) value growth rate, and moisture mobility in raw pork. Additionally, it improved the color and texture of raw pork, all of which effectively prolonged its shelf life. This study will encourage the application of PCA as a natural antibacterial agent in the food industry.


Assuntos
Carne de Porco , Carne Vermelha , Animais , Suínos , Micrococcus luteus , Trifosfato de Adenosina/farmacologia
5.
Int J Mol Sci ; 23(21)2022 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-36362410

RESUMO

Gamma-Aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the brain. It is produced by interneurons and recycled by astrocytes. In neurons, GABA activates the influx of Cl- via the GABAA receptor or efflux or K+ via the GABAB receptor, inducing hyperpolarization and synaptic inhibition. In astrocytes, the activation of both GABAA and GABAB receptors induces an increase in intracellular Ca2+ and the release of glutamate and ATP. Connexin 43 (Cx43) hemichannels are among the main Ca2+-dependent cellular mechanisms for the astroglial release of glutamate and ATP. However, no study has evaluated the effect of GABA on astroglial Cx43 hemichannel activity and Cx43 hemichannel-mediated gliotransmission. Here we assessed the effects of GABA on Cx43 hemichannel activity in DI NCT1 rat astrocytes and hippocampal brain slices. We found that GABA induces a Ca2+-dependent increase in Cx43 hemichannel activity in astrocytes mediated by the GABAA receptor, as it was blunted by the GABAA receptor antagonist bicuculline but unaffected by GABAB receptor antagonist CGP55845. Moreover, GABA induced the Cx43 hemichannel-dependent release of glutamate and ATP, which was also prevented by bicuculline, but unaffected by CGP. Gliotransmission in response to GABA was also unaffected by pannexin 1 channel blockade. These results are discussed in terms of the possible role of astroglial Cx43 hemichannel-mediated glutamate and ATP release in regulating the excitatory/inhibitory balance in the brain and their possible contribution to psychiatric disorders.


Assuntos
Astrócitos , Conexina 43 , Ratos , Animais , Conexina 43/metabolismo , Astrócitos/metabolismo , Receptores de GABA-A , Bicuculina/farmacologia , Animais Recém-Nascidos , Células Cultivadas , Ácido Glutâmico/farmacologia , Ácido gama-Aminobutírico/farmacologia , Trifosfato de Adenosina/farmacologia
6.
Int J Mol Sci ; 23(22)2022 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-36430877

RESUMO

Dry eye disease (DED) is a multifactorial disease with an incidence of approximately 50% worldwide. DED seriously affects quality of life and work. The prevalence of environmental DED (eDED) ranges from 35 to 48%. Conjunctival fluid secretion dysfunction may be one of the major causes of DED. Notably, the Cl- flux corresponds to the conjunctival fluid secretion and could be affected by ATP. Both the cystic fibrosis transmembrane conductance regulator (CFTR) and the Ca2+-activated Cl- channel (CaCC) are Cl- channels involved in epithelial fluid secretion. Conjunctival fluid secretion could be increased by activating P2Y2R (an ATP receptor) in DED. However, the role of the CaCC and CFTR channels regulated by P2Y2R in eDED remains unclear. In this study, we established a rabbit eDED model using a controlled drying system. A Ussing chamber was used to perform a conjunctival short-circuit current induced by ATP to evaluate the reactivity of the ion channels to the ATP. Our results revealed that eDED accompanied by conjunctival fluid secretion impairment was caused by a P2Y2R dysfunction, which is related to CaCC-CFTR signaling in the conjunctiva epithelium. Notably, the coupling effect of the ATP-induced CaCC-CFTR activation and intracellular Ca2+ may represent a promising therapeutic target for treating eDED.


Assuntos
Canais de Cloreto , Síndromes do Olho Seco , Animais , Coelhos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Qualidade de Vida , Células Epiteliais , Túnica Conjuntiva , Trifosfato de Adenosina/farmacologia
7.
Front Endocrinol (Lausanne) ; 13: 939699, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36387844

RESUMO

It has been reported that reactive oxygen species (ROS) derived from oxygen molecule reduction can interfere with the cross-talk between the hypothalamic-pituitary-gonadal (HPG) axis and other endocrine axes, thus affecting fertility. Furthermore, ROS have been linked to GnRH receptor signaling in gonadotropes involved in gonadotropin release. There has been evidence that ROS can interfere with the HPG axis and gonadotropin release at various levels. However, the direct effect of ROS on gonadotropin-releasing hormone (GnRH) neuron remains unclear. Thus, the objective of this study was to determine the effect of hydrogen peroxide (H2O2), an ROS source, on GnRH neuronal excitabilities in transgenic GnRH-green fluorescent protein-tagged mice using the whole-cell patch-clamp electrophysiology. In adults, H2O2 at high concentrations (mM level) hyperpolarized most GnRH neurons tested, whereas low concentrations (pM to µM) caused slight depolarization. In immature GnRH neurons, H2O2 exposure induced excitation. The sensitivity of GnRH neurons to H2O2 was increased with postnatal development. The effect of H2O2 on adult female GnRH neurons was found to be estrous cycle-dependent. Hyperpolarization mediated by H2O2 persisted in the presence of tetrodotoxin, a voltage-gated Na+ channel blocker, and amino-acids receptor blocking cocktail containing blockers for the ionotropic glutamate receptors, glycine receptors, and GABAA receptors, indicating that H2O2 could act on GnRH neurons directly. Furthermore, glibenclamide, an ATP-sensitive K+ (KATP) channel blocker, completely blocked H2O2-mediated hyperpolarization. Increasing endogenous H2O2 by inhibiting glutathione peroxidase decreased spontaneous activities of most GnRH neurons. We conclude that ROS can act as signaling molecules for regulating GnRH neuron's excitability and that adult GnRH neurons are sensitive to increased ROS concentration. Results of this study demonstrate that ROS have direct modulatory effects on the HPG axis at the hypothalamic level to regulate GnRH neuron's excitabilities.


Assuntos
Hormônio Liberador de Gonadotropina , Peróxido de Hidrogênio , Animais , Camundongos , Feminino , Hormônio Liberador de Gonadotropina/metabolismo , Peróxido de Hidrogênio/farmacologia , Espécies Reativas de Oxigênio , Neurônios/metabolismo , Camundongos Transgênicos , Receptores de GABA-A , Trifosfato de Adenosina/farmacologia
8.
Bull Exp Biol Med ; 173(5): 602-605, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36210415

RESUMO

We studied the effect of molecular hydrogen (H2) on the content of 2,3-diphosphoglyceric acid (2,3-DPG), ATP, malondialdehyde, and catalase activity in erythrocytes in chronic heart failure. Inhalation of 2% molecular hydrogen H2 was carried out for 40 min repeatedly (5 days) or once. Inhalation of H2 caused an increase in ATP concentration in both research groups, but was more pronounced after repeated inhalation. The content of 2,3-DPG increased after repeated exposure to H2. The increase in metabolic activity under the effect of H2 was accompanied by a decrease in malondialdehyde concentration and an increase in catalase activity. Thus, the application of H2 in chronic heart failure reduced oxidative stress and improved metabolism of erythrocytes, which contributes to improvement of microcirculation. This allows us to recommend H2 for protection against ischemic and reperfusion damage to the myocardium.


Assuntos
Insuficiência Cardíaca , Hidrogênio , 2,3-Difosfoglicerato , Trifosfato de Adenosina/farmacologia , Antioxidantes/farmacologia , Catalase , Eritrócitos , Insuficiência Cardíaca/tratamento farmacológico , Humanos , Hidrogênio/farmacologia , Hidrogênio/uso terapêutico , Malondialdeído , Estresse Oxidativo
9.
Front Endocrinol (Lausanne) ; 13: 994307, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36213280

RESUMO

Background context: Low back pain, affecting nearly 40% of adults, mainly results from intervertebral disc degeneration (IVDD), while the pathogenesis of IVDD is still not fully elucidated. Recently, some researches have revealed that necroptosis, a programmed necrosis, participated in the progression of IVDD, nevertheless, the underlying mechanism remains unclear. Purpose: To study the mechanism of necroptosis of Nucleus Pulposus (NP) cells in IVDD, focusing on the role of MyD88 signaling. Study design: The expression and co-localization of necroptotic indicators and MyD88 were examined in vivo, and MyD88 inhibitor was applied to determine the role of MyD88 signaling in necroptosis of NP cells in vitro. Methods: Human disc specimens were collected from patients receiving diskectomy for lumbar disc herniation (LDH) or traumatic lumbar fractures after MRI scanning. According to the Pfirrmann grades, they were divided into normal (Grades 1, 2) and degenerated groups (4, 5). Tissue slides were prepared for immunofluorescence to assess the co-localization of necroptotic indicators (RIP3, MLKL, p-MLKL) and MyD88 histologically. The combination of TNFα, LPS and Z-VAD-FMK was applied to induce necroptosis of NP cells. Level of ATP, reactive oxygen species (ROS), live-cell staining and electron microscope study were employed to study the role of MyD88 signaling in necroptosis of NP cells. Results: In vivo, the increased expression and co-localization of necroptotic indicators (RIP3, MLKL, p-MLKL) and MyD88 were found in NP cells of degenerated disc, while very l low fluorescence intensity in tissue of traumatic lumbar fractures. In vitro, the MyD88 inhibitor effectively rescued the necroptosis of NP cells, accompanied by increased viability, ATP level, and decreased ROS level. The effect of MyD88 inhibition on necroptosis of NP cells was further confirmed by ultrastructure of mitochondria shown by Transmission Electron Microscope (TEM). Conclusion: Our results indicated that the involvement of MyD88 signaling in the necroptosis of NP cells in IVDD, which will replenish the pathogenesis of IVDD and provide a novel potential therapeutic target for IVDD.


Assuntos
Degeneração do Disco Intervertebral , Núcleo Pulposo , Proteínas Adaptadoras de Transdução de Sinal/farmacologia , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Adulto , Humanos , Lipopolissacarídeos , Fator 88 de Diferenciação Mieloide/metabolismo , Fator 88 de Diferenciação Mieloide/farmacologia , Necroptose , Núcleo Pulposo/metabolismo , Núcleo Pulposo/patologia , Espécies Reativas de Oxigênio/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
10.
Arh Hig Rada Toksikol ; 73(3): 200-206, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36226819

RESUMO

Acrolein (AC) is one of the most toxic environmental pollutants, often associated with incomplete combustion of petrol, wood, and plastic, oil frying, and tobacco smoking, that causes oxidative damage to DNA and mitochondria. Considering that little is known about the protective effects of whey protein (WP) against AC-induced liver toxicity, the aim of our study was to learn more about them in respect to liver mitochondrial oxidative stress, respiratory enzymes, Krebs cycle enzymes, and adenosine triphosphate (ATP). To do that, we treated Sprague Dawley rats with daily doses of AC alone (5 mg/kg bw in 0.9 % NaCl solution), WP alone (200 mg/kg bw, in 0.9 % NaCl solution), or their combination by oral gavage for six days a week over 30 days. As expected, the AC group showed a drop in glutathione levels and antioxidant, transport chain, and tricarboxylic acid cycle enzyme activities and a significant rise in mitochondrial lipid peroxidation and protein carbonyl levels. Co-treatment with WP mitigated oxidative stress and improved enzyme activities. Judging by the measured parameters, WP reduced AC toxicity by improving bioenergetic mechanisms and eliminating oxidative stress.


Assuntos
Acroleína , Poluentes Ambientais , Acroleína/metabolismo , Acroleína/toxicidade , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Antioxidantes/metabolismo , Antioxidantes/farmacologia , Poluentes Ambientais/toxicidade , Glutationa/metabolismo , Peroxidação de Lipídeos , Fígado/metabolismo , Mitocôndrias/metabolismo , Estresse Oxidativo , Plásticos/metabolismo , Plásticos/farmacologia , Ratos , Ratos Sprague-Dawley , Cloreto de Sódio/metabolismo , Cloreto de Sódio/farmacologia , Proteínas do Soro do Leite/metabolismo , Proteínas do Soro do Leite/farmacologia
11.
Oxid Med Cell Longev ; 2022: 5424411, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36238646

RESUMO

Objective: Local radiotherapy may cause distant tumor regression via inducing immunogenic cell death (ICD). Here, we investigated the effect of curcumin on ionizing radiation-induced immunogenic cell death in normoxic or hypoxic glioma cells and its mechanism in vitro and vivo. Methods: Hypoxic or normoxic glioma cell apoptosis and the cell surface exposure of calreticulin (CRT) were detected by flow cytometry. Extracellular ATP and HSP70 were measured by chemiluminescence assay and ELISA, respectively. Endoplasmic reticulum (ER) stress protein levels were detected by western blot. Moreover, the induction of bona fide ICD was detected by vaccination assays in mice bearing glioma model. Spleen lymphocytes and tumor-infiltrating lymphocyte subsets were analyzed by flow cytometry and immunohistochemistry. Results: Curcumin incubation before X-ray irradiation significantly increased radiation-induced apoptosis rate in normoxic or hypoxic glioma cells. Curcumin enhanced radiation-induced CRT exposure, release of HSP70 and ATP, and ER stress signaling activity. After treatment with ER stress pathway inhibitors, cell apoptosis and CRT exposure induced by the combination treatment of curcumin and X-ray were reduced. In vaccination experiments, glioma cells irradiated by X-ray produced a strong immunogenic response rejecting tumor formation in 70% mice. In comparison, cells treated by curcumin and X-ray produced a stronger immune response rejecting tumor formation in 90% mice. The combination treatment increased the percentage of tumor-infiltrating CD4+, CD8+ T lymphocytes, and CD11c+ dendritic cells compared to X-ray irradiation alone. Conclusion: Ionizing radiation-induced normoxic or hypoxic glioma immunogenic cell death could be further enhanced by curcumin through activating the ER stress PERK-eIF2α and IRE1α-XBP1 signaling pathways.


Assuntos
Curcumina , Glioma , Trifosfato de Adenosina/farmacologia , Animais , Apoptose , Calreticulina , Linhagem Celular Tumoral , Curcumina/farmacologia , Curcumina/uso terapêutico , Estresse do Retículo Endoplasmático , Endorribonucleases , Proteínas de Choque Térmico , Morte Celular Imunogênica , Camundongos , Proteínas Serina-Treonina Quinases , Radiação Ionizante , Transdução de Sinais
12.
Gen Physiol Biophys ; 41(5): 447-455, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36222342

RESUMO

We aimed to explore the role of Sirt3 in the regulation of skeletal muscle mitophagy with hypoxic training. C57BL/6J mice were randomly divided into four groups: C group (control), HT group (mice performed a hypoxic training of living in an environment with an oxygen concentration of 13.8% and treadmill exercise under normoxia for 6 weeks), T group (mice were subjected to an intraperitoneal (i.p.) injection of the Sirt3 inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine (3-TYP) 50 mg/kg three times per week for 6 weeks) and THT group (the hypoxic training of HT group with i.p. injection of 3-TYP in T group). The results showed that 6 weeks of hypoxic training could improve ATP synthesis in skeletal muscle. After the combined intervention of 3-TYP injection and hypoxic training, Sirt3, FOXO3a, and SOD2 protein contents were still lower than those in hypoxic training group. Hypoxic training cannot improve the negative effect of Sirt3 inhibition on muscle PINK1/Parkin signal. This study demonstrated that Sirt3 plays a key role in mediating skeletal muscle mitophagy by hypoxic training. The results of our study also provided the first evidence that mitophagy caused by hypoxic training might be transduced through the Sirt3-FOXO3a signaling pathway.


Assuntos
Mitofagia , Sirtuína 3 , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Hipóxia/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Mitofagia/fisiologia , Músculo Esquelético/metabolismo , Oxigênio/metabolismo , Oxigênio/farmacologia , Proteínas Quinases/metabolismo , Proteínas Quinases/farmacologia , Piridinas/farmacologia , Sirtuína 3/metabolismo , Sirtuína 3/farmacologia , Ubiquitina-Proteína Ligases
13.
J Neuroinflammation ; 19(1): 256, 2022 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-36224611

RESUMO

BACKGROUND: Previous studies have demonstrated that purinergic receptors could be therapeutic targets to modulate the inflammatory response in multiple models of brain diseases. However, tools for the selective and efficient targeting of these receptors are lacking. The development of new P2X7-specific nanobodies (nbs) has enabled us to effectively block the P2X7 channel. METHODS: Temporary middle cerebral artery occlusion (tMCAO) in wild-type (wt) and P2X7 transgenic (tg) mice was used to model ischemic stroke. Adenosine triphosphate (ATP) release was assessed in transgenic ATP sensor mice. Stroke size was measured after P2X7-specific nbs were injected intravenously (iv) and intracerebroventricularly (icv) directly before tMCAO surgery. In vitro cultured microglia were used to investigate calcium influx, pore formation via 4,6-diamidino-2-phenylindole (DAPI) uptake, caspase 1 activation and interleukin (IL)-1ß release after incubation with the P2X7-specific nbs. RESULTS: Transgenic ATP sensor mice showed an increase in ATP release in the ischemic hemisphere compared to the contralateral hemisphere or the sham-treated mice up to 24 h after stroke. P2X7-overexpressing mice had a significantly greater stroke size 24 h after tMCAO surgery. In vitro experiments with primary microglial cells demonstrated that P2X7-specific nbs could inhibit ATP-triggered calcium influx and the formation of membrane pores, as measured by Fluo4 fluorescence or DAPI uptake. In microglia, we found lower caspase 1 activity and subsequently lower IL-1ß release after P2X7-specific nb treatment. The intravenous injection of P2X7-specific nbs compared to isotype controls before tMCAO surgery did not result in a smaller stroke size. As demonstrated by fluorescence-activated cell sorting (FACS), after stroke, iv injected nbs bound to brain-infiltrated macrophages but not to brain resident microglia, indicating insufficient crossing of the blood-brain barrier of the nbs. Therefore, we directly icv injected the P2X7-specific nbs or the isotype nbs. After icv injection of 30 µg of P2X7 specific nbs, P2X7 specific nbs bound sufficiently to microglia and reduced stroke size. CONCLUSION: Mechanistically, we can show that there is a substantial increase of ATP locally after stroke and that blockage of the ATP receptor P2X7 by icv injected P2X7-specific nbs can reduce ischemic tissue damage.


Assuntos
Receptores Purinérgicos P2 , Anticorpos de Domínio Único , Acidente Vascular Cerebral , Trifosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Caspase 1/metabolismo , Infarto da Artéria Cerebral Média/patologia , Interleucina-1beta/metabolismo , Camundongos , Microglia/metabolismo , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7/metabolismo , Anticorpos de Domínio Único/metabolismo , Acidente Vascular Cerebral/metabolismo
14.
J Neuroinflammation ; 19(1): 244, 2022 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-36195881

RESUMO

BACKGROUND: Neuropathic pain is still a challenge for clinical treatment as a result of the comprehensive pathogenesis. Although emerging evidence demonstrates the pivotal role of glial cells in regulating neuropathic pain, the role of Schwann cells and their underlying mechanisms still need to be uncovered. Pannexin 1 (Panx 1), an important membrane channel for the release of ATP and inflammatory cytokines, as well as its activation in central glial cells, contributes to pain development. Here, we hypothesized that Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain. METHODS: A mouse model of chronic constriction injury (CCI) in CD1 adult mice or P0-Cre transgenic mice, and in vitro cultured Schwann cells were used. Intrasciatic injection with Panx 1 blockers or the desired virus was used to knock down the expression of Panx 1. Mechanical and thermal sensitivity was assessed using Von Frey and a hot plate assay. The expression of Panx 1 was measured using qPCR, western blotting, and immunofluorescence. The production of cytokines was monitored through qPCR and enzyme-linked immunosorbent assay (ELISA). Panx1 channel activity was detected by ethidium bromide (EB) uptake. RESULTS: CCI induced persistent neuroinflammatory responses and upregulation of Panx 1 in Schwann cells. Intrasciatic injection of Panx 1 blockers, carbenoxolone (CBX), probenecid, and Panx 1 mimetic peptide (10Panx) effectively reduced mechanical and heat hyperalgesia. Probenecid treatment of CCI-induced mice significantly reduced Panx 1 expression in Schwann cells, but not in dorsal root ganglion (DRG). In addition, Panx 1 knockdown in Schwann cells with Panx 1 shRNA-AAV in P0-Cre mice significantly reduced CCI-induced neuropathic pain. To determine whether Schwann cell Panx 1 participates in the regulation of neuroinflammation and contributes to neuropathic pain, we evaluated its effect in LPS-treated Schwann cells. We found that inhibition of Panx 1 via CBX and Panx 1-siRNA effectively attenuated the production of selective cytokines, as well as its mechanism of action being dependent on both Panx 1 channel activity and its expression. CONCLUSION: In this study, we found that CCI-related neuroinflammation correlates with Panx 1 activation in Schwann cells, indicating that inhibition of Panx 1 channels in Schwann cells reduces neuropathic pain through the suppression of neuroinflammatory responses.


Assuntos
Carbenoxolona , Neuralgia , Trifosfato de Adenosina/farmacologia , Animais , Carbenoxolona/farmacologia , Carbenoxolona/uso terapêutico , Conexinas/genética , Conexinas/metabolismo , Citocinas/metabolismo , Etídio/metabolismo , Etídio/farmacologia , Etídio/uso terapêutico , Hiperalgesia/metabolismo , Lipopolissacarídeos/farmacologia , Camundongos , Proteínas do Tecido Nervoso/metabolismo , Neuralgia/metabolismo , Probenecid/metabolismo , Probenecid/farmacologia , Probenecid/uso terapêutico , RNA Interferente Pequeno/metabolismo , Células de Schwann
15.
Oxid Med Cell Longev ; 2022: 3119649, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36254232

RESUMO

Mitophagy, the selective removal of damaged mitochondria through autophagy, is crucial for mitochondrial turnover and quality control. Docosahexaenoic acid (DHA), an essential omega-3 fatty acid, protects mitochondria in various diseases. This study aimed to investigate the neuroprotective role of DHA in ischaemic stroke models in vitro and in vivo and its involvement in mitophagy and mitochondrial dysfunction. A mouse model of ischaemic stroke was established through middle cerebral artery occlusion (MCAO). To simulate ischaemic stroke in vitro, PC12 cells were subjected to oxygen-glucose deprivation (OGD). Immunofluorescence analysis, western blotting (WB), electron microscopy (EM), functional behavioural tests, and Seahorse assay were used for analysis. DHA treatment significantly alleviated the brain infarction volume, neuronal apoptosis, and behavioural dysfunction in mice with ischaemic stroke. In addition, DHA enhanced mitophagy by significantly increasing the number of autophagosomes and LC3-positive mitochondria in neurons. The Seahorse assay revealed that DHA increased glutamate and succinate metabolism in neurons after ischaemic stroke. JC-1 and MitoSox staining, and evaluation of ATP levels indicated that DHA-induced mitophagy alleviated reactive oxygen species (ROS) accumulation and mitochondrial injury. Mechanistically, DHA improved mitochondrial dynamics by increasing the expression of dynamin-related protein 1 (Drp1), LC3, and the mitophagy clearance protein Pink1/Parkin. Mdivi-1, a specific mitophagy inhibitor, abrogated the neuroprotective effects of DHA, indicating that DHA protected neurons by enhancing mitophagy. Therefore, DHA can protect against neuronal apoptosis after stroke by clearing the damaged mitochondria through Pink1/Parkin-mediated mitophagy and by alleviating mitochondrial dysfunction.


Assuntos
Lesões Encefálicas , Isquemia Encefálica , AVC Isquêmico , Fármacos Neuroprotetores , Acidente Vascular Cerebral , Trifosfato de Adenosina/farmacologia , Animais , Encéfalo/metabolismo , Isquemia Encefálica/tratamento farmacológico , Ácidos Docosa-Hexaenoicos/farmacologia , Ácidos Docosa-Hexaenoicos/uso terapêutico , Dinaminas/metabolismo , Glucose/farmacologia , Glutamatos/farmacologia , Camundongos , Mitofagia , Fármacos Neuroprotetores/farmacologia , Fármacos Neuroprotetores/uso terapêutico , Oxigênio/farmacologia , Proteínas Quinases/metabolismo , Ratos , Espécies Reativas de Oxigênio/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Succinatos/farmacologia , Ubiquitina-Proteína Ligases/metabolismo
16.
Int J Mol Sci ; 23(19)2022 Sep 30.
Artigo em Inglês | MEDLINE | ID: mdl-36232899

RESUMO

ATP-binding cassette subfamily G and tubulin pharmacological mechanisms decrease the effectiveness of anticancer drugs by modulating drug absorption and by creating tubulin assembly through polymerization. A series of natural and synthetic chalcones have been reported to have very good anticancer activity, with a half-maximal inhibitory concentration lower than 1 µM. By modulation, it is observed in case of the first mechanism that methoxy substituents on the aromatic cycle of acetophenone residue and substitution of phenyl nucleus by a heterocycle and by methoxy or hydroxyl groups have a positive impact. To inhibit tubulin, compounds bind to colchicine binding site. Presence of methoxy groups, amino groups or heterocyclic substituents increase activity.


Assuntos
Antineoplásicos , Chalcona , Chalconas , Acetofenonas/farmacologia , Trifosfato de Adenosina/farmacologia , Antineoplásicos/química , Proliferação de Células , Chalcona/farmacologia , Chalconas/química , Colchicina/metabolismo , Colchicina/farmacologia , Estrutura Molecular , Relação Estrutura-Atividade , Tubulina (Proteína)/metabolismo , Moduladores de Tubulina/química , Moduladores de Tubulina/farmacologia
17.
Int J Mol Sci ; 23(19)2022 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-36233228

RESUMO

As members of the family of nucleotide receptors, P2X7 receptors are of particular interest due to their unique structural and pharmacological characteristics. As ATP-gated ionic channels, P2X7 receptors in their activation elicit membrane depolarization; extracellular calcium influx; and activation of several downstream intracellular signaling pathways, some of them independent of the ionic channel activity. Further interactions of P2X7 receptors and cytoskeleton-related proteins have also been confirmed, and we previously described the effects of P2X7 receptor stimulation on the morphology of rat cerebellar astrocytes. In the present work, we used time-lapse video microscopy and atomic force microscopy (AFM) to elucidate the effects of P2X7 receptor stimulation on the morphology, migratory capabilities, and mechanical properties of rat cerebellar astrocytes in vitro. Stimulation of P2X7 receptors with the selective agonist BzATP specifically caused an increase in cell size, motility, and number of membrane protrusions of the astrocytes in culture. These effects were reverted when cells were previously treated with the competitive antagonist of P2X7R, A 438079. AFM analysis also showed an increase in cell stiffness and viscosity after P2X7 receptor stimulation. Surprisingly, these effects on the mechanical properties of the cell were not blocked by the treatment with the antagonist. Fluorescence microscopy analysis of the actin cytoskeleton showed an increase in actin stress fibers after BzATP treatment, an effect that again was not blocked by previous treatment with the antagonist, further confirming that the effects of P2X7 receptors on the cytoskeleton of astrocytes are, at least in part, independent of the ionic channel activity.


Assuntos
Astrócitos , Nucleotídeos , Actinas/metabolismo , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia , Animais , Astrócitos/metabolismo , Cálcio/metabolismo , Sinalização do Cálcio , Células Cultivadas , Nucleotídeos/metabolismo , Ratos , Receptores Purinérgicos P2X7/metabolismo
18.
Oxid Med Cell Longev ; 2022: 1652244, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36299604

RESUMO

Corilagin, a gallotannin, shows excellent antioxidant and anti-inflammatory effects. The NLRP3 inflammasome dysfunction has been implicated in a variety of inflammation diseases. However, it remains unclear how corilagin regulates the NLRP3 inflammasome to relieve gouty arthritis. In this study, bone marrow-derived macrophages (BMDMs) were pretreated with lipopolysaccharide (LPS) and then incubated with NLRP3 inflammasome agonists, such as adenine nucleoside triphosphate (ATP), nigericin, and monosodium urate (MSU) crystals. The MSU crystals were intra-articular injected to induce acute gouty arthritis. Here we showed that corilagin reduced lactate dehydrogenase (LDH) secretion and the proportion of propidium iodide- (PI-)stained cells. Corilagin suppressed the expression of N-terminal of the pyroptosis executive protein gasdermin D (GSDMD-NT). Corilagin restricted caspase-1 p20 and interleukin (IL)-1ß release. Meanwhile, corilagin attenuated ASC oligomerization and speck formation. Our findings confirmed that corilagin diminished NLRP3 inflammasome activation and macrophage pyroptosis. We further discovered that corilagin limited the mitochondrial reactive oxygen species (ROS) production and prevented the interaction between TXNIP and NLRP3, but ROS activator imiquimod could antagonize the inhibitory function of corilagin on NLRP3 inflammasome and macrophage pyroptosis. Additionally, corilagin ameliorated MSU crystals induced joint swelling, inhibited IL-1ß production, and abated macrophage and neutrophil migration into the joint capsule. Collectively, these results demonstrated that corilagin suppressed the ROS/TXNIP/NLRP3 pathway to repress inflammasome activation and pyroptosis and suggest its potential antioxidative role in alleviating NLRP3-dependent gouty arthritis.


Assuntos
Artrite Gotosa , Piroptose , Humanos , Inflamassomos/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Taninos Hidrolisáveis/farmacologia , Taninos Hidrolisáveis/uso terapêutico , Lipopolissacarídeos/farmacologia , Artrite Gotosa/tratamento farmacológico , Artrite Gotosa/metabolismo , Ácido Úrico/uso terapêutico , Antioxidantes/farmacologia , Nigericina/farmacologia , Nigericina/uso terapêutico , Imiquimode/farmacologia , Imiquimode/uso terapêutico , Propídio/farmacologia , Propídio/uso terapêutico , Nucleosídeos/farmacologia , Caspase 1/metabolismo , Inflamação/tratamento farmacológico , Inflamação/metabolismo , Interleucina-1beta/metabolismo , Anti-Inflamatórios/farmacologia , Trifosfato de Adenosina/farmacologia , Adenina/farmacologia , Lactato Desidrogenases
19.
J Sports Sci ; 40(17): 1981-1990, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-36251983

RESUMO

Citrulline malate (CM) has been shown to improve muscle performance in healthy participants during a single exercise session. Yet, within the framework of exercises repeated at close time interval, the consequences of CM ingestion on mechanical performance are controversial and the bioenergetics side remains undocumented. The aim of this double-blind placebo-controlled study was to evaluate in vivo the effect of short-term (7 doses in 48 h) oral administration of CM upon gastrocnemius muscle function and bioenergetics using non-invasive multimodal NMR techniques in healthy rats. The experimental protocol consisted of two 6-min bouts of fatiguing exercise spaced by an 8-min recovery period. CM treatment did not affect the basal bioenergetics status and increased the half-fatigue time during the first exercise bout. With exercise repetition, it prevented PCr cost alteration and decreased both the glycolytic ATP production and the contractile ATP cost in working muscle, but these changes were not associated to any improvement in mechanical performance. In addition, CM did not influence the replenishment of high-energy phosphorylated compounds during the post-exercise recovery periods. Therefore, short-term CM administration enhances muscle bioenergetics throughout fatiguing bouts of exercise repeated at close time interval but this enhancement does not benefit to mechanical performance.


Assuntos
Citrulina , Fadiga Muscular , Ratos , Animais , Fadiga Muscular/fisiologia , Citrulina/farmacologia , Citrulina/metabolismo , Músculo Esquelético/fisiologia , Metabolismo Energético , Fadiga , Método Duplo-Cego , Suplementos Nutricionais , Trifosfato de Adenosina/metabolismo , Trifosfato de Adenosina/farmacologia
20.
Int J Mol Sci ; 23(20)2022 Oct 11.
Artigo em Inglês | MEDLINE | ID: mdl-36292937

RESUMO

Bottom-up mechanokinetic models predict ensemble function of actin and myosin based on parameter values derived from studies using isolated proteins. To be generally useful, e.g., to analyze disease effects, such models must also be able to predict ensemble function when actomyosin interaction kinetics are modified differently from normal. Here, we test this capability for a model recently shown to predict several physiological phenomena along with the effects of the small molecular compound blebbistatin. We demonstrate that this model also qualitatively predicts effects of other well-characterized drugs as well as varied concentrations of MgATP. However, the effects of one compound, amrinone, are not well accounted for quantitatively. We therefore systematically varied key model parameters to address this issue, leading to the increased amplitude of the second sub-stroke of the power stroke from 1 nm to 2.2 nm, an unchanged first sub-stroke (5.3-5.5 nm), and an effective cross-bridge attachment rate that more than doubled. In addition to better accounting for the effects of amrinone, the modified model also accounts well for normal physiological ensemble function. Moreover, a Monte Carlo simulation-based version of the model was used to evaluate force-velocity data from small myosin ensembles. We discuss our findings in relation to key aspects of actin-myosin operation mechanisms causing a non-hyperbolic shape of the force-velocity relationship at high loads. We also discuss remaining limitations of the model, including uncertainty of whether the cross-bridge elasticity is linear or not, the capability to account for contractile properties of very small actomyosin ensembles (<20 myosin heads), and the mechanism for requirements of a higher cross-bridge attachment rate during shortening compared to during isometric contraction.


Assuntos
Actinas , Actomiosina , Actomiosina/metabolismo , Actinas/metabolismo , Contração Muscular , Miosinas/metabolismo , Contração Isométrica , Trifosfato de Adenosina/farmacologia , Amrinona , Modelos Biológicos
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