RESUMO
The body temperature of mice is higher at night than during the day. We show here that global deletion of acid-sensing ion channel 1a (ASIC1a) results in lower body temperature during a part of the night. ASICs are pH sensors that modulate neuronal activity. The deletion of ASIC1a decreased the voluntary activity at night of mice that had access to a running wheel but did not affect their spontaneous activity. Daily rhythms of thyrotropin-releasing hormone mRNA in the hypothalamus and of thyroid-stimulating hormone ß mRNA in the pituitary, and of prolactin mRNA in the hypothalamus and pituitary were suppressed in ASIC1a-/- mice. The serum thyroid hormone levels were however not significantly changed by ASIC1a deletion. Our findings indicate that ASIC1a regulates activity and signaling in the hypothalamus and pituitary. This likely leads to the observed changes in body temperature by affecting the metabolism or energy expenditure.
Assuntos
Canais Iônicos Sensíveis a Ácido , Temperatura Corporal , Animais , Camundongos , Canais Iônicos Sensíveis a Ácido/genética , Metabolismo Energético/genética , Hipotálamo , RNA MensageiroRESUMO
Acid-sensing ion channels (ASICs) are proton-gated ion channels that contribute to pain perception and neurotransmission. Being involved in sensing inflammation and ischemia, ASIC1a and ASIC3 are promising drug targets. Polyphenol tannic acid (TA) as well as green tea can interact with a variety of ion channels, but their effect on ASICs remains unknown. In addition, it is unknown whether they interact with ion channels via a common mechanism. Here, we show that TA is a potent modulator of ASICs. TA inhibited the transient current of rat ASIC3 expressed in HEK cells with an apparent IC50 of 2.2 ± 0.6 µM; it potentiated the sustained current and induced a slowly declining decay current. In addition, it produced an acidic shift of the pH-dependent activation of ASIC3 and inhibited the window current at pH 7.0. Moreover, TA inhibited the transient current of ASIC1a, ASIC1b, and ASIC2a. Pentagalloylglucose that is chemically identical to the central part of TA and a green tea extract both had effects on ASIC3 comparable to TA. TA and green tea inhibited inward currents generated by gramicidin channels, indicating interaction with the membrane. These results show that TA, pentagalloylglucose, and green tea modulate ASICs and identify alteration of the membrane as the potential common mechanism of this modulation. These properties will limit clinical application of these molecules.
Assuntos
Canais Iônicos Sensíveis a Ácido , Chá , Ratos , Animais , Taninos Hidrolisáveis , Concentração de Íons de HidrogênioRESUMO
BACKGROUND: Destruction of articular cartilage and bone is the main cause of joint dysfunction in rheumatoid arthritis (RA). Acid-sensing ion channel 1a (ASIC1a) is a key molecule that mediates the destruction of RA articular cartilage. Estrogen has been proven to have a protective effect against articular cartilage damage, however, the underlying mechanisms remain unclear. METHODS: We treated rat articular chondrocytes with an acidic environment, analyzed the expression levels of mitochondrial stress protein HSP10, ClpP, LONP1 by q-PCR and immunofluorescence staining. Transmission electron microscopy was used to analyze the mitochondrial morphological changes. Laser confocal microscopy was used to analyze the Ca2+, mitochondrial membrane potential (Δψm) and reactive oxygen species (ROS) level. Moreover, ASIC1a specific inhibitor Psalmotoxin 1 (Pctx-1) and Ethylene Glycol Tetraacetic Acid (EGTA) were used to observe whether acid stimulation damage mitochondrial function through Ca2+ influx mediated by ASIC1a and whether pretreatment with estrogen could counteract these phenomena. Furthermore, the ovariectomized (OVX) adjuvant arthritis (AA) rat model was treated with estrogen to explore the effect of estrogen on disease progression. RESULTS: Our results indicated that HSP10, ClpP, LONP1 protein and mRNA expression and mitochondrial ROS level were elevated in acid-stimulated chondrocytes. Moreover, acid stimulation decreased mitochondrial membrane potential and damaged mitochondrial structure of chondrocytes. Furthermore, ASIC1a specific inhibitor PcTx-1 and EGTA inhibited acid-induced mitochondrial abnormalities. In addition, estrogen could protect acid-stimulated induced mitochondrial stress by regulating the activity of ASIC1a in rat chondrocytes and protects cartilage damage in OVX AA rat. CONCLUSIONS: Extracellular acidification induces mitochondrial stress by activating ASIC1a, leading to the damage of rat articular chondrocytes. Estrogen antagonizes acidosis-induced joint damage by inhibiting ASIC1a activity. Our study provides new insights into the protective effect and mechanism of action of estrogen in RA.
Assuntos
Canais Iônicos Sensíveis a Ácido , Artrite Reumatoide , Condrócitos , Estrogênios , Mitocôndrias , Animais , Ratos , Canais Iônicos Sensíveis a Ácido/genética , Canais Iônicos Sensíveis a Ácido/metabolismo , Artrite Experimental , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Condrócitos/efeitos dos fármacos , Condrócitos/metabolismo , Ácido Egtázico/metabolismo , Ácido Egtázico/toxicidade , Estrogênios/farmacologia , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Espécies Reativas de Oxigênio , Cartilagem Articular/efeitos dos fármacos , Cartilagem Articular/patologiaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Bacopa monnieri L. (Scrophulariaceae) is commonly known as Brahmi and traditionally used as a neuroprotective herbal medicine. Recently, Bacopa monnieri exhibited significant therapeutic activity against animal model of neuropathic pain. However, the therapeutic potential of methanolic extract of Bacopa monnieri in experimental animal model is yet to establish. AIM OF THE STUDY: The present study was designed to evaluate the anti-nociceptive potential of standardized methanolic extract of Bacopa monnieri in experimental adult zebrafish (Danio rerio) model of pain. MATERIALS AND METHODS: The methanolic extract of Bacopa monnieri (BME) was standardized to bacoside-A using chromatographic method. Subsequently, BME (0.75, 1.25 and 5.0 mg/ml) was evaluated for anti-nociceptive activity using adult zebrafish model. RESULTS: Standardized BME showed antioxidant effect through radical quenching activity in in vitro study. BME at 1.25 mg/ml significantly decreased the nociceptive effect induced by different noxious agents like acetic acid where as BME at 2.5 mg/ml exhibited significant antinociceptive activity against glutamate, formalin, capsaicin, cinnamaldehyde when compared to control and sham group animals. CONCLUSION: BME exerted antinociceptive activity in adult zebrafish. It could be presumed that BME may involve glutamatergic receptor, ASIC and TRP channel activity in its anti-nociceptive effect. BME could be considered as a potential therapeutic option in the management of pain.
Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Analgésicos/farmacologia , Bacopa , Neuralgia , Extratos Vegetais/farmacologia , Canais de Potencial de Receptor Transitório/metabolismo , Animais , Antioxidantes/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Plantas Medicinais , Peixe-ZebraRESUMO
ABSTRACT: Prolotherapy is widely used in pain control and tissue repair in pain medicine. The classical mode is injection with hypertonic dextrose in muscle or perimysium. However, the analgesic mechanism is still not known. Here, we successfully established dextrose-mediated antinociception in a mouse model of fibromyalgia. The antinociceptive effects of dextrose injections were evaluated in a mouse model of fibromyalgia, in which bilateral chronic mechanical hyperalgesia was induced by unilateral intramuscular acid injection. The injectant (dextrose), dose (≥5%), and volume (>10 µL), but not osmolarity, were essential for the prolotherapy. Further studies showed that the activation of acid-sensing ion channel 1a (ASIC1a), neural activation, and the release of substance P from muscle afferents were required in the dextrose-induced reduction of mechanical hypersensitivity. Both pharmacological blockade and genetic deletion of ASIC1a or substance P as well as lidocaine abolished the dextrose-induced antinociception in mice with chronic hyperalgesia. Moreover, intramuscular dextrose injection induced phosphorylated extracellular signal-regulated kinase expression in dorsal root ganglion neurons expressing substance P; the phosphorylated extracellular signal-regulated kinase expression was inhibited by the ASIC1a antagonist PcTx1. The optimal settings for prolotherapy in fibromyalgia-like pain are dextrose dependent and volume dependent, and the peripheral antinociception involves ASIC1a and substance P signaling in muscle afferents. This study suggests a possible mechanism of action of dextrose prolotherapy in noninflammatory muscle pain such as fibromyalgia and provides insights into treating other types of chronic pain.
Assuntos
Analgesia , Fibromialgia , Proloterapia , Canais Iônicos Sensíveis a Ácido , Animais , Modelos Animais de Doenças , MAP Quinases Reguladas por Sinal Extracelular , Fibromialgia/tratamento farmacológico , Glucose , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Camundongos , Mialgia/tratamento farmacológico , Substância P/uso terapêuticoRESUMO
Depression is a prominent complex psychiatric disorder, usually complicated through expression of comorbid conditions, with chronic pain being among the most prevalent. This comorbidity is consistently associated with a poor prognosis and has been shown to negatively impact patient outcomes. With a global rise in this condition presenting itself, the importance of discovering long-term, effective, and affordable treatments is crucial. Electroacupuncture has demonstrated renowned success in its use for the treatment of pain and is a widely recognized therapy in clinical practice for the treatment of various psychosomatic disorders, most notably depression. Our study aimed to investigate the effects and mechanisms of Acid-Saline (AS) inducing states of chronic pain and depression comorbidity in the cerebellum, using the ST36 acupoint as the therapeutic intervention. Furthermore, the role of TRPV1 was relatedly explored through the use of TRPV1-/- mice (KO). The results indicated significant differences in the four behavioral tests used to characterize pain and depression states in mice. The AS and AS + SHAM group showed significant differences when compared to the Control and AS + EA groups in the von Frey and Hargreaves's tests, as well as the Open-Field and Forced Swimming tests. This evidence was further substantiated in the protein levels observed in immunoblotting, with significant differences between the AS and AS + SHAM groups when compared to the AS + EA and AS + KO groups being identified. In addition, immunofluorescence visibly served to corroborate the quantitative outcomes. Conclusively these findings suggest that AS-induced chronic pain and depression comorbidity elicits changes in the cerebellum lobules VI, VII, VIII, which are ameliorated through the use of EA at ST36 via its action on TRPV1 and related molecular pathways. The action of TRPV1 is not singular in CPDC, which would suggest other potential targets such as acid-sensing ion channel subtype 3 (ASIC3) or voltage-gated sodium channels (Navs) that could be explored in future studies.
Assuntos
Canais Iônicos Sensíveis a Ácido/genética , Dor Crônica/genética , Depressão/genética , Canais de Cátion TRPV/genética , Ácidos/toxicidade , Pontos de Acupuntura , Animais , Cerebelo/metabolismo , Cerebelo/patologia , Cerebelo/efeitos da radiação , Dor Crônica/induzido quimicamente , Dor Crônica/complicações , Dor Crônica/terapia , Comorbidade , Depressão/complicações , Depressão/patologia , Modelos Animais de Doenças , Eletroacupuntura , Humanos , Camundongos , Camundongos Knockout , Solução Salina/toxicidade , NataçãoRESUMO
Drugs used to treat pain are associated with adverse effects, increasing the search for new drugs as an alternative treatment for pain. Therefore, we evaluated the antinociceptive behavior and possible neuromodulation mechanisms of triterpene 3ß, 6ß, 16ß-trihydroxylup-20(29)-ene (CLF-1) isolated from Combretum leprosum leaves in zebrafish. Zebrafish (n = 6/group) were pretreated with CLF-1 (0.1 or 0.3 or 1.0 mg/mL; i.p.) and underwent nociception behavior tests. The antinociceptive effect of CFL-1 was tested for modulation by opioid (naloxone), nitrergic (L-NAME), nitric oxide and guanylate cyclase synthesis inhibitor (methylene blue), NMDA (Ketamine), TRPV1 (ruthenium red), TRPA1 (camphor), or ASIC (amiloride) antagonists. The corneal antinociceptive effect of CFL-1 was tested for modulation by TRPV1 (capsazepine). The effect of CFL-1 on zebrafish locomotor behavior was evaluated with the open field test. The acute toxicity study was conducted. CLF-1 reduced nociceptive behavior and corneal in zebrafish without mortalities and without altering the animals' locomotion. Thus, CFL-1 presenting pharmacological potential for the treatment of acute pain and corneal pain, and this effect is modulated by the opioids, nitrergic system, NMDA receptors and TRP and ASIC channels.
Assuntos
Analgésicos/farmacologia , Combretum/química , Locomoção/efeitos dos fármacos , Nociceptividade/efeitos dos fármacos , Dor/prevenção & controle , Triterpenos/farmacologia , Canais Iônicos Sensíveis a Ácido/metabolismo , Amilorida/farmacologia , Analgésicos/isolamento & purificação , Animais , Cânfora/farmacologia , Capsaicina/análogos & derivados , Capsaicina/farmacologia , Relação Dose-Resposta a Droga , Feminino , Ketamina/farmacologia , Locomoção/fisiologia , Masculino , Azul de Metileno/farmacologia , NG-Nitroarginina Metil Éster/farmacologia , Naloxona/farmacologia , Nociceptividade/fisiologia , Dor/metabolismo , Dor/fisiopatologia , Medição da Dor , Extratos Vegetais/química , Folhas de Planta/química , Receptores de N-Metil-D-Aspartato/metabolismo , Rutênio Vermelho/farmacologia , Canais de Cátion TRPV/metabolismo , Triterpenos/isolamento & purificação , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismoRESUMO
The pyroptosis is a causative agent of rheumatoid arthritis, a systemic autoimmune disease merged with degenerative articular cartilage. Nevertheless, the precise mechanism of extracellular acidosis on chondrocyte pyroptosis is largely unclear. Acid-sensing ion channels (ASICs) belong to an extracellular H+ -activated cation channel family. Accumulating evidence has highlighted activation of ASICs induced by extracellular acidosis upregulate calpain and calcineurin expression in arthritis. In the present study, to investigate the expression and the role of acid-sensing ion channel 1a (ASIC1a), calpain, calcineurin, and NLRP3 inflammasome proteins in regulating acid-induced articular chondrocyte pyroptosis, primary rat articular chondrocytes were subjected to different pH, different time, and different treatments with or without ASIC1a, calpain-2, and calcineurin, respectively. Initially, the research results showed that extracellular acidosis-induced the protein expression of ASIC1a in a pH- and time-dependent manner, and the messenger RNA and protein expressions of calpain, calcineurin, NLRP3, apoptosis-associated speck-like protein, and caspase-1 were significantly increased in a time-dependent manner. Furthermore, the inhibition of ASIC1a, calpain-2, or calcineurin, respectively, could decrease the cell death accompanied with the decreased interleukin-1ß level, and the decreased expression of ASIC1a, calpain-2, calcineurin, and NLRP3 inflammasome proteins. Taken together, these results indicated the activation of ASIC1a induced by extracellular acidosis could trigger pyroptosis of rat articular chondrocytes, the mechanism of which might partly be involved with the activation of calpain-2/calcineurin pathway.
Assuntos
Canais Iônicos Sensíveis a Ácido/fisiologia , Artrite Experimental , Calcineurina/metabolismo , Calpaína/metabolismo , Condrócitos , Piroptose , Animais , Artrite Experimental/mortalidade , Artrite Experimental/patologia , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Masculino , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Ratos , Ratos Sprague-DawleyRESUMO
Acid-sensing ion channels (ASICs) are voltage-independent cation channels that detect decreases in extracellular pH. Dysregulation of ASICs underpins a number of pathologies. Of particular interest is ASIC3, which is recognised as a key sensor of acid-induced pain and is important in the establishment of pain arising from inflammatory conditions, such as rheumatoid arthritis. Thus, the identification of new ASIC3 modulators and the mechanistic understanding of how these compounds modulate ASIC3 could be important for the development of new strategies to counteract the detrimental effects of dysregulated ASIC3 activity in inflammation. Here, we report the identification of novel ASIC3 modulators based on the ASIC3 agonist, 2-guanidine-4-methylquinazoline (GMQ). Through a GMQ-guided in silico screening of Food and Drug administration (FDA)-approved drugs, 5 compounds were selected and tested for their modulation of rat ASIC3 (rASIC3) using whole-cell patch-clamp electrophysiology. Of the chosen drugs, guanabenz (GBZ), an α2-adrenoceptor agonist, produced similar effects to GMQ on rASIC3, activating the channel at physiological pH (pH 7.4) and potentiating its response to mild acidic (pH 7) stimuli. Sephin1, a GBZ derivative that lacks α2-adrenoceptor activity, has been proposed to act as a selective inhibitor of a regulatory subunit of the stress-induced protein phosphatase 1 (PPP1R15A) with promising therapeutic potential for the treatment of multiple sclerosis. However, we found that like GBZ, sephin1 activates rASIC3 at pH 7.4 and potentiates its response to acidic stimulation (pH 7), i.e. sephin1 is a novel modulator of rASIC3. Furthermore, docking experiments showed that, like GMQ, GBZ and sephin1 likely interact with the nonproton ligand sensor domain of rASIC3. Overall, these data demonstrate the utility of computational analysis for identifying novel ASIC3 modulators, which can be validated with electrophysiological analysis and may lead to the development of better compounds for targeting ASIC3 in the treatment of inflammatory conditions.
Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Simulação por Computador , Guanabenzo/análogos & derivados , Guanabenzo/metabolismo , Guanidinas/metabolismo , Quinazolinas/metabolismo , Canais Iônicos Sensíveis a Ácido/química , Regulação Alostérica/efeitos dos fármacos , Regulação Alostérica/fisiologia , Animais , Células CHO , Cricetinae , Cricetulus , Relação Dose-Resposta a Droga , Avaliação Pré-Clínica de Medicamentos/métodos , Guanabenzo/química , Guanabenzo/farmacologia , Guanidinas/química , Guanidinas/farmacologia , Estrutura Secundária de Proteína , Quinazolinas/química , Quinazolinas/farmacologiaRESUMO
Rationale: Synovial inflammation is one of the main pathological features of rheumatoid arthritis (RA) and is a key factor leading to the progression of RA. Understanding the regulatory mechanism of synovial inflammation is crucial for the treatment of RA. Acid-sensing ion channel 1a (ASIC1a) is an H+-gated cation channel that promotes the progression of RA, but the role of ASIC1a in synovial inflammation is unclear. This study aimed to investigate whether ASIC1a is involved in the synovial inflammation and explore the underlying mechanisms in vitro and in vivo. Methods: The expression of ASIC1a and nuclear factor of activated T cells (NFATs) were analyzed by Western blotting, immunofluorescence, and immunohistochemistry both in vitro and in vivo. The Ca2+ influx mediated by ASIC1a was detected by calcium imaging and flow cytometry. The role of ASIC1a in inflammation was studied in rats with adjuvant-induced arthritis (AA). Inflammatory cytokine profile was analyzed by protein chip in RA synovial fibroblasts (RASF) and verified by a magnetic multi-cytokine assay and ELISA. The NFATc3-regulated RANTES (Regulated upon activation, normal T cell expressed and secreted) gene transcription was investigated by ChIP-qPCR and dual-luciferase reporter assay. Results: The expression of ASIC1a was significantly increased in human RA synovial tissues and primary human RASF as well as in ankle synovium of AA rats. Activated ASIC1a mediated Ca2+ influx to increase [Ca2+]i in RASF. The activation/overexpression of ASIC1a in RASF up-regulated the expression of inflammatory cytokines RANTES, sTNF RI, MIP-1a, IL-8, sTNF RII, and ICAM-1 among which RANTES was increased most remarkably. In vivo, ASIC1a promoted inflammation, synovial hyperplasia, articular cartilage, and bone destruction, leading to the progression of AA. Furthermore, activation of ASIC1a upregulated the nuclear translocation of NFATc3, which bound to RANTES promoter and directly regulated gene transcription to enhance RANTES expression. Conclusion: ASIC1a induces synovial inflammation, which leads to the progression of RA. Our study reveals a novel RA inflammation regulatory mechanism and indicates that ASIC1a might be a potential therapeutic target for RA.
Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Artrite Reumatoide/patologia , Cálcio/metabolismo , Quimiocina CCL5/metabolismo , Fatores de Transcrição NFATC/metabolismo , Membrana Sinovial/patologia , Idoso , Animais , Artrite Reumatoide/metabolismo , Células Cultivadas , Citocinas/metabolismo , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Humanos , Inflamação/metabolismo , Inflamação/patologia , Masculino , Pessoa de Meia-Idade , Ratos , Ratos Sprague-Dawley , Membrana Sinovial/metabolismoRESUMO
BACKGROUND: Fibromyalgia (FM) is a syndrome involving chronic pain, fatigue, sleep difficulties, morning stiffness and muscle cramping lasting longer than 3 months. The epidemiological prevalence is approximately 3-5% in women and increases with age. Antagonism of acid-sensing ion channel 3 (ASIC3) reportedly attenuates acid saline-induced FM pain in mice. AIMS: Whether pre-treatment with electroacupuncture (EA) or APETx2 can attenuate mechanical hyperalgesia in this murine model remains unknown. METHODS: Accordingly, we examined the analgesic effect of EA in a murine model of FM pain induced by dual injections of acid saline and investigated whether EA or APETx2 can attenuate FM pain via the ASIC3 channel. RESULTS: EA significantly reduced mechanical hyperalgesia in this model. ASIC3 antagonism, induced by injecting APETx2, also significantly reduced mechanical hyperalgesia. The expression of ASIC3 in the dorsal root ganglia, spinal cord and thalamus was increased after FM model induction. Over-expression of these nociceptive channels was attenuated by pre-treatment with EA or an ASIC3 antagonist. CONCLUSION: Our data reveal that both EA and ASIC3 blockade significantly reduce FM pain in mice via the ASIC3, Nav1.7 and Nav1.8 signalling pathways. Moreover, our findings support the potential clinical use of EA for the treatment of FM pain.
Assuntos
Bloqueadores do Canal Iônico Sensível a Ácido/farmacologia , Canais Iônicos Sensíveis a Ácido/metabolismo , Eletroacupuntura/métodos , Fibromialgia/terapia , Hiperalgesia/terapia , Manejo da Dor/métodos , Bloqueadores do Canal Iônico Sensível a Ácido/administração & dosagem , Animais , Terapia Combinada , Modelos Animais de Doenças , Feminino , CamundongosAssuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Canais Iônicos Sensíveis a Ácido/metabolismo , Eletroacupuntura , Fibromialgia/complicações , Dor Musculoesquelética/terapia , Solução Salina/administração & dosagem , Pontos de Acupuntura , Animais , Venenos de Cnidários/uso terapêutico , Modelos Animais de Doenças , Fibromialgia/metabolismo , Hiperalgesia/diagnóstico , Hiperalgesia/terapia , Camundongos , Dor Musculoesquelética/metabolismo , Solução Salina/química , Transdução de SinaisAssuntos
Trifosfato de Adenosina/toxicidade , Eletroacupuntura/métodos , Moxibustão/métodos , Manejo da Dor/métodos , Dor/induzido quimicamente , Dor/metabolismo , Canais Iônicos Sensíveis a Ácido/metabolismo , Terapia por Acupuntura/métodos , Animais , Masculino , Camundongos , Camundongos Knockout , Purinas/toxicidade , Ratos , Ratos Sprague-Dawley , Receptores Purinérgicos P2X7/deficiência , Roedores , Canais de Cátion TRPV/deficiênciaRESUMO
BACKGROUND: Remifentanil induces hyperalgesia, but the underlying mechanisms are not fully understood. Acid-sensing ion channel 3 (ASIC3) plays a regulatory role in the pain pathway. This study aimed to explore the effect of remifentanil administration on postoperative pain and on ASIC3 expression at the prespinal and supraspinal levels in a rat model. METHODS: Rats were randomly allocated to the control, incision, remifentanil, and remifentanilâ¯+â¯incision groups. Remifentanil was given by a 1-h intravenous infusion prior to plantar incision. Paw withdrawal mechanical threshold (PWMT) and paw withdrawal thermal latency (PWTL) were measured at different time points before and after incision to evaluate mechanical and thermal hyperalgesia, respectively. The dorsal root ganglion (DRG), hippocampus, and hypothalamus were obtained after sacrifice at 48â¯h post-incision for determination of the protein expression of ASIC3 using western blot. RESULTS: Remifentanil administration significantly induced mechanical and thermal hyperalgesia from 2 to 48â¯h after incision. In addition, remifentanil exposure remarkably stimulated ASIC3 protein expression in DRG, hippocampus, and hypothalamus of rats at 48â¯h after incision. CONCLUSION: Remifentanil-induced hyperalgesia is accompanied by increased ASIC3 expression at the DRG and supraspinal levels, implying a possible involvement of ASIC3 in remifentanil-induced hyperalgesia.
Assuntos
Canais Iônicos Sensíveis a Ácido/biossíntese , Gânglios Espinais/metabolismo , Hipocampo/metabolismo , Hiperalgesia/metabolismo , Hipotálamo/metabolismo , Remifentanil/toxicidade , Canais Iônicos Sensíveis a Ácido/genética , Analgésicos Opioides/toxicidade , Animais , Gânglios Espinais/efeitos dos fármacos , Expressão Gênica , Hipocampo/efeitos dos fármacos , Hiperalgesia/induzido quimicamente , Hiperalgesia/genética , Hipotálamo/efeitos dos fármacos , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Acid-sensing ion channels (ASICs), which are present in almost all types of neurons, play an important role in physiological and pathological processes. The ASIC1a subtype is the most sensitive channel to the medium's acidification, and it plays an important role in the excitation of neurons in the central nervous system. Ligands of the ASIC1a channel are of great interest, both fundamentally and pharmaceutically. Using a two-electrode voltage-clamp electrophysiological approach, we characterized lindoldhamine (a bisbenzylisoquinoline alkaloid extracted from the leaves of Laurus nobilis L.) as a novel inhibitor of the ASIC1a channel. Lindoldhamine significantly inhibited the ASIC1a channel's response to physiologically-relevant stimuli of pH 6.5-6.85 with IC50 range 150-9 µM, but produced only partial inhibition of that response to more acidic stimuli. In mice, the intravenous administration of lindoldhamine at a dose of 1 mg/kg significantly reversed complete Freund's adjuvant-induced thermal hyperalgesia and inflammation; however, this administration did not affect the pain response to an intraperitoneal injection of acetic acid (which correlated well with the function of ASIC1a in the peripheral nervous system). Thus, we describe lindoldhamine as a novel antagonist of the ASIC1a channel that could provide new approaches to drug design and structural studies regarding the determinants of ASIC1a activation.
Assuntos
Bloqueadores do Canal Iônico Sensível a Ácido/uso terapêutico , Canais Iônicos Sensíveis a Ácido/fisiologia , Anti-Inflamatórios/uso terapêutico , Derivados de Benzeno/uso terapêutico , Quinolinas/uso terapêutico , Ácido Acético , Bloqueadores do Canal Iônico Sensível a Ácido/farmacologia , Animais , Anti-Inflamatórios/farmacologia , Derivados de Benzeno/farmacologia , Feminino , Adjuvante de Freund , Temperatura Alta/efeitos adversos , Hiperalgesia/tratamento farmacológico , Hiperalgesia/etiologia , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Masculino , Camundongos , Oócitos/fisiologia , Dor/induzido quimicamente , Dor/tratamento farmacológico , Quinolinas/farmacologia , Xenopus laevisRESUMO
BACKGROUND: Central pro-inflammatory cytokine (PIC) signal is involved in neurological deficits after transient global ischemia induced by cardiac arrest (CA). The present study was to examine if blocking acid sensing ion channels (ASICs) using amiloride in the Central Nervous System can alleviate neurological deficits after the induction of CA and further examine the participation of PIC signal in the hippocampus for the effects of amiloride. METHODS: CA was induced by asphyxia and then cardiopulmonary resuscitation was performed in rats. Western blot analysis and ELISA were used to determine the protein expression of ASIC subunit ASIC1 in the hippocampus, and the levels of PICs. As noted, it is unlikely that this procedure is clinically used although amiloride and other pharmacological agents were given into the brain in this study. RESULTS: CA increased ASIC1 in the hippocampus of rats in comparison with control animals. This was associated with the increase in IL-1ß, IL-6 and TNF-α together with Caspase-3 and Caspase-9. The administration of amiloride into the lateral ventricle attenuated the upregulation of Caspase-3/Caspase-9 and this further alleviated neurological severity score and brain edema. Inhibition of central IL-6 and TNF-α also decreased ASIC1 in the hippocampus of CA rats. CONCLUSION: Transient global ischemia induced by CA amplifies ASIC1a in the hippocampus likely via PIC signal. Amiloride administered into the Central Nervous System plays a neuroprotective role in the process of global ischemia. Thus, targeting ASICs (i.e., ASIC1a) is suggested for the treatment and improvement of CA-evoked global cerebral ischemia.
Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Amilorida/uso terapêutico , Hipocampo/metabolismo , Interleucina-6/metabolismo , Ataque Isquêmico Transitório/tratamento farmacológico , Fator de Necrose Tumoral alfa/metabolismo , Canais Iônicos Sensíveis a Ácido/genética , Amilorida/farmacologia , Animais , Asfixia/complicações , Dano Encefálico Crônico/tratamento farmacológico , Dano Encefálico Crônico/etiologia , Dano Encefálico Crônico/prevenção & controle , Edema Encefálico/tratamento farmacológico , Edema Encefálico/etiologia , Caspase 3/biossíntese , Caspase 3/genética , Caspase 9/biossíntese , Caspase 9/genética , Avaliação Pré-Clínica de Medicamentos , Hidrazinas/farmacologia , Ataque Isquêmico Transitório/etiologia , Ataque Isquêmico Transitório/metabolismo , Masculino , Quinoxalinas/farmacologia , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Studies have shown that the etiology and pathogenesis of colorectal cancer are closely related to the tumor microenvironment, and the cancer tissue is still in the state of "energy deficit" and has to promote energy generation through high glycolysis. Rhus chinensis Mill is a Chinese herbal medicine used to treat various types of solid tumors in China. Colorectal cancer (CRC) is a heterogeneous disease group caused by abnormal changes in glucose metabolism resulted in lactic acid production, which remodels acidosis. AIM OF THE STUDY: Although previous studies have shown that the active compounds of Rhus chinensis Mill. can inhibit the proliferation of tumor cells, whether its triterpenoids could effectively regulate glycolysis involved in CRC have not been systematically investigated. MATERIALS AND METHODS: In this study, the extraction of triterpenoids extract from Rhus chinensis Mill. was obtained, and cell viability assay, the percentage of apoptosis for CRC cells were counted, and matrigel invasion assay and production of lactic acid and glucose uptake assay was determined. we further examined the expression of the key glycolytic enzymes and acid-sending ion channel (ASIC) family members of SW620 cells, and some key proteins in the glycolytic pathway were further verified. RESULTS: Notably, triterpenoids (TER) of Rhus chinensis Mill. showed effective anti-proliferative activity and significantly altered protein levels associated with CRC cell survival and glycolysis metabolism. TER could down-regulate the expression of ASIC2, in CRC SW620 cell line. Most importantly, the levels of ASIC2 and calcineurin/nuclear factor of activated T cells (NFAT) were also down-regulated by TER. Furthermore, inhibition of activated the ASIC2-mediated calcineurin/NFAT1 pathway and target gene transcript expression of MMP-2 and MMP-9 in parallel to reduce, and resulted in the reduced invasion ability by TER treatment. CONCLUSION: The potential pathways and targets that involved in glycolysis to excert the anti-CRC effects of main compounds in triterpenoids of Rhus chinensis Mill. were predicted by network pharmacology methods. Our findings thus provided rational evidence that inhibition of the ASIC2-induced calcineurin/NFAT pathway by triterpenoids in Rhus chinensis Mill. profoundly suppressed cell growth and invasion in CRC, which target alternative glycolysis in colorectal tumor cells, may be a useful adjuvant therapy in the treatment of colorectal cancer.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Neoplasias Colorretais/tratamento farmacológico , Rhus/química , Triterpenos/farmacologia , Canais Iônicos Sensíveis a Ácido/metabolismo , Antineoplásicos Fitogênicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Calcineurina/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Neoplasias Colorretais/patologia , Regulação para Baixo/efeitos dos fármacos , Glicólise/efeitos dos fármacos , Humanos , Medicina Tradicional Chinesa/métodos , Fatores de Transcrição NFATC/metabolismo , Extratos Vegetais/isolamento & purificação , Extratos Vegetais/farmacologia , Triterpenos/isolamento & purificação , Microambiente TumoralRESUMO
Acid-sensing ion channel (ASIC) serves important roles in the transmission of nociceptive information. To confirm the analgesic mechanism of dragon's blood resin, patch-clamp technique, in vivo animal experiments, and immunohistochemical staining were used to observe the effects of the three flavonoids (loureirin B, cochinchinemin A, and cochinchinemin B) isolated from dragon's blood resin on ASIC. Results showed that the three flavonoids exerted various inhibitory effects on ASIC currents in rat dorsal root ganglion (DRG) neurons. The combination of the three flavonoids with total concentration of 6.5 µM could decrease (53.8 ± 4.3%) of the peak amplitude and (45.8 ± 4.5%) of the sustained portion of ASIC currents. The combination of the three flavonoids was fully efficacious on complete Freud's adjuvant (CFA)-induced inflammatory thermal hyperalgesia at a dose of 6.5 mM similar with amiloride at 10 mM. The analgesic effects of the combination could be weakened by an ASIC activator 2-guanidine-4-methylquinazoline. CFA-induced hyperalgesia was accompanied by c-Fos up-regulation in DRG neurons, and the combination rescued thermal hyperalgesia through down-regulation of c-Fos and ASIC3 expression in CFA-induced inflammation. These collective results suggested that the flavonoids isolated from dragon's blood resin could be considered as the chemical compounds that exert analgesic effects on inflammatory thermal pain due to action on ASIC.
Assuntos
Canais Iônicos Sensíveis a Ácido/efeitos dos fármacos , Analgésicos/farmacologia , Flavonoides/farmacologia , Extratos Vegetais/análise , Animais , Gânglios Espinais/efeitos dos fármacos , Técnicas de Patch-Clamp , Ratos , Ratos Sprague-Dawley , Resinas Vegetais/farmacologiaRESUMO
Rheumatoid arthritis is an autoimmune disease with a poor prognosis. Pyroptosis is a type of proinflammatory programmed cell death that is characterised by the activation of caspase-1 and secretion of the proinflammatory cytokines interleukin (IL)-1ß/18. Previous reports have shown that pyroptosis is closely related to the development of some autoimmune diseases, such as rheumatoid arthritis. The decrease in the pH of joint fluid is a main pathogenic feature of RA and leads to excessive apoptosis in chondrocytes. Acid-sensitive ion channels (ASICs) are extracellular H+-activated cation channels that mainly influence Na+ and Ca2+ permeability. In this study, we investigated the role of Ca2+ in acid-sensing ion channel 1a-mediated chondrocyte pyroptosis in an adjuvant arthritis rat model. The expression of apoptosis-associated speck-like protein, NLRP3, caspase-1, ASIC 1a, IL-1ß and IL-18 was upregulated in the joints of rats compared with that in normal rats, but the expression of Col2a in cartilage was decreased. However, these changes were reversed by amiloride, which is an inhibitor of ASIC1a. Extracellular acidosis significantly increased the expression of ASIC1a, IL-1ß, IL-18, ASC, NLRP3 and caspase-1 and promoted the release of lactate dehydrogenase. Interestingly, Psalmotoxin-1 (Pctx-1) and BAPTA-AM inhibited these effects. These results indicate that ASIC1a mediates pyroptosis in chondrocytes from AA rats. The underlying mechanism may be associated with the ability of ASIC1a to promote [Ca2+]i and upregulate the expression of the NLRP3 inflammasome.
Assuntos
Canais Iônicos Sensíveis a Ácido/metabolismo , Artrite Experimental/metabolismo , Cálcio/metabolismo , Condrócitos/metabolismo , Piroptose/fisiologia , Canais Iônicos Sensíveis a Ácido/genética , Canais Iônicos Sensíveis a Ácido/fisiologia , Animais , Cartilagem Articular/citologia , Células Cultivadas , Técnicas de Silenciamento de Genes , Membro Posterior/fisiopatologia , Concentração de Íons de Hidrogênio , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
BACKGROUND: Millions of people worldwide are suffering from Alzheimer's disease (AD), and there are only symptomatic treatments available for this disease. Thus, there is a great need to identify drugs capable of arresting or reversing AD. Constituents of the spice turmeric, in particular, curcuminoids, seem to be very promising, as evident from in vitro experiments and tests using animal models of AD. However, most of the clinical trials did not reveal any beneficial effects of curcuminoids in the treatment of AD. These controversies, including conflicting results of clinical trials, are thought to be related to bioavailability of curcuminoids, which is low unless it is enhanced by developing a special formulation. However, there is growing evidence suggesting that other reasons may be of even greater importance, but these avenues are less explored. OBJECTIVE: Review relevant literature, and analyze potential reasons for the controversial results. METHODOLOGY: Recent in vitro and preclinical studies; clinical trials (without a limiting period) were searched in PubMed and Google Scholar. RESULTS: While recent in vitro and preclinical studies confirm the therapeutic potential of curcuminoids in the treatment of AD and cognitive dysfunctions, results of corresponding clinical trials remain rather controversial. CONCLUSION: The controversial results obtained in the clinical trials may be in part due to particularities of the curcuminoid formulations other than bioavailability. Namely, it seems likely that the various formulations differ in terms of their minor turmeric constituent(s). We hypothesize that these distinctions may be of key importance for efficacy of the particular formulation in clinical trials. A testable approach addressing this hypothesis is suggested.