RESUMO
Background: Cardiac arrhythmias are the main cause of sudden death due to Chronic Chagasic Cardiomyopathy (CCC). Here we investigated alterations in connexin 43 (Cx43) expression and phosphorylation in cardiomyocytes as well as associations with cardiac arrhythmias in CCC. Methods: C57Bl/6 mice infected with Trypanosoma cruzi underwent cardiac evaluations at 6 and 12 months after infection via treadmill testing and EKG. Histopathology, cytokine gene expression, and distribution of total Cx43 and its phosphorylated forms Cx43S368 and Cx43S325/328/330 were investigated. Human heart samples obtained from subjects with CCC were submitted to immunofluorescence analysis. In vitro simulation of a pro-inflammatory microenvironment (IL-1ß, TNF, and IFN-γ) was performed in H9c2 cells and iPSC-derived cardiomyocytes to evaluate Cx43 distribution, action potential duration, and Lucifer Yellow dye transfer. Results: Mice chronically infected with T. cruzi exhibited impaired cardiac function associated with increased inflammation, fibrosis and upregulated IL-1ß, TNF, and IFN-γ gene expression. Confocal microscopy revealed altered total Cx43, Cx43S368 and Cx43S325/328/330 localization and phosphorylation patterns in CCC, with dispersed staining outside the intercalated disc areas, i.e., in lateral membranes and the cytoplasm. Reduced co-localization of total Cx43 and N-cadherin was observed in the intercalated discs of CCC mouse hearts compared to controls. Similar results were obtained in human CCC heart samples, which showed Cx43 distribution outside the intercalated discs. Stimulation of human iPSC-derived cardiomyocytes or H9c2 cells with IL-1ß, TNF, and IFN-γ induced alterations in Cx43 localization, reduced action potential duration and dye transfer between adjacent cells. Conclusion: Heart inflammation in CCC affects the distribution and phosphorylation pattern of Cx43, which may contribute to the generation of conduction disturbances in Chagas disease.
Assuntos
Cardiomiopatia Chagásica , Conexina 43 , Camundongos Endogâmicos C57BL , Miócitos Cardíacos , Conexina 43/metabolismo , Conexina 43/genética , Animais , Cardiomiopatia Chagásica/metabolismo , Cardiomiopatia Chagásica/patologia , Cardiomiopatia Chagásica/imunologia , Cardiomiopatia Chagásica/parasitologia , Humanos , Camundongos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/parasitologia , Miócitos Cardíacos/patologia , Inflamação/metabolismo , Fosforilação , Masculino , Doença Crônica , Trypanosoma cruzi , Modelos Animais de Doenças , Linhagem Celular , Citocinas/metabolismo , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/parasitologia , Arritmias Cardíacas/imunologia , FemininoRESUMO
This study investigates whether hAFSCs can improve bladder function in partial bladder outlet obstruction (pBOO) rats by targeting specific cellular pathways. Thirty-six female rats were divided into sham and pBOO groups with and without hAFSCs single injection into the bladder wall. Cystometry, inflammation/hypoxia, collagen/fibrosis/gap junction proteins, and smooth muscle myosin/muscarinic receptors were examined at 2 and 6 weeks after pBOO or sham operation. In pBOO bladders, significant increases in peak voiding pressure and residual volume stimulated a significant upregulation of inflammatory and hypoxic factors, TGF-ß1 and Smad2/3. Collagen deposition proteins, collagen 1 and 3, were significantly increased, but bladder fibrosis markers, caveolin 1 and 3, were significantly decreased. Gap junction intercellular communication protein, connexin 43, was significantly increased, but the number of caveolae was significantly decreased. Markers for the smooth muscle phenotype, myosin heavy chain 11 and guanylate-dependent protein kinase, as well as M2 muscarinic receptors, were significantly increased in cultured detrusor cells. However, hAFSCs treatment could significantly ameliorate bladder dysfunction by inactivating the TGFß-Smad signaling pathway, reducing collagen deposition, disrupting gap junctional intercellular communication, and modifying the expressions of smooth muscle myosin and caveolae/caveolin proteins. The results support the potential value of hAFSCs-based treatment of bladder dysfunction in BOO patients.
Assuntos
Conexina 43 , Obstrução do Colo da Bexiga Urinária , Bexiga Urinária , Animais , Obstrução do Colo da Bexiga Urinária/metabolismo , Obstrução do Colo da Bexiga Urinária/patologia , Feminino , Ratos , Bexiga Urinária/metabolismo , Bexiga Urinária/fisiopatologia , Bexiga Urinária/patologia , Conexina 43/metabolismo , Transplante de Células-Tronco/métodos , Transdução de Sinais , Ratos Sprague-Dawley , Proteína Smad2/metabolismo , Modelos Animais de Doenças , Junções Comunicantes/metabolismo , Colágeno/metabolismoRESUMO
Connexin 43 (Cx43) is crucial for the development and homeostasis of the musculoskeletal system, where it plays multifaceted roles, including intercellular communication, transcriptional regulation and influencing osteogenesis and chondrogenesis. Here, we investigated Cx43 modulation mediated by inflammatory stimuli involved in osteoarthritis, i.e., 10 ng/mL Tumor Necrosis Factor alpha (TNFα) and/or 1 ng/mL Interleukin-1 beta (IL-1ß), in primary chondrocytes (CH) and osteoblasts (OB). Additionally, we explored the impact of synovial fluids from osteoarthritis patients in CH and cartilage explants, providing a more physio-pathological context. The effect of TNFα on Cx43 expression in cartilage explants was also assessed. TNFα downregulated Cx43 levels both in CH and OB (-73% and -32%, respectively), while IL-1ß showed inconclusive effects. The reduction in Cx43 levels was associated with a significant downregulation of the coding gene GJA1 expression in OB only (-65%). The engagement of proteasome in TNFα-induced effects, already known in CH, was also observed in OB. TNFα treatment significantly decreased Cx43 expression also in cartilage explants. Of note, Cx43 expression was halved by synovial fluid in both CH and cartilage explants. This study unveils the regulation of Cx43 in diverse musculoskeletal cell types under various stimuli and in different contexts, providing insights into its modulation in inflammatory joint disorders.
Assuntos
Condrócitos , Conexina 43 , Interleucina-1beta , Osteoartrite , Osteoblastos , Fator de Necrose Tumoral alfa , Humanos , Conexina 43/metabolismo , Conexina 43/genética , Condrócitos/metabolismo , Osteoblastos/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacologia , Osteoartrite/metabolismo , Osteoartrite/patologia , Osteoartrite/genética , Líquido Sinovial/metabolismo , Cartilagem Articular/metabolismo , Cartilagem Articular/patologia , Células Cultivadas , Idoso , Pessoa de Meia-Idade , Inflamação/metabolismo , Inflamação/genética , Inflamação/patologia , Cartilagem/metabolismo , Cartilagem/patologia , Artropatias/metabolismo , Artropatias/patologia , Artropatias/genéticaRESUMO
BACKGROUND: Atrial fibrillation (AF) is associated with increased risk of stroke and mortality. It has been reported that the process of atrial fibrosis was regulated by ß-catenin in rats with AF. However, pathophysiological mechanisms of this process in human with AF remain unclear. This study aims to investigate the possible mechanisms of ß-catenin in participating in the atrial fibrosis using human right atrial appendage (hRAA) tissues . METHODS: We compared the difference of ß-catenin expression in hRAA tissues between the patients with AF and sinus rhythm (SR). The possible function of ß-catenin in the development of AF was also explored in mice and primary cells. RESULTS: Firstly, the space between the membrane of the gap junctions of cardiomyocytes was wider in the AF group. Secondly, the expression of the gap junction function related proteins, Connexin40 and Connexin43, was decreased, while the expression of ß-catenin and its binding partner E-cadherin was increased in hRAA and cardiomyocytes of the AF group. Thirdly, ß-catenin colocalized with E-cadherin on the plasma membrane of cardiomyocytes in the SR group, while they were dissociated and accumulated intracellularly in the AF group. Furthermore, the expression of glycogen synthase kinase 3ß (GSK-3ß) and Adenomatous Polyposis Coli (APC), which participated in the degradation of ß-catenin, was decreased in hRAA tissues and cardiomyocytes of the AF group. Finally, the development of atrial fibrosis and AF were proved to be prevented after inhibiting ß-catenin expression in the AF model mice. CONCLUSIONS: Based on human atrial pathological and molecular analyses, our findings provided evidence that ß-catenin was associated with atrial fibrosis and AF progression.
Assuntos
Fibrilação Atrial , Fibrose , Átrios do Coração , Miócitos Cardíacos , beta Catenina , Humanos , Fibrilação Atrial/patologia , Fibrilação Atrial/metabolismo , beta Catenina/metabolismo , Animais , Átrios do Coração/metabolismo , Átrios do Coração/patologia , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Masculino , Glicogênio Sintase Quinase 3 beta/metabolismo , Caderinas/metabolismo , Junções Comunicantes/metabolismo , Pessoa de Meia-Idade , Camundongos , Feminino , Conexina 43/metabolismo , Camundongos Endogâmicos C57BL , IdosoRESUMO
Hypertrophic cardiomyopathy (HCM) is an inherited heart muscle disease that is characterised by left ventricular wall thickening, cardiomyocyte disarray and fibrosis, and is associated with arrhythmias, heart failure and sudden death. However, it is unclear to what extent the electrophysiological disturbances that lead to sudden death occur secondary to structural changes in the myocardium or as a result of HCM cardiomyocyte electrophysiology. In this study, we used an induced pluripotent stem cell model of the R403Q variant in myosin heavy chain 7 (MYH7) to study the electrophysiology of HCM cardiomyocytes in electrically coupled syncytia, revealing significant conduction slowing and increased spatial dispersion of repolarisation - both well-established substrates for arrhythmia. Analysis of rhythmonome protein expression in MYH7 R403Q cardiomyocytes showed reduced expression of connexin-43 (also known as GJA1), sodium channels and inward rectifier potassium channels - a three-way hit that reduces electrotonic coupling and slows cardiac conduction. Our data represent a previously unreported, biophysical basis for arrhythmia in HCM that is intrinsic to cardiomyocyte electrophysiology. Later in the progression of the disease, these proarrhythmic phenotypes may be accentuated by myocyte disarray and fibrosis to contribute to sudden death.
Assuntos
Cardiomiopatia Hipertrófica , Conexina 43 , Sistema de Condução Cardíaco , Miócitos Cardíacos , Cadeias Pesadas de Miosina , Conexina 43/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Humanos , Cardiomiopatia Hipertrófica/patologia , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/fisiopatologia , Cadeias Pesadas de Miosina/metabolismo , Cadeias Pesadas de Miosina/genética , Sistema de Condução Cardíaco/metabolismo , Sistema de Condução Cardíaco/fisiopatologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Miosinas Cardíacas/metabolismo , Miosinas Cardíacas/genética , Células Gigantes/metabolismo , Células Gigantes/patologia , Arritmias Cardíacas/patologia , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatologia , Potenciais de AçãoRESUMO
Major depressive disorder (MDD) is a form of glial cell-based synaptic dysfunction disease in which glial cells interact closely with neuronal synapses and perform synaptic information processing. Glial cells, particularly astrocytes, are active components of the brain and are responsible for synaptic activity through the release gliotransmitters. A reduced density of astrocytes and astrocyte dysfunction have both been identified the brains of patients with MDD. Furthermore, gliotransmission, i.e., active information transfer mediated by gliotransmitters between astrocytes and neurons, is thought to be involved in the pathogenesis of MDD. However, the mechanism by which astrocyte-mediated gliotransmission contributes to depression remains unknown. This review therefore summarizes the alterations in astrocytes in MDD, including astrocyte marker, connexin 43 (Cx43) expression, Cx43 gap junctions, and Cx43 hemichannels, and describes the regulatory mechanisms of astrocytes involved in synaptic plasticity. Additionally, we investigate the mechanisms acting of the glutamatergic, gamma-aminobutyric acidergic, and purinergic systems that modulate synaptic function and the antidepressant mechanisms of the related receptor antagonists. Further, we summarize the roles of glutamate, gamma-aminobutyric acid, d-serine, and adenosine triphosphate in depression, providing a basis for the identification of diagnostic and therapeutic targets for MDD.
Assuntos
Astrócitos , Conexina 43 , Transtorno Depressivo Maior , Plasticidade Neuronal , Humanos , Astrócitos/metabolismo , Transtorno Depressivo Maior/metabolismo , Transtorno Depressivo Maior/fisiopatologia , Plasticidade Neuronal/fisiologia , Animais , Conexina 43/metabolismo , Transmissão Sináptica/fisiologia , Ácido Glutâmico/metabolismo , Neuroglia/metabolismo , Ácido gama-Aminobutírico/metabolismo , Sinapses/metabolismo , Sinapses/fisiologiaRESUMO
Extracellular vesicles (EVs) have emerged as a promising drug delivery system. Connectosomes are a specialized type of EVs that contain connexins in their membranes. Connexin is a surface transmembrane protein that forms connexin hemichannels. When a connexin hemichannel on a connectosome docks with another connexin hemichannel of a target cell, they form a gap junction that allows direct intracellular delivery of therapeutic cargos from within the connectosome to the cytoplasm of the recipient cell. In the present study, we tested the feasibility of converting connectosomes into dry powders by (thin-film) freeze-drying to enable their potential storage in temperatures higher than the recommended -80 °C, while maintaining their activity. Connectosomes were isolated from a genetically engineered HeLa cell line that overexpressing connexin-43 subunit protein tagged with red fluorescence protein. To facilitate the testing of the function of the connectosomes, they were loaded with calcein green dye. Calcein green-loaded connectosomes were thin-film freeze-dried with trehalose alone or trehalose and a polyvinylpyrrolidone polymer as lyoprotectant(s) to produce amorphous powders with high glass transition temperatures (>100 °C). Thin-film freeze-drying did not significantly change the morphology and structure of the connectosomes, nor their particle size distribution. Based on data from confocal microscopy, flow cytometry, and fluorescence spectrometry, the connexin hemichannels in the connectosomes reconstituted from the thin-film freeze-dried powder remained functional, allowing the passage of calcein green through the hemichannels and the release of the calcein green from the connectosomes when the channels were opened by chelating calcium in the reconstituted medium. The function of connectosomes was assessed after one month storage at different temperatures. The connexin hemichannels in connectosomes in liquid lost their function when stored at -19.5 ± 2.2 °C or 6.0 ± 0.5 °C for a month, while those in dry powder form remained functional under the same storage conditions. Finally, using doxorubicin-loaded connectosomes, we showed that the connectosomes reconstituted from thin-film freeze-dried powder remained pharmacologically active. These findings demonstrate that (thin-film) freeze-drying represents a viable method to prepare stable and functional powders of EVs that contain connexins in their membranes.
Assuntos
Vesículas Extracelulares , Liofilização , Pós , Humanos , Vesículas Extracelulares/metabolismo , Vesículas Extracelulares/química , Células HeLa , Conexina 43/metabolismo , Trealose/química , Fluoresceínas/química , Povidona/química , Conexinas/metabolismo , Tamanho da PartículaRESUMO
This study used the brilliant cresyl blue (BCB) staining method to group buffalo oocytes (BCB+ and BCB-) and perform in vitro maturation, in vitro fertilization and embryo culture. At the same time, molecular biology techniques were used to detect gap junction protein expression and oxidative stress-related indicators to explore the molecular mechanism of BCB staining to predict oocyte developmental potential. The techniques of buffalo oocytes to analyse their developmental potential and used immunofluorescence staining to detect the expression level of CX43 protein, DCFH-DA probe staining to detect ROS levels and qPCR to detect the expression levels of the antioxidant-related genes SOD2 and GPX1. Our results showed that the in vitro maturation rate, embryo cleavage rate and blastocyst rate of buffalo oocytes in the BCB+ group were significantly higher than those in the BCB- group and the control group (p < .05). The expression level of CX43 protein in the BCB+ group was higher than that in the BCB- group both before and after maturation (p < .05). The intensity of ROS in the BCB+ group was significantly lower than that in the BCB- group (p < .05), and the expression levels of the antioxidant-related genes SOD2 and GPX1 in the BCB+ group were significantly higher than those in the BCB- group (p < .05). Brilliant cresyl blue staining could effectively predict the developmental potential of buffalo oocytes. The results of BCB staining were positively correlated with the expression of gap junction protein and antioxidant-related genes and negatively correlated with the reactive oxygen species level, suggesting that the mechanism of BCB staining in predicting the developmental potential of buffalo oocytes might be closely related to antioxidant activity.
Assuntos
Búfalos , Conexina 43 , Técnicas de Maturação in Vitro de Oócitos , Oócitos , Oxazinas , Estresse Oxidativo , Animais , Oócitos/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Feminino , Técnicas de Maturação in Vitro de Oócitos/veterinária , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismo , Superóxido Dismutase/genética , Glutationa Peroxidase/metabolismo , Glutationa Peroxidase/genética , Fertilização in vitro/veterinária , Técnicas de Cultura Embrionária/veterinária , Glutationa Peroxidase GPX1 , Desenvolvimento Embrionário/fisiologia , Coloração e Rotulagem , Antioxidantes/metabolismoRESUMO
AIM: Hyperhomocysteine has been recognized as an independent risk factor of multiple diseases, including several eye diseases. In this study, we aim to investigate whether increased homocysteine (Hcy) is related to cataracts, and to explore whether dysregulation of mTOR-mediated autophagy and connexin expression are underlying mechanisms. METHOD: We first developed a method of liquid chromatography tandem mass spectrometry to accurately measure serum concentrations of Hcy in 287 cataract patients and 334 healthy controls. Next, we treated human lens epithelial (HLC-B3) cells with Hcy at different concentrations and durations, and then analyzed expression of autophagy-related markers and connexins, as well as phosphorylated mTOR (p-mTOR) in these cells by Western blotting. Formation of autophagic vacuoles and intracellular Ca2+ in the Hcy-treated cells were observed by fluorescence microscopy. Further, we performed a rescue experiment in the Hcy-treated HLC-B3 cells by pre-incubation with rapamycin, an mTOR inhibitor. RESULTS: The serum levels of Hcy in patients with cataracts were significantly increased compared to those in healthy controls. In cultured HLC-B3 cells, expression of autophagy related markers (LC3B and Beclin1) and connexins (Cx43 and Cx50) was inhibited by Hcy treatment in a dose- and duration-dependent manner. Accumulation of Ca2+ in the Hcy-treated lens epithelial cells was observed as a consequence of reduced connexin expression. Meanwhile, expression of p-mTOR increased, representing up-regulation of the mTOR pathway. Importantly, inhibition of autophagy and connexin expression due to hyperhomocysteine was rescued via mTOR suppression by pretreatment with rapamycin in HLC-B3 cells. CONCLUSION: Our results demonstrate that hyperhomocysteine might promote cataract development through two mTOR-mediated pathways in the lens epithelial cells: 1) dysregulation of autophagy and 2) accumulation of intracellular calcium via decreased connexin expression.
Assuntos
Autofagia , Catarata , Conexinas , Homocisteína , Cristalino , Serina-Treonina Quinases TOR , Humanos , Catarata/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Autofagia/efeitos dos fármacos , Homocisteína/sangue , Masculino , Pessoa de Meia-Idade , Feminino , Conexinas/metabolismo , Cristalino/metabolismo , Cristalino/efeitos dos fármacos , Linhagem Celular , Cálcio/metabolismo , Idoso , Células Epiteliais/metabolismo , Células Epiteliais/efeitos dos fármacos , Conexina 43/metabolismo , Adulto , Proteína Beclina-1/metabolismoRESUMO
BACKGROUND: Empagliflozin (EMPA), a selective sodium-glucose cotransporter type 2 (SGLT2) inhibitor, has been shown to reduce major adverse cardiovascular events in patients with heart failure of different etiologies, although the underlying mechanism still remains unclear. Ponatinib (PON) is a multi-tyrosine kinase inhibitor successfully used against myeloid leukemia and other human malignancies, but its cardiotoxicity remains worrisome. Cardiac connexins (Cxs) are both substrates and regulators of autophagy and responsible for proper heart function. Alteration in connexin expression and localization have been described in patients with heart failure. AIMS: To assess whether EMPA can mitigate PON-induced cardiac dysfunction by restoring the connexin 43-autophagy pathway. METHODS AND RESULTS: Male C57BL/6 mice, randomized into four treatment groups (CNTRL, PON, EMPA, PON+EMPA) for 28 days, showed increased autophagy, decreased Cx43 expression as well as Cx43 lateralization, and attenuated systo-diastolic cardiac dysfunction after treatment with EMPA and PON compared with PON alone. Compared with CNTRL (DMSO), cardiomyocyte-differentiated H9c2 (dH9c2) cells treated with PON showed significantly reduced cell viability to approximately 20â¯%, decreased autophagy, increased cell senescence and reduced DNA binding activity of serum response factor (SRF) to serum response elements (SRE), which were paralleled by reduction in cardiac actin expression. Moreover, PON induced a significant increase of Cx43 protein and its S368-phosphorylated form (pS368-Cx43), as well as their displacement from the plasma membrane to the perinuclear and nuclear cellular region. All these effects were reverted by EMPA. CONCLUSION: EMPA attenuates PON-induced cardiotoxicity by reducing senescence, enhancing the SRE-SRF binding and restoring the connexin 43-autophagy pathway. This effect may pave the way to use of SGLT2 inhibitors in attenuating tyrosine-kinase inhibitor cardiotoxicity.
Assuntos
Autofagia , Compostos Benzidrílicos , Cardiotoxicidade , Conexina 43 , Glucosídeos , Imidazóis , Miócitos Cardíacos , Piridazinas , Animais , Masculino , Camundongos , Ratos , Autofagia/efeitos dos fármacos , Compostos Benzidrílicos/farmacologia , Cardiotoxicidade/etiologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Conexina 43/metabolismo , Glucosídeos/farmacologia , Imidazóis/farmacologia , Camundongos Endogâmicos C57BL , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Piridazinas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Inibidores do Transportador 2 de Sódio-Glicose/farmacologiaRESUMO
Connexin 43 (Cx43) is a gap junction protein that participates in small molecule exchange between adjacent cells. It is a predominant Cx within the mammalian ovary, where is associated with proper follicle development. The expression and regulation of Cx43 in the chicken ovary is largely unknown. The aim of the present study was to examine the expression of the Cx43 gene (GJA1) and protein as well as the immunolocalization of Cx43 in the laying hen ovary in relation to follicle development, and to examine how tamoxifen (TMX; an estrogen receptor modulator) treatment affects these factors. qRT-PCR and western blotting demonstrated differences in Cx43 mRNA transcript and protein abundances in ovarian white follicles, yellowish follicles, small yellow follicles, and the largest yellow preovulatory follicles (F3-F1). In general, Cx43 was more abundant in hierarchical than prehierarchical follicles and in granulosa cells compared with theca cells. Further, the response to TMX treatment depended on the stage of follicle development and the layer of the follicular wall. Ovarian regression following TMX treatment was accompanied by an increase in Cx43 expression in most ovarian tissues, which may impact the formation and function of Cx43 hemichannels. Overall, our results showed, for the first time, the differences in Cx43 mRNA and protein levels between ovarian follicles, suggesting the potential involvement of this gap junction protein in the regulation of ovarian follicle development and function. In addition, the results indicate a possible role for estradiol in regulation of Cx43 transcription and/or translation in the chicken ovary. Understanding the contribution of Cx43 in mechanisms underlying ovarian follicle development may be of considerable importance for poultry egg production.
Assuntos
Galinhas , Conexina 43 , Regulação da Expressão Gênica , Ovário , Tamoxifeno , Animais , Conexina 43/genética , Conexina 43/metabolismo , Feminino , Tamoxifeno/farmacologia , Galinhas/metabolismo , Ovário/metabolismo , Ovário/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Folículo Ovariano/efeitos dos fármacos , Folículo Ovariano/metabolismo , RNA Mensageiro/metabolismo , RNA Mensageiro/genéticaRESUMO
Spinal cord injury (SCI) is a severe condition with an extremely high disability rate. It is mainly manifested as the loss of motor, sensory and autonomic nerve functions below the injury site. High-frequency transcranial magnetic stimulation, a recently developed neuromodulation method, can increase motor function in mice with spinal cord injury. This study aimed to explore the possible mechanism by which transcranial magnetic stimulation (TMS) restores motor function after SCI. A complete T8 transection model of the spinal cord was established in mice, and the mice were treated daily with 15 Hz high-frequency transcranial magnetic stimulation. The BMS was used to evaluate the motor function of the mice after SCI. Western blotting and immunofluorescence were used to detect the expression of Connexin43 (CX43) and autophagy-related proteins in vivo and in vitro, and correlation analysis was performed to study the relationships among autophagy, CX43 and motor function recovery after SCI in mice. Western blotting was used to observe the effect of magnetic stimulation on the expression of mTOR pathway members. In the control group, the expression of CX43 was significantly decreased, and the expression of microtubule-associated protein 1 A/1b light chain 3 (LC3II) and P62 was significantly increased after 4 weeks of spinal cord transection. After high-frequency magnetic stimulation, the level of CX43 decreased, and the levels of LC3II and P62 increased in primary astrocytes. The BMS of the magnetic stimulation group was greater than that of the control group. High-frequency magnetic stimulation can inhibit the expression of CX43, which negatively regulates autophagic flux. HF-rTMS increased the expression levels of mTOR, p-mTOR and p-S6. Our experiments showed that rTMS can restore hindlimb motor function in mice after spinal cord injury via regulation of the Cx43-autophagy loop and activation of the mTOR signalling pathway.
Assuntos
Autofagia , Conexina 43 , Recuperação de Função Fisiológica , Traumatismos da Medula Espinal , Estimulação Magnética Transcraniana , Animais , Estimulação Magnética Transcraniana/métodos , Traumatismos da Medula Espinal/metabolismo , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/terapia , Recuperação de Função Fisiológica/fisiologia , Conexina 43/metabolismo , Autofagia/fisiologia , Camundongos , Serina-Treonina Quinases TOR/metabolismo , Camundongos Endogâmicos C57BL , Atividade Motora/fisiologia , Modelos Animais de Doenças , Masculino , FemininoRESUMO
In order to investigate the effectiveness and safety of miR-23b-3p in anti-seizure activity and to elucidate the regulatory relationship between miR-23b-3p and Cx43 in the nervous system, we have established a lithium chloride-pilocarpine (PILO) status epilepticus (SE) model. Rats were randomly divided into the following groups: seizure control (PILO), valproate sodium (VPA+PILO), recombinant miR-23b-3p overexpression (miR+PILO), miR-23b-3p sponges (Sponges+PILO), and scramble sequence negative control (Scramble+PILO) (n = 6/group). After experiments, we got the following results. In the acute phase, the time required for rats to reach stage IV after PILO injection was significantly longer in VPA+PILO and miR+PILO. In the chronic phase after SE, the frequency of spontaneous recurrent seizures (SRSs) in VPA+PILO and miR+PILO was significantly reduced. At 10 min before seizure cessation, the average energy expression of fast ripples (FRs) in VPA+PILO and miR+PILO was significantly lower than in PILO. After 28 days of seizure, Cx43 expression in PILO was significantly increased, and Beclin1expression in all groups was significantly increased. After 28 days of SE,the number of synapses in the CA1 region of the hippocampus was significantly higher in the VPA+PILO and miR+PILO groups compared to that in the PILO group. After 28 days of SE ,hippocampal necrotic cells in the CA3 region were significantly lower in the VPA+PILO and miR+PILO groups compared to those in the PILO group. There were no significant differences in biochemical indicators among the experimental group rats 28 days after SE compared to the seizure control group. Based on the previous facts, we can reach the conclusion that MiR-23b-3p targets and blocks the expression of hippocampal Cx43 which can reduce the formation of pathological FRs, thereby alleviating the severity of seizures, improving seizure-induced brain damage.
Assuntos
Conexina 43 , Hipocampo , MicroRNAs , Ratos Sprague-Dawley , Estado Epiléptico , Animais , Masculino , Ratos , Lesões Encefálicas/metabolismo , Conexina 43/metabolismo , Conexina 43/genética , Modelos Animais de Doenças , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , MicroRNAs/metabolismo , MicroRNAs/genética , Pilocarpina/toxicidade , Convulsões/metabolismo , Convulsões/induzido quimicamente , Estado Epiléptico/induzido quimicamente , Estado Epiléptico/metabolismoRESUMO
BACKGROUND: Accompanied by activation of the NOD-like receptor protein 3 (NLRP3) inflammasome, aberrant connexin 43 (Cx43) hemichannel-mediated ATP release is situated upstream of inflammasome assembly and inflammation and contributes to multiple secondary complications of diabetes and associated cardiometabolic comorbidities. Evidence suggests there may be a link between Cx43 hemichannel activity and inflammation in the diabetic kidney. The consequences of blocking tubular Cx43 hemichannel-mediated ATP release in priming/activation of the NLRP3 inflammasome in a model of diabetic kidney disease (DKD) was investigated. We examined downstream markers of inflammation and the proinflammatory and chemoattractant role of the tubular secretome on macrophage recruitment and activation. METHODS: Analysis of human transcriptomic data from the Nephroseq repository correlated gene expression to renal function in DKD. Primary human renal proximal tubule epithelial cells (RPTECs) and monocyte-derived macrophages (MDMs) were cultured in high glucose and inflammatory cytokines as a model of DKD to assess Cx43 hemichannel activity, NLRP3 inflammasome activation and epithelial-to-macrophage paracrine-mediated crosstalk. Tonabersat assessed a role for Cx43 hemichannels. RESULTS: Transcriptomic analysis from renal biopsies of patients with DKD showed that increased Cx43 and NLRP3 expression correlated with declining glomerular filtration rate (GFR) and increased proteinuria. In vitro, Tonabersat blocked glucose/cytokine-dependant increases in Cx43 hemichannel-mediated ATP release and reduced expression of inflammatory markers and NLRP3 inflammasome activation in RPTECs. We observed a reciprocal relationship in which NLRP3 activity exacerbated increased Cx43 expression and hemichannel-mediated ATP release, events driven by nuclear factor kappa-B (NFκB)-mediated priming and Cx43 hemichannel opening, changes blocked by Tonabersat. Conditioned media (CM) from RPTECs treated with high glucose/cytokines increased expression of inflammatory markers in MDMs, an effect reduced when macrophages were pre-treated with Tonabersat. Co-culture using conditioned media from Tonabersat-treated RPTECs dampened macrophage inflammatory marker expression and reduced macrophage migration. CONCLUSION: Using a model of DKD, we report for the first time that high glucose and inflammatory cytokines trigger aberrant Cx43 hemichannel activity, events that instigate NLRP3-induced inflammation in RPTECs and epithelial-to-macrophage crosstalk. Recapitulating observations previously reported in diabetic retinopathy, these data suggest that Cx43 hemichannel blockers (i.e., Tonabersat) may dampen multi-system damage observed in secondary complications of diabetes.
Assuntos
Nefropatias Diabéticas , Inflamassomos , Macrófagos , Proteína 3 que Contém Domínio de Pirina da Família NLR , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/genética , Nefropatias Diabéticas/metabolismo , Nefropatias Diabéticas/patologia , Humanos , Macrófagos/metabolismo , Macrófagos/efeitos dos fármacos , Inflamassomos/metabolismo , Conexina 43/metabolismo , Conexina 43/genética , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Trifosfato de Adenosina/metabolismo , Túbulos Renais Proximais/metabolismo , Túbulos Renais Proximais/patologiaRESUMO
Connexins (Cxs) are transmembrane proteins that assemble into gap junction channels (GJCs) and hemichannels (HCs). Previous researches support the involvement of Rho GTPases and actin microfilaments in the trafficking of Cxs, formation of GJCs plaques, and regulation of channel activity. Nonetheless, it remains uncertain whether distinct types of Cxs HCs and GJCs respond differently to Rho GTPases or changes in actin polymerization/depolymerization dynamics. Our investigation revealed that inhibiting RhoA, a small GTPase that controls actin polymerization, or disrupting actin microfilaments with cytochalasin B (Cyto-B), resulted in reduced GJCs plaque size at appositional membranes and increased transport of HCs to non-appositional plasma membrane regions. Notably, these effects were consistent across different Cx types, since Cx26 and Cx43 exhibited similar responses, despite having distinct trafficking routes to the plasma membrane. Functional assessments showed that RhoA inhibition and actin depolymerization decreased the activity of Cx43 GJCs while significantly increasing HC activity. However, the functional status of GJCs and HCs composed of Cx26 remained unaffected. These results support the hypothesis that RhoA, through its control of the actin cytoskeleton, facilitates the transport of HCs to appositional cell membranes for GJCs formation while simultaneously limiting the positioning of free HCs at non-appositional cell membranes, independently of Cx type. This dynamic regulation promotes intercellular communications and reduces non-selective plasma membrane permeability through a Cx-type dependent mechanism, whereby the activity of Cx43 HCs and GJCs are differentially affected but Cx26 channels remain unchanged.
Assuntos
Citoesqueleto de Actina , Conexina 26 , Conexina 43 , Junções Comunicantes , Proteína rhoA de Ligação ao GTP , Citoesqueleto de Actina/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismo , Junções Comunicantes/metabolismo , Conexina 43/metabolismo , Conexina 26/metabolismo , Humanos , Animais , Membrana Celular/metabolismo , Actinas/metabolismoRESUMO
BACKGROUND: In the fight against GBM, drug repurposing emerges as a viable and time-saving approach to explore new treatment options. Chlorpromazine, an old antipsychotic medication, has recently arisen as a promising candidate for repositioning in GBM therapy in addition to temozolomide, the first-line standard of care. We previously demonstrated the antitumor efficacy of chlorpromazine and its synergistic effects with temozolomide in suppressing GBM cell malignant features in vitro. This prompted us to accomplish a Phase II clinical trial to evaluate the efficacy and safety of adding chlorpromazine to temozolomide in GBM patients with unmethylated MGMT gene promoter. In this in vitro study, we investigate the potential role of chlorpromazine in overcoming temozolomide resistance. METHODS: In our experimental set, we analyzed Connexin-43 expression at both the transcriptional and protein levels in control- and chlorpromazine-treated GBM cells. DNA damage and subsequent repair were assessed by immunofluorescence of γ-H2AX and Reverse-Phase Protein microArrays in chlorpromazine treated GBM cell lines. To elucidate the relationship between DNA repair systems and chemoresistance, we analyzed a signature of DNA repair genes in GBM cells after treatment with chlorpromazine, temozolomide and Connexin-43 downregulation. RESULTS: Chlorpromazine treatment significantly downregulated connexin-43 expression in GBM cells, consequently compromising connexin-dependent cellular resilience, and ultimately contributing to cell death. In line with this, we observed concordant post-translational modifications of molecular determinants involved in DNA damage and repair pathways. Our evaluation of DNA repair genes revealed that temozolomide elicited an increase, while chlorpromazine, as well as connexin-43 silencing, a decrease in DNA repair gene expression in GBM cells. CONCLUSIONS: Chlorpromazine potentiates the cytotoxic effects of the alkylating agent temozolomide through a mechanism involving downregulation of Cx43 expression and disruption of the cell cycle arrest essential for DNA repair processes. This finding suggests that chlorpromazine may be a potential therapeutic strategy to overcome TMZ resistance in GBM cells by inhibiting their DNA repair mechanisms.
Assuntos
Clorpromazina , Conexina 43 , Reparo do DNA , Resistencia a Medicamentos Antineoplásicos , Glioblastoma , Temozolomida , Clorpromazina/farmacologia , Clorpromazina/uso terapêutico , Humanos , Temozolomida/farmacologia , Temozolomida/uso terapêutico , Glioblastoma/tratamento farmacológico , Glioblastoma/patologia , Glioblastoma/genética , Reparo do DNA/efeitos dos fármacos , Conexina 43/metabolismo , Conexina 43/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Sinergismo Farmacológico , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/patologia , Neoplasias Encefálicas/genéticaRESUMO
BACKGROUND AND AIMS: Sodium-glucose cotransporter 2 inhibitors (SGLT2is) can ameliorate arrhythmias; however, the mechanisms underlying their antiarrhythmic effect remain unclear. Therefore, we aimed to test the hypothesis that the SGLT2i empagliflozin (EMPA) ameliorates ventricular arrhythmias caused by myocardial infarction (MI) by inhibiting sympathetic remodeling. METHODS: Male nondiabetic Sprague-Dawley rats were divided into Sham ( n â=â10), MI ( n â=â13), low-EMPA (10âmg/kg/day; n â=â13), and high-EMPA (30âmg/kg/day; n â=â13) groups. Except for the Sham group, MI models were established by ligation of the left anterior descending coronary artery. After 4 weeks, the hearts were removed. Echocardiography, electrical stimulation, hematoxylin-eosin staining and Masson's staining, Western blotting, immunohistochemistry (IHC), and ELISA were performed. RESULTS: Except for left ventricular posterior wall thickness (LVPWT), EMPA treatment significantly ameliorated the left ventricular anterior wall thickness (LVAWT), interventricular septum thickness (IVST), left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), and left ventricular ejection fraction (LVEF) in MI rats; there was no statistical difference between the low-EMPA and high-EMPA groups. The threshold for ventricular fibrillation induction and myocardial fibrosis was significantly ameliorated in EMPA-treated rats, and there was no statistical difference between the high-EMPA and low-EMPA groups. EMPA decreased the expression of nerve growth factor (NGF), tyrosine kinase receptor A (TrkA), tyrosine hydroxylase, and growth-associated protein 43 (GAP43) in the left ventricular infarction margin myocardium of MI rats, especially in the high-EMPA group, with a statistically significant difference between the high-EMPA and low-EMPA groups. High-EMPA significantly decreased noradrenaline (NE) levels in the blood of MI rats; however, there was no statistical difference between the low-EMPA and MI groups. CONCLUSION: EMPA ameliorated the occurrence of ventricular arrhythmias in MI rats, which may be related to a reduction in sympathetic activity, inhibition of the NGF/TrkA pathway, inhibition of sympathetic remodeling, and improvement in cardiac function and cardiac structural remodeling.
Assuntos
Compostos Benzidrílicos , Modelos Animais de Doenças , Glucosídeos , Infarto do Miocárdio , Fator de Crescimento Neural , Ratos Sprague-Dawley , Transdução de Sinais , Inibidores do Transportador 2 de Sódio-Glicose , Sistema Nervoso Simpático , Remodelação Ventricular , Animais , Masculino , Compostos Benzidrílicos/farmacologia , Glucosídeos/farmacologia , Fator de Crescimento Neural/metabolismo , Infarto do Miocárdio/fisiopatologia , Infarto do Miocárdio/tratamento farmacológico , Infarto do Miocárdio/complicações , Infarto do Miocárdio/patologia , Infarto do Miocárdio/metabolismo , Sistema Nervoso Simpático/efeitos dos fármacos , Sistema Nervoso Simpático/fisiopatologia , Inibidores do Transportador 2 de Sódio-Glicose/farmacologia , Remodelação Ventricular/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Receptor trkA/metabolismo , Receptor trkA/antagonistas & inibidores , Proteína GAP-43/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos , Arritmias Cardíacas/etiologia , Arritmias Cardíacas/prevenção & controle , Arritmias Cardíacas/fisiopatologia , Arritmias Cardíacas/tratamento farmacológico , Ratos , Antiarrítmicos/farmacologia , Conexina 43RESUMO
OBJECTIVES: This study aims to investigate the role of gap junction mediated by connexin 43 (Cx43) in renal injury induced by periodontitis in rats. METHODS: Twelve SPF-grade Wistar male rats were divided into a control group and a periodontitis group by using a completely random number table method, with six rats in each group. The control group rats were not treated, while the periodontitis group rats were subjected to wire ligation of the neck of their bilateral maxillary first molars to construct a periodontitis model. After 8 weeks of modeling, the rats were examined for clinical indicators of the periodontium. micro-CT scanning of the maxilla reconstructed its 3D structure and analyzed the absorption of alveolar bone. Histopathological changes in periodontal and renal tissues were detected. MitoSOX red reagent was used to determine reactive oxygen species (ROS) content in renal tissues. A biochemical reagent kit was used to detect serum oxidative stress biomarkers. Real-time fluorescent quantitative-polymerase chain reaction (qRT-PCR) was employed to determine Cx43, nuclear factor kappa-B (NF-κB) , interleukin (IL)-1ß, IL-6, BCL2-Associated X (Bax), B-lymphomatoma-2 gene (Bcl-2), and Caspase-3 mRNA were determined. Western blot analysis was used to detect Cx43, NF-κB, IL-1ß, Bax, Bcl-2 and Caspase-3 protein. RESULTS: micro-CT 3D reconstruction showed significant bone resorption of the first molar alveolar bone in the periodontitis group rats and decreased height of the alveolar ridge. The distance from the enamel cementum boundary to the top of the alveolar ridge in the periodontitis group was significantly higher than that inthe control group. The histopathological results showed a large number of inflammatory cells that infiltrated the periodontal tissue of the periodontitis group, and the alveolar bone was significantly absorbed. Rats in the periodontitis group also exhibited mild thickening of the glomerular basement membrane, dilation of the Bowman's capsule, and destruction of the brush-like edge of the renal tubules in the renal tissue. The MitoSOX red staining results showed a significant increase in ROS content in the renal tissue of the periodontitis group. The biochemical test results showed that the levels of superoxide dismutase and glutathione in the serum of rats with periodontitis decreased, while that of malondialdehyde increased. The results of qRT-PCR and Western blot showed that the expression levels of Cx43, IL-1ß, IL-6, Bax, Caspase-3 mRNA and Cx43, IL-1ß, NF-κB, Bax, Caspase-3 proteins in the periodontitis group significantly increased compared with those in the control group, while the expression levels of Bcl-2 mRNA and protein decreased. CONCLUSIONS: Periodontitis may activate NF-κB signaling molecules by upregulating the expression of Cx43 in rat kidney tissues, leading to increased levels of inflammation and apoptosis and ultimately inducing kidney injury.
Assuntos
Conexina 43 , Modelos Animais de Doenças , Interleucina-6 , Estresse Oxidativo , Periodontite , Ratos Wistar , Animais , Masculino , Ratos , Perda do Osso Alveolar/metabolismo , Apoptose , Proteína X Associada a bcl-2/metabolismo , Caspase 3/metabolismo , Conexina 43/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Rim/metabolismo , Rim/patologia , NF-kappa B/metabolismo , Periodontite/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Microtomografia por Raio-XRESUMO
BACKGROUND AND PURPOSE: Icariin, an 8-prenylated flavonoid glycoside, is an anabolic agent that could exert rapid estrogenic actions via ligand-independent activation of estrogen receptor alpha (ERα) in osteoblastic cells to promote osteogenesis. However, relatively little is known about its direct cellular target, its protective effects, and cell adhesion activities in bone marrow stromal cells (BMSCs) against microgravity. In the present study, the effects of icariin on osteogenesis and cell adhesion under microgravity were examined with the involvement of integrin receptor α5ß1, connexin 43, and CAMs. STUDY DESIGN AND METHODS: Icariin was orally administered to 6-month-old ovariectomized (OVX) Sprague-Dawley (SD) rats for 3 months through daily intake of phytoestrogen-free rodent diets containing icariin at 2 different dosages (50 and 500 ppm). BMSCs were harvested for experiments and RNA-sequencing analysis to examine the mechanism of action of icariin and its direct cellular target in stimulating osteogenesis. RESULTS: The results revealed that icariin induced the expression of cell adhesion molecules (CAMs) and protected against microgravity-induced disruption of actin cytoskeleton and the loss of osteogenic activities in BMSCs through the activation of connexin-43 (Cx43) and Ras homolog family member A (RhoA) and Rac family small GTPase 1 (Rac1)-mediated signaling pathways. Computerized molecular docking techniques and the competitive solid-phase binding ELISA assay confirmed that icariin could be a direct ligand of integrin alpha 5 beta 1 (α5ß1), and it could also increase the protein expression of integrin α5ß1 for mechanosensing. CONCLUSION: Our findings suggest that icariin could directly activate cell adhesion signaling by binding to integrin α5ß1, which opens up new avenues for the development of integrin α5ß1 ligand as an agent to protect against unloading-induced bone loss.
Assuntos
Adesão Celular , Conexina 43 , Flavonoides , Integrina alfa5beta1 , Células-Tronco Mesenquimais , Osteogênese , Ratos Sprague-Dawley , Animais , Flavonoides/farmacologia , Osteogênese/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Integrina alfa5beta1/metabolismo , Células-Tronco Mesenquimais/efeitos dos fármacos , Feminino , Conexina 43/metabolismo , Ratos , OvariectomiaRESUMO
A robust and efficient cellular response to lysosomal membrane damage prevents leakage from the lysosome lumen into the cytoplasm. This response is understood to happen through either lysosomal membrane repair or lysophagy. Here we report exocytosis as a third response mechanism to lysosomal damage, which is further potentiated when membrane repair or lysosomal degradation mechanisms are impaired. We show that Connexin43 (Cx43), a protein canonically associated with gap junctions, is recruited from the plasma membrane to damaged lysosomes, promoting their secretion and accelerating cell recovery. The effects of Cx43 on lysosome exocytosis are mediated by a reorganization of the actin cytoskeleton that increases plasma membrane fluidity and decreases cell stiffness. Furthermore, we demonstrate that Cx43 interacts with the actin nucleator Arp2, the activity of which was shown to be necessary for Cx43-mediated actin rearrangement and lysosomal exocytosis following damage. These results define a novel mechanism of lysosomal quality control whereby Cx43-mediated actin remodelling potentiates the secretion of damaged lysosomes.