RESUMEN
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.
Asunto(s)
Conexina 43 , Obstrucción del Cuello de la Vejiga Urinaria , Vejiga Urinaria , Animales , Obstrucción del Cuello de la Vejiga Urinaria/metabolismo , Obstrucción del Cuello de la Vejiga Urinaria/patología , Femenino , Ratas , Vejiga Urinaria/metabolismo , Vejiga Urinaria/fisiopatología , Vejiga Urinaria/patología , Conexina 43/metabolismo , Trasplante de Células Madre/métodos , Transducción de Señal , Ratas Sprague-Dawley , Proteína Smad2/metabolismo , Modelos Animales de Enfermedad , Uniones Comunicantes/metabolismo , Colágeno/metabolismoRESUMEN
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.
Asunto(s)
Condrocitos , Conexina 43 , Interleucina-1beta , Osteoartritis , Osteoblastos , Factor de Necrosis Tumoral alfa , Humanos , Conexina 43/metabolismo , Conexina 43/genética , Condrocitos/metabolismo , Osteoblastos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Factor de Necrosis Tumoral alfa/farmacología , Interleucina-1beta/metabolismo , Interleucina-1beta/farmacología , Osteoartritis/metabolismo , Osteoartritis/patología , Osteoartritis/genética , Líquido Sinovial/metabolismo , Cartílago Articular/metabolismo , Cartílago Articular/patología , Células Cultivadas , Anciano , Persona de Mediana Edad , Inflamación/metabolismo , Inflamación/genética , Inflamación/patología , Cartílago/metabolismo , Cartílago/patología , Artropatías/metabolismo , Artropatías/patología , Artropatías/genéticaRESUMEN
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.
Asunto(s)
Búfalos , Conexina 43 , Técnicas de Maduración In Vitro de los Oocitos , Oocitos , Oxazinas , Estrés Oxidativo , Animales , Oocitos/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Femenino , Técnicas de Maduración In Vitro de los Oocitos/veterinaria , Especies Reactivas de Oxígeno/metabolismo , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa/genética , Glutatión Peroxidasa/metabolismo , Glutatión Peroxidasa/genética , Fertilización In Vitro/veterinaria , Técnicas de Cultivo de Embriones/veterinaria , Glutatión Peroxidasa GPX1 , Desarrollo Embrionario/fisiología , Coloración y Etiquetado , Antioxidantes/metabolismoRESUMEN
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.
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Cardiomiopatía Chagásica , Conexina 43 , Ratones Endogámicos C57BL , Miocitos Cardíacos , Conexina 43/metabolismo , Conexina 43/genética , Animales , Cardiomiopatía Chagásica/metabolismo , Cardiomiopatía Chagásica/patología , Cardiomiopatía Chagásica/inmunología , Cardiomiopatía Chagásica/parasitología , Humanos , Ratones , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/parasitología , Miocitos Cardíacos/patología , Inflamación/metabolismo , Fosforilación , Masculino , Enfermedad Crónica , Trypanosoma cruzi , Modelos Animales de Enfermedad , Línea Celular , Citocinas/metabolismo , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/parasitología , Arritmias Cardíacas/inmunología , FemeninoRESUMEN
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.
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Fibrilación Atrial , Fibrosis , Atrios Cardíacos , Miocitos Cardíacos , beta Catenina , Humanos , Fibrilación Atrial/patología , Fibrilación Atrial/metabolismo , beta Catenina/metabolismo , Animales , Atrios Cardíacos/metabolismo , Atrios Cardíacos/patología , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Masculino , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Cadherinas/metabolismo , Uniones Comunicantes/metabolismo , Persona de Mediana Edad , Ratones , Femenino , Conexina 43/metabolismo , Ratones Endogámicos C57BL , AncianoRESUMEN
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.
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Conexina 43 , Hipocampo , MicroARNs , Ratas Sprague-Dawley , Estado Epiléptico , Animales , Masculino , Ratas , Lesiones Encefálicas/metabolismo , Conexina 43/metabolismo , Conexina 43/genética , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Hipocampo/efectos de los fármacos , MicroARNs/metabolismo , MicroARNs/genética , Pilocarpina/toxicidad , Convulsiones/metabolismo , Convulsiones/inducido químicamente , Estado Epiléptico/inducido químicamente , Estado Epiléptico/metabolismoRESUMEN
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.
Asunto(s)
Compuestos de Bencidrilo , Modelos Animales de Enfermedad , Glucósidos , Infarto del Miocardio , Factor de Crecimiento Nervioso , Ratas Sprague-Dawley , Transducción de Señal , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Sistema Nervioso Simpático , Remodelación Ventricular , Animales , Masculino , Compuestos de Bencidrilo/farmacología , Glucósidos/farmacología , Factor de Crecimiento Nervioso/metabolismo , Infarto del Miocardio/fisiopatología , Infarto del Miocardio/tratamiento farmacológico , Infarto del Miocardio/complicaciones , Infarto del Miocardio/patología , Infarto del Miocardio/metabolismo , Sistema Nervioso Simpático/efectos de los fármacos , Sistema Nervioso Simpático/fisiopatología , Inhibidores del Cotransportador de Sodio-Glucosa 2/farmacología , Remodelación Ventricular/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Receptor trkA/metabolismo , Receptor trkA/antagonistas & inhibidores , Proteína GAP-43/metabolismo , Función Ventricular Izquierda/efectos de los fármacos , Arritmias Cardíacas/etiología , Arritmias Cardíacas/prevención & control , Arritmias Cardíacas/fisiopatología , Arritmias Cardíacas/tratamiento farmacológico , Ratas , Antiarrítmicos/farmacología , Conexina 43RESUMEN
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.
Asunto(s)
Conexina 43 , Modelos Animales de Enfermedad , Interleucina-6 , Estrés Oxidativo , Periodontitis , Ratas Wistar , Animales , Ratas , Periodontitis/metabolismo , Masculino , Conexina 43/metabolismo , Interleucina-6/metabolismo , FN-kappa B/metabolismo , Interleucina-1beta/metabolismo , Caspasa 3/metabolismo , Riñón/metabolismo , Riñón/patología , Microtomografía por Rayos X , Especies Reactivas de Oxígeno/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Pérdida de Hueso Alveolar/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , ApoptosisRESUMEN
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.
Asunto(s)
Autofagia , Conexina 43 , Recuperación de la Función , Traumatismos de la Médula Espinal , Estimulación Magnética Transcraneal , Animales , Estimulación Magnética Transcraneal/métodos , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/fisiopatología , Traumatismos de la Médula Espinal/terapia , Recuperación de la Función/fisiología , Conexina 43/metabolismo , Autofagia/fisiología , Ratones , Serina-Treonina Quinasas TOR/metabolismo , Ratones Endogámicos C57BL , Actividad Motora/fisiología , Modelos Animales de Enfermedad , Masculino , FemeninoRESUMEN
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.
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Citoesqueleto de Actina , Conexina 26 , Conexina 43 , Uniones Comunicantes , Proteína de Unión al GTP rhoA , Citoesqueleto de Actina/metabolismo , Proteína de Unión al GTP rhoA/metabolismo , Uniones Comunicantes/metabolismo , Conexina 43/metabolismo , Conexina 26/metabolismo , Humanos , Animales , Membrana Celular/metabolismo , Actinas/metabolismoRESUMEN
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.
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Nefropatías Diabéticas , Inflamasomas , Macrófagos , Proteína con Dominio Pirina 3 de la Familia NLR , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Nefropatías Diabéticas/metabolismo , Nefropatías Diabéticas/patología , Humanos , Macrófagos/metabolismo , Macrófagos/efectos de los fármacos , Inflamasomas/metabolismo , Conexina 43/metabolismo , Conexina 43/genética , Células Epiteliales/metabolismo , Células Epiteliales/patología , Adenosina Trifosfato/metabolismo , Túbulos Renales Proximales/metabolismo , Túbulos Renales Proximales/patologíaRESUMEN
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.
Asunto(s)
Clorpromazina , Conexina 43 , Reparación del ADN , Resistencia a Antineoplásicos , Glioblastoma , Temozolomida , Clorpromazina/farmacología , Clorpromazina/uso terapéutico , Humanos , Temozolomida/farmacología , Temozolomida/uso terapéutico , Glioblastoma/tratamiento farmacológico , Glioblastoma/patología , Glioblastoma/genética , Reparación del ADN/efectos de los fármacos , Conexina 43/metabolismo , Conexina 43/genética , Línea Celular Tumoral , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Sinergismo Farmacológico , Neoplasias Encefálicas/tratamiento farmacológico , Neoplasias Encefálicas/patología , Neoplasias Encefálicas/genéticaRESUMEN
Renal cell carcinoma (RCC) bone metastatis progression is driven by crosstalk between tumor cells and the bone microenvironment, which includes osteoblasts, osteoclasts, and osteocytes. RCC bone metastases (RCCBM) are predominantly osteolytic and resistant to antiresorptive therapy. The molecular mechanisms underlying pathologic osteolysis and disruption of bone homeostasis remain incompletely understood. We previously reported that BIGH3/TGFBI (transforming growth factor-beta-induced protein ig-h3, shortened to BIGH3 henceforth) secreted by colonizing RCC cells drives osteolysis by inhibiting osteoblast differentiation, impairing healing of osteolytic lesions, which is reversible with osteoanabolic agents. Here, we report that BIGH3 induces osteocyte apoptosis in both human RCCBM tissue specimens and in a preclinical mouse model. We also demonstrate that BIGH3 reduces Cx43 expression, blocking gap junction (GJ) function and osteocyte network communication. BIGH3-mediated GJ inhibition is blocked by the lysosomal inhibitor hydroxychloroquine (HCQ), but not osteoanabolic agents. Our results broaden the understanding of pathologic osteolysis in RCCBM and indicate that targeting the BIGH3 mechanism could be a combinational strategy for the treatment of RCCBM-induced bone disease that overcomes the limited efficacy of antiresorptives that target osteoclasts.
Asunto(s)
Apoptosis , Neoplasias Óseas , Carcinoma de Células Renales , Proteínas de la Matriz Extracelular , Uniones Comunicantes , Neoplasias Renales , Osteocitos , Osteocitos/metabolismo , Osteocitos/patología , Humanos , Animales , Neoplasias Óseas/secundario , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Neoplasias Óseas/tratamiento farmacológico , Carcinoma de Células Renales/patología , Carcinoma de Células Renales/metabolismo , Carcinoma de Células Renales/tratamiento farmacológico , Carcinoma de Células Renales/secundario , Apoptosis/efectos de los fármacos , Neoplasias Renales/patología , Neoplasias Renales/metabolismo , Neoplasias Renales/tratamiento farmacológico , Uniones Comunicantes/metabolismo , Uniones Comunicantes/patología , Proteínas de la Matriz Extracelular/metabolismo , Ratones , Progresión de la Enfermedad , Conexina 43/metabolismo , Línea Celular Tumoral , Factor de Crecimiento Transformador beta/metabolismo , Osteólisis/patología , Osteólisis/metabolismo , FemeninoRESUMEN
The increasing number of patients with depressive disorder is a serious socioeconomic problem worldwide. Although several therapeutic agents have been developed and used clinically, their effectiveness is insufficient and thus discovery of novel therapeutic targets is desired. Here, focusing on dysregulation of neuronal purinergic signaling in depressive-like behavior, we examined the expression profiles of ATP channels and ectonucleotidases in astrocytes of cerebral cortex and hippocampus of chronic social defeat stress (CSDS)-susceptible BALB/c mice. Mice were exposed to 10-d CSDS, and their astrocytes were obtained using a commercially available kit based on magnetic activated cell sorting technology. In astrocytes derived from cerebral cortex of CSDS-susceptible mice, the expression levels of mRNAs for connexin 43, P2X7 receptors and maxi anion channels were increased, those for connexin 43 and P2X7 receptors being inversely correlated with mouse sociability, and the expression of mRNAs for ecto-nucleoside triphosphate diphosphohydrase 2 and ecto-5'nucleotidase was decreased and increased, respectively. On the other hand, the alteration profiles of ATP channels and ectonucleotidases in hippocampal astrocytes of CSDS-susceptible mice were different from in the case of cortical astrocytes, and there was no significant correlation between expression levels of their mRNAs and mouse sociability. These findings imply that increased expression of ATP channels in cerebral cortex might be involved in the development of reduced sociability in CSDS-subjected BALB/c mice. Together with recent findings, it is suggested that ATP channels expressed by cortical astrocytes might be potential therapeutic targets for depressive disorder.
Asunto(s)
Astrocitos , Corteza Cerebral , Hipocampo , Ratones Endogámicos BALB C , Derrota Social , Estrés Psicológico , Animales , Astrocitos/metabolismo , Corteza Cerebral/metabolismo , Hipocampo/metabolismo , Estrés Psicológico/metabolismo , Masculino , Ratones , Receptores Purinérgicos P2X7/metabolismo , Receptores Purinérgicos P2X7/genética , Conexina 43/metabolismo , Conexina 43/genética , 5'-Nucleotidasa/metabolismo , 5'-Nucleotidasa/genética , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfatasas/genética , ARN Mensajero/metabolismo , ARN Mensajero/genéticaRESUMEN
Differences in structural and functional properties between oocytes and cumulus cells (CCs) may cause low vitrification efficiency for cumulus-oocyte complexes (COCs). We have suggested that the disconnection of CCs and oocytes in order to further cryopreservation in various ways will positively affect the viability after thawing, while further co-culture in vitro will contribute to the restoration of lost intercellular gap junctions. This study aimed to determine the optimal method of cryopreservation of the suspension of CCs to mature GV oocytes in vitro and to determine the level of mRNA expression of the genes (GJA1, GJA4; BCL2, BAX) and gene-specific epigenetic marks (DNMT3A) after cryopreservation and in vitro maturation (IVM) in various culture systems. We have shown that the slow freezing of CCs in microstraws preserved the largest number of viable cells with intact DNA compared with the methods of vitrification and slow freezing in microdroplets. Cryopreservation caused the upregulation of the genes Cx37 and Cx43 in the oocytes to restore gap junctions between cells. In conclusion, the presence of CCs in the co-culture system during IVM of oocytes played an important role in the regulation of the expression of the intercellular proteins Cx37 and Cx43, apoptotic changes, and oocyte methylation. Slow freezing in microstraws was considered to be an optimal method for cryopreservation of CCs.
Asunto(s)
Criopreservación , Células del Cúmulo , Uniones Comunicantes , Oocitos , Animales , Oocitos/metabolismo , Oocitos/citología , Criopreservación/métodos , Uniones Comunicantes/metabolismo , Células del Cúmulo/metabolismo , Células del Cúmulo/citología , Bovinos , Femenino , Conexina 43/metabolismo , Conexina 43/genética , Conexinas/metabolismo , Conexinas/genética , Vitrificación , Técnicas de Cocultivo/métodos , Supervivencia Celular , Técnicas de Maduración In Vitro de los Oocitos/métodosRESUMEN
Noncoding RNAs (ncRNAs) are a class of nucleotide sequences that cannot be translated into peptides. ncRNAs can function post-transcriptionally by splicing complementary sequences of mRNAs or other ncRNAs or by directly engaging in protein interactions. Over the past few decades, the pervasiveness of ncRNAs in cell physiology and their pivotal roles in various diseases have been identified. One target regulated by ncRNAs is connexin (Cx), a protein that forms gap junctions and hemichannels and facilitates intercellular molecule exchange. The aberrant expression and misdistribution of connexins have been implicated in central nervous system diseases, cardiovascular diseases, bone diseases, and cancer. Current databases and technologies have enabled researchers to identify the direct or indirect relationships between ncRNAs and connexins, thereby elucidating their correlation with diseases. In this review, we selected the literature published in the past five years concerning disorders regulated by ncRNAs via corresponding connexins. Among it, microRNAs that regulate the expression of Cx43 play a crucial role in disease development and are predominantly reviewed. The distinctive perspective of the ncRNA-Cx axis interprets pathology in an epigenetic manner and is expected to motivate research for the development of biomarkers and therapeutics.
Asunto(s)
Conexinas , ARN no Traducido , Humanos , ARN no Traducido/genética , ARN no Traducido/metabolismo , Animales , Conexinas/metabolismo , Conexinas/genética , MicroARNs/genética , MicroARNs/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/terapia , Regulación de la Expresión Génica , Enfermedades Cardiovasculares/genética , Enfermedades Cardiovasculares/metabolismo , Enfermedades Cardiovasculares/terapia , Uniones Comunicantes/metabolismo , Uniones Comunicantes/genética , Enfermedades del Sistema Nervioso Central/genética , Enfermedades del Sistema Nervioso Central/metabolismo , Enfermedades del Sistema Nervioso Central/terapiaRESUMEN
Air pollution (gases and particulate matter -PM) and child undernutrition are globally recognized stressors with significant consequences. PM and its components breach the respiratory alveolar-capillary barrier, entering the vasculature transporting not only harmful particles and its mediators but, altering vascular paracrine and autocrine functions. The aim of this study was to investigate the effects of Residual Oil Fly Ash (ROFA), on the vasculature of young animals with nutritional growth retardation (NGR). Weanling rats were fed a diet restricted 20% (NGR) compared to ad libitum intake (control-C) for 4 weeks. Rats were intranasally instilled with 1 mg/kg BW of ROFA. After 24h exposure, histological and immunohistochemical, biochemical and contractile response to NA/ACh were evaluated in aortas. ROFA induced changes in the tunica media of the aorta in all groups regarding thickness, muscular cells and expression of Connexin-43. ROFA increased TGF-ß1 and decreased eNOs levels and calcium channels in C and NGR animals. An increment in cytokines IL-6 and IL-10 was observed in C, with no changes in NGR. ROFA exposure altered the vascular contractile capacity. In conclusion, ROFA exposure could increase the risk for CVD through the alteration of vascular biochemical parameters, a possible step of the endothelial dysfunction.
Asunto(s)
Contaminación del Aire , Desnutrición , Animales , Ratas , Masculino , Desnutrición/fisiopatología , Desnutrición/complicaciones , Contaminación del Aire/efectos adversos , Óxido Nítrico Sintasa de Tipo III/metabolismo , Ceniza del Carbón/toxicidad , Ratas Wistar , Conexina 43/metabolismo , Material Particulado/toxicidad , Aorta/efectos de los fármacos , Factor de Crecimiento Transformador beta1/metabolismo , Contaminantes Atmosféricos/toxicidadRESUMEN
Neural precursor cells (NPCs) that persist in the postnatal/adult subventricular zone (SVZ) express connexins that form hemichannels and gap junctions. Gap junctional communication plays a role in NPC proliferation and differentiation during development, but its relevance on postnatal age remains to be elucidated. In this work we aimed to evaluate the effect of the blockade of gap junctional communication on proliferation and cell fate of NPCs obtained from the SVZ of postnatal rats. NPCs were isolated and expanded in culture as neurospheres. Electron microscopy revealed the existence of gap junctions among neurosphere cells. Treatment of cultures with octanol, a broad-spectrum gap junction blocker, or with Gap27, a specific blocker for gap junctions formed by connexin43, produced a significant decrease in bromodeoxyuridine incorporation. Octanol treatment also exerted a dose-dependent antiproliferative effect on glioblastoma cells. To analyze possible actions on NPC fate, cells were seeded in the absence of mitogens. Treatment with octanol led to an increase in the percentage of astrocytes and oligodendrocyte precursors, whereas the percentage of neurons remained unchanged. Gap27 treatment, in contrast, did not modify the differentiation pattern of SVZ NPCs. Our results indicate that general blockade of gap junctions with octanol induces significant effects on the behavior of postnatal SVZ NPCs, by reducing proliferation and promoting glial differentiation.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Uniones Comunicantes , Células-Madre Neurales , Neuroglía , Octanoles , Animales , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Células-Madre Neurales/citología , Proliferación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Ratas , Octanoles/farmacología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuroglía/citología , Células Cultivadas , Ventrículos Laterales/citología , Ventrículos Laterales/metabolismo , Ventrículos Laterales/efectos de los fármacos , Conexina 43/metabolismo , Ratas Wistar , Astrocitos/efectos de los fármacos , Astrocitos/metabolismo , Astrocitos/citología , Animales Recién Nacidos , HumanosRESUMEN
Osteoclast (OC) differentiation, vital for bone resorption, depends on osteoclast and precursor fusion. Osteoprotegerin (OPG) inhibits osteoclast differentiation. OPG's influence on fusion and mechanisms is unclear. Osteoclasts and precursors were treated with OPG alone or with ATP. OPG significantly reduced OC number, area and motility and ATP mitigated OPG's inhibition. However, OPG hardly affected the motility of precusors. OPG downregulated fusion-related molecules (CD44, CD47, DC-STAMP, ATP6V0D2) in osteoclasts, reducing only CD47 in precursors. OPG reduced Connexin43 phosphorylated forms (P1 and P2) in osteoclasts, affecting only P2 in precursors. OPG disrupted subcellular localization of CD44, CD47, DC-STAMP, ATP6V0D2, and Connexin43 in both cell types. Findings underscore OPG's multifaceted impact, inhibiting multinucleated osteoclast and mononuclear precursor fusion through distinct molecular mechanisms. Notably, ATP mitigates OPG's inhibitory effect, suggesting a potential regulatory role for the ATP signaling pathway. This study enhances understanding of intricate processes in osteoclast differentiation and fusion, offering insights into potential therapeutic targets for abnormal bone metabolism.
Asunto(s)
Adenosina Trifosfato , Diferenciación Celular , Osteoclastos , Osteoprotegerina , Osteoprotegerina/metabolismo , Osteoprotegerina/genética , Osteoclastos/metabolismo , Osteoclastos/citología , Animales , Adenosina Trifosfato/metabolismo , Ratones , Conexina 43/metabolismo , Conexina 43/genética , Fusión Celular , Antígeno CD47/metabolismo , Antígeno CD47/genética , Receptores de Hialuranos/metabolismo , Receptores de Hialuranos/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Resorción Ósea/metabolismo , Resorción Ósea/genética , Resorción Ósea/patología , Transducción de Señal , ATPasas de Translocación de Protón Vacuolares/metabolismo , ATPasas de Translocación de Protón Vacuolares/genética , Proteínas del Tejido NerviosoRESUMEN
BACKGROUND: Hydrogen (H2) has emerged as a potential therapeutic intervention for traumatic brain injury (TBI). However, the precise mechanism underlying H2's neuroprotective effects in TBI remain incompletely understood. METHODS: TBI mouse model was induced using the controlled cortical impact (CCI) method, and a cell model was established by exposing astrocytes to lipopolysaccharide (LPS). Cell viability was detected by CCK-8 kits. Cell apoptosis was measured by flow cytometry. ELISA was used to detect cytokine quantification. Protein and gene expression was detected by western blot and RT-PCR analysis. Co-immunoprecipitation (CO-IP) were employed for protein-protein interactions. Morris water maze test and rotarod test were applied for TBI mice. RESULTS: H2 treatment effectively inhibited the LPS-induced cell injury and cell apoptosis in astrocytes. NEDD4 expression was increased following H2 treatment coupled with enhanced mitophagy in LPS-treated astrocytes. Overexpression of NEDD4 and down-regulation of connexin 43 (CX43) mirrored the protective effects of H2 treatment in LPS-exposed astrocytes. NEDD4 interacts CX43 to regulates the ubiquitinated degradation of CX43. While overexpression of CX43 reversed the protective effects of H2 treatment in LPS-exposed astrocytes. In addition, H2 treatment significantly alleviated brain injury in TBI mouse model. CONCLUSION: H2 promoted NEDD4-CX43 mediated mitophagy to protect brain injury induced by TBI, highlighting a novel pathway underlying the therapeutic effects of H2 in TBI.