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Background: Myocardial inflammation and apoptosis induced by cirrhosis are among the primary mechanisms of cirrhotic cardiomyopathy. CD73, a common extracellular nucleotidase also known as 5'-nucleotidase, is associated with the progression of inflammation and immunity in multiple organs. However, the mechanism by which CD73 contributes to myocardial inflammation and apoptosis in cirrhosis remains unclear. Methods: In this study, a cirrhotic cardiomyopathy model in mice was established by bile duct ligation. Myocardial-specific overexpression of CD73 was achieved by tail vein injection of AAV9 (adeno-associated virus)-cTNT-NT5E-mCherry, and cardiac function in mice was assessed using echocardiography. Myocardial inflammation infiltration and apoptosis were evaluated through pathological observation and ELISA assays. The expression of CD73, A2AR, apoptotic markers, and proteins related to the NF-κB pathway in myocardial tissue were measured. Results: In the myocardial tissue of the cirrhotic cardiomyopathy mouse model, the expression of CD73 and A2AR increased. Overexpression of CD73 in the myocardium via AAV9 injection and stimulation of A2AR with CGS 21680 inhibited myocardial inflammation and cardiomyocyte apoptosis induced by cirrhosis. Additionally, overexpression of CD73 suppressed the activation of the NF-κB pathway by upregulating the expression of the adenosine receptor A2A. Conclusion: Our study reveals that the CD73/A2AR signaling axis mitigates myocardial inflammation and apoptosis induced by cirrhosis through negative feedback regulation of the NF-κB pathway.
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5'-Nucleotidasa , Cardiomiopatías , Cirrosis Hepática , Receptor de Adenosina A2A , Transducción de Señal , Animales , Masculino , Ratones , 5'-Nucleotidasa/metabolismo , Apoptosis , Cardiomiopatías/metabolismo , Cardiomiopatías/etiología , Cardiomiopatías/inmunología , Modelos Animales de Enfermedad , Retroalimentación Fisiológica , Proteínas Ligadas a GPI , Cirrosis Hepática/inmunología , Cirrosis Hepática/metabolismo , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Receptor de Adenosina A2A/metabolismoRESUMEN
Increased inflammatory responses and oxidative stress at the wound site following skin trauma impair healing. Furthermore, skin scarring places fibroblasts under severe mechanical stress and aggravates pathological fibrosis. A novel liposomal composite hydrogel is engineered for wound microenvironment remodeling, incorporating dual-loaded liposomes into gelatin methacrylate to create a nanocomposite hydrogel. Notably, tetrahydrocurcumin (THC) and hepatocyte growth factor (HGF) are encapsulated in the hydrophobic and hydrophilic layers of liposomes, respectively. The composite hydrogel maintains porous nanoarchitecture, demonstrating sustainable THC and HGF release and enhanced mechanical properties and biocompatibility. This system effectively promotes cell proliferation and angiogenesis and attenuates apoptosis. It decreases the expression of the inflammatory factors by inhibiting the high-mobility group box /receptor for advanced glycation end product/NF-κB (HMGB1/RAGE/NF-κB)pathway and increases macrophage polarization from M1 to M2 in vitro, effectively controlling inflammatory responses. It exhibits remarkable antioxidant properties by scavenging excess reactive oxygen species and free radicals. Most importantly, it effectively prevents scar formation by restraining the transforming growth factor beta (TGF-ß)/Smads pathway that downregulates associated fibrotic factors. It demonstrates strong therapeutic effects against inflammation and fibrosis in a rat skin wound model with biosafety, advancing the development of innovative hydrogel-based therapeutic delivery strategies for clinical scarless wound therapy.
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OBJECTIVES: MRSA is a major cause of hospital-acquired and community-acquired infections. Treatment options for MRSA are limited because of the rapid development of ß-lactam resistance. Combining antibiotics offers an affordable, time-saving, viable and efficient approach for developing novel antimicrobial therapies. Both amoxicillin and cefdinir are oral ß-lactams with indications for a wide range of bacterial infections and mild side effects. This study aimed to investigate the in vitro and in vivo efficacy of combining these two ß-lactams against MRSA strains. METHODS: Fourteen representative prevalent MRSA strains with diverse sequence types (STs) were tested with a combination of amoxicillin and cefdinir, using chequerboard and time-kill assays. The Galleria mellonella larvae infection model was used to evaluate the in vivo efficacy of this dual combination against the community-acquired MRSA (CA-MRSA) strain USA300 and the hospital-acquired MRSA (HA-MRSA) strain COL. RESULTS: The chequerboard assay revealed a synergistic activity of the dual amoxicillin/cefdinir combination against all tested MRSA strains, with fractional inhibitory concentration index (FICI) values below 0.5 and at least a 4-fold reduction in the MICs of both antibiotics. Time-kill assays demonstrated synergistic bactericidal activity of this dual combination against the MRSA strain USA300 and strain COL. Moreover, in vivo studies showed that the administration of amoxicillin/cefdinir combination to G. mellonella larvae infected with MRSA strains significantly improved the survival rate up to 82%, which was comparable to the efficacy of vancomycin. CONCLUSIONS: In vitro and in vivo studies indicate that the dual combination of amoxicillin/cefdinir demonstrates a synergistic bactericidal efficacy against MRSA strains of various STs. Further research is needed to explore its potential as a treatment option for MRSA infections.
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Amoxicilina , Antibacterianos , Sinergismo Farmacológico , Staphylococcus aureus Resistente a Meticilina , Pruebas de Sensibilidad Microbiana , Infecciones Estafilocócicas , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Animales , Antibacterianos/farmacología , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Amoxicilina/farmacología , Cefalosporinas/farmacología , Modelos Animales de Enfermedad , Cefdinir/farmacología , Larva/microbiología , Larva/efectos de los fármacos , Viabilidad Microbiana/efectos de los fármacos , Humanos , Mariposas Nocturnas/microbiología , Análisis de Supervivencia , Resultado del Tratamiento , Quimioterapia Combinada , beta-Lactamas/farmacologíaRESUMEN
Green tea is one of the main types of tea in China, and it has been widely consumed in the world. This study aims to investigate the potential mechanism by which the water extract of green tea (GTWE) may be effective in the treatment of alcohol-related hepatitis (ARH), utilizing a combination of network pharmacology, molecular docking, and experimental validation. Through network pharmacology analysis, seven active components and 45 potential targets were identified, with TLR4 being confirmed as the central target. Experimental findings demonstrate that GTWE exhibits significant efficacy in mitigating alcohol-induced liver inflammation and steatosis. Furthermore, the administration of GTWE has demonstrated significant efficacy in mitigating alcohol-induced intestinal inflammation and microbiota disturbance while concurrently restoring intestinal barrier function. Consequently, GTWE exhibits considerable potential as a pharmacological intervention and warrants further research and development as a lead compound for the treatment of ARH. Moreover, the prospective utilization of green tea in prolonged intakes exhibits potential as a prophylactic nutritive regimen against ARH.
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Microbioma Gastrointestinal , Hepatitis , Ratones , Animales , Té , Simulación del Acoplamiento Molecular , Estudios Prospectivos , Extractos Vegetales/farmacología , InflamaciónRESUMEN
In the peel of citrus (Rutaceae) fruit, hesperitin (Hesp), a flavanone glycoside chemical, is found naturally. Hesp has been found to have a wide range of pharmacological actions, including anti-inflammatory, antioxidant, antiviral, and anticancer properties, according to earlier research. However, nothing is known regarding its function in alcoholic liver steatosis and inflammation. In this study, we employed a network pharmacology approach to identify the TLR4 signaling pathway as a primary target of Hesp for the treatment of alcoholic steatohepatitis (ASH). Molecular docking results showed that Hesp bound to the representative target TLR4 and exhibited good affinity. In addition, Hesp inhibits the TLR4 target and consequently the NF-κB signaling pathway, which in turn slows the evolution of alcoholic steatohepatitis, according to further in vitro and in vivo tests. The results of this study preliminarily indicate that Hesp is an ideal drug candidate for the treatment of ASH.
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Hígado Graso Alcohólico , FN-kappa B , Ratones , Animales , FN-kappa B/metabolismo , Hígado Graso Alcohólico/tratamiento farmacológico , Receptor Toll-Like 4/metabolismo , Simulación del Acoplamiento Molecular , Transducción de SeñalRESUMEN
Alcohol-related liver disease (ALD) is the most common chronic liver disease worldwide; however, no effective treatment to prevent the progression of alcohol-related liver fibrosis (ALF) is available. CD73/NT5E, a nucleotidase, controls cellular homeostasis by combining extracellular purinergic signaling with intracellular kinase activity and gene transcription and is associated with cell proliferation, differentiation, and death. In this study, we demonstrated that CD73/NT5E had a more significant regulatory effect on the activation, proliferation, and apoptosis of HSCs compared with that of CD39/ENTPD1. We examined the expression of CD73/NT5E in the normal and fibrotic human livers. The absence of CD73/NT5E was protective in mouse models of ALF. In addition, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed that CD73/NT5E overexpression was related to the p53 signaling pathway, which regulates cell senescence. Proteins interacting with p53 were predicted using the STRING database. The overlap between proteomic analysis and STRING databases was for Aurora kinase A (AURKA), a cell cycle-regulated kinase. Coimmunoprecipitation (co-IP) assay and molecular docking confirmed that CD73/NT5E directly interacted with AURKA. We found that overexpression of CD73/NT5E inhibited AURKA ubiquitination, whereas p53 signaling was downregulated. Mechanistically, CD73/NT5E regulated ALF and the activation and senescence of stellate cells by binding to AURKA. These findings indicate that CD73/NT5E is a potential therapeutic target for ALF.
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Aurora Quinasa A , Células Estrelladas Hepáticas , Ratones , Animales , Humanos , Aurora Quinasa A/genética , Aurora Quinasa A/metabolismo , Células Estrelladas Hepáticas/metabolismo , Simulación del Acoplamiento Molecular , Proteómica , Proteína p53 Supresora de Tumor/genética , Cirrosis Hepática/genética , Cirrosis Hepática/metabolismo , 5'-Nucleotidasa/metabolismo , Proteínas Ligadas a GPI/metabolismoRESUMEN
CD73 is a membrane-bound glycoprotein that can dephosphorylate AMP to adenosine. Increasing evidence has shown that CD73 is involved in the occurrence and development of liver fibrosis. However, the potential mechanism by which CD73 affects the progression of alcohol-related liver fibrosis (ALF) remains unknown. This study aimed to examine the role and mechanism of CD73 in autophagy in HSC-T6 cells and its role in ALF in mice that treated with alcohol plus CCl4. We found that CD73 knockout reduced serum alanine aminotransferase and aspartate aminotransferase levels and decreased liver injury and collagen deposition. Furthermore, autophagy-related indicators were downregulated in the liver fibrosis tissues of CD73-/- (EtOH + CCl4) mice. In vitro, the expression of CD73 and autophagy increased in activated HSC-T6 cells. Autophagy inhibitor, 3-methyladenine, reduced autophagy and activation of acetaldehyde-induced HSC-T6 cells. When using CD73-siRNA, autophagy in HSC-T6 cells was found to be downregulated. However, the CD73 plasmid increased the activation and autophagy of hepatic stellate cells (HSCs). In addition, CD73 induced autophagy through the AMPK/AKT/mTOR pathway, which is characterized by an increase in the ratio of P-AMPKα/AMPKα and a decrease in the ratio of P-AKT/AKT and P-mTOR/mTOR. Our study found that CD73 promotes HSCs activation by regulating autophagy through the AMPK/AKT/mTOR signaling pathway.
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5'-Nucleotidasa , Células Estrelladas Hepáticas , Cirrosis Hepática Alcohólica , Transducción de Señal , Animales , Ratones , Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia , Etanol/metabolismo , Células Estrelladas Hepáticas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , 5'-Nucleotidasa/metabolismo , Cirrosis Hepática Alcohólica/patologíaRESUMEN
Three-dimensional carbon nanosheets (3D-CNS) were synthesized by salt template spray-drying method in order to solve the agglomeration of 2D nanocarbon by a traditional mixing method. MgB2 bulks doped with 3D-CNS with molar ratio composition of MgB2-x(3D-CNS)x (x = 0, 0.1 and 0.2) have been prepared by in situ sintering process. The microstructure, critical current density and flux pinning of the sintered samples have been investigated. Differing from the structure in previous studies, the 3D-CNS doping is more efficient for the refinement of the MgB2 grains due to the 3D network structures. The results clearly show that more active C releasing from 3D-CNS at high temperature can provide effective flux pinning centers by the substitution of C for B in MgB2 lattice. Furthermore, the lattice distortion and increased grain boundaries should be responsible for the enhancement of critical current density (Jc) at high magnetic fields as well as the increased irreversible magnetic field (Hirr). However, the positive action in Jc at low field has been extremely offset by the concentration of impurities at MgB2 grain boundaries such as released extra C without substitution and MgO, which is considered to further deteriorate the grain connectivity.
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Alcoholic liver disease is one of the most common chronic liver diseases in the world. It is a liver disease caused by prolonged heavy drinking and its main clinical features are nausea, vomiting, enlargement of the liver, and jaundice. Recent studies suggest that Kupffer cell-mediated inflammatory response is a core driver in the development of alcoholic steatohepatitis and alcoholic liver fibrosis. As a danger signal, extracellular ATP activates the assembly of NLPR3 inflammasome by acting on purine P2X7 receptor, the activated NLRP3 inflammasome prompts ASC to cleave pro-cCaspase-1 into active caspase-1in KCs. Active caspase-1 promotes the conversion of pro-IL-1ß to IL-1ß, which further enhances the inflammatory response. Here, we briefly review the role of the P2X7R-NLRP3 inflammasome axis in the pathogenesis of alcoholic liver disease and the evolution of alcoholic steatohepatitis and alcoholic liver fibrosis. Regulation of the inflammasome axis of P2X7R-NLRP3 may be a new approach for the treatment of alcoholic liver disease.
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This paper is concerned with the homing process in which a multi-underactuated AUV recovery system tracks the moving mother submarine in finite time in the presence of uncertain hydrodynamic parameters and unknown environmental disturbances. In the homing stage, underactuated AUVs and the moving mother submarine are treated as followers and the leader, respectively. The multi-underactuated AUV system finite time homing problem is converted to the leader-following finite-time formation control problem. The proposed leader-following formation strategy requires only the position information of the leader. The velocity of the leader can be designed as an additional degrees of freedom to stabilize position errors of the formation. A novel robust adaptive super-twisting sliding mode formation controller (ASTASMC) is proposed, specifically the super-twisting algorithm (STA) with adaptive uncertainty estimation. Wherein, the robust adaptive law can compensate for uncertainty with unknown upper bound in real time. Therefore, the proposed controller can not only enhance the tracking accuracy but also reduce the chattering. In addition, the finite-time convergence of estimation errors and tracking errors is rigorously proved. Finally, simulation results demonstrate the effectiveness of theoretical results.
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Alcoholic hepatitis is a serious form of liver damage. Inflammation is a key factor in alcoholic hepatitis and plays a key role in the progression of alcoholic liver disease. Adenosine receptor A2B (A2BAR) is a member of the adenosine receptor family and generally considered to be a negative regulator of the inflammatory response. We found that A2BAR was the most highly expressed adenosine receptor in ETOH-fed mouse liver tissue and was also highly expressed in primary Kupffer cells and ETOH-induced RAW264.7 cells. In addition, injection of BAY 60-6583 stimulated A2BAR, induced upregulation of the expression levels of cAMP, and reduced ETOH-induced steatosis and inflammation in mice. At the same time, knockdown of A2BAR in vitro increased the inflammatory response in RAW264.7 cells triggered by ETOH. After knockdown of A2BAR in vitro, the release of the inflammatory cytokines IL-6, IL-1ß and TNF-α was increased. After overexpression of A2BAR in vitro, the cAMP level was significantly increased, PKA expression was increased, the expression of phosphorylated proteins in the NF-kB signal transduction pathway was significantly affected, and the expression of the key phosphorylated protein p-P65 was decreased. However, after the simultaneous overexpression of A2BAR and inhibition of PKA, the expression of the key phosphorylated protein p-P65 was still significantly decreased. In addition, after the expression of A2BAR increased or decreased in RAW264.7 cells, AML-12 cells were cultured in the supernatant of RAW264.7 cells stimulated by ETOH, and the apoptosis rate was significantly changed by flow cytometry. These results suggest that A2BAR can reduce alcoholic steatohepatitis by upregulating cAMP levels and negatively regulating the NF-kB pathway. Overall, these findings suggest the significance of A2BAR-mediated inflammation in alcoholic liver disease.
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Hepatitis Alcohólica/tratamiento farmacológico , Macrófagos del Hígado/efectos de los fármacos , FN-kappa B/efectos de los fármacos , FN-kappa B/metabolismo , Receptor de Adenosina A2B/uso terapéutico , Receptores de AMP Cíclico/efectos de los fármacos , Receptores de AMP Cíclico/metabolismo , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Ratones , Ratones Endogámicos C57BLRESUMEN
BACKGROUND: Alcoholic liver disease (ALD) is liver damage caused by long-term drinking. Inflammation plays a central role in the progression of ALD. CD73 is a ubiquitously expressed glycosylphosphatidylinositol-anchored glycoprotein that is a key enzyme that converts ATP into adenosine. Evidence has shown that CD73 plays an important role in many diseases, but the role and mechanism of CD73 in alcohol-induced liver injury and inflammation is still unclear. METHODS: The alcohol-induced liver injury and inflammation mouse model was established. The rAAV9-CD73 was used to overexpress CD73. Isolation of primary macrophages (MΦ) from the liver was conducted. The effects of CD73 on alcohol-induced liver injury and inflammation were evaluated by quantitative realtime PCR, Western blotting, ELISA, and immunohistochemical assay. Flow cytometry was used to detect the cell cycle and apoptosis. RESULTS: Our results showed that overexpression of CD73 can reduce alcohol-induced liver damage, lipid accumulation, and the secretion of inflammatory cytokines. pEX3-CD73 can promote RAW264.7 cells proliferation and inhibit apoptosis via suppressing the activation of TLR4/MyD88/NF-κB signaling pathway. Inhibition of TLR4 further enhanced the anti-inflammatory effect of overexpression of CD73. CONCLUSION: Overexpression of CD73 can reduce alcohol-induced liver injury and inflammation. CD73 may serve as a potential therapeutic target for ALD.
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Alcoholic liver disease caused by chronic excessive drinking has become one of the most common types of liver disease. Alcohol-induced inflammatory immune responses play a central role in the development of alcohol-associated steatohepatitis. The content and expression of ATP and P2X4 in the livers of alcoholic steatohepatitis mice are significantly increased. The content of ATP increased by 20 percent and the expression of P2X4 receptor protein was 1.3 times higher than that in the livers of normal mice. Treatment with 5-BDBD, a P2X4 receptor-specific inhibitor, significantly reduced alcohol-induced liver inflammation and lipid deposition. In RAW264.7 cell experiments, 5-BDBD inhibited the expression of P2X4 and alleviated alcohol-induced inflammation, while the CD39-specific inhibitor POM-1 reduced extracellular ATP degradation and promoted the expression of P2X4, thereby exacerbating inflammation. After treatment with 5-BDBD, P2X4 receptor protein expression decreased by 0.2 times and after treatment with POM-1, P2X4 receptor protein expression increased by 0.1 times compared to the alcohol-stimulated group. In addition, inhibition of P2X4 expression in RAW264.7 cells reduced calcium influx in RAW264.7 cells. P2X4 may induce the activation of NLRP3 inflammasomes by mediating calcium influx, thus exacerbating the inflammatory response, and inhibition of P2X4 expression can effectively block this process. Conclusion: These results suggest that the ATP-P2X4 signaling pathway promotes the inflammatory response in alcoholic steatohepatitis and that CD39 may play a protective role in regulating P2X4 expression by hydrolyzing ATP. In conclusion, the CD39 and ATP-P2X4 signaling pathways may be potential therapeutic targets for alcoholic steatohepatitis.
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Hígado Graso Alcohólico , Hepatopatías Alcohólicas , Adenosina Trifosfato/metabolismo , Animales , Antígenos CD , Apirasa , Hígado Graso Alcohólico/metabolismo , Inflamasomas/metabolismo , Inflamación/metabolismo , Ratones , Receptores Purinérgicos P2X4RESUMEN
Intracellular calcium ion (Ca2+) in cytoplasm as an intracellular second messenger is involved in almost all important cellular activities of organisms. Generally its concentration ([Ca2+]i) is tested by live imaging followed image and data processes, in which much tedious and subjective manual work is involved. Here we show a computational approach of Deep Calcium following the principles of deep learning to predict the cytoplasmic Ca2+ ranges and calcium peaks in calcium curve of objective cells. To validate Deep Calcium, chondrocytes, bone marrow stromal cells (BMSCs) and osteoblastic like cells (MC3T3-E1) from both the tissue and cell samples as well as from spontaneous and mechanical stimulated calcium response patterns are used. The good performance comparing with other relative machine learning models, as well as consistency biological results with human experts are demonstrated. Deep Calcium provides references for other image and data processes of intracellular range determination and curve peak identification.
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Calcio , Aprendizaje Profundo , Calcio/metabolismo , Señalización del Calcio , Humanos , Iones , Aprendizaje AutomáticoRESUMEN
This paper proposes a robust cooperative trajectory tracking control scheme for an unactuated floating object with multiple vessels under environmental disturbances. The object and multiple vessels are connected by using towlines. The proposed control scheme consists of three parts: a virtual controller for the object, a control allocation algorithm and a distributed robust time-varying formation controller for vessels. The virtual controller is first designed to obtain the control forces of the object to track the reference trajectory. To compute the optimal tension of each towline, the control allocation algorithm is introduced. Then, the time-varying relative positions from the object to vessels are gained by using a nonlinear towline model and the towline attachment geometry. Furthermore, the distributed robust time-varying formation controller is devised for vessels based on dynamic surface control technique, an adaptive law and graph theory. It is proved that the tracking errors of the object and vessels are bounded. Simulations substantiate that the proposed method can achieve good cooperative control performance and robustness, and the unactuated object can track the reference trajectory with high accuracy.
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Phage therapy recently passed a key milestone with success of the first regulated clinical trial using systemic administration. In this single-arm non-comparative safety study, phages were administered intravenously to patients with invasive Staphylococcus aureus infections with no adverse reactions reported. Here, we examined features of 78 lytic S. aureus phages, most of which were propagated using a S. carnosus host modified to be broadly susceptible to staphylococcal phage infection. Use of this host eliminates the threat of contamination with staphylococcal prophage - the main vector of S. aureus horizontal gene transfer. We determined the host range of these phages against an international collection of 185 S. aureus isolates with 56 different multilocus sequence types that included multiple representatives of all epidemic MRSA and MSSA clonal complexes. Forty of our 78 phages were able to infect > 90% of study isolates, 15 were able to infect > 95%, and two could infect all 184 clinical isolates, but not a phage-resistant mutant generated in a previous study. We selected the 10 phages with the widest host range for in vitro characterization by planktonic culture time-kill analysis against four isolates:- modified S. carnosus strain TM300H, methicillin-sensitive isolates D329 and 15981, and MRSA isolate 252. Six of these 10 phages were able to rapidly kill, reducing cell numbers of at least three isolates. The four best-performing phages, in this assay, were further shown to be highly effective in reducing 48 h biofilms on polystyrene formed by eight ST22 and eight ST36 MRSA isolates. Genomes of 22 of the widest host-range phages showed they belonged to the Twortvirinae subfamily of the order Caudovirales in three main groups corresponding to Silviavirus, and two distinct groups of Kayvirus. These genomes assembled as single-linear dsDNAs with an average length of 140 kb and a GC content of c. 30%. Phages that could infect > 96% of S. aureus isolates were found in all three groups, and these have great potential as therapeutic candidates if, in future studies, they can be formulated to maximize their efficacy and eliminate emergence of phage resistance by using appropriate combinations.
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Bacteriófagos , Staphylococcus aureus Resistente a Meticilina , Infecciones Estafilocócicas , Bacteriófagos/genética , Genotipo , Humanos , Staphylococcus aureus Resistente a Meticilina/genética , Staphylococcus , Fagos de Staphylococcus/genética , Staphylococcus aureus/genéticaRESUMEN
CD39 is associated with diverse physiological and pathological processes, including cell proliferation and differentiation. Adenosine triphosphate (ATP) is hydrolysed to adenosine by different enzymes including ecto-nucleoside triphosphate diphosphohydrolase-1/ENTPD1 (CD39) and ecto-5'-nucleotidase (CD73), regulating many physiological and pathological processes in various diseases, but these changes and functions in alcoholic liver disease are generally unknown. In this study, an alcoholic liver disease model in vivo was induced by ethanol plus carbon tetrachloride(CCl4) administered to C57BL/6 mice, who were the intraperitoneally injected with the CD39 inhibitor sodium polyoxotungstate (POM1) or colchicine from the 5th week to the 8th week. Meanwhile, hepatic stellate cells were stimulated by acetaldehyde to replicate alcoholic liver fibrosis models in vitro. Exogenous ATP and POM1 were added in turn to the culture system. Pharmacological blockade of CD39 largely prevents liver damage and collagen deposition. We found that blockade or silencing of CD39 prevented acetaldehyde-induced proliferation of HSC-T6 cells and the expression of fibrogenic factors. Moreover, blockade or silencing of CD39 could block the activation of the adenosine A2A and adenosine A2B receptors and the TGF-ß/Smad3 pathway, which are essential events in HSC activation. Thus, blockade of CD39 to inhibit the transduction of ATP to adenosine may prevent HSC activation, alleviating alcoholic hepatic fibrosis. The findings from this study suggest ATP-adenosine signalling is a novel therapeutic and preventive target for alcoholic liver disease.
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Adenosina Trifosfato/metabolismo , Adenosina/metabolismo , Antígenos CD/metabolismo , Apirasa/metabolismo , Células Estrelladas Hepáticas/metabolismo , Hepatopatías Alcohólicas/etiología , Hepatopatías Alcohólicas/metabolismo , Transducción de Señal/efectos de los fármacos , 5'-Nucleotidasa/genética , 5'-Nucleotidasa/metabolismo , Acetaldehído/toxicidad , Animales , Antígenos CD/genética , Apirasa/antagonistas & inhibidores , Apirasa/genética , Tetracloruro de Carbono/toxicidad , Colchicina/farmacología , Citocinas/metabolismo , Modelos Animales de Enfermedad , Etanol/toxicidad , Técnicas de Silenciamiento del Gen , Humanos , Hepatopatías Alcohólicas/patología , Ratones Endogámicos C57BL , Cultivo Primario de Células , Ratas , Receptor de Adenosina A2A/metabolismo , Receptor de Adenosina A2B/metabolismo , Proteína smad3/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Compuestos de Tungsteno/farmacologíaRESUMEN
A major determinant of ß-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA) is the drug insensitive transpeptidase, PBP2a, encoded by mecA. Full expression of the resistance phenotype requires auxiliary factors. Two such factors, auxiliary factor A (auxA, SAUSA300_0980) and B (auxB, SAUSA300_1003), were identified in a screen against mutants with increased susceptibility to ß-lactams in the MRSA strain, JE2. auxA and auxB encode transmembrane proteins, with AuxA predicted to be a transporter. Inactivation of auxA or auxB enhanced ß-lactam susceptibility in community-, hospital- and livestock-associated MRSA strains without affecting PBP2a expression, peptidoglycan cross-linking or wall teichoic acid synthesis. Both mutants displayed increased susceptibility to inhibitors of lipoteichoic acid (LTA) synthesis and alanylation pathways and released LTA even in the absence of ß-lactams. The ß-lactam susceptibility of the aux mutants was suppressed by mutations inactivating gdpP, which was previously found to allow growth of mutants lacking the lipoteichoic synthase enzyme, LtaS. Using the Galleria mellonella infection model, enhanced survival of larvae inoculated with either auxA or auxB mutants was observed compared with the wild-type strain following treatment with amoxicillin. These results indicate that AuxA and AuxB are central for LTA stability and potential inhibitors can be tools to re-sensitize MRSA strains to ß-lactams and combat MRSA infections.
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Antibacterianos/farmacología , Lipopolisacáridos/metabolismo , Proteínas de la Membrana/metabolismo , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Proteínas de Unión a las Penicilinas/metabolismo , Ácidos Teicoicos/metabolismo , Amoxicilina/farmacología , Animales , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cefoxitina/farmacología , Pared Celular/metabolismo , ADN Bacteriano/genética , Farmacorresistencia Bacteriana , Humanos , Larva/microbiología , Proteínas de la Membrana/genética , Meropenem/farmacología , Staphylococcus aureus Resistente a Meticilina/genética , Pruebas de Sensibilidad Microbiana , Modelos Animales , Mariposas Nocturnas/microbiología , Mutación , Octoxinol/farmacología , Oxacilina/farmacología , Peptidoglicano/metabolismo , Fenotipo , Infecciones Estafilocócicas/tratamiento farmacológico , Infecciones Estafilocócicas/microbiología , Virulencia , Resistencia betalactámica , beta-Lactamas/farmacologíaRESUMEN
Staphylococcus aureus is a common skin commensal but is also associated with various skin and soft tissue pathologies. Upon invasion, S. aureus is detected by resident innate immune cells through pattern-recognition receptors (PRRs), although a comprehensive understanding of the specific molecular interactions is lacking. Recently, we demonstrated that the PRR langerin (CD207) on epidermal Langerhans cells senses the conserved ß-1,4-linked N-acetylglucosamine (GlcNAc) modification on S. aureus wall teichoic acid (WTA), thereby increasing skin inflammation. Interestingly, the S. aureus ST395 lineage as well as certain species of coagulase-negative staphylococci (CoNS) produce a structurally different WTA molecule, consisting of poly-glycerolphosphate with α-O-N-acetylgalactosamine (GalNAc) residues, which are attached by the glycosyltransferase TagN. Here, we demonstrate that S. aureus ST395 strains interact with the human Macrophage galactose-type lectin (MGL; CD301) receptor, which is expressed by dendritic cells and macrophages in the dermis. MGL bound S. aureus ST395 in a tagN- and GalNAc-dependent manner but did not interact with different tagN-positive CoNS species. However, heterologous expression of Staphylococcus lugdunensis tagN in S. aureus conferred phage infection and MGL binding, confirming the role of this CoNS enzyme as GalNAc-transferase. Functionally, the detection of GalNAc on S. aureus ST395 WTA by human monocyte-derived dendritic cells significantly enhanced cytokine production. Together, our findings highlight differential recognition of S. aureus glycoprofiles by specific human innate receptors, which may affect downstream adaptive immune responses and pathogen clearance.
Asunto(s)
Pared Celular/metabolismo , Células Dendríticas/inmunología , Glicosiltransferasas/metabolismo , Lectinas Tipo C/inmunología , Staphylococcus aureus/enzimología , Ácidos Teicoicos/química , Acetilgalactosamina/análogos & derivados , Acetilgalactosamina/química , Citocinas/metabolismo , Dermis/inmunología , Dermis/microbiología , Glicerofosfatos/química , Glicosiltransferasas/genética , Interacciones Huésped-Patógeno , Humanos , Macrófagos/inmunología , Mutación , Staphylococcus aureus/química , Staphylococcus aureus/inmunología , Staphylococcus aureus/patogenicidad , Staphylococcus lugdunensis/química , Staphylococcus lugdunensis/enzimologíaRESUMEN
Methicillin-resistant Staphylococcus aureus (MRSA) is a frequent cause of difficult-to-treat, often fatal infections in humans1,2. Most humans have antibodies against S. aureus, but these are highly variable and often not protective in immunocompromised patients3. Previous vaccine development programs have not been successful4. A large percentage of human antibodies against S. aureus target wall teichoic acid (WTA), a ribitol-phosphate (RboP) surface polymer modified with N-acetylglucosamine (GlcNAc)5,6. It is currently unknown whether the immune evasion capacities of MRSA are due to variation of dominant surface epitopes such as those associated with WTA. Here we show that a considerable proportion of the prominent healthcare-associated and livestock-associated MRSA clones CC5 and CC398, respectively, contain prophages that encode an alternative WTA glycosyltransferase. This enzyme, TarP, transfers GlcNAc to a different hydroxyl group of the WTA RboP than the standard enzyme TarS7, with important consequences for immune recognition. TarP-glycosylated WTA elicits 7.5-40-fold lower levels of immunoglobulin G in mice than TarS-modified WTA. Consistent with this, human sera contained only low levels of antibodies against TarP-modified WTA. Notably, mice immunized with TarS-modified WTA were not protected against infection with tarP-expressing MRSA, indicating that TarP is crucial for the capacity of S. aureus to evade host defences. High-resolution structural analyses of TarP bound to WTA components and uridine diphosphate GlcNAc (UDP-GlcNAc) explain the mechanism of altered RboP glycosylation and form a template for targeted inhibition of TarP. Our study reveals an immune evasion strategy of S. aureus based on averting the immunogenicity of its dominant glycoantigen WTA. These results will help with the identification of invariant S. aureus vaccine antigens and may enable the development of TarP inhibitors as a new strategy for rendering MRSA susceptible to human host defences.