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BACKGROUND: Inadequate nerve regeneration and an inhibitory local microenvironment are major obstacles to the repair of spinal cord injury (SCI). The activation and differentiation fate regulation of endogenous neural stem cells (NSCs) represent one of the most promising repair approaches. Metformin has been extensively studied for its antioxidative, anti-inflammatory, anti-aging, and autophagy-regulating properties in central nervous system diseases. However, the effects of metformin on endogenous NSCs remains to be elucidated. METHODS: The proliferation and differentiation abilities of NSCs were evaluated using CCK-8 assay, EdU/Ki67 staining and immunofluorescence staining. Changes in the expression of key proteins related to ferroptosis in NSCs were detected using Western Blot and immunofluorescence staining. The levels of reactive oxygen species, glutathione and tissue iron were measured using corresponding assay kits. Changes in mitochondrial morphology and membrane potential were observed using transmission electron microscopy and JC-1 fluorescence probe. Locomotor function recovery after SCI in rats was assessed through BBB score, LSS score, CatWalk gait analysis, and electrophysiological testing. The expression of the AMPK pathway was examined using Western Blot. RESULTS: Metformin promoted the proliferation and neuronal differentiation of NSCs both in vitro and in vivo. Furthermore, a ferroptosis model of NSCs using erastin treatment was established in vitro, and metformin treatment could reverse the changes in the expression of key ferroptosis-related proteins, increase glutathione synthesis, reduce reactive oxygen species production and improve mitochondrial membrane potential and morphology. Moreover, metformin administration improved locomotor function recovery and histological outcomes following SCI in rats. Notably, all the above beneficial effects of metformin were completely abolished upon addition of compound C, a specific inhibitor of AMP-activated protein kinase (AMPK). CONCLUSION: Metformin, driven by canonical AMPK-dependent regulation, promotes proliferation and neuronal differentiation of endogenous NSCs while inhibiting ferroptosis, thereby facilitating recovery of locomotor function following SCI. Our study further elucidates the protective mechanism of metformin in SCI, providing new mechanistic insights for its candidacy as a therapeutic agent for SCI.
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Proteínas Quinasas Activadas por AMP , Diferenciación Celular , Proliferación Celular , Ferroptosis , Metformina , Células-Madre Neurales , Ratas Sprague-Dawley , Traumatismos de la Médula Espinal , Metformina/farmacología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Traumatismos de la Médula Espinal/patología , Traumatismos de la Médula Espinal/metabolismo , Animales , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Proliferación Celular/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Ferroptosis/efectos de los fármacos , Proteínas Quinasas Activadas por AMP/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Transducción de Señal/efectos de los fármacos , Ratas , Especies Reactivas de Oxígeno/metabolismo , Recuperación de la Función/efectos de los fármacosRESUMEN
Spinal cord injury (SCI) occurs as a result of traumatic events that damage the spinal cord, leading to motor, sensory, or autonomic function impairment. Sarsasapogenin (SA), a natural steroidal compound, has been reported to have various pharmacological applications, including the treatment of inflammation, diabetic nephropathy, and neuroprotection. However, the therapeutic efficacy and underlying mechanisms of SA in the context of SCI are still unclear. This research aimed to investigate the therapeutic effects and mechanisms of SA against SCI by integrating network pharmacology analysis and experimental verification. Network pharmacology results suggested that SA may effectively treat SCI by targeting key targets such as TNF, RELA, JUN, MAPK14, and MAPK8. The underlying mechanism of this treatment may involve the MAPK (JNK) signaling pathway and inflammation-related signaling pathways such as TNF and Toll-like receptor signaling pathways. These findings highlight the therapeutic potential of SA in SCI treatment and provide valuable insights into its molecular mechanisms of action. In vivo experiments confirmed the reparative effect of SA on SCI in rats and suggested that SA could repair SCI by modulating the immune microenvironment. In vitro experiments further investigated how SA regulates the immune microenvironment by inhibiting the MAPK/NF-kB pathways. Overall, this study successfully utilized a combination of network pharmacology and experimental verification to establish that SA can regulate the immune microenvironment via the MAPK/NF-kB signaling pathway, ultimately facilitating functional recovery from SCI. Furthermore, these findings emphasize the potential of natural compounds from traditional Chinese medicine as a viable therapy for SCI treatment.
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In preclinical studies of spinal cord injury (SCI), behavioral assessments are crucial for evaluating treatment effectiveness. Commonly used methods include Basso, Beattie, Bresnahan (BBB) score and the Louisville swim scale (LSS), relying on subjective observations. The CatWalk automated gait analysis system is also widely used in SCI studies, providing extensive gait parameters from footprints. However, these parameters are often used independently or combined simply without utilizing the vast amount of data provided by CatWalk. Therefore, it is necessary to develop a novel approach encompassing multiple CatWalk parameters for a comprehensive and objective assessment of locomotor function. In this work, we screened 208 CatWalk XT gait parameters and identified 38 suitable for assessing hindlimb motor function recovery in a rat thoracic contusion SCI model. Exploratory factor analysis was used to reveal structural relationships among these parameters. Weighted scores for Coordination effectively differentiated hindlimb motor function levels, termed as the Coordinated Function Index (CFI). CFI showed high reliability, exhibiting high correlations with BBB scores, LSS, and T2WI lesion area. Finally, we simplified CFI based on factor loadings and correlation analysis, obtaining a streamlined version with reliable assessment efficacy. In conclusion, we developed a systematic assessment indicator utilizing multiple CatWalk parameters to objectively evaluate hindlimb motor function recovery in rats after thoracic contusion SCI.
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Contusiones , Traumatismos de la Médula Espinal , Ratas , Animales , Reproducibilidad de los Resultados , Marcha , Miembro Posterior , Recuperación de la Función , Médula Espinal/patología , Modelos Animales de EnfermedadRESUMEN
Introduction: Apicomplexan AP2 family of proteins (ApiAP2) are transcription factors (TFs) that regulate parasite growth and development, but little is known about the ApiAP2 TFs in Eimeria spp. ENH_00027130 sequence is predicted to encode a Eimeria necatrix ApiAP2 protein (EnApiAP2). Methods: The cDNAs encoding full-length and truncated EnApiAP2 protein were cloned and sequenced, respectively. Then, the two cDNAs were cloned into the pET28a(+) expression vector and expressed expressed in Escherichia coli BL21. The mouse polyclonal antibody (pAb) and monoclonal antibody (mAb) against recombinant EnApiAP2 (rEnApiAP2) and EnApiAP2tr (rEnApiAP2tr) were prepared and used to localize the native EnApiAP2 protein in E. necatrix, respectively. Finally, the recombinant pEGFP-C1-ΔNLS-EnApiAP2s (knockout of a nuclear localization sequence, NLS) and pEGFP-C1-EnApiAP2 plasmid were constructed and transfected into DF-1 cells, respectively, to further observe subcellular localization of EnApiAP2 protein. Results: The EnApiAP2 gene had a size of 5019 bp and encoded 1672 amino acids, containing a conserved AP2 domain with a secondary structure consisting of an α-helix and three antiparallel ß-strands. The rEnApiAP2 and rEnApiAP2tr were predominantly expressed in the form of inclusion bodies, and could be recognized by the 6×His tag mAb and the serum of convalescent chickens after infection with E. necatrix, respectively. The native EnApiAP2 protein was detected in sporozoites (SZ) and second generation merozoites (MZ-2) extracts, with a size of approximately 210 kDa. A quantitative real-time PCR (qPCR) analysis showed that the transcription level of EnApiAP2 was significantly higher in SZ than in MZ-2, third generation merozoites (MZ-3) and gametocytes (P<0.01). EnApiAP2 protein was localized in the nuclei of SZ, MZ-2 and MZ-3 of E. necatrix. The protein of EnApiAP2 was localized in the nucleus of the DF-1 cells, whereas the ΔNLS-EnApiAP2 was expressed in the cytoplasm, which further confirmed that EnApiAP2 is nucleoprotein. Discussion: EnApiAP2 protein encoded by ENH_00027130 sequence was localized in the nucleus of E. necatrix parasites, and relied on the NLS for migration to DF-1 cell nucleus. The function of EnApiAP2 need further study.
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Eimeria , Enfermedades de las Aves de Corral , Animales , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Pollos/genética , ADN Complementario/genética , Eimeria/genética , Eimeria/metabolismo , Proteínas de Microfilamentos/metabolismo , Proteínas Nucleares/metabolismo , Enfermedades de las Aves de Corral/parasitología , Esporozoítos/metabolismoRESUMEN
As a commonly used physical intervention, electrical stimulation (ES) has been demonstrated to be effective in the treatment of central nervous system disorders. Currently, researchers are studying the effects of electrical stimulation on individual neurons and neural networks, which are dependent on factors such as stimulation intensity, duration, location, and neuronal properties. However, the exact mechanism of action of electrical stimulation remains unclear. In some cases, repeated or prolonged electrical stimulation can lead to changes in the morphology or function of the neuron. In this study, immunofluorescence staining and Sholl analysis are used to assess changes in the neurite number and axon length to determine the optimal pattern and stimulation parameters of ES for neurons. Neuronal death and plasticity are detected by TUNEL staining and microelectrode array assays, respectively. mRNA sequencing and bioinformatics analysis are applied to predict the key targets of the action of ES on neurons, and the identified targets are validated by western blot analysis and qRT-PCR. The effects of alternating current stimulation (ACS) on neurons are more significant than those of direct current stimulation (DCS), and the optimal parameters are 3 µA and 20 min. ACS stimulation significantly increases the number of neurites, the length of axons and the spontaneous electrical activity of neurons, significantly elevates the expression of growth-associated protein-43 (GAP-43) without significant changes in the expression of neurotrophic factors. Furthermore, application of PI3K/AKT-specific inhibitors significantly abolishes the beneficial effects of ACS on neurons, confirming that the PI3K/AKT pathway is an important potential signaling pathway in the action of ACS.
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Fosfatidilinositol 3-Quinasas , Proteínas Proto-Oncogénicas c-akt , Proteínas Proto-Oncogénicas c-akt/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Neuronas/metabolismo , Transducción de Señal , Proyección Neuronal/fisiología , Células CultivadasRESUMEN
OBJECTIVE: This study aims to evaluate the efficacy and safety of spinal cord stimulation (SCS) compared to conventional medical management (CMM) for patients diagnosed with chronic pain. Furthermore, the study seeks to compare the utilization of analgesics, as well as the long-term outcomes in terms of quality of life and functional capacity. DATA SOURCES: We systematically searched Cochrane Library, Web of Science, PubMed, and EMBASE for randomized controlled trials from inception up to February 2022. REVIEW METHODS: Inclusion and exclusion criteria were set according to the PICOS criteria. We searched for studies in which SCS was compared with CMM alone for chronic pain. Two reviewers independently identified eligible studies and extracted data. Risk of bias assessments were performed according to Cochrane review criteria and Interventional Pain Management Techniques-quality Appraisal of Reliability and Risk of Bias Assessment (IPM-QRB) criteria. RESULTS: The present meta-analysis comprised eight studies and included a total of 893 patients. Our findings demonstrate that spinal cord stimulation (SCS) in combination with conventional medical management (CMM) is associated with a significant reduction in visual analogue scale (VAS) pain intensity (P = 0.0005) and decreased scores on the McGill Pain Questionnaire (MPQ) (P < 0.0001). Moreover, SCS plus CMM was found to improve patients' quality of life, as evidenced by improvements in SF-36 scores (P < 0.00001), EQ-5D utility index (P = 0.008), and Oswestry Disability Index (ODI) (P < 0.00001). Based on the results of four high-quality randomized controlled trials (RCTs), the level of evidence supporting the efficacy of SCS for the treatment of painful neuropathy is graded as level I to II. In contrast, there is currently only low-level evidence to support the use of high-frequency stimulation and other chronic pain conditions, which can be attributed to a lack of sufficient randomized controlled trials. LIMITATIONS: The principal limitation of our study is the significant heterogeneity observed among the cohorts investigated. The primary source of this heterogeneity is the fact that spinal cord stimulation is indicated for the treatment of multiple chronic pain conditions. Moreover, variations in the stimulation parameters, differences among manufacturers, and the specific surgical implantation settings contribute to the increased heterogeneity observed in our analyses. To address this issue, we conducted a subgroup analysis based on specific situations and performed evidence synthesis to mitigate the potential impact of heterogeneity. These approaches allow for a more precise interpretation of the results and a more accurate evaluation of the quality of the included studies. CONCLUSIONS: SCS is an effective treatment to relieve the pain level of chronic pain, decrease analgesic usage, and increase long-term quality of life and functional capacity.
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Dolor Crónico , Enfermedades del Sistema Nervioso Periférico , Estimulación de la Médula Espinal , Humanos , Dolor Crónico/terapia , Estimulación de la Médula Espinal/métodos , Resultado del Tratamiento , Manejo del Dolor/métodos , Analgésicos , Enfermedad Crónica , Médula EspinalRESUMEN
BACKGROUND: The growth and yield of pepper (Capsicum annuum L.) is often affected by the critical salt stress. Salicylic acid (SA) can improve plants' stress tolerance by promoting growth and regulating ion absorption and transportation. METHODS AND RESULTS: To uncover the alleviated mechanism of salt stress by SA in pepper, we conducted morphological, physiological, cytological, and transcriptomic analyses under a single SA treatment and NaCl with and without SA pre-treatment for 9 days. Seedlings under NaCl treatment showed yellow shrunken leaves, this tatus were alleviated by NS treatment (NaCl with SA pre-treatment). Compared with plants under NaCl treatment, those in the NS treatment showed reduced lipid peroxidation, and significantly increased contents of chlorophyll and osmotic regulators (proline, soluble sugars). Treatment with SA balanced the Na+/K+ ratio. We conducted transcriptome sequencing and identified differentially expressed genes (DEGs) contributing to alleviation of salt stress by SA in pepper. Besides photosynthesis related genes, GO and KEGG analyses revealed that the DEGs were enriched in 'sequence-specific DNA binding', 'transcription regulator activity' and 'DNA binding transcription factor activity' by GO terms. And our results showed that TFs, such as MYB, bZIP, BBX, AP2/ERF, NAC, etc., probably make a great contribution in the alleviation of salt stress by SA. CONCLUSIONS: These results reveal that SA can improve plants' stress tolerance by balancing ion absorption, gene expression and transcriptional regulation, which provide new ideas and resources for subsequent research on the mechanism of salt tolerance in pepper.
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Capsicum , Capsicum/genética , Transcriptoma/genética , Ácido Salicílico/farmacología , Ácido Salicílico/metabolismo , Cloruro de Sodio/farmacología , Cloruro de Sodio/metabolismo , Tolerancia a la Sal/genética , ADN/metabolismo , Estrés Fisiológico/genéticaRESUMEN
BACKGROUND: Circular RNAs have shown important regulatory roles in cardiovascular diseases, containing atherosclerosis (AS). We intended to explore the role of circ_0004104 in AS using oxidized low-density lipoprotein (ox-LDL)-induced vascular endothelial cells and its associated mechanism. METHODS: Real-time quantitative polymerase chain reaction and Western blot assay were conducted to analyze RNA levels and protein levels, respectively. Cell viability, apoptosis, angiogenic ability and inflammatory response were assessed by 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) assay, flow cytometry, capillary-like network formation assay and enzyme-linked immunosorbent assay, respectively. Cell oxidative stress was assessed using commercial kits. Dual-luciferase reporter assay, RNA immunoprecipitation assay and RNA-pull down assay were performed to verify the intermolecular interaction. RESULTS: ox-LDL exposure up-regulated the level of circ_0004104 in HUVECs. ox-LDL exposure suppressed cell viability and angiogenic ability whereas promoted the apoptosis, inflammation and oxidative stress of HUVECs partly through up-regulating circ_0004104. MicroRNA-328-3p (miR-328-3p) was confirmed as a target of circ_0004104. MiR-328-3p interference largely reversed circ_0004104 silencing-mediated effects in HUVECs upon ox-LDL exposure. MiR-328-3p interacted with the 3' untranslated region of tripartite motif 14, and circ_0004104 positively regulated TRIM14 expression by sponging miR-328-3p. TRIM14 overexpression largely overturned miR-328-3p accumulation-induced influences in HUVECs upon ox-LDL exposure. CONCLUSION: Circ_0004104 knockdown attenuated ox-LDL-induced dysfunction in HUVECs via miR-328-3p-mediated regulation of TRIM14.
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Aterosclerosis/metabolismo , Células Endoteliales de la Vena Umbilical Humana/efectos de los fármacos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Lipoproteínas LDL/toxicidad , MicroARNs/metabolismo , ARN Circular/metabolismo , Proteínas de Motivos Tripartitos/metabolismo , Apoptosis/efectos de los fármacos , Aterosclerosis/genética , Aterosclerosis/patología , Caspasa 3/metabolismo , Células Cultivadas , Citocinas/metabolismo , Regulación de la Expresión Génica , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Humanos , Mediadores de Inflamación/metabolismo , Péptidos y Proteínas de Señalización Intracelular/genética , MicroARNs/genética , Neovascularización Fisiológica/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , ARN Circular/genética , Proteínas de Motivos Tripartitos/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
BACKGROUND: The NAC (NAM, ATAF1/ATAF2, and CUC2) transcription factors belong to a large family of plant-specific transcription factors in monocot and dicot species. These transcription factors regulate the expression of stress tolerance-related genes that protect plants from various abiotic stresses, including drought, salinity, and low temperatures. RESULTS: In this study, we identified the CaNAC46 transcription factor gene in Capsicum annuum. Its open reading frame was revealed to comprise 921 bp, encoding a protein consisting of 306 amino acids, with an isoelectric point of 6.96. A phylogenetic analysis indicated that CaNAC46 belongs to the ATAF subfamily. The expression of CaNAC46 was induced by heat, cold, high salt, drought, abscisic acid, salicylic acid, and methyl jasmonate treatments. Thus, CaNAC46 may be important for the resistance of dry pepper to abiotic stresses. A subcellular localization analysis confirmed that CaNAC46 is localized in the nucleus. The overexpression of CaNAC46 improved the tolerance of transgenic Arabidopsis thaliana plants to drought and salt stresses. The CaNAC46-overexpressing lines had longer roots and more lateral roots than wild-type lines under prolonged drought and high salt stress conditions. Additionally, CaNAC46 affected the accumulation of reactive oxygen species (ROS). Moreover, CaNAC46 promoted the expression of SOD, POD, RD29B, RD20, LDB18, ABI, IAA4, and P5CS. The malondialdehyde contents were higher in TRV2-CaNAC46 lines than in wild-type plants in response to drought and salt stresses. Furthermore, the expression levels of stress-responsive genes, such as ABA2, P5CS, DREB, RD22, CAT, and POD, were down-regulated in TRV2-CaNAC46 plants. CONCLUSIONS: Under saline and drought conditions, CaNAC46 is a positive regulator that activates ROS-scavenging enzymes and enhances root formation. The results of our study indicate CaNAC46 is a transcriptional regulator responsible for salinity and drought tolerance and suggest the abiotic stress-related gene regulatory mechanisms controlling this NAC transcription factor are conserved between A. thaliana and pepper.
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Adaptación Fisiológica/genética , Arabidopsis/genética , Capsicum/genética , Sequías , Estrés Salino/genética , Estrés Fisiológico/genética , Factores de Transcripción , Regulación de la Expresión Génica de las Plantas , Filogenia , Plantas Modificadas Genéticamente/genética , SalinidadRESUMEN
The biosynthesis of histidine, a proteinogenic amino acid, has been extensively studied due to its importance in bacterial growth and survival. Histidinol-phosphate phosphatase (Hol-Pase), which is responsible for the penultimate step of histidine biosynthesis, is generally the last enzyme to be characterized in many bacteria because its origin and evolution are more complex compared to other enzymes in histidine biosynthesis. However, none of the enzymes in histidine biosynthesis, including Hol-Pase, have been characterized in Pseudomonas aeruginosa, which is an important opportunistic Gram-negative pathogen that can cause serious human infections. In our previous work, a transposon mutant of P. aeruginosa was found to display a growth defect on glucose-containing minimal solid medium. In this study, we found that the growth defect was due to incomplete histidine auxotrophy caused by PA0335 inactivation. Subsequently, PA0335 was shown to encode Hol-Pase, and its function and enzymatic activity were investigated using genetic and biochemical methods. In addition to PA0335, the roles of 12 other predicted genes involved in histidine biosynthesis in P. aeruginosa were examined. Among them, hisC2 (PA3165), hisH2 (PA3152), and hisF2 (PA3151) were found to be dispensable for histidine synthesis, whereas hisG (PA4449), hisE (PA5067), hisF1 (PA5140), hisB (PA5143), hisI (PA5066), hisC1 (PA4447), and hisA (PA5141) were essential because deletion of each resulted in complete histidine auxotrophy; similar to the case for PA0335, hisH1 (PA5142) or hisD (PA4448) deletion caused incomplete histidine auxotrophy. Taken together, our results outline the histidine synthesis pathway of P. aeruginosaIMPORTANCE Histidine is a common amino acid in proteins. Because it plays critical roles in bacterial metabolism, its biosynthetic pathway in many bacteria has been elucidated. However, the pathway remains unclear in Pseudomonas aeruginosa, an important opportunistic pathogen in clinical settings; in particular, there is scant knowledge about histidinol-phosphate phosphatase (Hol-Pase), which has a complex origin and evolution. In this study, P. aeruginosa Hol-Pase was identified and characterized. Furthermore, the roles of all other predicted genes involved in histidine biosynthesis were examined. Our results illustrate the histidine synthesis pathway of P. aeruginosa The knowledge obtained from this study may help in developing strategies to control P. aeruginosa-related infections. In addition, some enzymes of the histidine synthesis pathway from P. aeruginosa might be used as elements of histidine synthetic biology in other industrial microorganisms.
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Proteínas Bacterianas/genética , Histidina/metabolismo , Histidinol-Fosfatasa/genética , Pseudomonas aeruginosa/genética , Proteínas Bacterianas/metabolismo , Vías Biosintéticas , Histidinol-Fosfatasa/metabolismo , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/metabolismoRESUMEN
BACKGROUND: Salinity has obvious effects on plant growth and crop productivity. The salinity-responsive mechanisms have been well-studied in differentiated organs (e.g., leaves, roots and stems), but not in unorganized cells such as callus. High-throughput quantitative proteomics approaches have been used to investigate callus development, somatic embryogenesis, organogenesis, and stress response in numbers of plant species. However, they have not been applied to callus from monocotyledonous halophyte alkaligrass (Puccinellia tenuifora). RESULTS: The alkaligrass callus growth, viability and membrane integrity were perturbed by 50 mM and 150 mM NaCl treatments. Callus cells accumulated the proline, soluble sugar and glycine betaine for the maintenance of osmotic homeostasis. Importantly, the activities of ROS scavenging enzymes (e.g., SOD, APX, POD, GPX, MDHAR and GR) and antioxidants (e.g., ASA, DHA and GSH) were induced by salinity. The abundance patterns of 55 salt-responsive proteins indicate that salt signal transduction, cytoskeleton, ROS scavenging, energy supply, gene expression, protein synthesis and processing, as well as other basic metabolic processes were altered in callus to cope with the stress. CONCLUSIONS: The undifferentiated callus exhibited unique salinity-responsive mechanisms for ROS scavenging and energy supply. Activation of the POD pathway and AsA-GSH cycle was universal in callus and differentiated organs, but salinity-induced SOD pathway and salinity-reduced CAT pathway in callus were different from those in leaves and roots. To cope with salinity, callus mainly relied on glycolysis, but not the TCA cycle, for energy supply.
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Poaceae/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Estrés Salino , Antioxidantes/metabolismo , Metabolismo Energético/efectos de los fármacos , Osmorregulación/efectos de los fármacos , Proteínas de Plantas/metabolismo , Poaceae/efectos de los fármacos , Poaceae/enzimología , Poaceae/crecimiento & desarrollo , Mapeo de Interacción de Proteínas , Proteómica , Salinidad , Plantas Tolerantes a la Sal/efectos de los fármacos , Plantas Tolerantes a la Sal/enzimología , Plantas Tolerantes a la Sal/crecimiento & desarrollo , Plantas Tolerantes a la Sal/metabolismo , Cloruro de Sodio/toxicidadRESUMEN
BACKGROUND Qishen Yiqi Dropping Pills (QYDP) is a Chinese traditional medicine that has been applied to treat coronary heart disease and ischemic heart failure in China. However, few studies have explored whether QYDP exerted an effect on doxorubicin (Doxo)-induced cardiotoxicity. Hence, in this study we investigated the effect of QYDP on cardiotoxicity induced by doxorubicin (Doxo) and its potential mechanism. MATERIAL AND METHODS Male C57BL/6 mice (20-25 g, 8-10 weeks old) were randomly assigned to 4 groups: Control group, QYDP group, Doxo group, and QYDP+Doxo group. The mice were intraperitoneal injected with Doxo weekly for 4 weeks to mimic the chronic toxicity. Four weeks after Doxo injection, echocardiography was applied to evaluate the left ventricular (LV) function, and the structure of the cardiac muscle fibers was analyzed with anti-actinin-2 antibody staining by immunofluorescence. Moreover, TUNEL staining and western blot analysis of Bax protein, Bcl-2 protein, and cleaved caspase-3 protein expression levels were conducted to explore whether QYDP exerted effect on cardiac apoptosis. In addition, Masson trichrome staining and western blot analysis of alpha-SMA protein expression levels were used to evaluate whether QYDP exerted an effect on cardiac fibrosis. Western blots and quantitative real-time polymerase chain reaction were applied to detect the vascular endothelial growth factor (VEGF) protein and mRNA levels in the myocardial tissue, and anti-CD31 antibody staining by immunohistochemistry was employed to explore whether QYDP exerted an effect on cardiac angiogenesis. RESULTS QYDP effectively attenuated cardiac dysfunction and cardiac muscle fibers disruption in Doxo treated mice. Moreover, QYDP reduced myocardial apoptosis and myocardial fibrosis in Doxo treated mice, accompanied with elevated protein levels of VEGF and enhancement of myocardial microvessel density. CONCLUSIONS QYDP could protect against Doxo-induced cardiotoxicity, which may be closely associated with enhanced cardiac angiogenesis. Hence, QYDP could be a promising alternative for the treatment of Doxo-induced cardiotoxicity.
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Cardiotoxicidad/tratamiento farmacológico , Medicamentos Herbarios Chinos/farmacología , Corazón/efectos de los fármacos , Inductores de la Angiogénesis/metabolismo , Animales , Apoptosis/efectos de los fármacos , Cardiomiopatías/genética , China , Modelos Animales de Enfermedad , Doxorrubicina/farmacología , Doxorrubicina/toxicidad , Medicamentos Herbarios Chinos/metabolismo , Cardiopatías/metabolismo , Masculino , Medicina Tradicional China/métodos , Ratones , Ratones Endogámicos C57BL , Miocardio/metabolismo , Miocitos Cardíacos/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismo , Proteína X Asociada a bcl-2/metabolismoRESUMEN
QiShenYiQi dripping pills (QSYQ), a traditional Chinese medicine, are commonly used to treat coronary heart disease, and QSYQ was recently approved as a complementary treatment for ischemic heart failure in China. However, only few studies reported on whether QSYQ exerts a protective effect on heart failure induced by pressure overload. In this study, we explored the role of QSYQ in a mouse model of heart failure induced by transverse aortic constriction (TAC). Twenty-eight C57BL/6J mice were divided into four groups: Sham + NS group, Sham + QSYQ group, TAC + NS group, and TAC + QSYQ group. QSYQ dissolved in normal saline (NS) was administered intragastrically (3.5 mg/100 g/day) in the Sham + QSYQ and TAC + QSYQ groups. In the Sham + NS and TAC + NS groups, NS was provided every day intragastrically. Eight weeks after TAC, echocardiography, and cardiac catheterization were performed to evaluate the cardiac function, and immunofluorescent staining with anti-actinin2 antibody was performed to determine the structure of the myocardial fibers. Moreover, TUNEL staining and Masson trichrome staining were employed to assess the effects of QSYQ on cardiac apoptosis and cardiac fibrosis. Western blots and real-time polymerase chain reaction (PCR) were used to measure the expression levels of vascular endothelial growth factor (VEGF) in the heart, and immunohistochemical staining with anti-CD31 antibody was performed to explore the role of QSYQ in cardiac angiogenesis. Results showed that TAC-induced cardiac dysfunction and disrupted structure of myocardial fibers significantly improved after QSYQ treatment. Moreover, QSYQ treatment also significantly improved cardiac apoptosis and cardiac fibrosis in TAC-induced heart failure, which was accompanied by an increase in VEGF expression levels and maintenance of microvessel density in the heart. In conclusion, QSYQ exerts a protective effect on TAC-induced heart failure, which could be attributed to enhanced cardiac angiogenesis, which is closely related to QSYQ. Thus, QSYQ may be a promising traditional Chinese medicine for the treatment of heart failure induced by pressure overload such as hypertension.
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Our previous work showed that a plasmid-based chicken interleukin-7 (chIL-7) gene expression vector possessed potent adjuvant activity for a VP2 DNA vaccine against chicken infectious bursal disease virus (IBDV). Whether recombinant chIL-7 prepared in procaryotic expression system has the adjuvant activity for inactivated IBDV vaccine remains unknown. Here, we prepared recombinant chIL-7 using an E. coli expression system and analyzed its adjuvant activity for the inactivated IBDV vaccine. The results show that the recombinant chIL-7 was successfully prepared in E. coli using the pET20b vector, which possessed biological activity to stimulate mouse B lymphocyte proliferation. Co-administration of the chIL-7 with inactivated IBDV vaccine significantly increased specific serum antibody titers against IBDV, enhanced lymphocyte proliferation and IFN-γ and IL-4 productions, and increased protection against virulent IBDV infection.
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Infecciones por Birnaviridae/veterinaria , Pollos , Inmunogenicidad Vacunal , Virus de la Enfermedad Infecciosa de la Bolsa/inmunología , Interleucina-7/inmunología , Enfermedades de las Aves de Corral/prevención & control , Vacunas Virales/inmunología , Adyuvantes Inmunológicos/administración & dosificación , Animales , Infecciones por Birnaviridae/inmunología , Infecciones por Birnaviridae/prevención & control , Escherichia coli/genética , Interleucina-7/administración & dosificación , Enfermedades de las Aves de Corral/inmunología , Distribución Aleatoria , Proteínas Recombinantes/genética , Proteínas Recombinantes/inmunología , Vacunas de ADN/administración & dosificación , Vacunas de ADN/inmunología , Vacunas de Productos Inactivados/administración & dosificación , Vacunas de Productos Inactivados/inmunología , Vacunas Virales/administración & dosificaciónRESUMEN
We analyzed the effects of matrix metalloproteinase (MMP)-1, MMP-3, and MMP-12 on the degree of carotid atherosclerosis (CAS) and plaque stability, and investigated their correlations with cardiovascular and cerebrovascular events (CCEs). Two hundred CAS patients were enrolled. Carotid intima-media thickness (IMT) was measured using ultrasonic examination. Patients were divided into the no plaque group (NP group), stable plaque group (SP group), and vulnerable plaque group (VP group). The Crouse method was used for the evaluation of plaque scores. Additionally, 60 healthy subjects were enrolled as the control group. Serum triacylglycerol (TG), total cholesterol (TC), low density lipoprotein cholesterol (LDL-C), and high density lipoprotein cholesterol (HDL-C) were analyzed. The serum protein levels of MMP-1, MMP-3, and MMP-12 were measured by western blotting. The frequency of CCEs within 2 years was recorded, and its correlation with MMP-1, MMP-3, and MMP-12 was analyzed. The CAS plaque scores in the SP and VP groups were significantly increased compared with the NP group, and the difference between the SP and VP groups was significant. The levels of TC, TG, LDL-C, and HDL-C of CAS patients were significantly increased compared with those in the control group, but the differences in these indexes between the patient groups were not significant. Western blotting showed that the levels of MMP-1, MMP-3, and MMP-12 in the patient groups were significantly increased compared with those in the control group, and the protein levels in the VP group were significantly higher than those in the SP and NP groups. Additionally, the levels of MMP-1, MMP-3, and MMP-12 had significantly positive correlations with the occurrence of CCEs in CAS patients. In conclusion, MMP-1, MMP-3, and MMP-12 are positively correlated with CCEs in CAS patients. They can be used as markers for the clinical diagnosis and prognosis of CAS.
RESUMEN
OBJECTIVE: To generate recombinant Bacillus subtilis (B. subtilis) engineered for expression of porcine ß-defensin-2 (pBD-2) and cecropin P1 (CP1) fusion antimicrobial peptide and investigate their anti-bacterial activity in vitro and their growth-promoting and disease resisting activity in vivo. METHODS: The pBD-2 and CP1 fused gene was synthesized using the main codons of B. subtilis and inserted into plasmid pMK4 vector to construct their expression vector. The fusion peptide-expressing B. subtilis was constructed by transformation with the vector. The expressed fusion peptide was detected with Western blot. The antimicrobial activity of the expressed fusion peptide and the recovered pBD-2 and CP1 by enterokinase digestion in vitro was analyzed by the bacterial growth-inhibitory activity assay. To analyze the engineered B. subtilis on growth promotion and disease resistance, the weaned piglets were fed with basic diet supplemented with the recombinant B. subtilis. Then the piglets were challenged by enteropathogenic Escherichia coli (E. coli). The weight gain and diarrhea incidence of piglets were measured after challenge. RESULTS: The recombinant B. subtilis engineered for expression of pBD-2/CP1 fusion peptide was successfully constructed using the main codons of the B. subtilis. Both expressed pBD-2/CP1 fusion peptide and their individual peptides recovered from parental fusion peptide by enterokinase digestion possessed the antimicrobial activities to a variety of the bacteria, including gram-negative bacteria (E. coli, Salmonella typhimurium, and Haemophilus parasuis) and gram-positive bacteria (Staphylococcus aureus). Supplementing the engineered B. subtilis to the pig feed could significantly promote the piglet growth and reduced diarrhea incidence of the piglets. CONCLUSION: The generated B. subtilis strain can efficiently express pBD-2/CP1 fusion antimicrobial peptide, the recovered pBD-2 and CP1 peptides possess potent antimicrobial activities to a variety of bacterial species in vitro. Supplementation of the engineered B. subtilis in pig feed obviously promote piglet growth and resistance to the colibacillosis.
RESUMEN
Pseudomonas aeruginosa (PAO1) is an important opportunistic pathogen that thrives in various environments. It is known that the structural variations of the lipopolysaccharide (LPS), including lipid A moiety play an important role in encountering environmental changes. Genes PA3242 and PA0011 have recently been reported to be responsible for secondary-acylation of lipid A in P. aeruginosa. In this study, we confirmed that the PA3242-dependant secondary acylation affects the growth, antibiotic resistance and virulence of PAO1 and functions as a more predominant acyltransferase than PA0011. PA3242 mutant showed inhibited growth at 37 °C and inviability at 28 °C in rich medium LB. The inactivation of PA3242 leads to more sensitivity to a wide range of antibiotics than PAO1(ΔPA0011). Moreover, the virulence of PAO1(ΔPA3242) was attenuated more significantly than that of PAO1 and PAO1(ΔPA0011). The outer membrane integrity and stability of PAO1(ΔPA3242) were seriously compromised. Furthermore, PAO1(ΔPA3242) lost most of pilus and exhibited severely damaged cell envelope, which is probably responsible for the deficiency of swimming, swarming and twitching. These results partially explained the decreased antibiotic resistance and attenuated virulence of PAO1(ΔPA3242) compared to PAO1(ΔPA0011) and PAO1. Our study demonstrated that PA3242-dependent secondary acylation of lipid A plays a predominant role in growth, antibiotic resistance and virulence of PAO1 than PA0011.
Asunto(s)
Aciltransferasas/metabolismo , Farmacorresistencia Bacteriana , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/crecimiento & desarrollo , Acilación , Aciltransferasas/genética , Antibacterianos/farmacología , Medios de Cultivo/química , Eliminación de Gen , Lípido A/metabolismo , Locomoción , Viabilidad Microbiana/efectos de la radiación , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/efectos de la radiación , Temperatura , VirulenciaRESUMEN
Our previous work has demonstrated that the mammalian interleukin-7 (IL-7) gene can enhance the immunogenicity of DNA vaccine. Whether chicken IL-7 (chIL-7) possesses the ability to enhance the immunogenicity of VP2 DNA vaccine of infectious bursal disease virus (IBDV) remained unknown. To investigate this, we constructed a VP2 antigenic region (VP2366) gene and chIL-7 gene vectors, co-immunized chicken with these vectors and analyzed the effects of the chIL-7 gene on VP2366 gene immunogenicity. Results showed that co-administrated chIL-7 gene with VP2 DNA vaccine significantly increased specific serum antibody titers against IBDV, and enhanced lymphocyte proliferation and IFN-γ and IL-4 productions. More importantly, chIL-7 gene significantly increased VP2366 gene-induced protection against virulent IBDV infection, indicating that the chIL-7 gene possessed the capacity to enhance VP2366 DNA vaccine immunogenicity, and therefore might function as a novel adjuvant for IBDV VP2 DNA vaccine. Mechanically, chIL-7 could stimulate the common cytokine receptor γ chain (γc) expressions in vitro and in vivo, which might be involved in chIL-7 enhancement of the immunogenicity of VP2 DNA vaccine.
Asunto(s)
Infecciones por Birnaviridae/veterinaria , Pollos/inmunología , Virus de la Enfermedad Infecciosa de la Bolsa/inmunología , Interleucina-7/administración & dosificación , Enfermedades de las Aves de Corral/prevención & control , Vacunas de ADN/administración & dosificación , Adyuvantes Inmunológicos/administración & dosificación , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Infecciones por Birnaviridae/inmunología , Infecciones por Birnaviridae/prevención & control , Infecciones por Birnaviridae/virología , Inmunización , Inmunogenicidad Vacunal , Virus de la Enfermedad Infecciosa de la Bolsa/genética , Interferón gamma/inmunología , Interleucina-4/inmunología , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/virología , Análisis de Secuencia de ADN/veterinaria , Proteínas Estructurales Virales/genética , Proteínas Estructurales Virales/inmunologíaRESUMEN
Mammalian interleukin-7 (IL-7) is able to stimulate lymphocyte proliferation and maturation, and reverse immunosuppression. However, whether poultry IL-7 has similar functions remains unclear. Chicken infectious bursal disease virus (IBDV) causes serious immunosuppression in chicken due to virus-induced immune disorder. Whether chicken IL-7 (chIL-7) has the ability to restore the immunity during IBDV-induced immunosuppression is not clear. To test this, we amplified chIL-7 gene by RT-PCR, prepared recombinant chIL-7 using HEK293T cells and treated the chicken with the chIL-7 prior to IBDV infection. Our results indicate that chIL-7 promoted mouse B cell proliferation in vitro, and significantly reduced virus titer in bursal tissue and chicken morbidity of IBDV-infected chicken. Mechanically, chIL-7 induced chicken lymphocyte proliferation and interferon-γ production, but down-regulated TGF-ß expression, suggesting that chIL-7 has the ability to reverse IBDV-induced immunosuppression and might be a potential therapeutic agent for prevention and treatment of infectious bursal disease.
Asunto(s)
Infecciones por Birnaviridae/veterinaria , Pollos/genética , Interleucina-7/genética , Interleucina-7/uso terapéutico , Enfermedades de las Aves de Corral/tratamiento farmacológico , Secuencia de Aminoácidos , Animales , Proteínas Aviares/química , Proteínas Aviares/genética , Proteínas Aviares/metabolismo , Proteínas Aviares/uso terapéutico , Secuencia de Bases , Infecciones por Birnaviridae/tratamiento farmacológico , Infecciones por Birnaviridae/inmunología , Infecciones por Birnaviridae/virología , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Células HEK293 , Humanos , Virus de la Enfermedad Infecciosa de la Bolsa/fisiología , Interleucina-7/química , Interleucina-7/metabolismo , Ratones , Enfermedades de las Aves de Corral/inmunología , Enfermedades de las Aves de Corral/virología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/uso terapéuticoRESUMEN
BACKGROUND: Listeria monocytogenes (LM), a foodborne pathogen, can cause pregnancy failure in animals, especially in ruminants. Recent studies have shown that LM activates inflammasomes to induce IL-1ß release in macrophages, however, whether the inflammasome activation regulates LM-induced pregnancy failure remains largely unknown. Here we used mouse model to investigate the molecular mechanism by which LM-induced inflammsome activation contributes to LM-associated pregnancy failure RESULTS: We showed that wild-type, but not Listeriolysin O-deficient (Δhly) LM, significantly reduced mouse embryo survival, accompanied by the increase of IL-1ß release and caspase-1 activation. IL-1ß neutralization significantly reduced the LM-induced embryo losses, suggesting that LM-induced pregnancy failure was associated with LLO-induced inflammasome activation. To dissect the inflammasome sensor and components responsible for LM-induced caspase-1 activation and IL-1ß production, we used wild-type and NLRP3(-/-), AIM2(-/-), NLRC4(-/-), ASC(-/-), caspase-1(-/-) and cathepsin B(-/-) mouse macrophages to test the roles of these molecules in LM-induce IL-1ß production. We found that NLRP3 inflammasome was the main pathway in LM-induced caspase-1 activation and IL-1ß production. To explore the mechanism of LM-induced pregnancy failure, we investigated the effects of LM-infected macrophages on SM9-1 mouse trophoblasts. We found that the conditioned medium from LM-infected-macrophage or the recombinant IL-1ß significantly up-regulated TNFα, IL-6 and IL-8 productions in trophoblasts, suggesting that the LM-induced macrophage inflammasome activation increased trophoblast pro-inflammatory cytokine production, which was adverse to the animal pregnancy maintenance. CONCLUSIONS: Our data demonstrated that the LLO-induced NLRP3 inflammasome activation plays a key role in LM-induced pregnancy failure, and inflammasome-mediated macrophage dysregulation on trophoblasts might be involved in the pregnancy failure.