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PURPOSE: Melatonin has promising protective effects for retinopathy. However, its roles in retinopathy of prematurity (ROP) and the underlying mechanisms remain unknown. We aimed to explore its roles and mechanisms in a ROP model. METHODS: Hematoxylin and eosin staining were used to observe the morphology of the retina. Immunofluorescence was used to detect positive (Nrf2+ and VEGF+) cells. Immunohistochemistry was used to detect the level of nuclear expression of PCNA in retinal tissue. Transmission electron microscope (TEM) was used to observe the morphology and structure of pigment cells. qRT-PCR was used to assay the expression of miR-23a-3p, Nrf2, and HO-1. Western blotting was used to detect the expression of Nrf2, HO-1, ß-actin, and Lamin B1. RESULTS: Melatonin or miR-23a-3p antagomir treatment could ameliorate the Oxygen-induced pathological changes, increased the expression of Nrf2 and HO-1, SOD, and GSH-Px, and decreased the expression of VEGF, miR-23a-3p, MDA and the apoptosis in the ROP model. Further target prediction and luciferase reporter assays confirmed the targeted binding relationship between miR-23a-3p and Nrf2. CONCLUSION: Our study showed that melatonin could ameliorate H2O2-induced apoptosis and oxidative stress injury in RGC cells by mediating miR-23a-3p/Nrf2 signaling pathway, thereby improving retinal degeneration.
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Oxidative stress contributes greatly to doxorubicin (DOX)-induced cardiotoxicity. Down-regulation of nuclear factor erythroid 2-related factor 2 (Nrf2) is a key factor in DOX-induced myocardial oxidative injury. Recently, we found that mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1)-dependent k48-linked ubiquitination was responsible for down-regulation of myocardial Nrf2 in DOX-treated mice. Micafungin, an antifungal drug, was identified as a potential MALT1 inhibitor. This study aims to explore whether micafungin can reduce DOX-induced myocardial oxidative injury and if its anti-oxidative effect involves a suppression of MALT1-dependent k48-linked ubiquitination of Nrf2. To establish the cardiotoxicity models in vivo and in vitro, mice were treated with a single dose of DOX (15 mg/kg, i.p.) and cardiomyocytes were incubated with DOX (1 µM) for 24 h, respectively. Using mouse model of DOX-induced cardiotoxicity, micafungin (10 or 20 mg/kg) was shown to improve cardiac function, concomitant with suppression of oxidative stress, mitochondrial dysfunction, and cell death in a dose-dependent manner. Similar protective roles of micafungin (1 or 5 µM) were observed in DOX-treated cardiomyocytes. Mechanistically, micafungin weakened the interaction between MALT1 and Nrf2, decreased the k48-linked ubiquitination of Nrf2 while elevated the protein levels of Nrf2 in both DOX-treated mice and cardiomyocytes. Furthermore, MALT1 overexpression counteracted the cardioprotective effects of micafungin. In conclusion, micafungin reduces DOX-induced myocardial oxidative injury via suppression of MALT1, which decreases the k48-linked ubiquitination of Nrf2 and elevates Nrf2 protein levels. Thus, micafungin may be repurposed for treating DOX-induced cardiotoxicity.
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Cyprinid herpesvirus 2 (CyHV-2) is the pathogen of herpesviral hematopoietic necrosis (HVHN), causing the severe economic losses in farmed gibel carp (Carassius gibelio). Further exploration of the genome structure and potential molecular pathogenesis of CyHV-2 through complete genome sequencing, comparative genomics, and molecular characterization is required. Herein, the genome of a CyHV-2 YC-01 strain isolated from diseased gibel carp collected in Yancheng, Jiangsu Province, China was sequenced, then we analyzed the genomic structure, genetic properties, and molecular characterization. First, the complete YC-01 genome comprises 275,367 bp without terminal repeat (TR) regions, with 151 potential open reading frames (ORFs). Second, compared with other representative published strains of the genus Cyvirus, several evident variations are found in YC-01, particularly the orientation and position of ORF25 and ORF25B. ORF107 and ORF156 are considered as potential molecular genetic markers for YC-01. ORF55 (encoding thymidine kinase) might be used to distinguish YC-01 and ST-J1 from other CyHV-2 isolates. Third, phylogenetically, YC-01 clusters with the members of the genus Cyvirus (together with the other six CyHV-2 isolates). Fourth, 43 putative proteins are predicted to be functional and are mainly divided into five categories. Several conserved motifs are found in nucleotide, amino acid, and promoter sequences including cis-acting elements identification of YC-01. Finally, the potential virulence factors and linear B cell epitopes of CyHV-2 are predicted to supply possibilities for designing novel vaccines rationally. Our results provide insights for further understanding genomic structure, genetic evolution, and potential molecular mechanisms of CyHV-2.
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The mode and outcome of fish-virus interactions are influenced by many abiotic factors, among which water temperature is especially important in poikilothermic fish. Rare minnow Gobiocypris rarus is a eurythermal small cyprinid fish that is sensitive to infection with genotype II grass carp reovirus (GCRV). HSP70, a conservative and key player in heat shock response, is previously identified as an induced pro-viral factor during GCRV infection in vitro. Here, rare minnow was subjected to heat shock treatment (HST), 1 h treatment at 32 °C followed by reverting to a normal temperature of 24 °C, and subsequently challenged with GCRV-II at a dosage of 1 × LD50. The effect of HST on GCRV virulence in vivo was evaluated by calculating virus-associated mortality and viral load in both dead and survival fish. The results revealed that HST enhanced the mortality of rare minnow infected with GCRV; the fact that viral loads in the tissue samples of HST-treated fish were significantly higher than those in samples of the control group at 6, 8 d p.i. reflected a faster infection process due to HST. Quantitative gene expression analysis was further employed to show that the expression levels of Hsp70 in intestine and liver tissues from the HST group declined faster than muscle tissue after HST. HST W/O GCRV challenge upregulated proinflammatory cytokines such as MyD88 and Nf-κB, which was in consistence with the inflammation observed in histopathological analysis. This study shed light on the complexity of the interaction between fish abiotic and biotic stress response, which suggested that HST, an abiotic stress, could enhance the virulence of GCRV in Gobiocypris rarus that involved modulating the gene expression of host heat shock, as well as a pro-inflammatory response.
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Cyprinidae , Enfermedades de los Peces , Infecciones por Reoviridae , Reoviridae , Animales , Enfermedades de los Peces/virología , Reoviridae/patogenicidad , Reoviridae/genética , Reoviridae/fisiología , Virulencia , Infecciones por Reoviridae/virología , Infecciones por Reoviridae/veterinaria , Cyprinidae/virología , Carga Viral , Carpas/virología , Respuesta al Choque Térmico , Proteínas HSP70 de Choque Térmico/genética , Proteínas HSP70 de Choque Térmico/metabolismo , CalorRESUMEN
Aims: Downregulation of nuclear factor erythroid 2-related factor 2 (Nrf2) contributes to doxorubicin (DOX)-induced myocardial oxidative stress, and inhibition of mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1) increased Nrf2 protein level in rat heart suffering ischemia/reperfusion, indicating a connection between MALT1 and Nrf2. This study aims to explore the role of MALT1 in DOX-induced myocardial oxidative stress and the underlying mechanisms. Results: The mice received a single injection of DOX (15 mg/kg, i.p.) to induce myocardial oxidative stress, evidenced by increases in the levels of reactive oxidative species as well as decreases in the activities of antioxidative enzymes, concomitant with a downregulation of Nrf2; these phenomena were reversed by MALT1 inhibitor. Similar phenomena were observed in DOX-induced oxidative stress in cardiomyocytes. Mechanistically, knockdown or inhibition of MALT1 notably attenuated the interaction between Nrf2 and MALT1 and decreased the k48-linked ubiquitination of Nrf2. Furthermore, inhibition or knockdown of calcium/calmodulin-dependent protein kinase II (CaMKII-δ) reduced the phosphorylation of caspase recruitment domain-containing protein 11 (CARD11), subsequently disrupted the assembly of CARD11, B cell lymphoma 10 (BCL10), and MALT1 (CBM) complex, and reduced the MALT1-dependent k48-linked ubiquitination of Nrf2 in DOX-treated mice or cardiomyocytes. Innovation and Conclusion: The E3 ubiquitin ligase function of MALT1 accounts for the downregulation of Nrf2 and aggravation of myocardial oxidative stress in DOX-treated mice, and CaMKII-δ-dependent phosphorylation of CARD11 triggered the assembly of CBM complex and the subsequent activation of MALT1.
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Hydration plays a crucial role in cement composites, but the traditional methods for measuring hydration heat face several limitations. In this study, we propose a machine learning-based approach to predict hydration heat at specific time points for three types of cement composites: ordinary Portland cement pastes, fly ash cement pastes, and fly ash-metakaolin cement composites. By adjusting the model architecture and analyzing the datasets, we demonstrate that the optimized artificial neural network model not only performs well during the learning process but also accurately predicts hydration heat for various cement composites from an extra dataset. This approach offers a more efficient way to measure hydration heat for cement composites, reducing the need for labor- and time-intensive sample preparation and testing. Furthermore, it opens up possibilities for applying similar machine learning approaches to predict other properties of cement composites, contributing to efficient cement research and production.
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Large yellow croaker (Larimichthys crocea) is a vital marine-cultured species in China. Large yellow croaker iridovirus (LYCIV) can cause a high mortality rate in L. crocea. Rapid and convenient detection of LYCIV is an urgent demand for diagnosis. In this study, rapid and simple recombinase polymerase amplification (RPA), real-time RPA and RPA combined with lateral flow dipstick (RPA-LFD) methods were developed for the detection of LYCIV based on the conserved sequence of the LYCIV major capsid protein (MCP) gene. With these optimized RPA analyses, LYCIV detection could be completed within 20 min at 40°C. Both RPA and real-time RPA could detect viral DNA as low as 102 copies/µL, while the detection limit of RPA-LFD was 101 copies/µL, and there was no cross-reaction with other aquatic pathogens (KHV, CyHV-2, GCRV-JX01, SVCV, LCDV and LMBV). In practical evaluation of RPA, real-time RPA and RPA-LFD methods, the results showed consistency with the general PCR detection. In short, the developed RPA, real-time RPA and RPA-LFD analyses could be simple, rapid, sensitive and reliable methods for field diagnosis of LYCIV infection and have significant potential in the protection of LYCIV infection.
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Infecciones por Virus ADN , Enfermedades de los Peces , Iridovirus , Técnicas de Amplificación de Ácido Nucleico , Perciformes , Sensibilidad y Especificidad , Animales , Perciformes/virología , Enfermedades de los Peces/virología , Enfermedades de los Peces/diagnóstico , Infecciones por Virus ADN/veterinaria , Infecciones por Virus ADN/diagnóstico , Infecciones por Virus ADN/virología , Iridovirus/aislamiento & purificación , Iridovirus/genética , Técnicas de Amplificación de Ácido Nucleico/veterinaria , Técnicas de Amplificación de Ácido Nucleico/métodos , ADN Viral/genética , Proteínas de la Cápside/genéticaRESUMEN
Apoptosis is a physiological cell death phenomenon, representing one of the fundamental physiological mechanisms for maintaining homeostasis in living organisms. Previous studies have observed typical apoptotic features in Carassius auratus gibelio caudal fin cell (GiCF) infected with Cyprinid herpesvirus 2 (CyHV-2), and found a significant up-regulation of ccBAX expression in these infected cells. However, the specific apoptotic mechanism involved remains unclear. In this study, we utilized the GiCF cell line to investigate the apoptotic mechanism during CyHV-2 infection. Immunofluorescence staining revealed translocation of ccBAX into mitochondria upon CyHV-2 infection. Flow cytometry analysis demonstrated that overexpression of ccBAX expedited virus-induced apoptosis, characterized by heightened mitochondrial depolarization, increased transcriptional levels of Cytochrome c (Cyto c) in both the cytoplasm and mitochondria, and augmented Caspase 3/7 enzyme activity. Bax inhibitor peptide V5 (BIP-V5), an inhibitor interfering with the function of Bax proteins, inhibited Bax-mediated apoptotic events through the mitochondrial pathway and attenuated apoptosis induced by CyHV-2. In this study, it was identified for the first time that CyHV-2 induces apoptosis via the mitochondrial pathway in GiCF cells, bridging an important gap in our understanding regarding cell death mechanisms induced by herpesvirus infections in fish species. These findings provide a theoretical basis for comprehending viral apoptotic regulation mechanisms and the prevention and control of cellular pathologies caused by CyHV-2 infection.
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Enfermedades de los Peces , Infecciones por Herpesviridae , Herpesviridae , Animales , Proteína X Asociada a bcl-2 , Herpesviridae/fisiología , Apoptosis/genética , Mitocondrias , Carpa DoradaRESUMEN
Renal cell carcinoma is the most common and most lethal genitourinary tumor. The causes of renal clear cell carcinoma are complex and the heterogeneity of the tumor tissue is high, so patient outcomes are not very satisfactory. Exploring biomarkers in the progression of renal clear cell carcinoma is crucial to improve the diagnosis and guide the treatment of renal clear cell carcinoma. LRP6 is a co-receptor of the Wnt/ß-catenin signaling pathway, which is involved in cell growth, inflammation and cell transformation through activation of the Wnt/ß-catenin signaling pathway. Abnormal expression of LRP6 is associated with the malignant phenotype, metastatic potential and poor prognosis of various tumors. In this study, we found that LRP6 was abnormally highly expressed in a variety of tumors and significantly correlated with microsatellite instability, tumor mutation burden, and immune cell infiltration and immune checkpoint expression in a variety of tumors. Moreover, we found that LRP6 was significantly associated with the prognosis of renal clear cell carcinoma. Further we found a significant correlation between LRP6 and the expression of m6A-related genes and ferroptosis-related genes. Finally, we also found a significant correlation between the expression of LRP6 and the sensitivity to common drugs used in kidney clear cell carcinoma treatment. These results suggest that LRP6 is likely to be a potential target for kidney clear cell carcinoma treatment.
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Carcinoma de Células Renales , Neoplasias Renales , Humanos , Carcinoma de Células Renales/genética , Vía de Señalización Wnt , Neoplasias Renales/genética , Pronóstico , Biomarcadores , Riñón/metabolismo , beta Catenina/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/genética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad/metabolismoRESUMEN
White matter dysplasia (WMD) in preterm infants due to intrauterine inflammation is caused by excessive apoptosis of oligodendrocyte precursor cells (OPCs). In recent years, studies have found that excessive autophagy and apoptosis are highly interconnected and important in infection and inflammatory diseases in general. Therefore, in this study, we aimed to confirm whether regulation of autophagy by using the Akt phosphorylation agonist SC79 can inhibit abnormal apoptosis of OPCs and promote myelin maturation and white matter development in neonatal rats with WMD. We investigated the effect of inflammation on oligodendrocyte development in P0 neonatal rats by intracerebellar injection of LPS, and collected brain tissue at P2 and P5. Immunohistochemical and immunofluorescence staining were used to evaluate white matter damage, while immunofluorescence staining, terminal deoxynucleotidyl transferase dUTP nick end labeling analysis (TUNEL), and western blotting were used to evaluate autophagy and apoptosis. First, we observed that white matter development was arrested and white matter fiber maturation was impaired in LPS-inflicted pups compared with those in the sham-operated group. Second, treatment with SC79 reduced the levels of LC3II, caspase 3, caspase 9, and Bax/Bcl-2 and increased the levels of p62, p-Akt, and p-mTOR in the brain tissue of neonatal rats. Finally, SC79 treatment inhibited OPC apoptosis by increasing the binding of Beclin 1 to Bcl-2, which promoted OPC differentiation and maturation. However, the opposite results were observed after rapamycin administration. Taken together, our results suggest that SC79 can inhibit the abnormal apoptosis of OPCs caused by excessive autophagy through the Akt/mTOR pathway and that SC79 is a potential therapeutic agent for WMD in preterm infants.
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Células Precursoras de Oligodendrocitos , Sustancia Blanca , Humanos , Recién Nacido , Ratas , Animales , Sustancia Blanca/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células Precursoras de Oligodendrocitos/metabolismo , Lipopolisacáridos/farmacología , Recien Nacido Prematuro , Apoptosis , Serina-Treonina Quinasas TOR/metabolismo , Autofagia , Inflamación , Proteínas Proto-Oncogénicas c-bcl-2/metabolismoRESUMEN
PURPOSE/AIM: Bronchopulmonary dysplasia (BPD) is associated with poor survival in preterm infants. Intrauterine infection can aggravate the degree of obstruction of alveolar development in premature infants; however, the pathogenic mechanism remains unclear. In this study, we sought to determine whether pyroptosis could be inhibited by downregulating mammalian target of rapamycin (mTOR) activation and inducing autophagy in BPD-affected lung tissue. MATERIALS AND METHODS: We established a neonatal rat model of BPD induced by intrauterine infection via intraperitoneally injecting pregnant rats with lipopolysaccharide (LPS). Subsequently, mTOR levels and pyroptosis were evaluated using immunohistochemistry, immunofluorescence, TUNEL staining, and western blotting. The Shapiro-Wilk test was employed to assess the normality of the experimental data. Unpaired t-tests were used to compare the means between two groups, and comparisons between multiple groups were performed using analysis of variance. RESULTS: Pyroptosis of lung epithelial cells increased in BPD lung tissues. After administering an mTOR phosphorylation inhibitor (rapamycin) to neonatal rats with BPD, the level of autophagy increased, while the expression of autophagy cargo adaptors, LC3 and p62, did not differ. Following rapamycin treatment, NLRP3, Pro-caspase-1, caspase-1, pro-IL-1ß, IL-1ß, IL-18/Pro-IL-18, N-GSDMD/GSDMD, Pro-caspase-11, and caspase-11 were negatively regulated in BPD lung tissues. The opposite results were observed after treatment with the autophagy inhibitor MHY1485, showing an increase in pyroptosis and a significant decrease in the number of alveoli in BPD. CONCLUSIONS: Rapamycin reduces pyroptosis in neonatal rats with LPS-induced BPD by inhibiting mTOR phosphorylation and inducing autophagy; hence, it may represent a potential therapeutic for treating BPD.
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Displasia Broncopulmonar , Animales , Femenino , Humanos , Embarazo , Ratas , Autofagia , Displasia Broncopulmonar/tratamiento farmacológico , Displasia Broncopulmonar/metabolismo , Caspasas/metabolismo , Recien Nacido Prematuro , Interleucina-18/metabolismo , Fosforilación , Piroptosis , Sirolimus/farmacología , Sirolimus/uso terapéutico , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Nuclear factor e2-related factor 2 (Nrf2) plays a key role in cellular resistance to oxidative stress injury. Oxidative stress injury, caused by Nrf2 imbalance, results in increased pyroptosis, DNA damage, and inflammatory activation, which may lead to the arrest of alveolar development and bronchopulmonary dysplasia (BPD) in premature infants under hyperoxic conditions. We established a BPD mouse model to investigate the effects of tert-butylhydroquinone (TBHQ), an Nrf2 activator, on oxidative stress injury, pyroptosis, NLRP3 inflammasome activation, and alveolar development. TBHQ reduced abnormal cell death in the lung tissue of BPD mice and restored the number and normal structure of the alveoli. TBHQ administration activated the Nrf2/heme oxygenase-1 (HO-1) signaling pathway, resulting in the decrease in the following: reactive oxygen species (ROS), activation of the NOD-like receptor pyrin domain containing 3 (NLRP3) inflammasome, and IL-18 and IL-1ß expression and activation, as well as inhibition of pyroptosis. In contrast, after Nrf2 gene knockout in BPD mice, there was more severe oxidative stress injury and cell death in the lungs, there were TUNEL + and NLRP3 + co-positive cells in the alveoli, the pyroptosis was significantly increased, and the development of alveoli was significantly blocked. We demonstrated that TBHQ may promote alveolar development by enhancing Nrf2-induced antioxidation in the lung tissue of BPD mice and that the decrease in the NLRP3 inflammasome and pyroptosis caused by Nrf2 activation may be the underlying mechanism. These results suggest that TBHQ is a promising treatment for lung injury in premature infants with hyperoxia.
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Displasia Broncopulmonar , Hiperoxia , Lesión Pulmonar , Humanos , Ratones , Animales , Recién Nacido , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Displasia Broncopulmonar/tratamiento farmacológico , Lesión Pulmonar/tratamiento farmacológico , Piroptosis , Hiperoxia/complicaciones , Modelos Animales de EnfermedadRESUMEN
Impaired endothelium-dependent vasodilation in atherosclerosis is a high-risk factor for myocardial infarction and ischemic stroke, and inflammation, necroptosis and apoptosis contribute to endothelial dysfunction in atherosclerosis. Although DL-3-n-butylphthalide (NBP) has been widely used in treating ischemic stroke, its effect on endothelium-dependent vasodilation remains unknown. This study aims to explore whether NBP is able to improve endothelium-dependent vasodilation in atherosclerosis and the underlying mechanisms. Male ApoE-/- mice were fed with a high-fat diet (HFD) for 9-16 weeks to establish a model of atherosclerosis. NBP were given to the mice after eating HFD for 6 weeks and atorvastatin served as a positive control. The endothelium-dependent vasodilation, the blood flow velocity, the atherosclerotic lesion area, the serum levels of lipids, inflammatory cytokines and necroptosis-relevant proteins (RIPK1, RIPK3 and MLKL), and the endothelial necroptosis and apoptosis within the aorta were measured. Human umbilical vein endothelial cells (HUVECs) were incubated with oxidized low-density lipoprotein (ox-LDL) for 48 h to mimic endothelial injury in atherosclerosis, lactate dehydrogenase release, the ratio of necroptosis and apoptosis and the expression of necroptosis-relevant proteins were examined. Similar to atorvastatin, NBP improves endothelium-dependent vasodilation, decreases aortic flow velocity and reduces atherosclerotic lesion area in HFD-fed ApoE-/- mice, concomitant with a reduction in serum lipids, inflammatory cytokines and necroptosis-relevant proteins, and endothelial necroptosis and apoptosis. Consistently, NBP inhibited necroptosis and apoptosis in ox-LDL-treated HUVECs. Based on these observations, we conclude that NBP exerts beneficial effects on improving the endothelium-dependent vasodilation in atherosclerosis via suppressing inflammation, endothelial necroptosis and apoptosis.
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Aterosclerosis , Accidente Cerebrovascular Isquémico , Masculino , Humanos , Ratones , Animales , Dieta Alta en Grasa/efectos adversos , Vasodilatación , Atorvastatina/farmacología , Necroptosis , Aterosclerosis/metabolismo , Células Endoteliales de la Vena Umbilical Humana , Inflamación/metabolismo , Endotelio/metabolismo , Citocinas/metabolismo , Accidente Cerebrovascular Isquémico/metabolismo , Apoptosis , Apolipoproteínas E/genética , Ratones NoqueadosRESUMEN
Cyprinid herpesvirus 3 (CyHV-3) is the main pathogen of koi herpesvirus disease (KHVD), which has caused serious damage to the ornamental and food-producing carp industry. Effective and rapid on-site detection methods are needed for early diagnosis of CyHV-3. A lateral flow immuno-chromatographic assay (LFIA) using two specific anti-CyHV-3 monoclonal antibodies has been developed and validated for on-site detection of CyHV-3. MAb 3C9 was used to bio-conjugate CyHV-3 antigen with colloidal gold, and MAb 2A8 was used to capture antigen bound colloidal gold on the test line. The control line was lined with goat anti-mouse IgG to capture unbound colloidal gold to validate performance. The test results can be viewed within 10 min after putting the strip into CyHV-3 virus infection fluid. The lowest limit of detection for the LFIA test was found to be 1.5 × 104 copies/µL and it showed no cross-reactivity with other fish viral pathogens. The specificity of the strip was 100% when spleen and kidney tissues of CyHV-3-infected and healthy koi were validated at the field level. The LFIA strip will be an effective device for the early detection of CyHV-3 in the future.
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Carpas , Enfermedades de los Peces , Infecciones por Herpesviridae , Herpesviridae , Animales , Infecciones por Herpesviridae/diagnóstico , Infecciones por Herpesviridae/veterinariaRESUMEN
Cyprinid herpesvirus 2 (CyHV-2), the etiological agent of herpesvirus haematopoietic necrosis (HVHN) in carp and goldfish, has caused significant economic losses in the aquaculture industry. During viral infection, the host initiates a series of active or passive defences to regulate the process of virus infection. Apoptosis is a key component of active cellular defence, and members of the Bcl-2 family have been shown to play a critical role in the apoptotic process. However, the mechanism of action of the Bcl-2 family in inducing apoptosis during CyHV-2 infection remains unclear. In this study, we revealed the molecular mechanism of miRNA-mediated silver crucian carp BAX (ccBax) in CyHV-2-induced apoptosis for the first time and demonstrated that the overexpression of miR-124 suppressed ccBax expression and significantly down-regulated apoptosis in caudal fin cells of Carassius auratus gibelio (GiCF), while miR-124 inhibitors were the opposite. These studies indicated that miR-124 inhibits CyHV-2-induced apoptosis by reducing the expression of ccBax. Furthermore, the fact that transfection of miR-124 mimics promoted CyHV-2 replication, whereas miR-124 inhibitors inhibited CyHV-2 replication, indicated that miR-124 inhibited CyHV-2-induced apoptosis and contributed to viral replication. All these results suggested that miR-124 suppresses virus-induced apoptosis and promotes viral replication by targeting and regulating ccBax expression.
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Carpas , Enfermedades de los Peces , Infecciones por Herpesviridae , Herpesviridae , Animales , Carpas/genética , Infecciones por Herpesviridae/veterinaria , Proteína X Asociada a bcl-2 , Herpesviridae/genética , Carpa Dorada/genética , Apoptosis , Replicación ViralRESUMEN
Introduction: Ribonucleotide reductase (RR) is essential for the replication of the double-stranded DNA virus CyHV-2 due to its ability to catalyze the conversion of ribonucleotides to deoxyribonucleotides, and is a potential target for the development of antiviral drugs to control CyHV-2 infection. Methods: Bioinformatic analysis was conducted to identify potential homologues of RR in CyHV-2. The transcription and translation levels of ORF23 and ORF141, which showed high homology to RR, were measured during CyHV-2 replication in GICF. Co-localization experiments and immunoprecipitation were performed to investigate the interaction between ORF23 and ORF141. siRNA interference experiments were conducted to evaluate the effect of silencing ORF23 and ORF141 on CyHV-2 replication. The inhibitory effect of hydroxyurea, a nucleotide reductase inhibitor, on CyHV-2 replication in GICF cells and RR enzymatic activity in vitro was also evaluated. Results: ORF23 and ORF141 were identified as potential viral ribonucleotide reductase homologues in CyHV-2, and their transcription and translation levels increased with CyHV-2 replication. Co-localization experiments and immunoprecipitation suggested an interaction between the two proteins. Simultaneous silencing of ORF23 and ORF141 effectively inhibited the replication of CyHV-2. Additionally, hydroxyurea inhibited the replication of CyHV-2 in GICF cells and the in vitro enzymatic activity of RR. Conclusion: These results suggest that the CyHV-2 proteins ORF23 and ORF141 function as viral ribonucleotide reductase and their function makes an effect to CyHV-2 replication. Targeting ribonucleotide reductase could be a crucial strategy for developing new antiviral drugs against CyHV-2 and other herpesviruses.
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BACKGROUND: Cyprinid herpesvirus 2 (CyHV-2) is a pathogenic fish virus belonging to family Alloherpesviridae. The CyHV-2 gene encoding thymidine kinase (TK) is an important virulence-associated factor. Therefore, we aimed to investigate the biological function of open reading frame 55 (ORF55) in viral replication. METHODS: Purified CyHV-2 ORF55 protein was obtained by prokaryotic expression, and the interacting peptide was screened out using phage display. Host interacting proteins were then predicted and validated. RESULTS: ORF55 was efficiently expressed in the prokaryotic expression system. Protein and peptide interaction prediction and dot-blot overlay assay confirmed that peptides identified by phage display could interact with the ORF55 protein. Comparing the peptides to the National Center for Biotechnology Information database revealed four potential interacting proteins. Reverse transcription quantitative PCR results demonstrated high expression of an actin-binding Rho-activating protein in the latter stages of virus-infected cells, and molecular docking, cell transfection and coimmunoprecipitation experiments confirmed that it interacted with the ORF55 protein. CONCLUSION: During viral infection, the ORF55 protein exerts its biological function through interactions with host proteins. The specific mechanisms remain to be further explored.
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Bacteriófagos , Carpas , Enfermedades de los Peces , Infecciones por Herpesviridae , Herpesviridae , Animales , Sistemas de Lectura Abierta , Simulación del Acoplamiento Molecular , Herpesviridae/genética , Bacteriófagos/genéticaRESUMEN
BACKGROUND: Sepsis is a life-threatening disease with dominant mortality. Its early diagnosis and treatment can improve prognosis and reduce mortality. Long noncoding RNAs (lncRNAs) ATPase plasma membrane Ca2+ transporting 1 antisense RNA 1 (ATP2B1-AS1) is dysregulated and is involved in the progression of various diseases. Nevertheless, the role of ATP2B1-AS1 in sepsis remains unclear. METHODS: A human monocytic cell line, THP-1 cells, was stimulated to induce a model of sepsis in vitro. The levels of ATP2B1-AS1, miR-23a-3p, and TLR4 were assessed by real-time quantitative polymerase chain reaction. The role of ATP2B1-AS1 in cell apoptosis and inflammation was explored by flow cytometry, Western blot analysis and enzyme-linked immunosorbent serologic assay. The binding sites between ATP2B1-AS1 and miR-23a-3p, and between miR-23a-3p and TLR4 were predicted by BiBiServ and the Encyclopedia of RNA Interactomes (ENCORI) online sites, respectively, and confirmed by the luciferase assay. RESULTS: The level of ATP2B1-AS1 was increased in lipopolysaccharide (LPS)-treated THP-1 cells. LPS increased apoptosis ratio, relative protein expressions of pro-apoptotic factors, and relative messenger RNA (mRNA) level and concentrations of pro-inflammatory cytokines, but decreased the relative expression of anti-apoptosis protein and relative mRNA level and concentrations of anti-inflammatory factor. All these alterations were reversed with transfection of shATP2B1-AS1 into THP-1 cells. Moreover, ATP2B1-AS1 directly bound miR-23a-3p and negatively modulated the level of miR-23a-3p. Meanwhile, TLR4 was directly targeted by miR-23a-3p, and negatively and positively modulated by miR-23a-3p and ATP2B1-AS1, respectively. CONCLUSION: ATP2B1-AS1 aggravated apoptosis and inflammation by modulating miR-23a-3p/TLR4 axis in LPS-treated THP-1 cells.
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MicroARNs , Sepsis , Humanos , MicroARNs/genética , MicroARNs/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Lipopolisacáridos , Inflamación/metabolismo , Proliferación Celular/genética , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismoRESUMEN
Background: Sepsis is a life-threatening disease with dominant mortality. Its early diagnosis and treatment can improve prognosis and reduce mortality. Long noncoding RNAs (lncRNAs) ATPase plasma membrane Ca2+ transporting 1 antisense RNA 1 (ATP2B1-AS1) is dysregulated and is involved in the progression of various diseases. Nevertheless, the role of ATP2B1-AS1 in sepsis remains unclear. Methods: A human monocytic cell line, THP-1 cells, was stimulated to induce a model of sepsis in vitro. The levels of ATP2B1-AS1, miR-23a-3p, and TLR4 were assessed by real-time quantitative polymerase chain reaction. The role of ATP2B1-AS1 in cell apoptosis and inflammation was explored by flow cytometry, Western blot analysis and enzyme-linked immunosorbent serologic assay. The binding sites between ATP2B1-AS1 and miR-23a-3p, and between miR-23a-3p and TLR4 were predicted by BiBiServ and the Encyclopedia of RNA Interactomes (ENCORI) online sites, respectively, and confirmed by the luciferase assay. Results: The level of ATP2B1-AS1 was increased in lipopolysaccharide (LPS)-treated THP-1 cells. LPS increased apoptosis ratio, relative protein expressions of pro-apoptotic factors, and relative messenger RNA (mRNA) level and concentrations of pro-inflammatory cytokines, but decreased the relative expression of anti-apoptosis protein and relative mRNA level and concentrations of anti-inflammatory factor. All these alterations were reversed with transfection of shATP2B1-AS1 into THP-1 cells. Moreover, ATP2B1-AS1 directly bound miR-23a-3p and negatively modulated the level of miR-23a-3p. Meanwhile, TLR4 was directly targeted by miR-23a-3p, and negatively and positively modulated by miR-23a-3p and ATP2B1-AS1, respectively. Conclusion: ATP2B1-AS1 aggravated apoptosis and inflammation by modulating miR-23a-3p/TLR4 axis in LPS-treated THP-1 cells (AU)
Asunto(s)
Humanos , Apoptosis , Sepsis/patología , Inflamación/patología , Receptor Toll-Like 4/metabolismo , Células Cultivadas , Transfección , Citometría de Flujo , Western Blotting , Ensayo de Inmunoadsorción EnzimáticaRESUMEN
BACKGROUND: Bronchopulmonary dysplasia is a chronic lung disease commonly seen in preterm infants. It is characterized by delayed development of the alveoli and lung fibrosis. Protease-activated receptor 2 (PAR2) is an inflammatory driver that plays a proinflammatory role mainly through the P38 MAPK/NF-κB signaling pathway. METHODS: Newborn rat pups were kept under air or oxygen at >60% concentration. Lung tissues were collected at postnatal days (P) 1, 4, 7, and 10 to observe pathological changes and take measurements. RESULTS: In the hyperoxic group, P4 and P7 rats showed delayed alveolar development, septal thickening, and disturbances in alveolar structure.PAR2, P38 MAPK, NF-κB, and IL-18 expression at P4, P7, and P10 was significantly higher than in the air group. CONCLUSION: PAR2 is involved in lung injury induced by persistent hyperoxia. Activated PAR2 promotes IL-18 overexpression through the P38 MAPK/NF-κB signaling pathway, which may be an important mechanism of PAR2-mediated lung injury in bronchopulmonary dysplasia.