RESUMO
Aflatoxin B1 (AFB1) is extremely carcinogenic and can cause liver cancer in humans and animals with continued ingestion. As a natural compound, curcumin (Cur) exhibits excellent anti-inflammatory, and anti-cancer properties with few side effects. In this study, a total of 60 male mice (6-week-olds, 15 per group). After one week of acclimatization feeding, the mice were divided into control group (Con), AFB1 group, curcumin group (Cur), and AF+Cur group. The mice were gavaged with curcumin (Cur, 100 mg/kg) and/or AFB1 (0.75 mg/kg). To identify a new therapeutic target for AFB1-induced pyroptosis, we performed proteomic profiling for curcumin alleviating liver injury caused by AFB1 to further validate the targets through volcano plot analysis, Venn analysis, heatmap analysis, correlation, cluster analysis, GO and KEGG enrichment. AFB1 exposure resulted in the loss of hepatocyte membrane, swelling of the endoplasmic reticulum, and a significant increase in transaminase (ALT and AST) contents, while curcumin greatly improved these changes. We found that differentially expressed proteins are enriched in the endoplasmic reticulum membrane and identified ITPR2 as a target of curcumin that alleviates AFB1-induced liver injury by proteomics. Furthermore, ITPR2 expression was detected by immunofluorescence, and qRT-PCR for mRNA expression of genes downstream of ITPR2 (calpain1, calpain2, caspase-12, caspase-3). ITPR2-activated endoplasmic reticulum stress-related proteins (calpain1, calpaini2, bcl-2, BAX, cl-caspase-12, cl-caspase-3), apoptosis (PARP) and pyroptosis (DFNA5) related proteins were examined by western blotting. The analysis showed that it effectively prevents AFB1-induced pyroptosis by lowering endoplasmic reticulum stress via interfering with ITPR2 and its downstream proteins (calpain1, calpain2, bcl-2, Bax) and inhibiting caspase-12/caspase-3 pathway. Conclusively, this study applied proteomic profiling to elucidate ITPR2 as a new target, which might give a new perspective on the mechanism of curcumin alleviating AFB1-induced pyroptosis.
Assuntos
Curcumina , Piroptose , Masculino , Camundongos , Humanos , Animais , Caspase 3/metabolismo , Aflatoxina B1 , Curcumina/farmacologia , Proteína X Associada a bcl-2/metabolismo , Proteômica , Caspase 12/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Receptores de Inositol 1,4,5-TrifosfatoRESUMO
The purpose of this study was to investigate the role of epigenetic DNA methylation and CYPs expression in AFB1-exposed broiler liver and the protective effect of curcumin. Sixty-four one-day-old AA broilers were randomly divided into four groups, including control group, AFB1 group (1 mg/kg AFB1), curcumin + AFB1 group (1 mg/kg curcumin) and curcumin group (300 mg/kg curcumin). Histological observation, CYP450 enzyme activities, the expression levels of DNA methyltransferases and CYP450 enzymes, and the overall DNA methylation level in broiler liver were investigated. Dietary AFB1 was found to induce severe liver injury in broilers, upregulate the mRNA and protein expression of CYP450 enzymes (CYP1A1, CYP1A2 and CYP3A4) and the enzyme activities of CYP1A2 and CYP3A4. According to HPLC, qPCR and western blot analyses, the overall DNA methylation level and the mRNA and protein expression of DNA methyltransferases (DNMT1, DNMT3a and DNMT3b) in the liver were significantly increased after AFB1 exposure. Importantly, the Pearson test and correlation analysis data revealed that the overall DNA methylation level of broiler liver was positively correlated with DNMTs, while CYP1A1, CYP1A2 and CYP3A4 were negatively correlated. Surprisingly, curcumin supplementation strongly ameliorated AFB1-induced hepatotoxicity by restoring the histological changes, decreasing the expression and enzymatic activity of liver CYP450 enzymes (CYP1A1, CYP1A2, and CYP3A4), and increasing the overall DNA methylation level and the expression of DNMTs. Taken together, we concluded that curcumin could protect against AFB1-induced liver injury by mediating the effects of DNA methylation and CYPs expression.
Assuntos
Curcumina , Citocromo P-450 CYP1A2 , Animais , Citocromo P-450 CYP1A2/metabolismo , Aflatoxina B1/toxicidade , Curcumina/farmacologia , Citocromo P-450 CYP3A/metabolismo , Citocromo P-450 CYP1A1/metabolismo , Galinhas/metabolismo , Metilação de DNA , Fígado , Sistema Enzimático do Citocromo P-450/metabolismo , RNA Mensageiro/metabolismo , Metiltransferases/metabolismo , DNA/metabolismoRESUMO
This study set out to assess the mitigative effects of curcumin on AFB1-induced necroptosis and inflammation in chicken liver. Ninety-six one-day-old AA broiler chickens were separated into four groups, including control group, AFB1 (1 mg/kg) group, curcumin (300 mg/kg) + AFB1 (1 mg/kg) group and curcumin (300 mg/kg) group. After 28 days treatment, livers were collected for different experimental analyses. The morphological observation results showed obvious necrotic characteristics, including cell swelling, rupture of cell and mitochondrial membranes and inflammation in chicken livers. AFB1 exposure increased oxidative stress index (ROS and MDA) and decreased the antioxidant activity markers (SOD, CAT and GSH) and ATPase activities in chickens' liver. ELISA results showed that AFB1 exposure significantly induced the cytokines (TNF-α, iNOS, IL-6 and IL-1ß) release from the liver tissues. While, western blot and qRT-PCR results showed that the protein and mRNA expressions of inflammatory (TLR4/myd88/NF-κB) and necroptosis (RIPK1/RIPK3/MLKL) genes were up-regulated by AFB1 exposure. We suspect that signal crosstalk between TLR4 and TNF-α triggers inflammation and RIPK1/RIPK3 mediating necroptosis in AFB1-induced chicken liver injury. Curcumin can regulate the TLR4/RIPK signaling pathway, reduced oxidative stress biomarkers and inflammatory cytokines levels and attenuated the expression of necroptosis and inflammation genes altered by AFB1 to reduce necroptosis of chicken liver tissue. In conclusion, curcumin can protect against AFB1-induced necroptosis and inflammation by TLR4/RIPK pathway in chicken liver.
Assuntos
Aflatoxina B1 , Curcumina , Aflatoxina B1/toxicidade , Animais , Galinhas/metabolismo , Curcumina/farmacologia , Inflamação/induzido quimicamente , Inflamação/metabolismo , Fígado , Necroptose , Receptor 4 Toll-Like/genética , Receptor 4 Toll-Like/metabolismoRESUMO
Acute lung injury (ALI) is an inflammatory pulmonary disease that can easily develop into serious acute respiratory distress syndrome, which has high morbidity and mortality. However, the molecular mechanism of ALI remains unclear, and few molecular biomarkers for diagnosis and treatment have been identified. In this study, we aimed to identify novel molecular biomarkers using a bioinformatics approach. Gene expression data were obtained from the Gene Expression Omnibus database, co-expressed differentially expressed genes (CoDEGs) were identified using R software, and further functional enrichment analyses were conducted using the online tool Database for Annotation, Visualization, and Integrated Discovery. A protein-protein interaction network was established using the STRING database and Cytoscape software. Lipopolysaccharide (LPS)-induced ALI mouse model was constructed and verified. The hub genes were screened and validated in vivo. The transcription factors (TFs) and miRNAs associated with the hub genes were predicted using the NetworkAnalyst database. In total, 71 CoDEGs were screened and found to be mainly involved in the cytokine-cytokine receptor interactions, and the tumor necrosis factor and malaria signaling pathways. Animal experiments showed that the lung injury score, bronchoalveolar lavage fluid protein concentration, and wet-to-dry weight ratio were higher in the LPS group than those in the control group. Real-time polymerase chain reaction analysis indicated that most of the hub genes such as colony-stimulating factor 2 (Csf2) were overexpressed in the LPS group. A total of 20 TFs including nuclear respiratory factor 1 (NRF1) and two miRNAs were predicted to be regulators of the hub genes. In summary, Csf2 may serve as a novel diagnostic and therapeutic target for ALI. NRF1 and mmu-mir-122-5p may be key regulators in the development of ALI.
Assuntos
Lesão Pulmonar Aguda , Biologia Computacional , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Análise de Sequência com Séries de Oligonucleotídeos , Mapas de Interação de Proteínas , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/genética , Lesão Pulmonar Aguda/metabolismo , Animais , Modelos Animais de Doenças , Lipopolissacarídeos/toxicidade , Masculino , CamundongosRESUMO
Aflatoxin B1 (AFB1) is a potent hepatotoxic and carcinogenic agent. Curcumin possesses potential anti-inflammatory, anti-oxidative and hepatoprotective effects. However, the role of LncRNAs in the protective mechanisms of curcumin against AFB1-induced liver damage is still elusive. Experimental broilers were randomly divided into 1) control group, 2) AFB1 group (1 mg/kg feed), 3) cur + AFB1 group (1 mg/kg AFB1 plus 300 mg/kg curcumin diet) and 4) curcumin group (300 mg/kg curcumin diet). Liver transcriptome analyses and qPCR were performed to identify shifts in genes expression. In addition, histopathological assessment and oxidant status were determined. Dietary AFB1 caused hepatic morphological injury, significantly increased the production of ROS, decreased liver antioxidant enzymes activities and induced inflammation and apoptosis. However, dietary curcumin partially attenuated the abnormal morphological changes, oxidative stress, and apoptosis in liver tissues. Transcriptional profiling results showed that 34 LncRNAs and 717 mRNAs were differentially expressed with AFB1 and curcumin co-treatment in livers of broilers. Analysis of the LncRNA-mRNA network, GO and KEGG enrichment data suggested that oxidative stress, inflammation and apoptosis pathway were crucial in curcumin's alleviating AFB1-induced liver damage. In conclusion, curcumin prevented AFB1-induced oxidative stress, inflammation and apoptosis through LncRNAs. These results provide new insights for unveiling the protective mechanisms of curcumin against AFB1-induced liver damage.
Assuntos
Aflatoxina B1/toxicidade , Curcumina/farmacologia , Fígado/efeitos dos fármacos , Substâncias Protetoras/farmacologia , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Doença Hepática Crônica Induzida por Substâncias e Drogas/metabolismo , Doença Hepática Crônica Induzida por Substâncias e Drogas/patologia , Galinhas/metabolismo , Dieta , Inflamação/metabolismo , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , RNA Longo não Codificante/metabolismo , RNA Longo não Codificante/farmacologiaRESUMO
BACKGROUND: Apramycin is used exclusively for the treatment of Escherichia coli (E.coli) infections in swine around the world since the early 1980s. Recently, many research papers have demonstrated that apramycin has significant in vitro activity against multidrug-resistant E.coli isolated in hospitals. Therefore, ensuring the proper use of apramycin in veterinary clinics is of great significance of public health. The objectives of this study were to develop a wild-type cutoff for apramycin against E.coli using a statistical method recommended by Clinical and Laboratory Standards Institute (CLSI) and to investigate the prevalence of resistance genes that confer resistance to apramycin in E. coli. RESULTS: Apramycin susceptibility testing of 1230 E.coli clinical isolates from swine were determinded by broth microdilution testing according to the CLSI document M07-A9. A total number of 310 E.coli strains from different minimum inhibitory concentration (MIC) subsets (0.5-256 µg/mL) were selected for the detection of resistance genes (aac(3)-IV; npmA; apmA) in E. coli by PCR. The percentage of E. coli isolates at each MIC (0.5, 1, 2, 4, 8, 16, 32, 64, 128, and 256 µg/mL) was 0.08, 0.08, 0.16, 2.93, 31.14, 38.86, 12.85, 2.03, 1.46, and 10.41%. The MIC50 and MIC90 were 16 and 64 µg/mL. All the 310 E.coli isolates were negative for npmA and apmA gene, and only the aac(3)-IV gene was detected in this study. CONCLUSIONS: The wild-type cutoff for apramycin against E.coli was defined as 32 µg/mL. The prevelance of aac(3)-IV gene mainly concentrated in these MIC subsets 'MIC ≥ 64 µg/ mL', which indicates that the wild-type cutoff established in our study is reliable. The wild-type cutoff offers interpretion criteria of apramycin susceptibility testing of E.coli.
Assuntos
Farmacorresistência Bacteriana/genética , Infecções por Escherichia coli/veterinária , Escherichia coli/efeitos dos fármacos , Nebramicina/análogos & derivados , Animais , Antibacterianos/farmacologia , Escherichia coli/genética , Infecções por Escherichia coli/tratamento farmacológico , Testes de Sensibilidade Microbiana/veterinária , Nebramicina/farmacologia , Suínos , Doenças dos Suínos/tratamento farmacológico , Doenças dos Suínos/microbiologiaRESUMO
Mitophagy removes dysfunctional mitochondria and is known to play an important role in the pathogenesis of several diseases; however, the role of mitophagy in acute respiratory distress syndrome (ARDS) remains poorly understood. While we have previously demonstrated that polydatin (PD) improves lipopolysaccharide (LPS)-induced ARDS, the specific mechanism remains unclear. In present study, we aimed to determine whether PD activates Parkin-dependent mitophagy to protect against LPS-induced mitochondria-dependent apoptosis and lung injury. To establish the ARDS model, C57BL/6 mice were intratracheally injected with LPS (5 mg/kg) in vivo and Beas-2B cells were exposured to 0.5 mM LPS in vitro. Our results indicate that PD facilitates Parkin translocation to mitochondria and promotes mitophagy in ARDS-challenged mice and LPS-treated Beas-2B cells. However, PD-induced mitophagy was suppressed in Parkin-/- mice and Parkin siRNA transfected cells, indicating that PD activates Parkin-dependent mitophagy. Furthermore, the protective effects of PD against LPS-induced mitochondria-dependent apoptosis and lung injury were suppressed when Parkin was depleted both in vivo and in vitro. The inhibition of mitophagy with mitophagy inhibitor mitochondrial division inhibitor-1 in vivo and silencing of autophagy-related gene 7 in vitro also blocked the protective effects mediated by PD. Our data suggest that Parkin-dependent mitophagy induced by PD provides protection against mitochondria-dependent apoptosis in ARDS.
Assuntos
Apoptose/efeitos dos fármacos , Glucosídeos/uso terapêutico , Mitofagia/efeitos dos fármacos , Síndrome do Desconforto Respiratório/tratamento farmacológico , Estilbenos/uso terapêutico , Ubiquitina-Proteína Ligases/metabolismo , Animais , Avaliação Pré-Clínica de Medicamentos , Medicamentos de Ervas Chinesas/farmacologia , Medicamentos de Ervas Chinesas/uso terapêutico , Fallopia japonica , Glucosídeos/farmacologia , Masculino , Camundongos Endogâmicos C57BL , Fitoterapia , Síndrome do Desconforto Respiratório/metabolismo , Estilbenos/farmacologiaRESUMO
BACKGROUND: Improper use of antimicrobials results in poor treatment and severe bacterial resistance. Breakpoints are routinely used in the clinical laboratory setting to guide clinical decision making. Therefore, the objective of this study was to establish antimicrobial susceptibility breakpoints for danofloxacin against Escherichia coli (E.coli), which is an important pathogen of digestive tract infections. RESULTS: The minimum inhibitory concentrations (MICs) of 1233 E. coli isolates were determined by the microdilution broth method in accordance with the guidelines in Clinical and Laboratory Standards Institute (CLSI) document M07-A9. The wild type (WT) distribution or epidemiologic cutoff value (ECV) was set at 8 µg/mL with statistical analysis. Plasma drug concentration data were used to establish pharmacokinetic (PK) model in swine. The in vitro time kill test in our study demonstrated that danofloxacin have concentration dependent activity against E.coli. The PK data indicated that danofloxacin concentration in plasma was rapidly increased to peak levels at 0.97 h and remained detectable until 48 h after drug administration. The pharmacodynamic cutoff (COPD) was determined as 0.03 µg/mL using Monte Carlo simulation. To the best of our knowledge, this is the first study to establish the ECV and COPD of danofloxacin against E.coli with statistical method. CONCLUSIONS: Compared to the COPD of danofloxacin against E.coli (0.03 µg/mL), the ECV for E.coli seemed reasonable to be used as the final breakpoint of danofloxacin against E.coli in pigs. Therefore, the ECV (MIC ≤8 µg/mL) was finally selected as the optimum danofloxacin susceptibility breakpoint for swine E.coli. In summary, this study provides a criterion for susceptibility testing and improves prudent use of danofloxacin for protecting public health.
Assuntos
Antibacterianos/uso terapêutico , Infecções por Escherichia coli/veterinária , Escherichia coli/efeitos dos fármacos , Fluoroquinolonas/uso terapêutico , Doenças dos Suínos/tratamento farmacológico , Animais , Antibacterianos/administração & dosagem , Antibacterianos/sangue , Antibacterianos/farmacocinética , Infecções por Escherichia coli/tratamento farmacológico , Fluoroquinolonas/administração & dosagem , Fluoroquinolonas/sangue , Fluoroquinolonas/farmacocinética , Testes de Sensibilidade Microbiana/veterinária , Método de Monte Carlo , Suínos , Doenças dos Suínos/microbiologiaRESUMO
BACKGROUND: There are increasing evidence that left ventricle diastolic dysfunction is the initial functional alteration in the diabetic myocardium. In this study, we hypothesized that alogliptin prevents diastolic dysfunction and preserves left ventricular mitochondrial function and structure in diabetic rabbits. METHODS: A total of 30 rabbits were randomized into control group (CON, n = 10), alloxan-induced diabetic group (DM, n = 10) and alogliptin-treated (12.5 mg/kd/day for 12 weeks) diabetic group (DM-A, n = 10). Echocardiographic and hemodynamic studies were performed in vivo. Mitochondrial morphology, respiratory function, membrane potential and reactive oxygen species (ROS) generation rate of left ventricular tissue were assessed. The serum concentrations of glucagon-like peptide-1, insulin, inflammatory and oxidative stress markers were measured. Protein expression of TGF-ß1, NF-κB p65 and mitochondrial biogenesis related proteins were determined by Western blotting. RESULTS: DM rabbits exhibited left ventricular hypertrophy, left atrial dilation, increased E/e' ratio and normal left ventricular ejection fraction. Elevated left ventricular end diastolic pressure combined with decreased maximal decreasing rate of left intraventricular pressure (- dp/dtmax) were observed. Alogliptin alleviated ventricular hypertrophy, interstitial fibrosis and diastolic dysfunction in diabetic rabbits. These changes were associated with decreased mitochondrial ROS production rate, prevented mitochondrial membrane depolarization and improved mitochondrial swelling. It also improved mitochondrial biogenesis by PGC-1α/NRF1/Tfam signaling pathway. CONCLUSIONS: The DPP-4 inhibitor alogliptin prevents cardiac diastolic dysfunction by inhibiting ventricular remodeling, explicable by improved mitochondrial function and increased mitochondrial biogenesis.
Assuntos
Diabetes Mellitus Experimental/tratamento farmacológico , Cardiomiopatias Diabéticas/prevenção & controle , Inibidores da Dipeptidil Peptidase IV/farmacologia , Mitocôndrias Cardíacas/efeitos dos fármacos , Piperidinas/farmacologia , Uracila/análogos & derivados , Disfunção Ventricular Esquerda/prevenção & controle , Função Ventricular Esquerda/efeitos dos fármacos , Animais , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/fisiopatologia , Diástole/efeitos dos fármacos , Fibrose , Hipertrofia Ventricular Esquerda/etiologia , Hipertrofia Ventricular Esquerda/metabolismo , Hipertrofia Ventricular Esquerda/fisiopatologia , Hipertrofia Ventricular Esquerda/prevenção & controle , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Mitocôndrias Cardíacas/metabolismo , Mitocôndrias Cardíacas/patologia , Fator 1 Nuclear Respiratório/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Coativador 1-alfa do Receptor gama Ativado por Proliferador de Peroxissomo/metabolismo , Coelhos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Volume Sistólico/efeitos dos fármacos , Fatores de Transcrição/metabolismo , Uracila/farmacologia , Disfunção Ventricular Esquerda/etiologia , Disfunção Ventricular Esquerda/metabolismo , Disfunção Ventricular Esquerda/fisiopatologia , Pressão Ventricular/efeitos dos fármacos , Remodelação Ventricular/efeitos dos fármacosRESUMO
Aflatoxin B1 (AFB1) is an unavoidable environmental toxin. The accumulation of AFB1 and its metabolites in the liver poses a threat to both human and animal health. Curcumin exhibits anti-oxidative, anti-tumor, and anti-inflammatory properties. There is no report on the mechanism regarding how curcumin relived liver necroptosis in chickens induced by AFB1 based on the regulatory network of ceRNA. To explore this, we performed transmission electron microscopy and sequenced lncRNA and mRNA in chicken livers treated with AFB1 and/or curcumin for 28 d in vivo. We observed substantial alterations in the lncRNA and mRNA expression profiles within the chicken liver, indicating that curcumin can mitigate AFB1-induced necroptosis both in vivo and in vitro. Further analysis, including the establishment of an lncRNA-miRNA-mRNA network and the utilization of a dual luciferase reporter assay, revealed that LOC769044 acts as a competing endogenous RNA (ceRNA) for miR-1679. In addition, STAT1 was identified as a direct target of miR-1679. Modulating miR-1679 levels through overexpression, and silencing LOC769044 and STAT1, effectively reversed the necroptotic effects induced by AFB1, a reversal that was also observed with curcumin supplementation. In conclusion, our data demonstrate that curcumin alleviates AFB1-induced liver necroptosis through the LOC769044/miR-1679/STAT1 signaling axis. This study suggests that LOC769044 may serve as a novel therapeutic target for managing AFB1-mediated liver toxicity.
Assuntos
Aflatoxina B1 , Galinhas , Curcumina , Fígado , MicroRNAs , Necroptose , Fator de Transcrição STAT1 , Animais , Curcumina/farmacologia , Aflatoxina B1/toxicidade , MicroRNAs/metabolismo , MicroRNAs/genética , Necroptose/efeitos dos fármacos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fator de Transcrição STAT1/metabolismo , Fator de Transcrição STAT1/genética , Doenças das Aves Domésticas/induzido quimicamente , Doenças das Aves Domésticas/prevenção & controle , Proteínas Aviárias/metabolismo , Proteínas Aviárias/genéticaRESUMO
In this study, we examined the protective effects of curcumin against the AFB1-induced immune response of and pathological changes in broilers. Histopathology examinations showed that at day 28, AFB1 (5 mg/kg) exposure leads to severe histological changes in the spleen, thymus and bursa of Fabricius with a decrease in the number and karyoplasmic area ratio of plasma cells. Curcumin alleviated the AFB1-induced immune organs' damage as well as the changes in plasma cells in a dose-dependent manner. RT-PCR data showed that AFB1 significantly downregulated the IL-2 and IFN-γ mRNA expression levels in the thymus, spleen and bursa of Fabricius. However, curcumin supplementation improved the AFB1-induced immune organs' damage via upregulated cytokines' expression. Intriguingly, similar trends were noticed in abnormal morphological changes and the immune response at day 35 after the withdrawal of AFB1 and curcumin from the diet, suggesting the protective effects and immunomodulatory function against AFB1 in broilers. The current study provides a scientific experimental basis for the application of curcumin as a therapeutic drug or additive in animal husbandry productive practice.
Assuntos
Aflatoxina B1 , Curcumina , Aflatoxina B1/toxicidade , Animais , Galinhas , Curcumina/farmacologia , Suplementos Nutricionais , Imunidade , Interleucina-2/metabolismo , RNA Mensageiro/metabolismoRESUMO
Colistin is the last line of defense for the treatment of multidrug-resistant gram-negative bacterial infections. However, colistin resistance is gradually increasing worldwide, with resistance commonly regulated by two-component system and mcr gene. Thus, this study aimed to investigate molecular epidemiology and colistin-resistant mechanism of mcr-positive and mcr-negative Escherichia coli isolates from animal in Sichuan Province, China. In this study, a total of 101 colistin-resistant E. coli strains were isolated from 300 fecal samples in six farms in Sichuan Province. PCR was used to detect mcr gene (mcr-1 to mcr-9). The prevalence of mcr-1 in colistin-resistant E. coli was 53.47% (54/101), and the prevalence of mcr-3 in colistin-resistant E. coli was 10.89% (11/101). The colistin-resistant E. coli and mcr-1-positive E. coli showed extensive antimicrobial resistance profiles. For follow-up experiments, we used 30 mcr-negative and 30 mcr-1-positive colistin-resistant E. coli isolates and E. coli K-12 MG1655 model strain. Multi-locus sequence typing (MLST) of 30 strains carrying mcr-1 as detected by PCR identified revealed six strains (20%) of ST10 and three strains (10%) of each ST206, ST48, and ST155 and either two (for ST542 and 2539) or just one for all other types. The conjugation experiment and plasmid replicon type analysis suggest that mcr-1 was more likely to be horizontally transferred and primarily localized on IncX4-type and IncI2-type plasmid. The ST diversity of the mcr-1 indicated a scattered and non-clonal spreading in mcr-1-positive E. coli. Twenty-eight mcr-negative colistin-resistant E. coli isolates carried diverse amino acid alterations in PmrA, PmrB, PhoP, PhoQ, and MgrB, whereas no mutation was found in the remaining isolates. The finding showed the high prevalence of colistin resistance in livestock farm environments in Sichuan Province, China. Our study demonstrates that colistin resistance is related to chromosomal point mutations including the two-component systems PhoP/PhoQ, PmrA/PmrB, and their regulators MgrB. These point mutations may confer colistin resistance in mcr-negative E. coli. These findings help in gaining insight of chromosomal-encoded colistin resistance in E. coli.
RESUMO
Sepsis-induced acute lung injury (ALI) is a serious sepsis complication and the prevailing cause of death. Circulating plasma exosomes might exert a key role in regulating intercellular communication between immunological and structural cells, as well as contributing to sepsis-related organ damage. However, the molecular mechanisms by which exosome-mediated intercellular signaling exacerbate ALI in septic infection remains undefined. Therefore, we investigated the effect of macrophage-derived exosomal APN/CD13 on the induction of epithelial cell necrosis. Exosomal APN/CD13 levels in the plasma of septic mice and patients with septic ALI were found to be higher. Furthermore, increased plasma exosomal APN/CD13 levels were associated with the severity of ALI and fatality in sepsis patients. We found remarkably high expression of APN/CD13 in exosomes secreted by LPS-stimulated macrophages. Moreover, c-Myc directly induced APN/CD13 expression and was packed into exosomes. Finally, exosomal APN/CD13 from macrophages regulated necroptosis of lung epithelial cells by binding to the cell surface receptor TLR4 to induce ROS generation, mitochondrial dysfunction and NF-κB activation. These results demonstrate that macrophage-secreted exosomal APN/CD13 can trigger epithelial cell necroptosis in an APN/CD13-dependent manner, which provides insight into the mechanism of epithelial cell functional disorder in sepsis-induced ALI.
Assuntos
Lesão Pulmonar Aguda , Sepse , Lesão Pulmonar Aguda/complicações , Animais , Antígenos CD13/farmacologia , Células Epiteliais , Humanos , Pulmão , Macrófagos , Camundongos , Necroptose , Sepse/complicaçõesRESUMO
Improper use of antibiotics results in poor treatment and severe bacterial resistance. In this study, ultrafiltration probes were successfully placed in the ileum of piglets with the aid of anesthetic. After the fluoroquinolone antimicrobial drug danofloxacin (DAN) was intramuscularly administered, blood and ileum ultrafiltrate were collected at different time points and then determined by High Performance Liquid Chromatography (HPLC). Pharmacokinetics (PK) parameters for plasma and ileum ultrafiltrate were calculated by WinNonlin software. The DAN concentration in ileum ultrafiltrate was much higher than that in plasma during the period 1.2-48 h. The DAN concentration in plasma reached its maximum at 1.10 ± 0.03 h, but reached at 6.00 ± 0.00 h in the ileum ultrafiltrate. The mean Cmax of the ileum is 13.59 times that of plasma. The elimination half-life (T1/2ß) in the ileum ultrafiltrate (6.84 ± 1.49 h) was shorter than those in plasma (7.58 ± 3.20 h). The MIC, MBC and MPC of DAN in MH broth against Escherichia coli (O158) were 0.5 µg/mL, 0.5 µg/mL and 4 µg/mL, respectively. Both in vitro and ex vivo kill curves indicated that the killing mechanism of DAN against E. coli is concentration-dependent. The AUC/MPC ratio is 21.33 ± 2.14. Mean PK/PD index (AUC24h/MIC) for ileum ultrafiltrate that achieved bacteriostatic, bactericidal, and eradication were 99.85, 155.57, and 218.02 h, respectively. Three different dosages (1.49 mg/kg, 2.42 mg/kg, and 3.24 mg/kg) were calculated respectively based on AUC24h/MIC ratio above, which might provide a novel approach to the rational design of dosage schedules.
Assuntos
Infecções por Escherichia coli/tratamento farmacológico , Escherichia coli/efeitos dos fármacos , Fluoroquinolonas/farmacologia , Fluoroquinolonas/farmacocinética , Íleo/efeitos dos fármacos , Modelos Biológicos , Animais , Animais Recém-Nascidos , Anti-Infecciosos/farmacocinética , Anti-Infecciosos/farmacologia , Infecções por Escherichia coli/microbiologia , Íleo/microbiologia , Masculino , Suínos , Distribuição Tecidual , UltrafiltraçãoRESUMO
Mitophagy is involved in sepsis-induced acute lung injury (ALI). Bcl-2 family proteins play an important role in mitochondrial homeostasis. However, whether targeting Bcl-2 proteins (Bcl-2 and Bad) could influence mitophagy in ALI remains unclear. In this study, lipopolysaccharide (LPS) was used to induce injury in A549 cells and ALI in mice. LPS treatment resulted in elevated cell apoptosis, enhanced mitophagy, decreased Bcl-2 expression, increased Bad expression, and activation of PINK1/Parkin signaling in cells and lung tissues. Both Bcl-2 overexpression and Bad knockdown attenuated LPS-induced injury, inhibited cell apoptosis and mitophagy, and improved survival. Atg5 knockout (KO) inhibited LPS-induced cell apoptosis. Furthermore, Bcl-2 proteins regulated mitophagy by modulating the recruitment of Parkin from the cytoplasm to mitochondria via direct protein-protein interactions. These results were further confirmed in Park2 KO cells and Park2-/- mice. This is the first study to demonstrate that Bcl-2 proteins regulated mitophagy in LPS-induced ALI via modulating the PINK1/Parkin signaling pathway, promoting new insights into the mechanisms and investigation of therapeutic strategies for a septic patient with ALI.
Assuntos
Lesão Pulmonar Aguda/induzido quimicamente , Lipopolissacarídeos/efeitos adversos , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Ubiquitina-Proteína Ligases/genética , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Mitofagia , Transdução de Sinais , Ubiquitina-Proteína Ligases/metabolismoRESUMO
The aim of this study is to evaluate the safety and efficacy of gamithromycin (GAM) for the treatment of naturally occurring bacterial swine respiratory disease (SRD) administered IM. A total of 240 pigs (nine-weeks old) were selected from two sites in Heilongjiang Province of China. The pigs showed severe signs of respiratory disease. Among them, 120 pigs were randomly divided into 4 groups of low dose (3â¯mg/kg), middle dose (6â¯mg/kg), high dose (12â¯mg/kg) GAM IM injection and 2.5â¯mg/kg tulathromycin (TUL) IM injection (positive control group) for phase II clinical trial to screen effective therapeutic dose. The other 120 pigs were randomly divided into 2 groups of 6â¯mg/kg GAM IM injection and 2.5â¯mg/kg TUL IM injection (positive control group) for phase III clinical trial to further confirm the efficacy. Animals were clinically observed daily for 14â¯days after treatment initiation. The predominant pathogens present in pretreatment respiratory tract samples were Streptococcus suis (S. suis) and Actinobacillus pleuropneumoniae (A. pleuropneumoniae). Haemophilus parasuis (H. parasuis) and Pasteurella multocida (P. multocida) were also found in the respiratory tract. All isolates were subjected to in vitro sensitivity testing and the measured minimal inhibitory concentrations (MIC) of GAM were from 0.0625⯵g/mL to 8⯵g/mL. In all treatment groups, rectal temperature dropped and clinical index (mental status and respiratory symptom) significantly improved after treatment (Pâ¯≤â¯.05). As a result, 82.76% animals treated with the 6â¯mg/kg GAM injection were cured. This was significantly higher than that of 3â¯mg/kg GAM injection (Pâ¯≤â¯.05) and similar to that of 12â¯mg/kg GAM injection and 2.5â¯mg/kg TUL injection (Pâ¯>â¯.05) in phase II clinical trial. In phase III clinical trial, 80.70% of animals treated with the 6â¯mg/kg GAM injection were cured and the cure rate was similar to that of 2.5â¯mg/kg TUL injection (Pâ¯>â¯.05). In conclusion, we recommended a single dose (6â¯mg/kg) of GAM IM injection for the treatment of bacterial SRD.
Assuntos
Antibacterianos , Dissacarídeos , Compostos Heterocíclicos , Macrolídeos , Infecções Respiratórias , Doenças dos Suínos , Animais , Antibacterianos/administração & dosagem , Antibacterianos/uso terapêutico , Infecções Bacterianas/microbiologia , China , Dissacarídeos/administração & dosagem , Dissacarídeos/uso terapêutico , Relação Dose-Resposta a Droga , Compostos Heterocíclicos/administração & dosagem , Compostos Heterocíclicos/uso terapêutico , Macrolídeos/administração & dosagem , Macrolídeos/uso terapêutico , Infecções Respiratórias/tratamento farmacológico , Infecções Respiratórias/microbiologia , Infecções Respiratórias/veterinária , Suínos , Doenças dos Suínos/tratamento farmacológico , Doenças dos Suínos/microbiologiaRESUMO
BACKGROUND: Bupivacaine (BP) is commonly used as a local anaesthetic(LA) in the clinic, but it can also cause neurotoxicity, especially in patients with diabetes. Previous studies have found that high-glucose environments can aggravate BP-induced DNA damage in nerve cells. Ku70 is subunit of the DNA damage repair enzyme DNA-PK. This study was designed to determine whether high-glucose conditions enhance BP neurotoxicity and DNA damage by inhibiting Ku70 expression. METHODS: We examined the effect of BP on apoptosis and DNA damage in murine dorsal root ganglion (DRG) neurons under hyperglycaemic conditions. Untreated DRG cells and DRG cells pretreated with NU7441, a DNA-PK inhibitor, were cultured for 3 days under normal culture conditions or with 50â¯mM glucose, and the cells were then treated with BP for 3â¯h. DNA damage was investigated via comet assays, the ratio of early to late apoptotic cells was assessed by Annexin V-FITC/PI staining, and cell viability was measured by CCK-8 assays. The protein expression levels of DNA-PK, Ku70, Bax, Bcl-2 and γH2ax were measured by immunofluorescence or Western blotting. RESULTS: Compared to its effect under normal culture conditions, BP treatment led to decreased cell viability and increased DNA damage in DRG cells grown under high-glucose conditions. The rate of DRG cell apoptosis and the expression of γH2ax, the ratio of Bax to Bcl-2 also increased under the high-glucose conditions. Furthermore, Ku70 expression was inhibited. The DNA-PK inhibitor, NU7441, could significantly inhibit DNA-PK and Ku70 expression, simultaneously further aggravating BP-induced apoptosis and DNA damage under high-glucose conditions. CONCLUSION: These data indicate that hyperglycaemia may enhance BP-induced neurotoxicity and DNA damage by inhibiting the DNA repair protein Ku70.
Assuntos
Anestésicos Locais/toxicidade , Apoptose/efeitos dos fármacos , Bupivacaína/toxicidade , Cromonas/toxicidade , Inibidores Enzimáticos/toxicidade , Gânglios Espinais/efeitos dos fármacos , Glucose/toxicidade , Autoantígeno Ku/antagonistas & inibidores , Morfolinas/toxicidade , Síndromes Neurotóxicas/etiologia , Animais , Células Cultivadas , Dano ao DNA , Gânglios Espinais/enzimologia , Gânglios Espinais/patologia , Autoantígeno Ku/metabolismo , Camundongos , Síndromes Neurotóxicas/enzimologia , Síndromes Neurotóxicas/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
The regulation of intracellular mitochondria degradation is mediated by mitophagy. While studies have shown that mitophagy can lead to mitochondrial dysfunction and cell damage, the role of Mdivi-1 and mitophagy remains unclear in acute lung injury (ALI) pathogenesis. In this study, we demonstrated that Mdivi-1, which is widely used as an inhibitor of mitophagy, ameliorated acute lung injury assessed by HE staining, pulmonary microvascular permeability assay, measurement of wet/dry weight (W/D) ratio, and oxygenation index (PaO2/FiO2) analysis. Then, the mitophagy related proteins were evaluated by western blot. The results indicated that LPS-induced activation of mitophagy was inhibited by Mdivi-1 treatment. In addition, we found that Mdivi-1 protected A549 cells against LPS-induced mitochondrial dysfunction. We also found that Mdivi-1 reduced pulmonary cell apoptosis in the LPS-challenged rats and protected pulmonary tissues from oxidative stress (represented by the content of superoxide dismutase, malondialdehyde and lipid peroxides in lung). Moreover, Mdivi-1 treatment ameliorated LPS-induced lung inflammatory response and cells recruitment. These findings indicate that Mdivi-1 mitigates LPS-induced apoptosis, oxidative stress, and inflammation in ALI, which may be associated with mitophagy inhibition. Thus, the inhibition of mitophagy may represent a potential therapy for treating ALI.
Assuntos
Lesão Pulmonar Aguda/tratamento farmacológico , Mitocôndrias/metabolismo , Mitofagia/efeitos dos fármacos , Quinazolinonas/farmacologia , Células A549 , Lesão Pulmonar Aguda/induzido quimicamente , Lesão Pulmonar Aguda/patologia , Animais , Apoptose/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Lipopolissacarídeos/toxicidade , Masculino , Mitocôndrias/patologia , Estresse Oxidativo/efeitos dos fármacos , Ratos , Ratos Sprague-DawleyRESUMO
OBJECTIVE: This retrospective study was conducted to determine the prevalence and molecular epidemiology characteristics of carbapenem-resistant Escherichia coli (CRE). METHODS: A total of 593 Escherichia coli (E. coli) isolates were recovered from pigs and urban river from 2009 to 2014 in Heilongjiang Province of China. Forty CRE including 22 strains isolated from fecal samples of pigs and 18 strains isolated from water samples were selected. PCR detection of resistance determinants, multi-locus sequence typing (MLST), pulsed-field gel electrophoresis (PFGE), and phylogenetic groups were performed to characterize CRE isolates. Conjugation experiments, plasmid stability testing, PCR-based replicon typing (PBRT), and PCR mapping were conducted to analyze bla NDM-carrying plasmids. In vitro time-growth studies and competition experiments were carried out to assess the fitness impact of NDM carriage. RESULTS: Five NDM-1-positive E. coli isolates were identified from water samples. Genetic environment analysis revealed that a cluster of genes (ISAba125-bla NDM-1-ble MBL-ΔtrpF) was detected in all of the NDM-1-positive isolates. Conjugation assays showed that bla NDM-1 could be successfully transferred to E. coli J53 from 5 donor strains at frequencies of 4.6×10-5 to 2.6×10-2. The plasmids from all transconjugants belonged to different plasmid replicon types including IncA/C (n=2), IncFII (n=1) and IncX3 (n=2). In vitro time-growth studies revealed that bla NDM-1 did not have a significant impact on cell proliferation. Meanwhile, competition experiments showed that the acquisition of bla NDM-1 can place an energy burden on the bacterial host and incur fitness cost. However, plasmid stability testing showed that bla NDM-1-carrying plasmid remained stable in the hosts after seven passages without antimicrobial selection. CONCLUSION: The study revealed the early molecular epidemiology and dissemination characteristics of CRE. In addition, the overall antimicrobial resistance in E. coli recovered from water samples is higher than the strains isolated from fecal samples of pigs. Furthermore, we isolated and identified five NDM-1-producing E. coli strains from water samples.
RESUMO
INTRODUCTION: Atrial remodeling in the form of fibrosis is considered as the substrate for the development of atrial fibrillation (AF). The aim of this study was to investigate the effects of tolvaptan on chronic intermittent hypoxia (CIH) induced atrial remodeling and the mechanisms underlying such changes. METHODS: A total of 45 Sprague-Dawley rats were randomized into three groups: Control group, CIH group, CIH with tolvaptan treatment (CIH-T) group (n = 15). CIH rats were subjected to CIH 6 hour/d for 30 days, and CIH-T rats were administrated tolvaptan while they received CIH. After the echocardiography examination, rats were sacrificed in the 31 days. In each group, 5 rats were randomly selected for isolated heart electrophysiology testing, for other 10 rats, the tissues of atria were sampled for histological and molecular biological experiments, Masson's trichrome staining was used to evaluate the extent of atrial fibrosis, expression levels of microRNA-21 (miR-21), Sprouty-1 (Spry1), phosphatase, and tensin homolog (PTEN), extracellular regulated protein kinase (ERK), phospho-extracellular regulated protein kinase (p-ERK), matrix metalloprotein 9 (MMP-9), phosphoinositide 3-kinase (PI3K), protein kinase B (AKT), phospho-protein kinase B (p-AKT), nuclear factor-k-gene binding (NF-κB), phosphor-nuclear factor-k-gene binding (p-NF-κB) were measured. RESULTS: Compared to the Control rats, CIH rats showed higher atrial interstitial collagen deposition and AF inducibility, mRNA levels of miR-21, MMP-9, PI3K, AKT, and protein levels of ERK, p-ERK, MMP-9, NF-κB, p-NF-κB were significantly increased, whereas mRNA levels of Spry1, ERK, and protein levels of Spry1, PTEN, PI3K, AKT, p-AKT were significantly decreased. Treatment with tolvaptan attenuated CIH-induced atrial fibrosis, reduced AF inducibility, expression levels of miR-21 and its downstream factors were also improved. CONCLUSIONS: CIH-induced significant atrial remodeling in our rat model, which was attenuated by tolvaptan. These changes may be explained due to alterations in miR-21/Spry1/ERK/MMP-9, miR-21/PTEN/PI3K/AKT, and NF-κB pathways by tolvaptan.