RESUMO
Neurobrucellosis is a serious central nervous system (CNS) inflammatory disorder caused by Brucella, and outer membrane protein-31 (Omp31) plays an important role in Brucella infection. This study aims to determine whether Omp31 can induce autophagy in BV-2 microglia. Another goal of the study is to further examine the effect of autophagy on the nuclear transcription factor κB (NF-κB) p65 signaling pathway. We observed that Omp31 stimulated autophagy by increasing microtubule-associated protein 1 light chain 3B (LC3B-II) levels and inducing autophagosome formation at 6 h and 12 h. Concomitantly, Omp31 induced tumor necrosis factor-alpha (TNF-α) and interleukin-6 (IL-6) expression in a time-dependent manner but reduced the expression of TNF-α at 6 h. We utilized Omp31 with or without rapamycin or 3-methyladenine (3-MA) to treat BV-2 microglia, and it demonstrated further that Omp31 induced autophagy by promoting LC3B-II, Beclin-1 proteins expression and inhibiting the p62 protein levels. Furthermore, we explored the effects of autophagy on the NF-κB p65 pathway through western blot analysis, RT-qPCR assay, enzyme-linked immunosorbent assay (ELISA) and immunofluorescence. The data suggest that Omp31 as well as rapamycin, the autophagy inducer, can decrease TNF-α levels through the inhibition of the NF-κB p65 signaling pathway. Taken together, Omp31 can function as a catalyst in both autophagy induction and NF-κB p65 signal inhibition. Furthermore, Omp31-induced autophagy may inhibit the expression of TNF-α by negatively regulating NF-κB p65 signaling pathway.
Assuntos
Autofagia , Proteínas da Membrana Bacteriana Externa/metabolismo , Brucella/fisiologia , Brucelose/patologia , Microglia/patologia , NF-kappa B/antagonistas & inibidores , Animais , Proteínas da Membrana Bacteriana Externa/genética , Brucelose/metabolismo , Brucelose/microbiologia , Interleucina-6/metabolismo , Microglia/metabolismo , Microglia/microbiologia , Transdução de Sinais , Fator de Necrose Tumoral alfa/metabolismoRESUMO
Histidine triad nucleotidebinding protein (HINT) belongs to the histidine triad protein family. Recent studies have demonstrated that HINT1 and HINT2 both play a pivotal role in cancer growth. However, the functions of HINT3 in various types of cancer, including breast cancer (BRCA), have not yet been fully elucidated. In the present study, the role of HINT3 in BRCA was investigated. Based on The Cancer Genome Atlas and reverse transcriptionquantitative PCR analyses, HINT3 was found to be decreased in BRCA tissues. In vitro, HINT3 knockdown promoted the proliferation and colony formation of, and 5ethynyl2'deoxyuridine incorporation in MCF7 and MDAMB231 BRCA cells. By contrast, HINT3 overexpression suppressed DNA synthesis and the proliferation of both cell lines. Apoptosis was also found to be modulated by HINT3. In vivo, HINT3 ectopic expression attenuated the tumorigenesis of MDAMB231 and MCF7 cells in a mouse tumor xenograft model. Furthermore, HINT3 silencing or overexpression also enhanced or inhibited, respectively, the migratory capacity of the MCF7 and MDAMB231 cells. Finally, HINT3 upregulated phosphatase and tensin homolog (PTEN) at the transcriptional level, which resulted in the inactivation of AKT/mammalian target of rapamycin (mTOR) signaling both in vitro and in vivo. Taken together, the present study demonstrates that HINT3 inhibits the activation of the PTEN/AKT/mTOR signaling pathway, and suppresses the proliferation, growth, migration and tumor development of MCF7 and MDAMB231 BRCA cells.
Assuntos
Neoplasias da Mama , Proteínas Proto-Oncogênicas c-akt , Animais , Feminino , Humanos , Camundongos , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Carcinogênese/genética , Linhagem Celular Tumoral , Proliferação de Células/genética , Transformação Celular Neoplásica , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , PTEN Fosfo-Hidrolase/genética , PTEN Fosfo-Hidrolase/metabolismo , Transdução de Sinais/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/metabolismo , Ativação TranscricionalRESUMO
Neurobrucellosis is a chronic complication of human brucellosis that is caused by the presence of Brucella spp in the central nervous system (CNS) and the inflammation play a key role on the pathogenesis. Doxycycline (Dox) is a widely used antibiotic that induces apoptosis of bacteria-infected cells. However, the mechanisms of Brucella inhibition of microglial apoptosis and Dox induction of apoptosis are still poorly understood. In this study, we found that Brucella suis S2 strain (B. suis S2) increased calreticulin (CALR) protein levels and inhbited HMC3 cell apoptosis. Hence, we constructed two HMC3 cell line variants, one with stable overexpression (HMC3-CALR) and one with low expression of CALR (HMC3-sh-CALR). CALR was found to decrease levels of p-JNK and p-p53 proteins, as well as suppress apoptosis in HMC3 cells. These findings suggest that CALR suppresses apoptosis by inhibiting the JNK/p53 signaling pathway. Next, we treated HMC3, HMC3-CALR and HMC3-sh-CALR cell lines with B. suis S2 or Dox. Our results demonstrate that B. suis S2 restrains the JNK/p53 signaling pathway to inhibit HMC3 cell apoptosis via increasing CALR protein expression, while Dox plays the opposite role. Finally, we treated B. suis S2-infected HMC3 cells with Dox. Our results confirm that Dox induces JNK/p53-dependent apoptosis in B. suis S2-infected HMC3 cells through inhibition of CALR protein expression. Taken together, these results reveal that CALR and the JNK/p53 signaling pathway may serve as novel therapeutic targets for treatment of neurobrucellosis.
Assuntos
Brucella suis , Brucelose , Apoptose , Calreticulina , Doxiciclina , Humanos , Sistema de Sinalização das MAP Quinases , Microglia , Proteína Supressora de Tumor p53RESUMO
The lncRNAs have been made certain to take part in the development of most cancers in multiple ways. Here, our purpose is to making observation of the biological role and function of lncRNA CDKN2B-AS1 in human breast cancer. Twenty-eight pairs of breast cancer tissue and adjacent normal tissue from breast cancer patients were used to investigate the expression of CDKN2B-AS1 by qRT-PCR. And a lentivirus-shRNA guided CDKN2B-AS1 were to reduce its expression. The function of CDKN2B-AS1 was analyzed using a series of in vitro assays. Meanwhile, the xenograft model was used to further explicate the role of CDKN2B-AS1 in breast cancer. As for the results, there is a relative rich expression of CDKN2B-AS1 in breast cancer tissues compared with the corresponding adjacent normal tissues. Compared with the human breast epithelial cell line, the abundant expression of CDKN2B-AS1 in breast cancer cells were revealed as well. Then, knockdown CDKN2B-AS1 inhibited the malignant biological behaviors of MCF7 and T47D cells. In mechanism, CDKN2B-AS1 sponged the miR-122-5p to regulate STK39 expression. Furthermore, the inhibition effect with sh-CDKN2B-AS1 on breast cancer cells was alleviated by miR-122-5p inhibitor. Last, an in vivo model also confirmed that knockdown CDKN2B-AS1 retarded the growth of breast cancer. Our data concluded that knockdown of CDKN2B-AS1 suppresses the progression of breast cancer by miR-122-5p/STK39 axis.
Assuntos
Neoplasias da Mama , MicroRNAs/genética , Proteínas Serina-Treonina Quinases/genética , RNA Longo não Codificante/genética , Animais , Mama/metabolismo , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Progressão da Doença , Feminino , Técnicas de Silenciamento de Genes , Humanos , Camundongos , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Proteínas Serina-Treonina Quinases/metabolismo , RNA Longo não Codificante/metabolismoRESUMO
OBJECTIVE: To investigate the changes and clinical significances of intestinal fatty acid binding protein (I-FABP) and D-lactic acid levels in early intestinal injury of patients with sepsis and septic shock. METHODS: A prospective observational study was conducted. Thirty septic patients (septic group) and 30 septic shock patients (septic shock group) were admitted to the intensive care unit (ICU) of General Hospital of Ningxia Medical University from August 2018 to December 2018, and 20 healthy adults were served as healthy control group. Serum samples were collected within 24 hours after ICU admission in septic shock and septic groups, and in healthy control group during physical examination. The serum I-FABP, D-lactic acid, endotoxin, hypersensitive C-reactive protein (hs-CRP), interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α) and lactic acid (Lac) were determined. Gender and age of all subjects, and basic diseases, the main area of infection and acute physiology and chronic health evaluation II (APACHE II) scores within 24 hours after ICU admission of all patients were recorded. At the same time, the survival of the patients was followed up for 28 days. Spearman correlation analysis was used to analyze the correlation between serum I-FABP, D-lactic acid and other parameters. Risk factors of death in patients with sepsis and septic shock were screened by multivariate Logistic regression analysis of bicategorized variables. RESULTS: There was no significant difference in gender or age among the groups, as well as in the proportion of basic diseases, celiac infection or non-celiac infection between the sepsis group and the septic shock group, indicating that the general clinical baseline data among the groups were comparable. Serum levels of I-FABP and D-lactic acid in the sepsis group and the septic shock group were significantly higher than those in the healthy control group [I-FABP (µg/L): 27.46 (22.52, 34.39), 36.95 (29.82, 44.24) vs. 17.93 (14.65, 22.11), D-lactic acid (mg/L): 15.32 (9.84, 38.62), 27.95 (10.01, 47.69) vs. 9.38 (8.81, 14.48), all P < 0.01]. The serum level of I-FABP in the septic shock group was significantly higher than that in the sepsis group (P < 0.05), but the difference in serum D-lactic acid level between the two groups was not statistically significant (P > 0.05). Serum I-FABP level in the celiac infection group (n = 40) was significantly higher than that in the non-celiac infection group [n = 20; µg/L: 34.76 (27.46, 43.90) vs. 25.71 (20.55, 37.77), P < 0.01], but the difference in serum D-lactic acid level was not statistically significant [mg/L: 25.13 (9.83, 40.55) vs. 30.36 (10.17, 50.00), P > 0.05]. There was no significant difference in serum I-FABP or D-lactic acid levels between the survival group (n = 34) and the death group [n = 26; I-FABP (µg/L): 33.39 (25.20, 39.50) vs. 29.26 (22.50, 43.81), D-lactic acid (mg/L): 14.83 (9.71, 38.45) vs. 33.90 (11.93, 45.34), both P > 0.05]. Correlation analysis between serum I-FABP, D-lactic acid level and endotoxin, inflammatory factors, Lac and APACHE II score in septic and septic shock patients showed that only D-lactic acid was significantly positively correlated with TNF-α and Lac (r values were 0.455 and 0.406, respectively, both P < 0.01), while I-FABP was not correlated with endotoxin, inflammatory factors, Lac or APACHE II score. Multivariable Logistic regression analysis showed the APACHE II score was an independent risk factor to affect the prognosis (death for 28 days) of septic and septic shock patients [odds ratio (OR) = 1.248, 95% confidence interval (95%CI) = 1.091-1.427, P = 0.001], while I-FABP, D-lactic acid, endotoxin, hs-CRP, TNF-α, IL-6, and Lac had no impact on 28-day prognosis of patients. CONCLUSIONS: Serum I-FABP and D-lactic acid levels can evaluate early intestinal injury in patients with sepsis and septic shock, but neither of them is related to the prognosis of patients.
Assuntos
Proteínas de Ligação a Ácido Graxo/sangue , Mucosa Intestinal/metabolismo , Intestinos/lesões , Ácido Láctico/sangue , Sepse/complicações , APACHE , Adulto , Humanos , Prognóstico , Estudos Prospectivos , Sepse/sangueRESUMO
AIMS: The present study was aimed to investigate the neuroprotective effect of HSP70 against neuroinflammation in a rotenone-induced Parkinson's disease model. MATERIALS AND METHODS: In the present study, SH-SY5Y cells were treated with HSP70 (5-20â¯mg/L) for 72â¯h. Cell viability, reactive oxygen species (ROS) levels, mitochondrial membrane potential (MMP), levels of oxidative markers, mitochondrial fragmentation, apoptosis, and mRNA and protein expressions of signal transducer and activator of transcription (STAT)-3 and nuclear factor-kappa B (NF-κB) were assessed. KEY FINDINGS: Cells treated with 5, 10, 15, and 20â¯mg/L of HSP70 exhibited increased, by 61.7%, 70.3%, 84.6%, and 96.7%, respectively, in cell viability. ROS and lipid peroxidation levels decreased following treatment with HSP70, and reductions in glutathione (GSH), catalase, glutathione peroxidase (Gpx), and superoxide dismutase (SOD) levels were reversed following treatment with HSP70. Additionally, MMP levels were reduced by 29.7, 46.4, 79.5, and 125.2 relative units following treatment with 5-20â¯mg/L of HSP70, respectively. HSP70 treatment also decreased levels of fragmented mitochondria and apoptosis, and mRNA and protein expressions of NF-κB and STAT3 were reduced by >25%. SIGNIFICANCE: Taken together, these findings indicate that supplementation with HSP70s recovered cell viability and MMP and reduced levels of ROS, apoptosis, and mitochondrial fragmentation. Additionally, supplementation with HSP70 significantly reduced the expressions of STAT3 and NF-κB.
Assuntos
Regulação para Baixo/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/farmacologia , Inflamação/tratamento farmacológico , NF-kappa B/genética , Fármacos Neuroprotetores/farmacologia , Doença de Parkinson Secundária/tratamento farmacológico , Fator de Transcrição STAT3/genética , Apoptose/efeitos dos fármacos , Linhagem Celular , Humanos , Inflamação/genética , Doença de Parkinson Secundária/genética , RNA Mensageiro/genética , RotenonaRESUMO
Objective To construct a short hairpin RNA (shRNA) lentiviral of Mg2+/Mn2+-dependent protein phosphatase 1A (PPM1A) gene and establish mouse brain microglia cell line(BV2) which PPM1A was knocked down stably. Methods According to the Coding sequence (CDS) region of PPM1A gene, two pairs of short hairpin RNA (shRNA) sequences (shRNA1, shRNA2) were designed and cloned into GV493 vector, then it was transformed into competent Escherichia coli DH5α strain and cultured. The positive clones were picked for sequencing. Finally, lentiviral packaging and titer determination were performed. Two groups of lentiviruses were transfected into BV2 cells and screened with puromycin. Fluorescence microscopy and flow cytometry were used to observe the expression of green fluorescent protein (GFP), and the expression of PPM1A were evaluated by RT-qPCR and Western blot analysis. Results The PPM1A shRNA lentiviral expression vector was successfully constructed. The expression of GFP was more than 80%. Compared with the control group, the mRNA and protein levels of PPM1A were significantly decreased in shRNA1 and shRNA2 knockdown groups. Conclusion The PPM1A knockdown BV2 cell line was successfully constructed using the lentiviral shRNA.
Assuntos
Vetores Genéticos , Proteína Fosfatase 2C/metabolismo , Animais , Linhagem Celular , Lentivirus , Camundongos , Interferência de RNA , RNA Interferente Pequeno , TransfecçãoRESUMO
In order to improve the diagnosis of pathogenic bacteria in cerebrospinal fluid (CSF) with purulent meningitis, we developed a DNA microarray technique for simultaneous detection and identification of seven target bacterium. DNA were extracted from 24 CSF samples with purulent meningitis (or suspected purulent meningitis). The specific genes of each pathogen were chosen as the amplification target, performed the polymerase chain reaction (PCR), labeled with a fluorescence dye, and hybridized to the oligonucleotide probes on the microarray. There is no significant cross-hybridization fluorescent signal occurred in untargeted bacteria. There were 87.5% (21/24) positive results in DNA microarray compared with the 58.3% (14/24) of the CSF culture test. Of which 58.3% (14/24) of the patients with culture-confirmed purulent meningitis, 37.5% (9/24) patients who were not confirmed by culture test but were demonstrated by the clinical diagnosis and DNA microarray. Multiple bacterial infections were detected in 5 cases by the microarray. In addition, the number of gene copies was carried out to determine the sensitivity of this technique, which was shown to be 3.5 × 101 copies/µL. The results revealed that the microarray technique which target pathogens of the CSF specimen is better specificity, accuracy, and sensitivity than traditional culture method. The microarray method is an effective tool for rapidly detecting more target pathogens and identifying the subtypes of strains which can eliminate the impact of the different individuals with purulent meningitis for prompt diagnosis and treatment.
Assuntos
DNA Bacteriano/líquido cefalorraquidiano , Meningites Bacterianas/microbiologia , Análise de Sequência com Séries de Oligonucleotídeos , Análise Serial de Tecidos/métodos , Adolescente , Adulto , Idoso , Técnicas Bacteriológicas , Líquido Cefalorraquidiano/microbiologia , Criança , Pré-Escolar , DNA Bacteriano/isolamento & purificação , Feminino , Genes Bacterianos , Humanos , Lactente , Masculino , Meningites Bacterianas/líquido cefalorraquidiano , Meningites Bacterianas/diagnóstico , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase/métodos , Ribotipagem/métodos , Sensibilidade e Especificidade , Adulto JovemRESUMO
Objective To determine the influence of outer membrane protein 31 (Omp31) of B. melitensis on the formation of autophagosomes in BV-2 microglia cells. Methods BV-2 cells were treated for 6 hours with Omp31 at 0.17, 0.50, 1.50, 4.50, 13.50 µg/mL. Real-time quantitative PCR was used to detect the expression of microtubule associated protein 1 light chain 3B (LC3B) mRNA; Western blotting was applied to detect the expressions of LC3B II and LC3B proteins; transmission electron microscopy was performed to observe cell autophagy in each group. The levels of tumor necrosis factor α (TNF-α), interleukin 6 (IL-6) and IL-10 in the culture cell supernatant were determined with ELISA. Results Omp31 treatment resulted in an increase in the level of LC3B II protein, especially the most obvious increase in the ones treated by 0.5 µg/mL Omp31. The concentration of Omp31, being no more than 0.5 µg/mL, could promote the expression of LC3B mRNA, while more than 0.5 µg/mL Omp31, it could inhibit the expression of LC3B mRNA; 0.5 µg/mL Omp31 could induce the formation of more autophagosomes in BV-2 cells. Omp31 promoted the expressions of TNF-α and IL-6, and inhibited the expression of IL-10 in BV-2 cells. Conclusion Omp31 at the concentration of 0.5 µg/mL can significantly induce autophagy in BV-2 cells.
Assuntos
Autofagossomos/metabolismo , Autofagossomos/fisiologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Brucella melitensis/metabolismo , Microglia/metabolismo , Microglia/fisiologia , Animais , Linhagem Celular , CamundongosRESUMO
Omp25 protein, an outer membrane protein of Brucella, can cause damage to the central nervous system. As one type of macrophage, microglial cells play a role in immune surveillance and immune protection in the central nervous system; therefore, they are major targets of bacterial attack. The present study examined BV2 mouse microglial cells that were stimulated with different concentrations of Omp25 recombinant protein, and the secretion of inflammatory cytokines by the BV2 cells as well as their level of apoptosis were observed. The objective of the study was to preliminarily illustrate the possible mechanism that Omp25 uses to damage the central nervous system. Mouse BV2 microglial cells were incubated with different concentrations of Omp25 for 24 h, and an enzyme-linked immunosorbent assay (ELISA) was used to detect the secretion of the inflammatory cytokines interleukin (IL)-6, tumour necrosis factor (TNF)-α and HMGB1 (high mobility group box-1 protein); reverse transcription polymerase chain reaction (RT-PCR) was used to detect the expression of TLR4 (Toll-like receptor 4) mRNA; Annexin V-fluorescein isothiocyanate (FITC) double staining was used to detect apoptosis in the BV2 cells. After the BV2 cells were stimulated with different concentrations of Omp25, the levels of IL-6, TNF-α and HMGB1 was increased, and the difference was statistically significant compared with the control group (P<0.05). The secretion of TNF-α and HMGB1 showed a trend toward an initial increase followed by a decrease. The expression level of TLR4 mRNA was increased. Omp25 protein can inhibit apoptosis in BV2 cells. The outer membrane protein Omp25 of Brucella promotes microglial cells to secrete inflammatory cytokines and inhibit apoptosis. TLR4 may be involved in the immune response of the central nervous system to Brucella infection.