RESUMO
BACKGROUND: Current options to treat clinical relapse in inflammatory central nervous system (CNS) conditions such as cerebral ischemia-reperfusion injury are limited, and agents that are more effective are required. Disruption of the blood-brain barrier is an early feature of lesion formation that correlates with clinical exacerbation and facilitates the entry of inflammatory medium and inflammatory cells. Interleukin-1 receptor antagonist (IL-1RA) is a naturally occurring anti-inflammatory antagonist of the interleukin-1 (IL-1) family. The broad-spectrum anti-inflammatory effects of IL-1RA have been investigated against various forms of neuroinflammation. However, the effect of IL-1RA on blood-brain barrier disruption following ischemia-reperfusion has not been reported. METHODS: In this study, we investigated the effects of IL-1RA and a novel protein (IL-1RA-PEP) that was fused to IL-1RA with a cell penetrating peptide, on blood-brain barrier integrity, in male rats subjected to transient middle cerebral artery occlusion. RESULTS: After intravenous administration, IL-1RA-PEP (50 mg/kg) penetrated cerebral tissues more effectively than IL-1RA. Moreover, it preserved blood-brain barrier integrity, attenuated changes in expression and localization of tight junction proteins and matrix metalloproteinases, and enhanced angiogenesis in ischemic brain tissue. Further study suggested that the effects of IL-1RA-PEP on preserving blood-brain barrier integrity might be closely correlated with the p65/NF-κB pathway, as evidenced by the effects of the inhibitor JSH-23. CONCLUSIONS: Collectively, our results demonstrated that IL-1RA-PEP could effectively penetrate the brain of rats with middle cerebral artery occlusion and ameliorate blood-brain barrier disruption. This finding might represent its novel therapeutic potential in the treatment of the cerebral ischemia-reperfusion injury.
Assuntos
Barreira Hematoencefálica/metabolismo , Isquemia Encefálica/metabolismo , Cisteamina/análogos & derivados , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Peptídeos/metabolismo , Traumatismo por Reperfusão/metabolismo , Administração Intravenosa , Animais , Barreira Hematoencefálica/efeitos dos fármacos , Isquemia Encefálica/tratamento farmacológico , Cisteamina/administração & dosagem , Cisteamina/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/administração & dosagem , Masculino , Peptídeos/administração & dosagem , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/tratamento farmacológicoRESUMO
BACKGROUND: Current diagnostic methods for Schistosoma japonicum infection are insensitive for low-density infections. Therefore, a new diagnostic assay based on recombinase polymerase amplification (RPA) technology was established and assessed for field applification. METHODS: The S.japonicum RPA assay was developed to target highly repetitive retrotransposon SjR2 gene of S japonicum, and its sensitivity and specificity were assessed by serial dilution of S. japonicum genomic DNA and other related worm genomic DNA respectively. The RPA diagnostic validity was first evaluated in 60 fecal samples from healthy people and patients, and then compared with other diagnostic tests in 200 high-risk individuals living in endemic areas. RESULTS: The real time RPA assay could detect 0.9 fg S. japonicum DNA within 15 min and distinguish S. japonicum from other worms. The validity analysis of RPA for the detection of S. japonicum in stool samples from 30 S. japonicum-infected patients and 30 healthy persons indicated 100% sensitivity and specificity. When testing 200 fecal or serum samples from a high-risk population, the percentage sensitivity of RPA was 100%, whereas that of indirect hemagglutination assay (IHA) and enzyme-linked immunosorbent assay (ELISA) were 80.3% and 85.2% respectively. In addition, the RPA presented better consistency with the stool-based tests than IHA and ELISA. Overall, the RPA was superior to other detection methods with respect to detection time, sensitivity, and convenience. CONCLUSIONS: This is the first time we applied the RPA technology to the field evaluation of S. japonicum infection. And the results suggest that RPA-based assays can be used as a promising point-of-care test for the diagnosis of schistosomiasis.
Assuntos
Sistemas Automatizados de Assistência Junto ao Leito , Reação em Cadeia da Polimerase/métodos , Schistosoma japonicum/isolamento & purificação , Esquistossomose Japônica/diagnóstico , Adolescente , Adulto , Idoso , Animais , Estudos de Casos e Controles , China/epidemiologia , DNA de Helmintos/análise , Ensaio de Imunoadsorção Enzimática , Feminino , Testes de Hemaglutinação , Humanos , Masculino , Pessoa de Meia-Idade , Recombinases , Schistosoma japonicum/genética , Sensibilidade e Especificidade , Adulto JovemRESUMO
SAK-HV is an anti-atherosclerosis recombinant fusion protein developed by our lab. Our study determined that SAK-HV promoted macrophage proliferation, of which the mechanism was explored by both RAW264.7 cells and primary macrophages. Mass spectrometric analysis and co-immunoprecipitation were combined to screen the SAK-HV-interacting proteins in RAW264.7 cells. Confocal microscopy was adopted to detect the localization of SAK-HV in cells. The results indicated that SAK-HV triggered macrophage proliferation via the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinases (ERK) and c-Jun N-terminal kinases (JNK) pathways by its SAK-mutant functional domain. We screened out Uba1 as the SAK-HV-interacting protein in the RAW264.7 cells and discovered their co-localization in the cytoplasm and nucleus. Inhibiting Uba1 significantly decreased the SAK-HV-induced macrophage proliferation. Thus, we postulated an attractive model of ubiquitination, in which the interactions between Uba1 and specific E2 enzymes are blocked by its interaction with SAK-HV. Based on this model, we detected the decreased self-ubiquitination of MEKK1 after SAK-HV treatment and concluded that SAK-HV inhibits the self-ubiquitination of MEKK1 via its SAK-mutant functional domain to activate MAPK/ERK and JNK pathways, promoting macrophage proliferation. This conclusion highly supported our hypothesized model of ubiquitination at the level of Uba1, which may represent a novel paradigm to promote macrophage proliferation by using the E1 enzyme (Uba1) as a switch.
Assuntos
MAP Quinase Quinase Quinase 1/metabolismo , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Animais , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Camundongos , Mutação , Fosforilação , Domínios e Motivos de Interação entre Proteínas/genética , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Ubiquitinação/efeitos dos fármacosRESUMO
Receptor-interacting protein 2 (RIP2) is a member of the receptor interacting protein (RIP) family and plays an important role in the innate and adaptive immune responses. Overexpression of RIP2 mediates divergent signaling pathways including NF-κB activation and cell death. To further investigate the biological activity of RIP2 in vitro, a large amount of purified protein is required. For this purpose, the full length of RIP2 was cloned from human Ramos (human Burkitt lymphoma) tumor cells and inserted in a prokaryotic expression vector pET22b, and then the recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells. The expression of RIP2 was induced with IPTG. SDS-PAGE analysis showed that recombinant human RIP2 (rhRIP2) was mainly expressed as soluble fraction in the supernatant of the cell lysate. The recombinant protein was subsequently purified by His Trap FF crude to a purity of 90 %. MTT assay of the purified rhRIP2 showed its functional diversity in different cell lines, a specific inhibitory effect on MCF7 cells, but a promotion on the proliferation of Ramos cells. Furthermore, we identified that rhRIP2 could suppress activation of canonical NF-κB in MCF7 cells and activate non-canonical NF-κB signaling in Ramos cells, these data suggested that RIP2 participates in different signaling pathways contributing to its specific effects in vitro. Our results provided new clues to further explore the regulation mechanisms of RIP2 in tumorigenesis.
Assuntos
Expressão Gênica , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/genética , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/metabolismo , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Humanos , Células MCF-7 , NF-kappa B/metabolismo , Plasmídeos , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/isolamento & purificação , Proteína Serina-Treonina Quinase 2 de Interação com Receptor/farmacologia , Proteínas Recombinantes , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: The presence of transient receptor potential vanilloid 2 (TRPV2) in human peripheral blood cells may suggest a role under pathological conditions. The aim of this study was to explore the relationship between the expression profile of TRPV2 gene and childhood asthma in the north of China. The effects of allergens exposure on the expression of TRPV2 gene were also investigated. METHODS: Sixty asthmatics children confirmed by physician diagnosis and 60 healthy children as a control group were recruited. Serum total IgE and specific IgE were measured. Using quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR), TRPV2 was detected in total RNA extracted from peripheral blood lymphocytes. Student's t-test and chi-square test were used to analyze the relationship between TRPV2 transcript and different parameter variables on susceptibility of childhood asthma. Multiple logistic regression was used to analyze the associations between TRPV2 gene and allergens. RESULTS: The expression level of TRPV2 gene was increased 2.6 times in asthmatic children compared with controls (p < .01). The up-regulation of TRPV2 gene and sensitization to one of three the allergens-spring pollen, dust mite, and dog and cat hair-were correlated with childhood asthma. In addition, the hypersensitivity to spring pollen, cockroach, and dust mite and up-regulation of TRPV2 gene expression may be the risk factors for the childhood asthma in Beijing. CONCLUSIONS: The increased expression of TRPV2 gene in peripheral lymphocytes is closely correlated with childhood asthma in the north of China. This study provides a potential new biomarker of childhood asthma and lays the basis for further clarification of the pathogenesis underlying asthma.
Assuntos
Asma/metabolismo , Canais de Cátion TRPV/metabolismo , Alérgenos/imunologia , Asma/sangue , Asma/genética , Asma/imunologia , Biomarcadores/sangue , Estudos de Casos e Controles , Criança , Pré-Escolar , China , Feminino , Regulação da Expressão Gênica , Humanos , Imunoglobulina E/sangue , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/metabolismo , Modelos Logísticos , Masculino , RNA/química , RNA/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Canais de Cátion TRPV/sangue , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/imunologia , População UrbanaRESUMO
The potential of angiogenin (Ang) for clinical use has been highlighted in view of its important roles in inducing angiogenesis, facilitating cell proliferation, and inhibiting cell apoptosis. To produce soluble, correctly folded recombinant protein with a high yield, a DNA fragment encoding human Ang was inserted into eukaryotic expression vector pPIC9 and transformed into Pichia pastoris. The expression of recombinant human Ang (rhAng) accounted for about 70% of total secreted proteins. Purifying the Ang from the culture supernatant yielded 30 mg/L at 90% purity by chromatography with a SP Sepharose FF column. Biological assays indicated that rhAng can induce new blood-vessel formation, promote HeLa cell proliferation, increase Erk1/2 phosphorylation, and upregulate c-myc expression. Preparation of bioactive rhAng might lay the basis for further functional study, and might provide an effective strategy for large-scale production of soluble human Ang.
Assuntos
Membrana Corioalantoide/irrigação sanguínea , Expressão Gênica , Pichia/genética , Proteínas Recombinantes/isolamento & purificação , Ribonuclease Pancreático/isolamento & purificação , Animais , Bioensaio , Proliferação de Células/efeitos dos fármacos , Embrião de Galinha , Membrana Corioalantoide/efeitos dos fármacos , Cromatografia , Vetores Genéticos , Células HeLa , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Neovascularização Fisiológica , Fosforilação/efeitos dos fármacos , Fosforilação/genética , Pichia/metabolismo , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/farmacologia , Ribonuclease Pancreático/biossíntese , Ribonuclease Pancreático/farmacologia , Transformação GenéticaRESUMO
This study was aimed to rapidly identify and differentiate two main pathogens of the Mycobacterium tuberculosis complex: Mycobacterium tuberculosis subsp. tuberculosis and Mycobacterium bovis by a modified loop-mediated isothermal amplification (LAMP) assay. The reaction results could be evaluated by naked eye with two optimized closed tube detection methods as follows: adding the modified fluorescence dye in advance into the reaction mix so as to observe the color changes or putting a tinfoil in the tube and adding the SYBR Green I dye on it, then making the dye drop into the bottom of the tube by centrifuge after reaction. The results showed that the two groups of primers used jointly in this assay could successfully identify and differentiate Mycobacterium tuberculosis subsp. tuberculosis and Mycobacterium tuberculosis bovis. Sensitivity test displayed that the modified LAMP assay with the closed tube system could determine the minimal template concentration of 1 copy/µl, which was more sensitive than that of routine PCR. The advantages of this LAMP method for detection of the Mycobacterium tuberculosis complex included high specificity, high sensitivity, simplicity, and superiority in avoidance of aerosol contamination. The modified LAMP assay would provide a potential for clinical diagnosis and therapy of tuberculosis in the developing countries and the resource-limited areas.
Assuntos
Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Técnicas de Amplificação de Ácido Nucleico/métodos , Técnicas Bacteriológicas/métodos , DNA Bacteriano/genética , Humanos , Mycobacterium bovis/classificação , Mycobacterium tuberculosis/classificaçãoRESUMO
Interleukin-22 (IL-22), a member of the IL-10 cytokine family that is produced by activated Th22, Th1 and Th17 cells as well as natural killer cells, plays an important role in increase of innate immunity, protection from damage and enhancement of regeneration. Here, we examined the effects of IL-22 on acute liver failure model induced by d-galactosamine (GalN) and lipopolysaccharide (LPS). Administration of recombinant human IL-22 (rhIL-22) reduced the death rate markedly and prevented mice from severe hepatic injury, as evidenced by decreased serum alanine aminotransferase (ALT) and total bilirubin (T.Bil) activity as well as improved histological signs in liver. Furthermore, IL-22 treatment decreased the hepatic malondialdehyde (MDA) contents and increased the reduced glutathione levels. Serum tumor necrosis factor α (TNF-α) level and hepatic caspase-3 activity were significantly lower in mice administrated with IL-22. Moreover, IL-22 treatment significantly enhanced activation of STAT3 and up-regulated the expression of Bcl-xL, heme oxygenase-1 (HO-1) and redox factor-1 (Ref-1) in the liver injury induced by GalN/LPS. Collectively, these data indicate that IL-22 can provide critical protection against GalN/LPS-induced liver injury through anti-apoptotic, anti-oxidant and anti-inflammatory actions.
Assuntos
Galactosamina/toxicidade , Interleucinas/farmacologia , Lipopolissacarídeos/toxicidade , Falência Hepática Aguda/tratamento farmacológico , Alanina Transaminase/sangue , Animais , Sequência de Bases , Bilirrubina/sangue , Caspase 3/sangue , Primers do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Glutationa/metabolismo , Heme Oxigenase-1/genética , Interleucina-1/sangue , Interleucinas/administração & dosagem , Interleucinas/sangue , Fígado/metabolismo , Falência Hepática Aguda/induzido quimicamente , Falência Hepática Aguda/metabolismo , Masculino , Malondialdeído/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Fosforilação , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/sangue , Proteínas Recombinantes/farmacologia , Fator de Transcrição STAT3/metabolismo , Fator de Necrose Tumoral alfa/sangue , Proteína bcl-X/metabolismo , Interleucina 22RESUMO
The protective effects of interleukin-22 (IL-22) on acute alcohol-induced liver injury were investigated. Mice were gavaged with 7 doses of alcohol (56% wt/vol, 15.2 mL/kg of body weight for each dose) over the 24 h, and IL-22 (0.5 mg/kg BW) was given to the mice by injection into the tail vein 1 h after alcohol administration. The results indicated that acute alcohol administration caused prominent hepatic microvesicular steatosis and an elevation of serum transaminase activities, induced a significant decrease in hepatic glutathione in conjunction with enhanced lipid peroxidation, and increased hepatocyte apoptosis as well as hepatic TNF-alpha production. IL-22 treatment attenuated these adverse changes induced by acute alcohol administration. The protective effects of IL-22 on alcohol-induced hepatotoxicity were due mainly to its anti-inflammatory, anti-oxidant, and anti-apoptotic features.
Assuntos
Etanol/toxicidade , Fígado Gorduroso Alcoólico/prevenção & controle , Hepatite Alcoólica/prevenção & controle , Interleucinas/administração & dosagem , Fígado/patologia , Fator de Necrose Tumoral alfa/efeitos dos fármacos , Alanina Transaminase/sangue , Alanina Transaminase/efeitos dos fármacos , Alanina Transaminase/metabolismo , Animais , Antioxidantes/metabolismo , Apoptose/efeitos dos fármacos , Fígado Gorduroso Alcoólico/etiologia , Fígado Gorduroso Alcoólico/metabolismo , Fígado Gorduroso Alcoólico/patologia , Glutationa/efeitos dos fármacos , Glutationa/metabolismo , Hepatite Alcoólica/etiologia , Hepatite Alcoólica/metabolismo , Hepatite Alcoólica/patologia , Inflamação , Peroxidação de Lipídeos/efeitos dos fármacos , Fígado/citologia , Fígado/efeitos dos fármacos , Fígado/metabolismo , Masculino , Camundongos , Fator de Necrose Tumoral alfa/metabolismo , Interleucina 22RESUMO
Fibroblast growth factor 10 (FGF10) plays important roles in vertebrate limb development, lung branching morphogenesis, and epidermis regeneration. The receptor (FGFR2b) binding specificity is an essential element in regulating the diverse functions of FGF10. Analyzing the FGF10:FGFR2b complex we found that Thr-114 in beta4 of FGF10 could form specific interactions with D3 of FGFR2b. To investigate the role of Thr-114 played on functions of FGF10, two mutants of FGF10 were constructed, named TA (Thr-114-->Ala) and TR (Thr-114-->Arg), respectively. The biological activity assays showed that the receptor-binding affinity, the stimulating growth effect on rat tracheal epithelium (RTE) cells, and the inducing ability in receptor phosphorylation of both mutants were decreased, which were consistent with the interaction analysis of the TA:FGFR2b and TR:FGFR2b complexes. These results suggested that Thr-114 is a crucial functional residue for FGF10, and mutating Thr-114 to Ala or Arg would lead to great decrease in receptor-binding affinity and biological activity of FGF10.
Assuntos
Análise Mutacional de DNA , Fator 10 de Crescimento de Fibroblastos , Conformação Proteica , Treonina/metabolismo , Animais , Fator 10 de Crescimento de Fibroblastos/química , Fator 10 de Crescimento de Fibroblastos/genética , Fator 10 de Crescimento de Fibroblastos/metabolismo , Humanos , Camundongos , Modelos Moleculares , Células NIH 3T3 , Mutação Puntual , Ratos , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/química , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/genética , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Treonina/genéticaRESUMO
Angiopoietin-2 (Ang2) is a member of the Ang family. Its potential in clinical use has been highlighted for its important roles in angiogenesis during the individual development and the growth of tumors. Ang2 is difficult to be expressed in E. coli for its unique structure. The expressions of Ang2 in insect cells (Sf9) and Chinese hamster ovary (CHO) cell line have been reported, however, the large-scale production of Ang2 for application is still pendent. In this study, the expression of Ang2 in Pichia pastoris expression system was described for the first time. The cDNA encoding Ang2 was cloned from the rat vascular tissue by RT-PCR, and inserted in the eukaryotic expression vector pPIZαA, and then transformed into P. pastoris KM71H cells. The expression of recombinant rat Ang2 (rrAng2) was induced by methanol and accounted for about 75% of the total secreted proteins. The recombinant protein was subsequently purified by HisTrap FF crude with a purity of 90%. Functional analysis of the purified rrAng2 demonstrated a specific activity in promoting the survival of ECV304 cells and binding to the Tie2 receptor. Preparation of bioactive rrAng2 not only lays the basis for further functional study but also provides a new strategy for soluble and large-scale production of human Ang2.
Assuntos
Angiopoietina-2/isolamento & purificação , Angiopoietina-2/metabolismo , Pichia/metabolismo , Animais , Bioensaio , Linhagem Celular , Humanos , Plasmídeos , Ratos , Receptor TIE-2/metabolismo , Proteínas Recombinantes/metabolismoRESUMO
The heterologous nature of SAK, a thrombolytic drug, elicits high titers of neutralizing antibodies, which limits its clinical use. Here, we aim to establish a SAK mutant with equivalent activity to the wild type but reduced antigenicity, which may allow for multiple injections. Biosun software was used to predict SAK antigenic epitopes, and several main epitopes were modified by gene deletion and mutation. Ten SAK mutants were constructed, and their thrombolytic activity and immunogenicity were analyzed in vitro. SAK6, with a high expression level (45%), similar thrombolytic activity, and lower antibody reaction, was chosen for in vivo analysis in rhesus monkey. In the nearly 8-month experimental period, the antibody level of the SAK6 group was significantly lower than that of the SAK group. Moreover, only 5% of SAK activity was retained, whereas 75.6% of SAK6 activity was retained after incubating with respective antiserum. Overall, these results demonstrated that SAK6, established through comprehensive site-directed mutagenesis program, had identical thrombolytic activity to SAK, low immunogenicity, and less side effects, demonstrating its efficient clinical potential for thrombus disease.
Assuntos
Fibrinolíticos/química , Fibrinolíticos/imunologia , Metaloendopeptidases/química , Metaloendopeptidases/genética , Metaloendopeptidases/imunologia , Animais , Anticorpos , Modelos Animais de Doenças , Epitopos , Feminino , Fibrinolíticos/farmacologia , Macaca mulatta , Masculino , Metaloendopeptidases/farmacologia , Mutagênese , Mutagênese Sítio-Dirigida , Mutação , Ratos , Ratos Wistar , TromboseRESUMO
Keratinocyte growth factor-2 (KGF-2) plays an important role in vertebrate limb development, lung branching morphogenesis, regeneration and reconstruction of the epidermis. Previous studies have used the wild type factor. Here, we have constructed a double-site mutant of human KGF-2, named STEA. STEA possesses higher receptor binding affinity and promotes better proliferation activity on rat tracheal epithelium (RTE) cells than recombinant human KGF-2. These results suggest that the simultaneous mutation of Ser115 to Thr and Glu117 to Ala improves the biological activity of KGF-2.
Assuntos
Fator 10 de Crescimento de Fibroblastos/farmacologia , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Substituição de Aminoácidos , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Células Epiteliais/efeitos dos fármacos , Fator 10 de Crescimento de Fibroblastos/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Mutação de Sentido Incorreto , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Mucosa Respiratória/efeitos dos fármacosRESUMO
Four and a half LIM domain protein 3 (FHL3) is a member of the family of LIM proteins and is involved in myogenesis, cytoskeleton reconstruction, cell growth and differentiation. The full-length FHL3 cDNA was cloned from human spleen cDNA library and inserted in a prokaryotic expression vector pBV220 and then the recombinant plasmid was transformed into E. coli JM109. The expression of the recombinant protein was induced at 42 degrees C. SDS-PAGE analysis showed that recombinant human FHL3 (rhFHL3) was mainly expressed as an inclusion body. After purification by HisTrap FF crude, the rhFHL3 was renatured by dialysis against renaturing buffer and identified by Western blot analysis using human FHL3 polyclonal antibody. The MTT assay showed that the purified rhFHL3 could inhibit HepG2 cell growth but promote the proliferation of ECV304 cells. In addition, the expression of angiogenin (Ang) gene was increased when ECV304 cells were pretreated with rhFHL3.
Assuntos
Escherichia coli/genética , Expressão Gênica , Peptídeos e Proteínas de Sinalização Intracelular/isolamento & purificação , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , DNA Complementar/genética , DNA Complementar/isolamento & purificação , Biblioteca Gênica , Hepatócitos/efeitos dos fármacos , Humanos , Corpos de Inclusão , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas com Domínio LIM , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Baço , Sais de Tetrazólio/metabolismo , Tiazóis/metabolismoRESUMO
Staphylokinase (SAK) is an effective thrombolysis agent for therapy of myocardial infarction. We have constructed a fusion SAK variant (SAK-HV) with a thrombin-binding domain composed of 12 amino acids from hirudin and expressed it in Escherichia coli and purified the resultant protein. SAK-HV maintained fibrinolytic activity similar to SAK and had anticoagulant activity attributable to its hirudin segment. Measurement of thrombin-binding activity in vitro demonstrated that SAK-HV possessed binding activity with thrombin while SAK did not. SAK-HV might thus be a more potent thrombolytic agent with anticoagulation property than SAK.
Assuntos
Hirudinas/genética , Hirudinas/metabolismo , Metaloendopeptidases/genética , Metaloendopeptidases/metabolismo , Trombina/antagonistas & inibidores , Escherichia coli/genética , Expressão Gênica , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismoRESUMO
The aim of this study was to realize the oral delivery of SAK-HV protein and improve its oral bioavailability based on chitosan quaternary ammonium salt-PLGA microsphere. The results showed that the SAK-HV-loaded microsphere can overcome the multiple obstacles for oral adsorption and adhere effectively to the jejunal segment of a rat. The pharmacokinetic analysis of the oral drug-loaded microspheres in rats showed that the blood drug concentration of SAK-HV reached the peak value at 4 h after oral administration, and the relative oral bioavailability of SAK-HV was 3.4%. Additionally, after oral administration to the mice, a higher level of antibody against SAK-HV was produced on day 21 compared with that in the control and injection groups, and the antibody titre was 7.2 times that of the tail vein group. This work suggests that the microsphere of the chitosan quaternary ammonium salt-PLGA may be a promising drug delivery system for the oral administration of SAK-HV protein.
Assuntos
Quitosana/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Proteínas Serina-Treonina Quinases/química , Compostos de Amônio Quaternário/química , Proteínas Recombinantes de Fusão/química , Administração Oral , Animais , Portadores de Fármacos/química , Sistemas de Liberação de Medicamentos/métodos , Masculino , Camundongos Endogâmicos C57BL , Microesferas , Ratos , Ratos WistarRESUMO
BACKGROUND: Intestinal ischemia reperfusion injury is a frequent clinical damage, in which the oxidative stress and inflammation play an important role. Interleukin-1 receptor antagonist (IL-1Ra) is a natural anti-inflammatory factor, however, its effect on intestinal ischemia reperfusion injury remains unclear. METHODS: The rat model of intestinal I/R was induced by occlusion (for 60 min) and reopening (for 60 min) of superior mesenteric artery. The rats were randomly divided into the following 5 groups: sham-operation(S), model (I/R),10 mg/kgIL-1Ra + I/R (C1),20 mg/kgIL-1Ra + I/R (C2), and30 mg/kgIL-1Ra + I/R (C3). RESULTS: In this study it was the first time to confirm that IL-1Ra had a significant protection against the intestinal ischemia reperfusion injury. IL-1Ra not only effectively inhibited the expression of inflammatory factors (such as IL-1ß, IL-6 and TNF-α) and the activation of neutrophil in intestinal tissues, but also decreased the death of intestinal cells and the damages of intestinal tissues. Interestingly, besides anti-inflammation effect, it was also found that IL-1Ra possessed a significant inhibitory effect on the oxidative stress caused by ischemia/reperfusion injury. Furthermore, the expression of nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1), and the phosphorylation level of Nrf2 were greatly promoted by IL-1Ra. At the same time, IL-1Ra inhibited the mitogen-activated protein kinase (MAPKs) pathway. CONCLUSION: IL-1Ra had the protective effect against intestinal ischemia reperfusion injury, its mechanism included anti-inflammation and anti-oxidative stress in which the Nrf2/HO-1 pathway played an important role. The above-mentioned results may extend the clinical application of IL-1Ra in the treatment of intestinal ischemia reperfusion injury.
Assuntos
Heme Oxigenase-1/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Traumatismo por Reperfusão/metabolismo , Animais , Citocinas/metabolismo , Inflamação/imunologia , Inflamação/metabolismo , Masculino , Estresse Oxidativo/efeitos dos fármacos , Ratos Sprague-Dawley , Traumatismo por Reperfusão/imunologiaRESUMO
OBJECTIVE: To study the effects of interleukin 1 receptor antagonist (IL-Ira) on allergy asthma and its mechanism. METHODS: Thirty female SD rats underwent intraperitoneal and hypodermic injection of ovalbumin (OVA) on days 1 and 14, and then underwent spraying of OVA aerosol since day 21 for 7 days so as to provoke asthma, and then the rats were randomly divided into 3 equal groups: asthma model group, low dose IL-1ra treatment group undergoing intravenous injection of IL-1ra 6 mg/kg before each provocation (low dose treatment group), and high dose IL-1ra treatment group undergoing intravenous injection of IL-1ra 30 mg/kg before each provocation (high dose treatment group). Another 10 rats were used as normal controls. Twenty-four hours after the last provocation physiological monitoring equipment was used to detect the pulmonary function. Then the rats were killed. Bronchoalveolar lavage fluid (BALF) was collected. ELISA was used to detect the serum IgE content. The ratio of inflammatory cells from the BALF was calculated. Microscopy was conducted to observe the histopathology of lung. RT-PCR was used to examine the mRNA expression of NF-kappaB and signal transducer and activator of the transcription 6 (STAT6). RESULTS: The respiratory rate, expiratory flow, percentage of eosinophils in BALF inflammatory cells, peripheral blood IgE concentration, mRNA expression of STAT6 and NF-kappaB of the asthma group were (206 +/- 11) times/min, (77 +/- 8) microl/s, 24.8% +/- 1.3%, (72.5 +/- 8.1) ng/ml, 0.294 +/- 0.048, and 0.686 +/- 0.052 respectively, all significantly higher than those of the low dose treatment group [(183 +/- 9) times/min, (64 +/- 5) microl/s, 18.5% +/- 3.1%, (63.4 +/- 4.8) ng/ml, 0.229 +/- 0.038, and 0.613 +/- 0.062 respectively, all P < 0.05] and those of the high dose treatment group [(181 +/- 11) times/min, (57 +/- 4) microl/s, 14.7% +/- 2.1%, (41.4 +/- 7.4) ng/ml, 0.194 +/- 0.076, and 0.352 +/- 0.267, all P < 0.05]. The therapeutic effect of high dose treatment group is superior to that of low dose treatment group (P < 0.05). CONCLUSION: IL-1ra is significantly effective in treatment of allergic asthma, and its potential mechanism is through regulating both STAT6 mRNA and NF-kappaB mRNA expression simultaneously.
Assuntos
Asma/tratamento farmacológico , Asma/metabolismo , Proteína Antagonista do Receptor de Interleucina 1/uso terapêutico , Animais , Asma/etiologia , Modelos Animais de Doenças , Feminino , Hipersensibilidade/complicações , NF-kappa B/biossíntese , RNA Mensageiro , Ratos , Ratos Sprague-Dawley , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fator de Transcrição STAT6/biossínteseRESUMO
Macrophage migration plays an essential role in immune system and is also involved in many pathological situations. However, the regulatory mechanism of macrophage migration remains to be elucidated due to its diverse responses to various stimuli. SAK-HV, a multifunctional protein possessing thrombolytic and lipid-lowering activity, can selectively induce the macrophage proliferation. Here, we reported SAK-HV significantly triggered RAW264.7 cells migration through its functional domain of SAK-mutant by activating both c-jun N-terminal kinases (JNK) and nuclear factor-κB (NF-κB) pathways. Meanwhile, SAK-HV upregulated the expression of some effector proteins, among which only the expression of Monocyte chemoattractant protein-1 (MCP-1) was inhibited by the blockade of JNK and NF-κB pathways. Further research showed that MCP-1 promoted migration ultimately by interacting with Chemokine (C-C motif) Receptor 2 (CCR2) in an autocrine manner. In summary, SAK-HV induced RAW264.7 cells migration through its SAK-mutant domain, during which MCP-1 chemokine mediated by JNK and NF-κB pathways played a key role. These results revealed a novel effect of SAK-HV on modulating macrophage migration and also deepened the understanding of its pharmacodynamics.
Assuntos
Movimento Celular/fisiologia , Quimiocina CCL2/metabolismo , Animais , Movimento Celular/genética , Quimiocina CCL2/genética , Ensaio de Imunoadsorção Enzimática , Proteínas Quinases JNK Ativadas por Mitógeno/genética , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Proteínas Quinases Ativadas por Mitógeno/genética , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , Fosforilação/genética , Fosforilação/fisiologia , Células RAW 264.7 , RNA Interferente Pequeno/genética , Receptores CCR2/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transfecção , Cicatrização/genética , Cicatrização/fisiologiaRESUMO
The accumulations of excess lipids within liver and serum are defined as non-alcoholic fatty liver disease (NAFLD) and hyperlipemia respectively. Both of them are components of metabolic syndrome that greatly threaten human health. Here, a recombinant fusion protein (SAK-HV) effectively treated NAFLD and hyperlipemia in high-fat-fed ApoE-/- mice, quails and rats within just 14 days. Its triglyceride and cholesterol-lowering effects were significantly better than that of atorvastatin during the observation period. We explored the lipid-lowering mechanism of SAK-HV by the hepatic transcriptome analysis and serials of experiments both in vivo and in vitro. Unexpectedly, SAK-HV triggered a moderate energy and material-consuming liver proliferation to dramatically decrease the lipids from both serum and liver. We provided the first evidence that PGC-1α mediated the hepatic synthesis of female hormones during liver proliferation, and proposed the complement system-induced PGC-1α-estrogen axis via the novel STAT3-C/EBPß-PGC-1α pathway in liver as a new energy model for liver proliferation. In this model, PGC-1α ignited and fueled hepatocyte activation as an "igniter"; PGC-1α-induced estrogen augmented the energy supply of PGC-1α as an "ignition amplifier", then triggered the hepatocyte state transition from activation to proliferation as a "starter", causing triglyceride and cholesterol-lowering effects via PPARα-mediated fatty acid oxidation and LDLr-mediated cholesterol uptake, respectively. Collectively, the SAK-HV-triggered distinctive lipid-lowering strategy based on the new energy model of liver proliferation has potential as a novel short-period biotherapy against NAFLD and hyperlipemia.