RESUMO
Idiopathic pulmonary fibrosis (IPF) is a chronic, progressive pulmonary disease with an unclear pathogenesis and no available specific drug treatment. The principal etiological factors are lung inflammation caused by environmental factors, damage to alveolar epithelial cells, leading to epithelial-mesenchymal transition (EMT), and the abnormal proliferation of fibroblasts. Here, we have demonstrated that fibroblast growth factor 21 (FGF21) ameliorates IPF via the autophagy pathway. We administered FGF21 to bleomycin (BLM)-treated mice, which ameliorated their defects in lung function, reduced the accumulation of collagen, restored tissue structure, reduced the deposition of hydroxyproline, reduced the expression of collagen I and α-SMA and increased the expression of E-cadherin. The expression of LC3BII and the number of autophagosomes were significantly higher in the lungs. The expression of AKT and mTOR was significantly reduced by FGF21 treatment. We also determined the effects of FGF21 in A549 cells treated with TGF-ß, and found that FGF21 significantly inhibits activation of the AKT signaling pathway, thereby reducing TGF-ß-induced EMT and preventing the uncontrolled proliferation of fibroblasts. We conclude that FGF21 ameliorates IPF by inhibiting the PI3K-AKT-mTOR signaling pathway and activating autophagy, which provides a theoretical basis for FGF21 to be used for the treatment of IPF.
Assuntos
Autofagia , Bleomicina , Transição Epitelial-Mesenquimal , Fatores de Crescimento de Fibroblastos , Fibrose Pulmonar Idiopática , Fosfatidilinositol 3-Quinases , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Serina-Treonina Quinases TOR , Serina-Treonina Quinases TOR/metabolismo , Autofagia/efeitos dos fármacos , Animais , Fibrose Pulmonar Idiopática/tratamento farmacológico , Fibrose Pulmonar Idiopática/metabolismo , Fibrose Pulmonar Idiopática/patologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Humanos , Camundongos , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Bleomicina/efeitos adversos , Células A549 , Masculino , Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Pulmão/patologia , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos Endogâmicos C57BL , Fator de Crescimento Transformador beta/metabolismoRESUMO
In the healing of wounds, human-like collagen (hCol) is essential. However, collagen-based composite dressings have poor stability in vivo, which severely limits their current therapeutic potential. Based on the above, we have developed a recombinant fusion protein named hCol-ELP, which consists of hCol and an elastin-like peptide (ELP). Then, we examined the physicochemical and biological properties of hCol-ELP. The results indicated that the stability of the hCol-ELP fusion protein exhibited a more compact and homogeneous lamellar microstructure along with collagen properties, it was found to be significantly superior to the stability of free hCol. The compound hCol-ELP demonstrated a remarkable capacity to induce the proliferation and migration of mouse embryo fibroblast cells (NIH/3T3), as well as enhance collagen synthesis in human skin fibroblasts (HSF) when tested in vitro. In vivo, hCol-ELP demonstrated significant enhancements in healing rate and a reduction in the time required for scab removal, thereby exhibiting a scar-free healing effect. The findings provide a crucial theoretical foundation for the implementation of an hCol-ELP protein dressing in fields associated with the healing of traumatic injuries.
Assuntos
Elastina , Peptídeos , Camundongos , Animais , Humanos , Elastina/química , Peptídeos/farmacologia , Peptídeos/química , Colágeno/química , Cicatrização , Proteínas Recombinantes de Fusão/metabolismoRESUMO
Antibodies and labels were typically non-oriented conjugated in conventional immunochromatographic assays (ICAs). In this work, a C-terminal cysteine-tagged recombinant protein A (rPA) was conjugated in an oriented manner onto aggregation-induced emission fluorescence microsphere (AIEFM). The Fc fragment of anti-enrofloxacin monoclonal antibody (anti-ENR mAb) was then conjugated onto the rPA. The resulting oriented mAb-AIEFM probe was used in an ENR-ICA for the rapid detection of ENR, a widely abused animal drug. The ENR-ICA with the oriented probe saved 66.7% of anti-ENR mAb and 25% of ENR-bovine serum albumin, and had a limit of detection of 0.035 ng/mL, compared with 0.079 ng/mL for the non-oriented probe. The corresponding linear ranges of the ENR-ICA based on the oriented and non-oriented probes were 0.25-10 ng/mL and 0.1-2.5 ng/mL, respectively. This novel ICA based on the oriented probe has the potential to be used for sensitive and rapid detection in food safety.
Assuntos
Anticorpos , Animais , Enrofloxacina/análise , Microesferas , Imunoensaio , Fenômenos QuímicosRESUMO
Fibroblast growth factor 21 (FGF21) and glucagon-like peptide-1 (GLP-1) may be useful for the treatment of type 2 diabetes, obesity, and non-alcoholic fatty liver disease (NAFLD). Previous studies have shown that GLP-1 may synergize with FGF21 in the regulation of glucose and lipid metabolism. Currently, no approved drug therapy is available for non-alcoholic steatohepatitis (NASH). Here, we constructed and screened dual-targeting fusion proteins of GLP-1 and FGF21, connected by elastin-like polypeptides (ELPs), to investigate whether a combination of these two hormones would have therapeutic effects in models of NASH. The temperature phase transition and release of the hormones under physiological conditions were studied to identify a bifunctional fusion protein of FGF21 and GLP-1 (GEF) that was highly stable and showed sustained release. We further evaluated the quality and therapeutic efficacy of GEF in three mouse models of NASH. We successfully synthesized a novel recombinant bifunctional fusion protein with high stability and low immunogenicity. The GEF protein synthesized ameliorated hepatic lipid accumulation, hepatocyte damage, and inflammation; prevented the progression of NASH in the three models; reduced glycemia; and caused weight loss. This novel GEF molecule may be suitable for clinical use for the treatment of NAFLD/NASH and related metabolic diseases.
Assuntos
Hepatopatia Gordurosa não Alcoólica , Animais , Camundongos , Peptídeo 1 Semelhante ao Glucagon/genética , Peptídeo 1 Semelhante ao Glucagon/uso terapêutico , Peptídeo 1 Semelhante ao Glucagon/metabolismo , Fígado/metabolismo , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismoRESUMO
BACKGROUND: Sarcopenia, the age-related decline in skeletal muscle mass and function, diminishes life quality in elderly people. Improving the capacity of skeletal muscle differentiation is expected to counteract sarcopenia. However, the mechanisms underlying skeletal muscle differentiation are complex, and effective therapeutic targets are largely unknown. METHODS: The human Gene Expression Omnibus database, aged mice and primary skeletal muscle cells were used to assess the expression level of pyruvate dehydrogenase B (PDHB) in human and mouse aged state. d-Galactose (d-gal)-induced sarcopenia mouse model and two classic cell models (C2C12 and HSkMC) were used to assess the myogenic effect of PDHB and the underlying mechanisms via immunocytochemistry, western blotting, quantitative real-time polymerase chain reaction, RNA interference or overexpression, dual-luciferase reporter assay, RNA sequencing and untargeted metabolomics. RESULTS: We identified that a novel target PDHB promoted myogenic differentiation. PDHB expression decreased in aged mouse muscle relative to the young state (-50% of mRNA level, P < 0.01) and increased during mouse and primary human muscle cell differentiation (+3.97-fold, P < 0.001 and +3.79-fold, P < 0.001). Knockdown or overexpression of PDHB modulated the expression of genes related to muscle differentiation, namely, myogenic factor 5 (Myf5) (-46%, P < 0.01 and -27%, P < 0.05; +1.8-fold, P < 0.01), myogenic differentiation (MyoD) (-55%, P < 0.001 and -34%, P < 0.01; +2.27-fold, P < 0.001), myogenin (MyoG) (-60%, P < 0.001 and -70%, P < 0.001; +5.46-fold, P < 0.001) and myosin heavy chain (MyHC) (-70%, P < 0.001 and -69%, P < 0.001; +3.44-fold, P < 0.001) in both C2C12 cells and HSkMC. Metabolomic and transcriptomic analyses revealed that PDHB knockdown suppressed pyruvate metabolism (P < 0.001) and up-regulated ariadne RBR E3 ubiquitin protein ligase 2 (Arih2) (+7.23-fold, P < 0.001) in cellular catabolic pathways. The role of forkhead box P1 (FoxP1) (+4.18-fold, P < 0.001)-mediated Arih2 transcription was the key downstream regulator of PDHB in muscle differentiation. PDHB overexpression improved d-gal-induced muscle atrophy in mice, which was characterized by significant increases in grip strength, muscle mass and mean muscle cross-sectional area (1.19-fold to 1.5-fold, P < 0.01, P < 0.05 and P < 0.001). CONCLUSIONS: The comprehensive results show that PDHB plays a sarcoprotective role by suppressing the FoxP1-Arih2 axis and may serve as a therapeutic target in sarcopenia.
Assuntos
Sarcopenia , Idoso , Humanos , Camundongos , Animais , Sarcopenia/metabolismo , Mioblastos/metabolismo , Diferenciação Celular/genética , Oxirredutases/metabolismo , Oxirredutases/farmacologia , Piruvatos/metabolismo , Piruvatos/farmacologia , Proteínas Repressoras , Fatores de Transcrição Forkhead , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Fibroblast growth factor 21 (FGF-21) is an important regulator in glycolipid metabolism that is a promising drug candidate for treatment of diabetes and obesity. However, the productivity of recombinant hFGF-21 (rhFGF-21) in Escherichia coli (E. coli) is relatively low, which limits its clinical application. To meet the clinical demand and control the production cost, rhFGF-21 proteins were expressed in inclusion bodies (IBs) form in Rosetta (DE3) by high cell density fermentation in 50-L scale. Hollow fiber membrane filtration technology was used to enrich the bacteria, wash, denature and refold the IBs in the current report. The renatured proteins were purified by two-step affinity chromatography. Authenticity of the purified rhFGF-21 was confirmed by the N-and C-terminal sequence, disulfide bond composition and molecular weight analyses. Results showed that the average target protein and recovery of rhFGF-21 expressed in IBs form of three batches were more than those of the soluble form. Both the rhFGF-21 proteins from the two forms showed equal potency in improving the glucose uptake in HepG2 cells and anti-diabetic effect in db/db mice. In this study, an efficient method for preparation of FGF-21 was established. This novel process provides an important technical basis for the large-scale production of rhFGF-21.
Assuntos
Fatores de Crescimento de Fibroblastos/química , Fatores de Crescimento de Fibroblastos/farmacologia , Corpos de Inclusão/química , Redobramento de Proteína , Proteínas Recombinantes , Animais , Glicemia/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/metabolismo , Fermentação , Fatores de Crescimento de Fibroblastos/isolamento & purificação , Células Hep G2 , Humanos , Hipoglicemiantes/química , Hipoglicemiantes/isolamento & purificação , Hipoglicemiantes/farmacologia , Camundongos , Desnaturação Proteica , Estabilidade Proteica , Análise EspectralRESUMO
Onconase (ONC) as a novel anti-tumor drug has a significant killing effect on a variety of tumor cells. Drug delivery system mediated by transferrin (TF) and TF receptor (TfR), which can significantly increase the amount of drug uptake in the tumor cells, enhance the initiative target efficiency of drugs and reduce its toxic side effects. It has been widely used in drug delivery and clinical trials. In this study, the rONC-TFn was expressed in Escherichia coli by linking ONC with the N-terminal domain of TF (TFn). ELISA and competitive binding analysis demonstrated that rONC-TFn can bind to TfR. The rONC-TFn protein showed much higher cytotoxicity to the cultured HepG2 and Hela cells than rONC. These results suggested that the N-terminal domain protein of TF promoted the tumor targeting of ONC, and thus the rONC-TFn fusion protein may be further developed as a potential targeted anti-tumor drug.
Assuntos
Antineoplásicos/farmacologia , Proteínas Recombinantes de Fusão/metabolismo , Ribonucleases/farmacologia , Transferrina/metabolismo , Antineoplásicos/metabolismo , Ligação Competitiva , Sobrevivência Celular/efeitos dos fármacos , Cromatografia em Gel , Cromatografia por Troca Iônica , Relação Dose-Resposta a Droga , Sistemas de Liberação de Medicamentos , Eletroforese em Gel de Poliacrilamida , Ensaio de Imunoadsorção Enzimática , Escherichia coli/genética , Células HeLa , Células Hep G2 , Humanos , Modelos Biológicos , Domínios Proteicos , Receptores da Transferrina/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Ribonucleases/metabolismo , Transferrina/químicaRESUMO
A stable cell-line was established that expressed the recombinant avian antibody (rAb) against the infectious bursal disease virus (IBDV). rAb exhibited neutralization activity to IBDV-B87 strain in DF1 cells. The minimum rAb concentration required for inhibition of the cytopathic effect (CPE) was 1.563µg/mL. To test the efficacy of rAb, a 168-h cohabitation challenge experiment was performed to transmit the disease from the chickens challenged with vvIBDV (HLJ0504 strain) to three test groups of chickens, i.e. (1) chickens treated with rAb, (2) chickens treated with yolk antibody, and (3) non-treatment chickens. The survival rates of chickens treated with rAb, yolk antibody and without treatment were 73%, 67% and 20%, respectively. Another batch of chickens was challenged with IBDV (BC6/85 strain) and then injected with rAb (1.0mg/kg) 6, 24 and 36h post-challenge. Non-treatment chickens had 100% morbidity, whereas those administered with rAb exhibited only 20% morbidity. Morbidity was evaluated using clinical indicators and bursal histopathological section. This study provides a new approach to treating IBDV and the rAb represents a promising candidate for this IBDV therapy.
Assuntos
Anticorpos Antivirais/imunologia , Infecções por Birnaviridae/veterinária , Galinhas , Vírus da Doença Infecciosa da Bursa/imunologia , Doenças das Aves Domésticas/prevenção & controle , Proteínas Estruturais Virais/imunologia , Animais , Infecções por Birnaviridae/prevenção & controle , Linhagem Celular , Doenças das Aves Domésticas/imunologia , Doenças das Aves Domésticas/virologia , Proteínas Recombinantes , Vacinas Virais/imunologiaRESUMO
Newcastle disease virus (NDV) have shown oncolytic therapeutic efficacy in preclinical studies and are currently proved for clinical trials. We have previously reported, for the first time, NDV Anhinga strain has an efficient cancer therapeutic efficacy in hepatoma. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) functions as a cytokine to selectively kill various cancer cells without toxicity to most normal cells. Numerous studies have demonstrated the potential use of recombinant soluble TRAIL as a cancer therapeutic agent. In this study, we have showed administration of a recombinant NDV Anhinga strain expressing soluble TRAIL (NDV/Anh-TRAIL) results in an efficient suppression of hepatocellular carcinoma without significant toxicity. The results show that recombinant NDV Anhinga strain expressing soluble TRAIL is a promising candidate for hepatoma therapy.
Assuntos
Carcinoma Hepatocelular/terapia , DNA Recombinante/genética , Neoplasias Hepáticas/terapia , Vírus da Doença de Newcastle/genética , Vírus da Doença de Newcastle/fisiologia , Terapia Viral Oncolítica/métodos , Ligante Indutor de Apoptose Relacionado a TNF/genética , Animais , Apoptose , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Caspase 3/genética , Proliferação de Células , Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Células Hep G2 , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Camundongos , Camundongos Endogâmicos ICR , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Linfócitos T/imunologia , Replicação ViralRESUMO
Fibroblast growth factor 21 (FGF21) is a promising regulator of glucose and lipid metabolism with multiple beneficial effects including hypoglycemic and lipid-lowering. Previous studies have reported that FGF21 is expected to become a new drug for treatment of diabetes. Liraglutide and insulin glargine are the two representative anti-diabetic biological drugs. In the current study, we aim to compare the long-term pharmacological efficacy of mFGF21 (an FGF21 analogue), liraglutide and insulin glargine in type 2 diabetic db/db mice. Db/db mice were initially treated with three kinds of proteins (25nmol/kg/day) by subcutaneous injection once a day for 4weeks, then subsequently be treated with once every two days for next 4weeks. After 8weeks of treatments, the blood glucose levels, body weights, glycosylated hemoglobin levels, fasting insulin levels, serum lipid profiles, hepatic biochemical parameters, oral glucose tolerance tests and hepatic mRNA expression levels of several proteins (GK, G6P, GLUT-1 and GLUT-4) associated with glucose metabolism of the experimental mice were detected. Results demonstrated that three proteins could significantly decrease the fed blood glucose levels of db/db mice. After treatment for 1week, the fed blood glucose levels of db/db mice in liraglutide group were significantly lower than those in mFGF21 and insulin glargine groups. However, after 2weeks of administration, the long-lasting hypoglycemic effect of mFGF21 was superior to liraglutide and insulin glargine up to the end of the experiments. Compared with liraglutide and insulin glargine, mFGF21 significantly reduced the glycosylated hemoglobin levels and improved the ability on glycemic control, insulin resistance, serum lipid and liver function states in db/db mice after 8weeks treatments. In addition, mFGF21 regulated glucose metabolism through increasing the mRNA expression levels of GK and GLUT-1, and decreasing the mRNA expression level of G6P. But liraglutide and insulin glargine could only up-regulate the mRNA expression of GLUT-4. In summary, as a hypoglycemic drug for long-term treatment, mFGF21 has the potential to be an ideal drug candidate for the therapy of type 2 diabetes.
Assuntos
Diabetes Mellitus Tipo 2/tratamento farmacológico , Drogas em Investigação/uso terapêutico , Fatores de Crescimento de Fibroblastos/uso terapêutico , Hipoglicemiantes/uso terapêutico , Insulina Glargina/uso terapêutico , Liraglutida/uso terapêutico , Proteínas Mutantes/uso terapêutico , Animais , Biomarcadores/sangue , Glicemia/análise , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Drogas em Investigação/efeitos adversos , Drogas em Investigação/metabolismo , Drogas em Investigação/farmacologia , Endopeptidases/metabolismo , Feminino , Fatores de Crescimento de Fibroblastos/efeitos adversos , Fatores de Crescimento de Fibroblastos/genética , Fatores de Crescimento de Fibroblastos/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Células Hep G2 , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hiperglicemia/prevenção & controle , Hipoglicemiantes/efeitos adversos , Hipoglicemiantes/farmacologia , Insulina Glargina/efeitos adversos , Insulina Glargina/farmacologia , Resistência à Insulina , Liraglutida/efeitos adversos , Liraglutida/farmacologia , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Proteínas Mutantes/efeitos adversos , Proteínas Mutantes/genética , Proteínas Mutantes/farmacologia , Distribuição Aleatória , Proteínas Recombinantes/efeitos adversos , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Proteínas Recombinantes/uso terapêuticoRESUMO
Wound healing is a complex multifactorial process that relies on coordinated signaling molecules to succeed. Epidermal growth factor (EGF) is a mitogenic polypeptide that stimulates wound repair; however, precise control over its application is necessary to reduce the side effects and achieve desired therapeutic benefits. Moreover, the extensive oxidative stress during the wound healing process generally inhibits repair of the injured tissues. Topical applications of antioxidants like curcumin (Cur) could protect tissues from oxidative damage and significantly improve tissue remodeling. To achieve much accelerated wound healing effects, we designed a novel dual drug co-loaded in situ gel-forming nanoparticle/hydrogel system (EGF-Cur-NP/H) which acted not only as a supportive matrix for the regenerative tissue, but also as a sustained drug depot for EGF and Cur. In the established excisional full-thickness wound model, EGF-Cur-NP/H treatment significantly enhanced wound closure through increasing granulation tissue formation, collagen deposition, and angiogenesis, relative to normal saline, nanoparticle/hydrogel (NP/H), Cur-NP/H, and EGF-NP/H treated groups. In conclusion, this study provides a biocompatible in situ gel-forming system for efficient topical application of EGF and Cur in the landscape of tissue repair.
Assuntos
Curcumina/farmacologia , Fator de Crescimento Epidérmico/farmacologia , Hidrogel de Polietilenoglicol-Dimetacrilato/química , Nanopartículas/química , Regeneração/efeitos dos fármacos , Pele/efeitos dos fármacos , Células 3T3 , Animais , Morte Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Liberação Controlada de Fármacos , Células HEK293 , Humanos , Camundongos , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , Poliésteres/química , Fator de Crescimento Transformador beta1/metabolismo , Cicatrização/efeitos dos fármacosRESUMO
The aim of this study is to evaluate the role of fibroblast growth factor 21 (FGF21) in nonalcoholic fatty liver disease (NAFLD) and seek to determine if its therapeutic effect is through induction of autophagy. In this research, Monosodium L-glutamate (MSG)-induced obese mice or normal lean mice were treated with vehicle, Fenofibrate, and recombinant murine FGF21, respectively. After 5 weeks of treatment, metabolic parameters including body weight, blood glucose and lipid levels, hepatic and fat gene expression levels were monitored and analyzed. Also, fat-loaded HepG2 cells were treated with vehicle or recombinant murine FGF21. The expression levels of proteins associated with autophagy were detected by western blot, real-time PCR, and transmission electron microscopy (TEM). Autophagic flux was monitored by laser confocal microscopy and western blot. Results showed that FGF21 significantly reduced body weight (P < 0.01) and serum triglyceride, improved insulin sensitivity, and reversed hepatic steatosis in the MSG model mice. In addition, FGF21 significantly increased the expression of several proteins related to autophagy both in MSG mice and fat-loaded HepG2 cells, such as microtubule associated protein 1 light chain 3, Bcl-2-interacting myosin-like coiled-coil protein-1 (Beclin-1), and autophagy-related gene 5. Furthermore, the evidence of TEM revealed an increased number of autophagosomes and lysosomes in the model cells treated with FGF21. In vitro experimental results also showed that FGF21 remarkably increased autophagic flux. Taken together, FGF21 corrects multiple metabolic parameters on NAFLD in vitro and in vivo by inducing autophagy.
Assuntos
Autofagia , Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de Crescimento de Fibroblastos/farmacocinética , Hepatopatia Gordurosa não Alcoólica/tratamento farmacológico , Hepatopatia Gordurosa não Alcoólica/metabolismo , Animais , Células Hep G2 , Humanos , Camundongos , Camundongos Endogâmicos ICR , Camundongos Obesos , Hepatopatia Gordurosa não Alcoólica/patologiaRESUMO
Newcastle disease virus (NDV) have shown oncolytic therapeutic efficacy in preclinical study and are currently approved for clinical trials. NDV Anhinga strain which is a mesogenic strain should be classified as lytic strain and has a therapeutic efficacy in hepatocellular cancer. In this study, we evaluated the capacity of NDV Anhinga strain to elicit immune reaction in vivo and the possibility for using as a vaccine vector for expressing tumor therapeutic factors. Interleukin-2 (IL-2) could boost the immune response against the tumor cells. Therefore, we use NDV Anhinga strain as backbone to construct a recombinant virus (NDV/Anh-IL-2) expressing IL-2. The virus growth curve showed that the production of recombinant NDV/Anh-IL-2 was slightly delayed compared to the wild type. The NDV/Anh-IL-2 strain could express soluble IL-2 and effectively inhibit the growth of hepatocellular carcinoma in vivo. 60 days post-treatment, mice which were completely cured by previous treatment were well protected when rechallenged with the same tumor cell. From the H&E-stained sections, intense infiltration of lymphocyte was observed in the NDV Anhinga strain treated group, especially in NDV/Anh-IL-2 group. The NDV Anhinga strain could not only kill the tumor directly, but could also elicit immune reaction and a potent immunological memory when killing tumor in vivo. In conclusion, the Anhinga strain could be an effective vector for tumor therapy; the recombinant NDV/Anh-IL-2 strain expressing soluble IL-2 is a promising candidate for hepatoma therapy.
Assuntos
Carcinoma Hepatocelular/terapia , Interleucina-2/genética , Neoplasias Hepáticas/terapia , Vírus da Doença de Newcastle/genética , Animais , Vacinas Anticâncer , Carcinoma Hepatocelular/patologia , Linhagem Celular , Linhagem Celular Tumoral , Galinhas , Cricetinae , Feminino , Humanos , Neoplasias Hepáticas/patologia , Camundongos , Carga TumoralRESUMO
FGF-21 has become a potential drug candidate for the treatment of type 2 diabetes. Previous studies have demonstrated that PEGylation of FGF-21 could significantly increase its in vivo half-life and provide its long-lasting blood glucose-lowering effect. To accelerate the development of PEGylated FGF-21 for clinical application as a long-acting antidiabetes drug, we prepared ahmFGF-21 (FGF-21 mutant) and PEGylated ahmFGF-21 in Escherichia coli Rosetta (DE3) by high cell density fermentation at a 50-L scale and pilot-scale purification. The physical and chemical properties of the purified proteins were analyzed in this study, including purity, molecular weight, isoelectric point, bacterial endotoxin, PEGylated site and second structure. As well as the in vitro glucose uptake activity and in vivo anti-diabetic effect were evaluated. Under the optimal fermentation and purification conditions, the average bacterial yield and expression level of target protein of three batches attained 52.2±4.6g/L and 223.92±5.41mg/L, respectively. The purity of pilot product was above 98% by SDS-PAGE (non-reducing or reducing) and HPLC (SEC or RPC) analysis and the final yield of PEGylated ahmFGF-21 was 87.91±1.49mg/L, which indicated that the pilot-scale production process was relatively stable. N-terminal sequencing and circular dichroism (CD) spectroscopy results showed that modification site of PEGylated ahmFGF-21 was alanine at N-terminal and the second structure of ahmFGF-21 had no obvious changes after PEGylation. Compared with ahmFGF-21, the long-acting hypoglycemic effect of PEGylated ahmFGF-21 prepared in the pilot-scale production was significantly improved in type 2 diabetic db/db mice. Our results demonstrated that the pilot-scale production process of PEGylated ahmFGF-21 was successfully established, which was very important for the clinical application.
Assuntos
Fatores de Crescimento de Fibroblastos/química , Fatores de Crescimento de Fibroblastos/metabolismo , Polietilenoglicóis/química , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Animais , Reatores Biológicos , Glicemia/efeitos dos fármacos , Modelos Animais de Doenças , Escherichia coli/genética , Fermentação , Fatores de Crescimento de Fibroblastos/genética , Células Hep G2 , Humanos , Masculino , Camundongos , Projetos Piloto , Polietilenoglicóis/farmacologia , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologiaRESUMO
Fibroblast growth factor-21 (FGF-21) is a novel metabolic regulator and has the potential to become a powerful therapy to treat diabetes mellitus. However, we found that the clinical application of wild type FGF-21 was influenced by its low intrinsic bio-stability and poor hypoglycemic potency. In this study, The N-terminus of FGF-21 analog (mFGF-21) was PEGylated in a site-specific manner by 20kD methoxy poly-ethylene glycol-propionaldehyde (mPEG-ALD). PEGylated mFGF-21 was isolated by Capto Q anion exchange chromatography. The properties of PEGylated mFGF-21 including the in vitro bio-stability and biological activity were evaluated. As well as the anti-diabetic effect of PEGylated mFGF-21 were studied in streptozotocin (STZ)-induced type 1 diabetic mice. Results demonstrated that PEGylated mFGF-21 had a similar capacity of stimulating glucose uptake in HepG2 cells with mFGF-21 and PEGylation of mFGF-21 significantly enhanced the anti-protease ability and the long acting anti-diabetic effect in type 1 diabetic mice. Furthermore, the preliminary safety of PEGylated mFGF-21 following subcutaneously injection was assessed using healthy mice by measuring the body weight, histopathology and clinical biochemical parameters, and the results showed no subacute toxicity to major organs or tissues and no significant changes in physiological and biochemical parameters in healthy mice. Taken together, under the premise of remaining the in vitro biological activity of mFGF-21, PEGylation significantly improves the long lasting hypoglycemic effect of mFGF-21 in type 1 diabetic mice. Our valuation shows that PEGylated mFGF-21 is a potential drug for the effective treatment of type 1 diabetes.
Assuntos
Diabetes Mellitus Tipo 1/tratamento farmacológico , Fatores de Crescimento de Fibroblastos/farmacologia , Fatores de Crescimento de Fibroblastos/toxicidade , Hipoglicemiantes/farmacologia , Hipoglicemiantes/toxicidade , Polietilenoglicóis/química , Animais , Diabetes Mellitus Tipo 1/sangue , Diabetes Mellitus Tipo 1/metabolismo , Portadores de Fármacos/química , Estabilidade de Medicamentos , Fatores de Crescimento de Fibroblastos/química , Regulação da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Teste de Tolerância a Glucose , Hemoglobinas Glicadas/metabolismo , Células Hep G2 , Humanos , Hipoglicemiantes/química , Insulina/sangue , Masculino , Camundongos , Relação Estrutura-AtividadeRESUMO
Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator of glucose and lipid, which is safe, effective and independent on insulin. FGF21 is considered as a prospective anti-diabetic drug. The aim of this study was to express recombinant h-FGF21 in periplasmic space of Escherichia coli. The pET27b plasmid was used to create the expression vectors of h-FGF21 with a PelB secretion signal. The ph-FGF21 (periplasmic expression of h-FGF21) was successfully expressed in the periplasm of E. coli BL21 (DE3), and soluble ph-FGF21 was isolated by disruption of the outer membrane. After twice of ion exchange chromatography, the purity of ph-FGF21 was above 95% in an analysis with a gray analysis software. The molecular weight of ph-FGF21 was about 20 kDa in SDS-PAGE and Western blotting analysis. The activity of ph-FGF21 and ih-FGF21 (intracellular expression of h-FGF21) was observed in vitro in the glucose uptake assay in HepG2 cells. The activity was observed in type 2 diabetic db/db mice after short or long-term treatments. The results suggest that the ph-FGF21 has a consistent activity with ih-FGF21 in vitro and in vivo.
Assuntos
Fatores de Crescimento de Fibroblastos/biossíntese , Animais , Escherichia coli/metabolismo , Células Hep G2 , Humanos , Camundongos , Periplasma/metabolismo , Plasmídeos , Estudos Prospectivos , Proteínas Recombinantes/biossínteseRESUMO
Previous studies reveal that fibroblast growth factor 21 (FGF21) sensitizes insulin to achieve a synergy in regulating glucose metabolism. Here, we report that insulin sensitizes FGF21 in regulating both glucose and lipid metabolisms. db/db diabetic mice were subcutaneously administrated once a day for 6 weeks. Effective dose of insulin (1 U) could control blood glucose level of the db/db mice for maximum of 2 h, increased the body weight of the db/db mice and did not improve serum lipid parameters. In contrast, effective dose of FGF21 (0.5 mg/kg) could maintain blood glucose of the db/db mice at normal level for at least 24 h, repressed the weight gain of the mice and significantly improved lipid parameters. Ineffective doses of FGF21 (0.125 mg/kg) and insulin had no effect on blood glucose level of the db/db mice after 24 h administration, body weight or lipid parameters. However, combination of the two ineffective doses could maintain blood glucose level of the db/db mice for at least 24 h, suppressed weight gain and significantly improved lipid parameters. These results suggest that insulin sensitizes FGF21 in regulating both glucose and lipid metabolism. The results aimed to study the molecular basis of FGF21 sensitization indicates that combination of the two ineffective doses increased the mRNA expression of glut1, glut4, ß-Klotho, sirt1, pgc-1α, ucp-1 and AKT phosphorylation, decreased fasn. The results demonstrate that insulin sensitizes FGF21 through elevating the phosphorylation of common gene Akt and amplifying FGF21 downstream signaling, including increasing expression of glut1 sirt1, pgc-1α, ucp-1, and decreasing fasn expression. In summary, we reports herein for the first time that insulin sensitizes FGF21 to achieve a synergy in regulating glucose and lipid metabolism. Along with previous studies, we conclude that the synergistic effect between FGF21 and insulin is realized through mutual sensitization.
Assuntos
Glicemia/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Fatores de Crescimento de Fibroblastos/farmacologia , Insulina/farmacologia , Metabolismo dos Lipídeos/efeitos dos fármacos , Animais , Glicemia/metabolismo , Diabetes Mellitus Experimental/patologia , Sinergismo Farmacológico , Glucose/metabolismo , Resistência à Insulina , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Oncogênica v-akt/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
PURPOSE: In this study, we explored whether treatment with FGF-21 could prevent diethylnitrosamine (DEN) induced hepatocarcinogenesis in mice. METHODS & RESULTS: Hepatoma was induced by injection of DEN every three days for 18 weeks. For the prophylactic experiment, mice were firstly injected with FGF-21 for 2 weeks, then FGF-21 was administered to the mice once daily in association with DEN injection till the end of the experiment. The hepatoma incidence of mice treated with FGF-21 was 13.3%, while the incidence of mice treated with saline was 61.5%. To understand the mechanisms, we compared the expression of ßklotho (KLB) and oxidative stress level in the livers between the mice treated with FGF-21 and saline. We found that FGF-21 could suppress DEN-induced oxidative stress and up-regulate the expression of KLB in the livers. To confirm these results, we compared the expression of KLB in L02 cells stimulated with or without FGF-21. Besides, we established DEN-induced oxidative stress cell model to affirm the relationship between FGF-21 and DEN-induced oxidative stress in vitro. Results showed that FGF-21 increased the expression of KLB and diminished the DEN-induced oxidative stress in vitro in a dose dependent manner. CONCLUSION: Systemic administration of FGF-21 can prevent DEN-induced hepatocarcinogenesis via suppressing oxidative stress and increasing the expression of KLB.
Assuntos
Carcinogênese/efeitos dos fármacos , Carcinoma Hepatocelular/tratamento farmacológico , Fatores de Crescimento de Fibroblastos/farmacologia , Neoplasias Hepáticas/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Animais , Western Blotting , Carcinoma Hepatocelular/induzido quimicamente , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Dietilnitrosamina/farmacologia , Modelos Animais de Doenças , Esquema de Medicação , Imuno-Histoquímica , Proteínas Klotho , Neoplasias Hepáticas/induzido quimicamente , Neoplasias Hepáticas/patologia , Proteínas de Membrana/efeitos dos fármacos , Proteínas de Membrana/metabolismo , Camundongos , Neoplasias Experimentais/prevenção & controle , Distribuição Aleatória , Reação em Cadeia da Polimerase em Tempo Real/métodos , Valores de Referência , Sensibilidade e EspecificidadeRESUMO
Fibroblast growth factor 21 (FGF21), a recently identified member of the FGF superfamily, is mainly secreted from the liver and adipose tissues and plays an important role in improving metabolic syndrome and homeostasis. The aim of this study is to evaluate the role of FGF21 in alcoholic fatty liver disease (AFLD) and to determine if it has a therapeutic effect on AFLD. In this paper, we tested the effect of FGF21 on alcohol-induced liver injury in a murine model of chronic ethanol gavage and alcohol-treated HepG2 cells. Male KM mice received single dose of 5 g/kg ethanol gavage every day for 6 weeks, which induced significant fatty liver and liver injury. The alcohol-induced fatty liver cell model was achieved by adding ethanol into the medium of HepG2 cell cultures at a final concentration of 75 mM for 9 days. Results showed that treatment with recombinant FGF21 ameliorated alcoholic fatty liver and liver injury both in a murine model of chronic ethanol gavage and alcohol-treated HepG2 cells. In addition, FGF21 treatment down-regulated the hepatic expression of fatty acid synthetic key enzyme, activated hepatic AMPK-SIRT1 pathway and significantly down-regulated hepatic oxidative stress protein. Taken together, FGF21 corrects multiple metabolic parameters of AFLD in vitro and in vivo by activation of the AMPK-SIRT1 pathway.
Assuntos
Adenilato Quinase/metabolismo , Fígado Gorduroso Alcoólico/prevenção & controle , Fatores de Crescimento de Fibroblastos/uso terapêutico , Sirtuína 1/metabolismo , Animais , Sequência de Bases , Composição Corporal/efeitos dos fármacos , Linhagem Celular Tumoral , Primers do DNA , Ativação Enzimática , Fígado Gorduroso Alcoólico/enzimologia , Fígado Gorduroso Alcoólico/metabolismo , Comportamento Alimentar/efeitos dos fármacos , Fatores de Crescimento de Fibroblastos/farmacologia , Humanos , Lipídeos/sangue , Masculino , Camundongos , Reação em Cadeia da Polimerase , Espécies Reativas de Oxigênio/metabolismoRESUMO
Previous studies proposed that the synergistic effect of fibroblast growth factor-21 (FGF-21) and insulin may be due to the improvement of insulin sensitivity by FGF-21. However, there is no experimental evidence to support this. This study was designed to elucidate the mechanism of synergistic effect of FGF-21 and insulin in the regulation of glucose metabolism. The synergistic effect of FGF-21 and insulin on regulating glucose metabolism was demonstrated by investigating the glucose absorption rate by insulin resistance HepG2 cell model and the blood glucose chances in type 2 diabetic db/db mice after treatments with different concentrations of FGF-21 or/and insulin; The synergistic metabolism was revealed through detecting GLUT1 and GLUT4 transcription levels in the liver by real-time PCR method. The experimental results showed that FGF-21 and insulin have a synergistic effect on the regulation of glucose metabolism. The results of real-time PCR showed that the effective dose of FGF-21 could up-regulate the transcription level of GLUT1 in a dose-dependent manner, but had no effect on the transcription level of GLUT4. Insulin (4 u) alone could up-regulate the transcription level of GLUT4, yet had no effect on that of GLUT1. Ineffective dose 0.1 mg kg(-1) FGF-21 alone could not change the transcription level of GLUT1 or GLUT4. However, when the ineffective dose 0.1 mg x kg(-1) FGF-21 was used in combination with insulin (4 u) significantly increased the transcription levels of both GLUT1 and GLUT4, the transcription level of GLUT1 was similar to that treated with 5 time concentration of FGF-21 alone; the transcription level of GLUT4 is higher than that treated with insulin (4 u) alone. In summary, in the presence of FGF-21, insulin increases the sensitivity of FGF-21 through enhancing GLUT1 transcription. Vice versa, FGF-21 increases the sensitivity of insulin by stimulating GLUT4 transcription in the presence of insulin. FGF-21 and insulin exert a synergistic effect on glucose metabolism through mutual sensitization.