nattokinase and Disease-Models--Animal

The therapy of solid tumors is always hampered by the intrinsic tumor physical microenvironment (TPME) featured with compact and rigid extracellular matrix (ECM) microstructures. Herein, we introduce nattokinase (NKase), a thrombolytic healthcare drug, to comprehensively regulate the TPME for versatile enhancement of various therapy modalities. Intratumoral injection of NKase not only degrades the major ECM component fibronectin but also inhibits cancer-associated fibroblasts (CAFs) in generating fibrosis, resulting in decreased tumor stiffness, enhanced perfusion, and hypoxia alleviation. The NKase-mediated regulation of the TPME significantly promotes the tumoral accumulation of therapeutic agents, leading to efficient chemotherapy without inducing side effects. Additionally, the enhancement of tumor radiotherapy based on radiosensitizers was also achieved by the pretreatment of intratumorally injected NKase, which could be ascribed to the elevated oxygen saturation level in NKase-treated tumors. Moreover, a xenografted human breast MDB-MA-231 tumor model is established to evaluate the influence of NKase on chimeric antigen receptor (CAR)-T cell therapy, illustrating that the pretreatment of NKase could boost the infiltration of CAR-T cells into tumors and thus be a benefit for tumor inhibition. These findings demonstrate the great promise of the NKase-regulated TPME as a translational strategy for universal enhancement of therapeutic efficacy in solid tumors by various treatments.

Topics: Animals; Breast Neoplasms; Disease Models, Animal; Female; Humans; Immunotherapy, Adoptive; Neoplasms; Receptors, Chimeric Antigen; T-Lymphocytes; Tumor Microenvironment

Nattokinase with excellent anti-thrombotic, anti-inflammatory, anti-tumor, and anti-hypertension properties has been used in the development of several healthcare products in many countries. The probiotic Escherichia coli Nissle 1917 (EcN) with anti-inflammatory effect is commonly used to treat inflammatory bowel disease. To determine whether nattokinase could enhance the therapeutic efficacy of EcN in colitis, a recombinant E. coli Nissle 1917 strain (EcNnatto) with nattokinase-expressing ability was successfully constructed, and the protective effect of the engineered strain on mice with experimental chronic colitis was investigated. Although both EcN and EcNnatto strains substantially alleviated the clinical symptoms and pathological abnormalities in colitis mice by regulating gut flora and maintaining intestinal barrier function, the EcNnatto strain was found to perform better than the control strain, based on a further increase in colon length and a downregulation in pro-inflammatory cytokines (IL-6 and TNF-α). Nattokinase expressed in EcN attenuated DSS-induced epithelial damage and restored the mucosal integrity by upregulating the levels of tight junction proteins, including ZO-1 and occludin. The expression level of Lgr5, a marker of intestinal stem cells, was also increased. Moreover, constitutively expressed nattokinase in EcN reversed the gut microbial richness and diversity in colitis mice. Based on our findings, nattokinase could strengthen the capacity of EcN to treat intestinal inflammation.

Topics: Animals; Anti-Inflammatory Agents; Colitis; Dextran Sulfate; Disease Models, Animal; Escherichia coli; Escherichia coli Infections; Mice; Probiotics

To develop a dual-functional medicine for hypoglycemic and anti-thrombus.. The long-acting glucagon like peptide-1 (5×GLP-1) and nattokinase (NK) were cloned by SOE PCR and gained the GLP-1 and NK fusion polypeptide after transformed into E. coli. Use of mice models for the hypoglycemic and anti-thrombus activity of the fusion polypeptide. Balb/C mice were given the carrageenan by intraperitoneal injection to induce tail thrombus models. Type 2 diabetes mellitus mice model was used to research the hypoglycemic function of the fusion polypeptide.. Results showed that the fusion polypeptide could significantly prevent thrombus formation after oral administration. Continuous administration for 15 days, fasting blood glucose levels of the experimental group decreased to nearly normal levels.. The present study investigated the expression, purification and functional activity of the rolGLP-1 and NK fusion polypeptide, which provided a foundation for further studying the detailed pharmaceutical mechanism and drug development.

Topics: Animals; Blood Glucose; Diabetes Mellitus, Experimental; Disease Models, Animal; Glucagon-Like Peptide 1; Hypoglycemia; Insulin; Male; Mice; Mice, Inbred BALB C; Peptides; Subtilisins; Thrombosis

Nattokinase (NK), an active ingredient extracted from traditional food Natto, has been studied for prevention and treatment of cardiovascular diseases due to various vasoprotective effects, including fibrinolytic, antihypertensive, anti-atherosclerotic, antiplatelet, and anti-inflammatory activities. Here, we reported an antineovascular effect of NK against experimental retinal neovascularization.. The inhibitory effect of NK against retinal neovascularization was evaluated using an oxygen-induced retinopathy murine model. Expressions of Nrf2/HO-1 signaling and glial activation in the NK-treated retinae were measured. We also investigated cell proliferation and migration of human umbilical vein endothelial cells (HUVECs) after NK administration.. NK treatment significantly attenuated retinal neovascularization in the OIR retinae. Consistently, NK suppressed VEGF-induced cell proliferation and migration in a concentration-dependent manner in cultured vascular endothelial cells. NK ameliorated ischemic retinopathy partially via activating Nrf2/HO-1. In addition, NK orchestrated reactive gliosis and promoted microglial activation toward a reparative phenotype in ischemic retina. Treatment of NK exhibited no cell toxicity or anti-angiogenic effects in the normal retina.. Our results revealed the anti-angiogenic effect of NK against retinal neovascularization via modulating Nrf2/HO-1, glial activation and neuroinflammation, suggesting a promising alternative treatment strategy for retinal neovascularization.

Topics: Angiogenesis Inhibitors; Animals; Animals, Newborn; Blotting, Western; Cell Movement; Cell Proliferation; Dextrans; Disease Models, Animal; Fluorescein-5-isothiocyanate; Gliosis; Heme Oxygenase-1; Human Umbilical Vein Endothelial Cells; Humans; In Situ Nick-End Labeling; Membrane Proteins; Mice; Mice, Inbred C57BL; Neuroglia; NF-E2-Related Factor 2; Orbit; Retinal Neovascularization; Soy Foods; Subtilisins; Transfection

Nattokinase (NK, E.C. 3.4.21.62) is a serine protease produced by

Topics: Animals; Bacillus subtilis; Carcinoma, Hepatocellular; Complex Mixtures; Disease Models, Animal; Fermentation; Fibrinolytic Agents; Forkhead Box Protein M1; Hyaluronan Receptors; Liver; Liver Neoplasms; Mice; Mice, Inbred C57BL; Platelet Endothelial Cell Adhesion Molecule-1; Subtilisins; Vimentin

Chronic rhinosinusitis with nasal polyps (CRSwNP) is often comorbid with asthma and resistant to therapeutic interventions. We recently reported that excessive fibrin deposition caused by impairment of fibrinolysis might play pivotal role in forming nasal polyp. Nattokinase (NK), a serine protease produced by Bacillus subtilis, has been reported to be a strong fibrinolytic enzyme. NK could be a promising drug candidate for use in the treatment of both CRSwNP and asthma. The objective of this study was to investigate the effects of NK on nasal polyp tissues from patients with CRSwNP. The nasal discharge from patients with CRSwNP and sputum from subjects with asthma were also used to investigate whether NK influences the viscosity of mucus.. To examine the effects on NK on nasal polyp tissues, pieces of nasal polyps were incubated either with saline or NK (10-1000 FU/ml) at 37 °C for 24 h. We assessed the presence of fibrin in nasal polyp tissue incubated with NK by means of immunohistochemistry. To examine the effects of NK on nasal discharge and sputum from patients with CRSwNP and asthma, respectively, were incubated with NK solution at 37 °C for 1 h.. NK effectively shrinks the nasal polyp tissue through fibrin degradation. We also found that the viscosity of the nasal discharge and sputum from patients with CRSwNP and asthma, respectively, was significantly reduced by incubation with NK solution.. NK may be an effective alternative therapeutic option in patients with CRSwNP and comorbid asthma by causing fibrin degradation.

Topics: Adult; Aged; Animals; Asthma; Chronic Disease; Disease Models, Animal; Eosinophils; Female; Fibrin; Humans; Immunoglobulin E; Leukocyte Count; Male; Mice; Middle Aged; Mucus; Nasal Mucosa; Nasal Polyps; Proteolysis; Rhinitis; Sinusitis; Subtilisins; Viscosity

Serrapeptase (SP) and nattokinase (NK) are proteolytic enzymes belonging to serine proteases. In this study, we hypothesized that SP and NK could modulate certain factors that are associated with Alzheimer's disease (AD) pathophysiology in the experimental model. Oral administration of aluminium chloride (AlCl3) in a dose of 17 mg/kg body weight (bw) daily for 45 days induced AD-like pathology in male rats with a significant increase in brain acetylcholinesterase (AchE) activity, transforming growth factor β (TGF-β), Fas and interleukin-6 (IL-6) levels. Meanwhile, AlCl3 supplementation produced significant decrease in brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1 (IGF-1) when compared with control values. Also, AlCl3 administration caused significant decline in the expression levels of disintegrin and metalloproteinase domain 9 (ADAM9) and a disintegrin and metalloproteinase domain 10 (ADAM10) genes in the brain. Histological investigation of brain tissue of rat model of AD showed neuronal degeneration in the hippocampus and focal hyalinosis with cellular as well as a cellular amyloid plaques formation. Oral administration of SP or NK in a rat model of AD daily for 45 days resulted in a significant decrease in brain AchE activity, TGF-β, Fas and IL-6 levels. Also, the treatment with these enzymes produced significant increase in BDNF and IGF-1 levels when compared with the untreated AD-induced rats. Moreover, both SP and NK could markedly increase the expression levels of ADAM9 and ADAM10 genes in the brain tissue of the treated rats. These findings were well confirmed by the histological examination of the brain tissue of the treated rats. The present results support our hypothesis that the oral administration of proteolytitc enzymes, SP and/or NK, would have an effective role in modulating certain factors characterizing AD. Thus, these enzymes may have a therapeutic application in the treatment of AD.

Topics: ADAM Proteins; ADAM10 Protein; Alzheimer Disease; Animals; Brain; Brain-Derived Neurotrophic Factor; Cholinesterases; Disease Models, Animal; Disintegrins; fas Receptor; Insulin-Like Growth Factor I; Interleukin-6; Male; Peptide Hydrolases; Rats; Subtilisins; Transforming Growth Factor beta

By introducing aprN into Lactococcus lactis NZ9000, the genetically modified L. lactis NZ9000/pNZPNK successfully expressed the nattokinase. The safety assessment of this novel strain was based on allergenicity of pepsin digestion stability and murine model serologic identity. Subjecting to the GM strain and host to pepsin digestion, the soluble fractions and cell debris were fast degraded completely. Feeding with ovalbumin resulted in significantly higher production of IgG1 and IgE as compared to that of L. lactis NZ9000/pNZPNK or L. lactis NZ9000. Further, the serum IgG2a level increased dose-dependently at week 2 and induced immune reaction toward Th1 pathway. Secretion of cytokines IL-4 and IL-10 fed with lactococci was significantly lower than that of the OVA group. L. lactis NZ9000/pNZPNK did not increase the proliferation of type 2 helper T cells in spleen or induce allergenicity in BALB/c mice. On the basis of the results, the new GM lactic acid bacterium is regarded as safe to use.

Topics: Allergens; Animals; Base Sequence; Disease Models, Animal; DNA Primers; Enzyme-Linked Immunosorbent Assay; Immunoglobulins; Lactococcus lactis; Mice; Organisms, Genetically Modified; Pepsin A; Subtilisins

Thrombosis is characterized by congenital and acquired procatarxis. Nattokinase inhibits thrombus formation in vitro. However, in vivo evaluation of the therapeutic efficacy of nattokinase against thrombosis remains to be conducted. Subcutaneous nattokinase injections of 1 or 2 mg/ml were administered to the tails of rats. Subsequently, κ-carrageenan was intravenously administered to the tails at 12 h after nattokinase injections. The mean length of the infarcted regions in the tails of rats was significantly shorter in rats administered 2 mg/ml of nattokinase than those in control rats. Nattokinase exhibited significant prophylactic antithrombotic effects. Previously, the in vitro efficacy of nattokinase against thrombosis had been reported; now our study has revealed the in vivo efficacy of nattokinase against thrombosis.

Topics: Animals; Carrageenan; Disease Models, Animal; Female; Fibrinolytic Agents; Infarction; Rats; Rats, Wistar; Soy Foods; Subtilisins; Tail; Thrombosis

Although soy foods have been consumed for more than 1000 y, it is only in the past 20 y that they have made inroads into Western diets. We investigated the effect of dietary supplementation with natto extracts produced from fermented soybeans on intimal thickening of arteries after vessel endothelial denudation. Natto extracts include nattokinase, a potent fibrinolytic enzyme having four times greater fibrinolytic activity than plasmin. Intimal thickening was induced in the femoral arteries by intravenous infusion of rose bengal followed by focal irradiation with a transluminal green light. Dietary natto extract supplementation was started 3 wk before endothelial injury and continued for another 3 wk after. In ex vivo studies, euglobulin clot lysis times were measured 3 wk after the initial supplementation. Neointima formation and thickening were also initiated successfully. The intima media ratio 3 wk after endothelial injury was 0.15 +/- 0.03 in the control group. Dietary natto extract supplementation suppressed intimal thickening (0.06 +/- 0.01; P < 0.05) compared with the control group. Natto extracts shortened euglobulin clot lysis time, suggesting that their thrombolytic activities were enhanced. These findings suggest that natto extracts, because of their thrombolytic activity, suppress intimal thickening after vascular injury as a result of the inhibition of mural thrombi formation.

Topics: Animals; Cell Division; Dietary Supplements; Disease Models, Animal; Endothelium, Vascular; Fermentation; Glycine max; Male; Plant Extracts; Platelet Aggregation; Random Allocation; Rats; Rats, Sprague-Dawley; Subtilisins; Thrombosis; Tunica Intima

We have previously demonstrated that natto-extracts containing nattokinase (NK) inactivates plasminogen activator inhibitor type 1 and then potentiates fibrinolytic activity. In the present study, we investigated the effects of dietary supplementation with natto-extracts on neointima formation and on thrombolysis at the site of endothelial injury. Endothelial damage in the rat femoral artery was induced by intravenous injection of rose bengal followed by focal irradiation by transluminal green light. Dietary natto-extracts supplementation containing NK of 50 or 100 CU/body was started 3 weeks before endothelial injury and then continued for another 3 weeks. Intimal thickening in animals given supplementation was significantly (P<0.01) suppressed compared with controls and the intima/media ratio in animals with 50 and 100 CU/body NK and control group was 0.09 +/- 0.03, 0.09 +/- 0.06 and 0.16 +/- 0.12, respectively. Although femoral arteries were reopened both in control animals and those treated with NK within 8 hours after endothelial injury, mural thrombi were histologically observed at the site of endothelial injury. In the control group, the center of vessel lumen was reopened and mural thrombi were attached on the surface of vessel walls. In contrast, in NK-treated groups, thrombi near the vessel wall showed lysis and most of them detached from the surface of vessel walls. In conclusion, dietary natto-extracts supplementation suppressed intimal thickening produced by endothelial injury in rat femoral artery. These effects may partially be attributable to NK, which showed enhanced thrombolysis near the vessel wall.

Topics: Animals; Dietary Supplements; Disease Models, Animal; Femoral Artery; Fibrinolytic Agents; Glycine max; Male; Plant Extracts; Rats; Rats, Sprague-Dawley; Subtilisins; Thrombolytic Therapy; Thrombosis; Tunica Intima

The existence of a potent fibrinolytic enzyme (nattokinase, NK) in the traditional fermented food called 'natto', was reported by us previously. It was confirmed that oral administration of NK (or natto) produced a mild and frequent enhancement of the fibrinolytic activity in the plasma, as indicated by the fibrinolytic parameters, and the production of tissue plasminogen activator. NK capsules were also administered orally to dogs with experimentally induced thrombosis, and lysis of the thrombi was observed by angiography. The results obtained suggest that NK represents a possible drug for use not only in the treatment of embolism but also in the prevention of the disease, since NK has a proven safety and can be massproduced.

Topics: Administration, Oral; Adult; Animals; Disease Models, Animal; Dogs; Female; Fibrinolysis; Fibrinolytic Agents; Glycine max; Humans; Male; Middle Aged; Serine Endopeptidases; Subtilisins; Thrombosis; Time Factors; Tissue Plasminogen Activator