Abstract
Background/Aim: Nattokinase (NK) is a potent serine protease with various pharmacological properties that include thrombolytic, anti-inflammatory, and antioxidant activities. The aim of this study was to investigate the neuroprotective effects of NK in transient middle cerebral artery occlusion (MCAO)-induced cerebral ischemia-reperfusion injury and determine the mechanisms underlying the effects of NK.
Materials and Methods: Rats were administered NK (65 and 130 mg/kg body weight, p.o.) daily for seven days prior to MCAO surgery. The infarct volume, behavioral tests, clotting time, and antioxidant markers in the MCAO model rats were assessed. The involvement of various cellular pathways in the effects of NK was examined.
Results: NK treatment dose-dependently reduced infarct volume and prolonged clotting time in MCAO model rats. Transcription factor activity analysis revealed the involvement of nuclear factor erythroid 2-related factor 2 (Nrf2) activity and enhanced antioxidant enzyme expression following MCAO.
Conclusion: Oral nattokinase confers early neuroprotection after experimental cerebral ischemia through Nrf2-associated antioxidative modulation and a mild, transient anticoagulant effect, supporting its potential as an orally available adjunct in ischemic stroke management.
Introduction
Vascular diseases are major causes of death and disability worldwide. The incidence of cerebral infarction is high, and cerebral infarction considerably reduces the quality of life of patients (1). Middle cerebral artery occlusion (MCAO) is a common and severe form of cerebral infarction in which cerebral blood flow is blocked, leading to neuronal necrosis and marked neurological deficits (2). Vascular diseases impose substantial social and economic burdens as patients often require long-term rehabilitation and nursing care.
Current therapeutic approaches to MCAO, such as thrombolytic therapy and mechanical thrombectomy, effectively restore blood flow; however, the limitations of these approaches are their narrow therapeutic time windows (3, 4) and the risk of serious bleeding complications (5, 6). Therefore, safer and more effective treatment options are urgently needed for MCAO.
Natto is a traditional Japanese fermented food that contains an enzyme known as nattokinase (NK). NK is a protease produced by Bacillus subtilis var. natto during the fermentation process (7). The thrombolytic activity of NK was the first to be identified, which is achieved via activating plasminogens and promoting fibrinolysis (8). Orally administering NK substantially inhibited thrombus formation in a rat model (9). NK is absorbed by the intestinal tract and produces systemic thrombolytic effects, supporting the use of NK as an orally administered therapeutic agent (10).
NK exerts anti-inflammatory, antioxidant, and neuroprotective effects, in addition to thrombolytic effects. For instance, NK suppresses lipopolysaccharide-induced inflammation and oxidative stress via inhibiting NOX2 and TLR4 activation, thus reducing the production of ROS and the activation of NF-κB (11). Neuroprotective effects of nattokinase were found in cerebral infarction models (12). These findings suggest that NK is a multifunctional therapeutic candidate for preventing and treating ischemic stroke.
MCAO-related mortality is high and the sequelae are severe when early treatment is not received. Safe, sustainable, and orally administered treatments must be developed given the increasing prevalence of cerebrovascular diseases owing to the aging of the population. Basic research on the thrombolytic and neuroprotective properties of NK has progressed; however, evidence for the clinical application of NK is insufficient.
The aim of this study was to determine the therapeutic potential of NK in MCAO through focusing on the efficacy, safety, and mechanisms of action of NK. These findings can contribute to the development of practical treatment options for cerebral infarction and related thrombotic disorders to increase the quality of life of patients.
Materials and Methods
Animals and grouping. Female Long Evans rats weighing 212.6-331.1 g were purchased from the National Laboratory Animal Center. All animals were housed under controlled conditions (50-70% relative humidity, 20-24°C) with ad libitum access to food and a 12-hour light/dark cycle. The rats were randomly assigned to one of four groups, with five animals in each group: sham, MCAO, and two treatment-dose groups. Rats were orally administered deionized water (control) or one of two NK doses (65 mg/kg and 130 mg/kg body weight) once per day for seven consecutive days prior to surgery. A MCAO rat model was established to induce cerebral ischemia. The rats were continuously administered the drug after MCAO was established until sacrifice.
Ethics approval. All animal procedures were approved by the Animal Experiment Approval Provisions (IACUC-2017-SH-007) and performed in compliance with the Animal Experiment Regulations of Trineo Biotechnology Co., Ltd., and the Animal Experiment Committee Regulations (approval no. IACUC-2017-SH-007). The research facility was accredited by the Taiwan Accreditation Foundation and certified as a compliance-registered good laboratory practices facility for preclinical testing laboratories (certificate No. GLP-0031).
MCAO surgery. All procedures were performed under anesthesia induced by an intraperitoneal injection of 450 mg/kg chloralhydrate. The temperature of the rats was maintained constant during surgery using a heating pad. A midline neck incision was created, and the carotid artery was separated from the vagus nerve and isolated. The middle cerebral artery (MCA) was then exposed from the exposed area of the brain. A 10-0 surgical suture was used to ligate the MCA until fully occluded. The bilateral common carotid arteries were temporarily blocked with microvascular clips for 60 min, after which the common carotid arteries were unclipped to blood recirculation. All incisions were sutured using 4-0 surgical suture. The rats in the sham group were subjected to the same anesthesia and surgical procedures as the rats in the other groups but without occlusion.
Infarction volume analysis. The rats were sacrificed on either day 4 or 8 after the MCAO operation. Their brains were immediately removed for measuring the infarct volume. The brains were divided into coronal sections 2 mm thick starting from the frontal pole. The slices were incubated in a 2% 2,3,5-triphenyltetrazolium chloride solution at 37°C for 30 min before fixation with 10% formaldehyde. The infarct area was measured using Image J v1.50 software (NIH), and the area was multiplied by the section thickness to obtain the infarct volume as a percentage using the following formula: (total brain volume infarct brain volume)/total brain volume × 100.
Gait analysis. The gait of unforced moving rats was analyzed using CatWalk XT (Noldus Information Technology, Wageningen, The Netherlands). CatWalk XT quantifies animal footprints. The quantified gait parameters included the maximum contact mean intensity [arbitrary units (a.u.); the mean pressure exerted by one paw in contact with the floor at the point of maximum contact], stand (s; duration of paws in contact with glass plate), and cadence (steps/s; the frequency of steps during a trial). The cadence was calculated as the number of steps divided by the initial contact last step minus the initial contact of the first step using CatWalk XT software.
Blood coagulation tests. To determine the direct effects of NK dose on coagulation without MCAO, rats were randomly allocated into two groups, 13 and 65 mg/kg, with three rats in each group. The rats were orally administered NK once daily for five consecutive days. Blood was collected from the rats on days 0 and 5. The blood samples were collected from tail veins, and the plasma was separated from the blood. The plasma was stored until the blood coagulation tests to determine the prothrombin time (PT) and activated partial thromboplastin time (APTT).
For the MCAO experiments, blood samples were collected from rats 4 and 8 days after surgery. The clotting time, PT, APTT, and plasma tissue plasminogen activator (tPA) levels were measured to evaluate the effects of NK on coagulation after ischemic injury. The clotting time test was conducted by collecting a capillary blood sample in a microhematocrit glass capillary. The chronometer was started when the blood contacted the glass capillary tube. The blood was allowed to flow due to gravity between the two marks on the tube that were 45 mm apart by tilting the capillary tube alternately to +60° and −60° with respect to the horizontal plane until the blood ceased to flow, which was considered the reaction end point. The PT and APTT tests were performed using platelet-free plasma samples with an automated blood coagulation analyzer (Sysmex CA-600 series).
Biochemical analysis. The plasma tissue plasminogen activator (tPA) levels were measured using a tPA ELISA Kit (Abcam, Cambridge, UK) according to the provided instructions. Lactate dehydrogenase (LDH) activity was measured using an automated clinical chemistry analyzer (Toshiba TBA-25FR, Tokyo, Japan) using blood serum and brain tissue homogenates.
Transcription factor activity assay. The transcription factor-DNA binding activity assay for the Nrf2 in the brain tissue homogenates from each group was performed according to the instructions provided with the kit (Cayman Chemicals, Ann Arbor, MI, USA).
Briefly, 10 μg of nuclear protein was loaded into a well containing a specific dsDNA sequence and incubated overnight at 4°C. The primary antibodies against Nrf2 were loaded into each well, and the plates were incubated at room temperature for 1 h. Each well was flushed twice with a diluted wash buffer for 30 min each time. The samples were further incubated with secondary horseradish peroxidase-conjugated antibody for 1 h at room temperature. The transcription factor binding activity was quantified by adding a developing solution containing a 3,3′,5,5′-tetramethylbenzidine (TMB) to each well and incubating the wells for 45 min at room temperature. DNA binding was quantified using photospectrometry, with absorbance measurements recorded at 450 nm using a microplate reader (FLUOStar, BMG, Ortenberg, Germany).
Gene expression analysis. The gene expression levels were detected by collecting brain tissue homogenates from each group, from which RNA was extracted (Illustra RNAspin Mini Kit, GE Healthcare, Chicago, IL, USA), and cDNA was synthesized. The gene expression of four different antioxidant enzymes, heme oxygenase-1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1), glutathione S-transferase (GSTP1), and superoxide dismutase 3 (SOD3), was analyzed using a StepOne Plus Real-Time PCR System (Applied Biosystems, Carlsbad, CA, USA) according to the protocol provided by the manufacturer.
Statistical analysis. Data were collected from the experiments and are presented as mean and standard deviation (mean ± S.D.). Data were compared between groups using nonparametric one-tailed Mann-Whitney tests. Statistical significance was set at p<0.05.
Results
NK protected against ischemia-induced brain damage. The effects of NK on ischemia-induced brain injury were evaluated using rats subjected to MCAO as cerebral ischemia models. It has been reported that the APTT was prolonged after administering NK to healthy male patients (13). The appropriate NK dose was determined by administering NK to normal rats for five consecutive days. Blood was collected from the rats on days 0 and 5, and blood parameters were measured. Administering 65 mg/kg NK altered the APTT; however, this dose did not affect the PT (Figure 1).
Effects of nattokinase administration on prothrombin time (PT) and activated partial thromboplastin time (APTT) in each group of rats. Rats were orally administered 13 or 65 mg/kg nattokinase once per day for five consecutive days. PT (A) and APTT (B) blood coagulation test results. *p<0.05. All values are expressed as mean±S.D. (n=3).
NK was administered at doses of 65 and 130 mg/kg in vivo to investigate its efficacy and potential mechanisms of action. None of the rats showed any adverse clinical effects during the pre-MCAO dosing period (7 days) at 65 or 130 mg/kg NK (data not shown). One rat in each control group (days 4 and 8 after surgery) died before the scheduled sacrifice after MCAO. The remaining rats showed no severe adverse effects after MCAO.
The histopathological analysis revealed that the white infarct area in the MCAO group was substantially larger than that in the sham group (Figure 2). In contrast, the infarct size was markedly smaller in the NK-treated groups than in the MCAO control group. Administering NK at doses of 65 and 130 mg/kg notably reduced the infarct volume 4 and 8 days after MCAO compared with that at baseline. The neurological function of the rats was evaluated by testing their gait behavior using CatWalk XT before sacrifice, 8 days after surgery. The mean maximum left hind contact intensity in the 65 mg/kg group was significantly higher than that in the control group (Figure 3); however, the remaining parameters did not significantly differ among the groups (data not shown).
Changes in cerebral infarction volume in each study group. (A, B) Staining with 2,3,5-triphenyltetrazolium chloride was used to observe infarct volume in rats 4 (A) or 8 (B) days after stroke. (C, D) Infarct region volume in rats 4 (C) or 8 (D) days after stroke. *p<0.05. All values are expressed as mean±S.D. (n=4-5).
Effects of nattokinase treatment on gait traits. Mean intensity changes in maximum contact of four limbs, (A) RF and RH, (B) LR and LH, on day 8 after middle cerebral artery occlusion. All values are expressed as mean±S.D. (n=4-5).
These results demonstrate that orally administering NK reduced cerebral infarct volume and partially improved motor performance after ischemic injury, suggesting a mild but notable neuroprotective effect in MCAO rats.
NK prolonged clotting time in MCAO model rats. The effects of NK on the rat coagulation system were evaluated by measuring the clotting time, PT, APTT, and plasma tPA levels 4 and 8 days after MCAO. The clotting time was significantly prolonged in the 65 and 130 mg/kg groups compared with that in the controls 4 days after surgery (p<0.05), indicating a transient anticoagulant effect of NK (Figure 4). The APTT was also much longer in the 130 mg/kg group at this time point, whereas the PT and plasma tPA levels remained unchanged in all groups. No significant differences were observed in any of these parameters between any group and the controls 8 days after surgery (Figure 4). These results suggest that NK exerts a mild, transient anticoagulant effect, which is primarily reflected in an increase in clotting time rather than in alterations in classical coagulation factors or tPA-mediated fibrinolysis.
Effects of nattokinase treatment on blood coagulation. Clotting time, prothrombin time (PT), activated partial thromboplastin time (APTT) and tissue plasminogen activator (tPA) on day 4 (A) and day 8 (B) after middle cerebral artery occlusion. *p<0.05. All values are expressed as mean ± S.D. (n=4-5).
NK reduced LDH release in MCAO model rats. The extent of neuronal injury was evaluated by measuring the LDH levels in the serum of MCAO rats. The LDH activity was lower in the 130 mg/kg NK-treated group at 4 days after surgery compared with that in the control group. The LDH activity in the 65 mg/kg NK-treated group did not notably differ from that in the controls (Figure 5). These findings indicated that NK exerted a partial protective effect against ischemia-induced cytotoxicity, as evidenced by the reduction in the LDH released in the high-dose group.
Effects of nattokinase treatment on LDH activity in each group. Serum LDH levels on days 4 (A) and 8 (B) after middle cerebral artery occlusion. *p<0.05. All values are expressed as mean±S.D. (n=4-5).
NK promoted Nrf2 activity in MCAO model rats. Nrf2 is a key transcription factor activated under oxidative stress (14). ELISA was used to analyze the effect of NK treatment on the DNA-binding activity of Nrf2 in the brain. The Nrf2 activity in the 65 mg/kg NK group was significantly higher than that in the control group on day 8 (Figure 6).
Effects of nattokinase treatment on transcription factor Nrf2 activity in each group. Transcription factor Nrf2 activity on day 4 (A) and 8 (B) after middle cerebral artery occlusion. *p<0.05. All values are expressed as mean±S.D. (n=4-5).
NK regulated antioxidative enzyme expression in MCAO model rats. The effects of NK treatment on the gene expression of four different antioxidant enzymes, including HO-1, NQO1, GSTP1 and SOD3, were examined on days 4 and 8 after MCAO. The expression level of SOD3 was significantly higher in both NK treatment groups (65 mg/kg and 130 mg/kg) than in the control group 4 days after surgery (Figure 7). The GSTP1 expression level in the 130 mg/kg NK group was substantially higher than that in the control and 65 mg/kg NK group. However, no significant differences were found among the groups in the HO-1, NQO1, GSTP1 and SOD3 expression level on day 8 after surgery (Figure 7). The results showed that NK regulated Nrf2 pathway activation and dose-dependently affected the expression levels of SOD3 and GSTP1 in MCAO-induced ischemic cerebral injury in rats.
Effects of nattokinase treatment on antioxidant enzyme gene expression levels in each group. Quantitative analysis of relative expression levels of HO-1, GSTP1, NQO1, and SOD3 in rat brains day 4 (A) and 8 (B) after middle cerebral artery occlusion. *p<0.05. All values are expressed as mean±S.D. (n=4-5).
Discussion
Consistent with previous findings demonstrating the neuroprotective efficacy of NK in thrombolytic stroke models (12), the present study further confirms its potential to attenuate ischemic injury, likely through complementary vascular and antioxidant mechanisms. NK was administered in a previous study to Wistar rats 4 h and 1-2 days after MCAO reperfusion, which attenuated poststroke brain injury by reducing free radical levels and enhancing the expression of Janus kinase 1 and nitric oxide (12).
In our study, oral pretreatment with NK substantially reduced the infarct volume in MCAO rats compared with that in the control group, confirming the anti-ischemic effect of NK administration (Figure 2). NK treatment also produced limited improvements in gait parameters, transiently prolonged clotting time on day 4 after surgery, and did not alter PT or plasma tPA, decreased LDH levels on day 4 in the high-dose group, and increased Nrf2 DNA-binding activity in the rats. These results collectively indicate that NK treatment exerts partial and time-dependent neuroprotective effects.
These findings suggest that NK primarily exerts effects through endogenously modulating antioxidant enzyme expression rather than through direct thrombolysis. Activating Nrf2, a master regulator of the cellular response to oxidative stress, implies that NK enhances redox homeostasis during the acute postischemic phase, limiting secondary neuronal injury.
The Nrf2/HO-1 pathway plays a crucial role in the cellular defense against oxidative stress in vitro and in vivo (14). Nuclear Nrf2 is a transcription factor that induces the expression of several antioxidant enzymes, such as HO-1, NQO1, and GST. Consistent with this activation, the mRNA expression analysis of downstream antioxidant enzymes showed that the SOD3 and GSTP1 expression levels were substantially and dose-dependently up-regulated on day 4 after surgery, whereas the HO-1 and NQO1 expression levels remained unchanged. Similar findings were reported by Elbakry et al. (15), who demonstrated that NK attenuated hepatic and neural toxicity by activating the Nrf2/HO-1 pathway and suppressing inflammatory mediators in rats. This finding suggests that NK partially or phase-dependently activate the Nrf2 pathway, reflecting early antioxidative adaptation following ischemic insult.
The transient increase in clotting time observed on day 4 after surgery in the rats, in the absence of alterations in PT or tPA, indicated a mild anticoagulant shift that increases microcirculatory reperfusion without increasing the risk of systemic bleeding. This temporary effect aligns with previous reports describing NK fibrinolytic activity mediated through degrading fibrin and activating plasmin. However, our findings indicate that the physiological influence of NK on coagulation is modest and reversible.
In summary, NK is a multifunctional but moderate neuroprotectant that acts during the early reperfusion phase. Future investigations should verify the causal role of NK in activating Nrf2 using pharmacological inhibition or gene-silencing approaches, determine the pharmacokinetic-pharmacodynamic relationship of oral NK, and evaluate long-term outcomes of NK treatment, such as behavioral recovery and vascular remodeling.
Conclusion
Oral NK administration confers early phase neuroprotection after cerebral ischemia predominantly through Nrf2-mediated antioxidative modulation and a transient anticoagulant effect. These results support the potential of NK as a safe and orally available adjunct for preventing or attenuating injury due to ischemic stroke.
Footnotes
Authors’ Contributions
Conceptualization, T.T., J.Y., and M.K.; methodology, J.Y., K.H., and S.C.; formal analysis, T.T. and M.K.; supervision, T.T. and M K.; writing–original draft preparation, T.T. and M.K.; writing–review and editing, T.T., J.Y., K.H., S.C., A.I., and M.K. All Authors have read and agreed to the published version of the article.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
Funding
Not applicable.
Artificial Intelligence (AI) Disclosure
During the preparation of this manuscript, a large language model (ChatGPT, OpenAI) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning-based image enhancement tools.
- Received November 19, 2025.
- Revision received November 30, 2025.
- Accepted December 1, 2025.
- Copyright © 2026 The Author(s). Published by the International Institute of Anticancer Research.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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