Study On The Degradation Of β-amyloid Aggregates By Nattokinase

The accumulation and deposition of beta-amyloid protein(Aβ)in the brain,especially Aβ(Aβ42)with 42 amino acids,is a key factor in the pathogenesis of Alzheimer’s disease(AD).At present,the drugs or methods for the clinical treatment of AD can only relieve symptoms or have controversial efficacy.Therefore,it is urgent to find a safe and effective drug to prevent or treat AD.It has been shown that Aβ in the brain can be transported to the peripheral system and cleared through various pathways.Therefore,increasing the clearance rate of Aβ in peripheral blood and reducing its level in blood to reduce its deposition in the brain may be a good way to treat AD.Aβ42 aggregates,especially Aβ42 oligomers,not only damage the function of nerve cells,but also damage vascular endothelial cells in the soluble state or deposition on the blood vessel wall,which further aggravates the damage related to AD.Therefore,reducing or eliminating the effects of Aβ42 aggregates on vascular endothelial cells will also become an important goal for the prevention and treatment of AD.Nattokinase(NK)(EC 3.4.21.62)is a natural proteolytic enzyme,and its safety and thrombolytic effect have been widely studied and fully confirmed.Recently,it has been proposed that NK may degrade Aβ42 slowly under physiological conditions,indicating that NK may play a certain role in the prevention or treatment of Aβ42-related diseases such as AD.However,the role of NK in the targeted binding and degradation of different forms of Aβ42,especially the neurotoxic forms of Aβ42,and the reduction and clearance of Aβ42 load in the blood and even the brain are still very limited.Since there is no safe and effective drug or technology to prevent or treat AD,it is necessary to investigate the degradation effect of NK on different forms of Aβ42 aggregates and reveal its physiological significance for the prevention and treatment of AD.In this paper,we systematically studied the targeted binding and degradation of Aβ42 and its various aggregates by NK,the protective effect of the degradation of Aβ42 aggregates catalyzed by NK on vascular endothelial cells,and the effect of NK on Aβ levels or plaque load in blood and brain.The purpose of this study is to promote the research and development of drugs for the prevention and treatment of diseases related to Aβ42 aggregates,such as AD.The results of the research are as follows:1.NK has different abilities to capture different forms of Aβ42 within a certain time,among which the ability to capture Aβ42 oligomers is the strongest.Further analysis revealed that the binding of NK to different forms of Aβ42(monomer,oligomer,fiber)could be either noncatalytic or catalytic.2.We used a series of Aβ peptides and their aggregates to determine the substrate binding specificity of NK.The results showed that NK had the highest binding specificity for Aβ42 or Aβ40 and their aggregates,much higher than C-terminal truncated Aβ peptides such as Aβ35(including their aggregates),and almost no binding to Aβ28,Aβ9.It is concluded that the C-terminal region of Aβ42/Aβ40 from Gly29 to Val40 is likely to be an important region specifically targeted by NK,and the specific integrated conformation formed by this region should be the most effective target for NK to recognize and bind.3.On the basis of confirming the high binding efficiency of NK to Aβ42 aggregates,the degradation rate of substrate and product system of NK incubated with Aβ42 monomers,oligomers and fibers were analyzed.The results showed that NK mainly catalyzed the aggregates of Aβ42,and the ability of NK to degrade Aβ42 oligomers was higher than that of Aβ42 fibers.Therefore,compared with larger Aβ42 aggregates,the interaction between smaller Aβ42aggregates and NK may be more suitable for the requirements of NK catalysis,which should be caused by the steric hindrance of Aβ42 aggregates.And the results suggest that the specific integrated conformation between Aβ42 chains is the structural basis of NK’s catalytic effect on Aβ42,and this integrated conformation is more present in Aβ42 oligomers than in Aβ42 fibers,but not in Aβ42 monomers.On the other hand,degradation of Aβ42 or its aggregates by NK results in a hydrophilic Aβfragment(N-terminal fragment)and a hydrophobic Aβ fragment(C-terminal fragment),respectively.Based on the results of item 2 above,these hydrophilic Aβ fragments are likely to be generally hydrophilic Aβ peptides with 28 to 35 amino acid residues,while the hydrophobic Aβ fragments are less free due to their higher hydrophobicity.They may mainly aggregate with the C-terminal region of other Aβ42 chains to form incomplete Aβ42 aggregates or remain in the remaining substrate Aβ42 aggregates in situ.4.To determine whether NK catalyzed cleavage of chains in Aβ42 aggregates affects the structural stability of Aβ42 aggregates,we determined the extent of Aβ42 aggregation during NK incubation with three representative Aβ42 forms(monomer,oligomer,fiber).The results showed that the degradation of Aβ42 aggregates catalyzed by NK significantly slowed down or reduced the aggregation of Aβ42 or its microaggregates,and may also lead to the separation(or depolymerization)of incomplete Aβ42 aggregates,especially the Aβ42 fibers.In combination with the results of Item 3 of this paper,we speculate that NK catalyzed degradation of Aβ42aggregates directly reduces aggregation of Aβ42 or its small aggregates,and also destroys Aβ42oligomers,especially the stable integrated conformation of Aβ42 aggregates.5.Vascular endothelial cell was applied respectively to determine the effect of NK catalyzed degradation of Aβ42 aggregates on cell survival rate and damage rate.The results show that Aβ42 oligomers and Aβ42 fibers can cause a dose-dependent decrease in the viability of vascular endothelial cells,and the same dose of Aβ42 oligomers and Aβ42 fibers have similar negative effects on vascular endothelial cell viability.However,NK catalytic degradation of Aβ42aggregates significantly reduced or even eliminated the adverse effects of Aβ42 oligomers and Aβ42 fibers on vascular endothelial cell activity(or survival).The results of cell damage rate determination showed that both Aβ42 oligomers and Aβ42 fibers could induce the injury or death of vascular endothelial cells in a dose-dependent manner,and the presence of NK significantly alleviated the injury effect of Aβ42 oligomers and Aβ42 fibers on vascular endothelial cells.It is concluded that NK can protect the activity or survival rate of vascular endothelial cells by catalyzing the degradation of Aβ42 aggregates.6.We analyzed the effects of Aβ42 aggregates on transverse and radial migration of HUVECs by scratch healing assay and transwell migration assay,respectively.In the scratch healing assay(lateral migration),both Aβ42 oligomer and Aβ42 fiber could delay the scratch fusion of HUVECs,which showed that Aβ42 fiber had a more significant effect on the migration of target cells than Aβ42 oligomer.However,the addition of NK significantly increased the mobility of target cells.Therefore,NK catalyzed degradation of Aβ42 aggregates,especially Aβ42 fibers,has a significant protective effect on the lateral migration ability of vascular endothelial cells.On the other hand,the radial migration of collective cells determined by transwell assay showed that Aβ42 oligomers and Aβ42 fibers barely affected the ability of target cells to migrate in three-dimensional space,although the presence of NK still helped to maintain the radial migration of target cells.In conclusion,Aβ42 fibers were more effective than Aβ42oligomers in affecting or hinders vascular endothelial cell migration,especially radial migration,while the presence of NK significantly maintained or protected vascular endothelial cell migration from Aβ42 aggregates,especially Aβ42 fibers.7.The density and barrier performance of vascular endothelial cells are also highly dependent on their adhesion.Therefore,we analyzed the effect of Aβ42 aggregates on the adhesion ability of HUVECs.The results showed that both Aβ42 oligomers and Aβ42 fibers could decrease the adhesion rate of target cells in a concentration-dependent manner,that is,the adhesion ability of vascular endothelial cells was impaired,and the effect of Aβ42 oligomers was more severe than that of Aβ42 fibers.NK degradation of Aβ42 oligomers and Aβ42 fibers significantly mitigated the effect of Aβ42 aggregates on the adhesion rate of target cells,and even restored the adhesion ability(the re-adhesion rate)of target cells that were slightly damaged and active desorption.To determine whether the effect of Aβ42 aggregates on the adhesion rate of vascular endothelial cells was related to VE-cadherin,we further analyzed the VE-cadherin levels of HUVECs under the corresponding conditions.The results showed that Aβ42 aggregates significantly decreased the expression level of VE-cadherin in HUVECs.The catalytic degradation of NK on Aβ42 aggregates,especially Aβ42 oligomers,can reduce the effect of Aβ42 aggregates on the expression level of VE-cadherin to a certain extent,so as to maintain cell-to-cell adhesion.In addition,these results suggest that the effect of Aβ42 aggregates on VE-cadherin expression may be part of the mechanism by which they affect the adhesion ability of vascular endothelial cells.8.We investigated the effect of NK on Aβ levels and plaque loading in blood and brain using animal experiments.In vitro,NK decreased the level of Aβ in a concentration-dependent manner.In vivo,although NK was partially lost during the process of uptake and/or entry into the blood,a small amount of NK still entered the blood system and showed a good degradation effect on Aβ42/Aβ40 in a dose-dependent manner.We analyzed the cell morphology and Aβplaque burden in the hippocampus of AD mice.The results showed that although NK had no significant effect on the morphology of hippocampal cells,the load of Aβ plaques was reduced.These results suggest that NK may indirectly promote the outflow of Aβ in the brain by reducing the levels of peripheral Aβ42/Aβ40,and thus reduce the pathological changes in the hippocampus to a certain extent.In summary,we conclude that NK can degrade Aβ42 aggregates,inhibit the damage of Aβ42 aggregates to vascular endothelial cells,effectively reduce the level of Aβ in blood after entering the body,and reduce the burden of Aβ plaques in the brain to a certain extent.The results of this study provide experimental basis and theoretical guidance for the feasibility of NK in the prevention and treatment of AD.