Study On Intestinal Absorption Characteristics And Thrombolytic Activity Of Nattokinase

Nattokinase（NK）is a serine protease with a molecular weight of about 28 k Da.It consists of 275 amino acids in a fixed arrangement.It was first extracted and purified by Japanese scholars from the traditional Japanese food natto,discovered and named for the first time,and can degrade fibrin.Natto is considered a functional food because it can lower cholesterol levels in the blood,lower blood pressure,inhibit osteoporosis and be beneficial to the body.Nattokinase is considered one of the most potent functional ingredients found in natto,and the recommended daily oral dose is2000 Fibrin Degrading Units（FU）.Nattokinase is a strong plasmin.Compared with other plasmins,it has the advantages of oral administration,long half-life,high activity,no toxic side effects and low cost.Studies have shown that nattokinase could be absorbed orally through the small intestine and enter the blood circulation,but how nattokinase was absorbed by the small intestine into the blood circulation and could maintain the original biological activity needs further in-depth research.In this study,the Caco-2 cell monolayer model,the inverted intestinal sac model and ligated intestinal loops model in vivo were established to further explore the absorption process and mechanism of nattokinase through the small intestine.This research mainly includes an introduction and four chapters.The introduction part reviews the literature on nattokinase,the thrombolytic mechanism of nattokinase,the research status of nattokinase and the methods and principles used in this experiment,which provides theoretical support for subsequent research.In the first chapter,the MTT toxicity test and lactate dehydrogenase release（LDH）test confirmed that nattokinase was not toxic to Caco-2 cells and would not destroy the integrity of the cell membrane.The absorption of nattokinase by Caco-2 cells was determined by flow cytometry and fluorescence microscopy.The absorption of nattokinase in Caco-2 cells was decreased after low temperature treatment.In the second chapter,the Caco-2 cell monolayer model was successfully established,and the transmembrane resistance（TEER）value was（652±36）Ω·cm~2and the alkaline phosphatase（AKP）activity ratio was 2.01 to confirm that the integrity and polarity of the Caco-2 cell monolayer model met the standard.The transport of nattokinase across the Caco-2 cell monolayer model was investigated in two directions from the AP side to the BL side and from the BL side to the AP side.The absorption and transport mechanism of nattokinase was explored by adding three different endocytosis inhibitors,Chlorpromazine,Nystatin and EIPA.In the third chapter,the absorption characteristics of nattokinase through the small intestine were verified in vitro and in vivo by establishing an in vitro culture model of intestinal eversion and an in vivo intestinal ring ligation and perfusion model,respectively.Finally,Chapter six states the conclusions of this study,reflecting on the inadequacies of the study and making a reasonable outlook for the future.This study mainly draws the following four conclusions:1.Nattokinase was not toxic to Caco-2 cells and the absorption of nattokinase by Caco-2 cells was a time-and energy-dependent process.2.The transport amount of nattokinase across the Caco-2 cell monolayer model increased with time and was proportional to the concentration of nattokinase.The calculated P_appvalues were all greater than 1×10^-6cm/s,indicating that nattokinase was a well-absorbed biomacromolecule.3.The transport of nattokinase across Caco-2 cell monolayers involved the macropinocytosis pathway and related pathways mediated by clathrin or caveolae（or lipid rafts）.4.In vitro rat intestinal eversion culture model confirmed that nattokinase could be absorbed through the small intestine and had thrombolytic activity,and the amount of absorption increased with time.5.In vivo intestinal loop ligation perfusion model verified that nattokinase could penetrate the small intestinal wall and be absorbed into the body.The significance of this study is to have a comprehensive understanding of the oral absorption of nattokinase,and to provide a theoretical basis for the clinical application of nattokinase.It is clear that nattokinase can be absorbed into the body through the small intestine to exert thrombolytic activity and its absorption mechanism.