Study on the transport and internalization mechanism of dietary supplement nattokinase in the small intestine using animal and Caco-2 cell monolayer models

Abstract:1. Maintaining proper blood flow is critical to promoting good health. Nattokinase is a serine protease from Bacillus subtilis that has significant in vitro thrombolytic activity, but its mechanism as a dietary supplement to prevent thrombosis through intestinal absorption and transport is still unclear.2.The purpose of this study is to study the transport and internalization mechanism of NK in the small intestine using animal models and Caco-2 cell monolayer models.3.This study first evaluated the preventive effect of supplementing low dose (4000 FU (Fibrin Unit)/kg, n = 6), medium dose (8000 FU/kg, n = 6), and high dose (12000 FU/kg, n = 6) of nattokinase on carrageenan induced thrombosis in mice. Subsequently, we used the rat gut sac model, ligated intestinal loop model, and Caco-2 cell uptake model to study the intestinal transport mechanism of NK.4. Results indicate that NK is a moderately absorbed biomolecule whose transport through enterocytes is energy- and time-dependent. Chlorpromazine, nystatin and EIPA all inhibited the endocytosis of NK to varying degrees, indicating that the endocytosis of NK in Caco-2 cells involves macropinocytosis, clathrin-mediated and caveolae-mediated pathway. These findings offer a theoretical basis for investigating the mechanism of oral NK supplementation in greater depth.Keywords: NattokinaseCaco-2 cellsinverted intestinal sac modelintestinal absorptiontransport mechanismDisclaimerAs a service to authors and researchers we are providing this version of an accepted manuscript (AM). Copyediting, typesetting, and review of the resulting proofs will be undertaken on this manuscript before final publication of the Version of Record (VoR). During production and pre-press, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal relate to these versions also. Conflict of interestThe authors declare that there are no conflicts of interest.Additional informationFundingThis work is supported by Liaoning Province Science and Technology Innovation Leading Talent "Xing Liao Talents Program" Project [grant number XLYC2002045], The National Natural Science Foundation of China [grant number 82003655], Shenyang Science and Technology Plan Project [grant number 21-116-3-48], Shenyang Young and Middle‐aged Science and Technology Innovation Talent Support Program [grant number RC220277, RC210216], The Doctoral Research Startup Fund Guidance Program of Liaoning Province [grant number 2023-BS-084], and the Scientific Research Project from the Department of Education of Liaoning Province [grant number LJKZ0088, LQN201906, LJKMZ20220455].