The purpose of this study is to study the affinity of MFG-E8 and Oleic acid/linolic acid/linolic acid by molecular docking technology( Agu et al , 2023). We divided the project into four modules: cell damage model building module, protein structure modeling module, small molecule preparation module and docking analysis module. We used AlphaFold3 to model the target protein MFG-E8, and optimized the structures of MFG-E8 and oleic acid by using Maestro calculation software. The predicted protein structure and small molecules were optimized to ensure stability and minimize energy, which significantly increased the possibility and accuracy of successful docking. Subsequently, according to the semi-flexible docking method, the binding modes of three fatty acid molecules were analyzed. We expect to determine the best binding mode of MFG-E8 with three fatty acids, which will help us to understand their relieving effects on inflammation.

1. Esophageal epithelial cells (HEEpiC) were cultured in the DMEM medium containing 10%FBS and 1%penicillin/streptomycin,in a humidified incubator at 37℃with 5% CO2.The growth medium was replaced everyday and the cells were propagated every three days.For cells injury model construction(M group),the cells were seeded into 6-well plates and grew for 24 h,then the plates were put into the aseptic cell incubator for 1 h at 45℃with 5%CO2.Then the plates were sent back to the normal culture incubator at 37℃with 5%CO2.In treatment group(M+D group),before the cells were put into the special incubator at 45℃,the embedded functional protein complex was added into the medium with the concentration of 1μM,and the action time of functional protein complex was 1 h. Then, cell samples were collected.

2. For MFG-E8 is a soluble protein that can be expressed in Escherichia coli(E.coli), which is designed as follows. By cloning into a vector, expression plasmids encoding functional proteins are further generated and transformed into pET283a and Dh5a strains. Then, plasmids are extracted and constructed from positive clones and confirmed by sequencing. Next, extract the protein from the plasmid for purification.( Dong et al , 2023)

3. Introduce the overexpression vector into expression-deficient cells for the purpose of interaction validation or to apply stress treatment to the overexpressing cells.

1.Experiment results showed that inflammatory factors including NF-κB,TNF-αand IL-1βwere up-regulated in model group,which were sharply down-regulated with the treatment of three types of fatty acids,indicating that three types of fatty acids could suppress inflammation reaction in some degree,in the present heat injury model.

2. Results demonstrated that MFG-E8 protein could inhibit the expression levels of inflammatory factors,and three types of fatty acids could up-regulate the expression level of MFG-E8 protein,further proved that three types of fatty acids could increase the level of MFG-E8,and the latter factor could suppress inflammation.

3. Through Western blot analysis, we successfully verified the interaction between MFG-E8 and Oleic acid/linolic acid/linolic acid. Results demonstrated that the treatment of MFG-E8 protein complex with the concentration of 1μM significantly alleviated inflammatory reaction,embodying on the down-regulation of inflammatory factors,including TNF-αand IL-1βproteins.

4. Conduct morphological, protein, and molecular level assessments on cells successfully transfected with the plasmid to confirm the successful construction of the vector and the functionality of the protein pathway

1. Through experiments, we verified the potential interaction model between MFG-E8 and Oleic acid/linolic acid/linolic acid. The expression of functional protein (MFG-E8) in this pathway plays a key role in inhibiting inflammation and malignant lesions of esophageal tissue(Hua et al , 2022). The three fatty acids inhibited the inflammatory reaction in the heat injury model to some extent by reducing the level of inflammatory factors and increasing the level of functional protein (MFG-E8).

2. We found that three kinds of fatty acids can improve the level of MFG-E8, and the treatment of MFG-E8 protein complex with a concentration of 1 μ M can significantly alleviate the inflammatory reaction, which is manifested by the down-regulation of inflammatory factors TNF-α and IL-1 β. Based on these characteristics, we prepared a chemical tablet containing 1 μ M MFG-E8 protein, which is a functional protein with in vitro repair effect.

1.Agu P C , Afiukwa C A , Orji O U ,et al.Molecular docking as a tool for the discovery of molecular targets of nutraceuticals in diseases management[J].Scientific Reports, 2023, 13(1).DOI:10.1038/s41598-023-40160-2.

2.Dong Xiaohong, et al."MFG-E8 Alleviates Cognitive Impairments Induced by Chronic Cerebral Hypoperfusion by Phagocytosing Myelin Debris and Promoting Remyelination.."Neuroscience bulletin 40.4(2023):483-499.

3.Hua Xiaomin, et al."Protective Effect of MFG-E8 on Necroptosis-Induced Intestinal Inflammation and Enteroendocrine Cell Function in Diabetes."Nutrients 14.3(2022):604-604.