Isolation And Identification Of GDNPs And Their Intervention Effect And Mechanism In Inflammatory Bowel Disease

Ginseng is a valuable Chinese medicinal herb.In ancient Chinese medicine records,it is widely used to treat diseases such as spleen deficiency and diarrhea.Inflammatory bowel disease（IBD）is a chronic and highly recurrent intestinal disease that causes great distress to patients.At present,there is a lack of corresponding clinical treatment,and its occurrence factors are not only genetic,but also related to immunity,intestinal microorganisms and the environment.A combination of factors contributes to an abnormal mucosal immune response and impaired epithelial barrier function.A variety of plant exosomes of different origins provide new ideas for the treatment of IBD.In this paper,GDNPs were successfully extracted and their components were sequenced and analyzed by various technical means.The regulatory effects of GDNPs on macrophages,epithelial cells,stem cells and intestinal flora were elaborated.It helps to better understand GDNPs and promote the development of ginseng fresh medicine.Objective:This article aims to study the alleviating effect of GDNPs on inflammatory bowel disease,elucidate the specific mechanism of ginseng fresh medicine in the treatment of inflammatory bowel disease,and provide new ideas for clinical prevention and treatment.Method:1 In this study,GDNPs were separated and purified by ultracentrifugation and sucrose pad density gradient centrifugation.Detection of properties such as appearance,diameter and surface potential of GDNPs using transmission electron microscopy and particle sizer;Use gastric and intestinal juice to simulate the digestive process,and determine the particle size and potential after digestion;The lipid composition of exosomes was absolutely quantified by non-targeted lipid groups.Proteome sequencing analyzes the type and abundance of proteins contained therein;RNA from GDNPs was extracted and mi RNA sequencing performed;High Performance Liquid Chromatograph（HPLC）was used to determine the types and contents of ginsenosides in exosomes.2 In vitro cell experiments were used to verify the protective effect of GDNPs on immune cells-macrophages.Macrophages were cultured in vitro to determine their uptake effect on GDNPs;Establish a model of cellular inflammation induced by lipopolysaccharide（LPS）,determine the effect of GDNPs on the secretion of inflammatory factors and the recovery of reactive oxygen species（ROS）,superoxide anion（O₂^-）levels and mitochondrial membrane function.Western blot methods were used to detect changes in factors in inflammation-related pathways MAPK,TLR4/MYD88,JAK/STAT and oxidative stress-related signaling pathways p62-Nrf2-Keap1 and the effects of autophagy-related signaling pathways.3 In vitro culture of the intestinal epithelial cell line Caco2 to detect the effect of GDNPs on cell viability.Construct a cellular inflammation model to determine the effect of GDNPs on ROS,NO and inflammatory factor levels in intestinal epithelial cells.Western blotting analyzed the expression of inflammation-related pathway factors.At the same time,intestinal stem cells were extracted,and q RT-PCR and western blotting were used to detect the changes of intestinal stem cell markers and Wnt/β-catenin-related signaling pathway factors in crypt tissue.4 A mouse intestinal inflammation model was established using DSS,and intestinal histological morphological changes were analyzed by HE staining.The ELISA method detects TNF-α,IL-6 levels in serum,q RT-PCR and western blot detect the expression of inflammatory factors in intestinal tissues and related factors in signaling pathways.5 16Sr RNA sequencing technology analyzed the composition of intestinal flora between different experimental groups to determine the regulatory effect of GDNPs on the structure of intestinal microbiota in IBD mice.Results:1 After isolation and purification,GDNPs were successfully obtained,which showed a double-layer membrane structure and slightly concave under transmission electron microscopy.GDNPs have an average diameter of 256.2 nm and a surface charge of-39.0m V.After simulated gastrointestinal digestion,the particle size of GDNPs first decreased and then increased,and the surface charge also showed a change of first increasing and then decreasing.Lipid detection showed that there were as many as 559 lipid-rich species in GDNPs;Transcriptomics detects that known mi RNAs contain 29 mature bodies and 42precursors;Proteomics detected 219 proteins in GDNPs;GO and KEGG analysis showed that it is closely related to organelles such as mitochondria and ribosomes,amino acids,oxidative phosphorylation and terpene backbone biosynthesis,and MAPK signaling pathways.2 In vivo imaging and immunofluorescence results of small animals confirmed that GDNPs were mainly localized in the intestine and could be taken up by intestinal macrophages and stem cells.In vitro experiments,GDNPs can be taken up by RAW264.7cells and promote macrophage proliferation;in the macrophage inflammation model caused by LPS,GDNPs can significantly reduce the indicators of inflammation and oxidative stress caused by LPS,have a restorative effect on mitochondrial damage of cells,increase the number of mitochondria,and restore mitochondrial membrane potential;q RT-PCR results show that GDNPs can reduce the expression of inflammatory factors at the transcription level;WB results show that GDNPs can reduce the expression of inflammatory factors through p62-Nrf2-Keap1,MAPK TLR4/MYD88 and JAK/STAT signaling pathways reduced inflammation and oxidative stress induced in vitro,and increased the level of autophagy in cells.3 In the intestinal epithelial cell inflammation model,it was confirmed that GDNPs had no obvious toxicity to intestinal epithelial cells,while reducing LPS-induced ROS,NO levels and expression of inflammatory factors.After co-culture of GDNPs and ex vivo intestinal stem cells,the morphology and activity of stem cells were maintained,and the expression of stem cell markers Lgr5 and proliferation-related factors was significantly promoted.WB results showed that GDNPs may promote the proliferation of intestinal stem cells by regulating Wnt/β-catenin.4 In mouse models of inflammatory bowel disease,GDNPs improved survival and intestinal length in mice after modeling.HE results showed a decrease in inflammatory infiltrates and an increase in epithelial goblet cells after administration;The expression of inflammatory factors in mouse intestinal tissues was significantly reduced;At the same time,GDNPs also significantly reduced the levels of inflammatory factors（TNF-α,IL-6）in the serum of mice.WB results showed that GDNPs alleviated DSS-induced intestinal inflammation by affecting the Nrf2 signaling pathway,MAPK signaling pathway and autophagy levels.The results of intestinal microbiota analysis showed that the proportion of Bacteroides and Firmicutes increased after administration,and the proportion of Proteobacteria decreased compared with the module.This shows that GDNPs are beneficial in regulating homeostasis in the intestinal environment.Conclusion:1 Purified GDNPs were successfully obtained and their physicochemical properties and composition analyzed.2 The in vivo localization of GDNPs was determined,confirming that they can alleviate the inflammatory response and oxidative stress of macrophages through multiple signaling pathways.3 GDNPs can establish a repairing effect of the intestinal barrier by reducing intestinal epithelial cell inflammation and promoting intestinal stem cell proliferation.4 GDNPs improved the structure of intestinal flora,increased the proportion of Bacteroides and Firmicutes,and confirmed the therapeutic effect of GDNPs on mouse models of inflammatory bowel disease from multiple angles.