Effects Of Main Active Substances Of Saffron On Intestinal Immunity And Insulin Metabolism In Drosophila

The intestinal epithelium is the first line of defense against infection from outside harmful substances.The gut of Drosophila has become an ideal model for studying the mechanisms of intestinal immune regulation due to its simple structure and similar functions to humans.When the intestinal mucosal epithelial barrier of Drosophila is damaged,intestinal homeostasis is disrupted,which in turn promotes the proliferation and differentiation of intestinal stem cells to compensate for the damaged intestinal epithelial cells.Studies have shown that this process requires the coordinated regulation of Notch,JNK,JAK/STAT and other signaling pathways.In addition,insulin,a regulatory hormone secreted by islets of pancreatic β cells,is responsible for maintaining glucose homeostasis in the organism.Therefore,if insulin secretion is insufficient or insulin signal transduction is defective,metabolic disorder can be caused.When Drosophila melanogaster is fed with a high concentration of sucrose,it may have symptoms similar to human type 2 diabetes: insulin resistance,fat accumulation and shortened life span.Moreover,a long-term feeding of high-sugar diet can increase insulin-like peptide levels and can induce insulin resistance.Therefore,mutants related to insulin pathway can be used as a model to study human metabolic diseases.Safranal and Crocin are the main bioactive substances of Crocus sativus L.,among which Safranal has anti-inflammatory,anti-oxidation and anti-cancer pharmacological effects,while Crocin has anti-inflammatory,anti-cancer and nerve protection effects.However,other functions and regulatory mechanisms of saffron and its bioactive substances are still unclear,especially in the fields of intestinal immunity and insulin metabolism,and the detailed regulatory mechanisms have not been elucidated.Therefore,in order to explore the effects of the main bioactive substances of saffron on intestinal immunity and insulin metabolism,the following studies were carried out in this paper.First,in terms of intestinal immunity,flies fed with dextran sulfate sodium(DSS)or infected with Erwinia carotovora subsp.carotovora 15(Ecc15)were used as a model of intestinal injury.Various intestinal epithelial cells or signal transduction pathway specificity labeled flies and antibodies were usedd to analyze intestinal epithelial cell antimicrobial peptides(AMPs)levels,cell death numbers,reactive oxygen species(ROS)levels,gut integrity,and regulation of intestinal stem cell proliferation and differentiation.It was found that the number of intestinal stem cells(ISC cells),enteroblasts(EB cells)and enteroendocrines(EE cells)in the intestine increased significantly after feeding DSS.Adding Safranal could inhibit the excessive proliferation and differentiation of intestinal stem cells induced by DSS by regulating JNK,EGFR and JAK/STAT signaling activities.Crocin had no effect on the excessive proliferation of intestinal stem cells induced by DSS.DSS feeding or infecting Ecc15 caused a significant increase in the expression level of ROS and AMPs in the intestinal tract,as well as a large number of cell deaths.Adding Safranal could eliminate excessive ROS and AMPs,and inhibited cell death,thus protecting the integrity of the intestinal barrier.In addition,in terms of insulin metabolism,flies fed a high-sugar diet or insulin signaling pathway mutants were used as models of abnormal insulin metabolism.Cells,signal transduction pathway-specific markers and antibodies to analyze whether the flies fed a highsugar diet or growth rate,pupal volume,adipocyte area,lipid droplet area,Dilp2 levels,ROS levels,and intestinal epithelial homeostasis in insulin gene-deficient flies.We found that after feeding high-sugar diet,flies appeared insulin resistance characteristics,including growth retardation,pupa size reduction,adult size reduction,adipocyte area reduction,fat droplet area increase,brain Dilp2 level and fat body ROS level increase.Adding Crocin could alleviate insulin resistance characteristics;Safranal did not affect the growth and development of flies fed a high-sugar diet.The pupa volume of insulin pathway mutants decreased,the area of fat cells and fat droplets decreased,and the ROS level of fat bodies of larvae and adults increased.The addition of Crocin could increase the pupa volume,fat cell area and fat droplet area of mutants,and reduce the ROS level of fat bodies.After high-sugar diet feeding,a large amount of intestinal fat was accumulated,and the cell arrangement was disordered.After adding Crocin,the number of lipid droplets was reduced,and the cells were arranged in an orderly manner.The number of EB cells and EE cells in the intestine was significantly increased after high-sugar diet feeding,and after the addition of Crocin,the increase in the number of these cells was inhibited by regulating JNK and JAK/STAT signal activities to maintain the homeostasis of intestinal epithelial cells.Taken together,Safranal protects intestinal integrity by regulating JNK,EGFR and JAK/STAT signal pathways,inhibiting the proliferation and differentiation of intestinal stem cells,increasing ROS and AMPs levels,and cell death induced by DSS or Ecc15.Crocin can increase the pupa size and adult size of fed high-sugar diet flies or insulin pathway mutants,reduce fat storage and ROS level in fat body,and inhibit the proliferation and differentiation of intestinal EB cells,fat accumulation and abnormal cell arrangement by down-regulating JNK and JAK/STAT signals,thus maintaining intestinal homeostasis.This study can provide a theoretical basis for the clinical research of saffron-active substances in the treatment of intestinal injury and insulin metabolism-related diseases.