Exploring The Effects Of Iron Deficiency On Intestinal Cells Development Based On Piglet Intestinal Organoids

Iron is an important trace element in mammals and plays an important role in the process of growth and development.The iron content in newborn piglets is often difficult to meet their growth requirements,which can easily lead to diseases such as iron deficiency anaemia,inhibit the normal growth and development of piglets.Although recent studies have shown that iron deficiency affects intestinal function,the effects of iron deficiency on intestinal cell growth and differentiation and the related molecular mechanisms have yet to be explored.The intestinal organoid is a three-dimensional cellular aggregate similar to the in vivo structure formed by intestinal stem cells supported by matrigel and using their proliferation and differentiation ability,and has various cell types of intestinal epithelium.Therefore,this study established a piglet intestinal organoid culture system and explored the effects of iron deficiency on the growth and differentiation of piglet intestinal cells and the related molecular mechanisms based on the piglet intestinal organoids model,with the following main results:1.A piglet intestinal organoid model was successfully constructed.The extraction,culture,passaging,cryopreservation and resuscitation of piglet intestinal organoids were determined.2.We successfully constructed an iron deficiency model of intestinal organoids after treatment with 40 μmol/L DFO and found that iron deficiency significantly reduced the expansion rate and budding rate of intestinal organoids,indicating that iron deficiency could inhibit the growth and development of piglet intestinal organoids.3.Iron deficiency resulted in a significant decrease in the expression of intestinal stem cell related marker genes Lgr5,Sox9 and Ascl2 and secretory cells(Paneth cells and goblet cells)related genes such as Pbd3,Lyz,Muc2 and Muc4.Immunofluorescence staining revealed that iron deficiency significantly inhibited the expression of stem cell related protein Lgr5 and Paneth cell related protein Lyz.These results suggest that iron deficiency significantly inhibited stem cell proliferation and differentiation of secretory cells such as Paneth cells in the intestinal organoids.4.Iron deficiency reduces stem cell stemness in intestinal organoids by inhibiting the activation of the Wnt/β-catenin signalling pathway.Iron deficiency significantly inhibited the expression of Wnt3 a,Tcf4,β-catenin and Wnt signalling target genes C-myc,Cyclin D1 and Mmp7,while the Wnt signalling agonist CHIR99021 was able to reverse the inhibitory effect of DFO on the Wnt signalling pathway and restore stemness-related gene expression.These results suggest that iron deficiency inhibits the proliferation and stemness maintenance of stem cells in piglets through the Wnt/β-catenin pathway.In summary,this study has successfully constructed a piglet intestinal organoid model,which can efficiently simulate the real digestive and physiological functions of piglets in vitro.Based on this model,we found that iron deficiency inhibited the proliferation and stemness maintenance of stem cells in the organoids by suppressing the activation of the Wnt/β-catenin signalling pathway,thus inhibiting the growth and development of the intestinal organoids.