Effect Of Chronic Food-Derived Iron Overload On The Mucosal Barrier Of Mouse Colon And Its Mechanism

Iron is a trace element that is essential for biological processes such as oxygen transport,energy production,and DNA synthesis.Iron deficiency can cause nutritional disorders and diseases,and oral iron supplementation is the main way to treat iron deficiency and iron-deficiency anemia,but improper oral iron supplementation may cause many side effects.The colon contains the most abundant intestinal flora,and iron is a key element for the proliferation and survival of almost all bacteria.Therefore,when oral iron overdose is administered,changes in iron content in the colonic lumen may affect the microbial barrier of the colon;the colon has a certain capacity for iron absorption,and colonic iron overload due to excessive iron absorption may affect the physical barrier of the colon.The microbial barrier and the physical barrier are important components of the colonic mucosal barrier and play an important role in maintaining intestinal function,and it is of great importance to investigate the effect of iron overload on the colonic mucosal barrier for the health of the organism.However,there is still a lack of sufficient data on the effect of colonic iron overload on the colonic mucosal barrier,so this thesis further investigates the effect of iron overload on the colonic mucosal barrier based on a chronic food-borne iron overload mouse model induced by long-term gavage of excess ferric citrate(FC).Experiment.I The effects of changes in iron content on the structure and function of colonic flora in miceTo investigate the effect of long-term oral iron supplementation on the colonic microbial barrier in mice,forty healthy male C57BL/6N mice at 9 months were randomly divided into four groups,with a control group(Ctr)and three FC test groups of 1.25%,2.5% and 5%.The test groups were administrated with different concentrations of FC daily at 0.1 m L/10 g body weight ratio,and the Ctr group was treated with ultrapure water in the same way.The body weight and feed intake of mice were weighed at regular times each week,and the number and weight of mice defecating within 2 h were measured.After 16 weeks of continuous gavage,the mice were executed after anesthesia,and the colonic contents were collected and counted under aseptic cryogenic conditions,and the colonic flora composition profile was analyzed by 16 S r RNA high throughput sequencing.The results showed that:(1)Long-term oral administration of FC had no significant effect on body weight and feed intake of mice.However,in the middle and late stages of gavage,the weights and number of defecations in the 5% FC group were significantly reduced within 2h(P < 0.05).(2)Colonic flora Alpha diversity(Pielou’s evenness,Shannon and Simpson)was significantly higher in all FC groups(P < 0.05).(3)Further analysis revealed that the species composition of the colonic flora was significantly different depending on the variation of iron content.At the phylum level,the abundance of Firmicutes and Deferribacteres were significantly higher in the FC groups compared to the Ctr group(P <0.05);at the genus level,the abundance of Mucispirillum and Desulfovibrio were also differed significantly among groups(P < 0.05).(4)Metagenome Seq analysis showed that at the genus level,the pathogenic bacteria(Mucispirillum,Desulfovibrio,Helicobacter and Shigella),as well as the conditional pathogen(Acinetobacter)were enriched in the 5% FC group.Functional prediction revealed that the LPS hypersynthetic was significantly enhanced in the 5% FC group(P < 0.05),and Shigella played a key role in this study.The above results suggest that oral iron supplementation disrupts the microbial barrier of the colon by altering colonic flora diversity and species composition,and that enrichment of pathogenic and conditionally pathogenic bacteria leads to structural disorders and functional alterations of the colonic flora.Experiment.Ⅱ The effect of chronic foodborne iron overload on the colonic mucosal barrier in miceIn order to investigate the effect of chronic foodborne iron overload on the physical barrier of mouse colonic mucosa,sixty healthy male 9-month-old C57BL/6N mice were randomly grouped into 4 groups(15 mice per group),and a chronic foodborne iron overload model was established according to the same method as above.The results showed that:(1)Compared with the Ctr,the ferritin content in serum and peripheral tissues of mice in the FC group increased significantly(P < 0.05)with the increase of FC concentration,a large amount of ferritin accumulated in the colonic mucosal layer and submucosal layer,and the expression of colonic iron transport proteins(DMT1 and Tf R1)was significantly weakened(P < 0.05).(2)The levels of colonic antioxidants(SOD,GSH-Px,GSH and T-AOC)were significantly reduced(P < 0.05)and the levels of lipid peroxidation(MDA and 4-HNE)were significantly increased(P < 0.05)in colonic iron-overloaded mice compared with the Ctr.(3)Histopathological observations revealed different degrees of inflammatory cell infiltration,colonic gland atrophy,disintegration and significant reduction in the number of cupped cells with significant reduction in mucin in the colonic mucosal layer of chronic foodborne iron overload mice compared with the Ctr(P < 0.05);and significant thickening of the colonic smooth muscle layer with diffuse collagen fiber hyperplasia(P < 0.05).(4)Compared with the Ctr,the colon of chronic food-derived iron overload mice enhanced m RAN levels and protein expression of pro-inflammatory cytokines(IL-1β,IL-6,IL-8 and TNF-α)by activating the expression of genes upstream of the TLR4-NF-κB inflammatory signaling pathway(TLR4,MYD88 and NF-κB p-p65)(P < 0.05),thereby induced colonic inflammatory response in mice.(5)Compared with Ctr,the colon of chronic food-derived iron-overloaded mice showed enhanced expression of pro-apoptotic factors(Capase9,Caspase3 and Bax)and reduced expression of anti-apoptotic factor(Bcl2)(P < 0.05)resulting in activation of the Caspase9/3 mitochondrial apoptotic pathway,leading to apoptosis of colonic mucosal epithelial cells and lamina propria cells.These results suggest that in a chronic food-borne iron overload model,excess iron accumulation in the colon generates large amounts of ROS,causing colonic oxidative stress and oxidative damage,which then induces colonic inflammatory responses and apoptosis through activation of TLR4-NF-κB inflammatory signaling pathway and mitochondrial apoptotic pathway,respectively,weakening the tight junctions of the colon and disrupting the physical barrier of the colon.Experiment.Ш The direct effect of iron overload on colonic epithelial cells in vitroTo investigate the effects of iron overload on colon epithelial cells in vitro.Different concentrations of FC were used to stimulate the cultured human colon cancer cells(SW480)in vitro,respectively.The results showed that:(1)FC-50 μmol/L stimulated to SW480 cells for 24 h had no effect on cell survival,but FC-100 μmol/L treatment for 36 h significantly decreased the cell survival(P < 0.05).(2)When FC was stimulated at 50 μmol/L and FC-100 μmol/L,respectively,it caused iron overload in SW480 cells and generated a large amount of ROS(P < 0.05).(3)When iron overload was induced by stimulating cells with FC-50 μmol/L for 24 h,m RNA levels and protein levels of genes upstream of the NF-κB inflammatory signaling pathway(TLR4,My D88 and NF-κB p-p65)were significantly upregulated,leading to increased m RNA levels and protein expression of pro-inflammatory cytokines(IL-1β,IL-6 and TNF-α)(P < 0.05).(4)When cells were stimulated with FC-100 μmol/L for 36 h,the m RNA levels and protein expression of pro-apoptotic factors(Caspase9,Caspase3 and Bax)were significantly enhanced,whereas m RNA levels and protein expression of anti-apoptotic factors(Bcl2)were significantly attenuated(P < 0.05).The above results suggest that FC stimulation of SW480 cells induced iron overload by generating large amounts of ROS,activating NF-κB inflammatory signaling pathway and Caspase9/3 mitochondrial apoptotic pathway to induce inflammatory responses and apoptosis in cells,leading to damage of the physical barrier of the colonic mucosa.In conclusion,this study demonstrates that when oral over supplementation with iron,the iron content in the lumen of the mouse colon increases,causing pathogenic bacteria or conditionally pathogenic bacteria enrichment in the colon.The large amount of ROS generated by iron overload induced inflammatory damage and apoptosis of colonic epithelial cells through activation of NF-κB inflammatory signaling pathway and Capase9/3 mitochondrial apoptosis pathway,disrupting the physical barrier of colonic mucosa and increasing colonic permeability.Impairment of the microbial and physical barriers in the mouse colon ultimately leads to abnormal defecation function in mice.