The Modulation Effect Of Soluble Dietary Fiber On Intestinal Fibrosis Induced By Chronic Radiation Enteropathy

Background:Luminal stenosis secondary to intestinal fibrosis is the major complication of chronic radiation enteropathy and the main reason for surgery.In general,there is no effective treatment for intestinal fibrosis induced by chronic radiation enteropathy,and most drugs are unable to effectively reverse the fibrotic response in patients with chronic radiation enteropathy.Epithelial-to-mesenchymal transition(EMT)plays an significant role in the pathogenesis of fibrosis lesions,and has been confirmed in kidney,lung?liver and other organ injury lesions.However,few studies has revealed the effect of EMT in chronic inflammatory mediated intestinal fibrosis,especially in radiation-induced intestinal fibrosis.Recently,emerging evidences has shown that soluble dietary fiber has potential beneficial effect on intestinal and systemic immune,and has been demonstrated to be a radiation protective agents in preventing ionizing radiation induced intestinal stem cell depletion and decrease overall survival.However,the effect of soluble dietary fiber on intestinal fibrosis induced by chronic radiation enteropathy has never been reportedObjective:The purpose of the study was to investigate the effect of soluble dietary fiber on intestinal fibrosis induced by chronic radiation enteropathy,and to identify the role of EMT in the pathogenesis of intestinal fibrosis in a mouse model after irradiation.Methods:Wild-type and pVillin-Cre;B6 1 29(Cg)Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo transgenic mice on a C57BL/6J background were used in the experiment.Mices were divided into three groups as follows:1)control group;2)radiation group;3)radiation+pectin group(4%).After received a single abdominal dose of 10 Gy gamma-irradiation,we established a mouse model of chronic radiation enteropathy.Mices in each group were euthanized on the day prior to irradiation and 2,4,12 weeks after irradiation,respectively.Terminal ileum was stained with hematoxylin-eosin(H-E)to evaluate the histological changes and Masson's trichrome to assess the severity of collagen deposition.Immunofluorescence staining was performed to evaluate the EMT by observing the distribution of E-cadherin/vimentin,E-cadherin/a-SMA,EGFP/vimentin,EGFP/?-SMA co-localizing cells.Meanwhile,the influence of radiation and dietary pectin treatment on gut microbiota composition and SCFA concentration were investigated by using 16SrRNA Pyrosequencing technology and gas chromatography mass spectrometry(GC-MS)analysis,respectively.Results:After receiving a single dose of 10Gy abdominal radiation,H-E stained sections of terminal ileum showed remarkable thickening of submucosa layers and increased over time(19.9±0.8 vs 23.0±1.0?m,p<0.05 at 2w;20.3±0.9 vs 24.6±1.1?m,p<0.01 at 4w;and 21.2±0.7 vs 27.6±1.3um,p<0.001 at 12w for submucosa in mice from control group compared to radiation group,respectively).Dietary pectin significantly ameliorated the thickness of submucosa at 12 weeks after irradiation compared to radiation group(24.3±0.9 vs 27.6±1.3um,p<0.05).In the irradiated intestinal segment,late intestinal fibrosis which exhibited with marked collagen deposition in sub-mucosa area as disclosed by Masson trichrome staining was observed from 4 weeks after irradiation compared to controls(62.3±1.5 vs 55.3±1.5%,p<0.01),with the most severe lesion occurred at 12 weeks(69.0±1.7 vs 57.1±1.4%,p<0.001).When compared with mice in irradiation group,a significant reduction of submucosal blue-stained areas was observed at 12 weeks in pectin treatment group(64.2±1.4 vs 69.0±1.7%,p<0.05).Immunofluorescence staining showed the direct evidence of EMT involved in intestinal fibrosis induced by irradiation.We found that a-SMA+EGFP+and vimentin+/EGFP+co-expressing cells in mice from radiation group are most obvious at 2w(vimentin+/EGFP+:16.5±1.2/visual field;a-SMA+/EGFP+:8.6±1.2/visual field),and decreased at 4w and 12w(vimentin+/EGFP+:11.4±1.6 at 4w,7.7±1.7/visual field at 12w;a-SMA+/EGF+:7.2±1.0 at 4w,5.7±0.9/visual field at 12w,respectively).EMT cells were significantly decreased in pectin treatment group at 2 and 4 weeks after irradiation when compared with mice in radiation group(9.8±0.9 vs 16.6±1.2,p<0.001 at 2w;7.4 ±0.9 vs 11.4±1.6/visual field,p<0.05 at 4w for vimentin+/EGFP+cells;4.7±1.1 vs 8.6±1.2,p<0.05 at 2w;4.0±1.0 vs 7.2±1.0/visual field,p<0.05 at 4w for a-SMA+/EGFP+ cells,respectively).Irradiation treatment markedly decreased the diversity of intestinal microbiota,while dietary pectin treatment can decrease this downward trend.At the phylum level,the proportion of Firmicutes was decreased and Verrucomicrobia with Bacteroidetes increased after irradiation treatment,while dietary pectin treatment reversed this effect to some extent.The concentrations of total SCFA,acetate and butyrate in mice from radiation group at 2 weeks irradiation was markedly reduced compared with mice in controls(3.6±0.6 vs 2.0±0.3ug/mg,p=0.057 for total SCFA;1.8±0.1 vs 1.1±0.1 ug/mg,p<0.05 for acetate;0.8±0.1 vs 0.3±0.1 ug/mg,p<0.05 for butyrate;radiation vs control,respectively).However,dietary pectin treatment markedly increase the level of total SCFA,acetate,propionate and butyrate compared with mice in radiation group(5.0±0.7 vs 2.0±0.3ug/mg,p<0.05 for total SCFA;2.3±0.4 vs 1.1±0.1 ug/mg,p<0.05 for acetate;0.9±0.1 vs 0.5±0.1 ug/mg,p<0.05 for propionate;1.5±0.3 vs 0.3±0.1 ug/mg,p<0.05 for butyrate,respectively).Conclusions:EMT plays a significant role in the process of radiation-induced intestinal fibrosis.The soluble dietary fiber,pectin,reduced radiation-induced EMT and intestinal fibrosis possibly through modulating gut microbiota compositions and luminal SCFA concentrations.As a consequence,prophylactic use of soluble dietary fiber prior to radiotherapy can be used in clinical practice to protect against radiation-induced early and late intestinal complications.