Effect Of Fecal Supernatants From UC Patients And Dss-induced Colitis Mice On Intestinal Inflammation And Visceral Hypersensitivity In Mice

Objectives: To observe the effects of fecal supernatants from UC patients or DSS-induced colitis mice on intestinal inflammation,intestinal tight junction protein ZO-1,abdominal myoelectric activities（AMAs）,expression of substance P（SP）and calcitonin gene-related peptide（CGRP）in lumbosacral spinal in mice,and to explore the influence of fecal supernatants from UC on IBS-like symptoms in patients with UC in remission.Methods: 1.Sixty male C57BL/6 mice were randomly divided into three groups:（1）Group RP1（Recipient 1）:twenty-four mice,were randomly subdivided into 3 subgroups（n=8）.The mice in group A（control group,CON）were enemaed with 10% glycerol saline solution,in group B（UC patients’ fecal supernatants,pFS）with fecal supernatants from UC patients,and in group C（DSS-induced colitis mice’s fecal supernatants,cmFS）with fecal supernatants from DSS-induced colitis mice respectively（0.4ml/time,2 times/day,12 times in total）.（2）Group RP2（Recipient 2）: fourteen mice,were first enemaed with fecal supernatants from DSS-induced colitis mice（0.4ml/time,2 times/day,12 times in total）,and then subdivided into group D（NS group）and E（FMT group）（n=7）.The mice in group D were subcequently enemaed（0.4ml/time,2 times/day,2 times in total）with saline,and in Group E with fecal supernatants from health mice respectively.（3）Group DN（Donor）: twenty-two mice,15 of them received DSS orally,and then stool samples were collected for fecal supernatants preparation.The other 7 mice offered stool samples for normal fecal supernatants preparation.2.Fecal samples were collected from 2 patients with new diagnosed UC.3.The abdominal myoelectric activities（AMAs）in group RP1 and RP2 were assessed by recording their abdominal muscle response to colorectal distension（CRD）by electromyography at a specific time.The colonic histopathology were evaluated with HE stain.The IL-6,IL-1β and TNF-α cincentration in colonic mucosal tissue were detected by ELISA.The expression of tight junction protein ZO-1 in colon mucosal tissue,and the SP,CGRP in spine cord were detected respectively by immunohistochemistry.Results: 1.Fecal Supernatants from UC patients and DSS-induced colitis mice didn’t affect intestinal histology and cytokines（IL-6,IL-1β,TNF-α）cincentration in colonic mucosal tissue.The concentration of IL-6 in colonic mucosal tissue in groupA,B,C respectively was 80.29 ± 15.36,77.35 ± 8.87,91.84 ± 10.16 pg/ml（P=0.054）;the concentration of IL-1β was 93.66±23.06,94.86±18.26,91.05±18.18 pg/ml（P=0.927）;the concentration of TNF-α was 393.15 ± 60.57,337.76 ± 75.73,404.65 ± 74.38 pg/ml（P =0.153）.2.The fecal supernatants from UC patients or colitis mice can decrease the expressions of intestinal tight junction protein ZO-1:the expression of ZO-1 in group B（0.215±0.013,P_AB=0.001）and group C（0.187±0.021,P_AC=0.000）were significantly lower than in group A（0.311±0.035）.The fecal supernatants from healthy mice can increase the expression of ZO-1:group E（0.210±0.012）were significantly higher than in group D（0.173±0.008,P_DE=0.001）.3.The AMAs with CRD by 0.04、0.06、0.08 ml were significantly increased after the mice were enemaed with fecal supernatants from UC : the AMAs in group B（14.91±2.38,21.88±3.51and40.77±4.00μv）,groupC（13.25±3.96,26.62±4.28and39.07±5.03μv）were significantly higher than those in group A（6.74±1.88,14.94±2.60 and 20.02±2.70μv,P<0.05）.The AMAs in group E（FMT）（16.19±1.83,20.01±2.58μv）with CRD by 0.06,0.08 ml were significantly decreased than in group D（NS）（19.76±1.55,26.56±2.74μv,P₀.06ml= 0.029,P₀.08ml=0.003）.4.The fecal supernatants from UC can decrease the expressions of SP and CGRP in spinal cord :the expression of SP and CGRP in group B（0.094± 0.009,0.096±0.014）and group C（0.095 ± 0.008,0.097 ± 0.006）were significantly higher than in group A（0.065±0.004,0.062±0.008,p=0.000）.The fecal supernatants from normal mice can decrease the expression of SP,CGRP:group E（0.075 ± 0.006,0.077±0.005）were significantly lower than in group D（0.090±0.0 06,0.093 ±0.008,P = 0.000,0.004,respectively）.5.There was a negative relationship between ZO-1 and AMAs,and the mean optical density of SP,or CGRP showed a positive relationship with AMAs.Conclusions: These results indicated that the fecal supernatants from UC patients or colitis mice can decrease the expressions of intestinal tight junction protein ZO-1,and trigger visceral hypersensitivity with an increased expression of SP and CGRP in spinal cord.