Fusobacterium Nucleatum Can Cause Dysbiosis And Exacerbate Visceral Sensitivity In Maternal Separated Rats In A Colonization-independent Manner

BackgroundIrritable bowel syndrome(IBS)is a common functional gastrointestinal disorder characterized by abdominal pain and stool irregularities.The pathogenesis of IBS is complicated and is generally considered to be associated with visceral hypersensitivity,gastrointestinal motility abnormality,psychological stress,brain-intestinal axis dysfunction,immune system activation,intestinal microbiota dysbiosis and so on.Although there is a growing consensus on the association between the intestinal microbiota and IBS,the specific microbial signature of IBS remains elusive.A number of studies have shown that intestinal microbiota is associated with visceral hypersensitivity characterizing IBS patients.For example,many probiotics may relieve the symptoms in IBS patients.Moreover,the colonization of the gut microbiota from IBS patients in germ-free animals can induce visceral hypersensitivity.The abundance of Fusobacteria is related to the visceral hypersensitivity in maternal separation rats.In addition,Fusobacterium nucleatum(F.nucleatum),one species of Fusobacteria,is involved in the pathogenesis of a variety of gastrointestinal diseases.The abundance of F.nucleatum is found increasing in the tissue of colorectal cancer,inflammatory bowel disease,appendicitis;it can also invade the epithelial cells through the expression of some pathogenic factors.Visceral hypersensitivity was found in stress-related models.In addition,maternal separation(MS)rat model captured some intestinal dysbiosis features of IBS patients.Therefore,the MS rat model was constructed to investigate the effects of F.nucleatum on visceral hypersensitivity and the mechanism involved.Objective1.To investigate the effects of F.nucleatum on visceral hypersensitivity in normal rats and MS rats.1.To investigate the effects of F.nucleatum on intestinal microbiota in normal rats and MS rats.2.To investigate the colonization of F.nucleatum in the intestine and the influencing factors concerned.Methods1.Animal model construction:32 male Sprague Dawley(SD)rat pups were separated into MS group(n=16)and normal-breeding group(n=16).The rat pups in MS group was isolated from the dams for 3 h(from 9:00 a.m.to 12:00 a.m.)between postnatal day(PND)2 and PND 14,while the rat pups in normal-breeding group were nursed normally.After weaned on PND 22,the rats in the two group were randomly received gavage of F.nucleatum or normal saline.So all the experimental rats were divided into four groups:group D(MS and gavage of F.nucleatum),group M(MS and gavage of saline),group F(simply gavage of F.nucleatum)and group N(simply gavage of saline).The gavage was performed at week 4,5,6,7 and 8.The amount of F.nucleatum in group D and F was 109CFU and the volume of gavage was determined by the weight of rats(1 ml/100 g).The rats in group M and N was received normal saline at the same volume of the same time.2.Visceral sensitivity evaluation:The balloon fixed in the descending colon of the rats was injected of saline to constant volume(0.2,0.4,0.6,0.8,1.0,1.2,1.4,1.6 ml)at week 12.Visceral sensitivity was evaluated using the abdominal withdraw reflex(AWR)score in response to graded colorectal distension(CRD).To stand for the overall visceral sensitivity for each rat,visceral hypersensitivity index(VHI)was calculated by summing up the rank of AWR score at the balloon volume of 0.4ml,0.6ml,0.8ml,1.0ml,1.2ml and 1.4ml.3.Fecal microbiota sequencing:Rat stool was collected at the end of week 3,8,and 12 from birth to extract DNA,then the 16S rRNA gene sequencing was performed to investigate the influence of MS and gavage of F.nucleatum on intestinal microbiota of rats.4.Specific IgA detection:This part was learnt from western blot.The stool was diluted in normal saline of a certain proportion(1g/7ml).After being centrifuged,the stool supernatant was collected as the first antibody.F.nucleatum was re-suspended in normal-saline and the total protein was extracted by hypothermic ultrasonication.Excessive F.nucleatum protein of equal amount was separated in SDS-PAGE,and then was transferred onto PVDF membrane.After being blocked with non-fat milk,the membrane strips were incubated with stool supernatant and then the horseradish peroxidase(HRP)labeled with Goat Anti-Rat IgA alpha chain.Then the bands were detected by ECL solution.E.coli BL21(DE3)was used as control under the same procedure.5.Antigen identification and confirmation:The protein of F.nucleatum separated in SDS-PAGE was stained with coomassie brilliant blue.Then the protein of interest was extracted and identified by protein analysis system.The FomA recombinant E.coli was constructed to express protein FomA.The total protein of FomA recombinant E.coli was collected and the specific IgA to recombinant E.coli was detected as the procedure above.The protein was also identified after stained with coomassie brilliant blue.Results1.Visceral sensitivity evaluation:There was significant difference of visceral sensitivity among the four groups.The group D and M had higher visceral sensitivity than the group F and N respectively.The group D had higher visceral sensitivity than the group M.There is no significant difference between group F and group N.2.Dysbiosis of intestinal microbiota:The gavage of F.nucleatum and maternal separation could cause dysbiosis of intestinal microbiota in rats.They could both decrease the diversity of intestinal microbiota and change the structure the microbiota.However,the F.nucleatum was not found in the sequencing data.3.Specific IgA detection and antigen identification:There was no obvious reactive band from the stool supernatant of 4 groups at week 3.Two strong reactive antigen bands were found at the molecular mass of 40 kDa and 130 kDa in most samples of group D and F at week 8 and 12,while two relatively light reactive bands at the same positions were detected in most samples of group M and N.After the E.coli BL21(DE3)was used as control,the bands were relatively weak and the positions were different,indicating the specific IgA was generated.The results of protein identification indicated that the protein encoded by FomA gene was detected at both positions of 40 kDa and 130 kDa.After the western blot was performed to the FomA recombinant E.coli,the obvious band was appeared at 40 kDa.The following antigen identification further confirmed that protein FomA was expressed at the position of 40 kDa.Conclusion1.F.nucleatum can exacerbate the visceral sensitivity in maternal separation rats,but could not increase that in normal breeding rats.2.F.nucleatum and maternal separation can cause intestinal microbiota dysbiosis of rats.The gavage of F.nucleatum can change the structure of the intestinal microbiota in a colonization-independent manner.3.Administration of F.nucleatum induces the augment of specific IgA against F.nucleatum.Protein FomA is the target protein recognized by specific IgA.