Role And Mechanism Of TLR4/NF-?B Signaling Pathway In Diarrhea-predominant Irritable Bowel Syndrome

Background and ObjectiveIrritable bowel syndrome(IBS)is not a disease in nature,but is a syndrome defined by a series of symptoms(recurrent episodes of abdominal pain associated with defecation,accompanied by changes in bowel habits),which lacks of diagnostic molecular biomarkers and morphological diagnostic criteria.As one of the most common gastrointestinal dysfunctions,the prevalence of the disease is high and is increasing year by year,and the patients are mainly young and middle-aged.Due to the long-term repeated examination and treatment of patients,the medical expenses for the disease are huge,the family burden is heavy,and the social harm is great.According to changes in bowel habits,IBS can be classified into four main subtypes: diarrhea-predominant IBS(IBS-D),constipation-predominant IBS(IBS-C),mixed irritable bowel syndrome(IBS-M)and unsubtyped irritable bowel syndrome(IBS-U).Among them,IBS-D has received special attention from clinicians and researchers because of its high prevalence,high rate of diagnosis and high risk.The current evidence suggests that a variety of factors are involved in the promotion of IBS-D,gene or early life environment and stress interacts on visceral hyperesthesia,intestinal motility disorders,intestinal flora disorders,low degree of gut inflammation through brain-gut axis,leading to the onset of IBS-D,but the specific and precise mechanism remains unclear.There is a lack of clinically relevant morphological and biochemical abnormalities that explain the symptoms of IBS-D,and there are few studies on biomarkers or key targets of the disease.The treatment method is chosen mainly based on the severity of abdominal symptoms and the degree of psychological complications,from simple publicity and education,lifestyle adjustments to pharmacological treatment,but the effection is still controversial.Therefore,to study the pathogenesis and clarify the biomarkers or key targets of of IBS will help to guide clinical diagnosis and treatment,so as to relieve the pain of patients and reduce the burden of social care.The paper is to probe the role and mechanism of Toll-like receptor 4/ nuclear factor-?B(TLR4/NF-?B)signal pathway in diarrhea-predominant irritable bowel syndrome by animal experiment,and provide the theoretical basis for clarifying the mechanism and finding effective prevention and treatment measures of IBS-D.MethodsThe total of 30 SPF wistar rats(female,average weight:160±10g)were selected as the object,which was randomly assigned into three groups: IBS-D group(10 rats),pyrrolidine dithiocarbamate(PDTC)group(10 rats)and control group(10 rats).The rats of IBS-D group received acute and chronic stresses to build up IBS-D model(28 day);Rats in PDTC group received acute and chronic stresses combine with intraperitoneal injection of PDTC(50mg/kg/wk,once a week for 4week);Control group were 10 rats without any operation.The body weight,abdominal withdral reflex(AWR)pressure threshold and sucrose water intake of the three groups were recorded on the baseline,7th,14 th,21th and 28 th day.The wet stool rate and 1h defecation number were recorded from 28 th day to 31 th day.At the same time,we collected fecal sample of three groups,and determined of fecal flora(Bifidobacterium,Lactobacillus and Escherichia coli)by Real-time PCR;On 31 th day the protocol,blood sample were collected through inferior vena cava and centrifuged to obtain serum,serum inflammatory cytokines(IL-8,IL-10,TNF-?,MYD88)were detected by ELISA.Colonic specimens were taken and western blotting was used to detect TLR4,NF-?B and claudin-1 in intestinal tissues.Hematoxylin-eosin staining(HE)was performed on rat intestinal tissue specimens,and the microstructure of intestinal mucosa was observed using an optical microscope.Results1.At 4th week of experiment,there was a statistically significant difference(F=239.295,P=0.000)of 1h defecation number among IBS-D group(7.14±0.84),PDTC group(5.24±0.55)and control group(0.82±0.42).By pairwise comparison,the IBS-D group was significantly higher than PDTC(P<0.05)and the control group(P<0.05);comparison of wet feces rate: the difference among IBS-D group(12.15±0.85),PDTC group(7.25±0.58)and control group(0)was statistically significant(F=1072.929,P=0.000).Comparison between the two groups,control group(0)and PDTC group(7.25±0.58)were both significantly lower(P<0.05)than IBS-D group(12.15±0.85).2.On the baseline,there was no statistically significant difference of AWR(2 socre)thresholds among IBS-D group,PDTC group and control group.With the increase of time,the AWR pain threshold of the control group gradually increased,while IBS-D group was fluctuated and the overall trend was reduced.The AWR pain threshold in the PDTC group was fluctuated and the overall change was not obvious.From the 1st week to the 3rd week,there was a statistical difference among three groups(1st week: F=6.601,P=0.005,2nd week: F=17.476,P=0.000,3rd week: F=14.577,P=0.000).The AWR pain threshold in IBS-D group(27.00±3.68,26.00±2.26,28.33±2.58)was statistically lower(P=0.001,0.000,0.000)than that in control group(32.67±2.38,33.67±3.40,36.33±4.46),but there was no statistically significant difference between the IBS-D group and the PDTC group(P=0.180,0.232,0.349).At the 4th week,the AWR pain threshold increased in the PDTC group and the control group,while the IBS-D group was decreased.the AWR pain threshold was significantly different in the three groups(F=66.879,P=0.000).The results showed that the IBS-D group(27.17±1.50)AWR pain threshold was significantly lower(P<0.001)than PDTC group(30.33±2.56)and control group(38.67±2.33).3.On the baseline,there was no statistically significant difference in the maximum tolerance threshold of AWR among IBS-D group,PDTC group and control group.With the effect of stress factors,the AWR maximum tolerance threshold of the control group gradually increased,while it in IBS-D group and PDTC group was fluctuated and the overall trend was decreased.From the 1st week to the 3rd week,there was a statistical difference among three groups(1st week: F=4.158,P=0.027,2nd week: F=8.496,P=0.001,3rd week: F=16.390,P=0.000).Pairwise pairing comparison,there was a statistical difference between the IBS-D group and the control group(P=0.001,0.000,0.000)from 1st week to 3rd week,while there was a statistical difference between the IBS-D group and the PDTC group(P=0.014)only at 2nd week.At the 4th week,the AWR maximum tolerance threshold of the control group was increased,but that in the PDTC group and the IBS-D group was decreased.There was a statistically significant difference in the three groups(F=54.281,P=0.000).The results showed that the difference between the IBS-D group(37.67±2.80)and control group(51.00±2.90)was significantly significant(P<0.001)but not statistically significant(>0.05)between the IBS-D group and PDTC group(40.00±2.89).4.On the baseline,there was no significant difference in sucrose water intake among IBS-D group,PDTC group and control group.From 1st week to 3rd week,sucrose water intake increased gradually in the three groups.However,with the effect of stress factors,there was a statistical difference between the three groups(1st week: F=6.204,P=0.006,2nd week: F=6.521,P=0.005,3rd week: F=5.845,P= 0.008).At the 4th week,the sucrose water intake of the PDTC group and the control group continued to increase,while that in IBS-D group was decreased.There was a statistical differences(F=27.362,P=0.000)among three groups of rats.Comparison between the two groups showed that the sucrose water intake in the IBS-D group(14.1±1.81)was significantly lower than that in the PDTC group(16.4±1.58)and the control group(19.4±1.26),The difference was statistically significant(P < 0.001).5.On baseline,there was no significant difference of body weight among IBS-D group,PDTC group and control group.With the progress of the study,the body weight of the three groups continued to increase,but the increasing trend of the control group was more obvious.At the 4th week of the experiment,the difference of weights among IBS-D group(279.37±9.47),PDTC group(294.14±6.39)and control group(313.91±6.66)was statistically significant(F=49.915,P=0.000).The IBS-D group was significantly lower than the control group(P=0.000),and the difference between the IBS-D group(279.37±9.47)and the PDTC group(294.14±6.39)was statistically significant(P=0.000).6.The rat colon tissue specimens of three groups were observed in general,and there was no obvious edema,erosion,ulcer and hemorrhage in the appearance and internal mucosa.Then the colon tissue specimens were observed by light microscope(HE,×100),and there was no obvious edema,erosion,ulcer,and hemorrhage were observed.There was no or a small amount of neutrophil infiltration in the lamina propria and no obvious edema in the interstitial.7.The staining density of colon tissue TLR4 and NF-?B(p65)protein was detected by western blot and that in IBS-D group was higer than PDTC and control group.The gray values ratio of TLR4 and NF-?B(p65)in IBS-D group(1.52±0.08,1.88±0.29)were significantly higher than those in the PDTC(1.22±0.19,1.48±0.18)and control group(1.00±0.11,1.00±0.30)(P < 0.05).8.The expression of IL-8 and IL-10 in the intestinal tract of rats was detected by ELISA.The proinflammatory factor IL-8(pg/ml): there was statistical difference(F=7.954,P=0.002)among IBS-D group(2714.61±1450.23),PDTC group(1594.21±610.46)and control group(1135.97±311.03);The pairwise comparison:IBS-D group was higher than PDTC and control group(P <0.05);Inflammation inhibitor IL-10: There was a statistical difference(F=9.357,P=0.001)among IBS-D group(214.64±69.05),PDTC group(280.96±87.80)and control group(368.86±81.01).The IBS-D group was higher than the control group(P <0.05),but there was no statistically significant(P=0.108)difference between IBS-D group and PDTC group.9.The expression of TNF-? and MYD88 in the intestinal tract of rats was detected by ELISA.The proinflammatory TNF-?: The difference among IBS-D group(375.83±73.83),PDTC group(289.62±57.58)and control group(246.62±30.71)was statistically significant(F=13.374,P=0.000).The level of TNF-? in IBS-D group(375.83±73.83)was higher(P <0.05)than that in PDTC group(289.62±57.58)and control group(246.62±30.71);MYD88: There was a statistical difference(F=18.900,P=0.000)among IBS-D group(3435.11±581.21),PDTC group(2533.28±472.45)and control group(2028.20±495.27).Pairwise comparison,IBS-D group was higher than PDTC and control group(P <0.05).10.Image-Pro Plus 6.0 software was used to measure the thickness(?m)of mucosa under 40-fold magnification.The overall difference among IBS-D group(177.59±45.10),PDTC group(224.21±4.53)and control group(249.01±33.18)was statistically significant(F=12.50,P<0.001).The results showed that the mucosal thickness of the IBS-D group was significantly lower than that of the PDTC group and the control group,and the difference was statistically significant(P<0.001).11.The structure of rat colonic mucosa after HE staining was observed using an optical microscope(×200).In the control group,the intestinal epithelium was intact,neatly arranged,without loss of epithelial structure,with normal glands and crypt structure.Some rats in the IBS-D group had epithelial degeneration and necrosis,and the epithelium was separated from the lamina propria.Some of the lamina propria was destroyed,the crypt structure was missing,but the lesions were relatively limited and could not be found in all visual fields.The severity of destroyed intestinal mucosal barrier in PDTC group was less than that in IBS-D group.There was only a small amount of epithelial loss,necrosis,part of the epithelium separated from the lamina propria.12.Western blot was used to detect the expression of claudin-1 in rat intestinal tissue.The staining density of claudin-1 in IBS-D group was less than that in PDTC group and control group.13.Bifidobacterium: The difference among control group(4.41±0.50),PDTC group(3.91±0.43)and IBS-D group(3.73±0.51)was statistically significant(F=6.614,P=0.005).Bifidobacterium in IBS-D group(3.73±0.51)was significantly less(P < 0.05)than that in control group(4.41±0.50).The PDTC group(3.91±0.43)was more than the IBS-D group(3.73±0.51),but with no statistical difference(P>0.05);E.coli:The difference among IBS-D group(7.64±1.12),PDTC group(7.03±1.77)and control group(6.13±0.75)was statistically significant(F=4.110,P=0.028).The E.coli in IBS-D group(7.64±1.12)was statistically significant(P <0.05)more than that in the control group(6.13±0.75),and the PDTC group was less than IBS-D group(7.64±1.12),but without statistical difference(P>0.05);Lactobacillus : There was a statistical difference(F=7.760,P=0.002)among control group(6.91±0.28),PDTC group(6.64±0.32)and IBS-D group(6.21±0.54).The lactobacillus in IBS-D group was less than that in PDTC group and control group(P <0.05).The above results suggest:1.According to the characteristics of symptoms and diseases,IBS-D rat model was successed in building up via acute and chronic stress,which can meet requirements of our experiment's characteristics.2.The expression of NF-?B(p65)and TLR4 protein in intestinal mucosa of IBS-D rats were more than those of normal rats.3.Proinflammatory cytokines(IL-8 TNF-? and MYD88)in IBS-D rat were higher than that in normal rats,while anti-inflammatory factor(IL-10)was lower than normal rats.4.Intestinal probiotics(Bifidobacterium,Lactobacillus)in IBS-D rat were lower than normal rats,while harmful bacteria(Escherichia coli)was higher than normal rats.5.The mechanical barrier of the intestinal mucosa of IBS-D rats was destroyed(the thickness of the mucosa was less than normal,some of the structures were incomplete,and the expression of tight junction protein claudin-1 was decreased).6.PDTC played a role in blocking TLR4 / NF-?B signaling pathway.After intraperitoneal injection of PDTC,there was a significant reduction of expression of TLR4 and NF-?B(P65)protein.7.After blocking the TLR4 / NF-?B signaling pathway of IBS-D rat,symptoms were less severe than before;proinflammatory cytokines(IL-8,TNF-?,MYD88)were decreased;Probiotics(Bifidobacterium,Lactobacillus)were increased,Escherichia coli was increased,but probiotics(Lactobacillus)increased more significantly;The damage of the intestinal mucosal mechanical barrier was improved.Conclusion1.There were high expression of TLR4/NF-?B signaling pathway,imbalance of inflammatory cytokines,dysbacteriosis,and damage intestinal mucosa mechanical barrier in IBS-D rats.2.The TLR4/NF-?B signaling pathway plays a role in the pathogenesis and symptom improvement of IBS-D.The possible mechanism is to affect the release of inflammatory cytokines,correct the flora imbalance and protect the intestinal mucosal mechanical barrier.