Study On The Role Of CRF-R1 In The Treatment Of IBS-D By EA And The Signal Mechanism Of IEB

Objective:Diarrhea-predominant irritable bowel syndrome(IBS-D)is a common clinical disease with high incidence which mainly characterized by intestinal symptoms combined with psychological disorder.Our previous polycentric RCT have confirmed the positive effect of EA on treating both intestinal and emotional symptoms.On the clinical research basis,our trial aims at clarifying the mechanism of EA on the dual regulations to disorders of gut-brain interaction,by means of regulating the CRF and its receptor CRF-R1,on inhibiting the activity of the TNFα-NFκB-MLCK signaling pathway and restoring the TJs structure and functions.Eventually it can be provided a scientific basis for the treatment of IBS-D by EA.Methods:The IBS-D rat model was established under the CUMS and the administration by gavage of senna soaking agent.Then the rats which were determined to be successful under the model further randomly divided into control group,model group,model + EA group,model + pinaverium bromide group and model + NBI-27914 group.The EA group was alternately acupunctured on Tianshu(ST25),Zusanli(ST36),Sanyinjiao(SP6)and Taichong(LR3)for 15 minutes.The medicine group was treated by the administration by gavage with 2.7mg/mL pinaverium.The NBI-27914 group was injected on the abdomen with CRF-R1 antagonist NBI-27914.After 14 days,all the groups were respectively evaluated in the aspects of diarrhea index,SP%,OT% and visceral pain threshold.Following the above-referenced evaluation,the abdominal aorta blood,colon and hypothalamus samples were obtained.Next,fqRT-PCR was used to evaluate the relative expression of CRFmRNA and CRF-R1 mRNA of hypothalamus;double immunofluorescence labeling technique was used to evaluate the double staining cells of IMMC and CRF-R1;Western Blot was used to evaluate the protein expression of TNFα-NFκB-MLCK signaling pathway;immunohistochemistry was used to evaluate the expression of Claudin-1、ZO-1、Occludin in intestinal epithelium;ultraviolet spectrophotometry was used to evaluate the activity of plasma DAO;and transmission electron microscopy was used to evaluate the intercellular connections between intestinal epithelium.Results:After modeling,the visceral pain threshold of the model group was obviously decreased(P<0.01);the diarrhea index has significantly increased(P<0.01);the OT% and SP% were significantly decreased(P<0.01,P<0.05);and there was no obvious histopathological change in the ileum and colon.After 14 days’ intervention,the visceral pain thresholds of the EA group,the pinaverium bromide group and the NBI-27914 group were obviously higher than the model group(P<0.05,P<0.01,P<0.01);the diarrhea indexes were obviously lower than the model group(all P<0.01);the OT% were obviously higher than the model group(all P<0.01);but the SP% only of the EA group and the NBI-27914 group were obviously higher than the model group(P<0.05,P<0.01).Compared to the model group,the CRF and CRF-R1 mRNA expression in hypothalamus of the EA group,the pinaverium bromide group and the NBI-27914 group have all relatively decreased(all P<0.01);the the double staining cells of IMMC and CRF-R1 have all relatively decreased(all P<0.01);the expression of TNFα and NFκB and MLC phosphorylation have all relatively decreased(all P<0.01);the mean optical density of Claudin-1 and ZO-1 has decreased with statistical significance(all P<0.01);the mean optical density of Occludin have all relatively increased(P<0.05,P<0.01,P<0.01);the activity of plasma DAO have all relatively decreased(all P<0.01);under the transmission electron microscope,for the model group,only the vague tight junctions can be observed between the intestinal epithelial cells,while the intermediate junction,gap junctions and desmosomes are absent;for the EA group,the structures of the tight junction and intermediate junction between the intestinal epithelial cells are clear,while the amount of desmosomes obviously decreased;as to the medicine group,the structures of intermediate junction,gap junctions and desmosomes are relatively clear,but the gaps between the tight junctions have expanded;for the NBI-27914 group,both the tight junctions,intermediate junction,gap junctions and desmosomes between the intestinal epithelial cells are clear,and the amount of desmosomes is relatively large.In addition,in down-regulation of NF-κB and up-regulation of ZO-1,EA is better than pinaverium bromide(P<0.01).Conclusions:1.CUMS combine with the senna soaking agent can be successfully establishied the IBS-D rat model with intestinal symptoms combined with psychological disorder.2.EA can improve both the intestinal and emotional symptoms,as well as it can restore the intestinal epithelial barrier.The effect in anti-depression,up-regulate ZO-1 and down-regulate NF-κB of EA is superior to Pinaverium bromide.3.The improving effect of EA on both the emotional and intestinal symptoms may be relevant to down-regulation of the expression of CRF-R1 in PVN and IMMC.4.The improving effect of EA on the intestinal epithelial barrier is realized through inhibiting the TNFα-NFκB-MLCK signaling pathway activated by the CRF and CRF-R1,and thereby restoring the TJs structure and functions of the intestinal mucosa.