基于肥大细胞介导的NGF/TrkA通路探讨电针对IBS-D大鼠肠黏膜屏障的影响OA

Objective To observe the effect of electroacupuncture(EA)on the activation of mast cells(MCs)in the colon of rats with diarrhea-predominant irritable bowel syndrome(IBS-D)and to explore the underlying mechanisms of its protective effect on the intestinal mucosal barrier.Methods Of 74 offsprings from 6 pregnant SD rats,14 offsprings were randomly collected as a normal group,and the remaining 60 offsprings underwent IBS-D model prepared by maternal separation combined with acetic acid enema and chronic restraint stress.Fifty-six successfully modeled rats were randomly divided into a model group,a ketotifen group,an EA group,and an EA+ketotifen(combination)group,14 rats in each one.In the ketotifen group,the rats received the intragastric administration of ketotifen solution(10 mL/kg).In the EA group,acupuncture was delivered at bilateral"Tianshu"(ST25)and"Shangjuxu"(ST37)with disperse-dense wave,a frequency of 2 Hz/100 Hz and a current of 0.3 mA.In the combination group,the intervention measures of the above two groups were operated in combination.In each group,the intervention was delivered once daily for 7 consecutive days.Separately,before modeling,after modeling and after intervention completion,diarrhea score and the minimum injection volume for the abdominal withdrawal reflex(AWR)in rats were evaluated in each group.After intervention,HE staining was adopted to observe the morphology of colonic mucosa;scanning electron microscopy to examine mucosal ultrastructure;Toluidine blue staining to observe MCs degranulation in the colonic mucosa;ELISA to detect serum levels of calcitonin gene-related peptide(CGRP),diamine oxidase(DAO),D-lactate(D-LA),and lipopolysaccharide(LPS);fluorescence microplate assay to detect the serum concentration of 4 kDa fluorescein isothiocyanate-dextran(FD4);Western blot,real-time quantitative PCR(RT-qPCR),and immunofluorescence to detect the expression of nerve growth factor(NGF),tyrosine kinase receptor A(TrkA),and transient receptor potential vanilloid type 1(TRPV1)in colon tissue;and Pearson correlation coefficient to analyze the relationship between CGRP level and intestinal barrier function.Results Compared with the normal group,the model group showed the decrease in AWR minimum injection volume(P＜0.01),the increase in diarrhea score and MC degranulation rate(P＜0.01),the damage of colonic mucosal structure,obvious inflammatory cell infiltration,and the increase in serum levels of CGRP,DAO,D-LA,LPS,FD4 mean fluorescence intensity(MFI),as well as the protein,mRNA,and positive expression levels of NGF,TrkA,and TRPV1 in colon tissue(P＜0.01,P＜0.05).Compared with the model group,the ketotifen,EA,and combination groups exhibited the increase in AWR minimum injection volume(P＜0.01),the decline in diarrhea score and MC degranulation rate(P＜0.01),relatively intact intestinal mucosal structure,reduced inflammatory cell infiltration,and the decrease in serum levels of CGRP,DAO,D-LA,LPS,FD4 MFI,as well as the protein,mRNA,and positive expression levels of NGF,TrkA,and TRPV1 in colon tissue(P＜0.01).When compared with the ketotifen group,the EA group demonstrated lower FD4 MFI in serum and reduced protein expression of NGF,TrkA,and TRPV1 in colon(P＜0.01),and no statistically significant differences were presented in the other indicators(P＞0.05).In comparison with both the ketotifen and EA groups,the combination group showed the improvements in all the aforementioned indicators(P＜0.01,P＜0.05).Serum level of CGRP was positively correlated with the levels of DAO,D-LA,LPS,and FD4(P＜0.001).Conclusion Electroacupuncture may ameliorate intestinal mucosal barrier function and alleviate symptoms in IBS-D rats by inhibiting MC activation,modulating the NGF/TrkA pathway,and reducing serum levels of DAO,D-LA,LPS,and FD4.