Objective

2

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

2

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

2

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

2

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.