ObjectiveTo observe the effect of different stimulation frequencies of electroacupuncture （EA） of “Zusanli”（ST36） on the general state， visceral sensitivity， anxiety and depression， adrenocorticotropin releasing factor （CRF）， adrenocorticotropin releasing factor receptor 1 （CRFR1）， CRFR2 and mast cells in mice with irritable bowel syndrome （IBS）， so as to explore its mechanism underlying the improvement of IBS.MethodsMale C57BL/6 mice were randomly divided into blank group， model group， 2 Hz-EA group， 15 Hz-EA group and 2 Hz/15 Hz-EA group， with 9 mice in each. The IBS model was established by chronic unpredictable stress （CUMS）. EA （1 mA） was applied to bilateral ST36 for 15 min， once a day for 7 consecutive days. The general state， abdominal withdrawal reflex （AWR） scores for injection of air （0.25， 0.35 and 5 mL） into the colorectum-inserted air-balloon （for evaluating the visceral sensitivity）， open field test （OFT， for assessing the state of anxiety and depression） were observed. The number of CRFR1/tryptase- and CRFR2/tryptase-positive mast cells in the colon tissue was observed by immunofluorescence double labeling. The protein expression levels of CRF， CRFR1 and CRFR2 in the colon and hypothalamus tissues were measured by western blot.ResultsCompared with the blank group， the mice in the model group showed listlessness， easy irritation， loose stool attached around the anus， and an increase in the AWR scores at 0.25， 0.35 and 0.5 mL， number of colonic mast cells， number of colonic CRFR1- and CRFR2-positive mast cells， expression of hypothalamic CRF， CRFR1， colonic CRF， CRFR1 and CRFR2 proteins （P<0.05，P<0.01）， and a significant decrease in the dwelling time in the center area of OFT， and the expression of hypothalamic CRFR2 （P<0.01）. In contrast to the model group， the AWR scores of 2 Hz-EA group at 0.25 and 0.35 mL and 2 Hz/15 Hz-EA group at 0.25， 0.35 and 0.50 mL were significantly decreased （P<0.05， P<0.01）， and the dwelling time of the 3 EA groups was obviously increased （P<0.01）. In addition， the increased levels of the number of colonic mast cells， CRFR1- and CRFR2-positive mast cells， and the expression of hypothalamic CRF and CRFR1， colonic CRF， CRFR1 and CRFR2 proteins and the decreased level of hypothalamic CRFR2 protein expression were reversed by 2 Hz-EA， 15 Hz-EA and 2 Hz/15 Hz-EA （P<0.05， P<0.01）. The therapeutic effects of 2 Hz/15 Hz-EA were apparently superior to those of 2 Hz-EA and 15 Hz-EA in reducing the AWR scores， number of colonic mast cells and CRFR1- and CRFR-positive mast cells， down-regulating the expression levels of CRF， CRFR1 and CRFR2 proteins in both colon tissue and CRF and CRFR1 proteins in the hypothalamus tissue and in increasing the dwelling time in the center area and the expression of hypothalamic CRFR2 protein （P<0.05， P<0.01）.ConclusionEA of ST36 can improve the general state， visceral sensitivity， anxiety and depression in mice with IBS， which may be related to its functions in lowering the number of CRFR1- and CRFR2-positive mast cells in the colon， downregulating the expression of CRF and CRFR1 and upregulating the expression of CRFR2 protein in the hypothalamus. The effect of 2Hz/15Hz-EA is better than that of low frequency-EA and medium frequency-EA.

Electroacupuncture;Irritable bowel syndrome;Visceral sensitivity;Anxiety and depression;Corticotropin releasing factor