Establishment of the IBS rat model

The IBS rat model was established by colorectal distention (CRD) through mechanical and chemical stimulus as previously described [23]. An inflatable balloon (Shanghai Dinghuang Industrial Co., Ltd. China) was slowly inserted rectally about 2 cm into the descending colon of rats. The balloon was distended with 0.5 ml of air, for 1 min and then repeated after 30 min. The same distention was performed for 14 consecutive days between the age of 8 and 21 days. After 4 weeks rest, mustard oil (0.2 ml, 4%, Shanghai Zhixin Chemical Co., Ltd. China.) was injected into the descending colon from the anus once a day for 14 days.