Model preparation

SD rats were randomly divided into OIC (n=10), normal saline group (NSG; n=10), and normal control group (NCG; n=10). For the OIC group, rats were given i.p injection of loperamide hydrochloride dissolved in 0.9% normal saline (4 mg/kg; 1 ml/100 g) as previously described (26-29) twice/day for 7 days. For NSG, 0.9% of normal saline (1 ml/100 g) was i.p injected twice/day for 7 days; rats in the NCG did not receive any treatment.

Humane endpoints were as follows: i) Persistent diarrhea; ii) body weight loss >20% of the original weight; iii) the rat was curled up to the corner of the cage and struggled excessively during the drug administration and iv) peritonitis, such as abdominal muscle tension, accompanied by persistent high or low temperature or intestinal obstruction and intussusception characterized by increased abdominal circumference. Affected rats were euthanized. A total of three rats reached humane endpoints due to the abnormal increase of abdominal circumference.

Tissues used for the IF, WB and RT-PCR experiments were obtained from the distal colonic area of the gut that could not be effectively repaired after being collected. Rats were euthanized via cervical dislocation while still under anesthesia.