Huosha Oral Solution improves symptoms of irritable bowel syndrome with diarrhea by inhibiting activation of ERK signaling pathway and apoptosis of interstitial cells of Cajal in rats

Objective: To investigate the mechanisms by which Huosha Oral Solution inhibits apoptosis of interstitial cells of Cajal (ICC) and ERK pathway activation in a rat model of irritable bowel syndrome with diarrhea (IBS-D). Methods: Sixty newborn male Sprague-Dawley (SD) rats were purchased, from which 50 rats were randomly assigned to the model group for IBS-D modeling. The model group received interventions including maternal deprivation, chronic restraint, and diarrhea induced by senna leaf gavage. The remaining 10 rats were assigned to the normal group without intervention. Rectal dilatation was performed in the model group and the normal group, and the abdominal withdrawal reflex (AWR) scores under different pressures were evaluated. The 44 successfully modeled rats were randomized into 4 groups to receive oral gavage of 10 mL/kg of either Huosha Oral Solution at different concentrations or normal saline: the high-dose group (Huosha Oral Solution, 1 g/mL), the medium-dose group (Huosha Oral Solution, 0.75 g/mL), the low-dose group (Huosha Oral Solution, 0.5 g/mL), and the model group (normal saline, 10 mL/kg); there are 11 rats in each group. The fecal water content before and after gavage was evaluated and the form of feces was scored using the Bristol Stool Scale. Colon tissue from each group of rats was harvested and frozen at -80°C. The level of the neurotransmitter 5-hydroxytryptamine (5-HT) in the colon tissue of each group was determined by ELISA. The expression level of tyrosine kinase receptor c-kit protein (c-kit) in colon mucosa from each group was determined by immunohistochemistry. The protein levels of cleaved caspase-3, ERK1, p-ERK1, MEK1 and p-MEK1 in colon tissue from each group were determined by Western blot. The apoptosis of ICC in the colon from each group was detected by flow cytometry. Results: Six rats died during the modeling process; 44 rats were successfully modeled with a final success rate of 88.00% (44/50). The AWR scores at the pressures of 20, 40, 60, and 80 mmHg in the model group were significantly higher than those of the normal group (all P<0.05). Compared with the normal group, the Bristol Stool Scale scores, fecal water content, colonic 5-HT levels, apoptosis rate of ICC, as well as protein expression levels of p-ERK1, p-MEK1, and cleaved caspase-3 in the colon tissues significantly increased in the medium-dose, the low-dose, and the model groups (all P<0.05); while the c-kit protein expression in the colon mucosa decreased (all P<0.05). However, no significant differences were detected between the normal group and the high-dose group with respect to those parameters (all P<0.05). Compared with the model group, the Bristol Stool Scale scores, fecal water content, colonic 5-HT levels, apoptosis rate of ICC, as well as protein expression levels of p-ERK1, p-MEK1, and cleaved caspase-3 in the colon tissue significantly decreased in the high-dose, the medium-dose, and the low-dose groups (all P<0.05); while the c-kit protein expression in the colon mucosa increased (all P<0.05). The higher the dose, the more significant the effect (P<0.05). Conclusion: Huosha Oral Solution alleviates the symptoms of irritable bowel syndrome with diarrhea; it also inhibits the apoptosis of interstitial cells of cajal (ICCs) and ERK pathway activation in the rat model.