To study the effects of cinobufagin and its main component alkaloids on the contraction of colonic smooth muscle in rats in vitro and their influence on abnormal expression of channel proteins related to diarrhea in vivo.

Normal rat colon tissue samples were put into the bath to connect with the displacement tension transducer. After the tension of the colon sample was stabilized, different doses of cinobufagin and alkaloid solution were given to the colon sample, and the changes of the contraction tension of the colon sample were recorded. The rats were given the clinically equivalent dose of cinobufagin and alkaloid solution by gavage for 7 consecutive days, and the rats were killed on the 8th day. Colon tissues were taken, and the expressions of calcium channel proteins CaV1.2, CaV1.3, sodium channel proteins NHE2, NHE3, aquaporins AQP3, AQP8 were detected by Western blot to observe the changes.

The tension of colon specimen did not change significantly when the original solution was <0.10 mg (drug content<0.09 mg); 0.10~18.74 mg stock solution (0.09~16.21 mg) increased the tension of colon specimen. If the original solution was >19.25 mg (drug content >16.64 mg), the tension of colon specimen decreased. When alkaloids was <0.06 μg, the tension of colon specimen had no obvious change; 0.06~23.41 μg alkaloid increased the tension of colon specimen. When alkaloids was >24.03 μg, the tension of colon specimen decreased. Compared with the normal group, the sodium channel proteins NHE2, NHE3 and aquaporins AQP3, AQP8 in the cinobufotalin group decreased significantly (P<0.05), while the calcium channel protein CaV1.2 increased significantly (P<0.05). Sodium channel protein NHE3 and aquaporin AQP3 were significantly decreased in the alkaloid group (P<0.05). There was no significant difference in the expression of other proteins (P>0.05).

In the study of diarrhea mechanism, the results of colon tension test in vitro showed that a certain dose of cinobufagin could promote intestinal peristalsis and increase the incidence of diarrhea, and it could inhibit intestinal peristalsis when the dosage was higher than a certain level. The detection results of diarrhea-related proteins in the colon showed that the administration of cinobufotalin and alkaloid solution would cause changes in diarrhea-related channel proteins, and the number of alkaloid solutions that had significant effects on channel proteins was less than that of the original solution, suggesting that there were other components in the purgative component besides the alkaloids.