Effects of Hemp seed soft capsule on colonic ion transport in rats

ARTICLE HIGHLIGHTS

Research background

Constipation is one of the most common gastrointestinal disorders, which maintains a high incidence rate and greatly influences the life quality of patients. As reported, multiple factors and potential pathogenesis contribute to the occurrence and development of constipation. And, a variety of treatment methods have been adopted for constipation, including Traditional Chinese Medicine, in which Hemp seed soft capsule is one of the safe and effective Chinese patent medicines. However, the treatment mechanisms of Hemp seed soft capsule have been unclear, which restricts its popularization and application. Hence, the exploration of possible mechanisms about Hemp seed soft capsule for treating constipation is necessary.

Research motivation

This study aimed to investigate the effect of Hemp seed soft capsule on colonic ion transport and its related mechanisms in constipation rats, the findings of which will have an important significance for clarifying the pharmacological effect of Traditional Chinese Medicine.

Research objectives

The main objective of the study was to explore whether the regulation of the intestinal secretion was the possible mechanism of Hemp seed soft capsule for treating constipation. This research provided a new thought for us to study Hemp seed soft capsule, and more molecules related to the colonic secretion could be discussed in the future.

Research methods

The constipation rats were induced by oral administration of loperamide (3 mg/kg per day for 12 d). The Hemp seed soft capsule group was given Hemp seed soft capsule by gavage (0.126 g/kg per day for 7 d). The normal and constipation groups were treated with the same volume of distilled water. After treatment, the fecal wet weight and water content were measured. The basal short-circuit current (Isc) and resistance were acquired by an Ussing Chamber. Further, after the ion substitution or inhibitor application, the Isc induced by Hemp seed soft capsule was also measured.

The statistical analyses were performed by using SPSS 17.0 software and the differences among the three groups were analyzed using one-way analysis of variance followed by least-significant difference test to compare the differences between two groups. The differences among the two groups were analyzed using t-test. P < 0.05 was considered statistically significant.

Research results

In this study, it was found that Hemp seed soft capsule could increase the fecal wet weight and water content in constipation rats. Meanwhile, Hemp seed soft capsule could increase colonic secretion, and after the application of cAMP- dependent or Ca²⁺- dependent Cl^- channels inhibitor, NKCC inhibitor, Na⁺-HCO₃^- cotransporter inhibitor or Cl^-/HCO₃^- exchanger inhibitor, the colonic secretion induced by HSSC were decreased in experimental group than that in the control group, which demonstrated that cAMP- dependent and Ca²⁺- dependent Cl^- channels, Na⁺-K⁺-2Cl^- cotransporter (NKCC), Na⁺-HCO₃^- cotransporter or Cl^-/HCO₃^- exchanger may participate in the HSSC treatment. Meanwhile, after pretreatment with a neural pathway inhibitor (tetrodotoxin, atropine or hexamethonium), there were no differences between experimental mucosa and control mucosa, which implied that the secretagogue effect of HSSC was not dependent on the submucosal nervous system.

Though some results were obtained, the effect of Hemp seed soft capsule on real-time ion current and expression of ion channel protein should be explored in further studies, which will also be important for exploring the specific targets.

Research conclusions

The secretagogue effect of Hemp seed soft capsule for constipation may be achieved via the combined action of cAMP-dependent and Ca²⁺-dependent Cl^- channels, NKCC, Na⁺-HCO₃^- cotransporter or Cl^-/HCO₃^- exchanger. However, the submucosal neurons seem to not play a key role in the process. Decreased colonic secretion is found in constipation rats and Hemp seed soft capsule can reverse it. Besides, Cl^- and HCO₃^- participate in the regulative process. The effect of Hemp seed soft capsule for constipation can be achieved by increasing colonic secretion, which is related with the coaction of cAMP-dependent and Ca²⁺-dependent Cl^- channels, NKCC, Na⁺-HCO₃^- cotransporter or Cl^-/HCO₃^- exchanger. The discoveries in this study imply that Cl^-, HCO₃^- and related channels/cotransporters/exchangers participate in the effect of HSSC on constipation.

The new hypotheses proposed in this study is that regulation of the intestinal secretion was the possible mechanism of Hemp seed soft capsule for treating constipation. Ussing Chamber is the acknowledged method, although it is new, to be used in the discovery of the Hemp seed soft capsule therapeutic mechanism. Hemp seed soft capsule can increase the fecal wet weight and water content in constipation rats, while the colonic secretion is increased. Besides, cAMP-dependent and Ca²⁺-dependent Cl^- channels, NKCC, Na⁺-HCO₃^- cotransporter and Cl^-/HCO₃^- exchanger are involved in the effect of Hemp seed soft capsule on colonic secretion. The effect of Hemp seed soft capsule on constipation can be achieved by regulating the colonic secretion, which is related with cAMP-dependent and Ca²⁺-dependent Cl^- channels, NKCC, Na⁺-HCO₃^- cotransporter or Cl^-/HCO₃^- exchanger. Hemp seed soft capsule may increase the fecal water content in constipation patients and can be used as an effective laxative in clinical practice.

Research perspectives

In the study, we found Hemp seed soft capsule could relieve the symptom of constipation and increase the colonic secretion, which implied that the effect of Hemp seed soft capsule on constipation may be achieved by regulating colonic secretion. Further, it was found that Cl^- and HCO₃^- participated in the process, mediated via the related channels/cotransporters/exchangers. But, several questions remain unanswered, all of which can be discussed in the future (e.g., what specific pathways, receptors or molecules may be involved in the effect of Hemp seed soft capsule on colonic secretion, such as the 5-HT/5-HTR pathway or the dopamine pathway, or what structural and functional changes can be found in the ion transport protein on the effect of Hemp seed soft capsule, as the expression of cystic fibrosis transmembrane conductance regulator (CFTR, a cAMP- dependent Cl^- channel) protein).

In further studies, we will focus on the effect of Hemp seed soft capsule on Cl^- current and CFTR protein in constipation rats. The methods of patch-clamp and short-circuit current will be used to measure the Cl^- current. Immunofluorescence will be used to investigate the location and expression of the CFTR protein. Finally, western blot and PCR will be conducted to evaluate the expression of CFTR protein and phosphorylated CFTR protein.