Herbal plants and medicinal foods have recently received increased attention as novel therapeutic drugs for the treatment of constipation and its related diseases [10, 11, 25]. In an effort to develop and investigate drugs for the treatment of constipation, we investigated the therapeutic effects of AEtLP on Lop-induced constipated SD rats. The results clearly demonstrated that AEtLP has laxative effects, including elevation of stool and urine volumes and the recovery of histological changes induced by Lop in the transverse colon. Our data are the first to demonstrate that the laxative effects of AEtLP are tightly correlated with the down-regulation of mAchRs and their downstream signal, alteration of the ultrastructure, and mucin secretion in the transverse colon.
Several chemical compounds including Lop and morphine are widely applied to induce constipation in laboratory animals. Among these, Lop is well known to stimulate the extension of stool evacuation time and the delay of intestinal luminal transit through inhibition of water secretion  and smooth movement in the intestinal wall [27, 28]. Furthermore, Lop has been used to induce constipation in a variety of studies to determine the cause of constipation and identify novel compounds with therapeutic effects [6, 11, 25]. Although the dose and time for Lop treatment have varied among studies, constipation was successfully induced by treatment with 1.5 – 3 mg/kg body weight of Lop for 3–7 days [6, 11, 25, 29, 30]. In the present study, we used Lop to induce constipation and observed the human-like symptoms of constipation in SD rats injected with 4 mg/kg of Lop without any specific problems. Furthermore, Lop and racecadotril have been successfully applied to decrease the risk of dehydration and systemic or metabolic diseases such as diarrhea. Loperamide is a mu-opiate-receptor agonist that extends the orocecal and colonic transit times by increasing gut activity, disarranging electrical activity in the gut and delaying the passage of fluid through the small intestine, although it has some side effects such as severe allergic reaction, toxic megacolon, constipation, decreased urination and stomach bloating [31, 32]. Racecadotril is an indirect delta receptor agonist that accompanies antisecretory activity without an increase of activity in gut muscle [33, 34].
It should be noted that there are some limits and restrictions to the clinical translation of results obtained from Lop induced rats to human conditions, even though the experimental procedure for rat models which is easy and reproducible without negative side effects or histologically detectable damages are widely used for the evaluation and development of therapeutic drug . Chronic constipation is categorized into three groups by assessment of colonic transit and anorectal function; normal transit or irritable bowel syndrome, pelvic floor dysfunction (functional defecatory disorders) and slow transit constipation . Among patients with chronic constipation, the most prevalent form is normal transit (59%), followed by functional defecatory disorders (25%), slow transit (13%) and a combination of defecatory disorders and slow transit (3%) . However, models of pharmacological constipation induced by Lop can only simulate slow transit constipation showing less daily fecal excretion, lower water content, lower numbers of fecal pellets and thinner fecal mucus [23, 29, 38]. Therefore, our results obtained from the Lop-induced constipation model cannot completely translate all forms of chronic constipation detected in human patients. Additionally, further studies are needed to develop a novel model of phenotypes of all classes of chronic constipation using genetic engineering technology and transient injection of chemical substances.
Food intake and water consumption are considered to be important factors for evaluation of constipation symptoms and therapeutic effects. However, Lop-induced constipation models can have several different effects on food intake and water consumption. In some cases, administration of Lop induces decreases in food intake and water consumption , whereas some studies have shown that Lop treatment does not induce any change in food intake or water consumption . In our study, food intake was reduced in response to Lop administration, while water consumption was maintained. These findings are in agreement with those of a study conducted by Shimotoyodome et al. , although the rate of decrease in food intake differed among several studies. Furthermore, a variety of responses regarding food intake and water consumption in constipation models are induced by several herbal medicines and foods. Aqueous leaf extract of Aloe ferox Mill. only induces enhancement of water consumption, whereas food intake is consistently maintained without any significant alteration . In addition, food intake and water consumption can be significantly decreased by the administration of Ficus carica paste for 4 weeks . In the present study, AEtLP treatment did not induce any significant alteration of food intake or water consumption, although a slight decrease in water consumption was observed. These differences might be due to factors such as the innate tastes of the herbal medicines and foods used in each study.
A significant reduction in fecal excretion in Lop-induced rats is considered one of the key markers of constipation in most constipation studies. Previously, stool-related factors such as pellet number, weight, and water content were shown to be dramatically decreased in rats upon administration of Lop [6, 10, 11, 29]. However, these alterations were significantly recovered by plant extracts with laxative effects. Aloe ferox Mill., a widely used medicinal plant with healing properties, has been shown to improve the number, water content, and weight of stools in Lop-induced rats in a dose-dependent manner . Furthermore, leaf extracts of Mareya micrantha increased stool output of rats relative to a control group at doses of 200 and 400 mg/kg . In the present study, a similar effect on stool-related factors was observed in Lop + AEtLP-treated rats (Figure 2).
Furthermore, histological studies have demonstrated significant alterations in the intestines of constipation-induced animals. The thickness of the distal colon layer decreases significantly upon Lop treatment , and the average thickness of the mucus layer is thinner in Lop-treated rats than in control rats . As shown in Figure 3, similar results were observed in our study, such as decreases in crypt layer and muscle thickness in Lop-treated SD rats, although the detection site differed between them [11, 23]. Furthermore, our data indicated that villus length decreased significantly in the transverse colons of Lop-induced constipated rats. These histological changes have been shown to be recovered by herbal medicines and foods having laxative effects. AEtLP treatment showed similar results in the present study as those observed in response to treatment with Ficus carica paste, which induced increases in the thickness of the distal colon and areas of crypt epithelial cells in a dose-dependent manner. Specifically, the thickness of the crypt layer was restored to that of the No-treated group .
Mucin is an important component of luminal mucus, which protects the colorectal mucosa from a variety of mechanical and chemical damage [39, 40]. Secretion of mucin is usually detected by staining with alcian blue at pH 2.5. In this study, crypt epithelial cells and lumens were strongly stained with alcian blue in No-treated rats (Figure 3), which is agreement with the results of previous reports [23, 41] and may validate the present histochemical results. For example, Lop administration induces reduction of mucin storage in crypt epithelial cells by decreasing both the thickness and amount of the luminal mucus layer . Similar effects on mucin secretion were detected in the present study. As shown in Figure 3, the total level of mucin was significantly reduced in Lop-treated SD rats, but was recovered in Lop + AEtLP-treated rats. However, the amount of mucin per crypt lumen was maintained in all groups. Taken together, these findings indicate that one of the main reasons for the decrease in total mucin was the reduced thickness of the crypt layer.
mAChRs are a type of ACh receptor that mediate cholinergic signaling and are expressed on the surfaces of certain neurons and other cells, including heart and smooth muscle cells [41, 42]. These cells act as main receptors and are activated by acetylcholine secreted from postganglionic fibers in the parasympathetic nervous system. Upon activation, signals are transferred to G protein-receptor complexes via cytoplasmic domain interactions, stimulating phospholipase c, which then cleaves PIP2 into IP2 and DAG. The enhanced DAG then induces the activation of PKC, which is involved in receptor desensitization, modulation of membrane structure events, regulation of transcription, immune responses, regulation of cell growth, and learning and memory [43, 44]. mAChRs are classified into five subtypes (M1-M5) according to their tissue distribution and signal transduction mechanism . The role of these receptors has received great attention as they have emerged as key therapeutic drugs . Among the five mAChR subtypes, M1, M2, and M3 significantly influence intestinal activity, including motility and secretion . mAChR M2 has the following indirect function in cholinergic contraction: (1) mAChR M2 stimulation may reverse the relaxation of smooth muscle induced by isoproterenol via inhibition of adenylate cyclase , (2) mAChR M2 stimulation may participate in regulation of intracellular signaling originating from mAChR M3 , and (3) mAChR M2 may play a role in papillary dilation rather than papillary contraction as suggested by studies of mAChR M2−/−M3−/− mice . In this study, we selected mAChR M2 and M3 as a target protein to investigate the molecular mechanism behind the laxative effects of AEtLP. We successfully detected the level of mAChR M2 and M3 transcripts in the transverse colon by RT-PCR as shown in previous studies, although the origin of the samples and detection method used in the analyses was not the same . Furthermore, significant alterations of the level of mAChR M2 transcripts were observed under abnormal conditions, including ischemia, neuropathic pain, arrhythmia, and diabetes [49–52]. The results presented herein are the first to show that the level of mAChR M2 and M3 transcripts dramatically increased in the transverse colon during AEtLP treatment or Lop-induced constipation. These increases in mAChR M2 and M3 transcripts were decreased by AEtLP treatment. Furthermore, the expression pattern of PI-3K and PKC in the down-stream signaling pathway was very similar to that of mAChR M2 and M3. The increase in PI-3 K and PKC expression induced by Lop administration was inhibited by AEtLP treatment. Taken together, the alteration of mAChR M2 and M3 signaling pathway observed in the present study provide information that can be used in future investigations of the causes of constipation and selection of targets for constipation treatment.
The Crypt of Lieberkühn is an intestinal gland located in the epithelial lining of the small intestine and colon. The crypt is composed of three major cell types, enterocytes, goblet cells, and paneth cells. Enterocytes are simple columnar epithelial cells responsible for the digestion and absorption of ions, sugars, peptides, amino acids, lipids, vitamins, electrolytes, and water. Paneth cells are able to secrete various enzymes, including sucrase and maltase, along with enteropeptidase. Goblet cells are glandular, simple, columnar epithelial cells that secrete mucin dissolved in water to form mucus with apocrine and merocrine patterns [53, 54]. As shown in Figure 5, our results provide the first evidence that the ultrastructural changes in the three crypt cells are tightly correlated with the progression and recovery of Lop-induced constipation. After induction of constipation, lipid droplets containing mucin accumulated in the cytoplasm of goblet cells and enterocytes, whereas abundant granules were present in paneth cells. However, Lop + AEtLP treatment stimulated the secretion of lipid droplets into the crypt lumen in both goblet cells and enterocytes.