1. Introduction
Constipation is a common health problem and a predisposing factor for many conditions with high death rates. With changes in diet structure and the influence of psychological and social factors in recent years, the incidence of constipation has shown a clear upward trend, which has seriously affected human health and quality of life [
1]. Irritant drugs are generally used to promote defecation, but this treatment has side effects [
2,
3]. Reports have shown that constipation and diarrhea are associated with gut microbes. The relative abundance of pathogens (methanogenic archaea [
4] and clostridia [
5]) increases in patient with constipation, and the relative abundance of enteropathogenic bacteria (
Salmonella,
Shigella, enterotoxigenic
Escherichia coli, or
Vibrio cholera) increases in patients with diarrhea [
6,
7], which disturbs the ecological balance in the intestines [
8]. As research into the intestinal micro-ecology continues to develop, it is understandable to attempt treatment of constipation with intestinal micro-ecological therapy.
Increasing fiber intake or using laxatives is commonly recommended to alleviate or treat constipation. The intestinal microbiome is related to normal gastrointestinal (GI) functions such as GI motility, immune modulation, and drug metabolism [
9,
10,
11]. Clinical studies have shown that gut microbiota in constipation differs from that in healthy subjects [
12,
13] and is mainly manifested in the levels of
Bifidobacterium,
Lactobacillus and pathogenic bacteria. Most studies have shown that changes in the intestinal flora in the constipation group mainly involve a decrease in Bifidobacteria and Lactobacilli and an increase in pathogenic bacteria (methanogenic archaea [
4] and clostridia [
5]). Therefore, supplementation with probiotics has become a new method to treat constipation. Probiotics have been defined as living microbes that, when administered in adequate amounts, such as 10
6 to 10
9 colony-forming units (CFU), confer health benefits to the host [
14]. Some studies have supported the use of probiotics to prevent or treat constipation [
15,
16]. Some probiotic strains, either alone—
Bifidobacterium infantis 36524 or
Lactobacillus plantarum 299v—or combined—VSL#3 (
Bifidobacterium (
B. longum,
B. infantis, and
B. breve),
Lactobacillus (
L. acidophilus,
L. casei,
L. bulgaricus and
L. plantarum)), and
Streptococcus thermophiles—have been associated with significant alleviation of constipation [
17,
18], whereas others have proved ineffective [
19,
20]. The fundamental reason for the use of probiotics to treat constipation may be that the colonic microflora influences peristalsis of the colon [
21]. Therefore, it has been suggested that an imbalance in the colonic microflora plays a role in constipation. Furthermore, Bifidobacteria produce lactic acid and acetic acid, which decrease the pH in the colon. This lower pH enhances peristalsis and decreases the colonic transit time, which is beneficial in the treatment of constipation [
16,
22]. This latter hypothesis was confirmed by showing a decrease in the colonic transit time in healthy adults who consumed a supplement with
B. animalis [
23].
Numerous probiotic supplementation trials have been carried out in animals and humans to test the efficacy of probiotics against constipation [
15,
24,
25]. It has been shown that Bifidobacteria display inter-species differences in the alleviation of constipation [
24]. Bifidobacteria may comprise as much as 25% of the cultivable gut microflora.
B. adolescentis is recognized as one of the dominant anaerobes in adults and is considered to be beneficial to human health [
26]. Therefore, we hypothesized that
B. adolescentis could alleviate constipation and that inter-strain differences exist in the alleviation of constipation induced by loperamide in BALB/c mice.
Based on this background, the aims of this study were: (1) to determine whether B. adolescentis shows inter-strain differences in the alleviation of constipation induced by loperamide in mice; (2) to analyze the main reasons for the inter-strain differences, such as the basic biological characteristics of the strains, the concentration changes of short-chain fatty acids (SCFAs) in feces, and the changes in the fecal flora; and (3) to determine the changes in other indicators of constipation, including some parameters of the enteric nervous system, including motilin (MTL), gastrin (Gas), substance P (SP), endothelin (ET), somatostatin (SS) and vasoactive intestinal peptide (VIP).
3. Discussion
Constipation is a common functional GI disorder whose main clinical symptoms include difficulty with defecation, reduced defecation frequency, dry and hard stools and a prolonged GI emptying time [
27]. Patients with constipation have severe disturbances of intestinal flora and a large number of pathogen-produced nitrite amines, phenols, ammonia, azobenzene and carcinogenic substances. Direct contact with some of these harmful substances can cause inflammation of intestinal mucosal or even colon cancer. These toxins cannot be discharged in time and can be absorbed into the blood to induce breast cancer [
28]. Disruption of the intestinal microflora balance may modify the intestinal barrier function and influence health. A micro-ecological view showed that a sufficient quantity of Bifidobacteria in the gut can ferment oligosaccharides and produce acetic acid and lactic acid to promote intestinal peristalsis, excretion of feces, and alleviation of constipation [
29]. The fecal wet weight, the fecal water content, the time to the first black stool defecation and the rate of intestinal charcoal propulsion are important indices with which to evaluate the function of the GI tract.
The purpose of this study was to determine whether B. adolescentis exerts a strain-specific effect on constipation and the causes of these differences. Our study revealed that the three strains of B. adolescentis exerted a strain-specific effect on constipation caused by loperamide, and these differences were mainly caused by the fundamental properties of the strains and their effects on the intestinal flora and intestinal microenvironment.
A mouse constipation model was established by administration of loperamide, which is an agonist of μ-opioid receptors that prevents the release of acetylcholine and prostaglandin, resulting in inhibition of intestinal peristalsis and prolonged retention of the intestinal contents. Therefore, loperamide-induced constipation is considered a model of spastic constipation [
30]. In this study, the loperamide (control) group showed evident symptoms of constipation, including significant decreases in fecal wet weight, fecal water content, small intestinal transit rate, and time to the first black stool defecation in comparison to the normal group. These symptoms were relieved in the
B. adolescentis CCFM 669 and 667 treatment groups, but not in the
B. adolescentis CCFM 626 treatment group. This finding indicates that
B. adolescentis has a strain-specific effect on constipation.
There are three modes of small bowel movement: tension contraction, segmentation movement and peristalsis. The movement of intestinal contents is mainly promoted by peristalsis, which is detected by measuring the small intestine propulsion rate. A faster small intestine propulsion rate is conducive to the discharge of feces; otherwise, constipation is likely [
31]. Our study revealed that
B. adolescentis CCFM 667 and 669 relieve constipation by improving the small intestine propulsion rate. Although the high dose of
B. adolescentis CCFM 626 did not alleviate constipation, the low dose could, which showed that a larger dose did not lead to a better effect; therefore, screening for the optimum dose was the next task. Previous studies have confirmed the efficacy of treatment with
B. lactis DN-173010,
B. longum 46 (DSM 14583),
B. longum 2C (DSM 14579) and
B. lactis HN019 (DR10TM) on the frequency of defecation and stool consistency [
32,
33]. All of these studies indicated that Bifidobacteria could relieve constipation by improving the fecal status and the rate of intestinal propulsion.
The whole intestinal transit time is reflected by the time to the first black stool defecation, which is the sum of the transit times of the small and large intestine. The main cause of constipation is the long retention time of feces in the intestinal tract and excessive absorption of water. The time to the first black stool defecation is an important indicator to judge both the effect of treatment and prognosis. A shorter time to the first black stool defecation indicates a better effect; otherwise, the effect will be worse. Our study showed that the time to the first black stool defecation was the shortest in the group that received a high dose of
B. adolescentis CCFM 667 or 669 or phenolphthalein and the longest in the control and
B. adolescentis CCFM 626 groups. Thus, CCFM 667 and 669 could relieve constipation, whereas CCFM 626 could not. Published data hold that supplementation of
Bifidobacterium is associated with a slower whole intestinal transit time in the human gut [
34], a finding that was confirmed by Favretto [
35].
The water content of feces and the small intestinal transit rate were significantly greater in the low-dose B. adolescentis CCFM 669 group than those in the low-dose B. adolescentis CCFM 667 group, and the time to the first black stool defecation was similar to that in the B. adolescentis CCFM 667 group. These results revealed that B. adolescentis CCFM 667 relieves constipation by improving the colonic transit time and that B. adolescentis CCFM 669 relieves constipation by promoting small intestinal peristalsis. Furthermore, B. adolescentis CCFM 667 did not have adhesion ability, so it passed into the large intestine with chyme. It would quickly produce SCFAs from carbohydrates that could not be absorbed by the small intestine and would stimulate colon peristalsis, ultimately accelerating colonic transit time. The action of B. adolescentis CCFM 669 was completely contrary to that of B. adolescentis CCFM 667. It partly colonized in the small intestine, promoted intestinal peristalsis and improved the small intestinal transit rate. The remainder passed into the large intestine with chyme and played the same role as B. adolescentis CCFM 667.
The changes in the intestinal flora in mice were mainly detected by the changes in the bacteria in their feces. How do mouse intestinal flora change before and after constipation, and after supplementation with
B. adolescentis? The results clearly showed that the composition of the gut microbiota community both before and after intervention was dominated by two phyla, Bacteroidetes (which includes
Bacteroides and
Odoribacter) and Firmicutes (which includes
Clostridium,
Streptococcus,
Lactobacillus and
Dorea), whereas Actinobacteria (mainly
Bifidobacterium), Proteobacteria and Tenericutes played minor roles. In general, at the phylum level, the ratio of Firmicutes to Bacteroidetes increased and the abundance of other phyla decreased in the
B. adolescentis treatment groups. Similarly, at the genus level,
B. adolescentis did not increase the abundance of
Bifidobacterium in Actinobacteria but did increase the relative abundance of
Lactobacillus in Firmicutes and reduced the abundance of
Clostridium. Some species of the
Clostridium genera are associated with the emergence of intestinal complications such as colitis [
36], necrotizing enterocolitis and gastroenteritis [
37]. Therefore, the reduction of harmful
Clostridium after
B. adolescentis ingestion indicates a potential benefit for gut health. The intestinal flora comprises a complex and dynamic bacterial community that plays an important role in human health [
38]. Previous studies described the changes in the gut microbiota of patients with constipation, which are characterized by a relative decrease in the
Bifidobacterium and
Lactobacillus species and an increase in potentially pathogenic microorganisms [
39]. Previous studies reported no significant differences in the microbial profiles of patients with constipation and healthy subjects [
4]. The conflicting results might also relate to differences in the definition of constipation in the various studies. We measured objective indices such as the whole intestinal transit time rather than more subjective indices. For example, Khalif et al. [
39] observed little difference in the gut bacteria between healthy subjects and those with constipation, but only constipated subjects with a severely prolonged transit time were considered in the comparison. It is possible that symptom-based diagnosis such as that with a subjective index may not be sufficient to differentiate gut microbial differences between subjects with and without constipation. In brief, altering the composition of the gut microbiota community in patients with constipation to resemble that in normal subjects may decrease constipation-associated changes in GI function.
Microbes do not simply remain within the gut; they must be metabolically active to survive in that environment. Hence,
B. adolescentis would have an influence not only on the composition and numbers of various microbes, but also on their fermentation products, such as SCFAs. SCFAs are the final product of microbial fermentation in the mammalian colon, in which they represent the major organic anions. The ability of the bacteria to produce SCFAs is influenced by the number of bacteria, the pH and the substrate. The total amount and proportion of SCFAs produced by different substrates differ [
40]. SCFAs are involved in important physiological metabolic processes in vivo [
41]. Thus, measuring the content of SCFAs in the intestine has become the main method to detect differences between strains in the relief of constipation. Our study showed that
B. adolescentis increased the concentration of propionic acid and butyric acid to relieve constipation. Propionate is primarily used in the liver and has been suggested as a potential modulator of cholesterol synthesis and a precursor in lipo-neogenesis, which may influence body weight [
42]. Butyrate is the preferred energy source for colonocytes and thus is extensively metabolized by the colon. Butyrate and other SCFAs have also been shown to provide protection against colon cancer [
43]. Compared with other similar investigations, some have observed significantly higher levels of isobutyrate in samples from subjects with constipation than in those from healthy subjects [
4], and others reported increases in butyrate levels [
44]. These phenomena might be related to diet because SCFAs originate from the degradation of polysaccharides, which increases when the system operates with longer retention times [
45]. Our study incorporated dietary and gut transit time, and the results are more reliable. Further studies are also needed to provide direct evidence for increased propionate and butyrate acid and to examine the possible role of propionate and butyrate in the pathogenesis of constipation. As to the molecular mechanism of the beneficial effects of SCFAs, butyrate was reported to be a physiological regulator of major pathways of colonic epithelial cell maturation including cell cycle arrest, lineage-specific differentiation, and apoptosis [
46]. In addition, increased expression of genes that favor apoptosis (Bax, and Bak) and a reduced expression of counter-players that prevent apoptosis (Bcl-2, and Bcl-XL) were all reported to be the molecular mechanisms of SCFAs that mediate colorectal cancer [
47]. Compared to the in-depth investigation of the molecular mechanism of SCFAs intervention in colorectal cancer, few studies have reported molecular evidence of the mediation of SCFAs in constipation. It is believed that SCFAs may stimulate water and electrolyte absorption, potentiate the proliferation of epithelial cells, influence GI motility, increase mesenteric blood flow and exert other physiological effects [
41]. However, the molecular mechanism still needs to be investigated.
GI hormones such as MTL, Gas, SP, ET, SS, and VIP play important roles in the regulation of GI motility [
48] and have been implicated to different extents in normal and pathophysiological situations. MTL, Gas and SP are excitatory peptide neurotransmitters, whereas ET, SS and VIP are inhibitory peptide neurotransmitters. In this study, the control group showed evident constipation symptoms in relation to neurotransmitters, including significantly decreased levels of MTL, Gas and SP and increased levels of ET, SS and VIP in comparison to the normal group and the
B. adolescentis CCFM 669 and 667 groups, whereas no statistical differences were seen between the
B. adolescentis CCFM 626 group and the control group.
B. adolescentis not only improved the symptoms of constipation and changed the mice’s intestinal flora and microenvironment, but also affected the GI neurotransmitters related to constipation. MTL influences the transport of water and electrolytes, promotes gastric contractions and small intestine segmental movement, accelerates intestinal transfer time and increases colon movement. Gas stimulates the secretion of gastric acid and pepsinogen and promotes the growth of digestive tract mucosa, the contraction of the GI smooth muscle and the relaxation of the pyloric sphincter. SP adjust the contraction of the GI tract, intestinal motility and gastric acid secretion. Therefore, the promotion of the serum levels of MTL, Gas and SP accelerates intestinal peristalsis and the transport of contents. In our study, the levels of MTL, Gas and SP in the control group were significantly decreased compared with those in the normal and
B. adolescentis treatment groups. This phenomenon confirmed that the decreases in MTL, Gas and SP might be amongst the causes of constipation. These results are consistent with those of several other reports [
49,
50,
51].
ET, as a multifunctional peptide, can exert important effects on numerous aspects of cardiovascular, neuroendocrine and gastrointestinal function [
52]. Meanwhile, it plays an important role in the stability of vascular tension and maintains the basic cardiovascular system. SS inhibits the release of GI hormones, such as MTL and Gas, and the secretion of gastric acid, trypsin and amylase. Vecht [
53] revealed that SS could slow the small intestinal transit time significantly, whether during eating or fasting. VIP is a polypeptide composed of 28 amino acids whose function is to relax the GI tract and GI sphincter, and it significantly promotes the colon cancer induced by carcinogens in mice. Fasth et al. [
54] held that VIP is an important factor in the production of descending inhibition, resulting in slow transmission. Studies have found that the levels of ET, SS and VIP in control groups were higher than those in normal and
B. adolescentis treatment groups, which means that
B. adolescentis influenced the level of GI hormones, an index that could reflect the status of constipation.