Lactobacillus plantarum KSFY06 and geniposide counteract montmorillonite‐induced constipation in Kunming mice

Abstract Constipation is a common clinical manifestation of digestive system disorders and occurs worldwide. This study investigated the ability of Lactobacillus plantarum KSFY06 (LP‐KSFY06) to promote the action of geniposide in preventing montmorillonite‐induced constipation in Kunming mice, with the aim of providing a successful solution. The effects of LP‐KSFY06 and geniposide on constipation were measured, and the results showed that the protective effect of geniposide on constipation was enhanced by LP‐KSFY06 and that the combination resulted in increased weight, moisture content, and particle number of feces. The first black stool defecation time was decreased from 182 min to 87 min, which clearly indicates that defecating difficulty was alleviated in constipated mice. The synergic intervention of LP‐KSFY06 and geniposide (LP + G) assisted in maintaining the body weight of constipated mice. The LP + G intervention significantly increased serum levels of motilin (MTL, 167.8 pg/ml), acetylcholinesterase (AChE, 45.3 pg/ml), substance P (SP, 61.0 pg/ml), vasoactive intestinal peptide (VIP, 70.5 pg/ml), endothelin‐1 (ET‐1, 16.1 pg/ml), and gastrin (73.0 pg/ml) and remarkably decreased somatostatin (SS, 35.2 pg/ml) when compared to those indexes in the LP‐KSFY06 group and geniposide group. The LP + G treatment also significantly increased the mRNA expression of cluster of differentiation 117 (c‐Kit), stem cell factor (SCF), glial cell‐derived neurotrophic factor (GDNF), and remarkably downregulated the expression of inducible nitric oxide synthase (iNOS), transient receptor potential vanilloid‐1 (TRPV1), and cyclooxygenase‐2 (COX‐2). The experimental results showed that the combination treatment has the strongest prevention effect against constipation, and LP‐KSFY06 promotes the ability of geniposide to prevent constipation. Therefore, LP‐KSFY06 is a potential probiotic strain with the capacity to prevent montmorillonite‐induced constipation.

Unhealthy eating and excretion habits, lack of exercise, neurological disorders, or irregular metabolic conditions can trigger constipation, although the exact pathogenesis of constipation is still unclear (Schuster, Kosar, & Kamrul, 2015). The duration of stools retained in the large intestine is prolonged when constipation occurs, and fecal moisture is subsequently absorbed, resulting in dry feces that inhibit the movement of the bowel and induce intestinal imbalance and bowel pain (Chen, Wu, Liao, & Yang, 2010;Khalif, Quigley, Konovitch, & Maximova, 2005). Constipation is a risk factor for irritable bowel syndrome and colorectal cancer, as well as other gastrointestinal disorders (Okawa, Fukudo, & Sanada, 2019;Shimotoyodome, Meguro, Hase, Tokimitsu, & Sakata, 2000). Administration of laxatives and prokinetic agents (motilin agonists, 5-hydroxytryptamine modulators, opioid antagonists, and chloride-channel activators) are common clinical treatments for constipation (Shin et al., 2014). Longterm intake of anticonstipation agents, however, can cause drug dependence and is economically unfeasible due to direct and indirect health care expenses (Crowell, Harris, Lunsford, & Dibaise, 2009).
Laxatives can also cause side effects such as diarrhea, upset stomach, stomach cramping, and vomiting.
Constipation is generally not regarded as a disease. It is a multifactorial disorder that can be managed by exercise and consumption of a healthy diet and additional water (Webster, Tummala, Diva, & Lappalainen, 2016). Nutritional therapy for constipation consists of consumption of fruits, vegetables, whole grains, and cereals, which are rich in dietary fiber, and can increase fecal mass and accelerate the small intestine passage, which subsequently reduces colonic transit time (Karabudak, Koksal, & Macit, 2019;Wang, Sun, Zhou, & Zhao, 2015). Moreover, increasing numbers of studies indicate that probiotics (i.e., Lactobacillus and Bifidobacterium) decrease wholegut transit time and increase defecation frequency by increasing intestinal motility, as well as softening fecal consistency (Eskesen et al., 2015;. Probiotics are defined as living microorganisms, and only when administered in adequate amounts can they confer beneficial health effects upon the host (FAO/WHO, 2006). Lactic acid bacteria (LAB) are an important group of probiotics and include Lactobacillus spp., Bifidobacterium spp., and Enterococcus spp., which are widely used as food supplements. LAB directly or indirectly act on the large intestine and other organs by influencing the intestinal flora, modulating intestinal permeability and immunological parameters, and producing regulatory or bioactive metabolites (Markowiak & Slizewska, 2017).
Geniposide is an iridoid glycoside extracted from the fruit of Gardenia jasminoides that is widely used in Asian countries. As a natural bioactive constituent, it has been proven that geniposide is a curative substance for diseases of the digestive system (Qian, Suo, Yi, Li, & Zhao, 2017). Lactobacillus strains also can effectively alleviate constipation (Chen et al., 2020;Zhao et al., 2019). Therefore, this study evaluated the potential synergic effect of the combination of Lactobacillus plantarum KSFY06 (LP-KSFY06) and geniposide.
We attempted to determine if the bacterial strain has the ability to increase the effectiveness of geniposide, which provides a theoretical basis for further in-depth research, application, and industrial development.

| Experimental strain
Lactobacillus plantarum KSFY06 (LP-KSFY06) was isolated from naturally fermented yogurt from Xinjiang, China. It was identified using Basic Local Alignment Search Tool (BLAST) at the National Center for Biotechnology Information (NCBI) website.

| Design of a mouse constipation model
Fifty male, 6-week-old Kunming (KM) mice were purchased from the Experimental Animal Center of Chongqing Medical University (Chongqing, China). They were acclimatized for 1 week at room temperature of 25 ± 2°C with relative humidity 55 ± 5% and a 12 hr light/12 hr dark cycle, with diet and water ad libitum. Then, the mice were randomly divided into 5 groups (n = 10/group): (a) normal, (b) model, (c) LP-KSFY06, (d) geniposide, and (e) LP-KSFY06 and geniposide (LP + G). All groups were fed a standard rodent AIN-93G chow diet (Jiangsu Medicine Ltd.). The normal and model groups received a normal diet for 9 days, and the treatment groups received a normal diet with LP-KSFY06 (0.5 × 10 7 colony-forming units (CFU)/ kg body weight), geniposide (50 mg/kg body weight), and LP + G (LP-KSFY06 1 × 10 7 CFU/kg body weight and 50 mg/kg body weight). geniposide to prevent constipation. Therefore, LP-KSFY06 is a potential probiotic strain with the capacity to prevent montmorillonite-induced constipation.

| Gastrointestinal transit ratio and defecation time
All groups were fasted for 24 hr with free access to water. The model and intervention groups received an oral administration of 0.2 ml of 10% activated carbon that was dissolved in 10% gum arabic, while the normal group received oral administration of 10% gum arabic.
Half of the mice from each group were killed by CO 2 following 30 min of treatment, and small intestines were collected. The gastrointestinal transit ratio for each mouse was calculated as: The remaining 5 mice from each group were used to measure the defecation time of the first black stool.

| Morphology of small intestinal villi from mice
All mice were killed using CO 2 , and the blood, colon tissue, and small intestine were collected. The small intestine of each mouse was cut into lengths of 2 cm and then fixed in 10% formalin. After dehydrating for 48 hr, transparency treatment, wax immersion, embedding, sectioning, and hematoxylin and eosin (H&E) staining were performed to observe the morphological changes of these tissues under optical microscopy (BX43, Olympus) .

| Total RNA extraction and quantitative PCR assay
The small intestine tissue from each mouse was washed with normal saline and homogenized. Total RNA was extracted with TRIzol reagent according to the manufacturer's protocol (Thermo Fisher Scientific). The purity and concentration of the extracted total RNA were determined by ultra-micro spectrophotometry (Nano-100, All for Life Science), and then, the RNA was diluted to 1 μg/μl. Total RNA samples were used as templates to generate cDNAs by reverse transcription (Thermo Fisher Scientific). Then, cDNAs (2 μl) were mixed with 2× SYBR Premix Ex Taq II (10 μl), 50× ROX reference dye (0.4 μl), total primer (2 μl, 10 μmol/L, Note: Values presented are the mean ± standard deviation (n = 10/group).
Day 1-6: treatment period without induction of constipation; Day 7-9: treatment period with induction of constipation.
LP-KSFY06: mice treated with 0.5 × 10 7 CFU/kg of Lactobacillus plantarum KSFY06; Geniposide: mice treated with 50 mg/kg body weight of geniposide; LP + G: mice treated with 1.0 × 10 7 CFU/ kg of Lactobacillus plantarum KSFY06 and 50 mg/kg body weight of geniposide. a-e In the same column means significant differences (p < .05) according to Duncan's multiple range test.
TA B L E 1 Stool status of mice during the experiment

| Statistical analysis
In this study, the experiments were carried out in triplicate, and the mean values were obtained. All the data, presented as the mean ± standard deviation, were analyzed by SPSS 22 software (SPSS Inc., Chicago, IL, USA). Significant differences among groups, presented by p < .05, were analyzed by one-way analysis of variance (ANOVA). All figures were drawn using Origin 8.1 software.

| Body weights of mice
The changes in BW shown in Figure 1 illustrated that gradual growth of mice in all groups occurred during the first six days. From day 7, the BWs of the model group were significantly decreased due to constipation induced by montmorillonite clay. The LP-KSFY06 group exhibited the same tendency of BW. However, the treatment consisting of geniposide or LP + G inhibited weight loss compared with the model group (p < .05), and BW steadily increased.

| Effect of LP-KSFY06 and geniposide on fecal parameters
The fecal parameters shown in Table 1 indicate that montmorillonite significantly decreased the weight and water content of stool from 0.99 g to 0.49 g and 47% to 20%, respectively (p < .05). LP-KSFY06 treatment enhanced the stool moisture of the constipated mice (p < .05), while there was no effect on stool weight (p > .05). The geniposide or LP + G intervention significantly increased the stool weight and water content (p < .05).

| Gastrointestinal transit capability in constipation with different treatments
As shown in

| Determination of serum gastrointestinal motility-related biomarkers
The serum gastrointestinal motility-related biomarkers were determined and are shown in A more optimal regulatory effect was observed after LP + G treatment in constipated mice, and indexes of serum gastrointestinal motility were similar to those of the normal group.

| RNA expression in the mice small intestine
Compared with the normal group, the expression of c-Kit, SCF, and GDNF mRNA in the model group was upregulated, and TRPV1, iNOS, and COX-2 mRNA expression was downregulated (Figure 3). After intervention with LP-KSFY06, geniposide, or LP + G, the changes induced by constipation were significantly attenuated in all the experimental groups (all p < .05), except for LP-KSFY06 alone and its effects on TRPV1 and COX-2. The efficacy of LP + G was superior to that of sole treatments with LP-KSFY06 or geniposide. Moreover,

F I G U R E 3
The cluster of differentiation 117 (c-Kit), stem cell factor (SCF), glial cell-derived neurotrophic factor (GDNF), inducible nitric oxide synthase (iNOS), transient receptor potential vanilloid-1 (TRPV1), cyclooxygenase-2 (COX-2) mRNA expression in small intestine of mice. a-d The different letters mean that there are significant differences (p < .05) between every two groups between every two groups according to Duncan's multiple range test. LP-KSFY06: mice treated with 0.5 × 10 7 CFU/kg of Lactobacillus plantarum KSFY06; Geniposide: mice treated with 50 mg/kg body weight of geniposide; LP + G: mice treated with 1.0 × 10 7 CFU/kg of Lactobacillus plantarum KSFY06 and 50 mg/kg body weight of geniposide LP-KSFY06 improved the regulatory effect of geniposide on mRNA expression of some marker molecules in montmorillonite-induced constipation.

| D ISCUSS I ON
Constipation is a common problem in humans, and current treatments do not provide relief of symptoms. Studies have shown that constipation is associated with gut microbiota dysbiosis, and decreased abundance of Lactobacillus, Bifidobacteria, Prevotella, and Bacteroides might be a potential cause or a consequence of altered gut motility (Attaluri, Jackson, Valestin, & Rao, 2010;Wojtyniak, Horvath, Dziechciarz, & Szajewska, 2017). With the recognition of the gut microbiota, probiotics can be used to modify health and disease, even though the exact mechanisms remain unclear. LAB probiotics are widely used in the food industry and have gradually developed into relatively indispensable functional foods that are eaten on a daily basis (Filanniono, Cagno, & Gobbetti, 2018; Venegas-Ortega, Flores-Gallegos, Martinez-Hernandez, Aguilar, & Nevarez-Moorillion, 2019). Studies have shown that LAB can alter the sensation and motility of the gut (Quigley, 2007;Rousseaux et al., 2007), soften the stool, and change secretion, as well as produce lactic acid and/or short-chain fatty acids that can lower the intraluminal pH and promote defecation (Salminen & Salminen, 1997;Waller et al., 2011).
In the current study, combination treatment with LP-KSFY06 and geniposide (LP + G group) significantly enhanced levels of serum MTL, VIP, gastrin, AchE, and SP and inhibited serum ET-1 and SS, of which the effects were remarkably superior to those of treatments with LP-KSFY06 or geniposide alone. The serum levels of these biomarkers in the combination treatment group were similar to those in the normal group.
Apart from direct regulation of these neurotransmitters, LP-KSFY06 also affects RNA expression in the small intestine. The interstitial cells of Cajal (ICC) are nerve-like cells at the ends of motor neurons that maintained by c-Kit, which promotes intestinal motility in the gastrointestinal system (Bansil & Turner, 2018;Lee, Park, Kamm, & Talbot, 2005;Yu, Crowell, Tihan, & Lacy, 2002). Low levels of ICC can be observed in patients with constipation (Sabri, Barksdale, & Lorenzo, 2003;Su et al., 2019). LAB could improve constipation by upregulating the expression of c-Kit mRNA (Chen et al., 2020). SCF is a ligand for c-Kit and necessary for the appropriate development and survival of ICC in the intestine (Wedel et al., 2002). In previous studies, the mRNA expression of c-Kit and SCF in constipated mice or patients with constipation was decreased compared with that in normal mice and healthy individuals (Lee et al., 2005;Li et al., 2010).
Our results also showed that there were lower mRNA levels of c-Kit and SCF in model mice compared with those in the normal group.
Additionally, combination LP-KSFY06 and geniposide treatment enhanced the expression of c-Kit and SCF mRNA ( Figure 3).
Transient receptor potential cation channel subfamily V member 1 (TRPV1), a member of the TRPV group of transient receptor potential family of ion channels, is associated with the release of SP and closely related to defecation (Geppetti & Trevisani, 2004;Peng & Li, 2010), and its overexpression might indicate intestinal injury (Su et al., 2019). Treatment with LP-KSFY06 reduced the increased expression level of TRPV1 to some degree, while the combination of LP-KSFY06 and geniposide nearly eliminated the increased expression of TRPV1 in a manner similar to that in normal mice.
GDNF is a protein distributed in the gastrointestinal tract that modulates the growth and development of nerve cells (Allen, Watson, Shoemark, Barua, & Patel, 2013), repairs damaged nerve fibers, as well as promotes intestinal epithelial cell proliferation (Kalff, Schraut, Billiar, Simmons, & Bauer, 2000) and repairs the damaged intestinal tract (Su et al., 2019). As shown in Figure 3, the mRNA expression of GDNF in model mice was significantly inhibited by montmorillonite-induced constipation. The inhibitory effects were totally negated, and there were no differences among mice treated with LP-KSFY06, geniposide, or the combination.
Nitric oxide synthase (NOS) is the key to the produce endogenous NO from the esophagus to the anal sphincter. Activated inducible nitric oxide synthase (iNOS) hardly expresses in intestinal smooth muscle cells under normal circumstances which directly modulates intestinal dysmotility (Ma et al., 2019). However, a mass of NO derived from iNOS under inflammatory reaction will suppress the contractility of intestinal smooth muscle (Moojen et al., 1999;Toda & Okamura, 2016). Moreover, prostaglandin released by cyclooxygenase-2 (COX-2) also inhibits the intestinal smooth muscle contractility (Schwarz et al., 2001;Su et al., 2019). Inhibition of the expression of either iNOS or COX-2 can significantly enhance the motility of the small intestine (Wen et al., 2006). In the current study, the mRNA expression of iNOS and COX-2 significantly increased by 1.33-fold and 0.95-fold when compared to that in the normal group (Figure 3).
After treatment with the combination of LP-KSFY06 and geniposide, the mRNA expression of iNOS and COX-2 was significantly decreased. However, LP-KSFY06 showed no effect on regulating the expression of COX-2 mRNA.
In this study, oral administration of LP-KSFY06 and geniposide attenuated the montmorillonite-induced constipation in mice. In addition, L. plantarum KSFY06 enhanced the effect of geniposide on constipation. The combination of KSFY06 and geniposide effectively increased stool weight and water content and increased the activated carbon propulsive rate, shortened the defecation time of the first black stool, and repaired intestinal villi. In addition, serum levels of MTL, VIP, AchE, gastrin, and SP were increased, and ET-1 and SS were decreased to a greater degree after LP + G treatment compared with LP-KSFY06-or geniposide-treated mice. LP + G-treated mice also demonstrated significantly increased mRNA expression levels of c-Kit, SCF, and GDNF and decreased TRPV1, iNOS, and COX-2. These results suggest that LP-KSFY06 promoted geniposide attenuation of montmorillonite-induced constipation in mice, and LP-KSFY06 could be a potential agent that can be used for prevention or healing of constipation.

| CON CLUS IONS
LP-KSFY06 alleviated symptoms and reverted intestinal villi changes associated with constipation. Even though there were no differences in c-Kit, SCF, GDNF, TRPV1, iNOS, and COX-2 mRNA expression between the genisoside and LP + G groups, LP-KSFY06 improved the regulatory effect of geniposide on gastrointestinal motility-related biomarkers in constipated mice. This study indicated that L. plantarum KSFY06 may be an effective probiotic candidate with the ability to mitigate the adverse effects of constipation. The mechanism used by LP-KSFY06 to promote geniposide's action will be more closely examined, and clinical trials will be carried out to verify their effectiveness in humans.

CO N FLI C T O F I NTE R E S T
There are no conflicts of interest in this paper.

DATA AVA I L A B I L I T Y S TAT E M E N T
No data were used to support this study.