Lactobacillus plantarum PS128 Promotes Intestinal Motility, Mucin Production, and Serotonin Signaling in Mice

Lactobacillus plantarum PS128 has been reported as a psychobiotic to improve mental health through the gut–brain axis in experimental animal models. To explore its mechanism of action in the gut, this study aimed to analyze the effects of L. plantarum PS128 ingestion on naïve and loperamide (Lop)-induced constipation mice. We found that, in the two mouse models, the weight, number, and water content of feces in the L. plantarum PS128 group were higher than those in the vehicle control group. Histological observation revealed that L. plantarum PS128 increased the level of colonic mucins including the major mucin MUC2. In addition, the charcoal meal test showed that L. plantarum PS128 significantly increased the small intestine transit in naïve mice, but not in the Lop-treated mice. Since intestinal serotonin has been found to modulate motility, we further analyzed the expression of genes related to serotonin signal transduction in the small intestine of naïve mice. The results showed that L. plantarum PS128 significantly altered the expression levels of Tph1, Chga, Slc6a4, and Htr4, but did not affect the expression levels of Tph2, Htr3a, and Maoa. Furthermore, immunohistochemistry revealed that L. plantarum PS128 significantly increased the number of serotonin-containing intestinal cells in mice. Taken together, our results suggest that L. plantarum PS128 could promote intestinal motility, mucin production, and serotonin signal transduction, leading to a laxative effect in mice.


Introduction
Probiotics are defined as live microorganisms which when administered in adequate amounts confer a health benefit on the host [1]. In the market, probiotics have been extensively available as food products (cheese, yogurt, fermented milk, meat, and vegetables) or food supplements (capsules, tablets, and powders). Numerous studies have demonstrated the diverse beneficial effects of probiotics in the maintenance of gastrointestinal (GI) homeostasis [2,3], regulation of immune responses [4], and attenuation of metabolic dysfunction [5].
The gut-brain axis facilitates bidirectional communication between the GI tract and the brain or between the enteric nervous system (ENS) and central nervous system (CNS), which involves the neural, immune, and endocrine pathways [6]. A special class of probiotics, termed "psychobiotics," can improve the CNS-related functions and behaviors of the host through the gut-brain axis [7]. Moreover, psychobiotics have been demonstrated to improve the neurodegenerative and neurodevelopmental disorders, including autism spectrum disorder (ASD) and Parkinson's disease (PD) [8]. Though, psychobiotic effects are considered strain-specific, a given strain might exert several health-promoting effects in many cases [9].
Lactobacillus plantarum PS128 is a novel psychobiotic that alleviates depression-and anxiety-like behaviors [10, Chih-Ming Chen and Chien-Chen Wu contributed equally to this study. 11], visceral hypersensitivity [12], and neurobehavioral aspects of movement disorders [13,14] in experimental animals. Clinically, L. plantarum PS128 appears to ameliorate the opposition/defiance behaviors in children with ASD [15], enhance exercise performance in triathletes [16], and improve self-perceived stress and salivary cortisol levels in highly stressed information technology specialists [17]. These studies suggest that L. plantarum PS128 affects CNS-related functions through the gut-brain axis. However, the effect of L. plantarum PS128 on GI function, including motility and secretion, remains largely unknown.
In this study, we aimed to evaluate the effect of L. plantarum PS128 on fecal parameters, intestinal motility, and intestinal secretion in two mouse models, including naïve mice and loperamide (Lop)-induced constipation model mice. Since intestinal serotonin (5-hydroxytryptamine; 5-HT) is a well-known neurotransmitter that regulates GI motility [18], we further studied the effect of L. plantarum PS128 on the expression of genes related to serotonin signaling in the intestine.

Preparation of Lactobacillus plantarum PS128 Culture
Lactobacillus plantarum (recently re-classified as Lactiplantibacillus plantarum [19]) PS128 was prepared using the method described by Liao et al. [14]. In brief, L. plantarum PS128 was cultured at 37 °C in the de Man, Rogosa and Sharpe broth (Difco Corp., MD, USA) for 18 h. The bacterial culture was harvested by centrifugation at 6000 × g for 10 min. Before oral administration, the bacterial pellet was resuspended in sterile phosphate-buffered saline (PBS) to attain a final concentration of approximately 10 10 colonyforming units (CFUs)/mL.

Animals
Eight-week-old adult male ICR mice were purchased from the National Laboratory Animal Center, Taipei, Taiwan. All mice were maintained on a 12-h light/dark cycle in a humidity-controlled (55-65%) and temperature-controlled (22 ± 2 °C) environment with standardized laboratory chow and tap water ad libitum at the National Yang Ming Chiao Tung University Laboratory Animal Center. The use of animals and the procedures for animal handling and treatments were approved by the Institutional Animal Use and Care Committee (IACUC 1,060,606) at the National Yang Ming Chiao Tung University in Taiwan.

Experimental Design
As shown in Fig. 1, mice were divided into four groups (n = 10 per group). The PS128 group received daily oral gavage of PS128 of 10 9 CFU for 14 consecutive days while vehicle control (Veh) group received PBS (0.2 mL per day). The PS128 + Lop group received daily oral gavage of PS128 of 10 9 CFU for 14 consecutive days, and orally administered loperamide hydrochloride (5 mg/kg body weight; Sigma-Aldrich, St. Louis, MO, USA) on day 8 to 14 of the experiment [20]. In addition, the Veh + Lop group received daily oral gavage of 0.2 mL PBS for 14 consecutive days and the same treatment of Lop. To analyze fecal parameters, the wet weight and number of feces were measured for 3 h per day on day 8 to 14 of the experiment. Subsequently, the collected feces were dried at 70 °C for 18 h and weighed to determine the percentage of water content, which was calculated using the following formula: Fecal water content (%) = [(wet mass − dry mass)/wet mass] × 100. On day 15 of the experiment, the small intestinal transit rate of mice was determined by a charcoal meal test, and the mice were sacrificed for subsequent analysis.

Charcoal Meal Test
To measure the small intestinal transit rate in mice, a charcoal meal test was performed as previously described with minor modifications [21]. After fasting for 16 h with water ad libitum, mice were orally administered 0.2 mL of PBS or L. plantarum PS128 suspensions. After 1 h, mice were administered PBS or Lop. HCl by oral gavage followed by administration of charcoal meal (5% charcoal and 10% gum arabica) after 30 min. Fifteen minutes later, mice were sacrificed by cervical dislocation and the small intestine was removed from the stomach to the caecum to measure the distance traveled by the charcoal meal and total length of the intestine. The small intestine transit rate was calculated using the following formula: small intestine transit rate (%) = (distance traveled by the charcoal meal/total length of the intestine) × 100.

qRT-PCR Analysis
The total RNA in the ileum tissue was extracted, converted to cDNA, and subjected to quantitative reverse-transcriptionpolymerase chain reaction (qRT-PCR) analysis as previously described [22]. In brief, cDNA samples from each group were subjected to triplicate real-time PCR experiments with specific primers (Table 1) and KAPA SYBR FAST ABI PRISM Kit (KAPA Biosystems, Woburn, MA, USA) using the StepOnePlus Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). The targets included genes encoding tryptophan hydroxylase 1 and 2 (Tph1 and Tph2), chromogranin A (Chga), the serotonin transporter solute carrier family 6 member 4 (Slc6a4), 5-hydroxytryptamine receptor 3A and 5-hydroxytryptamine receptor 4 (Htr3a and Htr4), and monoamine oxidase A (Maoa). The target threshold cycle (Ct) was subtracted from the Ct for glyceraldehyde-3-phosphate dehydrogenase (Gapdh) to calculate ΔCt, and a relative quantification analysis was performed using the 2 −ΔΔCT method.

Histological Analysis
The paraffin-embedded distal colon and ileum tissue blocks were sectioned into 5-μm-thick slices and mounted on poly(lysine)-coated slides. After deparaffinization and rehydration, tissue sections were further subjected to alcian blue staining, and the expression levels of MUC2 and 5-HT were determined by immunohistochemical analysis. For alcian blue staining analysis, distal colon sections were rinsed with 3% acetic acid for 3 min and then incubated with 1% alcian blue solution (pH 2.5) for 15 min. After running tap water for 5 min, the sections were subjected to neutral red staining for 1 min. Slides were mounted and visualized under a microscope.
For immunohistochemical analysis, the expression levels of MUC2 and 5-HT in the distal colon and ileum were detected. The paraffin sections were deparaffinized, blocked with 3% hydrogen peroxide for 10 min, and subjected to antigen retrieval with microwaves in a 0.01 M citrate buffer for 15 min. The slides were then washed twice with PBS and incubated with MUC2 (1:100; ab76774, Abcam, UK) or 5-HT (1:1000; #20,080, Acris, Herford, Germany) antibodies. This was followed by incubation with the polymer conjugated peroxidase for 30 min using a polymer detection system (Zymed Laboratories, San Francisco, CA, USA). Finally, the color was developed using 3, 3-diaminobenzidine (Sigma, St. Louis, MO, USA). The slides were counterstained with Gill's hematoxylin (Sigma-Aldrich, St. Louis, MO, USA), then dehydrated, and mounted prior to microscopic reading.
Images were observed and photographed using a microscope equipped with a digital image system. Quantitative analyses were performed using ImageJ software, for alcian blue-and MUC2-positive area, or by counting 5-HTpositive cells under five different fields to calculate the mean ± standard error of means (SEM) per filter.

Statistical Analysis
Data were analyzed using GraphPad Prism (GraphPad Prism, version 7, La Jolla, CA, USA) and represented as means ± SEM. The changes in the fecal parameters over time between two groups (vehicle control and probiotic groups) were analyzed using two-way analysis of variance (ANOVA), mixed design. For multiple comparisons, oneway ANOVA with Tukey's post hoc test was used. Statistical significance was set at *P < 0.05.

Increased Fecal Output in Mice Treated with L. plantarum PS128
The effects of L. plantarum PS128 on fecal parameters were analyzed in naïve and Lop-treated mice. Compared with the vehicle control (Veh) group, PS128 ingestion resulted in increased fecal weight (Fig. 2a), number (Fig. 2b), and water content (Fig. 2c) in both mouse models, suggesting a laxative effect of L. plantarum PS128.

Histology of the Distal Colon
Alcian blue staining was performed to analyze mucin production in the distal colon (Fig. 3a). As shown in Fig. 3b, image quantification of the stained areas showed that, compared with the Veh group, PS128 significantly increased the amount of colonic mucin in naïve mice. In addition, treatment with Lop significantly reduced the amount of colonic mucin, which could be reversed by the ingestion of PS128. Furthermore, immunohistochemical detection and image quantification of MUC2, the major intestinal mucin, showed that PS128 significantly increased the level of MUC2 in both naïve and Lop-treated mice (Fig. 4). However, treatment with Lop did not affect the level of MUC2 in the distal colon.

L. plantarum PS128 Increases the Small Intestine Transit Time in Naïve Mice
To investigate whether L. plantarum PS128 affects intestinal motility, a charcoal meal test was performed. As shown in Fig. 5a, a representative photograph shows the total length of the small intestine and the distance traveled by the charcoal meal in the intestine. Compared with the Veh group, PS128 significantly increased the small intestinal transit rate in naïve mice (Fig. 5b). In addition, treatment with Lop significantly reduced the small intestinal transit rate. However, this reduction could not be ameliorated by PS128. Since PS128 only increased the intestinal motility in naïve mice, but not in Lop-treated mice, we focused on two groups of naïve mice to further analyze how PS128 affects the intestinal motility.

L. plantarum PS128 Modulates the Serotonin Signal Transduction in the Intestine
To further investigate the mechanism of action of L. plantarum PS128 on intestinal motility, qRT-PCR was performed to analyze the expression of genes related to serotonin signal transduction ( Table 1). As shown in Table 2, compared with the Veh group, PS128 ingestion significantly increased Tph1 expression and decreased the expression levels of Chga, Slc6a4, and Htr4. In addition, no other significant differences in the expression levels of Tph2, Htr3a, and Maoa were observed. Furthermore, we examined 5-HT expression in the ileum sections by immunohistochemical staining (Fig. 6a). Compared with the Veh group, the number of 5-HT-positive cells was significantly increased in the PS128 group (Fig. 6b).

Discussion
Emerging evidence has demonstrated that intestinal homeostasis affects CNS-related functions through the gut-brain axis in a bidirectional manner [23]. Patients with psychiatric and neurological disorders, including ASD [24], major depressive disorder [25], PD [26], and Alzheimer's disease [27], are commonly reported to have GI symptoms and imbalanced gut microbiota. Moreover, psychological stress leads to several GI symptoms and plays a crucial role in the development of irritable bowel syndrome (IBS) [28]. In contrast, dysregulated GI mucus secretion and increased intestinal permeability may lead to systemic inflammation and impairment of the blood-brain barrier (BBB), thus negatively influencing CNS-related functions [29]. Therefore, maintaining intestinal homeostasis and improving gut health may help to alleviate CNS disorders. Probiotic food supplementation can reduce stress-induced GI symptoms in volunteers [30], decrease depression scores, alter brain activity in patients with IBS [31], and improve symptoms associated with ASD and PD [8]. These probiotics that affect CNS-related functions and behaviors are classified as psychobiotics, including L. plantarum PS128, Bifidobacterium longum 1714 [32], B. longum NCC3001 [31], Lactobacillus rhamnosus JB-1 [33], Lactobacillus helveticus R0052, and B. longum R0175 [30]. Although these psychobiotics have been suggested to regulate CNS-related functions through the gut-brain axis, their regulation in the intestine remains largely unknown.
In this study, we used two mouse models, naïve and Lop-induced constipation mice, to investigate the effect of L. plantarum PS128 on intestinal homeostasis. Daily administration of L. plantarum PS128 appeared to increase the fecal output, colonic mucin production, and intestinal motility in naïve mice (Figs. 2, 3, 4, and 5), suggesting that L. plantarum PS128 can be used as a laxative food supplement. A recent clinical study has also shown that daily administration of L. plantarum PS128 for eight weeks significantly improved self-perceived stress and GI symptoms in highly stressed information technology specialists [17], which supported the results of this study. However, in mice with Lop-induced constipation, the laxative effect of and water content (c) of feces were measured for 3 h. Data were expressed as mean ± SEM. Differences between groups were analyzed using two-way ANOVA, mixed design. *P < 0.05 compared with the indicated groups (n = 10 per group) L. plantarum PS128 was relatively faint (Fig. 2). Lop has been widely used in animal experiments to reduce intestinal motility and colonic water secretion, thus prolonging the evacuation time of feces [34]. Lop treatment in the Sprague-Dawley rats also resulted in a thinner mucus layer at the fecal surface [35]. In this study, we found that treatment with Lop significantly reduced the level of colonic mucins stained with alcian blue (Fig. 3), which is a central feature of this constipation model [20,36]. Several studies have also shown that treatment with Lop reduced the expression of colonic MUC2 at mRNA [37,38] and protein levels [39]. However, the effect of Lop treatment on the expression of MUC2 cannot be observed in this study (Fig. 4). Possible reasons include that, first, alcian blue and MUC2 antibodies detect different targets, which may lead to different results. The staining of alcian blue is restricted mainly to acidic carbohydrates but not the protein core of mucin [40]. Second, although MUC2 is the major colonic mucin, the expression of other mucins, such as MUC3 and MUC6 [41], may affect the results. Third, there are some differences in the response of ICR mice derived from different sources to Lop treatment [36]. It is still unclear how Lop treatment may affect the expression and glycosylation of different colonic mucins.
As shown in Fig. 5, Lop significantly reduced the small intestinal motility; however, this reduction could not be reversed by the administration of L. plantarum PS128. These findings indicate that L. plantarum PS128 may be ineffective in patients with serious or chronic constipation. Chronic constipation is known to have many possible causes, including blockage of the colon or rectum, dysfunction of the ENS, difficulty in moving the pelvic muscle involved in elimination, and imbalance of hormones in the body. We assume that L. plantarum PS128 is only effective for specific subtypes of constipation, and this assumption requires further investigation. On the other hand, pharmacological therapy for constipation includes the use of bulking agents, osmotic agents, stool softeners, stimulant laxatives, lubricants, etc. [42], while non-pharmacological therapy includes increased intake of dietary fibers and water, increased physical activity, and supplementation of probiotics [43,44]. Fermented milk containing Lactobacillus casei Shirota increases bowel movement frequency and stool consistency in patients with Fig. 3 Oral administration of L. plantarum PS128 increased the amount of colonic mucus in mice. a Representative images of Alcian blue stained areas of distal colonic sections (some are indicated by black arrows). Bars, 50 μm. b Quantification using the ImageJ software for each group (n = 6 per group). Data were expressed as mean ± SEM and analyzed by one-way ANOVA with Tukey's post hoc test, and different superscript letters (a, b, c) differed significantly at P < 0.05 Fig. 4 Oral administration of L. plantarum PS128 increased the amount of colonic MUC2 in mice. a The distal colons were analyzed for the expression of MUC2 by immunohistochemistry (some are indicated by red arrows). Bars, 50 μm. b Quantification using the ImageJ software for each group (n = 6 per group). Data were expressed as mean ± SEM and analyzed by one-way ANOVA with Tukey's post hoc test, and different superscript letters (a, b) differed significantly at P < 0.05 constipation [45,46]. In addition, daily consumption of L. helveticus for one week alleviates constipation-related symptoms and reduces both fecal pH and intestinal transit time in patients with constipation-predominant IBS [47]. The efficacy of probiotic products is suggested to be both strainspecific and disease-specific [48], which should be proven by further evidence-based research. Clinically, whether L. plantarum PS128 is effective in constipation and PD-and ASD-complicated gut dysfunction remains to be studied.
GI epithelial cells sense luminal signals from the ingested food and microbiota, and then translate and deliver signals to exert local and systematic effects. One of the important signals is 5-HT, which originates from the enterochromaffin (EC) cells of the GI tract, which act as the major source of peripheral 5-HT by secreting 95% of total 5-HT in the body [49]. Although peripheral 5-HT does not cross the BBB [50], gut-derived 5-HT can be carried and released by circulating platelets, remain free in the serum, and interact with the CNS through the ENS, thus affecting various biological phenomena, including gut motility and secretion, bowel inflammation, bone development, and platelet aggregation [51]. In this study, we found that L. plantarum PS128 modulated the expression levels of genes related to serotonin signaling in the intestine ( Table 2). TPH1 is a key enzyme for 5-HT biosynthesis and Chromogranin A (ChgA) is colocalized with 5-HT in EC cell storage granules [52,53]. The upregulation of Tph1 and downregulation of ChgA suggested that there is an increase in the biosynthesis and storage of 5-HT in EC cells. Moreover, immunohistochemical analysis showed that L. plantarum PS128 increased the number of 5-HT-containing cells in the ileum (Fig. 6). These results support the previous finding that L. plantarum PS128 ingestion can increase 5-HT levels in the ileum, colon, and serum of rats [13]. Besides, a previous study has shown that heat-killed L. casei 327 promotes colonic 5-HT biosynthesis and GI motility in mice [54], which is similar to the effects exerted by L. plantarum PS128. However, heat-killed L. plantarum PS128 is ineffective in mice [11]. Thus, we suggest that L. plantarum PS128 regulates intestinal 5-HT biosynthesis and motility through mechanisms different from those of heat-killed L. casei 327, which may include bacterial secretory molecules, heat-labile constituents, and specific metabolic activities. In addition, L. plantarum PS128 downregulates the expression of Slc6a4 and Htr4 in the ileum (Table 2), which suggested that 5-HT reuptake would be reduced and may compromise 5-HT 4 receptor signaling. Correlated expressions of Slc6a4 and Htr4 in the mouse colon have been previously reported, which is relevant in the pathogenesis of visceral hypersensitivity by influencing local 5-HT abundance/availability [55]. Moreover, a previous study also reported that L. plantarum PS128 alleviated  Table 2 Gene expression of 5-HT related genes in the ileum of naïve ICR mice Gene expression levels of Tph1, Tph2, Chga, Slc6a4, Htr3a, Htr4, and Maoa were quantified by real-time PCR relative to the expression of Gapdh. Values were expressed as mean ± SEM and analyzed using an unpaired t test * P < 0.05; **P < 0.01; ***P < 0.001 versus the vehicle control group visceral hypersensitivity induced by subcutaneous injection of 5-hydroxytryptophan (5-HTP), a precursor of 5-HT, in rats [56]. Therefore, modulation of the peripheral serotonin signal transduction may be a crucial action mechanism of L. plantarum PS128, and this requires further investigation. This study had several limitations. First, the psychobiotic effects of L. plantarum PS128 on the two mouse models were not evaluated; thus, PS128 regulation in the intestine cannot be directly correlated to the host behavior and previous findings in the CNS. Second, although the qRT-PCR results showed that PS128 modulated the expression of 5-HT-related genes in the intestine, the expression level of the protein was not analyzed. Moreover, the mechanism by which PS128 modulates the intestinal serotonin signaling remains largely uncharacterized. Third, the specific role of gut microbiota in this process remains unknown. Future studies of L. plantarum PS128 action mechanisms should focus on both the gut and the brain, and experiments using vagotomy or 5-HT agonists/antagonists should be conducted to obtain a more comprehensive understanding of these mechanisms.

Conclusions
In this study, we found that daily administration of the psychobiotic strain L. plantarum PS128 could increase the fecal output, colonic mucin production, and small intestinal motility in mice. Moreover, L. plantarum PS128 appeared to promote serotonin signal transduction in the intestine, which might indirectly affect the CNS-related functions and host behaviors through the gut-brain axis.
Author Contribution All authors contributed to the study conception and design. Material preparation, data collection, and analysis were performed by Chin-Lin Huang, Min-Yu Chang, and Shih-Hsuan Cheng. The first draft of the manuscript was written by Chin-Lin Huang, Chien-Chen Wu, and Chih-Ming Chen, and all authors commented on previous versions of the manuscript. The study was supervised by Ching-Ting Lin and Ying-Chieh Tsai. All authors read and approved the final manuscript.
Funding This study was supported by a grant (RD10601) from the Bened Biomedical R&D Project.

Availability of Data and Material
The datasets generated during and/or analysed during the current study are available from the corresponding author on reasonable request.

Declarations
Ethics Approval The use of animals and the procedures for animal handling and treatments were approved by the Institutional Animal Use and Care Committee (IACUC 1,060,606) at the National Yang Ming Chiao Tung University in Taiwan.

Conflict of Interest
Chih-Ming Chen, Chien-Chen Wu, Chin-Lin Huang, Min-Yu Chang, and Shih-Hsuan Cheng are employees of Bened Biomedical Co., Ltd. Ying-Chieh Tsai owns stock from Bened Biomedical Co., Ltd. The views presented in this article reflect those of the authors and not necessarily those of the funder. Ching-Ting Lin declares no conflicts of interest.
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