Effect of the probiotic Lactobacillus plantarum IS-10506 on BDNF and 5HT stimulation: role of intestinal microbiota on the gut-brain axis.

Background and Objectives
Microbial communities residing in the gut play a major role in the communication between the gut and the brain through neural, immune, and hormonal routes. Changes in abundance of beneficial intestinal bacteria can affect health of individuals. Conversely, drugs, disease, diet and other factors can alter the gut microbiome. However, there is limited information on the effect of exogenous factors on gut microbiota. In this study, we investigated whether a beneficial bacterium, the probiotic Lactobacillus plantarum IS-10506, can stimulate the gut-brain axis using Wistar rats.


Materials and Methods
The animals were divided into two groups: one received L. plantarum IS strain 10506 supplementation, while the control group received no treatment. Activation of the gut-brain axis was evaluated by immunohistochemical analysis of intestinal and brain serotonin (5-HT) and brain neurotrophin (NT), serotonin transporter (5-HTT), and brain-derived neurotrophic factor (BDNF) levels.


Results
The results showed that BDNF (p< 0.000), NT (p< 0.000007), and 5-HTT (p< 0.000007) expression was upregulated in the brain along with intestinal 5-HT (p< 0.000) level in rats treated with L. plantarum strain IS-10506 relative to the control group.


Conclusion
The probiotic L. plantarum IS-10506 stimulates the gut-brain axis and can potentially promote brain development and function.


INTRODUCTION
The digestive tract and brain are intimately connected (1). Microbial communities residing in the gut play a major role in the communication between the gut and the brain (1)(2)(3). In humans, the 200-600 IRAN. J. MICROBIOL. Volume 11 Number 2 (April 2019) 145-150 http://ijm.tums.ac.ir million neurons of the enteric nervous system respond to chemical and mechanical stimuli from the gastrointestinal tract and combine this information with that transmitted by the brain via the vagus nerve (i.e., cranial nerve X) (4,5). Signals between these two organs are bidirectional but most (90%) are retrograde and keep the brain informed of gut activity. The communication between the brain's emotional and cognitive centres and peripheral intestinal function is known as the gut-brain axis (1, 2, 6, 7). Data from mice and humans have shown that gut microbiota influence the gut-brain axis; disruption of gut microbiota communities alters the expression of the neuromodulator brain-derived neurotrophic factor (BDNF), the growth factor neurotrophin (NT), and the serotonin (5-HT) transporter (5-HTT) in the hippocampus and amygdala (8)(9)(10). Additionally, clinical studies have reported that changes in gut microbiota lead to observable changes in mood or behaviour; conversely, ingestion of probiotics can positively affect brain function in healthy individuals (6,(11)(12)(13).
In the present study, we investigated whether the probiotic bacterium Lactobaccillus plantarum strain IS-10506, which is prevalent in Indonesia and is a typical resident of the intestine, can influence the gut-brain axis.

Animals.
Male Wistar rats (8-12 weeks old; 100-120 g) were divided into two groups (n = 10 each): animals in the first served as untreated controls, and the second group received probiotic supplementation (L. plantarum IS-10506) for 7 days at 2.67 × 10 9 CFU/ day in 0.25 ml saline. At the end of the experiment, rats were sacrificed by cervical dislocation and the brain and small intestine (ileum) were immediately dissected. The intestinal contents were removed, and the tissues were washed with phosphate-buffered saline (PBS) and prepared for immunohistochemistry. This study was approved by the Animal Care and Use Committee of Ethics Committee of the Veterinary Faculty of Universitas Airlangga, Surabaya, Indonesia.
Probiotic supplementation. Microencapsulated L. plantarum strain IS-10506 (GenBank accession no. DQ860148) was packed in an aluminium foil sachet at the Pharmacy Installation of Dr. Soetomo Hospital (Surabaya, Indonesia) and dissolved in 1.5 ml sterile water, and administered to rats via a gastric tube once daily for 7 days at a dose of 2.67 × 10 9 CFU/day. Probiotic viability was assessed 1 week prior to the treatment.
Statistical analysis. Differences between groups were evaluated with a paired sample t test and oneway analysis of variance for normally distributed data. Significance was set at p< 0.05.  [10][11][12]: in each case, the number of immunopositive cells was higher in the treatment group than in control rats.

DISCUSSION
A growing body of evidence indicates that gutbrain communication is influenced by the gut microbiome (5,12,14). The results of the present study demonstrate that a probiotic bacterium, which is a typical gut microbe, influences neurotransmitter and            neurotrophin expression in the brain and intestine. 5-HT is released from enterochromaffin cells of the gastric mucosa in response to various stimuli including signals from gut microbiota (15)(16)(17). Although 5-HT is a well-known neurotransmitter that is associated with emotion and behaviour, about 90% of 5-HT in the body is produced in the digestive tract where it is involved in the detection of resource availability and regulation of bowel movement. In platelets, 5-HT contributes to haemostasis and in the central nervous system (CNS), it regulates mood, cognition, appetite, and sleep. 5-HT is also associated with bone metabolism and organ development (8,9,17). Gut microbiota stimulates host intestinal cells to produce 5-HT. An imbalance of 5-HT in the brain can lead to depression, whereas peripheral dysregulation of this neurotransmitter has been linked to a variety of diseases (1,2,7,12,15,16,(18)(19)(20)(21). In this study we found that treatment with L. plantarum IS-10506 caused an increase in brain 5-HT level in rats relative to the control group.
BDNF is the most abundant and widely distributed growth factor in the CNS and is important for neuronal survival, migration, and differentiation; axonal and dendritic growth; synapse formation; regulation of synaptic plasticity and behaviour (1,10,14,16,22); and hippocampal neurogenesis (23). We found here that brain BDNF expression in the hippocampus was enhanced by L. plantarum IS-10506 treatment relative to untreated rats, implying that probiotic supplementation can enhance brain development, especially memory and brain plasticity. 5-HTT may contribute to the maintenance of BDNF equilibrium and brain function. Gut microbes are thought to regulate 5-HTT expression (24). Previous studies have shown that the supernatant of Lactobacillus rhamnosus GG (LGG) cultures increases 5-HTT expression in mouse intestine (25), whereas Lactobacillus acidophilus and Bifidobacterium longum culture supernatants have the same effect in intestinal epithelial cells (2). In this study, the number of hippocampal neurons expressing 5-HTT was higher in rats that received L. plantarum IS-10506 supplementation than in those without treatment, suggesting that probiotics enhance serotonergic function in the brain.
NTs constitute a family of proteins that promote the survival, development, and function of neurons, in part by inducing BDNF release from hippocampal neurons (26). We observed that L. plantarum IS-10506 treatment increased the level of NT in the brain, which corresponded to the elevation in BNDF level. Thus, L. plantarum IS-10506 supplementation may enhance neuronal survival, differentiation, and growth.

CONCLUSION
The results of this study indicate that L. plantarum IS-10506 treatment can increase intestinal 5-HT and brain 5-HTT, BDNF, and NT expression in adult rats. These findings suggest that probiotics can promote brain development and function and offer a model for investigating the effects of exogenous factors on the gut-brain axis.