Neurogastroenterology & Motility Neurogastroenterol Motil (2015) 27, 1239–1248
doi: 10.1111/nmo.12615
Lactobacillus rhamnosus GG supernatant upregulates serotonin transporter expression in intestinal epithelial cells and mice intestinal tissues Y. M. WANG ,* X. Z. GE ,* W. Q. WANG ,* T. WANG ,* H. L. CAO ,* B. L. WANG †
& B. M. WANG *
*Department of Gastroenterology and Hepatology, Tianjin Medical University General Hospital, Tianjin, China †Key Research Laboratory of Hormone and Development Affiliated to the Ministry of Health, Institute of Endocrinology, Tianjin Medical University, Tianjin, China
Key Messages
• Lactobacillus rhamnosus GG (LGG) is one of the best-studied Lactobacillus strains in clinical trials, which can effectively reduce the incidence of diarrhea without side effects.
• SERT mRNA and SERT-P levels in LGG-s treated HT-29, Caco-2 cells, and intestinal epithelial cells were higher than those in the control.
• LGG-s can upregulate SERT mRNA and SERT-P levels in intestinal epithelial cells and mice intestinal tissues. • LGG-s might improve intestinal motility and gastrointestinal sensation by regulating SERT expression is speculative.
Abstract Background The role that probiotics play in relieving irritable bowel syndrome (IBS) has been demonstrated; however, the mechanism by which IBS is affected remains unclear. In this study, serotonin transporter (SERT) mRNA and serotonin transporter protein (SERT-P) levels in HT-29, Caco-2 cells, and mice intestinal tissues were examined after treatment with Lactobacillus rhamnosus GG supernatant (LGG-s). Methods HT-29 and Caco-2 cells were treated with different concentrations of LGG-s for 12 and 24 h and C57BL/6 mice received supplements of different concentrations for 4 weeks. SERT mRNA and SERT-P levels were detected by real-time PCR and Western blotting. Key Results SERT mRNA and SERTP levels in HT-29 and Caco-2 cells were higher than those in the control 24 h after treatment. Undiluted
LGG-s upregulated SERT mRNA levels by 9.4-fold in the first week, which dropped in the second week. The double-diluted LGG-s upregulated SERT mRNA by 2.07-fold in the first week; levels dropped to 1.75-fold within the second week and under base expression levels by the third week, while they again climbed to 1.56-fold in the fourth week. The triple-diluted LGG-s could not upregulate SERT mRNA expression until the end of the fourth week. The SERT-P levels in the double-diluted LGG-s group were higher than that in the control but fluctuated with time. SERT-P levels in the triple-diluted LGG-s were higher than that in the control in the last 2 weeks and increased with time. Conclusions & Inferences LGG-s can upregulate SERT mRNA and SERT-P levels in intestinal epithelial cells and mice intestinal tissues. Keywords serotonin transporter, SERT mRNA, SERT protein, Lactobacillus rhamnosus supernatant, intestinal tissues.
Address for Correspondence B. M. Wang, Department of Gastroenterology and Hepatology, Tianjin Medical University Hospital, No. 154, Anshan Road, District, Tianjin 300052, China. Tel: +86 02260362608; fax: +86 02260362608; e-mail: [email protected] Received: 28 December 2014 Accepted for publication: 12 May 2015
© 2015 John Wiley & Sons Ltd
Abbreviations: 5-HT, 5-hydroxytryptamine; 5-HTTL PR, 5-HTT-linked polymorphic region; 5-HTT, serotonin transporter gene; C-IBS, IBS with constipation; D-IBS, IBS with diarrhea; GAPDH, glyceraldehyde-3phosphate dehydrogenase; IBS, irritable bowel
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is speculative. The purpose of this study was to investigate the effects of LGG-s on the expression of SERT mRNA and SERT protein (SERT-P).
syndrome; LGG-s, Lactobacillus rhamnosus GG supernatant; PI-IBS, postinfectious IBS; SERT-P, SERT protein; SERT, serotonin transporter.
MATERIALS AND METHODS INTRODUCTION Bacterial culture, LGG-s
Serotonin transporter (SERT) is a universally existing cross-membrane transport protein that plays a key role in 5-hydroxytryptamine (5-HT) reuptake. The human SERT gene (5-HTT) is located on chromosome 17q11, named solute carrier family 6 member 4, which is composed of 14–15 exons.1 The 5-HTT-linked polymorphic region (5-HTTLPR) is located upstream of the transcription start site, which plays a key role in the etiology of irritable bowel syndrome (IBS) by modulating the transcriptional activity of the 5-HTT.1 5-HT is an important gastrointestinal hormone that modulates intestinal fluid secretion, gut motility, and gastrointestinal sensation.2–5 Serum 5-HT levels decrease in patients with IBS with constipation (C-IBS) and increase in patients with IBS with diarrhea (D-IBS),6 but is inactivated after reuptake by SERT in intestinal or nerve cells. Downregulation of SERT is implicated in the pathophysiology of various functional gut disorders. Faure et al.7 found that SERT mRNA was lower in children with IBS than in the control. In our previous study, we found that the S/S genotype results in lower SERT expression and causes 5-HT accumulation at the affected site. Ordinarily, 5-HT can activate the intestinal functions of motion and secretion, which are often related to D-IBS.8 Chen et al.9 found that transgenic mice with targeted deletion of SERT frequently exhibit diarrhea interspersed with transient constipation. Lactobacillus rhamnosus GG (LGG) is one of the best-studied Lactobacillus strains in clinical trials. Oksanen et al.10 found that LGG can effectively reduce the incidence of diarrhea without side effects. LGG also has been shown to induce remission and prevent recurrence of IBD in humans11 and in nonhuman colitis models.12 Yan et al.13 purified two soluble proteins from LGG supernatant (LGG-s) and demonstrated that LGG-s and soluble proteins produced by LGG-s activated Akt, inhibited cytokineinduced epithelial cell apoptosis, and promoted cell growth in human and mouse colon epithelial cells and cultured mouse colon explants. The role of probiotics on IBS has been proposed over the past decade and the suggestion that probiotics can relieve IBS symptoms has been confirmed by many studies.14,15 Whether LGG-s improves intestinal motility and gastrointestinal sensation by regulating SERT expression
LGG (ATCC 53103 and CGMCC1 3724) were obtained from the China General Microbiological Culture Collection Center. LGG was incubated in Lactobacillus MRS broth at 37 °C for 24 h, diluted in MRS broth, and incubated again at 37 °C to reach log phase with the density determined as 0.5 at A600. The culture suspension was then centrifuged at 5000 g for 10 min. The supernatant was removed and filtered through 0.22-lm filters.13
Cell culture and cell treatment The human colonic epithelial carcinoma cell line, HT-29, was grown in 1640 media supplemented with 10% fetal bovine serum at 37 °C. The Caco-2 cell line, a continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells, were grown in DMEM media supplemented with 10% fetal bovine serum and 1.0% nonessential amino acid at 37 °C. Cells were serum starved (0.5%) at 37 °C for approximately 24 h before the experiments and then treated with LGG-s (supernatant-to-cell media ratios: 1 : 100, 1 : 20, and 1 : 10) for 12 and 24 h. The control was treated with MRS broth.
Animal studies Forty-eight female C57BL/6N mice (5–8 weeks old) were obtained from Vital River Laboratories Animal Technology Co., Ltd., Beijing, China. Four groups of mice were used, one designated as the control group (n = 12) and the other three as the experimental groups (n = 12 for each group). The mice were kept in cages in a room at 22 °C with a 12 : 12-h light/dark cycle and had free access to a normal mouse-chow diet. The mice of the control group were fed 1.0 mL/day MRS broth through a gavage needle; the mice of the other three groups were given 1.0 mL/day double-diluted, triple-diluted, or undiluted LGG-s through a gavage needle. Mice were maintained on the treatments for 1.0, 2.0, 3.0, and 4.0 weeks during the experiment. Three mice of each group were sacrificed and the colons were removed for later use each week. The protocol was approved by the Animal Use and Care Committee of Tianjin Medical University.
Real-time polymerase chain reaction Total RNA was extracted from HT-29 cells, Caco-2 cells, and 50 mg colon tissue from each of the sacrificed mice with Trizol according to the manufacturer’s instructions (Life, Hilden, Germany). First-strand cDNA was synthesized using the iScriptcDNA synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Quantitative real-time (QRT) PCR was performed on an ABI One plus setup PCR thermocycler; RT-PCR was performed on the SYBR Green PCR Master Mix (Roche Applied Science, Mannheim, Germany). The PCR were set up in a volume of 20 lL containing 1.0 lL cDNA, 10 lL 29 iQ SYBR Green Supermix (Roche Applied Science), and 0.6 lL each of the forward and reverse primers. The sequences of forward and reverse primers are
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(ATCC53103). No significant differences were found for SERT mRNA and SERT-P expression in HT-29 cells in any of the groups treated for 12 h (p > 0.05). Both LGG-s (ATCC53103) and LGG-s (CGMMCC1.3724) can upregulate SERT mRNA and SERT-P expression; however, the effects of LGG-s (ATCC53103) were significant, so LGG-s (ATCC53103) was chosen for further animal experiments (Figs. 1 and 2). Serotonin transporter mRNA levels in Caco-2 cells treated with 1 : 100, 1 : 20, and 1 : 10 concentrations of LGG-s (ATCC53103) for 24 h were 1.60-, 1.40-, and 1.66-fold higher than that in the control group, respectively (p = 0.03, p = 0.022, p = 0.000). The increase in SERT mRNA levels and SERT-P levels in Caco-2 cells in response to 24-h incubation in different concentrations of LGG-s (ATCC53103) were similar to that of HT29 cells, although the effect is modest compared to that of HT-29 cells at 1 : 10 dilution. In addition,
listed in Table 1. For normalization purposes, glyceraldehyde-3phosphate dehydrogenase (GAPDH) was measured as an internal control. Relative mRNA expression was calculated using the 2DDCt method.
Western blotting Proteins were extracted from HT-29 cells, Caco-2 cells, and colonic tissues, and levels were quantified using a BCA protein concentration assay kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were incubated with SERT antibody ab181034, b-actin (Abcam Biotech Company, Cambridge, UK) and horseradish peroxidase-labeled rabbit antirabbit IgG (EarthOx Life Sciences, Millbrae, CA, USA). After incubating with secondary antibodies, the immunoreactive bands were visualized using an enhanced chemiluminescence method (GE Healthcare, Buckinghamshire, UK) and exposure to X-ray film. b-Actin was used as an inner control.
Statistical analyses A
Statistical analysis was carried out using SPSS 19.0 for Windows (SPSS, Chicago, IL, USA). One-way ANOVA and post hoc tests (Dunnett’s T3 and Dunnett’s C) were used to compare the values of RT-PCR. For all analyses, p = 0.05 was defined as statistically significance.
RESULTS Effects of LGG-s on SERT mRNA and protein expression in HT-29 and Caco-2 cells Serotonin transporter mRNA levels in HT-29 cells treated with 1 : 100, 1 : 20, and 1 : 10 concentrations of LGG-s (ATCC53103) for 24 h were 1.46-, 1.47-, and 2.67-fold higher than that in the control group, respectively (p = 0.01, p = 0.01, p = 0.000). As with SERT mRNA levels, SERT-P levels were significantly increased in response to 24-h incubation of HT-29 cells at different concentrations of LGG-s
B
Table 1 Primer sequences for RT-PCR Gene
Sequences (forward/reverse 50 –30 )
hSERT
Sense: 50 -AAT GGG TAC TCA GCA GTT CC-30 Antisense: 50 -CCA CAG CAT AGC CAA TCA C-30 Sense: 50 -ACA GCA ACT CCC ATT CTT-30 Antisense: 50 -TCC AGG GTT TCT TAC TCC-30 Sense: 50 -TGGGCGCTCTACTACCTCAT-30 Antisense: 50 -ATGTTGTCCTGGGCGAAGTA-30 Sense: 50 -AGGTCGGTGTGAACGGATTTG-30 Antisense: 50 -TGTAGACCATGTAGTTGAGGTCA-30
hGAPDH mSERT mGAPDH
Figure 1 Effects of LGG-s on SERT mRNA expression in HT-29 cells. (A) SERT mRNA levels in HT-29 cells treated with different concentration LGG-s for 12 h. (B) SERT mRNA levels in HT-29 cells treated with different concentration LGG-s for 24 h.
hSERT, human serotonin transporter; hGAPDH, human glyceraldehyde-3-phosphate dehydrogenase; mSERT, mouse serotonin transporter; mGAPDH, mouse glyceraldehyde-3-phosphate dehydrogenase.
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A
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D Figure 2 Effects of LGG-s on SERT protein expression in HT-29 cells. (A) SERT protein levels in HT-29 cells treated with different concentration of LGG-s for 12 h. (B) SERT protein levels in HT-29 cells treated with different concentration of LGG-s for 24 h. (C) Quantitative analysis of the protein levels of SERT in HT-29 cells treated with different concentration of LGG-s for 12 h. (D) Quantitative analysis of the protein levels of SERT in HT-29 cells treated with different concentration of LGG-s for 24 h. Error bars represent the mean SD.
expression levels decreased in the third week and were lower than that of control (p = 0.002) but increased slightly in the fourth week (p > 0.05). The doublediluted LGG-s1 upregulated SERT mRNA by 2.07-fold by the end of the first week but levels slowly dropped to 1.75-fold within the second week and to less than the base expression levels during the third week (p = 0.000, p = 0.006, p = 0.008, respectively). By the fourth week, SERT mRNA levels were 1.56-fold higher than that of the control in the double-diluted LGG-s group (p = 0.007). The triple-diluted LGG-s1 did not upregulate SERT mRNA expression within the first to third weeks, but did slightly upregulate SERT mRNA expression (p = 0.004, p < 0.05) to nearly equal that of the double-diluted LGG-s1 by the end of the fourth week (Fig. 5).
unlike HT-29 cells, there was no change in effects with different concentrations of LGG-s. No significant differences were found in SERT mRNA and SERT-P expression in Caco-2 cells in any of the groups treated for 12 h (p > 0.05). LGG-s (CGMMCC1.3724) cannot upregulate SERT mRNA and SERT-P expression in Caco-2 cells (Figs. 3 and 4).
Effects of LGG-s1 on SERT mRNA and SERT-P expression in mice intestinal tissues Undiluted LGG-s1 significantly upregulated SERT mRNA expression levels by 9.4-fold by the end of the first week (p = 0.000, p < 0.05), but levels decreased rapidly to the base expression levels of the control by the end of the second week. The SERT mRNA
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cation.16–22 Irritable bowel syndrome can be subtyped according to the predominant bowel-movement type as C-IBS, D-IBS, and IBS with alternating symptoms of both constipation and diarrhea. Postinfectious IBS (PI-IBS) accounts for 7.0–30% of IBS in patients and often exhibits characteristics of diarrhea, which has a history of acute gastrointestinal infection.23 The mechanisms underlying the development of PI-IBS are believed to be associated with changes in intestinal permeability, persistent lowgrade inflammation, and dysregulation of gut microbiota.24–27 Probiotics and prebiotics have been proved as effective clinical therapies for D-IBS and PI-IBS28,29 and LGG has been one of the most widely studied probiotic strains that is used in a variety of commercially available probiotic products. One open-labeled, randomized, controlled trial enrolled 200 children who had acute, watery diarrhea and demonstrated that the use of LGG resulted in faster improvement in stool consistency compared to that of the control group,30 demonstrating that LGG can significantly reduce the frequency and severity of abdominal pain in children with IBS.31 In recent years, there have been numerous publications that have confirmed the effects of LGG on IBS.32–36 The possible mechanisms by which LGG works on IBS are as follows: regulating intestinal flora,37–39 improving intestinal barrier function31 normalizing levels of interleukins (such as IL-10/IL-2),40 and increasing the visceral pain threshold, and reducing colonic epithelial cells, which carry M opioid receptors and cannabinoid receptors.41 Whether and how probiotics regulate SERT expression remains unclear. In our present study, LGG-s1 upregulated SERT expression in HT-29 cells and Caco-2 cells, which was significantly time dependent. LGG-s diluted by 1 : 100, 1 : 20, and 1 : 10 did not increase either SERT mRNA or SERT-P levels in HT-29 cells and Caco-2 cells when treated for 12 h; however, SERT mRNA and SERT-P levels did increase when treated for 24 h. As to LGG-s2, the upregulation effect on HT-29 was slight with no statistically significance and there was no effect on Caco-2 cells. We believe that there might be some differences in LGG-s1 and LGG-s2, and will perform some screen works on the differential proteins expressed between LGG-s1 and LGG-s2. The results suggest that LGG-s1 can upregulate SERT expression in HT-29 cells and Caco-2 cells; by doing so, LGG-s1 might play a key role in intestinal motility by regulating SERT expression in intestinal tissues. The mouse experiment was designed to verify whether LGG-s1 can upregulate SERT expression in the intestine.
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Figure 3 Effects of LGG-s on SERT mRNA expression in Caco-2 cells. (A) SERT mRNA levels in Caco-2 cells treated with different concentration LGG-s for 12 h. (B) SERT mRNA levels in Caco-2 cells treated with different concentration LGG-s for 24 h. Results are means SE; *significant.
The SERT-P levels in the undiluted LGG-s1 group were nearly equal to that of the control within 1– 4 weeks. The SERT-P levels of the double-diluted LGG-s1 group were higher than that of the control, and gradually increased in the first, second, and fourth weeks but decreased in the third week. The SERT-P levels of the triple-diluted LGG-s1 were higher than that of the control in the last 2 weeks and increased over time (Fig. 6).
DISCUSSION Irritable bowel syndrome is one of the most frequent lower gastrointestinal tract disorders resulting in abdominal pain or discomfort accompanied by altered bowel habits, including alterations in stool frequency and/or form or appearance, especially disordered defe-
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Figure 4 Effects of LGG-s on SERT protein expression in Caco-2 cells. (A) Protein levels of SERT in Caco-2 cells treated for 12 h analyzed by Western blot. (B) Quantitative analysis of the protein levels of SERT treated with different concentration of LGG-s in Caco-2 cells treated for 12 h. (C) Protein levels of SERT in Caco-2 cells treated for 24 h analyzed by Western blot. (D) Quantitative analysis of the protein levels of SERT treated with different concentration of LGG-s in Caco-2 cells treated for 24 h. LGG-s1, LGG-s(ATCC53103), LGG-s2, LGG-s(CGMCC1.3724). Results are means SE; *significant.
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D Figure 5 Effects of LGG-s1 on SERT mRNA expression in mice intestinal epithelial cells. (A) SERT mRNA levels in mice intestinal epithelial cells at the first week. (B) SERT mRNA levels in mice intestinal epithelial cells at the second week. (C) SERT mRNA levels in mice intestinal epithelial cells at the third week. (D) SERT mRNA levels in mice intestinal epithelial cells at the fourth week.
and lipids as well as other small molecules. Studies by Yan et al.13 showed that p40 and p75 in LGG-s activate Akt, inhibit cytokine-induced epithelial cell apoptosis, and promote cell growth in human and mouse colon
Undiluted LGG-s1 was found to significantly increase SERT mRNA expression in mice intestinal tissues, while SERT-P levels were no different. LGG-s is a relatively complex system with a series of proteins
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Figure 6 Effects of LGG-s1 on SERT-P expression in mice intestinal epithelial cells. (A) Form up to down represents the SERT-P level at the first, second, third and fourth week analyzed by Western blot, respectively. Lane a: control; Lane b: triple dilution LGG-s1; Lane c: double dilution LGG-s1; Lane d: undiluted LGG-s1. (B) Form up to down represents the quantitative analysis of SERT-P level at the first, second, third and fourth week analyzed by Western blot, respectively. Results are mean SE; *significant.
increased to about 2.07-fold, they peaked, after which the complex system of SERT mRNA expression might be readjusted by the interaction of 5-HT and SERT-P. Previous studies have indicated that the accumulation of 5-HT inhibited the expression of SERT in Caco-2 cells.43,44 We presumed that there might exist some differences between in vivo and in vitro studies. The regulation mechanism in vivo might be more complicated, which needs to be explored with further research. For the triple-diluted LGG-s1 group, no marked effects on SERT mRNA expression were found within the first and second weeks, but levels increased the fourth week. SERT-P was higher than that of the control in the third and fourth weeks. The presumption from this is that the low concentration of LGG-s1 could participate in the same upregulating action as the high concentration of LGG-s1 when the key components accumulate to an operative concentration over time. LGG-s1 is a relatively complex system that contains a series of proteins and other molecules. We speculate that some macromolecular substances, such
epithelial cells and cultured mouse colon explants. We presumed that there might be some substances in LGG-s that can balance SERT-P translation. A concern with the double-diluted LGG-s group was that SERT mRNA and SERT-P levels increased in the first and second weeks, decreased the third week, and increased again the fourth week. Kerckhoffs et al.42 indicated that excessive accumulation of free and active 5-HT might lead to subsequent increases in SERT expression. When SERT expression increased within the first and second weeks, the 5-HT content of the affected site was accordingly excessively reuptaken by the overexpressed SERT. The lower levels of 5-HT then led to lower levels of SERT expression in the third week. We presume that the increases of SERT mRNA and SERT-P in the fourth week were associated with three aspects. Firstly, the accumulation of 5-HT content stimulates the upregulation of SERT mRNA and SERT-P expression. Secondly, double-diluted LGG-s1 might exhibit a high performance–effect upregulation of SERT expression when there is a higher accumulation of 5-HT. Thirdly, when the SERT mRNA levels
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have biased the results of the study. We will further investigate how and through what signal transduction pathway LGG-s1 upregulates SERT mRNA and SERTP expression. On the basis of our findings, we will do additional experiments using an IBS animal model to verify the impacts of LGG-s1 on IBS. In conclusion, we found that LGG-s1 upregulates SERT expression in HT-29 cells and mice intestinal tissues. By LGG-s1 increasing SERT expression levels and consequently decreasing 5-HT levels, a potential mechanism is provided by which probiotics can be used for therapy in IBS patients.
as p40 and p75, might act as regulative factors. Some might upregulate SERT mRNA and SERT-P expression, while others might downregulate SERT mRNA and SERT-P expression. In this study, we demonstrated that LGG-s1 upregulates SERT mRNA and SERT-P levels, which might contribute to the mechanisms by which probiotics relieve IBS symptoms. Kerckhoffs et al.45 indicated that SERT mRNA expression is reduced in patients with IBS syndrome of the large intestine. In addition, our previous research indicated that patients with the S/S and L/S genotypes have low SERT expression levels that lead to D-IBS.8 Thus, LGG-s1 can be used for IBS patients with lower SERT mRNA and SERT-P expression to exhibit diarrhea-predominant symptoms. Epidermal growth factor (EGF), an important 53amino acid polypeptide and growth hormone secreted by the salivary glands, liver, pancreas, kidneys, and intestine,46 upregulates SERT expression in human intestinal tissues through the EGF receptor (R).47 Cui et al.48 indicated that EGF’s upregulating of SERT is associated with visceral hypersensitivity in IBS. One study suggested that p40 prevents cytokine-induced apoptosis in intestinal epithelial cells through an EGFRdependent mechanism.49 We speculate that LGG-s1 upregulates through the EGFR signaling pathway, but this needs to be verified by additional research. There were some limitations to this study. Firstly, this was a small-sample preliminary experiment, and IBS models were not be used. All these factors might
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FUNDING This study was fully supported by a grant from the National Natural Science Foundation of China (30871151 and 81300272).
DISCLOSURE The authors have stated that they have no conflicts of interest associated with this study.
AUTHOR CONTRIBUTION YMW and XZG contributed equally to the manuscript; YMW, XZG, TW, HLC and BMW designed the study and performed the research; WQW and BLW contributed essential laboratory technical instruction. The final version of this article was professionally edited by BMW.
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patients with irritable bowel syndrome. Gastroenterology 2011; 141: 1782–91. Jeffery IB, O’Toole PW, Ohman L, Claesson MJ, Deane J, Quigley EM, Simren M. An irritable bowel syndrome subtype defined by species-specific alterations in faecal microbiota. Gut 2012; 61: 997–1006. Ko SJ, Han G, Kim SK, Seo JG, Chung WS, Ryu B, Kim J, Yeo I et al. Effect of Korean herbal medicine combined with a probiotic mixture on diarrheadominant irritable bowel syndrome: a double-blind, randomized, placebocontrolled trial. Evid Based Complement Alternat Med 2013; 2013: 824605. Lorenzo-Zuniga V, Llop E, Suarez C, Alvarez B, Abreu L, Espadaler J, Serra J. I.31, a new combination of probiotics, improves irritable bowel syndrome-related quality of life. World J Gastroenterol 2014; 20: 8709–16. Aggarwal S, Upadhyay A, Shah D, Teotia N, Agarwal A, Jaiswal V. Lactobacillus GG for treatment of acute childhood diarrhoea: an open labelled, randomized controlled trial. Indian J Med Res 2014; 139: 379–85. Francavilla R, Miniello V, Magista AM, De Canio A, Bucci N, Gagliardi F, Lionetti E, Castellaneta S et al. A randomized controlled trial of Lactobacillus GG in children with functional abdominal pain. Pediatrics 2010; 126: e1445–52. Hosseini A, Nikfar S, Abdollahi M. Are probiotics effective in management of irritable bowel syndrome? Arch Med Sci 2012; 8: 403–5. Whelan K, Quigley EM. Probiotics in the management of irritable bowel syndrome and inflammatory bowel disease. Curr Opin Gastroenterol 2013; 29: 184–9. Hosseini A, Nikfar S, Abdollahi M. Probiotics use to treat irritable bowel syndrome. Expert Opin Biol Ther 2012; 12: 1323–34. Clarke G, Cryan JF, Dinan TG, Quigley EM. Review article: probiotics for the treatment of irritable bowel syndrome–focus on lactic acid bacteria. Aliment Pharmacol Ther 2012; 35: 403–13. Rogers NJ, Mousa SA. The shortcomings of clinical trials assessing the efficacy of probiotics in irritable bowel syndrome. J Altern Complement Med 2012; 18: 112–9. Nobaek S, Johansson ML, Molin G, Ahrne S, Jeppsson B. Alteration of
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intestinal microflora is associated with reduction in abdominal bloating and pain in patients with irritable bowel syndrome. Am J Gastroenterol 2000; 95: 1231–8. Kajander K, Myllyluoma E, RajilicStojanovic M, Kyr€ onpalo S, Rasmussen M, J€arvenp€a€a S, Zoetendal EG, de Vos WM et al. Clinical trial: multispecies probiotic supplementation alleviates the symptoms of irritable bowel syndrome and stabilizes intestinal microbiota. Aliment Pharmacol Ther 2008; 27: 48–57. Lyra A, Krogius-Kurikka L, Nikkila J, Malinen E, Kajander K, Kurikka K, Korpela R, Palva A. Effect of a multispecies probiotic supplement on quantity of irritable bowel syndrome-related intestinal microbial phylotypes. BMC Gastroenterol 2010; 10: 110. O’Mahony L, McCarthy J, Kelly P, Hurley G, Luo F, Chen K, O’Sullivan GC, Kiely B et al. Lactobacillus and bifidobacterium in irritable bowel syndrome: symptom responses and relationship to cytokine profiles. Gastroenterology 2005; 128: 541–51. Rousseaux C, Thuru X, Gelot A, Barnich N, Neut C, Dubuquoy L, Dubuquoy C, Merour E et al. Lactobacillus acidophilus modulates intestinal pain and induces opioid and cannabinoid receptors. Nat Med 2007; 13: 35–7. Kerckhoffs AP, Ter Linde JJ, Akkermans LM, Samsom M. Trypsinogen IV, serotonin transporter transcript levels and serotonin content are increased in small intestine of irritable bowel syndrome patients. Neurogastroenterol Motil 2008; 20: 900–7. Esmaili A, Nazir SF, Borthakur A, Yu D, Turner JR, Saksena S, Singla A, Hecht GA et al. Enteropathogenic E. coli infection inhibits intestinal serotonintransporter (SERT) function and expression. Gastroenterology 2009; 137: 2074–83. Iceta R, Aramayona JJ, Mesonero JE, Alcalde AI. Regulation of the human serotonin transporter mediated by long-term action of serotonin in Caco-2 cells. Acta Physiol (Oxf) 2008; 193: 57–65. Kerckhoffs AP, ter Linde JJ, Akkermans LM, Samsom M. SERT and TPH-1 mRNA expression are reduced in irritable bowel syndrome patients regardless of visceral sensitivity state in large intestine. Am J Physiol Gastrointest Liver Physiol 2012; 302: G1053–60.
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46 Carpenter G, Cohen S. Epidermal growth factor. J Biol Chem 1990; 265: 7709–12. 47 Gill RK, Anbazhagan AN, Esmaili A, Kumar A, Nazir S, Malakooti J, Alrefai WA, Saksena S. Epidermal growth factor upregulates serotonin transporter in human intestinal tissues via transcriptional mechanisms.
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Am J Physiol Gastrointest Liver Physiol 2011; 300: G627–36. 48 Cui XF, Zhou WM, Yang Y, Zhou J, Li XL, Lin L, Zhang HJ. Epidermal growth factor upregulates serotonin transporter and its association with visceral hypersensitivity in irritable bowel syndrome. World J Gastroenterol 2014; 20: 13521–9.
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doi: 10.1111/nmo.12615
Lactobacillus rhamnosus GG supernatant upregulates serotonin transporter expression in intestinal epithelial cells and mice intestinal tissues Y. M. WANG ,* X. Z. GE ,* W. Q. WANG ,* T. WANG ,* H. L. CAO ,* B. L. WANG †
& B. M. WANG *
*Department of Gastroenterology and Hepatology, Tianjin Medical University General Hospital, Tianjin, China †Key Research Laboratory of Hormone and Development Affiliated to the Ministry of Health, Institute of Endocrinology, Tianjin Medical University, Tianjin, China
Key Messages
• Lactobacillus rhamnosus GG (LGG) is one of the best-studied Lactobacillus strains in clinical trials, which can effectively reduce the incidence of diarrhea without side effects.
• SERT mRNA and SERT-P levels in LGG-s treated HT-29, Caco-2 cells, and intestinal epithelial cells were higher than those in the control.
• LGG-s can upregulate SERT mRNA and SERT-P levels in intestinal epithelial cells and mice intestinal tissues. • LGG-s might improve intestinal motility and gastrointestinal sensation by regulating SERT expression is speculative.
Abstract Background The role that probiotics play in relieving irritable bowel syndrome (IBS) has been demonstrated; however, the mechanism by which IBS is affected remains unclear. In this study, serotonin transporter (SERT) mRNA and serotonin transporter protein (SERT-P) levels in HT-29, Caco-2 cells, and mice intestinal tissues were examined after treatment with Lactobacillus rhamnosus GG supernatant (LGG-s). Methods HT-29 and Caco-2 cells were treated with different concentrations of LGG-s for 12 and 24 h and C57BL/6 mice received supplements of different concentrations for 4 weeks. SERT mRNA and SERT-P levels were detected by real-time PCR and Western blotting. Key Results SERT mRNA and SERTP levels in HT-29 and Caco-2 cells were higher than those in the control 24 h after treatment. Undiluted
LGG-s upregulated SERT mRNA levels by 9.4-fold in the first week, which dropped in the second week. The double-diluted LGG-s upregulated SERT mRNA by 2.07-fold in the first week; levels dropped to 1.75-fold within the second week and under base expression levels by the third week, while they again climbed to 1.56-fold in the fourth week. The triple-diluted LGG-s could not upregulate SERT mRNA expression until the end of the fourth week. The SERT-P levels in the double-diluted LGG-s group were higher than that in the control but fluctuated with time. SERT-P levels in the triple-diluted LGG-s were higher than that in the control in the last 2 weeks and increased with time. Conclusions & Inferences LGG-s can upregulate SERT mRNA and SERT-P levels in intestinal epithelial cells and mice intestinal tissues. Keywords serotonin transporter, SERT mRNA, SERT protein, Lactobacillus rhamnosus supernatant, intestinal tissues.
Address for Correspondence B. M. Wang, Department of Gastroenterology and Hepatology, Tianjin Medical University Hospital, No. 154, Anshan Road, District, Tianjin 300052, China. Tel: +86 02260362608; fax: +86 02260362608; e-mail: [email protected] Received: 28 December 2014 Accepted for publication: 12 May 2015
© 2015 John Wiley & Sons Ltd
Abbreviations: 5-HT, 5-hydroxytryptamine; 5-HTTL PR, 5-HTT-linked polymorphic region; 5-HTT, serotonin transporter gene; C-IBS, IBS with constipation; D-IBS, IBS with diarrhea; GAPDH, glyceraldehyde-3phosphate dehydrogenase; IBS, irritable bowel
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is speculative. The purpose of this study was to investigate the effects of LGG-s on the expression of SERT mRNA and SERT protein (SERT-P).
syndrome; LGG-s, Lactobacillus rhamnosus GG supernatant; PI-IBS, postinfectious IBS; SERT-P, SERT protein; SERT, serotonin transporter.
MATERIALS AND METHODS INTRODUCTION Bacterial culture, LGG-s
Serotonin transporter (SERT) is a universally existing cross-membrane transport protein that plays a key role in 5-hydroxytryptamine (5-HT) reuptake. The human SERT gene (5-HTT) is located on chromosome 17q11, named solute carrier family 6 member 4, which is composed of 14–15 exons.1 The 5-HTT-linked polymorphic region (5-HTTLPR) is located upstream of the transcription start site, which plays a key role in the etiology of irritable bowel syndrome (IBS) by modulating the transcriptional activity of the 5-HTT.1 5-HT is an important gastrointestinal hormone that modulates intestinal fluid secretion, gut motility, and gastrointestinal sensation.2–5 Serum 5-HT levels decrease in patients with IBS with constipation (C-IBS) and increase in patients with IBS with diarrhea (D-IBS),6 but is inactivated after reuptake by SERT in intestinal or nerve cells. Downregulation of SERT is implicated in the pathophysiology of various functional gut disorders. Faure et al.7 found that SERT mRNA was lower in children with IBS than in the control. In our previous study, we found that the S/S genotype results in lower SERT expression and causes 5-HT accumulation at the affected site. Ordinarily, 5-HT can activate the intestinal functions of motion and secretion, which are often related to D-IBS.8 Chen et al.9 found that transgenic mice with targeted deletion of SERT frequently exhibit diarrhea interspersed with transient constipation. Lactobacillus rhamnosus GG (LGG) is one of the best-studied Lactobacillus strains in clinical trials. Oksanen et al.10 found that LGG can effectively reduce the incidence of diarrhea without side effects. LGG also has been shown to induce remission and prevent recurrence of IBD in humans11 and in nonhuman colitis models.12 Yan et al.13 purified two soluble proteins from LGG supernatant (LGG-s) and demonstrated that LGG-s and soluble proteins produced by LGG-s activated Akt, inhibited cytokineinduced epithelial cell apoptosis, and promoted cell growth in human and mouse colon epithelial cells and cultured mouse colon explants. The role of probiotics on IBS has been proposed over the past decade and the suggestion that probiotics can relieve IBS symptoms has been confirmed by many studies.14,15 Whether LGG-s improves intestinal motility and gastrointestinal sensation by regulating SERT expression
LGG (ATCC 53103 and CGMCC1 3724) were obtained from the China General Microbiological Culture Collection Center. LGG was incubated in Lactobacillus MRS broth at 37 °C for 24 h, diluted in MRS broth, and incubated again at 37 °C to reach log phase with the density determined as 0.5 at A600. The culture suspension was then centrifuged at 5000 g for 10 min. The supernatant was removed and filtered through 0.22-lm filters.13
Cell culture and cell treatment The human colonic epithelial carcinoma cell line, HT-29, was grown in 1640 media supplemented with 10% fetal bovine serum at 37 °C. The Caco-2 cell line, a continuous line of heterogeneous human epithelial colorectal adenocarcinoma cells, were grown in DMEM media supplemented with 10% fetal bovine serum and 1.0% nonessential amino acid at 37 °C. Cells were serum starved (0.5%) at 37 °C for approximately 24 h before the experiments and then treated with LGG-s (supernatant-to-cell media ratios: 1 : 100, 1 : 20, and 1 : 10) for 12 and 24 h. The control was treated with MRS broth.
Animal studies Forty-eight female C57BL/6N mice (5–8 weeks old) were obtained from Vital River Laboratories Animal Technology Co., Ltd., Beijing, China. Four groups of mice were used, one designated as the control group (n = 12) and the other three as the experimental groups (n = 12 for each group). The mice were kept in cages in a room at 22 °C with a 12 : 12-h light/dark cycle and had free access to a normal mouse-chow diet. The mice of the control group were fed 1.0 mL/day MRS broth through a gavage needle; the mice of the other three groups were given 1.0 mL/day double-diluted, triple-diluted, or undiluted LGG-s through a gavage needle. Mice were maintained on the treatments for 1.0, 2.0, 3.0, and 4.0 weeks during the experiment. Three mice of each group were sacrificed and the colons were removed for later use each week. The protocol was approved by the Animal Use and Care Committee of Tianjin Medical University.
Real-time polymerase chain reaction Total RNA was extracted from HT-29 cells, Caco-2 cells, and 50 mg colon tissue from each of the sacrificed mice with Trizol according to the manufacturer’s instructions (Life, Hilden, Germany). First-strand cDNA was synthesized using the iScriptcDNA synthesis kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Quantitative real-time (QRT) PCR was performed on an ABI One plus setup PCR thermocycler; RT-PCR was performed on the SYBR Green PCR Master Mix (Roche Applied Science, Mannheim, Germany). The PCR were set up in a volume of 20 lL containing 1.0 lL cDNA, 10 lL 29 iQ SYBR Green Supermix (Roche Applied Science), and 0.6 lL each of the forward and reverse primers. The sequences of forward and reverse primers are
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(ATCC53103). No significant differences were found for SERT mRNA and SERT-P expression in HT-29 cells in any of the groups treated for 12 h (p > 0.05). Both LGG-s (ATCC53103) and LGG-s (CGMMCC1.3724) can upregulate SERT mRNA and SERT-P expression; however, the effects of LGG-s (ATCC53103) were significant, so LGG-s (ATCC53103) was chosen for further animal experiments (Figs. 1 and 2). Serotonin transporter mRNA levels in Caco-2 cells treated with 1 : 100, 1 : 20, and 1 : 10 concentrations of LGG-s (ATCC53103) for 24 h were 1.60-, 1.40-, and 1.66-fold higher than that in the control group, respectively (p = 0.03, p = 0.022, p = 0.000). The increase in SERT mRNA levels and SERT-P levels in Caco-2 cells in response to 24-h incubation in different concentrations of LGG-s (ATCC53103) were similar to that of HT29 cells, although the effect is modest compared to that of HT-29 cells at 1 : 10 dilution. In addition,
listed in Table 1. For normalization purposes, glyceraldehyde-3phosphate dehydrogenase (GAPDH) was measured as an internal control. Relative mRNA expression was calculated using the 2DDCt method.
Western blotting Proteins were extracted from HT-29 cells, Caco-2 cells, and colonic tissues, and levels were quantified using a BCA protein concentration assay kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China). The protein samples were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrophoretically transferred to polyvinylidene difluoride membranes. The membranes were incubated with SERT antibody ab181034, b-actin (Abcam Biotech Company, Cambridge, UK) and horseradish peroxidase-labeled rabbit antirabbit IgG (EarthOx Life Sciences, Millbrae, CA, USA). After incubating with secondary antibodies, the immunoreactive bands were visualized using an enhanced chemiluminescence method (GE Healthcare, Buckinghamshire, UK) and exposure to X-ray film. b-Actin was used as an inner control.
Statistical analyses A
Statistical analysis was carried out using SPSS 19.0 for Windows (SPSS, Chicago, IL, USA). One-way ANOVA and post hoc tests (Dunnett’s T3 and Dunnett’s C) were used to compare the values of RT-PCR. For all analyses, p = 0.05 was defined as statistically significance.
RESULTS Effects of LGG-s on SERT mRNA and protein expression in HT-29 and Caco-2 cells Serotonin transporter mRNA levels in HT-29 cells treated with 1 : 100, 1 : 20, and 1 : 10 concentrations of LGG-s (ATCC53103) for 24 h were 1.46-, 1.47-, and 2.67-fold higher than that in the control group, respectively (p = 0.01, p = 0.01, p = 0.000). As with SERT mRNA levels, SERT-P levels were significantly increased in response to 24-h incubation of HT-29 cells at different concentrations of LGG-s
B
Table 1 Primer sequences for RT-PCR Gene
Sequences (forward/reverse 50 –30 )
hSERT
Sense: 50 -AAT GGG TAC TCA GCA GTT CC-30 Antisense: 50 -CCA CAG CAT AGC CAA TCA C-30 Sense: 50 -ACA GCA ACT CCC ATT CTT-30 Antisense: 50 -TCC AGG GTT TCT TAC TCC-30 Sense: 50 -TGGGCGCTCTACTACCTCAT-30 Antisense: 50 -ATGTTGTCCTGGGCGAAGTA-30 Sense: 50 -AGGTCGGTGTGAACGGATTTG-30 Antisense: 50 -TGTAGACCATGTAGTTGAGGTCA-30
hGAPDH mSERT mGAPDH
Figure 1 Effects of LGG-s on SERT mRNA expression in HT-29 cells. (A) SERT mRNA levels in HT-29 cells treated with different concentration LGG-s for 12 h. (B) SERT mRNA levels in HT-29 cells treated with different concentration LGG-s for 24 h.
hSERT, human serotonin transporter; hGAPDH, human glyceraldehyde-3-phosphate dehydrogenase; mSERT, mouse serotonin transporter; mGAPDH, mouse glyceraldehyde-3-phosphate dehydrogenase.
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A
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D Figure 2 Effects of LGG-s on SERT protein expression in HT-29 cells. (A) SERT protein levels in HT-29 cells treated with different concentration of LGG-s for 12 h. (B) SERT protein levels in HT-29 cells treated with different concentration of LGG-s for 24 h. (C) Quantitative analysis of the protein levels of SERT in HT-29 cells treated with different concentration of LGG-s for 12 h. (D) Quantitative analysis of the protein levels of SERT in HT-29 cells treated with different concentration of LGG-s for 24 h. Error bars represent the mean SD.
expression levels decreased in the third week and were lower than that of control (p = 0.002) but increased slightly in the fourth week (p > 0.05). The doublediluted LGG-s1 upregulated SERT mRNA by 2.07-fold by the end of the first week but levels slowly dropped to 1.75-fold within the second week and to less than the base expression levels during the third week (p = 0.000, p = 0.006, p = 0.008, respectively). By the fourth week, SERT mRNA levels were 1.56-fold higher than that of the control in the double-diluted LGG-s group (p = 0.007). The triple-diluted LGG-s1 did not upregulate SERT mRNA expression within the first to third weeks, but did slightly upregulate SERT mRNA expression (p = 0.004, p < 0.05) to nearly equal that of the double-diluted LGG-s1 by the end of the fourth week (Fig. 5).
unlike HT-29 cells, there was no change in effects with different concentrations of LGG-s. No significant differences were found in SERT mRNA and SERT-P expression in Caco-2 cells in any of the groups treated for 12 h (p > 0.05). LGG-s (CGMMCC1.3724) cannot upregulate SERT mRNA and SERT-P expression in Caco-2 cells (Figs. 3 and 4).
Effects of LGG-s1 on SERT mRNA and SERT-P expression in mice intestinal tissues Undiluted LGG-s1 significantly upregulated SERT mRNA expression levels by 9.4-fold by the end of the first week (p = 0.000, p < 0.05), but levels decreased rapidly to the base expression levels of the control by the end of the second week. The SERT mRNA
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cation.16–22 Irritable bowel syndrome can be subtyped according to the predominant bowel-movement type as C-IBS, D-IBS, and IBS with alternating symptoms of both constipation and diarrhea. Postinfectious IBS (PI-IBS) accounts for 7.0–30% of IBS in patients and often exhibits characteristics of diarrhea, which has a history of acute gastrointestinal infection.23 The mechanisms underlying the development of PI-IBS are believed to be associated with changes in intestinal permeability, persistent lowgrade inflammation, and dysregulation of gut microbiota.24–27 Probiotics and prebiotics have been proved as effective clinical therapies for D-IBS and PI-IBS28,29 and LGG has been one of the most widely studied probiotic strains that is used in a variety of commercially available probiotic products. One open-labeled, randomized, controlled trial enrolled 200 children who had acute, watery diarrhea and demonstrated that the use of LGG resulted in faster improvement in stool consistency compared to that of the control group,30 demonstrating that LGG can significantly reduce the frequency and severity of abdominal pain in children with IBS.31 In recent years, there have been numerous publications that have confirmed the effects of LGG on IBS.32–36 The possible mechanisms by which LGG works on IBS are as follows: regulating intestinal flora,37–39 improving intestinal barrier function31 normalizing levels of interleukins (such as IL-10/IL-2),40 and increasing the visceral pain threshold, and reducing colonic epithelial cells, which carry M opioid receptors and cannabinoid receptors.41 Whether and how probiotics regulate SERT expression remains unclear. In our present study, LGG-s1 upregulated SERT expression in HT-29 cells and Caco-2 cells, which was significantly time dependent. LGG-s diluted by 1 : 100, 1 : 20, and 1 : 10 did not increase either SERT mRNA or SERT-P levels in HT-29 cells and Caco-2 cells when treated for 12 h; however, SERT mRNA and SERT-P levels did increase when treated for 24 h. As to LGG-s2, the upregulation effect on HT-29 was slight with no statistically significance and there was no effect on Caco-2 cells. We believe that there might be some differences in LGG-s1 and LGG-s2, and will perform some screen works on the differential proteins expressed between LGG-s1 and LGG-s2. The results suggest that LGG-s1 can upregulate SERT expression in HT-29 cells and Caco-2 cells; by doing so, LGG-s1 might play a key role in intestinal motility by regulating SERT expression in intestinal tissues. The mouse experiment was designed to verify whether LGG-s1 can upregulate SERT expression in the intestine.
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Figure 3 Effects of LGG-s on SERT mRNA expression in Caco-2 cells. (A) SERT mRNA levels in Caco-2 cells treated with different concentration LGG-s for 12 h. (B) SERT mRNA levels in Caco-2 cells treated with different concentration LGG-s for 24 h. Results are means SE; *significant.
The SERT-P levels in the undiluted LGG-s1 group were nearly equal to that of the control within 1– 4 weeks. The SERT-P levels of the double-diluted LGG-s1 group were higher than that of the control, and gradually increased in the first, second, and fourth weeks but decreased in the third week. The SERT-P levels of the triple-diluted LGG-s1 were higher than that of the control in the last 2 weeks and increased over time (Fig. 6).
DISCUSSION Irritable bowel syndrome is one of the most frequent lower gastrointestinal tract disorders resulting in abdominal pain or discomfort accompanied by altered bowel habits, including alterations in stool frequency and/or form or appearance, especially disordered defe-
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Figure 4 Effects of LGG-s on SERT protein expression in Caco-2 cells. (A) Protein levels of SERT in Caco-2 cells treated for 12 h analyzed by Western blot. (B) Quantitative analysis of the protein levels of SERT treated with different concentration of LGG-s in Caco-2 cells treated for 12 h. (C) Protein levels of SERT in Caco-2 cells treated for 24 h analyzed by Western blot. (D) Quantitative analysis of the protein levels of SERT treated with different concentration of LGG-s in Caco-2 cells treated for 24 h. LGG-s1, LGG-s(ATCC53103), LGG-s2, LGG-s(CGMCC1.3724). Results are means SE; *significant.
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D Figure 5 Effects of LGG-s1 on SERT mRNA expression in mice intestinal epithelial cells. (A) SERT mRNA levels in mice intestinal epithelial cells at the first week. (B) SERT mRNA levels in mice intestinal epithelial cells at the second week. (C) SERT mRNA levels in mice intestinal epithelial cells at the third week. (D) SERT mRNA levels in mice intestinal epithelial cells at the fourth week.
and lipids as well as other small molecules. Studies by Yan et al.13 showed that p40 and p75 in LGG-s activate Akt, inhibit cytokine-induced epithelial cell apoptosis, and promote cell growth in human and mouse colon
Undiluted LGG-s1 was found to significantly increase SERT mRNA expression in mice intestinal tissues, while SERT-P levels were no different. LGG-s is a relatively complex system with a series of proteins
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Figure 6 Effects of LGG-s1 on SERT-P expression in mice intestinal epithelial cells. (A) Form up to down represents the SERT-P level at the first, second, third and fourth week analyzed by Western blot, respectively. Lane a: control; Lane b: triple dilution LGG-s1; Lane c: double dilution LGG-s1; Lane d: undiluted LGG-s1. (B) Form up to down represents the quantitative analysis of SERT-P level at the first, second, third and fourth week analyzed by Western blot, respectively. Results are mean SE; *significant.
increased to about 2.07-fold, they peaked, after which the complex system of SERT mRNA expression might be readjusted by the interaction of 5-HT and SERT-P. Previous studies have indicated that the accumulation of 5-HT inhibited the expression of SERT in Caco-2 cells.43,44 We presumed that there might exist some differences between in vivo and in vitro studies. The regulation mechanism in vivo might be more complicated, which needs to be explored with further research. For the triple-diluted LGG-s1 group, no marked effects on SERT mRNA expression were found within the first and second weeks, but levels increased the fourth week. SERT-P was higher than that of the control in the third and fourth weeks. The presumption from this is that the low concentration of LGG-s1 could participate in the same upregulating action as the high concentration of LGG-s1 when the key components accumulate to an operative concentration over time. LGG-s1 is a relatively complex system that contains a series of proteins and other molecules. We speculate that some macromolecular substances, such
epithelial cells and cultured mouse colon explants. We presumed that there might be some substances in LGG-s that can balance SERT-P translation. A concern with the double-diluted LGG-s group was that SERT mRNA and SERT-P levels increased in the first and second weeks, decreased the third week, and increased again the fourth week. Kerckhoffs et al.42 indicated that excessive accumulation of free and active 5-HT might lead to subsequent increases in SERT expression. When SERT expression increased within the first and second weeks, the 5-HT content of the affected site was accordingly excessively reuptaken by the overexpressed SERT. The lower levels of 5-HT then led to lower levels of SERT expression in the third week. We presume that the increases of SERT mRNA and SERT-P in the fourth week were associated with three aspects. Firstly, the accumulation of 5-HT content stimulates the upregulation of SERT mRNA and SERT-P expression. Secondly, double-diluted LGG-s1 might exhibit a high performance–effect upregulation of SERT expression when there is a higher accumulation of 5-HT. Thirdly, when the SERT mRNA levels
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have biased the results of the study. We will further investigate how and through what signal transduction pathway LGG-s1 upregulates SERT mRNA and SERTP expression. On the basis of our findings, we will do additional experiments using an IBS animal model to verify the impacts of LGG-s1 on IBS. In conclusion, we found that LGG-s1 upregulates SERT expression in HT-29 cells and mice intestinal tissues. By LGG-s1 increasing SERT expression levels and consequently decreasing 5-HT levels, a potential mechanism is provided by which probiotics can be used for therapy in IBS patients.
as p40 and p75, might act as regulative factors. Some might upregulate SERT mRNA and SERT-P expression, while others might downregulate SERT mRNA and SERT-P expression. In this study, we demonstrated that LGG-s1 upregulates SERT mRNA and SERT-P levels, which might contribute to the mechanisms by which probiotics relieve IBS symptoms. Kerckhoffs et al.45 indicated that SERT mRNA expression is reduced in patients with IBS syndrome of the large intestine. In addition, our previous research indicated that patients with the S/S and L/S genotypes have low SERT expression levels that lead to D-IBS.8 Thus, LGG-s1 can be used for IBS patients with lower SERT mRNA and SERT-P expression to exhibit diarrhea-predominant symptoms. Epidermal growth factor (EGF), an important 53amino acid polypeptide and growth hormone secreted by the salivary glands, liver, pancreas, kidneys, and intestine,46 upregulates SERT expression in human intestinal tissues through the EGF receptor (R).47 Cui et al.48 indicated that EGF’s upregulating of SERT is associated with visceral hypersensitivity in IBS. One study suggested that p40 prevents cytokine-induced apoptosis in intestinal epithelial cells through an EGFRdependent mechanism.49 We speculate that LGG-s1 upregulates through the EGFR signaling pathway, but this needs to be verified by additional research. There were some limitations to this study. Firstly, this was a small-sample preliminary experiment, and IBS models were not be used. All these factors might
REFERENCES 1 Lesch KP, Balling U, Gross J, Strauss K, Wolozin BL, Murphy DL, Riederer P. Organization of the human serotonin transporter gene. J Neural Transm Gen Sect 1994; 95: 157–62. 2 Barbara G, Cremon C. Serine proteases: new players in diarrhoea-predominant irritable bowel syndrome. Gut 2008; 57: 1035–7. 3 Gershon MD. Serotonin and its implication for the management of irritable bowel syndrome. Rev Gastroenterol Disord 2003; 3 (Suppl. 2): S25–34. 4 Gershon MD, Liu MT. Serotonin and neuroprotection in functional bowel disorders. Neurogastroenterol Motil 2007; 19 (Suppl. 2): 19–24. 5 Gershon MD, Tack J. The serotonin signaling system: from basic understanding to drug development for functional GI disorders. Gastroenterology 2007; 132: 397–414.
FUNDING This study was fully supported by a grant from the National Natural Science Foundation of China (30871151 and 81300272).
DISCLOSURE The authors have stated that they have no conflicts of interest associated with this study.
AUTHOR CONTRIBUTION YMW and XZG contributed equally to the manuscript; YMW, XZG, TW, HLC and BMW designed the study and performed the research; WQW and BLW contributed essential laboratory technical instruction. The final version of this article was professionally edited by BMW.
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