Lactobacillus plantarum ZS2058 produces CLA to ameliorate DSS-induced acute colitis in mice

Abstract

Lactobacillus plantarum ZS2058 is an efficient producer of conjugated linoleic acid (CLA) in vitro. To investigate whether L. plantarum ZS2058 produces CLA in vivo and exerts beneficial effects through CLA, an acute colitis model was induced with dextran sodium sulfate (DSS) in C57BL6/J mice. The mice were treated with L. plantarum ZS2058, L. plantarum ST-III, CLA, or vehicle 7 days before modeling until the end of modeling which lasts for seven days. Compared to L. plantarum ST-III, L. plantarum ZS2058 significantly inhibited the increase of the disease activity index (DAI), colon shortening and myeloperoxidase activity in colitic mice. L. plantarum ZS2058 treatment improved the histological damage, protected the colonic mucous layer integrity and significantly attenuated the expression of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6), while up-regulated the expression of colonic anti-inflammatory cytokine IL-10 and nuclear receptor PPARγ. Furthermore, colonic CLA concentrations were significantly increased in response to L. plantarum ZS2058 treatment, which demonstrates that L. plantarum ZS2058 prevents colitis via producing CLA locally.

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Introduction

Inflammatory bowel disease (IBD) is a relapsing, chronic, mucosal immune-associated illness, comprising two major disorders, Crohn's disease (CD) and ulcerative colitis (UC); however, the exact pathogenesis of IBD remains unclear.¹ Existing treatments for IBD, such as antibiotics, corticosteroids, and immuno-modulators, are moderately successful, but they present strong side effects; hence, they are not suitable for long term therapy.^2–5 Therefore, it is necessary and important to find novel treatments. Probiotics have received more attention in recent years as a new safe and effective therapy against IBD via different mechanism.^6–9 However, numerous studies have suggested that the therapeutic effects of probiotics on IBD were strain-dependent.^7,8,10–13

Certain probiotics or gut microorganisms can produce many different metabolites associated with health, such as functional lipids, which include short chain fatty acids (SCFA) like butyrate, acetate and propionate, and polyunsaturated fatty acids (PUFA). The beneficial PUFA from gut microbiota regulate mucosal immunity and improve intestine health,^6,14,15 especially conjugated linoleic acid (CLA) and conjugated linolenic acid (CLNA) isomers.^16–18 CLA is a class of PUFA that contains a conjugated double bond. CLA has a strong ability to regulate immunity, and this effect has already been demonstrated in a great variety of inflammation-related disorders, including IBD.^14,19,20 cis9, trans11-CLA (c9, t11-CLA) and trans10, cis12-CLA (t10, c12-CLA) are the major beneficial CLA isomers.²¹ One of the mechanisms underlying the various biologically beneficial effects of CLA is the fact that they modulate the expression or activity of peroxisome proliferator-activated receptor gamma (PPARγ).^14,21–23 Compared to c9, t11-CLA, t10, c12-CLA has a weaker PPARγ activating ability.^24,25 However, the t10, c12-CLA isomer is thought to be specifically responsible for anti-obesity effects,^23,26 whereas c9, t11-CLA is more likely to be responsible for anti-inflammatory effects.²¹ In studies of immune-associated disorders such as IBD, CLA suppressed inappropriate colonic and uncontrolled intestinal mucosal inflammation through a PPARγ-dependent mechanism.^14,27 Trefoil factor family (TFF), which plays a role in mucus stabilization by interacting with or cross linking mucins to contribute to the gel layer,²⁸ could be activated by CLA through modulation of PPARγ in DSS-induced colitis in mice.²⁹

The ability of producing CLA occurred in many lactic acid bacteria including L. bulgaricus, L. plantarum, L. acidophilus, L. reuteri, B. breve, etc.^18,30 And there are some evidence indicate that probiotics could produce CLA in vivo.⁶

Therefore, we hypothesize that probiotics that are able to generate CLA might exert anti-inflammatory effects in vivo. To test this hypothesis, the effect of two similar Lactobacillus strains, L. plantarum ZS2058 and L. plantarum ST-III, was assessed in DSS-induced colitis in mice. L. plantarum ZS2058 is a high CLA producer isolated from sauerkraut, and the major CLA isomer produced by L. plantarum ZS2058 is c9, t11-CLA, whereas L. plantarum ST-III, also isolated from sauerkraut, is unable to synthesize CLA.^18,31

Materials and methods

Preparation and administration of lactobacilli

L. plantarum ZS2058 and L. plantarum ST-III were cultured anaerobically in de Man, Rogosa and Sharpe (MRS) medium at 37 °C for 16 h. Subsequently, the culture was centrifuged (7000 × g, 10 min at 4 °C), the cell pellets were collected and washed twice with phosphate buffer solution (PBS, pH 7.4), and then re-suspended at 1 × 10¹¹ CFU mL⁻¹ in 13% skim milk aqueous solution. One-milliliter aliquots were lyophilized and lactic acid bacteria powder was stored at 4 °C prior to use. Each mouse was inoculated intragastrically with 1 × 10⁹ CFU L. plantarum ZS2058, L. plantarum ST-III or vehicle (freeze-dried 13% wt/v skim milk in PBS) once daily from days 1 to 14.

Animals and treatment

Four-week-old male C57BL6/J mice were housed in the Animals Housing Unit of Jiangnan University at a room temperature of 23–25 °C with a 12 h light/dark cycle. The mice were group-housed in standard laboratory cages; standard laboratory chow and sterile water were provided ad libitum. All experimental protocols were approved by the Animal Ethics Committee of Jiangnan University, China, and were performed according to the ethical guidelines of the European Community guidelines (Directive 2010/63/EU).

The experimental protocols were as follows: forty C57BL6/J male mice were distribute into 5 groups (n = 8) randomly: control, DSS + vehicle, DSS + ZS2058, DSS + ST-III, DSS + CLA. Lactic acid bacteria or vehicle (freeze-dried 13% wt/v skim milk in PBS) was administered once a day by gavage from days 1 to 14. Experimental colitis was induced from days 7 to 14 with 2% DSS as described below. The CLA group was given standard laboratory chow with 1% (wt/wt) CLA (Qingdao PengYang Co., Ltd., Qingdao, China) and vehicle from days 1 to 14. The concentration of CLA was chosen based on the Bassaganya-Riera's research, and this concentration equivalent to an optimal therapeutic dosage of 45–80 mg per day per mouse.¹⁴

Induction and assessment of colitis

Acute colitis was induced by administering 2% dextran sodium sulfate (DSS) salt, molecular weight 36 000–50 000, (MP Biomedicals, Aurora, OH) to mice for 7 days.³² Briefly, DSS was added to drinking water to a concentration of 2% and mice were allowed to drink freely; DSS solution was replaced daily.

During DSS treatment, the disease activity index (DAI) was assessed daily based on a modified scoring system which was comprised of weight loss, stool consistency, and hematochezia,^33,34 occult blood in the feces was measured using an Occult Blood kit (Nanjing Jiancheng Co., Ltd., Nanjing, China). The scale standard is shown in Table 1.

Table 1 Standard for evaluation of the disease activity index (DAI)^a

Weight loss (%)	Stool consistency^b	Occult blood or gross bleeding	Score
a Disease activity index is a summation of the three parameters weight loss, stool consistency, and hematochezia.b Normal stool: well-shaped stool; loose stool: unformed pasty stool that does not stick to the anus; diarrhea: liquid stool that is attached to the anus.
0	Normal	Negative	0
1–5	Loose stool	Negative	1
6–10	Loose stool	Hemoccult positive	2
11–15	Diarrhea	Hemoccult positive	3
>15	Diarrhea	Gross bleeding	4

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At the end of the DSS challenge, the mice were euthanized and the colon was removed. Colon length was measured and colon contents was carefully collected. 1 cm distal colon segments were fixed in Carnoy's solution (ethanol : chloroform : acetic acid 6 : 3 : 1, vol/vol/vol) for 2 h at 4 °C, and then embedded in paraffin, sectioned (5 μm) and stained with H&E for histological examination. Colonic pathology slides were scanned by Pannoramic MIDI Digital Slide Scanner (3D-Histech Co., Ltd., Budapest, Hungary) and images were captured. The severity of colonic histological injury of each mouse (n = 8 mice per group) was scored using a modified scoring system taking into account the degree of inflammation, mucosal damage, crypt damage and range of pathological changes (Table 2).^33,35

Table 2 Standard for evaluation of histological injury

Inflammation	Mucosal injury	Crypt distortion	Area of lesions (%)	Score
None	None	None	None	0
Mild	Mucous layer	1/3	1–25	1
Moderate	Submucosa	2/3	26–50	2
Severe	Muscularis and serosa	100%	51–75	3
—	—	Entire crypt and surface epithelium destroyed	76–100	4

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Assessment of myeloperoxidase activity

The change in myeloperoxidase activity in the colon was assessed by a Myeloperoxidase Test Kit (Nanjing Jiancheng Co., Ltd., Nanjing, China) according to the manufacturer's instructions. The methods that prepare samples was as follows: briefly, frozen colons at −80 °C were weighed and cut into pieces in normal saline solution, then colonic tissue homogenate was prepared using Ultra-turrax T8 high-speed dispersion machine (IKA, Staufen, German).

Alcian blue staining

Gastrointestinal tract was protectively covered by mucous layer that major component is secretory mucin glycoproteins, and the MUC2 is the main mucin glycoprotein synthesized and secreted by goblet cells.^36–38 Additionally, mucin can be stained blue by alcian blue.³⁹ So, the extent of colonic mucous layer damage was investigated by performing alcian blue staining using a modified method of Steedman.³⁹ Briefly, Carnoy's solution-fixed paraffin-embedded colon tissue was sectioned (5 μm), then sections of each mouse were stained with alcian blue and nuclear fast red. Mucin and nuclei are dyed blue and reddish pink, respectively.

RNA extraction and quantitative real-time polymerase chain reaction

Total RNA was extracted from each colonic tissue sample using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. 1 μg total RNA was used to generate complementary DNA (cDNA) with the PrimeScript™ 1st Strand cDNA Synthesis Kit (Takara, Tokyo, Japan) based on the manufacturer's instructions. Quantitative real-time polymerase chain reaction was performed with the Bio-Rad CFX Connect™ Real-time System (Bio-Rad, Hercules, CA). The results were analyzed by the method of 2^−ΔΔCt. The PCR primer pairs used in the study are shown in ESI Table S1.†

Fatty acid analysis

Lipids were extracted from colonic contents, blood and liver samples with chloroform : methanol (2 : 1 vol/vol). Fatty acid methyl esters (FAMEs) were prepared using 2 mL 0.5 M sodium hydroxide methanol solution for 5 min at 100 °C, followed by 2 mL 14% (w/w) solution of boron trifluoride methanol (Sigma, St. Louis, MO) for 5 min at 100 °C. FAMEs were recovered with hexane and analyzed using a gas chromatograph (GC2010 plus, Shimadzu, Kyoto, Japan) fitted with a QP2010 ultra mass spectrometer (Shimadzu, Kyoto, Japan) using an Rtx-WAX column (30 m × 0.25 mm i.d. with 0.25 μm thickness) (Restek Corporation, Bellefonte, PA). The temperature programming for gas chromatography was as follows: the column was programmed at 40 °C for 5 min initially, and increased to 120 °C at a rate of 20 °C min⁻¹, then increased to 190 °C at a rate of 5 °C min⁻¹ and held for 5 min, then finally increased to 220 °C at a rate of 5 °C min⁻¹ and held for 17 min. Electron energy and ion source temperature of mass chromatography were 70 eV and 200 °C, respectively.

Statistical analysis

All data are presented as mean ± SEM of 8 animals per group. Statistical significance of the results was analyzed by one-way analysis of variance (ANOVA) followed by Bonferroni's post hoc test or Kruskal Wallis test with Dunn's post hoc test. A p-value <0.05 was regarded as significant. Statistical analyses were performed using SPSS 19.0 software (SPSS Inc, Chicago, IL) or GraphPad Prism 5 (GraphPad Software, Inc).

Results

L. plantarum ZS2058 alleviates DSS-induced colitis in mice

During DSS treatment, typical colitis-associated clinical symptoms appeared in mice that were challenged with 2% DSS and treated with vehicle; these mice presented the highest weight loss, diarrhea, gross bleeding, and DAI level among all the groups (Fig. 1A). L. plantarum ZS2058 and CLA alleviated the DSS-induced clinical symptoms significantly, but L. plantarum ST-III failed to prevent the onset of colitis (Fig. 1A). Colon length was measured (Fig. 1B) after mice were euthanized. L. plantarum ZS2058 prevented colon shortening induced by DSS to a degree similar to the CLA group, but the colon length of mice treated with L. plantarum ST-III was not statistically different from the DSS treated vehicle group.

Fig. 1 Lactobacillus plantarum ZS2058 alleviates the symptoms of DSS-induced colitis in mice. (A) Disease activity index (DAI), (B) colon length (C) histological examination, scale bars, 200 μm, (D) colonic histological injury, (E) myeloperoxidase activity. ^$$$: p < 0.001, compared to healthy control group; *: p < 0.05, **: p < 0.01, ***: p < 0.001, compared to DSS treated group; ^#: p < 0.05, ^##: p < 0.01, ^###: p < 0.001, compared to L. plantarum ZS2058 treated group. All data are presented as mean ± SEM (n = 8 mice per group).

Images from all the tissue sections were captured (Fig. 1C), and the histological injury score of each mouse was evaluated (Fig. 1D). According to our histopathological analysis, DSS-treated mice showed signs of intense inflammation: severe inflammatory cellular infiltration in the colonic mucosa, submucosal edema, loss and hyperplasia of crypts, and severe epithelial structure damage (Fig. 1C). By contrast, mice treated with L. plantarum ZS2058 and CLA present mild tissue damage and inflammatory behavior induced by DSS. However, histological injury in the L. plantarum ST-III treatment group remained severe (Fig. 1C).

Myeloperoxidase (MPO) activity of the colon reflects the degree of neutrophil infiltration.⁴⁰ Our results show that L. plantarum ZS2058, ST-III and CLA inhibited the increased MPO activity induced by DSS; overall, MPO activity in the L. plantarum ZS2058 group was lower than in the L. plantarum ST-III group, but there was no significant difference between the two groups (Fig. 1E).

L. plantarum ZS2058 regulates inflammatory cytokine expression in colonic tissue

The mRNA levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in colonic tissue samples were increased significantly upon DSS treatment. L. plantarum ZS2058 and CLA attenuated this increase, but L. plantarum ST-III failed to alter the mRNA level of TNF-α, IL-1β, and IL-6 (Fig. 2A–C) compared with vehicle. L. plantarum ZS2058 and CLA induced the up-regulation of colonic anti-inflammatory cytokine IL-10 mRNA expression significantly, furthermore, L. plantarum ZS2058 significantly up-regulated colonic PPARγ mRNA expression, CLA also the expression of PPARγ, but its influence differed slightly from that of DSS group; however, L. plantarum ST-III did not exhibit this property (Fig. 2D and E).

Fig. 2 Effects of Lactobacillus plantarum ZS2058 on TNF-α (A), IL-1β (B), IL-6 (C), IL-10 (D) and PPARγ (E) mRNA levels in colonic tissue. *: p < 0.05, **: p < 0.01, ***: p < 0.001. All data are presented as mean ± SEM (n = 8 mice per group).

L. plantarum ZS2058 protects the colonic mucous layer and epithelium structure

The extent of colonic mucous layer integrity was investigated via alcian blue staining (Fig. 3A); goblet cells of DSS-treated mice were almost entirely destroyed, and the colonic mucous layer on the colonic epithelium had disappeared. L. plantarum ZS2058 and CLA protected the goblet cells and the integrity of the colonic mucous layer (Fig. 3A). However, mice inoculated with L. plantarum ST-III exhibited fewer goblet cells and a heavily damaged mucous layer (Fig. 3A).

Fig. 3 Effects of Lactobacillus plantarum ZS2058 on the mucous layer and mRNA expression of TJ-related genes in colon. (A) Representative alcian blue staining pictures of each group, (B) mRNA expression of MUC2, (C) mRNA expression of E-cadherin 1, (D) mRNA expression of ZO-1, (E) mRNA expression of Claudin 3. *: p < 0.05, **: p < 0.01, ***: p < 0.001. All data are presented as mean ± SEM (n = 8 mice per group).

MUC2 (Mucin-2, which is secreted by the goblet cells and identified as the predominant secreted mucin glycoprotein³⁸), E-cadherin 1, ZO-1 (zonula occludens-1) and Claudin 3 mRNA expression in colonic tissue was assessed to investigate the influence of L. plantarum and CLA on the colonic physical barrier and epithelium structure at the transcriptional level. DSS significantly reduced the mRNA expression of MUC2, but treatment with L. plantarum ZS2058 and CLA significantly restored the mRNA expression of MUC2 (Fig. 3B). The relative mRNA expression of MUC2 in mice treated with L. plantarum ST-III was not significantly different form the vehicle group; it is noteworthy that this was consistent with our alcian blue staining results (Fig. 3A and B). E-cadherin 1, ZO-1 and Claudin 3 are tight junctions (TJ)-related genes which are important for maintaining the epithelial structure. Treatment with DSS down-regulated the mRNA expression of E-cadherin 1, ZO-1 and Claudin 3, but there were not significantly differences between the group DSS and vehicle. By contrast, treatment with L. plantarum ZS2058 and CLA alleviated the down-regulation of E-cadherin 1, ZO-1 and Claudin 3 mRNA expression to different extents; however, L. plantarum ST-III did not prevent the down-regulation of E-cadherin 1 and Claudin 3 mRNA expression induced by DSS colitis. L. plantarum ST-III only dramatically up-regulated the mRNA expression of ZO-1 (Fig. 3C–E).

L. plantarum ZS2058 treatment increased the CLA concentration in the colonic contents

CLA was analyzed in the colonic contents, and was significantly increased in the L. plantarum ZS2058 and CLA groups, whereas no significant statistical difference was found between the L. plantarum ST-III group and the vehicle group. Additionally, L. plantarum ZS2058 treatment significantly increased the CLA concentration in the colonic contents compared to L. plantarum ST-III (Fig. 4A). The CLA content in blood and liver was also analyzed (Fig. 4B and C). However, only CLA treatment significantly increased CLA levels in blood and liver samples (Fig. 4B and C).

Fig. 4 CLA concentration in the colonic contents, blood and liver. (A) Colonic contents (A), blood (B) and liver tissue (C). **: p < 0.01. All data are presented as mean ± SEM (n = 8 mice per group).

Discussion

The current IBD therapies are not suitable for long term treatment because of their adverse effects such as increased cancer susceptibility and infection.^41–45 Probiotics have attracted more attention as a new strategy against IBD due to their health-promoting benefits.⁴⁶ Therefore, among the numerous probiotics, it is necessary to find effective organisms that can fight IBD. Some lactic acid bacteria can metabolize a variety of bioactive compounds with physiological properties and one of which is CLA. The ability for CLA production by lactic acid bacteria was assessed in many publications. For instance, Yang et al. analyzed a number of food-grade lactobacilli and in which L. plantarum and L. bulgaricus were the most efficient CLA-producers.¹⁸ A mixture of probiotics, VSL#3, which contains four strains of Lactobacillus (L. casei, L. plantarum, L. bulgaricus and L. acidophilus), three strains of Bifidobacterium (B. longum, B. breve and B. infantis) and Streptococcus thermophilus, was reported that could produce CLA in vivo, unfortunately, which strain in the VSL#3 could produce CLA is still unknown.⁶ And the mechanism underlying the health-regulation effects of CLA-producing probiotics are still unclear. The present study was designed to test the effects of an efficient CLA-producer L. plantarum ZS2058 on inflammation, colonic mucous layer and TJ-related genes in colitis mice.

The clinical symptoms of DSS-induced colitis which composed of weight loss, diarrhea and gross bleeding were significantly alleviated by L. plantarum ZS2058, and leading to a lower DAI (Fig. 1A). Additionally, these clinical symptoms are equally important clinical symptoms for IBD patients.⁴⁷ By contrast, L. plantarum ST-III did not alleviate DSS colitis effectively, resulting in a similar DAI as in the vehicle group (Fig. 1A). CLA could prevent the onset of colitis efficiently, and the current results are consistent with those from Bassaganya-Riera and colleagues.^6,19 Additionally, treatment with L. plantarum ZS2058 increased CLA concentrations in the colonic contents, while L. plantarum ST-III had no influence on CLA concentrations (Fig. 4A), therefore, the increase of CLA levels in intestine content might be due to the administration of ZS2058 which indicates that this CLA-producer could generate CLA in vivo. Our results demonstrated that L. plantarum ZS2058 alleviated colitis at least in part via CLA.

L. plantarum ZS2058 and CLA significantly mitigated crypt disruption, submucosal edema, and epithelial structure damage induced by DSS (Fig. 1C). Furthermore, the changes in lymphocyte infiltration following treatment with CLA or CLA-producing Lactobacillus were consistent with the changes in MPO activity (Fig. 1E), which could reflects the degree of neutrophil infiltration in the colon. Moreover, since MPO is also a marker for oxidative stress,^48,49 it is possible that CLA alleviated the status of colonic oxidative stress in mice, and mechanisms of inhibiting DSS-induced colitis might be associated with CLA produced by L. plantarum ZS2058 in colon which improved oxidative stress.

PPARγ is one of the three isoforms of PPAR that belong to the nuclear receptors superfamily,⁵⁰ and previous research indicates that PPARγ regulates the expression of genes involved in inflammation, adipogenesis and lipid metabolism.^51,52 Furthermore, PPARγ is considered to be a central inhibitor of colitis^53,54 and can be activated by CLA.¹⁹ Our results showed that L. plantarum ZS2058 and CLA increased the mRNA level of PPARγ in the colon, while L. plantarum ST-III did not result in a similar change (Fig. 2E); this suggests that L. plantarum ZS2058 produced CLA locally, which then up-regulated the expression of PPARγ.

Although the exact pathogenesis of IBD remains unclear, previous studies indicated that the pathogenesis of IBD involved uncontrolled and hyper-activated intestinal mucosal inflammation.^53,54 TNF-α is a key pro-inflammatory cytokine produced by macrophages, T cells and epithelial cells in the pathogenic process of IBD,^55,56 moreover, TNF-α is an activator of NF-κB which could further promote the secretion of TNF-α and other pro-inflammatory cytokines such as IL-1β and IL-6.^57–59 Additionally, PPARγ reduces the activation of NF-κB through an IκB-α-dependent mechanism.⁶⁰ The change in mRNA expression of pro-inflammatory cytokines TNF-α, IL-1β and IL-6 was assessed in this study, as well as that of anti-inflammatory cytokine IL-10. Treatment with L. plantarum ZS2058 and CLA significantly down-regulated the expression of TNF-α, IL-1β, IL-6 (Fig. 2A–C) and up-regulated the PPARγ mRNA level (Fig. 2E), whereas L. plantarum ST-III failed to reduce significantly the mRNA level of TNF-α, IL-1β, or IL-6. This suggests that the improvement in colitis symptoms by L. plantarum ZS2058 was probably mediated by a mechanism associated with the activation of PPARγ and the inhibition of NF-κB, resulting in a lower secretion of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6). Notably, L. plantarum ZS2058 up-regulated the mRNA level of anti-inflammatory cytokine IL-10 (Fig. 2D), which was up-regulated by CLA as well, and this result is in accordance with previous studies.^61,62 Therefore, we believe that L. plantarum ZS2058 improves the immune response in colitic mice via a CLA-dependent mechanism.

Goblet cells are vital for the secretion of trefoil factors and mucins that help sustain the mucosal barrier.^37,63 L. plantarum ZS2058 and CLA up-regulated MUC2 (Fig. 3B) and protected the colonic mucous layer integrity and goblet cells (Fig. 3A). In addition, our results showed that DSS down-regulated ZO-1 and Claudin 3 (Fig. 3D and E), in line with previous results.^34,64 L. plantarum ZS2058 and CLA could up-regulate E-cadherin 1, ZO-1, and Claudin 3 (Fig. 3C–E), thus maintaining the enteric epithelium structure, whereas L. plantarum ST-III had no improvement except for ZO-1, which might be an alternative mechanism for their protective effect against DSS-induced colitis. Additionally, there are few pieces of information about connection of CLA and tight junctions (TJ) in vivo. To our knowledge, there are some studies to investigate the effects of CLA on epithelial permeability in vitro usage of Caco-2 cells, and the results demonstrated that CLA increased the paracellular permeability, reduced the transepithelial electrical resistance (TEER) across the Caco-2 monolayers, and enhanced transepithelial calcium transport.^65,66 Those results indicated that enhanced permeability of the intestinal epithelium could be induced by CLA, but this may increase intestinal infection in disease situation. However, our results indicate that CLA protects the enteric epithelium structure from the DSS-induced colitis in vivo. Furthermore, in this study, the molecular expression of ZO-1 was up-regulated, this observation is in accordance with Eileen' studies which operated in vitro with Caco-2 cells.⁶⁷

In conclusion, L. plantarum ZS2058 could significantly ameliorate DSS-induced colitis in mice by mechanisms which include up-regulating anti-inflammation factors and inhibiting pro-inflammation factors, as well as improving the tight junctions to sustain the mucosal barrier. CLA played a key role in these effects. This may not only help us understand the mechanisms by which probiotics benefit human health, but it also implies that metabolites of probiotics can be important indicator for finding new probiotics that are beneficial for human health. Additionally, whether L. plantarum ZS2058 can modulate the damaged gut microbiota resulting from colitis to alleviate IBD needs further investigation.

Acknowledgements

This research was supported by the National Natural Science Foundation of China (No. 31571810, 31530056, 31125021), the Program for New Century Excellent Talents (NCET-13-0831), the Program for Changjiang Scholars and Innovative Research Team in University (IRT1249), the Fundamental Research Funds for the Central Universities (No. JUSRP51320B), and the National Natural Science Foundation of Jiangsu Province (No. BK20150141, BK20150132).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c5ra24491a

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