The ameliorative effect of a Lactobacillus strain with good adhesion ability against dextran sulfate sodium-induced murine colitis

Abstract

The objective of this study was to effectively screen out a Lactobacillus strain with excellent adhesion ability and ameliorative effect on the disease symptoms of a murine ulcerative colitis model. The auto-aggregation rate (R_AA), co-aggregation rate with Escherichia coli O157 (R_CA), and cell surface hydrophobicity (A_HC) of 17 Lactobacillus strains were measured for primary selection. The results indicate that Lactobacillus plantarum AR326 displayed the topmost overall aggregation performance among all strains examined. A positive and linear relationship between R_CA and R_AA values was observed for 15 Lactobacillus strains with R_AA = 16–46% and R_CA = 15–27%. For adhesion ability to human adenocarcinoma HT-29 cells, five representative Lactobacillus strains showed a good, positive dependence on R_AA, where L. plantarum AR326 and L. fermentum AR184 showed a higher adhesion ability than the others. Using the 5 (6)-carboxyfluorescein diacetate N-succinimidyl ester (cFDA SE)-labeling technique, L. plantarum AR326 was confirmed to adhere to and colonize well in the intestinal mucosa of mice mainly in the ileum and colon. Finally, L. plantarum AR326 at the dosage applied (daily 2 × 10⁹ cfu per mouse) could attenuate murine dextran sulfate sodium (DSS)-induced colitis by effectively decreasing the body weight loss, disease activity index, colon length shortening, myeloperoxidase activity, and colon epithelial damage of experimental animals. The protective effects involved the restoration of the tight junction protein expression and reduction of the abnormal expression of pro-inflammatory cytokines. In conclusion, L. plantarum AR326 can be used as promising probiotics to ameliorate DSS-induced colitis.

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1. Introduction

Some Lactobacillus strains have been used in food products as probiotics due to their beneficial effects on human health.^1–4 The functionality of probiotic Lactobacillus strains has been reported such as restoring the balance of the gastro-intestinal microbiota, reducing intestinal inflammation, and helping to manage inflammatory bowel disease (IBD).^5–7

Inflammatory bowel disease (IBD) is a chronic relapsing disorder that encompasses a range of intestinal pathologies, e.g. the most common ulcerative colitis and Crohn's disease.^4,8 For in vivo animal studies, the dextran sulfate sodium (DSS)-induced ulcerative colitis model has been widely used as a representative IBD model, because of its simplicity and similarities to human IBD.⁸ Several studies have been conducted using the strategy of oral administration with probiotic lactobacilli to prevent or ameliorate ulcerative colitis in vivo. Three Lactobacillus plantarum strains (21, K68, and CLP-0611) have been reported to ameliorate experimental colitis in mice or rats to a good effect.^9–11L. plantarum NCIMB8826 can reduce the weight loss and colon length shortening in DSS-induced colitis mice, without significant impact on reducing disease activity index (DAI) and histological damage.¹² Two L. fermentum strains (BR11 and CCTCC M206110) are proved to be effective for alleviating DSS-induced colitis in mice or rats.^12,13 However, L. rhamnosus attenuated insignificantly the severity of DSS-induced colitis, in comparison with Bifidobacterium breve that attenuated significantly.¹⁴ Exceptionally, L. crispatus CCTCC M206119 appeared to aggravate DSS-induced colitis in BALB/c mice.¹²

Generally, different Lactobacillus strains may exert positive or opposite effects on the disease severity of ulcerative colitis in vivo. This diversity is strain-specific and closely related to several key factors, especially the prerequisite one, the adhesion ability of Lactobacillus strains in the gut.^15,16 The other factors include the dosage, survival rate, and immunomodulatory or anti-inflammatory activities of the Lactobacillus strains applied.

For saving the cost of screening the strains with good probiotic potential in vivo, three adhesion-related abilities in vitro are necessarily considered together: firstly, the adhesion ability to the intestinal epithelial cells that could be mimicked by human adenocarcinoma cell lines such as HT-29 or Caco-2;^17–19 secondly, the auto-aggregation ability that is potentially and positively correlated with lactobacilli adhesion and colonization in the intestinal tract;^19–22 and thirdly, the co-aggregation ability between lactobacilli and pathogens (e.g. Escherichia coli), which has been shown to be a competitively excluding activity against pathogen adhesion to the gut.^23,24 The role of the adhesion ability of the Lactobacillus strains to the intestinal epithelial cells in ameliorating the colitis-associated symptoms of animals has not yet been discovered in the literature. Discovering Lactobacillus strains with high adhesion ability and excellent colitis-ameliorating potential in vivo is crucial for the probiotic therapeutic industry and still under investigation.

Accordingly, in this study, several Lactobacillus strains with excellent adhesion abilities to HT-29 cells were screened from 17 Lactobacillus strains with characterized auto-aggregation rates, co-aggregation rates, and hydrophobicity. One Lactobacillus strain was specially selected for studying its colonization and distribution in the mouse intestine and its ameliorative effects on the disease severity of mice with DSS-induced colitis. The colonization and distribution of the selected Lactobacillus strain in the mouse intestine were monitored with the 5 (6)-carboxyfluorescein diacetate N-succinimidyl ester (cFDA SE)-labeling technique.²⁵

2. Materials and methods

2.1 Bacterial strains, cells, animals and culture conditions

Bacterial strains were isolated from fermented pickles and dairy products. The identification of these isolates was based on the sequences of the 16S rRNA gene. To amplify the 16S rRNA gene, the universal primers 27F (5′-AGAGT TTGATCCTGGCTCA-3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used. The 16s rRNA gene sequence was queried against the NCBI genetic database using the BLAST search algorithm. The Mega software package, version 7.0, was used for the phylogenetic tree construction based on the neighbor-joining method. Sixteen strains were identified as Lactobacillus species. These isolated Lactobacillus strains, Lactobacillus rhamnosus GG (LGG) (Jiangnan University, Wuxi, Shanghai) and Escherichia coli O157 (provided by Professor Qing Liu, University of Shanghai for Science and Technology, Shanghai) were stored in Shanghai Engineering Research Center of Food Microbiology (Shanghai, China). All Lactobacillus strains were anaerobically cultured in de Man, Rogosa and Sharpe (MRS) broth at 37 °C for 20 h before centrifugation (8000g for 3 min) for the following experiments. Escherichia coli O157 was aerobically cultured in brain heart infusion broth (BHIB) at 37 °C for 18 h before centrifugation for the co-aggregation experiments.

The human adenocarcinoma cell line HT29 was purchased from the Cell Resources Center of Shanghai Institutes for Biological Sciences (Shanghai, China). HT29 cells were cultivated in 1640 medium (Gibco™, Thermo Fisher Scientific, Grand Island, USA) containing 10% fetal bovine serum at 37 °C under 5% CO₂. The passage number was 4 for all experiments performed. The culture medium was refreshed every second day.

Female ICR mice were purchased from Shanghai SLRC Laboratory Animal Co. Ltd (Shanghai, China). The mice were housed in an air-conditioned room with the laboratory temperature maintained at 23 ± 2 °C and a relative humidity of 50 ± 10%. All mice were housed for acclimatization for at least one week before the following experiments, and the procedures were performed according to institutional and governmental regulations about the use of experimental animals. All animal procedures were performed in accordance with the Guidelines for Care and Use of Laboratory Animals of Shanghai Jiao Tong University and approved by the Animal Ethics Committee of Shanghai Jiao Tong University.

2.2 Detection of Lactobacilli auto-aggregation

An auto-aggregation assay was carried out as described previously with modification.²⁶ A bacterial pellet was washed with phosphate buffered saline (PBS) twice and resuspended in PBS to an initial absorbance (A₀) of 0.25 ± 0.05 at 600 nm. The bacterial suspension was maintained in PBS at 37 °C for 5 h in a thermostatic incubator and then measured for absorbance at 600 nm (noted as A_t). The measurement was conducted in triplicate. The auto-aggregation rate of Lactobacilli (R_AA) was then calculated using eqn (1).

R_AA = [1 − (A_t/A₀)] × 100% (1)

where A₀ and A_t are the absorbances of Lactobacilli suspensions at a staying time of 0 and t h, respectively.

2.3 Detection of Lactobacilli co-aggregation

A co-aggregation assay for lactobacilli and E. coli O157 was carried out according to Collado et al.²⁶ with modification. Lactobacilli and E. coli suspensions were gently mixed in an isovolumetric manner. The mixed bacterial suspension was maintained at 37 °C for 5 h and its absorbance examined at 600 nm. The measurement was conducted in triplicate. The co-aggregation rate (R_CA) was calculated using eqn (2).

R_CA = {[(A_lacto + A_{E. coli})/2 − A_mix]/(A_lacto + A_{E. coli})/2} × 100% (2)

where A_lacto and A_{E. coli} are the initial absorbances of lactobacilli and E. coli suspensions, respectively, and A_mix is the absorbance of the lactobacilli–E. coli mixed suspension after maintaining for 5 h.

2.4 Cell surface hydrophobicity measurement

Cell surface hydrophobicity was determined as the affinity to hydrocarbons, using the xylene extraction method according to Collado et al.²⁶ Xylene (3 mL, Sinopharm Chemical Reagent Co., Ltd, Shanghai, China) was added into a test tube containing 3 mL Lactobacillus suspension (A₀ = 0.25 ± 0.05). The mixture was well mixed for 3 min by vortex oscillation, followed by 1 h of incubation at room temperature before absorbance measurement at 600 nm. The measurement was conducted in triplicate. The strain surface hydrophobicity (A_HC) was calculated according to eqn (3).

A_HC = [(A₀ − A)/A₀] × 100% (3)

where A₀ is the initial absorbance of the Lactobacillus suspension at a holding time of 0 h, and A is the absorbance of the aqueous phase of the Lactobacillus–xylene suspension after 1 h of incubation at room temperature.

2.5 Lactobacillus adhesion experiment on HT29 cells

The concentration of the Lactobacillus suspension was adjusted to 10⁸ cfu mL⁻¹. HT29 cells were cultured on 12-well plates. When they grew to cover 80%–90% of the cell culture plates, they were rinsed with PBS twice and added to 1 mL of Lactobacillus suspension and 1 mL of 1640 culture medium per well. Then, they were placed in a cell incubator at 37 °C for 2 h, followed by successive PBS rinsing twice, methanol fixation for 20 min, PBS rinsing twice, Gram staining, and microscopic observation. Twenty views were randomly chosen for shooting. The number of lactobacilli adhered to HT29 cells was calculated on the basis of 100 cells. The experiment was repeated in triplicate.

2.6 Animal experiment

2.6.1 Fluorescent labelling of L. plantarum AR326. A fluorescent probe was prepared as described in the previous research.²⁵ The concentration of the Lactobacillus suspension was adjusted to 10¹⁰ cfu mL⁻¹. The bacterial suspension was then mixed with cFDA-SE (50 μM in ethanol) in an isovolumetric manner, followed by incubation in a 37 °C water bath for 20 min in the dark. The cell mass was collected by centrifugation at 8000g for 3 min and rinsed with PBS twice to remove unreacted cFDA SE. Flow cytometry (FACSCalibur, Becton, Dickinson and Company, New Jersey, USA) was used to detect cell fluorescent labelling percentage. The percentage of fluorescent-labelled positive alive cells was 99.74%.

2.6.2 Fluorescent labelling of L. plantarum AR326. Female ICR mice at 7 weeks of age were divided into two groups. In the experimental group, the mice were orally administered with fluorescent-labelled L. plantarum AR326 once at a dosage of 2 × 10⁹ cfu in 0.2 mL. For the control group, the mice were orally administered with sterilized PBS solution (0.2 mL). On the 1^st, 2^nd, 3^rd, 4^th, 5^th, and 6^th day post administration, five mice in each group were sacrificed by cervical dislocation. The duodenum, jejunum, ileum and colon were taken and further split vertically to remove the large particles inside. Then 150 μL cm⁻¹ PBS was used to rinse repeatedly. The bacteria in the washing liquid were collected and fixed with 0.75% oxymethylene for bacteria number analysis with flow cytometry.²⁵ Data were measured in five replications.

2.6.3 Mice with DSS-induced colitis and L. plantarum intervention. Female ICR mice at 7 weeks of age were divided into three groups: normal control, DSS-induced colitis, and L. plantarum intervention groups. The treatment schedule is depicted in Fig. 1. The normal group took a normal diet and drank water freely for the whole experimental period (14 days). The DDS-induced colitis group was fed with 3% DSS-containing drinking water by drinking freely for the first 5 days and then with a normal diet and water for the next 7 days of the experiment. In the L. plantarum intervention group, the above DSS-treated mice were orally administered with 2 × 10⁹ cfu of L. plantarum AR326 in 0.2 mL by oral gavage daily for the next 7 days of the experiment. At the end of the experiments, the mice were sacrificed by cervical dislocation for the following histological examinations.

Fig. 1 Feeding schedules for normal, DSS-induced colitis, and L. plantarum intervention models using ICR mice.

The disease activity index (DAI) is a clinical parameter reflecting the severity of weight loss, stool consistency and faecal bleeding of experimental animals.²⁷ Accordingly, the severity of the DSS-induced disease was determined by calculating the body weight change, disease activity index (DAI), colon length, and myeloperoxidase (MPO) activity.¹⁴ Body weight change was evaluated as a score: 0, normal, without change; 1, 1–5% weight loss; 2, 6–10% weight loss; 3, 11–15% weight loss; and 4, >15% weight loss. DAI was determined from the following eqn (4).

DAI = (body weight change + stool consistency + faecal bleeding)/3 (4)

where stool consistency was evaluated as a score: 0 = normal; 2 = soft with normal form; and 4 = loss of form/diarrhea. Faecal bleeding was evaluated as a score: 0 = no blood; 2 = detectable blood with the colorectal test kit (Baso Diagnostics Inc., Zhuhai, China); and 4 = visible blood without test. Colon length was measured based on the fact that DSS-induced colitis in mice can be characterized by colonic shortening.⁸ MPO activity was measured using the MPO assay kit according to the manufacturer's protocol (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). MPO activity was measured in units per g of fresh colon tissue, where one unit of MPO was defined as the amount needed to degrade 1.0 μmol of hydrogen peroxide per minute at 37 °C.

Colonic histology was assessed by H&E staining. The colon tissues were fixed in 10% neutral buffered formalin for 24 h and embedded in paraffin. Sections of 6 μm for each sample were stained with hematoxylin and eosin (H&E) for light microscopic evaluation. The samples were observed at ×100 magnification. The severity of colonic histological injury was scored using a scoring system taking into account the severity of inflammation (from 0 to 3 as the maximum score), crypt damage (from 0 to 5 as the maximum score) and ulcerations (from 0 to 3 as the maximum score).²⁸ Values were added to give a maximal histological score of 11.²⁸ All data were examined in five replications.

2.7 Quantitative real-time PCR

The total cellular RNA was extracted using TRIzol reagent (Sangon Biotech, Shanghai, China) according to the manufacturer's instructions. Quantitative real-time PCR amplification was performed using 2xSYBR® Premix Ex Taq (Takara Bio (Dalian) Co., Ltd, Dalian, China) to detect MUC2, E-cadherin 1, zonula occludens-1 (ZO-1), claudin 3, TNF-α, IL-1β, IL-6, and β-actin gene expression. The results were analysed by the method of 2^−ΔΔCt. The PCR primer pairs used in the study are shown in Table S1.†

2.8 Statistical analysis

Significant differences among data were examined with the one-way ANOVA program and Duncan's test of Statistical Product and Service Solutions (SPSS Inc., Chicago, USA). The significance of the correlation coefficient was also examined. Data are presented as the means with standard deviations and differ significantly as presented with different letters at P < 0.05. SigmaPlot 12.3 software (Systat Software Inc., CA, USA) was used for plotting and obtaining linear relationships.

3. Results

3.1 Auto-aggregation, co-aggregation and hydrophobicity

The sixteen isolated strains were identified to the species level (Table 1). They belong to the Lactobacillus species. Fig. 2 shows the phylogenetic tree based on the 16S rRNA gene sequences showing the relationships of the sixteen isolated Lactobacillus species and LGG. Lactobacillus brevis, Lactobacillus fermentum and L. plantarum are grouped within a single branch, separately. The position of Lactobacillus casei and Lactobacillus paracasei in the phylogenetic tree reflects their close evolutionary relationship. The strains, even in the same species, are separated well in the phylogenetic tree. These indicated that these strains are different and probably have different properties.

Fig. 2 Phylogenetic tree based on the 16S rRNA gene sequences showing the relationships of these strains.

Table 1 Auto-aggregation rates, co-aggregation rates, and hydrophobicity of 17 Lactobacillus strains examined

Strain	Auto-aggregation rate, RAA (%)	Co-aggregation rate, RCA (%)	Hydrophobicity, AHC (%)
a Data are presented as means ± standard deviations (n = 3). Data followed with different capital letters differed significantly (P < 0.05).
L. brevis AR281	41.7 ± 0.2^A ^a	27.5 ± 1.1^A	24.6 ± 1.3^D
L. brevis AR282	24.6 ± 3.8^CD	22.0 ± 6.2^ABCDE	90.7 ± 2.7^A
L. casei AR341	23.8 ± 1.6^CD	18.1 ± 4.3^DE	81.1 ± 2.2^A
L. casei AR342	13.7 ± 5.5^E	8.6 ± 0.9^F	88.6 ± 5.5^A
L. paracasei AR320	16.4 ± 5.4^DE	15.1 ± 2.2^E	81.8 ± 5.4^A
L. paracasei AR335	20.7 ± 4.5^DE	19.0 ± 4.1^CDE	83.1 ± 2.1^A
L. paracasei AR338	21.5 ± 4.5^DE	6.1 ± 1.1^F	83.9 ± 2.9^A
L. fermentum AR184	42.4 ± 1.0^A	23.5 ± 2.3^ABCD	86.8 ± 4.7^A
L. fermentum AR189	17.5 ± 1.3^DE	19.3 ± 4.1^CDE	83.6 ± 5.9^A
L. plantarum AR113	38.3 ± 0.6^AB	23.0 ± 1.1^ABCD	81.3 ± 3.2^A
L. plantarum AR171	23.0 ± 3.2^CD	19.9 ± 1.1^BCDE	89.0 ± 6.1^A
L. plantarum AR192	22.6 ± 4.1^CD	18.9 ± 1.1^CDE	85.1 ± 7.0^A
L. plantarum AR270	24.5 ± 5.1^CD	20.3 ± 2.4^BCDE	83.5 ± 2.3^A
L. plantarum AR307	41.9 ± 1.1^A	26.5 ± 0.2^AB	45.0 ± 3.5^C
L. plantarum AR326	45.5 ± 1.4^A	25.4 ± 1.8^ABC	15.0 ± 0.9^D
L. plantarum AR348	20.8 ± 4.1^DE	16.5 ± 2.4^DE	89.1 ± 5.5^A
L. rhamnosus GG (LGG)	30.5 ± 0.5^BC	23.3 ± 1.4^ABCD	66.2 ± 3.5^B

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The auto-aggregation rate (R_AA), co-aggregation rate (R_CA), and hydrophobicity (A_HC) of 17 strains of the lactobacilli examined were measured, including LGG as the positive control. The results are tabulated in Table 1. Generally, the obtained R_AA values ranged from 13.7% (L. casei AR342) to 45.5% (L. plantarum AR326). The five strains that showed an R_AA value significantly (P < 0.05) higher than that of LGG (30.5%) included L. brevis AR 281, L. fermentum AR184, and L. plantarum AR 113, AR307, and AR326. The R_CA values ranged from 6.1% to 27.5%; the highest values were for L. brevis AR281 and L. plantarum AR307 and AR326 which were greater than that for LGG (23.3%). The A_HC values ranged from 15.0% (L. plantarum AR326) to 90.7% (L. brevis AR282). Most of the strains, except L. brevis AR281 and L. plantarum AR307 and AR326, showed an average A_HC value of 81–91%, significantly (P < 0.05) greater than that of LGG (66.2%) and hardly correlating to their R_AA or R_CA values. Generally, the R_AA, R_CA, and A_HC values were partly strain-specific. Both aggregation rates for the strains from the L. plantarum group (R_AA = 21–46%; R_CA = 17–27%) were greater than those from the L. casei group (including L. paracasei) (R_AA = 14–24%; R_CA = 6–19%).

Fig. 3A illustrates that, for most of the Lactobacillus examined except L. casei AR342 and L. paracasei AR338 with extraordinarily low R_CA values, the obtained R_CA value appeared to linearly and positively correlate with the R_AA value, following the equation R_CA = 0.321 × R_AA + 11.9 (R² = 0.811, P < 0.05). The seven L. plantarum strains detected were also committed to a similar relationship (R_CA = 0.325 × R_AA + 11.5, R² = 0.895, P < 0.05; equation not shown). For affinity to hydrocarbons (A_HC) as an index for cell surface hydrophobicity (Fig. 3B), the A_HC values varied with R_AA in a roughly negative way for all strains (A_HC = −1.63 × R_AA + 119, R² = 0.527, P < 0.05). It is interesting to note that those strains with R_AA > 30% could be grouped into high-A_HC (L. fermentum AR184 and L. plantarum AR113) and low-A_HC (L. brevis AR281 and L. plantarum AR307 and AR326) groups, implying distinct cell surface hydrophobicity or compositions.

Fig. 3 Changes in the co-aggregation rate (R_CA) (A) and affinity to hydrocarbons (A_HC, an index for hydrophobicity) (B) with the auto-aggregation rate (R_AA) for the Lactobacillus strains studied. AR342 = L. casei AR342; AR338 = L. paracasei AR338. Data are presented as means ± standard deviations (n = 3) for two variables correlated.

3.2 Adhesion of selected Lactobacillus strains onto HT29 cells

From different species, three strains with high R_AA and R_CA values (L. brevis AR281, L. fermentum AR184, and L. plantarum AR326) and one strain with a low R_AA or R_CA (L. casei AR342) were selected for studying adhesion to HT-29 cells, in reference to LGG as the positive control. The results are given in Table 2. It is shown that the cell adhesion abilities were in the range of 171–430 bacterial counts per 100 cells, significantly (P < 0.05) greatest for L. plantarum AR326 and L. fermentum AR184, followed by L. brevis AR281 and LGG, and the least for L. casei AR342. Interestingly, the adhesion abilities of these five lactobacilli were closely and positively correlated with R_AA (AA = 8.17 × R_AA + 38.5, R² = 0.909, P < 0.05, Fig. 4A), rather than with R_CA, and negatively correlated with A_HC, except for L. fermentum AR184 (AA = −3.24 × A_HC + 456, R² = 0.969, P < 0.05, Fig. 4B). Obviously, the strain with a high R_AA value exhibited high adhesion ability to HT-29 cells, implying high adhesion potential to the intestinal epithelial cells.

Fig. 4 Relationships between adhesion ability to auto-aggregation rates (R_AA) (A) and affinity to hydrocarbons (A_HC) (B) for five representative Lactobacillus strains studied as shown in Table 2, including L. brevis AR281, L. casei AR342, L. fermentum AR184, L. plantarum AR326, and L. rhamnosus GG (LGG). In the equations, AA = adhesion ability. Data are presented as means ± standard deviations (n = 3) for two variables correlated.

Table 2 Adhesion abilities of 5 selected Lactobacillus strains onto HT29 cells

Strain	Adhesion ability (bacterial count per 100 cells)
a Data are presented as means ± standard deviations (n = 3). Data followed with different capital letters differed significantly (P < 0.05).
L. brevis AR281	350 ± 49^B ^a
L. casei AR342	171 ± 19^D
L. fermentum AR184	418 ± 31^A
L. plantarum AR326	430 ± 32^A
L. rhamnosus GG (LGG)	245 ± 25^C

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From the above in vitro experiments, L. plantarum AR326 had the best overall performance, i.e., maximal auto-aggregation, co-aggregation with E. coli, and adhesion ability to HT-29 cells. Accordingly, it was selected for the following in vivo experiments.

3.3 Colonization and distribution of L. plantarum AR326 in the mouse intestines

The number changes of cFDA SE-labelled L. plantarum AR326 distributed in different intestinal parts of the mice after oral administration once at a dosage of 2 × 10⁹ cfu in 0.2 mL are depicted in Fig. 5, with respect to the post administration day. It is indicated that L. plantarum AR326 survived and colonized very well inside mouse intestines. On day 1 after oral administration, the detected level of positive L. plantarum AR326 was high up to 7.80 × 10⁴ in the ileum, followed by 4.45 × 10⁴ in the colon, and ∼2 × 10³ in the jejunum and duodenum. This suggests that the AR326 colonized well in the ileum and colon. The bacterial count reduced slowly with the day post oral administration and still remained at 2 × 10⁴ in the ileum and colon on the sixth day post administration. Generally, the colonization potential of L. plantarum AR326 was in the order of ileum > colon > jejunum and duodenum.

Fig. 5 Changes in the fluorescence-labelled L. plantarum AR326 number adhered on various sections of the intestinal tract in ICR mice with respect to time after oral administration (lactobacilli dosage = 10¹⁰ CFU mL⁻¹ in 0.2 mL on day 1). Data are represented as means ± standard deviations (n = 5). Data indicated with different letters differ significantly (P < 0.05).

3.4 Intervention of L. plantarum AR326 on mice with DDS-induced colitis

The intervention effects of orally administered L. plantarum AR326 on the severity of DDS-induced colitis in mice were evaluated from the viewpoint of body weight loss, disease activity index (DAI), colon length, myeloperoxidase (MPO) activity, and histopathology, where the DAI value was the average score of body weight loss, stool consistency, and faecal bleeding scores for the experimental animals according to Tao et al.²⁷ Mice were fed according to the schedule in Fig. 1.

Fig. 6A depicts the body weight changes of normal, DSS-treated, and DSS-treated plus L. plantarum administered mice with respect to feeding time. In contrast to the normal group, the body weight of the DSS-treated mice decreased significantly after the 6^th day (i.e. post DSS treatment), maximized on the 9^th day, and recovered gradually thereafter. With L. plantarum intervention, the DSS-treated mice showed body weight recovery partly and significantly after the 8^th day until the end of the experiment. Clearly, L. plantarum intervention at the dosage applied could effectively reduce the body weight loss of DSS-treated mice. For the DAI scores of mice where the normal DAI = 0–0.25 (Fig. 6B), the DAI score of DSS-treated mice increased linearly and remarkably with the feeding time during the first 9 days, maximized at 3.1, and decreased gradually thereafter. With L. plantarum intervention, the DAI decreased on the 6^th day (i.e. the first day of L. plantarum administration) and maintained the reduced DAI score during the rest of experimental time, in contrast to the positive control. The final DAI score of the L. plantarum intervention group on the 14^th day was less than 1.0, approaching the normal one. This suggests that oral administration with L. plantarum AR326 at the dosage applied did reduce the DAI score of ICR mice with DSS-induced colitis.

Fig. 6 Effects of L. plantarum AR326 intervention on the body weight (A) and disease activity index (DAI) score (B) of ICR mice with DSS-induced colitis (lactobacilli dosage = 2 × 10⁹ CFU per mice). Data are represented as means ± standard deviations (n = 5). Data indicated with different letters differ significantly (P < 0.05).

Colonic shortening is well known as one of the characteristics of DSS-induced colitis in mice.⁸Fig. 7A shows that the DSS group exhibited a significant colon length reduction (4.8 ± 0.2 cm) at the end of the experiment (14^th day), in contrast to the normal one (6.3 ± 0.2 cm) (P < 0.05). Promisingly, the L. plantarum intervention group displayed a colon length of 5.83 ± 0.49 cm, significantly (P < 0.05) longer than the DSS counterpart and resembled the normal one. Fig. 7B depicted that the MPO activity, a marker of polymorphonuclear neutrophil primary granules,²⁹ increased significantly (P < 0.05) in the DSS group (0.60 U g⁻¹) by an increment almost double that of the normal one (0.22 U g⁻¹). In contrast to the DSS group, the L. plantarum intervention group showed a significantly (P < 0.05) decreased MPO activity. Evidently, the intervention of L. plantarum AR326 at the dosage applied could recover the DSS-damaged colon length to a normal level and alleviate the DSS-induced hyper-activated MPO activity of the DSS group to a significantly decreased level. The decreased MPO activity implies the reduced number and distribution of neutrophils in the tissues, i.e. decreased neutrophil infiltration and inflammatory symptoms.²⁹Fig. 8 shows the pathological examination of the colons with H&E staining. Epithelial erosion, disappearance of crypts, and cellular infiltration were observed in the DSS group. The evaluation of colons from the L. plantarum intervention group revealed a significant reduction in epithelial erosion and inflammatory cellular infiltration compared to the DSS group and the epithelium morphology resembled that observed in samples from the normal group (Fig. 8A). The score value measured for DSS-treated mice was significantly higher than that of the normal group (Fig. 8B). In contrast, the score value of the L. plantarum intervention group was lower than that of the DSS group (Fig. 8B). These further confirmed that L. plantarum AR326 can attenuate DSS-induced colitis.

Fig. 7 Effects of L. plantarum AR326 intervention on the colon length (A) and myeloperoxidase (MPO) activity (B) of ICR mice with DSS-induced colitis (lactobacilli dosage = 2 × 10⁹ CFU per mice). Data are represented as means ± standard deviations (n = 5). Means with different letters differ significantly (P < 0.05).

Fig. 8 Effects of L. plantarum AR326 intervention on the colon histology. (A) The representative section of the colons was determined by H&E staining (magnification 100×). (B) Associated histological scores. Data are represented as means ± standard deviations (n = 5). Means with different letters differ significantly (P < 0.05).

3.5 Intervention of L. plantarum AR326 on the expression of the tight junction proteins and inflammatory cytokines

DSS administration could induce changes in the expression of tight junction proteins and increased the expression of pro-inflammatory cytokines.^5,10 To determine whether the intervention of L. plantarum AR326 could maintain the integrity of the intestinal epithelial barrier, we examined the expression of tight junction proteins, including MUC2, ZO-1, claudin 3 and E-cadherin 1 using quantitative real-time PCR. Compared to the normal group, the DSS group showed significant reductions in these genes (P < 0.05, Fig. 9). In contrast to the DSS group, the L. plantarum intervention group could recover the expression of these tight junction proteins to a normal level (Fig. 9).

Fig. 9 The mRNA levels of MUC2 (A), ZO-1 (B), claudin 3 (C) and E-cadherin 1 (D) were determined using quantitative real-time PCR in the colon. Data are represented as means ± standard deviations (n = 5). Means with different letters differ significantly (P < 0.05).

Previous studies have demonstrated that the pro-inflammatory cytokines TNF-α, IL-1β and IL-6 are associated with the severity of colitis.^5,10,30 Accordingly, we evaluated the mRNA expression of colonic TNF-α, IL-1β, and IL-6 using quantitative real-time PCR. In the DSS group, the levels of these pro-inflammatory cytokines were significantly higher than that of the normal group (P < 0.05, Fig. 10). The abnormal increases of these proinflammatory cytokines induced by DSS were significantly attenuated by the intervention of L. plantarum AR326 (P < 0.05, Fig. 10). The cytokine levels in the L. plantarum intervention group were comparable to those of the normal group (Fig. 10).

Fig. 10 The mRNA levels of TNF-α (A), IL-1β (B) and IL-6 (C) were determined using quantitative real-time PCR in the colon. Data are represented as means ± standard deviations (n = 5). Means with different letters differ significantly (P < 0.05).

4. Discussion

In this study, a positively linear relationship between auto-aggregation (R_AA) and co-aggregation (R_CA) rates was observed for 15 Lactobacillus strains with R_AA = 16–46% and R_CA = 15–27%, except for L. casei AR342 and L. paracasei AR338 with a very low R_CA value (6–8%) (Fig. 3A). This relationship had not been discovered before for the 22 Lactobacillus strains,¹⁹ which show variations in R_AA (24–41%) and R_CA (21–32%) mostly comparable to those found in this study. For co-aggregation with E. coli O157, the obtained R_CA values for the L. casei group (L. casei and L. paracasei strains) in this study (R_CA = 6–19%) and the study by Tuo et al.¹⁹ (R_CA = 22–32%) are lower than the findings for L. paracasei subsp. paracasei BGSJ2-8 (R_CA ∼ 45%).³¹

Based on five representative Lactobacillus strains with diverse R_AA values (14–46%) (Fig. 4A), the adhesion ability to HT-29 cells appeared to change as a linear function of R_AA with a positive slope and high correlation coefficient. This correlation is evidently better than those for the 22 Lactobacillus strains adhering to Caco-2 cells, another type of human adenocarcinoma cell, and for Lactobacillus and Bifidobacterium strains adhering to Caco-2 cells.^19,20,22

As for the hydrophobicity index (A_HC), its dependence on R_AA is generally insignificant for the Lactobacillus strains of high A_HC values (>60%), but significant for medium- to low-A_HC strains as found in this study (Fig. 3B) and the study by Tuo et al.¹⁹ The adhesion ability relied linearly on the A_HC value for L. brevis AR281, L. plantarum AR326, LGG, and L. casei AR342, rather than L. fermentum AR184 (Fig. 4B). This suggests that the adhesion ability is partly species-specific and cannot be described by hydrophobicity alone, supporting the viewpoint of previous reports.^19,20,32 This further explains why different reports conclude different correlations between adhesion ability and hydrophobicity for lactobacilli.^19,20,32,33

Evidently, there is likely another cell surface factor besides hydrophobicity that is important for auto-aggregation, co-aggregation, and adhesion abilities. And, R_AA, more than A_HC, may be a good index for evaluating the in vivo adhesion ability and colonization potential of lactobacilli.

Using cFDA-SE labelled L. plantarum AR326 for verifying the real adhesion capability and colonization of lactobacilli in mouse intestine, L. plantarum AR326 was confirmed to adhere to and colonize well in the intestinal mucosa of mice, mainly in the ileum, followed by the colon, and partly in the jejunum and duodenum (Fig. 5). This distribution is similar to the case of L. casei²⁵ and provides direct evidence that L. plantarum AR326 is able to colonize well in the intestinal tract for a long time. This prolonged adhesion and colonization favour probiotic effects.

In this study, after DSS treatment daily for 5 days, oral administration with L. plantarum AR326 did reduce significantly the body weight loss and DAI score, recover the DSS-damaged colon length to a normal level, and significantly reduced the DSS-induced hyper-activated MPO activity and histopathological changes of ICR mice with DSS-induced colitis. The histological examination showed that oral administration with L. plantarum AR326 reversed the mucosal erosion and disruption of intestinal epithelial integrity that were induced by DSS. These results were consistent with the recovery of the expression of tight junction proteins (MUC2, ZO-1, claudin 3 and E-cadherin 1) by L. plantarum AR326. The pro-inflammatory cytokines TNF-α, IL-1β and IL-6 are associated with the severity of colitis.^5,10,30 The levels of pro-inflammatory cytokines increased in IBD patients and also in DDS-induced colitis animal models.^10,34 Previous studies have demonstrated that reducing the expression of cytokines, such as TNF-α, IL-1β or IL-6, can attenuate the development of colitis clinically and in DSS-induced experimental colitis.^35,36Lactobacilli and Bifidobacterium have been reported to mediate the inflammatory role by the inhibition of pro-inflammatory cytokines, such as TNF-α, IL-1β and IL-6.^{4,5,10,11,13,27,30} In the present study, the levels of pro-inflammatory cytokines (TNF-α, IL-1β and IL-6) increased in the DSS-induced mice and oral administration with L. plantarum AR326 significantly attenuated these cytokine levels. In summary, our results suggest that L. plantarum AR326 attenuates the severity of colitis by restoring the tight junction protein expressions and by inhibiting pro-inflammatory mediators.

Lactobacillus plantarum AR326 was discovered to have the topmost overall performance in aggregation and adhesion abilities among the selected strains. Also, L. plantarum AR326 was confirmed to adhere to and colonize well in the intestinal mucosa of mice for a long time. The daily dosage of L. plantarum AR326 used in this study (2 × 10⁹ cfu per mouse, i.e. 1 × 10¹¹ cfu per kg bw, for 8 days, after DSS treatment) is effective for ameliorating the disease symptoms associated with DSS-induced colitis of ICR mice. Comparatively, oral administration with L. fermentum BR11 at a daily dosage of (1.1–2.3) × 10¹¹ cfu per kg bw for 7 days prior to DSS treatment and during the 7-day DSS treatment alleviates the disease symptoms of male Sprague Dawley rats with DSS-induced colitis, with an effectiveness better than that of LGG.¹³ A similar effectiveness was also discovered in the case of L. plantarum K68 at a daily dosage of ∼5 × 10¹⁰ cfu per kg bw and with the same intervention schedule for female BALB/c mice with DSS-induced colitis.¹⁰

Oral administration of L. fermentum CCTCC M206110 at a daily dosage of 6 × 10⁹ cfu per kg bw for 3 days prior to DSS treatment and during DSS treatment for 6 days also alleviates effectively the disease symptoms of female BALB/c mice with DSS-induced colitis, with an effectiveness greater than that of L. plantarum NCIMB8826 which can only reduce the extents of weight loss and colon length shortening, rather than alleviating DAI and histological damage.¹² Pre-oral administration of LGG NutRes1 at a dosage of ∼4 × 10¹⁰ cfu per kg bw once every second day five times prior to DSS treatment shows mildly ameliorative effects on colon length shortening, histological score, and feces scores, but not MPO, for female C57BL/6 mice with DSS-induced colitis, with an effectiveness significantly less than that of Bifidobacterium breve NutRes204.¹⁴ Besides, intra-gastric injection of L. plantarum 21 at a daily dosage of 8 × 10¹⁰ cfu per kg bw for 2 weeks for female Wistar rats¹¹ and L. plantarum CLP-0611 at a daily dosage of 4 × 10¹⁰ cfu per kg bw for 3 days for male ICR mice⁹ can ameliorate effectively the disease symptoms of trinitrobenzenesulfonic acid-induced colitis.

5. Conclusions

In this study, seventeen Lactobacillus strains were evaluated for their auto-aggregation rate (RAA), co-aggregation rate (RCA), and cell surface hydrophobicity (AHC), illustrating a positive, linear RCA–RAA relationship for most of the Lactobacillus strains examined except L. casei AR342 and L. paracasei AR338 with extraordinarily low RCA values. Lactobacillus plantarum AR326 was discovered to have the topmost overall performance in aggregation and adhesion abilities among the selected strains concerned. This strain was found to colonize and sustain very well in the ileum and colon of ICR mice. In this study, L. plantarum AR326 could effectively alleviate the disease symptoms of ICR mice with DSS-induced colitis by reducing body weight loss, disease activity index, colon length shortening, MPO activity and histological damage. Further studies showed that the intervention of L. plantarum AR326 could restore the tight junction protein expression and reduce the abnormal expression of pro-inflammatory cytokines. In conclusion, L. plantarum AR326 can be considered as an excellent probiotic with high potential for ameliorating DSS-induced colitis. It can be applied as a nutraceutical supplement or pharmaceutical adjuvant.

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the National Natural Science Foundation of China (No. 31771956) and the “Shu Guang” project supported by Shanghai Municipal Education Commission.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c8fo01453a

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