Journal of Applied Microbiology ISSN 1364-5072

ORIGINAL ARTICLE

Lactobacillus salivarius Ren prevent the early colorectal carcinogenesis in 1, 2-dimethylhydrazine-induced rat model J. Zhu1,2, C. Zhu1,2, S. Ge1,2, M. Zhang3, L. Jiang1,2, J. Cui1,2 and F. Ren1,2 1 Beijing Laboratory for Food Quality and Safety, Key Laboratory of Functional Dairy, College of Food Science and Nutritional Engineering, China Agricultural University, Beijing, China 2 Beijing Higher Institution Engineering Research Centre of Animal Product, China Agricultural University, Beijing, China 3 School of Food and Chemical Engineering, Beijing Technology and Business University, China,

Keywords aberrant crypt foci, colorectal cancer, denaturing gradient gel electrophoresis, intestinal microflora, probiotic, short-chain fatty acids. Correspondence Fazheng Ren, China Agricultural University, P.O. Box 287, No. 17 Qinghua East Road, Beijing 100083, China. E-mail: [email protected] 2014/0166: received 26 January 2014, revised 3 March 2014 and accepted 17 March 2014 doi:10.1111/jam.12499

Abstract Aims: The objective of this study was to investigate the impact of Lactobacillus salivarius Ren (LS) on modulating colonic micro flora structure and influencing host colonic health in a rat model with colorectal precancerous lesions. Methods and Results: Male F344 rats were injected with 1, 2dimethylhydrazine (DMH) and treated with LS of two doses (5 9 108 and 1 9 1010 CFU kg 1 body weight) for 15 weeks. The colonic microflora profiles, luminal metabolites, epithelial proliferation and precancerous lesions [aberrant crypt foci (ACF)] were determined. A distinct segregation of colonic microflora structures was observed in LS-treated group. The abundance of one Prevotella-related strain was increased, and the abundance of one Bacillusrelated strain was decreased by LS treatment. These changes were accompanied by increased short-chain fatty acid levels and decreased azoreductase activity. LS treatment also reduced the number of ACF by c. 40% and suppressed epithelial proliferation. Conclusions: Lactobacillus salivarius Ren improved the colonic microflora structures and the luminal metabolisms in addition preventing the early colorectal carcinogenesis in DMH-induced rat model. Significance and Impact of the Study: Colonic microflora is an important factor in colorectal carcinogenesis. Modulating the structural shifts of microflora may provide a novel option for preventing colorectal carcinogenesis. This study suggested a potential probiotic-based approach to modulate the intestinal microflora in the prevention of colorectal carcinogenesis.

Introduction The human intestinal tract contains about 1014 bacteria that have a significant impact on the host physiology and pathology (Sobhani et al. 2011). Patients with colorectal cancer (CRC) have different intestinal microflora structures compared with the healthy population (Guarner and Malagelada 2003; Thompson-Chagoyan et al. 2007; Turnbaugh et al. 2008). The disordered microflora, especially several unfavourable bacteria are associated with a Journal of Applied Microbiology © 2014 The Society for Applied Microbiology

high risk of CRC (Collins et al. 2011). Previous studies using various CRC animal models concluded that colorectal microflora is involved in the aetiology of carcinogenesis (Dove et al. 1997; Kado et al. 2001; Uronis et al. 2009). Therefore, monitoring and modulating the structural shifts of intestinal microflora related to CRC may lead to a discovery of new methods for CRC prevention. Probiotics are widely used in the prevention of intestinal infections or diseases related to intestinal disorders because of their abilities to modulate the intestinal 1

LS prevent colorectal carcinogenesis

microflora (Gareau et al. 2010). Whether such an effect operates in the CRC-associated microflora remains to be shown. Studies reported that consumption of Lactobacillus or Bifidobacteria suppresses CRC in experimentally induced models (Urbanska et al. 2009; Leu et al. 2010; Chen et al. 2012). One possible mechanism is the modulation of microlflora metabolisms including short-chain fatty acids (SCFAs) and bacterial enzymes (Arthur and Jobin 2011; MacDonald and Wangner 2012). SCFAs, mainly acetate, propionate and butyrate, enhance cellular differentiation and reduce proliferation in colorectal cancer cell lines. Bacterial enzymes are capable of activating carcinogens, which may promote the process of tumorigenesis (Ruemmele et al. 2003; Yu et al. 2010). However, few studies focus on the connections between the microflora structure and metabolism. Aberrant crypt foci (ACF) induced by 1, 2-dimethylhydrazine (DMH) have been established as a surrogate biomarker of CRC, considering its high correlation to carcinogenesis and its recapitulation of pathologic and molecular features of potential patients with CRC (Mc Lellan and Bird 1991; Bird and Good 2000; Corpet and Tach
e 2002; Rudolph et al. 2005). A previous study suggested that the gut microflora structures of DMH-treated and control rats were similar at an early stage, but segregated after ACF formation, indicating ACF endpoint as a suitable biomarker. In the present study, we use this model to study the role of Lactobacillus salivarius Ren (LS) in modulating the structural shifts of colonic microflora and influencing the host health changes. LS were formerly shown to regulate the colonic microflora disorder caused by the carcinogen 4-nitroquinoline-1-oxide (4NQO) and reduce the incidence of oral cancer (Zhang et al. 2011, 2013). In this study, the efficiency of LS in influencing the colonic microflora profiles, luminal metabolites and epithelial processes was determined in the context of colorectal carcinogenesis. Materials and methods Animals and experimental procedure A total of 50 male F344 rats aged 5 weeks were obtained from Vital River Co., Beijing, China. All rats were housed in an animal-holding room under controlled conditions of 21  2°C, 50  10% humidity and 12 h light/dark cycle. Rats were fed purified basal rodent diets (Vital River Co) and had free access to water. After arrival, animals were quarantined for 1 week and divided randomly into five experimental groups. All experimental animal care and treatment followed the guidelines set by the Institutional Animal Care and Use Committee. 2

J. Zhu et al.

The experimental protocol for this study is shown in Fig. 1. The experimental groups were as follows: group one, saline; group two, high-dose LS (1 9 1010 CFU kg 1 body weight, c. 2 9 109 CFU for each rat); group three, DMH + saline; group four, DMH + low-dose LS (5 9 108 CFU kg 1 body weight, c. 1 9 108 CFU for each rat); group five, DMH + high-dose LS (1 9 1010 CFU kg 1 body weight, c. 2 9 109 CFU for each rat). After 2 weeks of oral inoculation with LS, rats received subcutaneous injections of DMH (Sigma chemicals Co., St Louis, MO; 30 mg kg 1 body weight in saline, pH = 6
5) once per week for 10 weeks. Inoculation with LS was maintained until week 15. LS (China General Microbiological Culture Collection 3606) were isolated from faecal samples of healthy centenarians, which were cultured in MRS broth (LuQiao, Beijing, China) at 37°C until late log phase, re-suspended in saline solution and supplied every day. Body weight and food intake were measured weekly. Rats were killed by CO2 asphyxiation. After laparotomy, all organs were examined. The colorectum was excised and opened longitudinally. The contents were collected and stored at 4°C for immediate analysis of SCFA concentrations and bacterial enzyme activities. Genomic DNA was extracted for microflora analysis. The colon and rectum were fixed flat in 10% buffered formalin using filter papers with the mucosa on the upper side. Quantification of ACF and measurement of cell proliferation Topographic analysis of the colorectal mucosa was performed after 24 h of fixation according to the method described by Bird (1995). The colon and rectum were stained with a 0
2% methylene blue solution for 10 min. The mucosal side of the colorectum was exposed on a microscopic slide, and viewed under a light microscope for ACF counting. The total number and size (number of component crypts in each ACF) of ACF in the entire colorectum were recorded. 0

2

12

15 (weeks) No treatment LS high DMH alone DMH + LS low dose DMH + LS high dose

Figure 1 Experimental protocol of 1, 2-dimethylhydrazine (DMH)induced colorectal tumorigenesis. Lactobacillus salivarius Ren (LS) low dose (5 9 108 CFU kg 1 body weight per day); LS high dose (1 9 1010 CFU kg 1 body weight per day). ( ) DMH injection, 30 mg kg 1 body weight; ( ) sacrifice for ACF analysis.

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Colon samples stored in 10% formalin were cut from distal segments and embedded in paraffin. The immunohistochemical staining of proliferating cell nuclear antigen (PCNA) was performed on paraffin-embedded sections using a specific rat monoclonal antibody against PCNA (Novus Biological, Littleton, CO). Images were viewed under a light microscope by using a 40 9 objective. PCNA-positive cells and the total number of cells were counted in 20 randomly chosen intact crypts, and the percentage of positively stained cells was calculated. Microbiology Genomic DNA was extracted by using the QIAamp stool mini kit (Qiagen, Du¨sseldorf, Germany) according to the manufacturer’s instructions. Then, genomic DNA was amplified using universal bacterial 16S rRNA primers aimed at the V3 variable region by a touchdown PCR reaction as described earlier (Femia et al. 2002). PCR products were examined by denaturing gradient gel electrophoresis (DGGE) (D-code Universal Mutation Detection System, Bio-Rad, Hercules, CA). The gel was silver stained and scanned using a CanoScan Lide 100 scanner (Canon, Tokyo, Japan). Gel images were analysed using QUANTITY ONE software (ver. 4.2, Bio-Rad), and the relative intensity of each band was calculated. Principal component analysis (PCA) and the linear model of redundancy analysis (RDA) were performed using CANOCO 4.5 (Biometrics, Wageningen, Netherlands). Bands corresponding to the strains of interest were excised from DGGE gels. DNA was extracted and amplified as previously described (Zhang et al. 2011). The resulting PCR products were cleaned with the Universal DNA Purification kit (Tiangen, Beijing, China) and cloned into Escherichia coli TOP10 competent cells (Tiangen) with the pMD18-T Simple Vector (Takara, Tokyo, Japan). Plasmid DNA was isolated from the E. coli cells using the TIANprep Mini Plasmid kit (Tiangen, China), and sequenced by Invitrogen Biotech Co. Ltd (Beijing, China) using M13F primers. The obtained sequences were compared with known sequences in the GenBank database using the BLASTN algorithm. SCFA analysis Samples of colorectal contents were homogenized in internal standard solution (heptanoic acid, 1
0 mmol l 1), and centrifuged at 3000 g for 10 min at 4°C. The supernatant was distilled, and 1 ll of the distillate was injected into the gas chromatograph (SHIMADZU GC-14, kyoto, Japan) equipped with a flame ionization detector and a capillary column (HP-FFAP, 25 m 9 0
32 mm, 0
5 lm). The initial oven temperature was 50°C and was increased by Journal of Applied Microbiology © 2014 The Society for Applied Microbiology

LS prevent colorectal carcinogenesis

10°C min 1 to 140°C and then increased to 240°C at 30°C min 1. The injector and detector were both set to 230°C. A standard SCFA mixture containing acetate, propionate and butyrate was used for calculation. Results are expressed as lmol g 1 faeces. Bacterial enzyme activities Samples of colorectal contents were homogenized in the phosphate-buffered saline of 0
1 mol l 1 and centrifuged at 3000 g for 10 min at 4°C. The supernatant was used for the assessment of b-glucosidase, b-glucuronidase and azoreductase as described by Goossens et al. (2003). Statistical analysis Quantitative data are expressed as mean  SD. Variables were analysed using either Student’s t-test for comparisons between means or one-way analysis of variance and Sidak post hoc comparisons (SPSS ver. 17.0, SPSS, Chicago, IL). The criterion for statistical significance was set at P < 0
05 for each endpoint. Results General observations and epithelial processes No observable changes in the health status of rats were detected during the experiment. The toxic effects of the DMH treatment were reflected in the significant decrease in final mean body weight of the DMH group (Table 1). Administration of LS had no significant effect on body weight, relative liver weights and relative kidney weights, indicating no toxic effect by consuming LS. No significant alterations of food consumption were observed in any of the groups during the experimental period, indicating that LS administration and DMH injection did not suppress the caloric intake. Colorectal ACF was identified and analysed by methylene blue staining under a light microscope. All rats developed ACF in the colorectum after DMH treatment, and no ACF formation was observed in saline-injected animals. There were significant differences in the final ACF formation between the DMH-treated group (147
5  36
2) and the LS-treated group (89
6  18
7 at low dose, P < 0
05; 87
9  19
4 at high dose, P < 0
05) (Fig. 2a). Approximately 40% reduction was observed in comparison between the DMH-treated and LS-treated groups. The number of high multiplicity ACF (ACF with more than three aberrant crypts) is thought to be the best predictor of cancer development (McLellan et al. 1991; Pretlow et al. 1992). LS administration also decreased the number of high multiplicity ACF (Fig. 2b). 3

LS prevent colorectal carcinogenesis

J. Zhu et al.

Table 1 Mean body weight, relative liver weight and relative kidney weight of rats in each group* Experimental group†

No. of rats

Body weight (g)

DMH DMH+LS low DMH+LS high No treatment LS high

10 10 10 10 10

307
8 313
2 313
4 323
6 322
7

    

Relative liver weight (g 100 g 1 body wt)

Food intake (g)

17
9a 19
0ab 16
8ab 22
3b 23
6b

17
31 16
53 16
07 17
71 16
87

    

1
24a 1
46a 1
38a 1
38a 0
95a

2
74 3
23 3
18 2
99 2
87

    

0
21ab 0
34bc 0
34bc 0
21ab 0
30ab

Relative kidney weight (g 100 g 1 body wt) 0
62 0
63 0
60 0
59 0
61

    

0
10ab 0
04ab 0
05a 0
05a 0
06a

*Data are presented as the mean  SD. One-way ANOVA analysis was performed among the five groups. Values not sharing common superscript (a–c) are significant with each other at P < 0
05. †DMH, 1, 2-dimethylhydrazine (30 mg kg 1 body weight); LS low (Lactobacillus salivarius Ren at 5 9 108 CFU kg 1 body weight per day); LS high (Lactobacillus salivarius Ren at 1 9 1010 CFU kg 1 body weight per day).

(c)

LS–

LS+

DMH–

70 #

60 50 40

*

*

*

*

30 20 10 0 C

on t LS rol hi gh D M H LS lo LS w hi gh

DMH+

(d) Ratio of PCNA-positive cells (%)

Total ACF/colorectum

Larger ACF/colorectum

(a) 220 (b) 24 21 200 180 18 160 15 140 12 120 9 100 6 80 3 60 40 0 DMH alone DMH+LS low DMH+LS high DMH alone DMH+LS low DMH+LS high

DMH

Colonic cell proliferation of rats in each group was evaluated by PCNA staining in complete, well-orientated distal colonic crypts. As shown in Fig. 2d, the DMH-treated group showed a significantly higher ratio of PCNApositive cells than the untreated group. In addition, rats treated with low or high-dose LS showed a lower PCNApositive index than the DMH alone group. Effect of Lact. salivarius Ren on microflora PCR–DGGE analysis of healthy, DMH-initiated and LS-treated rats for predominant bacteria was used to identify the structural response in the colonic microflora (Fig. 3a). PCA score plots (PC1 vs PC2) based on 4

Figure 2 Effects of Lactobacillus salivarius Ren (LS) on ACF formation and cell proliferation of colonic mucosa. Colons of rats from each group were removed, fixed, stained with methylene blue. ACF (a) and larger ACF with more than three crypts in each focus (b) were counted. The median (central thick lines), 25 and 75% quartile ranges (box width), and upper and lower limits (error bar) of each group are shown in the box plot. *Values are significantly different in comparison with DMH alone group, P < 0
05. (c) Proliferation cells were detected in distal colon crypts of rats by immunohistochemical analysis of proliferating cell nuclear antigen (PCNA). Rats were supplemented with LS (LS+) or not (LS-) after DMH (DMH+) or saline treatment (DMH-). (d) Ratio of PCNA-positive cells in crypt column from each group was quantified. Data are expressed as mean  SD. *P < 0
05, significantly different from DMH alone group. † P < 0
05, significantly different from control group. One-way analysis of variance and Sidak post hoc comparisons were performed.

the abundance of DGGE bands suggested a distinct segregation in the microflora structures of rats among three groups (Fig. 3b). When considering DMH initiation and LS treatment as environmental variables, the first axis of the PCA explained 19
5% of the total variation, and the first and second axes together explained 31
7%. Seven DGGE bands were identified as significant variables distinguishing DMH-induced rats from untreated rats based on the RDA test. Similarly, 14 DGGE bands were identified as variables distinguishing LS-treated rats from DMH-induced rats. These DGGE bands were identified by sequencing the eluted DNA, and the results were summarized in Table 2. The abundance of two Journal of Applied Microbiology © 2014 The Society for Applied Microbiology

J. Zhu et al.

(a)

LS prevent colorectal carcinogenesis

No treatment

DMH alone

DMH + LS high

(b)

–0·8

PC2 (12·2%)

0·8

1 2 3 4 5 6 7 81 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 M

–0·8

1·0

PC1 (19·5%)

Figure 3 Structural segregation analysis of gut microbiota in rats from different group. (a) 16s rRNA gene V3 region PCR-denaturing gradient gel electrophoresis (DGGE) profiles of faecal samples obtained from healthy rats (No treatment group), 1, 2-dimethylhydrazine (DMH)-treated rats (DMH alone group) and Lactobacillus salivarius Ren (LS)-treated group (DMH + LS high group). M represents the marker for DGGE analysis. (b) Principal component analysis (PCA) score plots (PC1 vs PC2) based on the PCR–DGGE profiles among no treatment ( ), DMH alone ( ) and DMH +LS high ( ) groups. The first and the second axes explained 1 9
5 and 12
2% of total variation, respectively.

Clostridium strains, two Bacteroidetes strains and one Firmicutes strain was correlated with DMH initiation. One Proteobacterium strain (B60) and one Ruminococcaceae strain (B14) were increased after DMH treatment. LS treatment decreased the prevalence of one Bacillus strain (B33) and one Ruminococcaceae strain (B14). The amounts of one Bacteroides strain (B6), one Lachnospiraceae strain

(B21), one Prevotella strain (B47) and one Clostridium strain (B50) were increased after LS treatment. Effect of Lact. salivarius Ren on faecal variables Short-chain fatty acid concentrations in the colonic content are shown in Table 3. All of the three SCFAs were

Table 2 Summary of identified bacterial strains significantly associated with 1, 2-dimethylhydrazine (DMH) initiation or Lactobacillus salivarius Ren (LS) treatment by redundancy analysis (RDA)* Band NO.

Closest relative

6 7 21 41 47 50 33 14 60 27 11 19 9 23 58 10 37 49

Bacteroides sp. XO77B42 Clostridium sp. Clone-27 Lachnospiraceae bacterium P43 Clostridium phytofermentans ISDg Prevotella sp. 152R-1a Clostridium bifermentans strain IBUN 188 Bacillus subtilis BSn5 Uncultured Ruminococcaceae bacterium clone Rs2-1 Uncultured Proteobacterium clone CTF2-180 Uncultured Lachnospiraceae bacterium clone MS176A1 Uncultured Clostridiales bacterium clone M_Fe_Clo108 Uncultured Clostridiales bacterium M_Fe_Clo045 Uncultured Bacteroidales bacterium Uncultured Bacteroidetes bacterium clone M0035_077 Uncultured Bacteroidetes bacterium clone TF2-36 Uncultured Firmicutes bacterium clone p1k11pool Uncultured Firmicutes bacterium clone CF2-123 Uncultured Firmicutes bacterium clone CM2-35

DMH

LS

% identity

Accession No.

+

100 96 97 98 97 100 100 98 99 98 100 100 98 99 99 100 99 100

AM230647.1 AB622841.1 AB730753.1 NR_074652.1 DQ278861.1 DQ680018.1 CP002468.1 JN653025.1 GU958264.1 EF707682.1 AB702872.1 AB702896.1 AB702761.1 EF071352.1 GU957853.1 HM104945.1 GU959067.1 GU959466.1

+ + + + + + + + + + + +

*Symbols ‘+’ and ‘ ’ indicate an increase or decrease in the abundance of the bacteria strain in response to environmental variables.

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Table 3 Short-chain fatty acid (SCFA) levels and bacterial enzyme activities in the colorectum of rats from different groups* DMH Variable

No treatment

LS high

Saline

LS low

LS high

1

Short-chain fatty acids (lmol g faeces) Acetate Propionate Butyrate Total SCFA Bacterial enzyme activities b-glucosidase (lmol h 1 mg 1 faeces) b-glucuronidase (lmol h 1 mg 1 faeces) Azoreductase (lmol h 1 g 1 faeces)

54
08 10
96 5
87 70
91

   

10
37† 1
88† 1
97† 12
20†

0
48  0
07 0
69  0
19 2
72  0
55†

66
62 12
51 9
53 88
66

   

5
60† 3
26† 1
88†,‡ 5
21†,‡

0
57  0
09 0
61  0
07 1
89  0
29†,‡

37
92 6
76 2
72 49
21

   

5
68‡ 1
05‡ 1
32‡ 6
25‡

0
50  0
08 0
77  0
11 4
13  0
72‡

43
13 7
08 5
64 52
93

   

10
71 2
36 2
93† 11
58‡

0
53  0
10 0
70  0
15 2
30  0
31†

47
38 9
51 7
91 64
80

   

4
58† 2
34† 2
46† 5
98†

0
61  0
15 0
72  0
14 2
07  0
38†,‡

*Values are mean  SD. DMH, 1, 2-dimethylhydrazine, LS, Lactobacillus salivarius Ren. †Significantly different from DMH alone group by unpaired t-test, P < 0
05. ‡Significantly different from control group by unpaired t-test, P < 0
05.

decreased after DMH treatment. Total SCFA concentrations were significantly higher in the high-dose group of LS compared with the DMH-treated group. Colonic butyrate concentration was significantly increased in both high-dose and low-dose groups of LS compared with DMH-treated group. Acetate and propionate concentrations were significantly increased only in the high-dose group of LS versus DMH-treated group. For rats without DMH injections, supplementation of LS increased the effect on butyrate and total SCFA concentrations compared with untreated counterpart. There was a significant increase in azoreductase activity of DMH-treated rats when compared to untreated rats (Table 3). LS treatment restored the activity to a lower level, with a stronger effect in the high-dose group than the low dose. However, the activities of b-glucosidase and b-glucuronidase were not significantly affected by either DMH or LS treatment. Discussion In our previous study, we demonstrated that LS counteracted unfavourable changes of colonic microflora induced by 4NQO (Zhang et al. 2011). By decreasing proliferation and increasing apoptosis of cancerous oral cells, LS also suppressed 4NQO-induced oral cancer (Zhang et al. 2013). These two studies suggested potential abilities of LS in modulating microflora and inhibiting cancer development. In the present study, we used a DMH-initiated rat model to evaluate the colonic microflora changes and the following metabolites changes by dietary consumption of LS. We also found that LS may significantly inhibit the formation of ACF, especially larger ACF (with more than three aberrant crypts per focus), suggesting the protective properties of LS supplement against colorectal carcinogenesis. 6

The PCA score plots of DGGE fingerprints indicated that colonic microflora compositions were significantly different in DMH-treated and untreated rats when a large number of ACF developed in their colons. This segregation between DMH-treated and untreated rats was consistent with a previous study, in which they identified Ruminococcus-like and Allobaculum-like bacteria as key variables for discrimination of two groups (Wei et al. 2010). In our study, we also found that one bacterial strain closely related to the Ruminococcaceae family was more abundant in DMH-treated rats. Differently, we found one Proteobacterium-related bacterial strain increased in the DMH-treated rats. This is consistent with a previous study showing increased levels of Proteobacteria in the adenoma samples of CRC patients (Shen et al. 2010). The underlying mechanisms are yet to be elucidated. Lactobacillus salivarius Ren played an important role in structuring colonic microfloral communities. The PCA analysis suggested that microbial community of LS-treated rats was varied from both the DMH-treated and untreated rats. Further investigation revealed 14 bacterial strains related to LS treatment. The relative abundance of one Prevotella-related strain was increased after LS treatment. This Prevotella-related strain was previously reported to be associated with high butyrate production in a study investigating the interaction between bovine ruminal microflora diversity and fermentation measurements (Hernandez-Sanabria et al. 2010). Conversely, LS treatment reduced the abundance of one Bacillus-related strain. The Bacillus species potentially produce azoreductase, which may play an important role in the initial stage of carcinogenesis (Rafil et al. 1990; Misal et al. 2011). Therefore, LS treatment improved the colonic microfloral structure in DMH-initiated rats, which potentially decreased the risk of colorectal carcinogenesis. Journal of Applied Microbiology © 2014 The Society for Applied Microbiology

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It should be noted that, in the PCA analysis, the first and the second axes only explained 31
7% of all variation, indicating that unaccounted reasons may affect the microfloral communities. Inter-individual variation may contribute to these effects. However, a relatively low percentage of explained variance is commonly found in the literature (Fuentes et al. 2008; Janczyk et al. 2010). The mechanisms by which colonic microflora influences the process of carcinogenesis may largely rely on the metabolisms produced by micro-organisms. In the present study, we focused on bacterial enzyme and SCFA. Investigations have revealed that the intestinal microflora can convert latent carcinogens into bioactive forms through the action of various enzymes (Arthur and Jobin 2011). Therefore, increased enzyme activity results in a high risk of DNA mutation at the initial stage of colorectal carcinogenesis (Misal et al. 2011). In this study, b-glucosidase and b-glucuronidase showed no alterations in responses to different treatments, but the azoreductase activity reduced in the LS-treated group, indicating a possible protective effect against carcinogenesis by decreasing levels of carcinogen activation and DNA mutation. This phenomenon might be partly associated with the fact of decreased Bacillus-related strain that produces azoreductase. Short-chain fatty acids, in particular butyrate, can induce differentiation, suppress proliferation and enhance apoptosis in colonic cancer cell lines (Maslowski et al. 2009; Havenaar 2011). In our study, we observed decreased SCFAs in the DMH-induced group and a reverse after LS consumption. The presence of SCFA may influence the colonic epithelial cells, resulting in a protective effect against colorectal carcinogenesis. By using immunohistochemical stain of PCNA, we found a decreased proliferation of colonic epithelial cells in LStreated rats, which may reduce the risks of mutations related to CRC development (Vogelstein 1988). The proportions of SCFAs are usually determined by colonic micro-organisms that ferment dietary carbohydrates. In the present study, we identified one Prevotella-related bacteria to be more abundant in LS-treated rats. This result may partly explain the increased production of SCFAs in LS-treated group. The present study showed that Lact. salivarius Ren can significantly modulate the microflora changes in the DMH-initiated rat model. These changes were accompanied with increased short-chain fatty acid levels and decreased azoreductase activity. We also determined that ACF formation was decreased by consumption of LS. ACF is accepted as a precancerous lesion of CRC (Mc Lellan and Bird 1991; Rudolph et al. 2005). People or animals with ACFs in their colons are still seemingly healthy, giving great challenge for screening or Journal of Applied Microbiology © 2014 The Society for Applied Microbiology

LS prevent colorectal carcinogenesis

surveillance of CRC in early stages (Bird and Good 2000; Corpet and Tach
e 2002). Dietary supplement of probiotics is a potential method for protection against colorectal carcinogenesis. Results in present study suggested a potential therapeutic approach based on the modulation of intestinal microflora by probiotic in the prevention of colorectal carcinogenesis. Acknowledgements This study was supported by Ministry of Science and Technology of China (2012BAD12B08, 2011AA100903) and the Beijing Municipal Commission of Education Co-constructed Program. Conflict of Interest The authors have no conflict of interests. References Arthur, J.C. and Jobin, C. (2011) The struggle within: microbial influences on colorectal cancer. Inflamm Bowel Dis 17, 396–408. Bird, R.P. (1995) Role of aberrant crypt foci in understanding the pathogenesis of colon cancer. Cancer Lett 93, 55–71. Bird, R.P. and Good, C.K. (2000) The significance of aberrant crypt foci in understanding the pathogenesis of colon cancer. Toxicol Lett 112, 395–402. Chen, C.C., Lin, W.C., Kong, M.S., Shi, H.N., Walker, W.A., Lin, C.Y., Huang, C.T., Lin, Y.C. et al. (2012) Oral inoculation of probiotics Lactobacillus acidophilius NCFM suppresses tumour growth both in segmental orthotopic colon cancer and extra-intestinal tissue. Br J Nutr 107, 1623–1634. Collins, D., Hogan, A.M. and Winter, D.C. (2011) Microbial and viral pathogens in colorectal cancer. Lancet Oncol 12, 504–512. Corpet, D.E. and Tach
e, S. (2002) Most effective colon cancer chemopreventive agents in rats: a systematic review of aberrant crypt foci and tumor data, ranked by potency. Nutr Cancer 43, 1–21. Dove, W.F., Clipson, L., Gould, K.A., Luongo, C., Marshall, D.J., Moser, A., Newton, M.A. and Jacoby, R.F. (1997) Intestinal neoplasia in the ApcMin mouse: independence from the microbial and natural killer (beige locus) status. Cancer Res 57, 812–814. Femia, A.P., Luceri, C., Dolara, P., Giannini, A., Biggeri, A., Salvadori, M., Clune, Y., Colins, K.J. et al. (2002) Antitumorigenic activity of the prebiotic insulin enriched with oligofrucose in combination with the probiotics Lactobacillus rhamnosus and Bifidobacterium lactis on azoxymethane-induced colon carcinogenesis in rats. Carcinogenesis 23, 1953–1960. 7

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Fuentes, S., Egert, M., Jim
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