CN106573044B - Use of eubacterium for preventing and treating colorectal cancer-related diseases - Google Patents

Abstract

The use of microorganisms, in particular eubacterium strains, for the treatment and prevention of colorectal cancer-related diseases is disclosed. Compositions comprising a eubacterium strain are described.

Description

Use of eubacterium for preventing and treating colorectal cancer-related diseases

Cross Reference to Related Applications

Is free of

FIELD

The present invention relates to microbiology, in particular, the invention relates to bacterial strains of the genus Eubacterium for the treatment and prevention of colorectal cancer-related diseases in the present application, and further relates to compositions comprising Eubacterium and uses thereof.

Background

Colorectal cancer (CRC) is the third most common form of cancer, and is the second leading cause of cancer-related death in the western world (Schetter AJ, Harris CRC (2011) alternatives of microRNAs con tribution Carcinogenesis.Semin Oncol 38: 734-. Many people are diagnosed with CRC and many die worldwide each year from the disease. Although current strategies, including surgery, radiation therapy and chemotherapy, have significant clinical value for CRC, the recurrence and metastasis of post-operative cancer has hindered the success of these treatment modalities.

Chronic intestinal inflammation occurring in Inflammatory Bowel Disease (IBD) induces sustained damage along the gastrointestinal tract and increased mucosal permeability, playing an important role in the development of colorectal cancer (CRC). IBD, along with the hereditary syndromes of familial adenomatous polyposis and hereditary non-polyposis, is the first three high risk conditions for CRC (XieJ, Itzkowitz SH (2008) cancer inf laboratory bowel disease. world J Gastroenterol 14: 378-389, incorporated herein by reference). One of The two major clinical manifestations of IBD in patients with Ulcerative Colitis (UC), The relative risk of developing CRC correlates with The extent and duration of The disease (Eaden JA, Absrams KR, Mayberry JF (2001) The risk of clinical cancer in The scientific community: a meta-analysis. Gut48: 526-. In IBD patients, this risk increases by 0.5-1.0% annually after 8-10 years (Munkholm P (2003) Review article: the occupancy and presence of clinical cancer inhibition bone disease. organism pharmaceutical Heat 18 Suppl 2: 1-5, incorporated herein by reference).

The Human intestinal microflora, which contains about 100 trillion microorganisms, plays a critical role in maintaining host health (in the gastrointestinal tract and the whole body) by absorbing metabolites such as vitamins and short chain fatty acids (Moore WE, Holdeman LV (1974) Human functional flora: the normal flora of 20 Japanese-Hawaians. apple Microbiol 27: 961-979, incorporated herein by reference). Recent studies have demonstrated that specific strains of bacteria are involved in the regulation of intestinal homeostasis, delivering regulatory signals to the epithelial, mucosal immune systems and neuromuscular activity of the intestine (Shanahan F (2004) biology in inflammatory bowel disease-therapeutic ratio and roll. AdvDrug Deliv Rev 56: 809-818, incorporated herein by reference). In addition, some commensals and pathogenic organisms of the human intestinal microbiome are essential in the pathogenesis of IBD and CRC. Therefore, manipulation of intestinal bacterial composition and local metabolites through the use of probiotics has been explored as a promising approach for therapeutic intervention of CRC. Probiotics are live microbial feeding additives that beneficially affect the health of the host. They rely on the introduction of specific foreign strains into the gut microflora. For the study of CRC, the inventors performed analyses in the field of metagenomics, presenting data from deep metagenomic analysis of CRC intestinal microbiome.

Summary of The Invention

Embodiments of the present disclosure seek to address, at least to some extent, at least one of the problems presented in the prior art.

The present invention is based on the following findings of the present inventors:

the assessment and characterization of intestinal microbiota has become a major area of research in human diseases, including colorectal cancer (CRC), which is one of the most common causes of death in all types of cancer. To analyze intestinal microbial content in CRC patients, the inventors performed the protocol of the metagenomic-Wide association study (MGWAS) (Qin, j. et al a metagenome-Wide association study of gutmicrobiotia in type 2diabetes. nature 490,55-60(2012), incorporated herein by reference), which is based on deep shotgun sequencing of intestinal microbial DNA from 128 chinese individuals. The present inventors identified 2 probiotics. The inventors then validated animal experiments with probiotics associated with colitis and CRC, respectively. The results of animal experiments demonstrated the ability of Eubacterium ventriosum (Eubacterium ventriosum) and Eubacterium shigella (Eubacterium elegans) to effectively prevent and treat colitis and colorectal cancer.

Brief Description of Drawings

These and other aspects and advantages of the present disclosure will become apparent and more readily appreciated from the following description, taken in conjunction with the accompanying drawings, wherein:

FIG. 1 shows the P-value associated statistical distribution of all microbial genes in this study. Correlation analysis of CRC P-value distribution identified disproportionate over-expression of strongly related markers at lower P-values, where most genes followed the expected P-value distribution under the null hypothesis. This indicates that significant markers may represent true rather than false associations.

FIG. 2 the protective effect of stomach-administered Eubacterium ventriosum ATCC27560, Eubacterium ventriosum L2-12, Eubacterium ventriosum STAFF1042, Eubacterium picking ATCC27750, Eubacterium picking STAFF1020, Eubacterium picking TSDC10.2-1.1 on DSS-induced colitis C57BL/6J in (A) weight loss, (B) disease Activity index and (C) Colon length, respectively. Data are presented as mean ± sem. Different asterisks indicate significant differences (× P <0.05, × P <0.01, × P < 0.001).

FIG. 3 quantification using ELISA (A) TNF-. alpha.and (B) IL-10 in the colon obtained at day 15 after induction of colitis. Data are presented as mean ± sem. Different asterisks indicate significant differences (. + -. P <0.05,. + -. P <0.01,. + -. P <0.001)

FIG. 4 therapeutic effects of Eubacterium ventriosum ATCC27560, Eubacterium ventriosum L2-12, Eubacterium ventriosum STAFF1042, Eubacterium collecticum ATCC27750, Eubacterium collecticum STAFF1020, and Eubacterium collecticum TSDC10.2-1.1 on (A) disease activity index and (B) colon histopathology, respectively. Different asterisks indicate significant differences (. + -. P <0.05,. + -. P <0.01,. + -. P <0.001)

FIG. 5 Eubacterium ventriosum ATCC27560, Eubacterium ventriosum L2-12, Eubacterium ventriosum STAFF1042, Eubacterium shiitaginii ATCC27750, Eubacterium shiitaginii STAFF1020, Eubacterium shiitaginii TSDC10.2-1.1 regulate colon gene expression (relative to β -actin) in BABL/C mice, respectively. Data are presented as mean ± sem. Different asterisks indicate significant differences (× P <0.05, × P <0.01, × P < 0.001).

Detailed Description

The terms used herein have the meanings commonly understood by those of ordinary skill in the art to which the present invention pertains. Terms such as "a," "an," and "the" are not intended to refer to only a singular entity, but include the general class of which a particular embodiment may be used for illustration. The terms used herein are used to describe specific embodiments of the invention, but their usage does not limit the invention unless outlined in the claims.

In one aspect, the present application provides a eubacterium pick or eubacterium ventriosum for use in the prevention or treatment of colitis and/or colorectal cancer. In another aspect, the present application provides the use of a eubacterium shiitanum or eubacterium ventriosum for the manufacture of a medicament for the prevention or treatment of colitis and/or colorectal cancer. In another aspect, the present application provides a method for preventing or treating colitis and/or colorectal cancer, comprising administering to a patient in need thereof an effective amount of mycobacterium leii or eubacterium ventriosum. In another aspect, the present application provides a kit comprising a mycobacterium tryanus or a mycobacterium ventriosum and instructions for its use as a medicament. The present application also provides a pharmaceutical composition comprising a Eubacterium shiitake or Eubacterium ventriosum and a pharmaceutically acceptable carrier.

Preferably, the shigella is a strain selected from the group consisting of: the bacteria selected from the group consisting of Bacillus typhii ATCC27750, Bacillus typhii STAFF1020 and Bacillus typhii TSDC 10.2-1.1. Preferably, the eubacterium ventriosum is a strain selected from the group consisting of: eubacterium ventriosum ATCC27560, Eubacterium ventriosum L2-12 and Eubacterium ventriosum STAFF 1042. In one embodiment, the c.shigella or c.ventriculi, or the metabolite thereof, is administered in the form of live c.shigella or c.ventriculi.

The invention is further illustrated in the following non-limiting examples. Parts and percentages are by weight and degrees are in degrees celsius unless otherwise indicated. It will be apparent to those of ordinary skill in the art that, while indicating preferred embodiments of the invention, these examples are given by way of illustration only and that the reagents are commercially available.

Examples

Example 1 identification of Probiotics from 128 individuals in China

1.1 sample Collection and DNA extraction

Stool samples from 128 subjects, including 74 colorectal cancer patients and 54 healthy controls (table 1), were collected at the wils king Hospital (Prince of Wales Hospital) with informed consent. To be eligible for inclusion in the study, the individual must meet the following stool sample collection criteria: 1) no antibiotics or other medications, no special diet (diabetic, vegetarian, etc.) and normal lifestyle (no additional stress) for at least 3 months; 2) at least 3 months after any medical intervention; 3) there was no history of colorectal surgery, or any kind of cancer, or inflammatory or infectious diseases of the intestine. Subjects were asked to collect stool samples at home in standardized containers prior to colonoscopy and to store the samples immediately in a home freezer. The frozen samples were then transported in insulated polystyrene foam containers to the wales king hospital and immediately stored at-80 ℃ until use.

Stool samples were thawed on ice and DNA extraction was performed using qiagen qiaamp DNA pool Mini Kit according to the manufacturer's instructions. The extract is treated with DNase-free RNase to eliminate RNA contamination. The amount of DNA was determined using a NanoDrop spectrophotometer, a Qubit fluorometer (with Quant-iTTMdsDNA BR assay kit) and gel electrophoresis.

Table 1 baseline characteristics of colorectal cancer cases and controls in group I. BMI: a body mass index; eGFR: epidermal growth factor receptor; DM: type 2diabetes.

Parameter(s)	Control group (n ═ 54)	Case (n ═ 74)
			Age (age)	61.76	66.04
Sex (M: F)	33:21	48:26
			BMI	23.47	23.9
eGFR	72.24	74.15
			DM(％)	16(29.6％)	29(39.2％)
Intestine type (Enterotipe) (1: 2: 3)	26:22:6	37:31:6
			Disease stage (1: 2: 3: 4)	n.a.	16:21:30:7
Position (near end: far end)	n.a.	13:61

1.2DNA library construction and sequencing

The DNA library construction was performed according to the manufacturer's instructions (Illumina HiSeq 2000 platform). The present inventors used the same workflow as previously described for cluster generation, template hybridization, isothermal amplification, linearization, blocking and denaturation, and hybridization of sequencing primers (Qin, j. et al a methodome-wide association study of gutmicrobiotia in type 2diabetes 490,55-60(2012), incorporated herein by reference).

The inventors constructed a double-ended (PE) library with an insert size of 350bp for each sample, followed by high throughput sequencing to obtain approximately 30,000,000PE reads of length 2X100 bp. High quality reads were extracted by filtering low quality reads with 'N' bases, adaptor contamination and human DNA contamination from the raw data, and by simultaneously pruning the read low quality terminal bases. 751,000,000 metagenomic reads (high quality reads) were generated (5,860,000 reads per individual on average, table 2).

1.3 reading mapping

The inventors mapped high quality reads to a gene catalog (table 2) to a published reference intestinal gene catalog established by european and chinese adults (Qin, j. et al a methods-wide association study of gutmicrobiotita in type 2diabetes. nature 490,55-60(2012), incorporated herein by reference) (identity ≧ 90%), based on which they derived a gene profile (gene profile) using the same method of the T2D article (paper) disclosed in Qin et al (2012, supra). From a reference gene catalog (gene catalog) such as Qin et al (2012, supra), the inventors derived a subset of 2,110,489(2.1M) genes that appeared in at least 6 of all 128 hong kong samples.

Table 2 summary of metagenomic data and mapping to reference gene catalogue. The fourth column reports the results from the Wilcoxon rank sum test.

Parameter(s)	Control	Cases of disease	P-value
				Average raw readings	60162577	60496561	0.8082
After deleting low quality readings	59423292(98.77％)	59715967(98.71％)	0.831
				After deleting the reading of people	59380535±7378751	58112890±10324458	0.419
Refractive index	66.82％	66.27％	0.252

1.4 analysis of factors affecting the intestinal microbiota Gene Profile

To ensure robust comparison of gene content for 128 metagenomes (robust compare), the inventors created a set of 2,110,489(2.1M) genes present in at least 6 subjects, and used these 210 ten thousand genes to generate 128 gene abundance profiles (abundance profiles). The inventors used a permutation multivariate analysis of variance (PERMANOVA) test to assess the effect of different features (including age, BMI, eGFR, TCHO, LDL, HDL, TG, gender, DM, CRC status, smoking status and location) on the gene profile of the 2.1M gene. The inventors performed analysis using the method implemented in the "vegan" packet in R, and obtained aligned p-values by 10,000 alignments. The present inventors also corrected the multiplex test using "p.adjust" in R using the Benjamini-Hochberg method to obtain a q value of each gene.

When the inventors performed permutation-multivariate analysis of variance (PERMANOVA) on 13 different covariates, only CRC status was significantly correlated with these gene profiles (q 0.0028, Table 3), showing a stronger association (q 0.15) than the second best determinant BMI (body mass index). Thus, the data indicate changes in the genetic composition in the microbiome of CRC patients.

TABLE 3 PERMANOVA analysis using microbial gene profiles. Assays were performed to test whether clinical parameters and colorectal cancer (CRC) status had significant impact on intestinal microbiota (q < 0.05). BMI: a body mass index; DM: type 2 diabetes; HDL: high density lipoprotein; TG: a triglyceride; eGFR: epidermal growth factor receptor; TCHO: total cholesterol; LDL; low density lipoprotein.

1.5 CRC-related genes identified by MGWAS

1.5.1 identification of colorectal cancer-associated genes. The present inventors performed metagenomic association analysis (MGWAS) to identify genes that contributed to altered genetic composition in CRC. To identify associations between metagenomic profiles and colorectal cancer, a two-sided Wilcoxon rank-sum test was used in 2.1M (2,110,489) gene profiles. The inventors obtained 140,455 gene markers, which were enriched in cases or controls with P <0.01 (FIG. 1).

1.5.2 estimate false positive rate (FDR). Instead of the sequential P-value exclusion method, the inventors applied the "qvalue" method proposed in the previous studies (J.D. storage, R.Tibshirani, Statistical design for genetic materials, proceedings of the National Academy of Sciences of the United States of America 100, 9440 (8/5/2003), incorporated herein by reference) to estimate FDR. In MGWAS, a statistical hypothesis test was performed on a large number of characteristics of 140,455 genes. The false positive rate (FDR) was 11.03%.

1.6 data Spectrum construction

Based on the above obtained sequencing reads of 128 microbiota, the inventors examined the taxonomic differences between control and CRC-related microbiota to identify dysbiosis-causing microbiota. To this end, the inventors used a taxonomic profile (taxonomic profile) derived from three different methods, as supporting evidence from multiple methods would strengthen the association. First, the present inventors mapped the metagenomic reads to 4650 microbial genomes in the IMG database (version 400) and estimated the abundance of the microbial species (denoted IMG species) included in the database. Second, the present inventors estimated the abundance of molecular operational taxonomic units (mOTU) at the species level using universal phylogenetic marker genes (universal phylogenetic marker genes). Third, the inventors organized 140,455 genomes identified by MGWAS into Metagenomic Linkage Groups (MLGs) representing gene clusters derived from the same genome, annotated MLGs at the species level using the IMG database as much as possible, grouped MLGs based on these species, and then estimated the abundance of these species (denoted as MLG species).

1.6.1 Species annotation of the IMG genome (Specifes annotation)

For each IMG genome, the inventors identified the corresponding NCBI taxonomic classification at the species and genus level using the NCBI taxonomic classifier identifier provided by the IMG, using the NCBI taxonomic dump file (taxonomy dump file). Genomes without the corresponding NCBI species name are retained in their original IMG names, most of which are unclassified.

1.6.2 Gene profiling

The inventors mapped high quality reads to a gene directory to a published reference intestinal gene directory established by european and chinese adults (Qin, j. et al a metagenome-wide association study of gut microbiota type 2diabetes. nature 490,55-60(2012), incorporated herein by reference) (identity ≧ 90%), based on which they derived the gene profiles using the same method as the T2D article disclosed in Qin et al (2012, supra).

1.6.3mOTU Spectrum

Clean reads (high quality reads in example 1) were aligned to the mtotu reference (79268 sequences total) using default parameters (s.sanagawa et al, Metagenomic profiling using elementary genomic markers genes. nature methods 10,1196(Dec, 2013), incorporated herein by reference). The mOTU was identified at the 549 species level, including 307 annotated species and 242 mOTU linkage groups with no representative genome, which were presumed to be either Firmicutes or Bacteroidetes.

1.6.4 IMG species and IMG genus spectra

From download from http: jgi-psf.org (v.m. markowitz et al, IMG: the Integrated microbiological Genomes database and compatible analysis system. nucleic acids research 40, D115(Jan, 2012), incorporated herein by reference) extracts bacterial, archaeal and fungal sequences. A total of 522,093 sequences were obtained and the SOAP reference index (reference index) was constructed based on 7 equal-sized chunks (chunk) of the original file. Clean readings were aligned to a reference using a SOAP aligner (R.Li et al, SOAP 2: an improvedpaltrafast tool for short read alignment, Bioinformatics 25,1966(Aug 1, 2009), incorporated herein by reference) version 2.22, using the parameters "-m 4-s 32-r 2-n 100-x 600-v 8-c 0.9-p 3". SOAP coverage software (coverage software) was then used to calculate the read coverage for each genome, normalized by genome length, and further normalized for the relative abundance of each individual sample. The spectrum is generated based on only the read of the unique map.

1.7 identification of colorectal cancer-related species

Based on the identified gene profile of 140,455 colorectal cancer-associated markers, the present inventors constructed colorectal cancer-associated MLGs using the methods described in previous type 2diabetes studies (e.g., Qin et al 2012, supra). All genes were aligned to the reference genome of IMG database v400 to obtain genome level annotations. MLG is assigned to a genome if > 50% of constitutive genes are annotated to the genome, otherwise it is referred to as unclassified. A total of 87 MLGs with a gene number exceeding 100 were selected as colorectal cancer-associated MLGs. These MLGs are grouped based on species annotations of these genomes to construct MLG species.

To estimate the relative abundance of MLG species, the inventors estimated the average abundance of genes of MLG species after removing the 5% lowest and 5% highest abundance genes. The relative abundance of the IMG species is estimated by summing the abundance of IMG genomes belonging to the species.

These analyses identified 30 IMG species, 21 mtou and 86 MLG species that were significantly associated with CRC status (Wilcoxon rank sum test, q)<0.05; see tables 4, 5). In all three methods (Wilcoxon rank sum test-IMG: q. 0.0414; mOTU: q. 0.012757; MLG: q. 5.446X 10)^-4) Of the three, eubacterium ventriosum was consistently enriched in the control microbiota and enriched for eubacterium collectici according to two methods (Wilcoxon rank sum test-IMG: q is 0.069; MLG: q ═ 0.00031). These results indicate that both species have a great capacity as probiotics in the prevention and treatment of CRC related diseases.

Parvimonas micra (q), on the other hand<1.80x10^-5)，Peptostreptococcus stomatis(q<1.80x10^-5)，Solobacterium moorei(q<0.004331) and Fusobacterium nucleatum (q) are provided<0.004565) consistently enriched in the CRC patient microbiota in all three methods. Stores are associated with oral cancer (S.Pullalkar et al, Comparison of oral microbiota in biological of tissues with oral tissue cell Carcinoma. BMCmicrobiology 12,144(2012), incorporated herein by reference), S.moorei is associated with bacteremia (R.M.Pedersen, H.M.Holt, U.S.Justesen, Solobacterium moorei. bacteriosis: identification, antibacterial Suscibility, and clinical bacteriology. journal of clinical microbiology 49,2766 (7 months 2011), incorporated herein by reference). Recent studies using 16S rRNA sequencing have observed significant enrichment of Fusobacterium nucleatum in CRC tumor samples (m.castellarin et al, Fusobacterium nuclear infection previous in human tumor genome research 22,299(Feb, 2012), incorporated herein by reference), and this bacterium has been shown to have a viscosity (g.bachrach, c.iancuulovici, r.naor, e.i.weiss, fluorenscence bated tumors)of bacillus tuberculosis aggregation and of bacillus adherence to mammarian cells fems microbiology letters 248,235(Jul 15,2005) incorporated herein by reference), invasive (y.w. han et al, Interactions beta genetic bacteria and human oral epithelial cells: bacillus nucleuses to and innovations epithelial cells, infection and immunity 68,3140(Jun, 2000), and proinflammatory agents (a.d. kostic et al, bacillus nucleuses Intestinal tissues and models the turbine-immunity microorganism, cell Host Microbe 14,207(2013), incorporated herein by reference). These results confirm this association in a new population with different genetic and cultural origins. However, p.microra (obligate anaerobic bacteria) which can cause oral infections such as fusobacterium nucleatum (g.fundqvist, taxomonom, ecology, and pathogenesis of the root flora. organic surgery, organic media, and organic pathology 78,822(Oct, 1994), the highly significant enrichment in CRC-related microorganisms is a new finding p.microra is involved in the etiology of periodontal disease (periodontosis) (b.h. et al, polyportroscopicus microorganisms and tissue genetics introduction and genetics. journal of periodontal disease 71,209 (2000. 2), which is incorporated herein by reference, and which produces a broad range of proteins and proteins as an energy source (pest, protein, T.T.Larsen, M.B.Edlund, Ptostreptococcus microhas a unique requirement for capacity for use in the context of water from the family of organic microbiology and technology 8,42(Feb, 1993), incorporated herein by reference), which promotes Tumor growth and proliferation of colon cancer cells (C.Szabo et al, Tumor-derived hydrogen sulfate, produced by nuclear pathway-beta-synthesis, stilts bioenergetics,cell promotion, and genetics in cancer, proceedings of the National Academy of Sciences of the United states of America 110,12474 (23/7/2013), incorporated herein by reference. Micra is involved in the pathogenesis of CRC or whether its enrichment is the result of CRC-related changes in the colon and/or rectum. However, it may represent an opportunity for noninvasive diagnosis of biomarkers for CRC.

Example 2 validation in animal experiments

To verify the ability of probiotics, Eubacterium ventriosum and Eubacterium shilicii, in the prevention and treatment of CRC-related diseases, the present inventors performed animal experiments.

2.1 validation in animal experiments related to colitis

2.1.1 methods

Ten to twelve week old C57BL/6J mice were prepared for DSS-induced colitis studies under SPF conditions. After acclimation, a portion of mice were freely obtained drinking water containing 3.5-5% DSS (dextran sulfate sodium) for 5 days for colitis induction. Eubacterium ventriosum ATCC27560, Eubacterium ventriosum L2-12, Eubacterium ventriosum STAFF1042, Eubacterium fastidiosa ATCC27750, Eubacterium fastidiosa STAFF1020, and Eubacterium fastidiosa TSDC10.2-1.1 (10)⁹-10¹⁰cfu/ml, freshly prepared daily) was administered to the mice by intragastric gavage daily from day 1 to day 14 after induction of colitis, respectively. The control group of non-colitis and colitis received the vehicle in the stomach (500 μ l/50g.bw per day, each group n ═ 10).

C57BL/6J mice were purchased from southern medical university laboratory animals center, China. Eubacterium ventriosum ATCC27560 and Eubacterium shiitanum ATCC27750 were purchased from American Type Culture Collection (ATCC).

Eubacterium ventriosum L2-12: estelle Deville, Freda M.McIntosh, Sylvia H.Duncan and R.John Wallace (3 months 2007). Metabolim of Linear Acid by Human Gut bacterium, Differencen Routes for Biosynthesis of jointed Linear Acid, journal of bacteriology 189 (6): 2566-2570.

Eubacterium ventriosum STAFF1042, Eubacterium shikimi STAFF 1020: kageyama, A et al, ranking of human biological bacteria species and related species by treated PCRmethods, Microbiology and Immunology (2001)45 (4): 315-318.

The bacteria of the genus Pectibacterium TSDC10.2-1.1 is available from the university of Washington (USA) of St.Louis as http:// www.genomesonline.org/projects? And id 47012.

Mice were observed daily and clinical measurements were recorded. Body weight and disease activity index (table 6) (Alex P, Zachos NC, Nguyen T, Gonzales L, ChenTE, Conklin, LS et al 2009 distentine patterns identified from multiple profiles of muscle DSS and TNBS-induced pathology. inflamboul Dis; 15 (3): 341-52, incorporated herein by reference) were evaluated before DSS administration and weekly after intragastric feeding, and inflammation was monitored at day 15 after induction of colitis when mice were sacrificed by cervical dislocation.

TABLE 6 clinical Scoring criteria

The colon was removed, the fat-free and mesentery excised, carefully opened, and washed with PBS (phosphate buffered saline). The colon length was measured. Colonic injury and inflammation were assessed blindly according to Wallace criteria. The colons were also collected in liquid nitrogen and stored at-80 ℃ for ELISA (enzyme linked immunosorbent assay) examination.

2.1.2 results

2.1.2.1 the severity of DSS-induced colitis in mice was reduced by Eubacterium ventriosum ATCC27560, Eubacterium ventriosum L2-12, Eubacterium ventriosum STAFF1042, Eubacterium pickeri ATCC27750, Eubacterium pickeri STAFF1020, and Eubacterium pickeri TSDC10.2-1.1, respectively.

The inventors explored the anti-inflammatory effects in vivo by testing the ability of the bacteria Mycobacterium picking ATCC27750, STAFF1020, TSDC10.2-1.1 and Eubacterium ventriosum L2-12, STAFF1042, ATCC27560 to restore acute colitis induced by DSS, respectively. After DSS treatment, severe acute inflammation was observed in the colitis control group, and no significant protection was observed in mice receiving sterile culture medium. In contrast, each daily intragastric administration of 6 live strains of eubacterium resulted in significant reduction of colitis with reduced weight loss (table 7, fig. 2A) and a reduction of the disease activity index associated with colitis on days 7 and 14 after intragastric gavage (data spectrum on day 7 is similar to that on day 14 and not shown here). And the partial colon length was normalized compared to the media-treated group (table 8, fig. 2B, 2C). There was no difference in body weight and disease activity index at the beginning of intragastric gavage. Data are presented as mean ± sem.

TABLE 7 body weight data (W0: body weight before DSS treatment; W1: body weight on day 7 after intragastric gavage)

ATCC27560 represents eubacterium ventriosum ATCC 27560; l2-12 represents Eubacterium ventriosum L2-12, STAFF1042 represents Eubacterium ventriosum STAFF1042, ATCC27750 represents Eubacterium collectici ATCC27750, STAFF1020 represents Eubacterium collectici STAFF1020, and TSDC10.2-1.1 represents Eubacterium collectici TSDC 10.2-1.1.

TABLE 8 data of clinical measurements related to colitis

2.1.2.2 Eubacterium ventriosum ATCC27560, Eubacterium ventriosum L2-12, Eubacterium ventriosum STAFF1042, Eubacterium fastigii ATCC27750, Eubacterium fastigii STAFF1020, and Eubacterium fastigii TSDC10.2-1.1 respectively regulate the expression of colon inflammation

Colonic pro-inflammatory TNF-alpha (tumor necrosis factor alpha) cytokines and anti-inflammatory IL-10 (interleukin-10) were quantified by ELISA. TNF-alpha levels were increased compared to the colitis-free control group, whereas IL-10 levels were hardly changed in colitis mice. After treatment with the suspension of Mycobacterium picking ATCC27750, the secretion of TNF- α (FIG. 3A, Table 8) was significantly lower than that of the colitis control and vehicle groups. Meanwhile, IL-10 secretion was induced in the colon of mice treated with ATCC27750 (FIG. 3B, Table 8). A similar phenomenon was observed in C57Bl/6J mice dosed with Eubacterium ventriosum ATCC27560, Eubacterium ventriosum L2-12, Eubacterium ventriosum STAFF1042, Eubacterium fastigii STAFF1020 and Eubacterium fastigii TSDC10.2-1.1 (FIG. 3, Table 8).

2.2 validation in animal experiments relating to colorectal cancer

2.2.1 methods

BABL/c mice were stimulated with azoxymethane (AOM, 10mg/kg) at 10 weeks of age, followed by treatment with drinking water containing 2.0% Dextran Sodium Sulfate (DSS) for 7 days, beginning at weeks 2, 5 and 8, to induce colitis-associated colorectal cancer (CRC). Then, the strains were treated with Eubacterium ventriosum ATCC27560, L2-12, STAFF1042, and Eubacterium pickeri ATCC27750, STAFF1020, TSDC10.2-1.1 (10) respectively from week 9 of the study⁹-10¹⁰cfu/ml, freshly prepared daily) or control medium until sacrifice by cervical dislocation at week 11 (0.1 ml/50g.bw per day, n ═ 15 per group). BABL/c mice were purchased from southern medical university laboratory animals center, China.

Disease activity index was calculated as described previously, and mice undergoing AOM + DSS stimulation were evaluated before intragastric gavage and weekly after intragastric gavage.

For histopathological examination, after necropsy, colon sections were fixed in 10% buffered neutral formalin, subsequently embedded in paraffin, then sectioned (6 μm) and stained with H & E (hematoxylin and eosin). The tissue slides were examined in an Olympus microscope (lympus america inc., Dulles, VA). Specimens were blinded histologically and scored 1-4 for adenomas and adenocarcinomas.

For RNA isolation and real-time polymerase chain reaction of cytokines, tTotal RNA from the colon was isolated using the QiagenRNA isolation kit (Qiagen) according to the manufacturer' S instructions, then used to generate cDNA templates using the iScript cDNA synthesis kit (Bio-Rad, Hercules, Calif.) and real-time RT-PCR was performed as described previously (Bassagaya-Riera J, Reynolds K, Martino-Catt S, Cui Y, Hennighausen L, et al (2004), Activation of PPAR gamma and delta by conjugated linear enzyme detection from experimental amino acid tissue patent. Gastroen-technology 127: 777-791, incorporated herein by reference). mRNA expression of CD36 and PPAR γ (peroxisome proliferator-activated receptor γ) was assessed by real-time quantitative PCR.

2.2.2 results

2.2.2.1 following daily administration of Eubacterium ventriosum L2-12 or Eubacterium ventriosum STAFF1042 or Eubacterium ventriosum ATCC27560 or Eubacterium collectins STAFF1020 or Eubacterium collectins TSDC10.2-1.1 or Eubacterium collectins ATCC27750 for 2 weeks, the disease activity index was significantly lower than that of the CRC control group (Table 9), while no difference was shown in the inflammation-driven colorectal cancer model prior to intragastric gavage (data not shown here). And the effect of the treatment of 6 eubacterium strains, respectively, on the colon histopathology of the experimental azoxymethane-induced colorectal cancer was examined, and all these treatments reduced adenoma and adenocarcinoma formation compared to the control (figure 4, table 9).

2.2.2.2 Eubacterium ventriosum L2-12 or Eubacterium ventriosum STAFF1042 or Eubacterium ventriosum ATCC27560 or Eubacterium collecticum STAFF1020 or Eubacterium collecticum TSDC10.2-1.1 or Eubacterium collecticum ATCC27750 modulate the expression of colonic inflammation and oncogenic markers, respectively.

Mice stimulated with azoxymethane and DSS were euthanized at the tumor-bearing stage of the disease. Those mice treated with either Mycobacterium picker ATCC27750 or Mycobacterium picker STAFF1020 or Mycobacterium picker TSDC10.2-1.1 or Mycobacterium ventri L2-12 or Mycobacterium ventri STAFF1042 or Mycobacterium ventri ATCC27560 showed increased mRNA expression of CD36 and PPAR γ in the colon compared to the vehicle treated control group (FIG. 5, Table 9).

TABLE 9 data of clinical measurements related to CRC

In summary, the results of this study demonstrate the ability of Eubacterium ventriosum L2-12 or Eubacterium ventriosum STAFF1042 or Eubacterium ventriosum ATCC27560 or Eubacterium collectins STAFF1020 or Eubacterium collectins TSDC10.2-1.1 or Eubacterium collectins ATCC27750 to improve both colitis and colorectal cancer.

Although illustrative embodiments have been shown and described, it will be understood by those skilled in the art that the above embodiments are not to be construed as limiting the present disclosure and that changes, substitutions and alterations can be made thereto without departing from the spirit, principles and scope of the present disclosure.

Claims (5)

1. Use of shiitake bacterium in the manufacture of a medicament for the prevention or treatment of colitis and/or colorectal cancer.

2. The use according to claim 1, wherein the Mycobacterium is a strain selected from the group consisting of: the bacteria selected from the group consisting of Bacillus typhii ATCC27750, Bacillus typhii STAFF1020 and Bacillus typhii TSDC 10.2-1.1.

3. The use according to claim 1 or claim 2, wherein the Mycobacterium is administered in the form of live Mycobacterium monocytogenes.

4. A kit for use in the prevention or treatment of colitis and/or colorectal cancer comprising a mycobacterium pickles and instructions for its use as a medicament, wherein the mycobacterium pickles is a strain selected from the group consisting of: the bacteria selected from the group consisting of Bacillus typhii ATCC27750, Bacillus typhii STAFF1020 and Bacillus typhii TSDC 10.2-1.1.

5. A pharmaceutical composition for preventing or treating colitis and/or colorectal cancer comprising a shiitake bacterium and a pharmaceutically acceptable carrier, wherein the shiitake bacterium is a strain selected from the group consisting of: the bacteria selected from the group consisting of Bacillus typhii ATCC27750, Bacillus typhii STAFF1020 and Bacillus typhii TSDC 10.2-1.1.

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