HK1209364A1

HK1209364A1 - Use of actinomyces naeslundii in the treatment or prevention of rheumatoid arthritis or related diseases

Info

Publication number: HK1209364A1
Application number: HK15110193.7A
Authority: HK
Inventors: 冯强; 张东亚; 贾慧珏; 刘传; 肖亮; 王俊
Original assignee: 深圳华大基因科技有限公司; 深圳华大生命科学研究院
Priority date: 2014-09-30
Filing date: 2015-10-16
Publication date: 2016-04-01
Also published as: CN104546941A; CN104546941B

Abstract

The invention provides application of actinomyces naeslundii in treating or preventing rheumatoid arthritis or related diseases thereof, and further provides a pharmaceutical composition, a medicine, a food and feed containing the actinomyces naeslundii. Applied to animals, the actinomyces naeslundii can be used for effectively developing an anti-inflammatory efficacy, so that the symptom of redness and swelling of joint is effectively improved and the symptom of arthritis is remarkably relieved; and the actinomyces naeslundii can be effectively used for treating or preventing rheumatoid arthritis or related diseases thereof.

Description

Application of Actinomyces naeslundii in treating or preventing rheumatoid arthritis or related diseases thereof

Technical Field

The invention relates to the field of microbiology, in particular to application of Actinomyces naeslundii in treating or preventing rheumatoid arthritis or related diseases thereof, and also relates to a pharmaceutical composition, a medicament, food and feed containing the Actinomyces naeslundii.

Background

Rheumatoid Arthritis (RA), abbreviated as rheumatoid, is a systemic autoimmune disease that mainly affects the synovial membrane of joints, and secondly connective tissues such as serosa, heart, lung, blood vessels, nerves, etc. Acute phase inflammation is mainly manifested as synovial angioedema and synovitis, and if the acute inflammatory reaction is not diagnosed and treated in time, the acute inflammatory reaction is resolved, exudation is gradually absorbed, pannus is formed in synovium, and synovial cells are remarkably proliferated. Which in turn causes destruction of articular cartilage, inflammation, necrosis of bone tissue and proliferation of fibrous connective tissue. Calcification then occurs with fibrotic and bony arthrosclerosis, and the joint cavity is significantly narrowed or completely disappeared. Joint dysfunction caused by this stage of chronic inflammation process is largely difficult to recover, and can cause permanent disability. 1 person in every 100-150 persons worldwide suffers from RA, and the prevalence rate in China is 0.32% -0.36%. RA can develop at all ages, with adults often occurring in middle-aged women, with a ratio of about 1:3 between men and women. In 2003, the WHO reported that under good medical conditions, the annual treatment cost per person was about $ 6000, and the indirect losses caused by the disease were not negligible.

At present, the western medicine mainly adopts methods such as drug therapy, gene therapy and the like. The common western medicines mainly comprise non-steroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, slow-acting drugs, immunosuppressants, biological agents and the like. However, western medicines generally have the disadvantages of high price and large toxic and side effects, so that the application of the western medicines is limited. The gene therapy has high selectivity, can treat diseases from a molecular level, and can open up a new way for treating RA patients by limiting the long-term high-level local expression of genes. However, gene therapy is still in the initial stage, and still has more problems. Further studies are needed to find out whether the ideal vector is lacking, different hosts have different immune responses to the vector, and whether the introduced gene can be stably expressed in the body. In addition, the safety, treatment cost and ethical problems of gene therapy are also problems to be solved by the research.

There is evidence that gut microbiota are linked to systemic immune responses and RA. Lactic Acid Bacteria (LAB) can reduce the level of proinflammatory cytokine without affecting the regulation of the level of the cytokine, thereby playing an anti-inflammatory role and relieving RA symptoms. Therefore, it is presumed that some therapeutic means for restoring the intestinal micro-ecology to a normal state may exert a certain effect of alleviating RA symptoms while restoring the functions of the intestinal tract and the immune system. Research proves that LAB (Bacillus coagulans GBI-30,6086) can be a safe and efficient auxiliary drug for relieving RA symptoms

Because the etiology and pathogenesis of RA are not completely understood, the treatment of RA is advanced to a certain extent, but is still in the stage of controlling or relieving symptoms so far, and no effective treatment or cure method is available. Therefore, there is an urgent need in the art to develop an effective new drug for treating and preventing rheumatoid arthritis and related diseases without toxic and side effects.

Disclosure of Invention

The present invention is directed to solving, at least to some extent, one of the technical problems in the related art. To this end, it is an object of the present invention to propose the use of Actinomyces naeslundii for the treatment or prevention of rheumatoid arthritis or a disease related thereto.

In a first aspect of the invention, the invention provides the use of Actinomyces naeslundii for the manufacture of a medicament for the treatment or prevention of rheumatoid arthritis or a disease related thereto. The inventor finds that the medicine can effectively exert anti-inflammatory effect by providing animals, effectively improve symptoms of redness and swelling of joints, and obviously relieve symptoms of arthritis, namely effectively treat or prevent rheumatoid arthritis or related diseases thereof.

According to an embodiment of the invention, the Actinomyces naeslundii is Actinomyces naeslundii. Therefore, the efficacy of treating or preventing rheumatoid arthritis or related diseases thereof can be effectively exerted.

According to an embodiment of the present invention, the Actinomyces naeslundii is at least one selected from the group consisting of Actinomyces naeslundii ATCC43146, Actinomyces naeslundii ATCC12104 and Actinomyces naeslundii W1527. Therefore, the effect of treating or preventing rheumatoid arthritis or related diseases is better.

In a second aspect of the invention, the invention provides a pharmaceutical composition. According to an embodiment of the invention, the pharmaceutical composition comprises: actinomyces naeslundii; and pharmaceutically acceptable auxiliary materials. The inventor surprisingly finds that the pharmaceutical composition can obviously inhibit the generation of arthritis, obviously reduce redness and swelling of joints, obviously relieve the symptoms of the arthritis and flexibly move animals by providing the pharmaceutical composition for the animals, and indicates that the pharmaceutical composition can effectively treat or prevent the rheumatoid arthritis or related diseases thereof.

According to an embodiment of the present invention, when the pharmaceutical composition is in a solid state, the pharmaceutical composition comprises 1 × 10 to 1 × 10²⁰cfu/g of said Actinomyces naeslundii, when said pharmaceutical composition is in a liquid state, said pharmaceutical composition comprises 1X 10-1X 10²⁰cfu/mL of said Actinomyces naeslundii. Therefore, the effects of inhibiting the occurrence of arthritis and improving the symptoms of arthritis are remarkable, if the content of Actinomyces naeslundii is too low, the effect of treating or preventing rheumatoid arthritis is not satisfactory, and if the content of Actinomyces naeslundii is too high, the effect of treating or preventing rheumatoid arthritis is not remarkably improved, and waste is caused.

According to a preferred embodiment of the present invention, when the pharmaceutical composition is in a solid state, the pharmaceutical composition comprises 1 × 10⁴-1×10¹⁵cfu/g of said actinomyces naeslundii, said pharmaceutical composition comprising 1x 10 when said pharmaceutical composition is in a liquid state⁴-1×10¹⁵cfu/mL of said Actinomyces naeslundii.

According to another preferred embodiment of the present invention, when the pharmaceutical composition is in a solid state, the pharmaceutical composition comprises 1 × 10⁶-1×10¹¹cfu/g of said actinomyces naeslundii, when said pharmaceutical composition is in a liquid state, saidThe pharmaceutical composition comprises 1 × 10⁶-1×10¹¹cfu/mL of said Actinomyces naeslundii.

According to an embodiment of the present invention, the pharmaceutically acceptable excipient is at least one selected from the group consisting of a carrier, an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative, a sweetener, and a flavor.

According to an embodiment of the present invention, the pharmaceutically acceptable excipient is at least one selected from the group consisting of lactose, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil.

In a third aspect of the invention, the invention provides a medicament. According to an embodiment of the invention, the medicament comprises: the pharmaceutical composition as described hereinbefore. The medicine can obviously inhibit the generation of arthritis, obviously reduce redness and swelling of joints, obviously relieve the symptoms of the arthritis and enable animals to move flexibly by providing the medicine for the animals, and shows that the medicine can effectively treat or prevent the rheumatoid arthritis or related diseases thereof.

According to an embodiment of the present invention, the drug is in the form of at least one formulation selected from the group consisting of granules, capsules, tablets, powders, oral liquids, suspensions, and emulsions. Thus, administration is facilitated.

In a fourth aspect of the invention, a food product is provided. According to an embodiment of the invention, the food product comprises: actinomyces naeslundii; and a dietetically acceptable auxiliary material. The inventor finds that the food can obviously inhibit the generation of arthritis, obviously reduce redness and swelling of joints, obviously relieve arthritis symptoms and enable animals to move flexibly by providing the food for the animals, and shows that the food can effectively treat or prevent the rheumatoid arthritis or related diseases thereof.

It should be noted that the kind of the term "food" used in the present invention is not particularly limited, and may be any known food, including but not limited to dairy products, cookies, pastries, beverages, health products, and the like.

According to an embodiment of the invention, when the food product is in solid form, the food product comprises 1x 10-1 x 10²⁰cfu/g of said Actinomyces naeslundii, said food product comprising 1X 10-1X 10 when said food product is in liquid form²⁰cfu/mL of said Actinomyces naeslundii. Therefore, the effects of inhibiting the occurrence of arthritis and improving the symptoms of arthritis are remarkable, if the content of Actinomyces naeslundii is too low, the effect of treating or preventing rheumatoid arthritis is not satisfactory, and if the content of Actinomyces naeslundii is too high, the effect of treating or preventing rheumatoid arthritis is not remarkably improved, and waste is caused.

According to a preferred embodiment of the invention, when the food product is in solid form, the food product comprises 1x 10⁴-1×10¹⁵cfu/g of said Actinomyces naeslundii, said food product comprising 1x 10 when said food product is in liquid form⁴-1×10¹⁵cfu/mL of said Actinomyces naeslundii.

According to another preferred embodiment of the invention, when the food product is in solid form, the food product comprises 1x 10⁶-1×10¹¹cfu/g, said food product comprising 1x 10 when said food product is in a liquid state⁶-1×10¹¹cfu/mL of said Actinomyces naeslundii.

According to an embodiment of the present invention, the bromatologically acceptable auxiliary is at least one selected from the group consisting of a carrier, an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative, a sweetener, and a flavor.

According to an embodiment of the present invention, the dietetically acceptable auxiliary is at least one selected from the group consisting of lactose, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

According to an embodiment of the invention, the food product is in the form of at least one formulation selected from the group consisting of a solid, a dairy product, a solution product, a powder product and a suspension product.

In a fifth aspect of the invention, the invention provides a feed. According to an embodiment of the invention, the feed comprises: actinomyces naeslundii. The inventor finds that the feed can obviously inhibit the generation of arthritis, obviously reduce redness and swelling of joints of animals, obviously relieve arthritis symptoms and flexibly move the animals by providing the feed for the animals, and shows that the feed can effectively treat or prevent the rheumatoid arthritis or related diseases thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows the macroscopic scoring results of the effects of microbial inoculum 1, microbial inoculum 2 and microbial inoculum 3 on the onset of arthritis in mice according to one embodiment of the invention;

FIG. 2 shows the results of the effects of bacterial agent 1, bacterial agent 2 and bacterial agent 3 on the proliferation of splenocytes from arthritic mice in vitro, according to an embodiment of the present invention; and

FIG. 3 shows the results of the effects of bacterial agent 1, bacterial agent 2 and bacterial agent 3 on cytokines of arthritic mice, according to an embodiment of the present invention.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, molecular cloning is generally performed according to conventional conditions such as Sambrook et al: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press, 1989), or according to the protocol of microorganisms: the conditions described in the handbook of experiments (James Cappuccino and Natalie Sherman eds., Pearson Edurion Press) or as recommended by the manufacturer.

It is to be noted that the term "animal" used herein is not particularly limited, and may be any animal having intestinal tract, preferably mammal, more preferably human, rabbit or mouse.

According to the embodiment of the invention, the test animal is molded to form an arthritis model animal, and the model animal can obviously improve the redness and swelling of the joint of the animal and relieve the symptoms of arthritis by taking the Actinomyces naeslundii ATCC43146, the Actinomyces naeslundii ATCC12104 and the Actinomyces naeslundii W1527. According to the present example, arthritic DBA/1J mice treated with the strains Actinomyces naeslundii ATCC43146, Actinomyces naeslundii ATCC12104, and Actinomyces naeslundii W1527, respectively, had a marked regression of joint redness and swelling and a marked reduction in arthritic symptoms compared to the untreated control group. Thus, it was suggested that the strain can be used for the prevention and treatment of rheumatoid arthritis or its related diseases.

According to another preferred embodiment of the present invention, when the pharmaceutical composition is in a solid state, the pharmaceutical composition comprises 1 × 10⁶-1×10¹¹cfu/g, when the pharmaceutical composition is in a liquid state, the pharmaceutical composition comprises 1x 10⁶-1×10¹¹cfu/mL of said Actinomyces naeslundii.

According to an embodiment of the invention, when the feed is in solid state, the feed comprises 1x 10 to 1x 10²⁰cfu/g of said Actinomyces naeslundii, said feed comprising 1X 10-1X 10 when said feed is in a liquid state²⁰cfu/mL of said Actinomyces naeslundii. Therefore, the effects of inhibiting the generation of arthritis and improving the symptoms of arthritis are obvious, if the actinomyces naeslundiiThe effect of treating or preventing rheumatoid arthritis is not ideal due to too low bacteria content, and if the content of actinomyces naeslundii is too high, the effect of treating or preventing rheumatoid arthritis is not obviously improved, so that waste is caused.

According to a preferred embodiment of the invention, when the feed is in solid form, the feed comprises 1x 10⁴-1×10¹⁵cfu/g of said Actinomyces naeslundii, said feed comprising 1x 10 when said feed is in a liquid state⁴-1×10¹⁵cfu/mL of said Actinomyces naeslundii.

According to another preferred embodiment of the invention, when the feed is in solid form, the feed comprises 1x 10⁶-1×10¹¹cfu/g, when the feed is in a liquid state, the feed comprises 1x 10⁶-1×10¹¹cfu/mL of said Actinomyces naeslundii.

Example 1 identification of beneficial bacteria for the treatment or prevention of rheumatoid arthritis

1.1 sample Collection and DNA extraction

Stool, tartar and saliva samples of 212 volunteers were collected from Beijing collaborating hospitals, and the sample information is shown in Table 1, wherein the samples were divided into a training group and a test group. The training group included 157 volunteers, of which 77 were untreated RA cases and 80 were healthy (controls). In the experimental group, 34 of the volunteers formed 17 "case-healthy" pairs, 8 of which were related bloody, and 9 of which were not; the other 21 volunteers treated RA cases for DMARDs (disease-modifying anti-inflammatory drugs).

Stool samples were taken, then cryopreserved and rapidly transferred to-80 ℃ for storage, and DNA extraction was performed (ref Qian, J.et. A. reagent-wide association study of gut microbiota in type 2 diabetes. Nature 490, 55-60 (2012)). When a sample of tartar was collected, 3. mu.l of tartar was scraped from the tooth surface using a pair of medical forceps, transferred to 200. mu.l of 1 Xlysis buffer (10mM Tris,1mM EDTA, 0.5% Tween 20, 200. mu.g/ml proteinase K), incubated at 55 ℃ for 2 hours, then incubated at 95 ℃ for 10 minutes, the lysis reaction was terminated, and finally stored at-80 ℃. When saliva samples were collected, 100. mu.l of sample was taken into a collection tube containing 100. mu.l of 2 Xlysis buffer, and the scraped pharyngeal surface sample was also added to the tube, and the subsequent lysis step was the same as for tartar samples, and was also stored at-80 ℃. And (3) carrying out refrigerated transportation on the tartar sample and the saliva sample to Shenzhen Hua Dageneinstitute, and carrying out DNA extraction with reference to the feces sample.

When volunteers were first admitted, phenotypic information was collected according to standard procedures. Fecal samples from 21 DMARD treatment cases were included in only 212 fecal samples used to construct the gut microbiome gene set and were not analyzed here. The research is approved by ethical review committees of Beijing coordination Hospital and Shenzhen Huada Gene institute.

TABLE 1 sample information

1.2 metagenomic sequencing and Assembly

And constructing a sequencing library by using the extracted DNA sample, and performing bidirectional (Paired-end) metagenome sequencing (the insert fragment is 350bp and the read length is 100bp) on an Illumina sequencing platform. The data generated by sequencing was filtered (quality-controlled, to remove adapter contaminating sequences, to remove low quality sequences and to remove host genome contaminating sequences) and reassembled using the SOAPdenovo software (v 2.04).

Mean host genome contamination for stool, tartar and saliva samples were 0.37%, 5.55% and 40.85%, respectively.

1.3 Gene set construction

For the assembled fragments (contigs), gene prediction was performed using GeneMark software (v2.7d), followed by redundancy removal (identity above 95%, coverage above 90% without gaps (gaps)) using BLAT software, resulting in a non-redundant gene set comprising 3,800,011 genes for 212 stool samples, including 21 DMARD treated samples; for 203 oral samples (including 105 tartar samples and 98 saliva samples), a non-redundant gene set containing 3,234,997 genes was obtained. The gene set of the fecal sample is further supplemented to the published reference gene set of the intestinal microorganisms containing more than 430 genes by using BLAT software (the comparison similarity is more than 95%, and the comparison coverage is more than 90%, refer to the document Qin, j.et al.2012 above), and finally a new gene set containing more than 590 genes is obtained.

The high quality sequencing fragments (reads) were aligned to intestinal or oral reference gene sets to obtain the relative abundance of the genes (ref Qin, j.et al.2012, supra).

1.4 species Classification Annotation and abundance calculation

Species classification of the predicted genes is performed by comparison with an IMG (v400) database (refer to Qin, j.et al.2012, mentioned above), and species classification at the phylum level (the comparison similarity is 65% or more and the comparison coverage is 70% or more), species classification at the genus level (the comparison similarity is 85% or more) and species classification (the comparison similarity is 95% or more) are obtained. The relative abundance of this species was calculated using the relative abundance of the genes (see Qin, j. et al 2012, supra) and statistical tests (p <0.05) were performed using the rank-sum test (Wilcoxon rank-sum test) to determine species with significant differences in relative abundance between cases and controls.

1.5 metagenomic Association analysis

By comparing the fecal microbiome gene sets (microbiomes) of the cases to the controls, significant differences were found in the relative abundance of 83,858 genes (p <0.01, rank sum test, FDR 0.3285), marker genes, among 3,110,085 genes (each gene occurring in at least 6 samples, sample n-157, without the 21 DMARD-treated cases described in table 1). Further clustering according to the relative abundance difference of these marker genes in the sample to obtain the Metagenomic Linkage Group (Metagenomic Linkage Group, MLG, one MLG is similar to one species, and the clustering method is referred to the document Qin, j.et al.2012). In the same manner, from 2,247,835 genes of tartar (each gene is at least present in 6 samples, and sample n is 105), 209,820 marker genes (p <0.01, rank sum test, FDR is 0.072) were selected and clustered to obtain the MLG of tartar. From 2,404,726 genes in saliva (each gene appeared in at least 6 samples, sample n ═ 98), 206,399 marker genes (p <0.01, rank sum test, FDR ═ 0.088) were selected and clustered to obtain saliva MLG. In addition, 86158 marker genes (p <0.05, paired Wilcoxon rank-sum test, FDR ═ 0.912) were selected from 1,538,688 genes (each gene appeared in at least 6 samples, sample n ═ 64) by comparing genes of fecal microbes before and after DMARD treatment and clustered into fecal MLGs of DMARD treatment cases.

Based on the classification and relative abundance of MLGs constitutive genes, MLGs species classification and construction of MLGs relative abundance spectra are performed (see Qin, J.et al.2012, supra). If the classification is to the species level, it is required that 90% of genes in MLGs can be aligned to the genome of the species (the alignment similarity is more than 95%, and the alignment coverage is more than 70%). To classify the genes at the genus level, 80% of the genes in MLGs are required, and the similarity to the genus, whether in DNA or protein sequence, is higher than 80%. MLGs can be further clustered based on Kendall's correlation between abundance of all samples (regardless of case-control status). The results of the species classification of intestinal MLG, salivary MLG, tartar MLG and DMARD treatment-related MLG are shown in tables 2, 3, 4, 5, respectively.

TABLE 2 species Classification information of intestinal MLG

TABLE 3 species Classification information of salivary MLG

TABLE 4 species Classification information of tartar MLG

TABLE 5 species Classification information of MLG associated with DMARD treatment

Example 2 animal Experimental validation

To verify the determination of beneficial bacteria for the treatment or prevention of rheumatoid arthritis in table 2, table 3, table 4, table 5 of example 1, the inventors performed animal experiments for each species and verified the efficacy using the strains available for that species.

Experimental methods

1. Molding and administration

SPF (specific pathogen free) grade male DBA/1J mice 6-8 weeks old are purchased from Shanghai laboratory animal center of Chinese academy of sciences, and are bred in IVC (Integrated laboratory cage) mouse cage system (purchased from Von laboratory animal facilities, Suzhou) with indoor temperature of 22-24 ℃, humidity of 40-60%, daily illumination of 12h, and free feeding and drinking water. After 2 weeks of adaptive feeding, the cells were randomly divided into 5 groups (model control group, microbial inoculum 1 group, microbial inoculum 2 group, microbial inoculum 3 group, normal control group), and 12 cells were administered to each group. Actinomyces naeslundii ATCC43146 (inoculum 1) and Actinomyces naeslundii ATCC12104 (inoculum 2) were purchased from ATCC (American Type Culture Collection); actinomyces naeslundii W1527 (inoculum 3) was derived from the Atlanta disease Control Center (Center for disease Control, Atlanta, Ga.). The strain was cultured in modified meat culture Medium (ATCC Medium 1490) anaerobically at 37 deg.C for 24-48h, and the experiment was started after 16S rDNA sequencing. The strain information is shown in Table 6.

TABLE 6 information on the strains

Numbering	Bacterial name
		Bacterial agent 1	Actinomyces naeslundii ATCC 43146
Bacterial preparation 2	Actinomyces naeslundii ATCC 12104
		Bacterial agent 3	Actinomyces naeslundii W1527

Reference is made to the moulding process of the method of s.yoshino (s.yoshino, e.sasatomi, m.ohsawa, Bacterial lipopolypharmaceutical as an adjuvant to indole autoimmumus extract in mic, 2000) briefly:

from the 1 st day of experiment, fresh culture solutions (10) of gavage 1, bacteria 2 and bacteria 3 are respectively filled with 500 mul/50 g.BW dosage of bacteria 1 (bacteria 1+ CIA/LPS), bacteria 2 (bacteria 2+ CIA/LPS) and bacteria 3 (bacteria 3+ CIA/LPS)⁹-10¹⁰cfu/ml), model control group (M + CIA/LPS) and normal control group (M + AA + PBS) were separately intragastric with equal volume of sterile culture medium and administered for 28 days every other day. On days 1 and 21, generation of arthritis was induced in 3 model groups (microbial inoculum 1 group, microbial inoculum 2 group, and microbial inoculum 3 group) and a model control group of mice by using combination of type II Collagen (CIA) and Lipopolysaccharide (LPS). The specific operation is as follows: 100mg of type II collagen (obtained from articular cartilage of calf, Funakoshi Co) was dissolved in 100ml of 5mM Acetic Acid (AA), stirred overnight at 4 ℃ and injected intraperitoneally into mice. Then, 1. mu.g of lipopolysaccharide (Shanghai Biotech research institute) of E.coli 011: B4 was dissolved in 0.1ml of Phosphate Buffer Solution (PBS), and administered by intraperitoneal injection. The normal control group was injected i.p. with the same volume of 5mM acetic acid and PBS buffer at the same time point. The paw status of the mice was observed daily and the arthritis index was scored weekly. At 35 days after the start of the experiment, 6 cells were randomly selected from each group and sacrificed, and spleen cells were taken out for the subsequent experiment. The remaining mice were continued to observe the progression of the arthritic course and sacrificed by cervical dislocation 50 days after the start of the experiment.

2. Evaluation of arthritis

The severity of arthritis was macroscopically scored visually according to the increase in erythema and edema in tissues surrounding the joint to obtain an arthritis index, and specific scoring criteria were described as follows: 0: no signs of arthritis; 1: swelling and/or redness of 1 joint; 2: swelling and/or redness of 2 joints; 3: greater than 2 joints swelling and/or redness; 4: severe joint inflammation occurs throughout the paw. The arthritis index per mouse was the sum of the 4 paw scores per animal.

3. Effect of microbial inoculum 1, microbial inoculum 2 and microbial inoculum 3 on collagen-induced in vitro proliferation of splenocytes of arthritis mice

And randomly selecting 6 mice in each of a model group (a microbial inoculum 1 group, a microbial inoculum 2 group and a microbial inoculum 3 group), a model control group and a normal control group, and conventionally separating splenocytes. For the subsequent procedures, reference was made to the method of zhao xiaohui, etc. (study on the therapeutic effect and mechanism of sialon bone extract on bovine type II collagen-induced arthritis in mice, zhao xiaohui, yue hui lan, meili juan, shoan, pottery yan ze, 2008, month 5): the cell concentration was adjusted to 4X 10 by using 10% calf serum in RPMI 1640 medium⁶cell/ml. Mu.l of cell suspension and 100. mu.l of collagen antigen were added to each well of a 96-well plate. Cells in 5% CO₂The culture was carried out in an incubator at 37 ℃ and 25. mu.l of the solution was added to each well 8 hours before the completion of the culture³H-thymine nucleotide. The culture was continued until the end of the experiment. Collecting cells on a glass fiber membrane by using a cell collector, adding a scintillation fluid, and reading cell DNA on a beta counter³The amount of H-thymidine is expressed as counts per minute (cpm) of cell proliferation.

4. Influence of microbial inoculum 1, microbial inoculum 2 and microbial inoculum 3 on inflammatory cytokines of arthritic mice

Spleen cells were isolated by the conventional method, and the cell concentration was adjusted to 4X 10 with 10% calf serum in RPMI 1640 medium⁶cell/ml, 1 ml/well spleen cell suspension in 24-well tissue culture plates containing 1mM glutamine, 100U/ml penicillin, 100mg/ml streptomycin, 5X 10^-5M2-mercaptoethanol and 1% heat-inactivated autologous serum. After 48h, the supernatants were collected and stored at-70 ℃ for detection of the levels of cytokines IL-12, IFN-. gamma., TNF-. alpha.and IL-10 using an ELISA kit (purchased from Shanghai enzyme-linked Biotechnology Ltd.).

Results of the experiment

1. The effects of the microbial inoculum 1, the microbial inoculum 2 and the microbial inoculum 3 on the attack of arthritis of mice.

The macroscopic scoring results of the effects of the microbial inoculum 1, the microbial inoculum 2 and the microbial inoculum 3 on the onset of arthritis in mice are shown in table 7 and figure 1. According to the experimental results, after the microbial inoculum 1, the microbial inoculum 2 and the microbial inoculum 3 are administrated for 4 weeks, compared with a model control group, the generation of the arthritis of a CIA mouse can be obviously inhibited, the foot swelling of the mouse is obviously reduced, the arthritis symptom is obviously relieved, and the mouse moves more flexibly.

TABLE 7 Effect of bacterial agent 1, bacterial agent 2 and bacterial agent 3 on the onset of arthritis in mice (6 mice per group)

2. Influence of microbial inoculum 1, microbial inoculum 2 and microbial inoculum 3 on in vitro proliferation of splenocytes of arthritic mice

The results of the effects of the microbial inoculum 1, the microbial inoculum 2 and the microbial inoculum 3 on the in vitro proliferation of spleen cells of arthritic mice are shown in a table 8 and a figure 2, wherein the same letters are marked to indicate that the difference of the numerical values between the two groups is not significant through Tukey's Test detection (p is more than 0.05). According to the test results, by filling the microbial inoculum 1, the microbial inoculum 2 and the microbial inoculum 3, the proliferation reaction of splenocytes of arthritis mice stimulated by antigen in vitro is obviously reduced compared with a model control group under the condition of adding the antigen, and each model group also shows the splenocyte proliferation inhibition effect with different degrees under the condition of not adding the antigen.

TABLE 8 Effect of inoculum 1, inoculum 2 and inoculum 3 on the in vitro proliferation of splenocytes from arthritic mice (6 mice per group)

3. Effect of microbial inoculum 1, microbial inoculum 2 and microbial inoculum 3 on cytokine of arthritis mice

The results of the effects of the microbial inoculum 1, the microbial inoculum 2 and the microbial inoculum 3 on the cytokine of the arthritis mouse are shown in a table 9 and a figure 3, wherein the same letter marks indicate that the values between the two groups are not significantly different (p is more than 0.05) through Tukey's Test detection. According to the experimental result, the ELISA detection can find that the secretion of the cytostatic factor IL-10 of the mice in 3 model groups is obviously reduced (p is less than 0.05), and the secretion of inflammatory factors IL-12, TNF-alpha and IFN-gamma is obviously increased (p is less than 0.05) compared with that of a normal control group; compared with the model control group, the mouse cells in the model group have significantly increased IL-10 secretion (p <0.05), significantly reduced IL-12, IFN-gamma and TNF-alpha secretion (p <0.05), and no significant difference among 3 model groups (p > 0.05). The fact that the bacterium agent 1, the bacterium agent 2 and the bacterium agent 3 can inhibit the secretion of inflammatory factors IL-12, IFN-gamma and TNF-alpha in spleen cell culture supernatant through gastric perfusion and improve the secretion of the inflammatory factors IL-10 shows that the bacterium agent 1, the bacterium agent 2 and the bacterium agent 3 have obvious inhibition effect on hyperfunction of spleen cell immune function of arthritic mice.

TABLE 9 Effect of inoculum 1, inoculum 2 and inoculum 3 on cytokines in arthritic mice (6 mice per group)

Conclusion

The results show that the microbial inoculum 1, the microbial inoculum 2 and the microbial inoculum 3 can obviously inhibit the incidence of the mouse arthritis, relieve the related symptoms of the CIA mouse and show better anti-inflammatory action on the CIA mouse rheumatoid arthritis model.

In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.

Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.

Claims

1. Use of Actinomyces naeslundii for the preparation of a medicament for the treatment or prevention of rheumatoid arthritis or a disease related thereto,

optionally, the Actinomyces naeslundii is Actinomyces naeslundii,

optionally, the Actinomyces naeslundii is at least one selected from the group consisting of Actinomyces naeslundii ATCC43146, Actinomyces naeslundii ATCC12104, and Actinomyces naeslundii W1527.

2. A pharmaceutical composition, comprising:

actinomyces naeslundii; and

a pharmaceutically acceptable auxiliary material, and a pharmaceutically acceptable auxiliary material,

optionally, the Actinomyces naeslundii is Actinomyces naeslundii,

3. The pharmaceutical composition of claim 2, wherein when the pharmaceutical composition is in a solid state, the pharmaceutical composition comprises 1x 10 to 1x 10²⁰cfu/g, preferably 1X 10⁴-1×10¹⁵cfu/g, more preferably 1X 10⁶-1×10¹¹cfu/g of said Actinomyces naeslundii,

when the pharmaceutical composition is in a liquid state, the pharmaceutical composition comprises 1X 10 to 1X 10²⁰cfu/mL, preferably 1X 10⁴-1×10¹⁵cfu/mL, more preferably 1X 10⁶-1×10¹¹cfu/mL of said Actinomyces naeslundii.

4. The pharmaceutical composition according to claim 2, wherein the pharmaceutically acceptable excipient is at least one selected from the group consisting of a carrier, an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative, a sweetener, and a flavor,

optionally, the pharmaceutically acceptable adjuvant is at least one selected from the group consisting of lactose, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil.

5. A medicament, comprising:

the pharmaceutical composition of any one of claims 2-4.

6. The pharmaceutical according to claim 5, wherein the pharmaceutical is in the form of at least one formulation selected from the group consisting of granules, capsules, tablets, powders, oral liquids, suspensions, and emulsions.

7. A food product, comprising:

actinomyces naeslundii; and

auxiliary materials which are acceptable in the food science,

optionally, the Actinomyces naeslundii is Actinomyces naeslundii,

8. The food product of claim 7, wherein the food product comprises 1x 10 to 1x 10 when the food product is in a solid state²⁰cfu/g, preferably 1X 10⁴-1×10¹⁵cfu/g, more preferably 1X 10⁶-1×10¹¹cfu/g, of said Actinomyces naeslundii,

when the food product is in liquid form, the food product comprises 1X 10-1X 10²⁰cfu/mL, preferably 1X 10⁴-1×10¹⁵cfu/mL, more preferably 1X 10⁶-1×10¹¹cfu/mL of said Actinomyces naeslundii.

9. The food according to claim 7, wherein the sitologically acceptable auxiliary is at least one selected from the group consisting of a carrier, an excipient, a diluent, a lubricant, a wetting agent, an emulsifier, a suspension stabilizer, a preservative, a sweetener, and a flavor,

optionally, the dietetically acceptable auxiliary is at least one selected from the group consisting of lactose, glucose, sucrose, sorbitol, mannose, starch, gum arabic, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil,

optionally, the food product is in the form of at least one formulation selected from the group consisting of a solid, a dairy product, a solution product, a powder product and a suspension product.

10. A feed, comprising: the microorganism is an actinomyces naeslundii strain,

optionally, the Actinomyces naeslundii is Actinomyces naeslundii,