Application of clostridium praeparatum in preparation of medicine for treating ankylosing spondylitis

Abstract

The invention belongs to the technical field of biological medicine, and provides application of clostridium pralaense in preparation of medicine for treating ankylosing spondylitis. According to the application disclosed by the invention, an AS animal model is combined to determine that the F.prausnitzi can prevent the AS progress; experiments find that after intragastric intervention is performed on the AS mouse with the clostridium pralaense, the AS intestinal flora tends to be balanced, the level of inflammatory factors in AS bodies is reduced, inflammatory damage to peripheral joints and spines of the AS mouse is relieved, and the progress of AS model mouse diseases is inhibited, so that a new strategy can be provided for AS treatment through targeted regulation and control of the intestinal flora homeostasis by the clostridium pralaense. In combination with experimental exploration, the following conclusion is drawn: F.prausnitzii can regulate and control the homeostasis of intestinal flora and inhibit the in-vivo inflammatory response of AS, so that the progress of AS is prevented, and the purpose of treating AS is achieved.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and more specifically, to the application of Faecalibacterium prausnitzii in the preparation of a drug for treating ankylosing spondylitis. Background Art

[0002] Ankylosing spondylitis (AS) is a chronic inflammatory autoimmune disease. The clinical treatment drugs for AS are mainly non-steroidal anti-inflammatory drugs, steroid hormones, biological agents, etc. Although they can alleviate clinical symptoms to a certain extent, they are also accompanied by more adverse reactions, and long-term use is likely to cause damage to the liver and kidneys. In addition, the current treatment methods are mainly to control the disease, relieve pain, and prevent and treat deformities, and there is no radical cure. Due to the lack of timely and effective treatment, some patients develop to the late stage of the disease, resulting in spinal stiffness, joint deformities and disability, and even bedridden for life, unable to take care of themselves.

[0003] Therefore, it is particularly important to strengthen the research on the pathogenesis of AS and find effective clinical diagnosis and treatment methods, and it is urgent to develop an effective drug for the treatment of AS. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems existing in the above-mentioned prior art. To this end, the present invention proposes the use of Faecalibacterium prausnitzii in the preparation of a drug for treating ankylosing spondylitis. The present invention is evaluated through an AS model and found that Faecalibacterium prausnitzii has the effect of inhibiting inflammation by regulating intestinal flora and thus preventing the progression of AS.

[0005] The first aspect of the present invention provides the use of Faecalibacterium prausnitzii in the preparation of a medicament for treating ankylosing spondylitis.

[0006] Specifically, the use of Faecalibacterium prausnitzii in the preparation of drugs for treating ankylosing spondylitis.

[0007] Application of Faecalibacterium prausnitzii in the preparation of medicines for treating intestinal flora disorders.

[0008] Application of Faecalibacterium prausnitzii in the preparation of medicines for treating intestinal flora disorders in patients with ankylosing spondylitis.

[0009] Application of Faecalibacterium prausnitzii in the preparation of drugs for treating ankylosing spondylitis by regulating intestinal flora disorders.

[0010] There are about trillions of bacteria in the human intestine, which constitute a complex microbial community system called "intestinal flora", which plays a key role in maintaining human health. Faecalibacterium prausnitzi (F.prausnitzii) is an important butyrate-producing probiotic in the intestine. The present invention studies the inflammatory cascade reaction in AS caused by intestinal flora disorders, finds out the key therapeutic targets, and finds that Faecalibacterium prausnitzii can inhibit the inflammatory response in AS by regulating the intestinal flora, thereby preventing the progression of AS.

[0011] A second aspect of the present invention provides a drug for treating ankylosing spondylitis.

[0012] A medicine for treating ankylosing spondylitis, comprising Faecalibacterium prausnitzii as a main raw material component.

[0013] Preferably, the dosage form of the drug is one of powder, capsule, tablet, emulsion and suspension.

[0014] The third aspect of the present invention provides an intestinal flora homeostasis regulator.

[0015] An intestinal flora homeostasis regulator comprises Faecalibacterium prausnitzii as a main raw material component.

[0016] Preferably, the intestinal flora homeostasis regulator is an intestinal flora homeostasis regulator for patients with ankylosing spondylitis.

[0017] Preferably, the dosage form of the intestinal flora homeostasis regulator is one of powder, capsule, tablet, emulsion and suspension.

[0018] A fourth aspect of the present invention provides a drug for treating ankylosing spondylitis by regulating intestinal flora disorder.

[0019] A drug for treating ankylosing spondylitis by regulating intestinal flora disorder, comprising Faecalibacterium prausnitzii as a main raw material component.

[0020] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0021] The present invention uses an AS animal model for research. Through experiments, it is found that oral administration of F. prausnitzi to AS mice can make the intestinal flora of AS tend to balance, reduce the level of inflammatory factors in the AS body, reduce the inflammatory damage of the peripheral joints and spine of AS mice, and inhibit the progression of the disease in AS model mice. It is further clarified that F. prausnitzi can prevent the progression of AS, and thus proposes to target and regulate the homeostasis of intestinal flora through F. prausnitzi, providing a new strategy for treating AS. Combined with a series of experimental research on the AS animal model, the present invention draws the following conclusions: F. prausnitzii can inhibit the inflammatory response in the AS body by regulating the homeostasis of intestinal flora, thereby preventing the progression of AS and achieving the purpose of treating AS. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a diagram showing the effect of F. prausnitzii on the joint index and swelling degree of AS mice;

[0023] Figure 2 This is a diagram showing the effect of F. prausnitzii on the pathology of joints and spine tissues in AS mice;

[0024] Figure 3 This is a graph showing the effect of F. prausnitzii on the levels of serum inflammatory factors in AS mice;

[0025] Figure 4 This is the β-diversity map of the intestinal flora of each group of mice sequenced by 16S rDNA;

[0026] Figure 5 This is the abundance composition diagram of the intestinal flora of each group of mice sequenced by 16S rDNA at the phylum level;

[0027] Figure 6 This figure shows the effect of F. prausnitzii on ileum histology and intestinal barrier function in AS mice. DETAILED DESCRIPTION

[0028] In order to make the technical scheme of the present invention more clearly understood by those skilled in the art, the following embodiments are listed for illustration. It should be noted that the following embodiments do not limit the protection scope of the present invention.

[0029] Unless otherwise specified, the raw materials, reagents or devices used in the following examples can be obtained from conventional commercial sources or by existing known methods.

[0030] Application effect verification

[0031] In order to clarify whether intervention with F. prausnitzii can inhibit the inflammatory cascade and thus prevent the progression of AS, the following study combined the AS animal model and evaluated the therapeutic effect of F. prausnitzii through microbiome and molecular biology methods. The specific experiments and their results are as follows.

[0032] 1. Experimental methods: Using AS animal models to evaluate the efficacy of F. prausnitzii in inhibiting AS progression

[0033] The specific method of constructing the AS mouse model is as follows: using the proteoglycan (PG) immunization method, cartilage proteoglycan (PG) is selected to induce polyarthritis in BALB / c mice, and this mouse model is used as an animal model of AS. This model is currently the only systemic autoimmune mouse model that invades the axial skeleton. On days 0, 21, and 42, mice were intraperitoneally injected with 100 mg of emulsified PG and 2 mg of dimethyldioctadecyl ammonium bromide (DDA) for induction. This model mouse can develop ankylosing of the spine and limbs and swelling of the joints, which can provide a strong guarantee for verifying that F. prausnitzii alleviates the onset and progression of AS.

[0034] Each group of mice was raised in an SPF environment (SPF environment, namely Specific Pathogen Free, refers to the completely sterile environment of experimental animals) and divided into the following three groups (10 mice / group). The following corresponding gavage treatments were given at the 10th week after PG induction. The entire experimental period lasted 14 weeks. Among them, the mice in the F. prausnitzii intervention group were gavaged (low concentration 1×10 9 CFU / mL) 0.2mL F. prausnitzii bacterial solution, once a day for 4 weeks, and the other groups were given an equal volume of normal saline instead. The details are as follows:

[0035] a. Control group (denoted as Control group): wild-type normal mice + normal saline;

[0036] b. AS mouse model group (referred as AS group): AS mice + normal saline;

[0037] cFprausnitzii intervention group (denoted as F.prausnitzii group): AS mice + F.prausnitzii.

[0038] Next, the following tests were carried out:

[0039] ①Regularly observe the systemic lesions of mice in each group and regularly record the arthritis index scores

[0040] After the experiment began, the mice in each group were observed daily for manifestations such as diarrhea, joint swelling, hair loss, etc., and their body weights were measured weekly. The severity of peripheral arthritis was evaluated by observing the changes in the mice's paws daily. The following arthritis index scoring criteria were used for scoring (0 - 4 points): 0 = normal, without arthritis; 1 = focal mild swelling and / or redness of one joint; 2 = moderate redness and swelling of one or more joints in the foot; 3 = obvious swelling and erythema of all joints and ankles; 4 = severe swelling of the entire paw. The disease severity and progression were determined based on the cumulative scores of all four paws of each mouse (maximum score 16 points), and the paw thickness of each mouse was measured using a vernier caliper. After the mice were sacrificed at the end of the 14th week, the hind feet and spinal tissues were dissected, then fixed in 4% paraformaldehyde, and further decalcified in 10% Titriplex EDTA for one month. The decalcified tissues were embedded in paraffin, cut into 5-μm thin sections and stained with HE (i.e., hematoxylin-eosin staining).

[0041] Ankle joint pathological examination and pathological scoring: After sectioning the ankle joints of the mice, HE staining was performed to observe the histopathological changes and conduct histopathological scoring. The scoring was carried out from three aspects: synovial inflammation, cartilage degeneration, and bone erosion.

[0042] The synovial inflammation scoring was as follows: 0 = normal; 1 = mild synovial proliferation, 2 - 4 layers of synovial cells, with a small amount of inflammatory cell infiltration; 2 = moderate synovial thickening, more than 4 layers of synovial cells, and a moderate number of infiltrating cells in the synovial layer; 3 = severe synovial proliferation, with a large number of inflammatory cells infiltrating into the synovium.

[0043] The cartilage degeneration scoring was as follows: 0 = normal; 1 = mild cartilage damage; 2 = moderate cartilage damage, with evidence of synovial cell invasion into the damaged cartilage; 3 = severe cartilage damage.

[0044] The bone erosion scoring was as follows: 0 = normal; 1 = mild loss of cortical bone in a few areas; 2 = moderate loss of cortex and trabecular bone; 3 = severe erosion, extensive bone damage.

[0045] The histological scoring of the intervertebral joints was graded from 1 - 4: 1 = accumulation of inflammatory cells around the intervertebral disc (IVD) and / or infiltration of the annulus fibrosus; 2 = mild inflammation, IVD absorption / erosion (<50% of the IVD); 3 = severe inflammation of the IVD, severe destruction and disappearance of the intervertebral space (>50%); 4 = complete cartilage / bony ankylosis. The overall score of each mouse was calculated by averaging the scores of 10 intervertebral joints of each mouse.

[0046] ② Detect the expression levels of inflammatory factors in each group

[0047] At the end of the 14th week, mice in each group were anesthetized with Avertin and blood was collected by cardiac puncture. After blood coagulation, serum samples were separated by centrifugation at 1500g for 10 min and stored in a refrigerator at -80°C. The concentrations of inflammatory factors IL-17, IL-23, IL-1β, and IL-18 in serum were detected by the steps of the ELISA kit instructions.

[0048] ③ Detect the fecal flora of each group of mice by 16S rDNA sequencing to explore the regulatory effect of F. prausnitzii on the intestinal flora of AS

[0049] At the end of the 14th week of the experiment, 12 hours after gavage, the feces of mice in each group were collected. The feces were collected in a sterilized clean bench. The abdomen of the mice was gently massaged to encourage them to defecate on the clean bench. The feces excreted by the mice were collected into sterile cryopreservation tubes, and the sample was not less than 0.2g per tube. At the same time, the sterile cryopreservation tubes were marked and stored in a -80℃ refrigerator within 2 hours after sample collection. Illumina HiSeq PE250 was used to perform 16S rDNA sequencing analysis on the bacterial DNA. UPARSE was used to control the quality of the raw data. Usearch software was used to remove chimeras and cluster the data. When clustering in Usearch, the reads were first sorted from large to small according to abundance, and OTUs (Operational Taxonomic Units) were obtained through standard clustering with a similarity of 97%. Each OTU was considered to represent a species. R software was used for principal coordinate analysis (PCoA), species abundance analysis, and heat map analysis. The rank sum test (Kruskal-Wallis) method was used to analyze the significant differences between species in different groups. LEfSe was used to use linear discriminant analysis (LDA) to estimate the influence of the abundance of each component (species) on the difference effect, and to find out the communities or species that had a significant difference in sample division. The threshold of LEfSe analysis was LDA score > 2. PICRUS software was used to predict the function analysis of intestinal flora from 16S rDNA data.

[0050] ④HE staining was used to detect the morphological changes of the ileum of each group of mice, and the intestinal permeability was evaluated by DX-4000-FITC probe to evaluate the effect of F. prausnitzii on the intestinal barrier function of AS mice

[0051] a. In vivo intestinal permeability test: At the 14th week of the experiment, mice were fasted for 6 h without water supplementation, and then given 4 kDa dextran labeled with fluorescein isothiocyanate (DX-4000-FITC, Sigma-Aldrich) (600 mg / kg body weight, 125 mg / mL) by gavage. Blood samples (300 μL) were collected from behind the eye sockets 1 h later, and serum was prepared for fluorescence measurement. The FITC concentration was analyzed using a fluorescence spectrophotometer at an excitation wavelength of 485 nm and an emission wavelength of 535 nm.

[0052] b. Intestinal tissue pathology: At 14 weeks, the mice were killed and the terminal ileum and colon tissues were quickly removed. One part was used to extract RNA and total protein, and the other part was fixed, routinely dehydrated, paraffin-embedded, and made into pathological tissue sections. The tissue morphology and pathological scores were observed after HE staining: 1) Hematoxylin-eosin staining: routine dewaxing, hematoxylin staining, 1% hydrochloric acid alcohol differentiation, eosin staining, water washing, graded alcohol dehydration, xylene transparent, and neutral gum sealing; 2) Microscopic observation of intestinal inflammatory cell infiltration and intestinal barrier integrity.

[0053] ⑤Statistical evaluation of the effect of F. prausnitzii in inhibiting AS progression

[0054] Statistical analysis of all test results was performed using SPSS 22.0 statistical software. Data were expressed as mean ± standard deviation (X ± SD), and one-way ANOVA was used for data between multiple groups. Bonferroni method was used for pairwise comparisons between groups. P value < 0.05 was considered statistically significant. The effect of F. prausnitzii in inhibiting the progression of AS was evaluated.

[0055] 2. Experimental Results

[0056] Effects of F. prausnitzii on joint index and swelling of AS mice in each group Figure 1 As shown, Figure 1 (A) is the joint index of mice in each group (n=10 / group). Figure 1 (B) is the thickness of the paws of mice in each group (n=10 / group). Figure 1 (C) is a typical picture of the hind paws of mice in each group at the end of the 14th week (scale bar is 2 mm). Values ​​are expressed as mean ± standard deviation (#P<0.05, ##P<0.01 compared with the control group; *P<0.05, **P<0.01 compared with the AS group). As can be seen from the figure, the AS group mice gradually developed redness and swelling of the feet and the joint index gradually increased from the 7th week of induction ( Figure 1 A) Increased claw thickness ( Figure 1B), while the control group did not show this symptom, indicating that the AS mouse model was successfully established. However, after intragastric administration of F. prausnitzii, the redness and swelling of the mice's feet could be significantly alleviated ( Figure 1 C), compared with the AS group mice, the joint index and paw thickness of the F. prausnitzii treatment group were significantly reduced (P<0.05). Figure 1 Where Time is time, Weeks is weeks, Arthritis scores is joint index, and Paw thickness is paw thickness.

[0057] like Figure 2 As shown, Figure 2 (A) HE staining images of typical ankle joint sections of mice in each group. The AS group was characterized by synovial hyperplasia, inflammatory cell infiltration (black arrows), cartilage degradation (yellow arrows), and bone erosion (red arrows) (scale bar, 500 μm). Figure 2 (B) HE staining images of typical sections of intervertebral joints in each group. The AS group is characterized by severe inflammatory cells gathering around the intervertebral disc (black arrow), excessive bone matrix formation (yellow arrow), and partial intervertebral disc destruction (red arrow) (scale bar, 50 μm). Figure 2 (C) Pathological scores of ankle joints of mice in AS group and F. prausnitzii group (n=6). Figure 2 (D) is the pathological score of intervertebral joints in AS group and F. prausnitzii group (n=6). Values ​​are expressed as mean ± standard deviation (*P<0.05, **P<0.01 compared with AS group). Pathological HE staining results of ankle joints showed that the ankle joint tissue structure of mice in the control group was normal, without synovial hyperplasia and inflammation, no signs of cartilage degeneration and bone erosion, and normal joint space. Compared with the control group mice, the AS group mice showed obvious arthritis, which was characterized by obvious synovial hyperplasia, inflammatory cell infiltration, cartilage erosion and bone erosion ( Figure 2 A. Figure 2 C). Compared with the AS group mice, the joint synovial hyperplasia score, cartilage degeneration score and bone erosion score of the F.prausnitzii group mice were significantly reduced after treatment with F.prausnitzii (P<0.05), indicating that arthritis was inhibited to a certain extent. At the same time, it was observed that the intervertebral tissue structure of the control group mice was normal, the annulus fibrosus and nucleus pulposus were intact, and there was no inflammatory cell infiltration around them; while the intervertebral structure of the AS mice was destroyed, with extensive accumulation of inflammatory cells around the intervertebral disc, excessive bone matrix formation, and destruction of the intervertebral space; in the mice treated with F.prausnitzii, the inflammatory cells around the intervertebral disc decreased, and the formation of bone matrix and the destruction of the intervertebral disc were significantly reduced ( Figure 2 B. Figure 2D, P<0.01). Figure 2 Histological score of arthritis is the histological score of arthritis, Histological score of vertebral joints is the histological score of vertebral joints, Synovia inflammation is synovial inflammation, Cartilage degradation is cartilage degradation, and Bone erosion is bone erosion.

[0058] The expression levels of inflammatory factors IL-17, IL-23, IL-1β and IL-18 in the serum of mice in each group were detected by ELISA. Figure 3 As shown, Figure 3 (A)-(D) are the results of IL-17, IL-23, IL-1β, and IL-18, respectively. Values ​​are expressed as mean ± standard deviation (n = 10; ## P<0.01 compared with the control group; * P<0.05, ** P<0.01 compared with the AS group). As shown in the figure, compared with the control group, the levels of inflammatory factors IL-17, IL-23, IL-1β and IL-18 in the serum of mice in the AS group were significantly increased (P<0.01). However, compared with the AS group, the levels of serum IL-17, IL-23, IL-1β and IL-18 in the F. prausnitzii group were significantly decreased (P<0.05), indicating that F. prausnitzii can reduce the levels of inflammatory factors in AS mice.

[0059] like Figure 4 As shown, β diversity analysis can be used to evaluate the differences in microbial community structure between different groups. The principal coordinates analysis (PCoA) method is used to show the differences between samples. Each point represents each sample, and different colors represent samples belonging to different groups. The intestinal flora of mice in each group is analyzed based on the Weighted UniFrac principal coordinates analysis (PCoA) diagram ( Figure 4 ) It can be seen that the AS group and the control group samples were significantly separated, indicating that the bacterial community structures of the two groups were quite different, while the distance between the F. prausnitzii group and the control group was reduced, indicating that the bacterial community structures of the two groups were highly similar. Figure 4 Weighted Unifrac means weighted uniform.

[0060] like Figure 5As shown in the figure, at the phylum level, Bacteroidetes, Firmicutes, Proteobacteria, and Verrucomicrobia were the main flora in the intestinal tract of each group of mice, accounting for about 99%. In the control group, the highest proportion of Bacteroidetes was 60.9%, followed by Firmicutes at 33.8%. Compared with the control group, the proportion of Firmicutes in the AS group increased to 57.12% (p<0.05), and the proportion of Bacteroidetes decreased to 36.48% (p<0.05). After AS mice were treated with F. prausnitzii, the proportion of Bacteroidetes increased to 69.2%, and the proportion of Firmicutes decreased to 27.6%. The ratio of Bacteroidetes to Firmicutes in the control group was 1.8, which decreased to 0.63 in the AS group. After treatment with F. prausnitzii, the ratio of Bacteroidetes to Firmicutes in the F. prausnitzii group increased to 2.5. Figure 5 In the Phylum Level Barplot, Relative Abundance is the relative abundance, Candidatus Saccharibacteria is the saccharifying bacteria of Candida, Deferribacteres is the iron-removing bacteria, Actinobacteria is the Actinobacteria, Tenericutes is the phylum, Spirochaetes is the phylum Spirulina, Cyanobacteria / Chloroplast is the cyanobacteria / chloroplast, and Other is others.

[0061] The intestinal barrier function of each group of mice was evaluated as follows Figure 6 As shown, Figure 6 (A) is the HE staining result of ileum in each group (scale bar is 50 μm). Figure 6 (B) is the DX-400-FITC level in the serum of each group (n=10). Pathological observation under light microscope showed that the villi of the ileum mucosa of the control group were dense, complete and neatly arranged ( Figure 6 A). Compared with the control group, the arrangement of small intestinal villi in the ileum of mice in the AS group was disordered, the villi became shorter, and the depth of crypts increased ( Figure 6 B). After treatment with F. prausnitzii, the morphological changes of the ileum of mice in the F. prausnitzii group were significantly improved compared with those in the AS group. The intestinal permeability was evaluated by using a fluorescently labeled tracer called DX-4000-FITC. When the intestine is damaged, DX-4000-FITC in the intestine can enter the blood. Therefore, by measuring the concentration of DX-4000-FITC in the blood, the intestinal permeability can be evaluated. The results showed that compared with the control group, the serum DX-4000-FITC level in the AS group mice was significantly increased ( Figure 6 B, P<0.05), indicating that the intestinal permeability of AS mice increased. Compared with the AS group, the serum DX-4000-FITC level of mice was reduced after treatment with F. prausnitzii, and the difference was statistically significant (P<0.05), indicating that F. prausnitzii can reduce the intestinal permeability of AS mice. Figure 6 Gut permeability refers to intestinal permeability.

Claims

1. Application of Faecalibacterium prausnitzii in the preparation of drugs for the treatment of ankylosing spondylitis.

2. Application of Faecalibacterium prausnitzii in the preparation of drugs for the treatment of intestinal flora disorders.

3. The use of Faecalibacterium prausnitzii in the preparation of drugs for treating intestinal flora disorders in patients with ankylosing spondylitis.

4. The use of Faecalibacterium prausnitzii in the preparation of drugs for treating ankylosing spondylitis by regulating intestinal flora disorders.

5. A drug for treating ankylosing spondylitis, characterized in that: The method comprises using Faecalibacterium prausnitzii as a main raw material component.

6. The drug according to claim 5, characterized in that The dosage form of the medicine is one of powder, capsule, tablet, emulsion and suspension.

7. A regulator of intestinal flora homeostasis, characterized in that: The method comprises using Faecalibacterium prausnitzii as a main raw material component.

8. The intestinal flora homeostasis regulator according to claim 7, characterized in that The intestinal flora homeostasis regulator is an intestinal flora homeostasis regulator for patients with ankylosing spondylitis.

9. The intestinal flora homeostasis regulator according to claim 7, characterized in that The dosage form of the intestinal flora homeostasis regulator is one of powder, capsule, tablet, emulsion and suspension.

10. A drug for treating ankylosing spondylitis by regulating intestinal flora disorder, characterized in that: The method comprises using Faecalibacterium prausnitzii as a main raw material component.

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