Parabacteroides coumarini and application thereof

Abstract

The invention belongs to the technical field of biological medicines and microorganisms, and particularly relates to a parabacteroides couchneri MRD02 and application thereof, the parabacteroides couchneri is Parabacteroides couchneri MRD02, the preservation number is GDMCC No.64793, and the preservation unit is Guangdong Province Microbial Culture Collection Center. The invention further discloses a preparation method of the parabacteroides couchneri MRD02 and application of the parabacteroides couchneri MRD02. The Parabacteroides goldsteinii MRD02 disclosed by the invention can be used for remarkably inhibiting joint swelling and systemic inflammation of rheumatoid arthritis of mice, and the inhibiting effect of the Parabacteroides goldsteinii MRD02 is remarkably superior to that of other parabacteroides strains; meanwhile, due to the fact that the usage amount of the Parabacteroides goldsteinii MRD02 is small, gastrointestinal reactions of nausea, vomiting or diarrhea are not found in the Parabacteroides goldsteinii MRD02, and anaphylactic reactions are also not found in the Parabacteroides goldsteinii MRD02.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and microbial technology, and specifically relates to a Parabacteroides archaeopteris and an application thereof. Background Art

[0002] Inflammatory arthritis is a general term for a class of diseases characterized by joint inflammation, with the main manifestations including joint pain, swelling, fever, redness, and limited joint function. This inflammation is caused by abnormal activation of the immune system, which leads to the accumulation of inflammatory cells and inflammatory mediators in the joints. Inflammatory arthritis includes ankylosing spondylitis, rheumatoid arthritis and gouty arthritis. Among them, rheumatoid arthritis, also known as rheumatoid arthritis, or RA for short, is an inflammatory, chronic, systemic autoimmune disease with synovial inflammation as the main pathological manifestation. From an immunological perspective, the production of autoantibodies and the presence of effector T cells in the blood and synovial structures are the prominent features of rheumatoid arthritis, and rheumatoid factor and anti-citrullinated protein antibodies are the main indicators of RA. From a histological perspective, rheumatoid masses are the hallmarks of rheumatoid arthritis, accompanied by synovial tissue hyperplasia, neovascularization, and infiltration by activated heterogeneous inflammatory cells. Activated osteoclasts and chondrocytes play a direct role in the development of joint structural damage. Cytokines also participate in the interaction between cells in the inflammatory network.

[0003] One hypothesis regarding the pathogenesis of rheumatoid arthritis is that the disease begins at mucosal sites as a result of the interaction of the mucosal immune system with an abnormal local microbiota, which then involves the synovial joints. Alterations in the composition of the intestinal microbiota in patients with preclinical and established RA suggest that intestinal dysbiosis plays a role in the development and persistence of RA.

[0004] The drugs currently used to treat rheumatoid arthritis mainly include nonsteroidal anti-inflammatory drugs, disease-modifying antirheumatic drugs, biologics, and glucocorticoids. In addition to drug therapy, stem cell transplantation, immune purification therapy, gene therapy, and surgical therapy are all used in the treatment of RA. However, the clinical phenotype of RA patients is heterogeneous, and the treatment effects are not the same. In addition, drugs have different side effects such as kidney damage, liver toxicity, and gastrointestinal reactions. In addition, RA-related complications increase the clinical demand for adverse prognostic factors and individualized treatment. In addition, the use of probiotics in the prior art for the treatment of rheumatoid arthritis is prone to cause gastrointestinal reactions such as nausea, vomiting, or diarrhea due to the large amount of probiotics used; in rare cases, for individuals with low or abnormal immune function, too many probiotics may stimulate the immune system and cause allergic reactions. Therefore, there is an urgent need to provide a new strategy for the treatment of rheumatoid arthritis. Summary of the invention

[0005] In order to provide a new strategy for treating rheumatoid arthritis, the present invention provides a Parabacteroides archaeopteris.

[0006] The technical solution adopted by the present invention is:

[0007] The invention provides a Parabacteroides goldsteinii, wherein the Parabacteroides goldsteinii is Parabacteroides goldsteinii MRD02, the preservation number is GDMCC No.64793, and the preservation unit is Guangdong Province Microbial Culture Collection Center.

[0008] The present invention also provides a use of the Parabacteroides gibberellus, wherein the Parabacteroides gibberellus is used to prepare a preparation for preventing, treating or alleviating inflammatory arthritis.

[0009] Preferably, the inflammatory arthritis includes any one of ankylosing spondylitis, rheumatoid arthritis and gouty arthritis.

[0010] Preferably, the preparation consists of Parabacteroides gibberellii, a drug for treating inflammatory arthritis and a pharmaceutically acceptable excipient; or consists of Parabacteroides gibberellii and a pharmaceutically acceptable excipient.

[0011] Preferably, the Parabacteroides grus is bacterial powder or bacterial liquid of Parabacteroides grus.

[0012] Preferably, the content of the Parabacteroides giusei in the preparation is not less than 1×10 7 CFU / mL.

[0013] Preferably, the drug for treating inflammatory arthritis includes at least one of non-steroidal anti-inflammatory drugs, glucocorticoids, sodium hyaluronate, medical chitosan and growth factors.

[0014] Preferably, the non-steroidal anti-inflammatory drug comprises at least one of aspirin, ibuprofen, naproxen and diclofenac.

[0015] Preferably, the pharmaceutically acceptable excipients include at least one of a filler, a flavoring agent, a binder, a disintegrant, a lubricant, an antacid and a nutritional enhancer.

[0016] Preferably, the filler comprises any one of microcrystalline cellulose, starch, lactose and mannitol.

[0017] Preferably, the flavoring agent includes any one of steviol glycoside, aspartame and sucrose.

[0018] Preferably, the binder comprises any one of starch slurry and hydroxypropyl methylcellulose.

[0019] Preferably, the disintegrant includes at least one of starch, microcrystalline cellulose and low-substituted hydroxypropyl cellulose.

[0020] Preferably, the lubricant includes any one of magnesium stearate, talc and polyethylene glycol.

[0021] Preferably, the antacid comprises any one of calcium carbonate and aluminum hydroxide.

[0022] Preferably, the nutritional enhancer includes any one of vitamin A, vitamin C and vitamin D.

[0023] Preferably, the acceptable dosage form of the preparation includes one of tablets, capsules, granules, injections, pills, powders, pastes and oral liquids.

[0024] The biomaterial sample preservation information involved in the present invention is as follows:

[0025] ParabacteroidesgoldsteiniiMRD02 was deposited in Guangdong Provincial Microbiological Culture Collection on July 1, 2024. The proposed taxonomic name of Parabacteroides goldsteinii MRD02 is Parabacteroidesgoldsteinii, the deposit number is GDMCC No.64793, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The present invention provides a Parabacteroides goldsteinii MRD02, wherein the Parabacteroides goldsteinii MRD02 has a deposit number of GDMCC No. 64793 and is deposited in the Guangdong Provincial Microbial Culture Collection Center. The Parabacteroides goldsteinii MRD02 of the present invention can significantly inhibit joint swelling and systemic inflammation in mice with rheumatoid arthritis, and its inhibitory effect is significantly better than other Parabacteroides strains; at the same time, because the amount of the Parabacteroides goldsteinii MRD02 used is small, no gastrointestinal reactions such as nausea, vomiting or diarrhea are found in the present invention, and no allergic reactions are found.

[0028] The Parabacteroides goldsteinii MRD02 of the present invention helps maintain the intestinal barrier function, reduces the release of inflammatory mediators, and thus affects the pathogenesis of arthritis. Compared with traditional nonsteroidal anti-inflammatory drugs or disease-modifying anti-rheumatic drugs, the Parabacteroides goldsteinii MRD02 of the present invention, as a probiotic, has fewer side effects and is safer for long-term use. It can be used as a supplement to traditional treatment and combined with other drugs to enhance the therapeutic effect, especially in some refractory cases.

[0029] Parabacteroides goldsteinii is a species of the genus Parabacteroides, originally isolated from the feces of healthy adults, formerly named Bacteroides goldsteinii, strictly anaerobic, indole negative, Parabacteroides goldsteinii can reduce intestinal inflammation, enhance colon mitochondrial and ribosome activity, restore abnormal host amino acid metabolism in serum, and inhibit lung inflammation. Oral administration of the Parabacteroides goldsteinii of the present invention to mice can reduce obesity in mice, increase heat production in adipose tissue, enhance intestinal integrity, and reduce inflammation and insulin resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1 The swelling of the hind paws of mice in different groups after immunotherapy, from left to right: normal group Con, model group Mod, L-Pg group, M-Pg group, H-Pg group, positive control group methotrexate MTX.

[0031] Figure 2 Changes in the joints of mice after immunotherapy. A: Arthritis index; B: Number of swollen joints.

[0032] Figure 3 Changes of thymus and spleen indexes, A: thymus index; B: spleen index.

[0033] Figure 4 These are X-rays of the ankle joints of mice in different groups after immunotherapy, from left to right: normal group Con, model group Mod, L-Pg group, M-Pg group, H-Pg group, and positive control group MTX.

[0034] Figure 5 The histopathological data of the ankle joints of mice in different groups after immunotherapy, A to F are: normal group Con, model group Mod, L-Pg group, M-Pg group, H-Pg group, positive control group MTX; G: ankle joint histopathological score

[0035] Figure 6 is the content of inflammatory factor IL-6 in the serum of mice in different groups after immunotherapy.

[0036] Figure 7 is the content of inflammatory factor TNF-α in the serum of mice in different groups after immunotherapy.

[0037] Figure 8 is the content of inflammatory factor IL-1β in the serum of mice in different groups after immunotherapy.

[0038] Fig. 9 is the content of inflammatory factor IL-10 in the serum of mice in different groups after immunotherapy.

[0039] Fig.10 is the content of inflammatory factor IL-18 in the serum of mice in different groups after immunotherapy. DETAILED DESCRIPTION

[0040] In order to enable those skilled in the art to better understand and implement the technical solution of the present invention, the present invention is further described below in conjunction with specific embodiments and drawings.

[0041] In the description of the present invention, unless otherwise specified, all reagents used are commercially available and all methods used are conventional techniques in the art.

[0042] The inventive concept of the present invention:

[0043] The drugs currently used to treat rheumatoid arthritis mainly include nonsteroidal anti-inflammatory drugs, disease-modifying antirheumatic drugs, biologics, and glucocorticoids. In addition, certain probiotics can also be used to treat rheumatoid arthritis, but because the amount of probiotics used is large, it is easy to cause gastrointestinal reactions such as nausea, vomiting, or diarrhea; in rare cases, for individuals with weakened or abnormal immune function, too many probiotics may stimulate the immune system and cause allergic reactions. Therefore, there is an urgent need to provide a new strategy for the treatment of rheumatoid arthritis.

[0044] Based on this, the present invention provides a Parabacteroides goldsteinii for the treatment of rheumatoid arthritis without obvious side effects. The Parabacteroides goldsteinii is Parabacteroides goldsteinii MRD02, with a preservation number of GDMCC No. 64793, and the preservation unit is Guangdong Provincial Microbial Culture Collection Center.

[0045] Since the Parabacteroides goldsteinii MRD02 described in the present invention is a Parabacteroides goldsteinii, it will be referred to as MRD02 hereinafter.

[0046] The experimental materials used in the present invention are as follows:

[0047] (1) The strain described in the present invention was isolated from the feces of C57 mice.

[0048] (2) Liquid Thioglycollate Medium Formula:

[0049] Each liter of culture medium contains: 15 g of trypsin-hydrolyzed casein, 5.0 g of yeast extract powder, 5 g of glucose, 0.5 g of sodium thioglycolate, 0.5 g of L-cystine, 2.5 g of sodium chloride, 0.001 g of resazurin and 0.75 g of agar, the solvent is water, and the pH is adjusted to 7.1.

[0050] (3) Primers for amplifying the 16s rRNA gene of the strain.

[0051] SEQ ID NO. 1: 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'.

[0052] SEQ ID NO. 2: 1492R: 5'-GGTTACCTTGTTACGACTT-3'.

[0053] Example 1

[0054] A Parabacteroides archaeopteris, specifically:

[0055] (1) Isolation and identification of Parabacteroides archaeopteris strain MRD02.

[0056] Strain MRD02 was isolated anaerobically from fresh feces of C57 mice in liquid thioglycollate medium.

[0057] Molecular identification method: The full sequence analysis of 16s rRNA was used. The 16s rRNA gene sequence of the MRD02 strain is shown in SEQ ID NO.3 in the sequence list, and the homology with the 16s rRNA sequence of Parabacteroides goldsteinii MTS01 is 98.75%. Therefore, the identification and classification was named Parabacteroides goldsteinii, the corresponding Chinese name was Parabacteroides goldsteinii, and the strain was named Parabacteroides goldsteinii MRD02.

[0058] SEQ ID NO.3:

[0059]

[0060] (2) Cultivation and preservation of MRD02.

[0061] Culture method: The MRD02 strain was activated once in liquid thioglycollate medium, then inoculated into liquid thioglycollate medium at a 1% inoculation rate and cultured anaerobically at 37°C for 7 days.

[0062] Storage method: Resuspend the MRD02 bacterial solution and glycerol in a volume ratio of 1:4, cover with paraffin oil, freeze quickly in liquid nitrogen and store at -80℃.

[0063] Example 2

[0064] The use of Parabacteroides in the preparation of a preparation for preventing, treating or alleviating rheumatoid arthritis is as follows:

[0065] (1) Parabacteroides archaeopteris MRD02 improves the inflammatory response in mice with collagen-induced rheumatoid arthritis.

[0066] Sixty 8-week-old SPF male DBA / 1 mice were used, and the temperature was 24°C, the humidity was 60%, the light was on for 12 hours per day, and they were free to eat and drink. After one week of adaptive feeding, the experimental animals were randomly divided into five groups, namely the normal group Con, the model group Mod, the positive control group MTX, the low-concentration L-Pg group, the medium-concentration M-Pg group, and the high-concentration H-Pg group; the positive control group used methotrexate, the L-Pg group used 10 7 CFU / mL of MRD02 and M-Pg groups were used 10 8 The MRD02 and H-Pg groups used 10 CFU / mL 9 CFU / mL of MRD02, there were 10 mice in each group, and the specific grouping and experimental treatments are shown in Table 1.

[0067] In order to induce rheumatoid arthritis using bovine type II collagen and Freund's complete adjuvant, it is necessary to prepare a complete emulsion, and mix and emulsify the bovine type II collagen solution and Freund's complete adjuvant in equal volumes. The specific modeling method of rheumatoid arthritis is to perform two immunizations. The first immunization is inoculated at multiple points in the back skin, and 100 μL of emulsifier containing 50 μg of type II collagen is injected intradermally from the distal end to the proximal end. The day of the first immunization is recorded as day 0; the second immunization time is the 21st day, and the second immunization is still inoculated with 100 μL of mixed emulsifier, collagen plus incomplete Freund's adjuvant 1:1 mixed emulsifier, the preparation method of the emulsifier is the same as the first immunization, and the needle is inserted 2 cm away from the base of the mouse tail.

[0068] After the first immunization, the mice were scored for joint index every 2 days. The score was based on a five-point scale of 0 to 4: 0 for normal; 1 for erythema and slight swelling in the ankle joint; 2 for erythema and slight swelling in the ankle joint, metatarsal joint, and palmar joint; 3 for erythema and moderate swelling from the ankle joint to the metatarsophalangeal joint or palmar joint; 4 for erythema and severe swelling from the ankle joint to the toe joint. All 4 paws were measured, and the highest score for each mouse was 16 points.

[0069] After the first immunization, the mice were observed for joint swelling and deformity, and arthritis scores were performed every 2 days, with each limb scoring from 0 to 4 points: 0 for no swelling in the joint; 1 for obvious / slight swelling in the toe joint; 2 for moderate swelling in the ankle joint; 3 for severe swelling in the entire joint; 4 for swelling in all paws including the ankle joint, with ankylosis. A score of 1 or above was considered to be an onset of disease.

[0070] Table 1 Experimental animal groups and treatment methods

[0071]

[0072] Swelling of the hind paws Figure 1 , arthritis index and joint swelling number are shown in Figure 2 , thymus index and spleen index see Figure 3 Oral administration of MRD02 can relieve RA symptoms in a dose-dependent manner in these three aspects. One week after booster immunization, arthritis symptoms appeared on d27, and the inflammation peaked around d36. High concentrations of Pg can significantly reduce the inflammatory response during the onset of joint disease.

[0073] (2) The effect of Parabacteroides gibberellin MRD02 on ankle joint symptoms in mice with rheumatoid arthritis.

[0074] X-rays are commonly used to observe joints and can show bone structure and some joint diseases. For example, in the diagnosis of arthritis, X-rays can help evaluate bone changes, joint space narrowing and calcification. By observing the X-rays of the ankle joints of each group of mice, we can see Figure 4 Compared with the Con group, the Mod group showed narrowing or even disappearance of the articular cartilage gap, swelling of the surrounding soft tissue, increased density, bone hyperplasia, and severe deformity of the toe joints. Both MRD02 and methotrexate can effectively reduce inflammatory changes. Histopathological data show that Figure 5In the Con group, the joint space was normal, the synovial cells did not proliferate, no pannus was observed, the cartilage surface was smooth and flat, the chondrocytes were neatly arranged, and there was no inflammatory cell infiltration. In the Mod group, a large number of inflammatory cells were observed in the synovium, cartilage hyperplasia caused the joint cavity to narrow significantly, the chondrocyte structure was destroyed, and pannus was generated. Compared with the Mod group, all concentration gradient groups of MRD02 alleviated the pathological changes of joint tissues, with obvious joint space, smooth cartilage surface, reduced bone erosion, and significantly reduced inflammatory cells in the synovium. Among them, the high-dose MRD02 group had the most obvious improvement effect, and the inhibition of inflammatory cell infiltration and bone destruction was better than the methotrexate group.

[0075] (3) Effect of Parabacteroides gibberellin MRD02 on serum cytokine levels in rats with rheumatoid arthritis

[0076] At the end of the experiment, mice were fasted but not watered for 12 h, blood samples were collected, and after standing at room temperature for 30 min, they were centrifuged at 3000 g for 15 min at room temperature. The supernatant was taken and the content of pro-inflammatory factors in serum was determined using mouse ELISA kits purchased from Shanghai Jianglai Biotechnology Co., Ltd. (TNF-α No. JL20242, IL-6 No. JL20268, IL-1β No. JL18442, IL-18 No. JL20253, IL-10 No. JL20228). The operation was performed according to the instructions to determine the concentrations of inflammation-related cytokines IL-1β, IL-6, TNF-α, IL-10 and IL-18.

[0077] The results are as follows Figure 6 to Figure 10 As shown, the mice in the model group were in a severe inflammatory state, with serum IL-1β, IL-6, TNF-α, and IL-18 levels significantly higher than those in the normal group, and IL-10 significantly lower than those in the normal group. MRD02 can significantly reduce serum IL-1β, IL-6, and TNF-α levels, and significantly increase IL-10 levels. High concentrations of MRD02 have similar effects to the positive control methotrexate, and the results show that MRD02 can affect the increase in inflammatory factors and the decrease in anti-inflammatory factors caused by arthritis.

[0078] All images: Values ​​are means±SD. * p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001vsmodel. # p<0.05, ## p<0.01, ### p<0.001, #### P<0.0001vs Con.

[0079] The technical features of the above-described embodiments may be arbitrarily combined. To make the description concise, not all possible combinations of the technical features in the above-described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0080] The above-mentioned embodiments only express several implementation methods of the present invention, and the description is relatively specific and detailed, but it cannot be understood as limiting the scope of the invention patent. It should be pointed out that for ordinary technicians in this field, several modifications and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A Parabacteroides archaeopteris, characterized in that The Parabacteroides goldsteinii is Parabacteroides goldsteinii MRD02, with a preservation number of GDMCC No. 64793, and the preservation unit is Guangdong Provincial Microbiological Culture Collection Center.

2. The use of Parabacteroides archaeopteris as claimed in claim 1, characterized in that: The Parabacteroides archaeopteris is used for preparing a preparation for preventing, treating or alleviating inflammatory arthritis.

3. The use according to claim 2, characterized in that The inflammatory arthritis includes any one of ankylosing spondylitis, rheumatoid arthritis and gouty arthritis.

4. The use according to claim 2, characterized in that The preparation consists of Parabacteroides gibberellus, a drug for treating inflammatory arthritis and a pharmaceutically acceptable excipient; or consists of Parabacteroides gibberellus and a pharmaceutically acceptable excipient.

5. The use according to claim 4, characterized in that The Parabacteroides giusenae is bacterial powder or bacterial liquid of Parabacteroides giusenae.

6. The use according to claim 4, characterized in that The content of Parabacteroides archivus in the preparation is not less than 1×10 7 CFU / mL.

7. The use according to claim 4, characterized in that The drug for treating inflammatory arthritis includes at least one of non-steroidal anti-inflammatory drugs, glucocorticoids, sodium hyaluronate, medical chitosan and growth factors.

8. The use according to claim 7, characterized in that The non-steroidal anti-inflammatory drug includes at least one of aspirin, ibuprofen, naproxen and diclofenac.

9. The use according to claim 4, characterized in that The pharmaceutically acceptable excipients include at least one of a filler, a flavoring agent, a binder, a disintegrant, a lubricant, an antacid and a nutritional enhancer.

10. The use according to claim 2, characterized in that The acceptable dosage forms of the preparation include one of tablets, capsules, granules, injections, pills, powders, pastes and oral liquids.

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