Application of lactobacillus helveticus CCFM1440 in relieving rheumatoid arthritis

Abstract

The invention discloses application of lactobacillus helveticus CCFM1440 in relieving rheumatoid arthritis, and belongs to the technical field of microorganisms. A strain of lactobacillus helveticus CCFM1440 is screened, the lactobacillus helveticus CCFM1501 can produce cell wall protease and aromatic transaminase at high yield, and has the effect of relieving rheumatoid arthritis, which is specifically reflected in that the thickness of a posterior claw of a rat with rheumatoid arthritis is reduced, and knee joint injury is improved; according to the present invention, the content of pro-inflammatory cytokines such as IL-1beta, IL-6, IL-17A and TNF-alpha in the serum of the rheumatoid arthritis rat can be reduced, the content of arthritis specific antibodies and matrix metalloproteinase in the serum of the rheumatoid arthritis rat can be reduced, the intestinal flora of the rheumatoid arthritis rat can be improved, and the abundance of the bacillus thuringiensis and the Rombout can be reduced;

Description

Technical Field

[0001] This invention relates to the application of Lactobacillus helveticus CCFM1440 in relieving rheumatoid arthritis, and belongs to the field of microbial technology. Background Technology

[0002] Rheumatoid arthritis (RA) is a persistent autoimmune disease characterized by persistent inflammation of the synovium, leading to cartilage degeneration, bone loss, and ultimately joint deformities and functional impairment. Approximately 1% of the global population is affected by RA. Currently, treatment for rheumatoid arthritis commonly uses traditional medications such as antirheumatic drugs, corticosteroids, and nonsteroidal anti-inflammatory drugs (NSAIDs), as well as some novel biologics; however, all of these medications have limitations.

[0003] Studies have shown that the gut microbiota of rheumatoid arthritis (RA) patients exhibits significant alterations compared to healthy individuals. The gut microbiota can influence the immune system and may trigger or exacerbate RA through various mechanisms. Probiotics, being beneficial live microorganisms, have shown promise in the treatment of RA. Numerous studies have demonstrated that specific probiotic strains, such as Lactobacillus and Bifidobacterium, can alleviate RA symptoms and improve patient outcomes. These probiotics help restore the balance of the gut microbiota, enhance intestinal barrier function, and modulate the immune system. Different strains can have varying effects on immune system function, potentially influencing the development of RA. Therefore, screening for probiotics with rheumatoid arthritis-relieving properties could contribute to providing new approaches to RA treatment. Summary of the Invention

[0004] [Technical Issues] The technical problem to be solved by this invention is to provide a strain of Lactobacillus helveticus that can alleviate rheumatoid arthritis. Lactobacillus helveticus ).

[0005] [Technical Solution] To solve the technical problem of this invention, this invention provides a strain of Lactobacillus helveticus (… Lactobacillus helveticus CCFM1440, the Lactobacillus helveticus ( Lactobacillus helveticus CCFM1440 was deposited on September 2, 2024, at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65124, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0006] This invention also provides Lactobacillus helveticus ( Lactobacillus helveticus The use of CCFM1440 in the preparation of medicines for the prevention and / or treatment of rheumatoid arthritis.

[0007] In one embodiment of the present invention, the pharmaceutical product contains the aforementioned Lactobacillus helveticus ( Lactobacillushelveticus The bacterial count of CCFM1440 should not be less than 3 × 10⁻⁶. 9 CFU / mL or 3×10 9 CFU / g.

[0008] In one embodiment of the present invention, the pharmaceutical product contains the above-mentioned Lactobacillus helveticus ( Lactobacillus helveticus CCFM1440, drug carriers and / or pharmaceutical excipients.

[0009] In one embodiment of the present invention, the drug carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.

[0010] In one embodiment of the present invention, the pharmaceutical excipient comprises excipients and / or additives.

[0011] In one embodiment of the present invention, the dosage form of the drug is granules, capsules, tablets, pills, or oral liquid.

[0012] In one embodiment of the present invention, the medicine has at least one of the following functions: (1) Reduce joint thickness in individuals with rheumatoid arthritis; (2) Reduce the levels of pro-inflammatory cytokines IL-1β, IL-6, IL-17A, and TNF-α in the serum of individuals with rheumatoid arthritis; (3) Reduce the level of arthritis-specific antibodies in the serum of individuals with rheumatoid arthritis; (4) Reduce the level of matrix metalloproteinases in the serum of individuals with rheumatoid arthritis; (5) Regulate the gut microbiota of individuals with rheumatoid arthritis.

[0013] The present invention also provides a method for preparing a medicine for the prevention and / or treatment of rheumatoid arthritis, wherein the Lactobacillus helveticus CCFM1440 is fermented in a culture medium and the bacterial cells are collected.

[0014] In one embodiment of the present invention, the above-mentioned Lactobacillus helveticus ( Lactobacillus helveticus CCFM1440 was inoculated into the culture medium at an inoculation rate of 2-4% of the total mass of the culture medium, and cultured at 37°C for 24-48 h to obtain the culture solution; the culture solution was centrifuged to obtain the bacterial cells.

[0015] In one embodiment of the present invention, the culture medium is MRS culture medium.

[0016] The present invention also provides the aforementioned Lactobacillus helveticus ( Lactobacillus helveticus Application of CCFM1440 in the preparation of health products that regulate intestinal flora.

[0017] In an embodiment of the present application, the adjusting the intestinal flora refers to down-regulating the relative abundance of Ochrobactrum Turicibacter and / or Romatia Romboutsia in the intestine.

[0018] [beneficial effects] 1. The present application screens a Lactobacillus helveticus (Lactobacillus helveticus) Lactobacillus helveticus CCFM1440, which has the effect of relieving rheumatoid arthritis, specifically embodied in: Lactobacillus helveticus (1) significantly reducing the thickness of the paw of a rheumatoid arthritis rat and improving pathological damage; (2) significantly reducing the content of pro-inflammatory cytokines IL-1β, IL-6, IL-17A and TNF-α in the serum of a rheumatoid arthritis rat; (3) significantly reducing the content of arthritis-specific antibodies in the serum of a rheumatoid arthritis rat; (4) significantly reducing the content of matrix metalloproteinases in the serum of a rheumatoid arthritis rat; (5) regulating the intestinal flora of a rheumatoid arthritis rat at the genus level. Therefore, the Lactobacillus helveticus (Lactobacillus helveticus)

[0019] CCFM1440 has great application prospects in the preparation of products (such as food or drugs, etc.) for preventing and / or treating rheumatoid arthritis. Lactobacillus helveticus

[0020] 2. Lactobacillus helveticus (Lactobacillus helveticus) Lactobacillus helveticus is a kind of probiotic bacteria, which has been included in the "List of Bacteria that Can be Used in Food" issued by the National Health and Health Commission, so the Lactobacillus helveticus (Lactobacillus helveticus) Lactobacillus helveticus CCFM1440 screened by the present application will not bring potential safety hazards to patients with rheumatoid arthritis (Rheumatoid Arthritis, RA).

[0021] 3. The cultivation process of Lactobacillus helveticus (Lactobacillus helveticus) Lactobacillus helveticus only needs culture medium and control of some culture conditions, and the cost is relatively low, compared with expensive biological agents, which will not bring too much economic burden to patients with rheumatoid arthritis (Rheumatoid Arthritis, RA).

[0022] Biological material preservation A Lactobacillus helveticus (Lactobacillus helveticus) Lactobacillus helveticus CCFM1440, taxonomically named Lactobacillus helveticus ​It was deposited on September 12, 2024 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 65124, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description

[0023] Figure 1 Different strains of Lactobacillus helveticus ( Lactobacillus helveticus ) cell wall protease activity.

[0024] Figure 2 Different strains of Lactobacillus helveticus ( Lactobacillus helveticus The activity of aromatic transaminases; different letters in the figure indicate significant differences. p <0.05).

[0025] Figure 3 Lactobacillus helveticus ( Lactobacillus helveticus Effects of CCFM1440 on paw thickness in rats with rheumatoid arthritis; different letters in the figure indicate significant differences. p <0.05).

[0026] Figure 4 Lactobacillus helveticus ( Lactobacillus helveticus Effects of CCFM1440 on pathological sections of the knee joint of rats with rheumatoid arthritis.

[0027] Figure 5 Lactobacillus helveticus ( Lactobacillus helveticus The effect of CCFM1440 on serum IL-1β levels in rheumatoid arthritis rats; in the figure: different letters above the bars indicate significant differences. p <0.05).

[0028] Figure 6 Lactobacillus helveticus ( Lactobacillus helveticus The effect of CCFM1440 on serum IL-6 levels in rheumatoid arthritis rats; in the figure: different letters above the bars indicate significant differences. p <0.05).

[0029] Figure 7 Lactobacillus helveticus ( Lactobacillus helveticus The effect of CCFM1440 on serum IL-17A levels in rheumatoid arthritis rats; in the figure: different letters above the bars indicate significant differences. p <0.05).

[0030] Figure 8 Lactobacillus helveticus ( Lactobacillus helveticus The effect of CCFM1440 on serum TNF-α levels in rheumatoid arthritis rats; in the figure: different letters above the bars indicate significant differences.p <0.05).

[0031] Figure 9 : Effect of L. helveticus (LH) CCFM1440 on CII-total IgG in serum of rheumatoid arthritis rats; in the figure: different letters on the column chart represent significant difference (P < 0.05). Lactobacillus helveticus <0.05). p

[0032] Figure 10 : Effect of L. helveticus (LH) CCFM1440 on CII-IgG1 in serum of rheumatoid arthritis rats; in the figure: different letters on the column chart represent significant difference (P < 0.05). Lactobacillus helveticus <0.05). p

[0033] Figure 11 : Effect of L. helveticus (LH) CCFM1440 on CII-IgG2a in serum of rheumatoid arthritis rats; in the figure: different letters on the column chart represent significant difference (P < 0.05). Lactobacillus helveticus <0.05). p

[0034] Figure 12 : Effect of L. helveticus (LH) CCFM1440 on CII-IgG2b in serum of rheumatoid arthritis rats; in the figure: different letters on the column chart represent significant difference (P < 0.05). Lactobacillus helveticus <0.05). p

[0035] Figure 13 : Effect of L. helveticus (LH) CCFM1440 on MMP-2 in serum of rheumatoid arthritis rats; in the figure: different letters on the column chart represent significant difference (P < 0.05). Lactobacillus helveticus <0.05). p

[0036] Figure 14 : Effect of L. helveticus (LH) CCFM1440 on MMP-3 in serum of rheumatoid arthritis rats; in the figure: different letters on the column chart represent significant difference (P < 0.05). Lactobacillus helveticus <0.05). p

[0037] Figure 15 : Effect of L. helveticus (LH) CCFM1440 on MMP-9 in serum of rheumatoid arthritis rats; in the figure: different letters on the column chart represent significant difference (P < 0.05). Lactobacillus helveticus <0.05). p

[0038] Figure 16 : Effect of L. helveticus (LH) CCFM1440 on MMP-9 in serum of rheumatoid arthritis rats; in the figure: different letters on the column chart represent significant difference (P < 0.05).​​​​​​​Lactobacillus helveticus CCFM1440 showed an effect on the presence of Zurich bacilli in the feces of rheumatoid arthritis rats. Turicibacter The effect of abundance; in the figure: different letters above the bars indicate significant differences ( p <0.05).

[0039] Figure 17 Lactobacillus helveticus ( Lactobacillus helveticus CCFM1440 showed that *Romebutzimia* spp. in the feces of rheumatoid arthritis rats ( Romboutsia The effect of abundance; in the figure: different letters above the bars indicate significant differences ( p <0.05). Detailed Implementation

[0040] The present invention will be further described below with reference to specific embodiments and accompanying drawings.

[0041] The female Wistar rats used in the following examples were purchased from Zhejiang Vital River Co., Ltd.; the bovine type II collagen solution and Freund's incomplete adjuvant used in the following examples were purchased from Chondrex; the ELISA kits for detecting IL-1β, IL-6, IL-17A, and TNF-α used in the following examples were purchased from Wuhan Elite Biotech Co., Ltd.; the ELISA kits for total type II collagen-specific antibody IgG, type II collagen-specific antibody IgG1, type II collagen-specific antibody IgG2a, and type II collagen-specific antibody IgG2b were purchased from Nanjing Senbega Co., Ltd.; and the ELISA kits for matrix metalloproteinase-2, matrix metalloproteinase-3, and matrix metalloproteinase-9 were purchased from Shanghai Enzyme Linked Synthetic Laboratory Co., Ltd.

[0042] The culture media involved in the following examples are as follows: MRS solid medium: tryptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 5 g / L, diammonium citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L, agar 15 g / L.

[0043] MRS liquid culture medium: tryptone 10 g / L, beef extract 10 g / L, glucose 20 g / L, sodium acetate 2 g / L, yeast extract 5 g / L, diammonium hydrogen citrate 2 g / L, K2PO4·3H2O 2.6 g / L, MgSO4·7H2O 0.1 g / L, MnSO4 0.05 g / L, Tween 80 1 mL / L.

[0044] Example 1: Screening and strain identification of Lactobacillus helveticus 1. Screening The sample was derived from the dairy products of Qula in Aba prefecture of Sichuan province, and the sample was stored in a -80℃ refrigerator in 30% (v / v) glycerol. After the sample was taken out and thawed, the sample was mixed in a sterile environment, 0.5 mL of the sample was taken and added into a 10 mL centrifuge tube containing 4.5 mL of physiological saline, and 10 -1 dilution liquid was obtained. The above dilution step was repeated, and 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 dilution liquids were obtained in turn. 100 μL of different gradient gradient dilution liquids was taken and spread on MRS solid culture medium, and incubated at 37℃ for 72 h to obtain dilution spread plates. Typical colonies on the dilution spread plates were picked and streaked on MRS solid culture medium, and incubated at 37℃ for 48 h to obtain purified colonies. The purified colonies were inoculated into MRS liquid culture medium, and incubated at 37℃ for 48 h to obtain CCFM1440.

[0045] 2. Identification The genome of CCFM1440 was extracted, and 16S amplification was performed. The 16S rDNA amplification conditions were as follows: 95℃ for 5 min; 35 cycles (95℃ for 30 s, 55℃ for 30 s, 72℃ for 2 min); and 72℃ for 10 min. The amplification primers were 27F: (5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R: (5'-TACGGCTACCTTGTTACGACTT-3'). The purification and sequence alignment of the amplification product were performed according to the method described in the literature (Turroni F et al. Exploring the Diversitym of the Bifidobacterial Population in the Human Intestinal Tract [J]. Appl Environ Microb. 2009; 75 (6) : 1534-45). The 16S rDNA of CCFM1440 was amplified and sequenced (by Suzhou Jinyuzhi Biological Technology Co., Ltd.), and the 16S rDNA sequence of CCFM1440 obtained by sequencing analysis was compared in NCBI. The results showed that the strain was Lactobacillus helveticus, and was named Lactobacillus helveticus (L. helveticus) CCFM1440. Lactobacillus helveticus ) CCFM1440.

[0046] Example 2: Preparation of Lactobacillus helveticus bacterial suspension The Lactobacillus helveticus CCFM1440 bacterial solution was prepared as follows: Lactobacillus helveticus CCFM1440 obtained in Example 1 was streaked on MRS solid medium and incubated at 37℃ for 48 h to obtain single colonies. The colonies were white and round, small in size, smooth in edge and convex in middle. The single colonies were picked and inoculated into MRS liquid medium and incubated at 37℃ for 24 h to obtain an activation liquid. The activation liquid was inoculated into MRS liquid medium at an inoculation amount of 1% (v / v) and incubated at 37℃ for 24 h to obtain a first-stage seed liquid. The first-stage seed liquid was inoculated into MRS liquid medium at an inoculation amount of 1% (v / v) and incubated at 37℃ for 24 h to obtain a second-stage seed liquid. The second-stage seed liquid was inoculated into 1 L of MRS liquid medium at an inoculation amount of 1% (v / v) and incubated at 37℃ for 24 h to obtain a bacterial liquid. The bacterial liquid was centrifuged at 6000 g for 20 min to collect the precipitate. The precipitate was washed twice with a physiological saline buffer and then centrifuged at 6000 g for 10 min to obtain bacterial cells. The bacterial cells were resuspended in physiological saline to a cell concentration of 3 x 10 9 CFU / mL to obtain a Lactobacillus helveticus bacterial liquid. In subsequent experiments, the glycerol was discarded by centrifugation, the bacterial cells were washed once with physiological saline and resuspended in physiological saline.

[0047] Example 3: Determination of carbohydrate utilization of Lactobacillus helveticus Lactobacillus helveticus was streaked on MRS solid medium and incubated at 37℃ for 48 h. Single colonies were picked and inoculated into MRS liquid medium and incubated at 37℃ for 24 h to obtain an activation liquid. The activation liquid was inoculated into MRS liquid medium at an inoculation amount of 1% (v / v) and incubated at 37℃ for 24 h for 3 times of activation.

[0048] MRS liquid medium without glucose was prepared (no glucose was added to MRS, and cysteine was added).

[0049] Preparation of bromocresol purple mother liquor: anhydrous ethanol was used for dissolution, and water was used for constant volume. For example, when preparing 20 mL of a bromocresol purple solution, 0.1 g of bromocresol purple was dissolved in a 50 mL centrifuge tube using 4 mL of anhydrous ethanol (which needed to be repeatedly blown), and then the volume was made up to 20 mL. The concentration of the mother liquor was 0.5%; 15 mL of the mother liquor was added to 1 L of the medium.

[0050] Preparation of sugar solution (freshly prepared): the sugar concentration in this experiment was 1 g / 100 mL. For example, 1% sugar solution required 0.5 g of sugar; at this time, 5 mL of water was needed to completely dissolve the sugar, and then 45 mL of MRS liquid medium without glucose was added. The sugar solution mother liquor needed to be filtered to remove bacteria.

[0051] Qualitative analysis by 96-well plate method: 200 μL of sugar solution was first added to the wells, then 2 μL of bacterial solution was added, and the color change was observed after 24 h of culture. Yellow color change indicated that the strain could utilize the carbon source for metabolism. The results are shown in Table 1. Lactobacillus helveticus CCFM1440 could utilize trehalose, while the model strain Lactobacillus helveticus ATCC15009 could not.

[0052] Table 1 Carbon source metabolism

[0053] Wherein: + indicates that the carbon source can be utilized, and - indicates that the carbon source cannot be utilized.

[0054] Example 4: Determination of protease activity in Lactobacillus helveticus MRS medium Preparation of bacterial cell suspension: Take the bacterial solution grown in MRS liquid medium for 16 h, add trisodium citrate to a final concentration of 0.25 mol / L, centrifuge at 6000 x g at 4°C for 10 min, resuspend the bacterial slurry with a mixture of 0.9% NaCl and 0.25 mol / L trisodium citrate, centrifuge at 6000 x g at 4°C for 10 min, wash twice with 0.9% NaCl and adjust the bacterial slurry to OD600 = 1.0 with 0.1 mol / L phosphate buffer (pH 6.5) for use.

[0055] Preparation of cell-free extract (CFE): Take the bacterial solution grown in MRS liquid medium for 16 h, centrifuge at 4000 x g at 4°C for 10 min, resuspend with 50 mmol / L sodium phosphate buffer pH 7.5 and centrifuge, repeat three times, resuspend the bacterial cells in the same buffer to a final volume of 2 mL. The cells were broken by liquid nitrogen freezing and thawing method, the bacterial cells with buffer were placed in a mortar, a certain volume of liquid nitrogen was added, and the freezing and thawing was repeated three times, then the liquid was collected by centrifugation at 10000 x g at 4°C for 10 min, the supernatant was collected and stored at -80°C for use.

[0056] Determination of cell wall protease activity: Prepare TBS solution, FTC-casein stock solution and FTC-casein working solution according to the instructions of the fluorescent protease detection kit, add 100 μL of sample to the fluorescent special 96-well plate, then add 100 μL of FTC-casein working solution, and repeat three times in parallel. Place the 96-well plate in a 37°C incubator for 24 h and detect with an enzyme marker, and the cell wall protease (CEP) activity of the strain is directly represented by the fluorescence intensity.

[0057] Determination of intracellular aromatic transaminase activity: 20 mmol / L L-phenylalanine, 10 mmol / L α-ketoglutarate, 0.5 mmol / L disodium EDTA, 0.05 mmol / L 5'-phosphopyridoxal were mixed in 100 mL borate buffer (pH 8.5). 150 μL of the mixture and 100 μL of CFE were added to each well of a 96-well plate, and incubation was performed at 37°C for 12 h. Absorbance was measured at 290 nm. The amount of phenylalanine converted to phenylpyruvic acid was defined as AraT activity, and 1 enzyme activity unit (U) refers to the amount of enzyme that converts 1 μmol of phenylpyruvic acid per minute under standard conditions. A standard curve of sodium phenylpyruvate was prepared: solutions of sodium phenylpyruvate at different concentrations (0.1 ~ 6 mmol / L) were prepared, and absorbance was measured at 290 nm to obtain a standard curve. There was no positive control, no commercial enzyme, and the negative control contained only CFE without phenylalanine; the blank control used buffer instead of CFE; and the protein content was determined in triplicate using a BCA kit.

[0058] The results of the above experiments are shown in Tables 1 to 3, respectively. Figures 1-2 .

[0059] Figure 1 It is shown that, compared with ATCC15009 (fluorescence intensity 33) and CCFM1096 (fluorescence intensity 40), the fluorescence intensity of CCFM1440 is 83.2, indicating that it has higher cell wall protease activity.

[0060] Figure 2 It is shown that the activity of ATCC15009 is 12.7 nmol of phenylpyruvic acid (per mg of protein), the activity of CCFM1096 is 5.9 nmol of phenylpyruvic acid (per mg of protein), and the activity of CCFM1440 is 30.17 nmol of phenylpyruvic acid (per mg of protein), i.e., the activities of ATCC15009 and CCFM1096 are 17.6 and 8.2 U / mg, respectively, while the activity of CCFM1440 is 41.83 U / mg, indicating that it has higher aromatic transaminase activity.

[0061] Example 5: Effect of Lactobacillus helveticus CCFM1440 on the thickness of the paw of a rheumatoid arthritis rat Eighteen 5-week-old female Wistar rats of the SPF (Specific Pathogen Free) level were raised in a room with a temperature of 22 ~ 24°C, a humidity of 40 ~ 60%, and a 12 h / 12 h day-night alternation, with free access to food and water, for 1 week, and then randomly divided into 3 groups, each group of 6 rats, i.e., a normal group, a model group, and a CCFM1440 group of Lactobacillus helveticus CCFM1440 administered by gavage. Lactobacillus helveticus ) CCFM1440.

[0062] The experiment lasted for five weeks (35 days): from one week before modeling to the end of the experiment, the normal group and the model group were given 1 mL of sterile normal saline solution with a concentration of 0.9% (w / w) by gavage every day, and the CCFM1440 group was given 1 mL of Lactobacillus helveticus solution with a concentration of 3x10 9 CFU / mL by gavage every day. Lactobacillus helveticus ) CCFM1440 bacterial solution; on the first day of the second week (day 8), the bovine type II collagen solution (Chondrex, 20022) was mixed with Freund's incomplete adjuvant (Chondrex, 7002) in equal volumes to form a complete emulsion, and the rats were anesthetized with isoflurane, fixed, and the entire tail base was disinfected with 75% alcohol. The rats were then immunized for the first time, and 0.2 mL of the complete emulsion was accurately injected subcutaneously 1.5 cm from the tail base. One week later (day 15), the same treatment was used for booster immunization, i.e., 0.2 mL of the complete emulsion was accurately injected subcutaneously 2.0 cm from the tail base. The normal group of rats was only injected with the same volume of sterile normal saline using the same method.

[0063] After modeling, the joint thickness of each group of rats was measured by a micrometer, and hematoxylin-eosin pathological sections of the rat knee joints were prepared (for details, see reference: Shan, J., et al., Integrated Serum and Fecal Metabolomics Study of Collagen-Induced Arthritis Rats and the Therapeutic Effects of the Zushima Tablet. Front Pharmacol, 2018. 9: p. 891.)The results are shown in Fig. 3-4 .

[0064] As can be seen from Fig. 3 , joint swelling began about 12 days after the first immunization, and the joint thickness of the model group rats was significantly higher than that of the normal group rats on day 35. The joint thickness of the CCFM1440 group rats was lower than that of the model group rats, among which the joint thickness of the control group, the model group, and the CCFM1440 group rats was 5.55, 8.44, and 6.94 mm, respectively.

[0065] As can be seen from Fig. 4 , the knee joints of the model group rats were severely damaged, with a large area of cartilage surface covered by proliferative tissue, forming a pannus, and a large number of inflammatory cells mainly composed of lymphocytes, macrophages, and granulocytes were observed. CCFM1440 can significantly alleviate the pathological damage of the knee joint.

[0066] It can be seen that Lactobacillus helveticus CCFM1440 can assist in the treatment and relief of rheumatoid arthritis.

[0067] Example 6: Effect of Lactobacillus helveticus CCFM1440 on the levels of pro-inflammatory factors in the serum of rheumatoid arthritis rats The experimental grouping and treatment were as described in Example 5. After the experiment, blood was collected from the rats, and they were euthanized. Rat serum was collected, and the levels of pro-inflammatory factors IL-1β, IL-6, IL-17A, and TNF-α in the serum of each group were measured using an ELISA kit. The results are shown in [Figure 5]. Fig. 5-8 .

[0068] like Fig. 5 As shown, the concentration of IL-1β in the serum of rats in the model group was 55 pg / mL, which was significantly higher than that in the normal group (16 pg / mL). Compared with the model group, the level of IL-1β in the serum of rats in the CCFM1440 group was significantly lower, at 21 pg / mL, which was comparable to that in the control group.

[0069] like Fig. 6 As shown, the concentration of IL-6 in the serum of rats in the model group was 166 pg / mL, which was significantly higher than that in the normal group (55 pg / mL); compared with the model group, the serum IL-6 level in rats in the CCFM1440 group was significantly lower, at 84 pg / mL.

[0070] like Fig. 7 As shown, the concentration of IL-17A in the serum of rats in the model group was 120 pg / mL, which was significantly higher than that in the normal group (37 pg / mL); compared with the model group, the level of IL-17A in the serum of rats in the CCFM1440 group was significantly lower, at 54 pg / mL.

[0071] like Fig. 8 As shown, the concentration of TNF-α in the serum of rats in the model group was 418 pg / mL, which was significantly higher than that in the normal group (107 pg / mL); compared with the rats in the model group, the level of TNF-α in the serum of rats in the CCFM1440 group was significantly lower, at 215 pg / mL.

[0072] It is evident that Lactobacillus helveticus CCFM1440 can reduce the levels of pro-inflammatory factors IL-1β, IL-6, IL-17A and TNF-α in the serum of rats with rheumatoid arthritis.

[0073] Example 7: Effect of Lactobacillus helveticus CCFM1440 on the content of type II collagen-specific antibody IgG subtype in the serum of rheumatoid arthritis rats The experimental grouping and treatment were as described in Example 5. After the experiment, blood was collected from rats, and the rats were euthanized. Rat serum was collected, and the levels of total type II collagen-specific antibody IgG, type II collagen-specific antibody IgG1, type II collagen-specific antibody IgG2a, and type II collagen-specific antibody IgG2b in each group of rat serum were measured using an ELISA kit. The results are shown in [Figure 1]. Fig. 9-12 .

[0074] The level of type II collagen-specific antibodies reflects the severity of arthritis symptoms in rats.

[0075] like Fig. 9 As shown, the concentration of total type II collagen-specific antibody IgG in the model group rats was 6.48 μg / L, which was significantly higher than that in the normal group (3.48 μg / L); compared with the model group rats, the level of total type II collagen-specific antibody IgG in the serum of the CCFM1440 group rats was significantly lower, at 4.01 μg / L.

[0076] like Fig. 10 As shown, the concentration of type II collagen-specific antibody IgG1 in the model group rats was 7.42 μg / L, which was significantly higher than that in the normal group (4.19 μg / L); compared with the model group rats, the level of type II collagen-specific antibody IgG1 in the serum of the CCFM1440 group rats was significantly lower, at 4.71 μg / L.

[0077] like Fig. 11 As shown, the concentration of type II collagen-specific antibody IgG2a in the model group rats was 4.07 μg / L, which was significantly higher than that in the normal group (2.26 μg / L); compared with the model group rats, the level of type II collagen-specific antibody IgG2a in the serum of the CCFM1440 group rats was significantly lower, at 2.50 μg / L.

[0078] like Fig. 12 As shown, the concentration of type II collagen-specific antibody IgG2b in the model group rats was 2.78 μg / L, which was significantly higher than that in the normal group (0.95 μg / L); compared with the model group rats, the level of type II collagen-specific antibody IgG2b in the serum of CCFM1440 group rats was significantly lower, at 1.41 μg / L.

[0079] It is evident that Lactobacillus helveticus CCFM1440 can reduce the levels of total type II collagen-specific antibody IgG, type II collagen-specific antibody IgG1, type II collagen-specific antibody IgG2a, and type II collagen-specific antibody IgG2b in the serum of rats with rheumatoid arthritis, thereby alleviating the symptoms of arthritis in rats.

[0080] Example 8: Effect of Lactobacillus helveticus CCFM1440 on the content of matrix metalloproteinases in the serum of rheumatoid arthritis rats The experimental grouping and treatment refer to Example 5. After the experiment, the rats were taken blood and sacrificed, and the rat serum was taken. The content of matrix metalloproteinase-2, matrix metalloproteinase-3 and matrix metalloproteinase-9 in the serum of each group of rats was determined by ELISA kit, and the detection results are shown in Fig. 13-15 .

[0081] Matrix metalloproteinases (MMPs) are a family of enzymes responsible for the degradation of extracellular matrix. Under normal circumstances, MMPs maintain the process of tissue remodeling and repair, but in the development of rheumatoid arthritis, the activity of MMPs is imbalanced, which leads to excessive degradation of extracellular matrix in joints and surrounding tissues, resulting in joint destruction, and MMP-3 is considered to be the most important protease leading to cartilage degradation, which can be used as an important indicator for early diagnosis of RA.

[0082] As shown in Fig. 13 , the concentration of MMP-2 in the serum of the model group rats was 180.73 ng / mL, which was significantly higher than that of the normal group (105.52 ng / mL); compared with the model group rats, the level of MMP-2 in the serum of CCFM1440 group rats was significantly reduced, which was 128.84 ng / mL.

[0083] As shown in Fig. 14 , the concentration of MMP-3 in the serum of the model group rats was 136.16 ng / mL, which was significantly higher than that of the normal group (70.37 ng / mL); compared with the model group rats, the level of MMP-3 in the serum of CCFM1440 group rats was significantly reduced, which was 103.66 ng / mL.

[0084] As shown in Fig. 15 , the concentration of MMP-9 in the serum of the model group rats was 53.22 ng / mL, which was significantly higher than that of the normal group (28.65 ng / mL); compared with the model group rats, the level of MMP-9 in the serum of CCFM1440 group rats was significantly reduced, which was 32.94 ng / mL.

[0085] Example 9: Effect of Lactobacillus helveticus CCFM1440 on the abundance of fecal flora in rheumatoid arthritis rats The experimental grouping and treatment refer to Example 5. After the modeling, the feces of rats were collected, the genomic DNA in the feces was extracted, the V3-V4 region was specifically PCR amplified, 16S rDNA sequencing was performed, and the change of fecal flora was analyzed, and the analysis results are shown in Fig. 16-17 .

[0086] As shown in Fig. 16-17As shown, compared with the normal group rats, the fecal microbiota of the model group rats underwent significant changes. Among them, the genus *Zurichobacterium* in the feces of the model group rats (…) Turicibacter The relative abundance of *Lactobacillus helveticus* (CCFM1440) was significantly increased in rats compared to the normal group, and *Lactobacillus zurichensis* (CCFM1440) significantly downregulated the abundance of *Lactobacillus* spp. in rat feces. Turicibacter The relative abundance of *Lactobacillus helveticus* CCFM1440 was significantly downregulated compared to the model group. Romboutsia The relative abundance of *Lactobacillus helveticus* CCFM1440 was reduced to the level of the normal group. This indicates that *Lactobacillus helveticus* CCFM1440 can improve gut microbiota dysbiosis in rheumatoid arthritis rats.

[0087] Although the present invention has been disclosed above with reference to preferred embodiments, it is not intended to limit the present invention. Anyone skilled in the art can make various modifications and alterations without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be determined by the claims.

Claims

1. Lactobacillus helveticus ( Lactobacillus helveticus The use of CCFM1440 in the preparation of medicaments for the prevention and / or treatment of rheumatoid arthritis is characterized by, The Lactobacillus helveticus CCFM1440 has been preserved in Guangdong Microbial Culture Collection Center on September 12, 2024, with the preservation number of GDMCC No: 65124, and the preservation address of 5th floor, Building 59, Guangzhou, Guangdong, China.

2. Use according to claim 1, characterized in that, The number of bacteria of Lactobacillus helveticus CCFM1440 of claim 1 in the medicine is not less than 3×10 9 CFU / mL or 3×10 9 CFU / g.

3. Use according to claim 2, wherein the compound is ###0002### The medicine contains the Lactobacillus helveticus CCFM1440 of claim 1, a pharmaceutical carrier and / or a pharmaceutical excipient.

4. Use according to claim 3, wherein the compound is ###0002### The pharmaceutical carrier includes one or more of the fillers, binders, wetting agents, disintegrants, lubricants, and flavoring agents commonly used in medicine.

5. The use according to claim 4, wherein the compound is ###0002### The pharmaceutical excipient includes excipients and / or additional agents.

6. The use according to claim 5, wherein the compound is ###0002### The dosage form of the medicine is granules, capsules, tablets, pills, or oral liquids.

7. Use according to claim 6, wherein The medicine has at least one of the following functions: (1) reducing the joint thickness of a rheumatoid arthritis individual; (2) reducing the content of pro-inflammatory cytokines IL-1β, IL-6, IL-17A, and TNF-α in the serum of a rheumatoid arthritis individual; (3) reducing the content of arthritis-specific antibodies in the serum of a rheumatoid arthritis individual; (4) reducing the content of matrix metalloproteinases in the serum of a rheumatoid arthritis individual; (5) regulating the intestinal flora of a rheumatoid arthritis individual.

8. A method of manufacturing a medicinal product for the prophylaxis and / or treatment of rheumatoid arthritis, characterized in that, The Lactobacillus helveticus CCFM1440 of claim 1 is fermented in a culture medium, and the bacterial cells are collected.

9. The method of claim 8, wherein, The culture medium includes but is not limited to MRS medium.

10. Lactobacillus helveticus of claim 1 for use in the preparation of a health product for modulating the gut microbiota. Lactobacillus helveticus ) CCFM1440 for use in the preparation of a health product for modulating the gut microbiota.

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