Lactobacillus helveticus capable of relieving rheumatoid arthritis and application of lactobacillus helveticus
By screening out Lactobacillus helveticus CCFM1501, preparing microbial preparations and pharmaceutical forms for application, the treatment limitations of rheumatoid arthritis have been overcome, significantly reducing arthritis symptoms and improving joint damage and immune response.
Patent Information
- Application Number
- CN202511440522.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-10-10
- Publication Date
- 2026-01-13
Smart Images
Figure CN121320147A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to a strain of Lactobacillus helveticus that can alleviate rheumatoid arthritis and its applications, belonging to the field of microbial technology. Background Technology
[0002] Rheumatoid arthritis (RA) is a chronic systemic autoimmune disease characterized by synovial hyperplasia, synovitis, and erosion of the joint bone and cartilage. These changes lead to joint swelling, symmetrical pain, and stiffness, ultimately resulting in joint deformities, limited mobility, and a decreased quality of life. Current RA treatments can be broadly categorized into four types: glucocorticoids, nonsteroidal anti-inflammatory drugs (NSAIDs), antirheumatic drugs (ART), and some novel biologics; however, all of these medications have certain limitations.
[0003] The gut microbiota is closely linked to rheumatoid arthritis (RA) and may trigger or exacerbate RA through various mechanisms. Studies have shown that supplementing with specific probiotics has anti-inflammatory effects, helping to increase daily activities and alleviate symptoms in RA patients. Using probiotics as adjunctive therapy for RA patients offers a novel strategy in the treatment of chronic, systemic, autoimmune inflammatory diseases. However, different strains can have varying effects on immune system function, potentially influencing the development of RA. Therefore, screening for probiotics that alleviate rheumatoid arthritis has significant clinical implications. 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 CCFM1501, the Lactobacillus helveticus ( Lactobacillus helveticus CCFM1501 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 30, 2025, with accession number GDMCC No: 66449. The deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
[0006] The present invention also provides a product containing the aforementioned Lactobacillus helveticus ( Lactobacillus helveticus CCFM1501 microbial preparation.
[0007] In one embodiment of the present invention, the number of *Lactobacillus helveticus* CCFM1501 cells in the microbial preparation is not less than 3 × 10⁻⁶. 9 CFU / mL or 3×10 9 CFU / g.
[0008] In one embodiment of the present invention, the method for preparing the microbial preparation is to use the above-mentioned Lactobacillus helveticus (… Lactobacillus helveticus CCFM1501 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 a culture solution; the culture solution was centrifuged to obtain bacterial cells; the bacterial cells were resuspended in physiological saline to obtain a microbial preparation.
[0009] In one embodiment of the present invention, the bacterial cells are resuspended in physiological saline and then freeze-dried to obtain a microbial preparation.
[0010] In one embodiment of the present invention, the culture medium is MRS culture medium.
[0011] The present invention also provides a medicament for the prevention and / or treatment of rheumatoid arthritis, said medicament containing the above-mentioned Lactobacillus helveticus ( Lactobacillus helveticus CCFM1501.
[0012] In one embodiment of the present invention, the pharmaceutical product contains the aforementioned Lactobacillus helveticus ( Lactobacillus helveticus The bacterial count of CCFM1501 should not be less than 3 × 10⁻⁶. 9 CFU / mL or 3×10 9 CFU / g.
[0013] In one embodiment of the present invention, the pharmaceutical product contains the above-mentioned Lactobacillus helveticus ( Lactobacillus helveticus CCFM1501, drug carriers and / or pharmaceutical excipients.
[0014] 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.
[0015] In one embodiment of the present invention, the pharmaceutical excipient comprises excipients and / or additives.
[0016] In one embodiment of the present invention, the number of *Lactobacillus helveticus* CCFM1501 cells in the pharmaceutical product is not less than 3 × 10⁻⁶. 9 CFU / mL or 3×10 9 CFU / g.
[0017] The present invention also provides the use of the aforementioned Lactobacillus helveticus CCFM1501 in the preparation of medicaments for the prevention and / or treatment of rheumatoid arthritis.
[0018] The present invention also provides the application of the aforementioned Lactobacillus helveticus CCFM1501 in the preparation of cell wall proteases, amino acid peptidases and / or aromatic transaminases.
[0019] [Beneficial Effects] 1. This invention screened out a strain of Lactobacillus helveticus (… Lactobacillus helveticus CCFM1501, this Lactobacillus helveticus ( Lactobacillus helveticus CCFM1501 has the function of producing high levels of cell wall proteases, amino acid peptidases and / or aromatic transaminases.
[0020] Meanwhile, the Lactobacillus helveticus ( Lactobacillus helveticus CCFM1501 has the effect of relieving rheumatoid arthritis, specifically in the following ways: (1) Significantly reduced the thickness of the hind paw and improved knee joint injury in rats with rheumatoid arthritis; (2) Significantly reduced the levels of pro-inflammatory cytokines IL-1β, IL-6, IL-17A, and TNF-α in the serum of rats with rheumatoid arthritis; (3) Significantly increased the serum levels of the anti-inflammatory cytokine IL-10 in rats with rheumatoid arthritis; (4) Significantly reduced the content of arthritis-specific antibodies in the serum of rats with rheumatoid arthritis.
[0021] (5) Significantly increased the relative mRNA expression levels of tight junction proteins ZO-1 and Occludin in the colon of rats with rheumatoid arthritis.
[0022] Therefore, Lactobacillus helveticus ( Lactobacillus helveticus CCFM1501 has great potential for application in the preparation of products (such as pharmaceuticals) for the prevention and / or treatment of rheumatoid arthritis.
[0023] 2. Lactobacillus helveticus ( Lactobacillus helveticus Lactobacillus helveticus (Lactobacillus) is a type of probiotic and is currently included in the "List of Microbial Strains that Can Be Used in Food" issued by the Ministry of Health. Therefore, the Lactobacillus helveticus (Lactobacillus) screened in this invention... Lactobacillus helveticus CCFM1501 does not pose any potential safety risks to patients with rheumatoid arthritis (RA).
[0024] 3. Lactobacillus helveticus ( Lactobacillus helveticus The cultivation process of this product only requires the control of culture medium and some culture conditions, and the cost is relatively low. Compared with expensive biological agents, it will not impose too much economic burden on patients with rheumatoid arthritis (RA).
[0025] Preservation of biological materials A strain of Lactobacillus helveticus ( Lactobacillus helveticus CCFM1501, taxonomically named Lactobacillus helveticus It was deposited on May 30, 2025 at the Guangdong Provincial Center for Microbial Culture Collection, with accession number GDMCC No: 66449, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. Attached Figure Description
[0026] Figure 1 Different strains of Lactobacillus helveticus ( Lactobacillus helveticus Cell wall protease activity; different letters in the figure indicate significant differences. p <0.05).
[0027] Figure 2 Different strains of Lactobacillus helveticus ( Lactobacillus helveticus The activity of aminopeptidase PepN in the figure; different letters in the figure indicate significant differences. p <0.05).
[0028] Figure 3 Different strains of Lactobacillus helveticus ( Lactobacillus helveticus The activity of aminopeptidase PepX in the figure; different letters in the figure indicate significant differences. p <0.05).
[0029] Figure 4 Different strains of Lactobacillus helveticus ( Lactobacillus helveticus The activity of aminopeptidase PepC in the figure; different letters in the figure indicate significant differences. p <0.05).
[0030] Figure 5 Different strains of Lactobacillus helveticus ( Lactobacillus helveticus The activity of aromatic transaminases; different letters in the figure indicate significant differences. p <0.05).
[0031] Figure 6 Lactobacillus helveticus ( Lactobacillus helveticus Effects of CCFM1501 on paw thickness in rats with rheumatoid arthritis; different letters in the figure indicate significant differences. p <0.05).
[0032] Figure 7 Lactobacillus helveticus ( Lactobacillus helveticus Effects of CCFM1501 on pathological sections of the knee joint of rats with rheumatoid arthritis.
[0033] Figure 8 Lactobacillus helveticus ( Lactobacillus helveticusThe effect of CCFM1501 on serum IL-1β levels in rheumatoid arthritis rats; in the figure: different letters above the bars indicate significant differences. p <0.05).
[0034] Figure 9 Lactobacillus helveticus ( Lactobacillus helveticus The effect of CCFM1501 on serum IL-6 levels in rheumatoid arthritis rats; in the figure: different letters above the bars indicate significant differences. p <0.05).
[0035] Figure 10 Lactobacillus helveticus ( Lactobacillus helveticus The effect of CCFM1501 on serum IL-17A levels in rheumatoid arthritis rats; in the figure: different letters above the bars indicate significant differences. p <0.05).
[0036] Figure 11 Lactobacillus helveticus ( Lactobacillus helveticus The effect of CCFM1501 on serum TNF-α levels in rheumatoid arthritis rats; in the figure: different letters above the bars indicate significant differences. p <0.05).
[0037] Figure 12 Lactobacillus helveticus ( Lactobacillus helveticus The effect of CCFM1501 on serum IL-10 levels in rheumatoid arthritis rats; in the figure: different letters above the bars indicate significant differences. p <0.05).
[0038] Figure 13 Lactobacillus helveticus ( Lactobacillus helveticus The effect of CCFM1501 on serum CII-total IgG in rheumatoid arthritis rats; in the figure: different letters above the bars indicate significant differences. p <0.05).
[0039] Figure 14 Lactobacillus helveticus ( Lactobacillus helveticus The effect of CCFM1501 on serum CII-IgG1 in rheumatoid arthritis rats; in the figure: different letters above the bars indicate significant differences. p <0.05).
[0040] Figure 15 Lactobacillus helveticus ( Lactobacillus helveticus The effect of CCFM1501 on serum CII-IgG2a in rheumatoid arthritis rats; in the figure: different letters above the bars indicate significant differences. p<0.05).
[0041] Figure 16 Lactobacillus helveticus ( Lactobacillus helveticus The effect of CCFM1501 on serum CII-IgG2b in rheumatoid arthritis rats; in the figure: different letters above the bars indicate significant differences. p <0.05).
[0042] Figure 17 Lactobacillus helveticus ( Lactobacillus helveticus Effect of CCFM1501 on the relative mRNA expression of tight junction protein ZO-1 in the colon of rats with rheumatoid arthritis; In the figure: different letters above the bars indicate significant differences (p<0.05).
[0043] Figure 18 Lactobacillus helveticus ( Lactobacillus helveticus Effect of CCFM1501 on the relative mRNA expression of Occludin, a tight junction protein in the colon of rats with rheumatoid arthritis; In the figure: different letters above the bars indicate significant differences (p<0.05).
[0044] Figure 19 Lactobacillus helveticus ( Lactobacillus helveticus Effect of CCFM1501 on acetic acid content in feces of rats with rheumatoid arthritis; In the figure: different letters above the bars indicate significant differences (p<0.05).
[0045] Figure 20 Effect of Lactobacillus helveticus CCFM1501 on butyrate content in feces of rats with rheumatoid arthritis; In the figure: different letters above the bars indicate significant differences (p<0.05). Detailed Implementation
[0046] The present invention will be further described below with reference to specific embodiments and accompanying drawings.
[0047] 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, TNF-α, and IL-10 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 RNA extraction kit, reverse transcription kit, and qPCR kit were purchased from Nanjing Novizan Co., Ltd.
[0048] In this invention, unless otherwise specified, "%" or percentage used to describe concentration or proportion refers to weight percentage.
[0049] The culture media involved in the following examples are as follows: MRS liquid culture medium: 10 g tryptone, 10 g beef extract, 5 g yeast powder, 20 g glucose, 2 g diammonium citrate, 5 g sodium acetate, 2 g dipotassium hydrogen phosphate, 0.5 g magnesium sulfate heptahydrate, 0.25 g manganese sulfate monohydrate, 1 mL Tween 80, add water to 1000 mL.
[0050] MRS solid medium is obtained by adding 1.5% agar based on the total weight of the liquid medium to the above.
[0051] Example 1: Screening and strain identification of Lactobacillus helveticus 1. Screening The samples were derived from dairy products in Xining, Qinghai Province, and stored in 30% (v / v) glycerol at -80°C. After thawing at low temperature, the samples were aseptically mixed and 0.5 mL was added to a 10 mL centrifuge tube containing 4.5 mL of physiological saline to obtain 10... -1 Diluent, repeat the above dilution steps to obtain 10 -2 10 -3 10 -4 10 -5 10 -6 Diluents: 100 μL of each gradient dilution was spread onto MRS solid medium and incubated at 37°C for 72 h to obtain dilution plates. Typical colonies from the dilution plates were streaked onto MRS solid medium and incubated at 37°C for 48 h to obtain purified colonies. Purified colonies were inoculated into MRS liquid medium and incubated at 37°C for 48 h to obtain CCFM1501.
[0052] 2. Identification The genome of CCFM1501 was extracted and 16S amplified. The 16S rDNA amplification conditions were: 95℃ for 5 min; 35 cycles (95℃ for 30 s, 55℃ for 30 s, 72℃ for 2 min); 72℃ for 10 min. Amplification primers: 27F: (5'-AGAGTTTGATCCTGGCTCAG-3'), 1492R: (5'-TACGGCTACCTTGTTACGACTT-3'). The purification and sequence alignment of the amplification products were performed according to the method described in the literature (Turroni F et al. Exploring the Diversity of the Bifidobacterial Population in the Human Intestinal Tract[J]. Appl Environ Microb. 2009; 75(6):1534–45). The 16S rDNA of CCFM1501 was amplified and sequenced (by Suzhou Genewiz Biotechnology Co., Ltd.). The 16S rDNA sequence of CCFM1501 obtained from the sequencing analysis was compared with NCBI, and the results showed that this strain is Lactobacillus helveticus, and it was named Lactobacillus helveticus. Lactobacillus helveticus CCFM1501.
[0053] Example 2: Preparation of Lactobacillus helveticus suspension Lactobacillus helveticus CCFM1501 bacterial culture was prepared as follows: The Lactobacillus helveticus CCFM1501 obtained in Example 1 was streaked on MRS solid medium and cultured at 37°C for 48 hours to obtain single colonies. The colonies were milky white with smooth edges. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37℃ for 24 h to obtain an activated solution; the activated solution was inoculated into MRS liquid medium at an inoculation rate of 1% (v / v) and cultured at 37℃ for 24 h to obtain a primary seed culture. The primary seed culture was inoculated into MRS liquid medium at an inoculation rate of 1% (v / v) and cultured at 37°C for 24 h to obtain the secondary seed culture. The secondary seed culture was inoculated into 1 L of MRS liquid medium at an inoculation rate of 1% (v / v) and cultured at 37°C for 24 h to obtain bacterial culture. The bacterial culture was centrifuged at 6000 g for 20 min, and the precipitate was collected. The precipitate was washed twice with physiological saline buffer, and then centrifuged again at 6000 g for 10 min to obtain bacterial cells. The Lactobacillus helveticus bacterial cells were resuspended in physiological saline to a cell concentration of 3 × 10⁻⁶ cells / mL. 9 CFU / mL was used to obtain Lactobacillus helveticus culture. For subsequent experiments, the culture was centrifuged to remove glycerol, washed once with physiological saline, and then resuspended in physiological saline.
[0054] Example 3: Determination of carbohydrate utilization by Lactobacillus helveticus Lactobacillus helveticus was streaked on MRS solid medium and cultured at 37°C for 48 h. Single colonies were picked and inoculated into MRS liquid medium and cultured at 37°C for 24 h to obtain an activated solution. The activated solution was inoculated into MRS liquid medium at an inoculation rate of 1% (v / v) and cultured at 37°C for 24 h, and the activation was repeated 3 times.
[0055] Prepare glucose-free MRS liquid culture medium (MRS does not contain glucose, but contains cysteine).
[0056] Preparation of bromocresol violet stock solution: Dissolve in anhydrous ethanol, then bring to volume with water. For example, to prepare 20 mL of bromocresol violet solution, add 0.1 g of bromocresol violet to a 50 mL centrifuge tube, dissolve in 4 mL of anhydrous ethanol (repeatedly pipetting is required), and then bring to volume 20 mL. The stock solution concentration is 0.5%; add 15 mL of stock solution to 1 L of culture medium.
[0057] Preparation of sugar solution (prepare and use immediately): The sugar concentration in this experiment was 1 g / 100 mL; taking a 50 mL aliquot as an example, a 1% sugar solution requires 0.5 g of sugar; at this point, 5 mL of water is needed to completely dissolve the sugar, and then 45 mL of glucose-free MRS liquid culture medium is added. The stock solution needs to be filtered and sterilized.
[0058] Qualitative analysis was performed using a 96-well plate method: 200 μL of sugar solution was added to each well, followed by 2 μL of bacterial culture. After culturing for 24 h, the color change was observed. A yellow color indicated that the strain could utilize this carbon source for metabolism. The results are shown in Table 1. *Lactobacillus helveticus* CCFM1501 can utilize trehalose, while the type strain *Lactobacillus helveticus* ATCC15009 cannot.
[0059] Table 1 Carbon source metabolism
[0060] Where: + indicates that the carbon source can be utilized, and - indicates that the carbon source cannot be utilized.
[0061] Example 4: Determination of protease activity in Lactobacillus helveticus MRS medium Preparation of bacterial cell suspension: Take the bacterial suspension grown in MRS liquid medium for 16 h, add trisodium citrate to a final concentration of 0.25 mol / L, centrifuge at 6000 ×g at 4℃ for 10 min, resuspend the bacterial sludge in a mixture of 0.9% NaCl and 0.25 mol / L trisodium citrate, centrifuge at 6000 ×g at 4℃ for 10 min, wash twice with 0.9% NaCl, and adjust the bacterial sludge to OD600 = 1.0 with 0.1 mol / L phosphate buffer (pH 6.5) for later use.
[0062] Preparation of cell-free extract (CFE): Take bacterial culture grown in MRS liquid medium for 16 h, centrifuge at 4000 × g, 4℃ for 10 min, resuspend in 50 mmol / L sodium phosphate buffer (pH 7.5) and centrifuge again. Repeat this process three times, resuspending the bacterial cells in the same buffer to a final volume of 2 mL. Disrupt the cells using liquid nitrogen freeze-thaw. Place the buffer containing the bacterial cells in a mortar, add a certain volume of liquid nitrogen, and repeat the freeze-thaw process three times. Collect the liquid, centrifuge at 10000 × g, 4℃ for 10 min, collect the supernatant, and store at -80℃ for later use.
[0063] Assay for cell wall protease activity: TBS solution, FTC-casein stock solution, and FTC-casein working solution were prepared according to the instructions of the fluorescent protease assay kit. 100 μL of sample was added to each well of a fluorescent 96-well plate, followed by 100 μL of FTC-casein working solution, repeated three times in parallel. The 96-well plates were incubated at 37°C for 24 h and then analyzed using a microplate reader. The cell wall protease (CEP) activity of the strain was directly expressed as fluorescence intensity.
[0064] Determination of the activities of three intracellular amino acid peptidases: The chromogenic substrates L-lysine-p-nitroaniline (L-Lys-pNA), H-arginine-p-nitroaniline (H-Arg-pNA), and H-glycyl-proline-p-nitroaniline (H-Gly-Pro-pNA) were prepared into 1 mmol / L solutions using 50 mmol / L sodium phosphate buffer (pH 7.5). 50 μL of substrate and 50 μL of CFE were added to each well of a 96-well plate, and the plates were incubated at 37°C for 30 min. The absorbance was measured at 405 nm. Each sample was measured three times. The activity of the amino acid peptidases was defined as the amount of p-nitroaniline released per unit amount of enzyme per minute. One unit of enzyme activity (U) refers to the amount of enzyme that converts 1 μmol of p-nitroaniline per minute under standard conditions. Construction of the p-nitroaniline standard curve: p-nitroaniline solutions of different concentrations (20–160 μmol / L) were prepared, and absorbance was measured at 405 nm to obtain the standard curve. This experiment had no positive control; no commercially available enzymes were used; a buffer solution was used instead of CFE as the blank control; protein content was measured three times in parallel using a protein quantification (BCA) kit.
[0065] Determination of intracellular aromatic transaminase activity: 20 mmol / L L-phenylalanine, 10 mmol / L α-ketoglutarate, 0.5 mmol / L disodium EDTA, and 0.05 mmol / L pyridoxal 5'-phosphate 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 the plates were incubated at 37°C for 12 h. The absorbance was measured at 290 nm. AraT activity is defined as the amount of phenylalanine converted to phenylpyruvate. One unit of enzyme activity (U) refers to the amount of enzyme that converts 1 μmol of phenylpyruvate per minute under standard conditions. Construction of sodium phenylpyruvate standard curve: Sodium phenylpyruvate solutions of different concentrations (0.1 ~ 6 mmol / L) were prepared, and the absorbance was measured at 290 nm to obtain a standard curve. This experiment had no positive control, no commercial enzymes, and the negative control contained only CFE and no phenylalanine; the blank control used buffer instead of CFE; the protein content was measured three times in parallel using the BCA kit.
[0066] The experimental results are shown below. Figures 1 - 5 .
[0067] Figure 1 The results showed that, compared with ATCC15009 (fluorescence intensity 33) and CCFM1096 (fluorescence intensity 40), CCFM1501 had a fluorescence intensity of 79, indicating that it had higher cell wall protease activity.
[0068] Figure 2The results show that, regarding PepN activity, ATCC15009 has 46 nmol of p-nitroaniline (per min per mg protein), CCFM1096 has 19 nmol of p-nitroaniline (per min per mg protein), and CCFM1501 has 180 nmol of p-nitroaniline (per min per mg protein). That is, the activities of ATCC15009 and CCFM1096 are 0.046 and 0.019 U / mg, respectively, while the activity of CCFM1501 is 0.18 U / mg, indicating that it has higher amino acid peptidase (PepN) activity.
[0069] Figure 3 The results show that, regarding PepX activity, ATCC15009 has 22.4 nmol of p-nitroaniline (per min per mg protein), CCFM1096 has 10.6 nmol of p-nitroaniline (per min per mg protein), and CCFM1501 has 47.6 nmol of p-nitroaniline (per min per mg protein). That is, the activities of ATCC15009 and CCFM1096 are 0.0224 and 0.0106 U / mg, respectively, while the activity of CCFM1501 is 0.0476 U / mg, indicating that it has higher amino acid peptidase (PepX) activity.
[0070] Figure 4 The results show that, regarding PepC activity, ATCC15009 has 53.5 nmol of p-nitroaniline (per min per mg protein), CCFM1096 has 15.8 nmol of p-nitroaniline (per min per mg protein), and CCFM1501 has 165.2 nmol of p-nitroaniline (per min per mg protein). That is, the activities of ATCC15009 and CCFM1096 are 0.0535 and 0.0158 U / mg, respectively, while the activity of CCFM1501 is 0.1652 U / mg, indicating that it has higher amino acid peptidase (PepC) activity.
[0071] Figure 5 The results showed that ATCC15009 had 12.7 mmol phenylpyruvate (per mg protein), CCFM1096 had 5.9 mmol phenylpyruvate (per mg protein), and CCFM1501 had 27.93 mmol phenylpyruvate (per mg protein). This means that ATCC15009 and CCFM1096 had activities of 17.6 and 8.2 U / mg, respectively, while CCFM1501 had an activity of 38.8 U / mg, indicating that it had higher aromatic transaminase activity.
[0072] Example 5: Effect of Lactobacillus helveticus CCFM1501 on hind paw thickness in rats with rheumatoid arthritis 1. Laboratory animals Six-week-old female SPF-grade Wistar rats were housed in IVC cages, with six rats per cage. The room temperature was 22-24°C and the humidity was 40-60%. The rats were housed in a 12-hour / 12-hour day-night cycle with free access to food and water.
[0073] 2. Experimental Methods (1) Establishment of a rat model of rheumatoid arthritis The experiment lasted five weeks, starting one week before modeling and continuing until the end of the experiment. The second and third weeks were the modeling period. On the first day of modeling, equal volumes of bovine type II collagen solution (Chondrex, 20022) and Freund's incomplete adjuvant (Chondrex, 7002) were mixed and emulsified to form a complete emulsion. Rats were anesthetized with isoflurane, fixed, and their tail base was disinfected with 75% alcohol. The rats were then given their first immunization (on day 8). For the first immunization, 0.2 mL of the complete emulsion was accurately aspirated and injected subcutaneously at a distance of 1.5 cm from the tail base. One week later, a booster immunization was performed using the same method, i.e., 0.2 mL of the complete emulsion was accurately aspirated and injected subcutaneously at a distance of 2.0 cm from the tail base. Rats in the normal group were injected with the same volume of sterile saline using the same method.
[0074] (2) Experimental grouping and drug administration The animals were randomly divided into 3 groups, with 6 animals in each group. The 3 groups were: normal group, model group, and group treated with Lactobacillus helveticus via gavage. Lactobacillus helveticus The CCFM1501 group consisted of rats in the normal and model groups who were administered 1 mL of physiological saline by gavage daily, and rats in the CCFM1501 group who were administered 1 mL of 3×10⁻⁶ saline by gavage daily. 9 CFU / mL CCFM1501 bacterial solution was administered via gavage from the first week to the end of the fifth week, for a total of 35 days.
[0075] After modeling, the joint thickness of each group of rats was measured using a micrometer, and hematoxylin-eosin pathological sections of the rat knee joints were prepared. The measurement results are shown below. Figures 6 - 7 .
[0076] Figure 6 This indicates that joint swelling began approximately 12 days after the initial immunization, and by day 35, the joint thickness in the model group rats was significantly greater than that in the normal group rats. p <0.05), the joint thickness of rats in the CCFM1501 group was significantly lower than that in the model group ( p<0.05), among which, the joint thickness of rats in the control group, model group and CCFM1501 group was 5.55, 8.44 and 7.16 mm, respectively.
[0077] Depend on Figure 7 It can be seen that the knee joints of the model group rats were severely damaged, with the cartilage surface covered by a large area of proliferating tissue, forming pannus, and a large number of inflammatory cells, mainly lymphocytes, macrophages and granulocytes, were infiltrated; CCFM1501 can significantly alleviate the pathological damage of the knee joint.
[0078] It is evident that Lactobacillus helveticus CCFM1501 can be used as an adjunct treatment to alleviate the symptoms of rheumatoid arthritis.
[0079] Example 6: Effect of Lactobacillus helveticus CCFM1501 on serum cytokine levels in rats with rheumatoid arthritis The animal experimental methods are described in Example 5.
[0080] Cytokines are typically secreted by leukocytes, exerting paracrine or autocrine effects to regulate various responses such as cell differentiation, activation, migration, and survival. Serum cytokine levels reflect the level of systemic inflammatory immune responses, and measuring their concentrations can, to some extent, reflect the body's inflammatory state.
[0081] 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, and the level of anti-inflammatory factor IL-10 in the serum of each group of rats were measured using an ELISA kit. The results are shown below. Figures 8 - 12 .
[0082] like Figure 8 As shown, the serum IL-1β concentration in the model group rats was 55 pg / mL, which was significantly higher than that in the normal group (16 pg / mL). p <0.05); Compared with the model group rats, the serum IL-1β level in the CCFM1501 group rats was significantly reduced ( p The concentration of the novel coronavirus was 28 pg / mL (<0.05), representing a decrease of 49.1%.
[0083] like Figure 9 As shown, the serum IL-6 concentration in the model group rats was 166 pg / mL, which was significantly higher than that in the normal group (55 pg / mL). p <0.05); Compared with the model group rats, the serum IL-6 level in the CCFM1501 group rats was significantly reduced ( p The concentration of the novel coronavirus was 86 pg / mL (<0.05), representing a decrease of 48.2%.
[0084] like Figure 10As shown, the serum IL-17A concentration in the model group rats was 120 pg / mL, which was significantly higher than that in the normal group (37 pg / mL). p <0.05); Compared with the model group rats, the serum IL-17A level in the CCFM1501 group rats was significantly reduced ( p The concentration of the novel coronavirus was 84 pg / mL (<0.05), a decrease of 30.0%.
[0085] like Figure 11 As shown, the serum TNF-α concentration in the model group rats was 418 pg / mL, which was significantly higher than that in the normal group (107 pg / mL). p <0.05); Compared with the model group rats, the serum TNF-α level in the CCFM1501 group rats was significantly reduced ( p The concentration of the novel coronavirus was 252 pg / mL (<0.05), a decrease of 39.7%.
[0086] like Figure 12 As shown, the serum IL-10 concentration in the model group rats was 69 pg / mL, which was significantly higher than that in the normal group (61 pg / mL). p <0.05); Compared with the model group rats, the serum IL-10 level in the CCFM1501 group rats was significantly increased ( p The concentration of the novel coronavirus was 102 pg / mL (<0.05), an increase of 47.8%.
[0087] Therefore, Lactobacillus helveticus CCFM1501 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, and increase the level of anti-inflammatory factor IL-10 in the serum of rats.
[0088] Example 7: Effect of Lactobacillus helveticus CCFM1501 on the content of type II collagen-specific antibody IgG subtype in the serum of rheumatoid arthritis rats The animal experimental methods are described in Example 5.
[0089] Anti-CII IgG antibodies are specific antibodies against type II collagen. In the CIA model, the production of anti-CII IgG is crucial for disease development, inducing or exacerbating arthritis, and can be used to assess the severity of rheumatoid arthritis. Reducing the production of autoantibodies can effectively block the progression of arthritis.
[0090] After the experiment, blood was collected from the rats, and they 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 below.Figure 13-16 .
[0091] like Figure 13 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). p <0.05); Compared with the model group rats, the level of total type II collagen-specific antibody IgG in the serum of CCFM1501 group rats was significantly reduced ( p The concentration of the novel coronavirus was 4.08 μg / L (<0.05), a decrease of 37.0%.
[0092] like Figure 14 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). p <0.05); Compared with the model group rats, the level of type II collagen-specific antibody IgG1 in the serum of CCFM1501 group rats was significantly reduced ( p The concentration of the novel coronavirus was 5.46 μg / L (<0.05), a decrease of 26.4%.
[0093] like Figure 15 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). p <0.05); Compared with the model group rats, the level of type II collagen-specific antibody IgG2a in the serum of CCFM1501 group rats was significantly reduced ( p The concentration of the novel coronavirus was 2.96 μg / L (<0.05), a decrease of 27.3%.
[0094] like Figure 16 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). p <0.05); Compared with the model group rats, the level of type II collagen-specific antibody IgG2b in the serum of CCFM1501 group rats was significantly reduced ( p The concentration of the novel coronavirus was 2.17 μg / L (<0.05), a decrease of 21.9%.
[0095] It is evident that Lactobacillus helveticus CCFM1501 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.
[0096] Example 8: Effect of Lactobacillus helveticus CCFM1501 on the relative mRNA expression levels of tight junction proteins ZO-1 and Occludin in the colon of rheumatoid arthritis rats. The animal experimental methods are described in Example 5.
[0097] Tight junctions are composed of various transmembrane protein families, cytoplasmic protein families, and filamentous actin-binding proteins. Among them, Occludin and ZO-1 are components of transmembrane and cytoplasmic proteins, respectively, and are important components for maintaining the barrier function of intestinal cells. Decreased barrier function promotes the pathogenesis of clinical rheumatoid arthritis (RA), therefore, restoring intestinal barrier function can play a role in alleviating RA.
[0098] After the experiment, rat colons were collected, ground in liquid nitrogen, and total RNA was extracted using an RNA extraction kit. The RNA was then reverse transcribed into cDNA and detected using quantitative PCR (qPCR). The results are shown below. Figure 17-18 .
[0099] like Figure 17 As shown, compared with the normal group, the relative expression level of colonic tight junction protein ZO-1 mRNA in the model group rats was significantly reduced, decreasing by 72.6%. Lactobacillus helveticus CCFM1501 could increase the relative expression level of colonic tight junction protein ZO-1 mRNA. p <0.05), which is 2.2 times higher than the model group.
[0100] like Figure 18 As shown, compared with the normal group, the relative expression level of Occludin mRNA in the colon of rats in the model group was significantly reduced by 67.3%. Lactobacillus helveticus CCFM1501 can increase the relative expression level of Occludin mRNA in the colon. p <0.05), which is 2.8 times higher than the model group, reaching a level comparable to the control group.
[0101] It is evident that Lactobacillus helveticus CCFM1501 can increase the relative mRNA expression levels of the colonic tight junction proteins ZO-1 and Occludin in rats with rheumatoid arthritis, thereby improving the intestinal barrier and alleviating rheumatoid arthritis.
[0102] Example 9: Lactobacillus helveticus CCFM1501 increases short-chain fatty acid content in rheumatoid arthritis rats. The animal experimental methods are described in Example 5.
[0103] Short-chain fatty acids (SCFAs) are fatty acids with fewer than six carbons produced by gut microbiota metabolism, including acetic acid and butyric acid. SCFAs enter the body via the bloodstream and exert a wide range of biological functions, such as regulating immune system function, maintaining the intestinal mucosal barrier, and inhibiting intestinal inflammation, which are of great significance for rheumatoid arthritis (RA).
[0104] After the experiment, rat feces were collected and stored at -80 ℃ for subsequent experiments. After sample pretreatment, the content of short-chain fatty acids in the feces was measured using GC-MS. The results are shown below. Figures 19-20 .
[0105] like Figure 19 As shown, compared with the normal group, the acetic acid content in the feces of rats in the model group was significantly reduced by 54.8%; compared with the model group rats, Lactobacillus helveticus CCFM1501 could increase the acetic acid content in the feces of rats (p<0.05), by 39.3%.
[0106] like Figure 20 As shown, compared with the normal group, the butyric acid content in the feces of rats in the model group was significantly reduced by 74.7%; compared with the model group rats, Lactobacillus helveticus CCFM1501 could increase the butyric acid content in the feces of rats (p<0.05), which was 4.97 times higher.
[0107] It is evident that Lactobacillus helveticus CCFM1501 can increase and improve the content of short-chain fatty acids in rats with rheumatoid arthritis, thereby alleviating rheumatoid arthritis.
[0108] 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. A strain of Lactobacillus helveticus ( Lactobacillus helveticus CCFM1501, characterized in that, The Lactobacillus helveticus CCFM1501 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on May 30, 2025, with accession number GDMCCNo: 66449, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.
2. A microbial preparation containing Lactobacillus helveticus CCFM1501 as described in claim 1.
3. The microbial preparation according to claim 2, characterized in that, In the aforementioned microbial preparation, the bacterial count of *Lactobacillus helveticus* CCFM1501 as described in claim 1 is not less than 3 × 10⁻⁶. 9 CFU / mL or 3×10 9 CFU / g.
4. A pharmaceutical product containing Lactobacillus helveticus CCFM1501 as described in claim 1 or containing the microbial preparation as described in claim 2 or 3.
5. The medicine as described in claim 4, characterized in that, The drug contains the Lactobacillus helveticus CCFM1501 as described in claim 1, a drug carrier, and / or pharmaceutical excipients.
6. The pharmaceutical product as described in claim 5, characterized in that, The drug carrier includes one or more of the following commonly used in medicine: fillers, adhesives, wetting agents, disintegrants, lubricants, and flavoring agents.
7. The pharmaceutical product as described in claim 6, characterized in that, The pharmaceutical excipients include excipients and / or additives.
8. The pharmaceutical product according to any one of claims 4 to 7, characterized in that, In the aforementioned medicine, the bacterial count of *Lactobacillus helveticus* CCFM1501 as described in claim 1 is not less than 3 × 10⁻⁶. 9 CFU / mL or 3×10 9 CFU / g.
9. The use of Lactobacillus helveticus CCFM1501 as described in claim 1 in the preparation of a medicament for the prevention and / or treatment of rheumatoid arthritis.
10. The use of Lactobacillus helveticus CCFM1501 as described in claim 1 in the preparation of cell wall proteases, amino acid peptidases, and / or aromatic transaminases.