Use of heat-inactivated bifidobacterium longum ZFML0012 in relieving rheumatoid arthritis

Abstract

The application belongs to the technical field of microorganisms and particularly relates to application of heat-inactivated Bifidobacterium longum ZFML0012 in relieving rheumatoid arthritis. TNF-α 、 iNOS 、 IL-1β The mRNA expression level of the heat-inactivated Bifidobacterium longum ZFML0012 bacterial liquid can be lowered, and the mRNA expression level of the heat-inactivated Bifidobacterium longum ZFML0012 bacterial liquid can be raised. IL-10 The taxonomic name of the Bifidobacterium longum ZFML0012 is Bifidobacterium longum ( Bifidobacterium longum ) ZFML0012, and the preservation number is CCTCC NO: M 20252539. The heat-inactivated Bifidobacterium longum ZFML0012 bacterial liquid has anti-inflammatory activity on inflammation induced by lipopolysaccharide, and the heat-inactivated Bifidobacterium longum ZFML0012 bacterial liquid can improve joint injury and immune imbalance.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to the application of heat-inactivated Bifidobacterium longum ZFML0012 in relieving rheumatoid arthritis. Background Technology

[0002] Rheumatoid arthritis (RA) is a chronic autoimmune disease primarily characterized by inflammation of the synovial membrane, cartilage destruction, and bone erosion, which can lead to joint deformities and loss of function in severe cases. Currently, nonsteroidal anti-inflammatory drugs (NSAIDs), glucocorticoids, and disease-modifying antirheumatic drugs (DMARDs) are commonly used in clinical practice; however, long-term use often results in adverse reactions such as gastrointestinal disturbances, liver and kidney damage, and increased risk of infection. Therefore, developing safe and effective intervention strategies for RA is of significant clinical importance.

[0003] The link between gut microbiota and rheumatoid arthritis (RA) is becoming increasingly clear. The gut is an important immune site in the human body, home to a large number of microorganisms. RA patients commonly exhibit gut microbiota dysbiosis, characterized by a decrease in beneficial bacteria (such as Bifidobacteria and Bacteroides) and an increase in pathogenic bacteria (such as Prevotella). This dysbiosis can damage the intestinal mucosal barrier, increase intestinal permeability, and allow bacterial metabolites, endotoxins, and even activated inflammatory cells to enter the bloodstream and reach the joints. This leads to the infiltration of joint tissues by immune cells such as T cells, B cells, and macrophages, producing various pro-inflammatory cytokines, exacerbating joint inflammation, and ultimately causing tissue damage. Therefore, anti-inflammatory therapy has become an important direction in the basic and clinical treatment of RA, and maintaining gut microbiota homeostasis is of great significance for alleviating joint inflammation.

[0004] The invention CN116574659A, titled "A Bifidobacterium infantis strain that can alleviate rheumatoid arthritis and its application," states that the bacterial culture of Bifidobacterium infantis subspecies CCFM1269 can be used to prepare drugs to alleviate rheumatoid arthritis. It can significantly relieve joint swelling, reduce serum IL-6, TNF-α, IL-22, and IL-23 levels, regulate the content of type II collagen-specific antibodies, osteoprotegerin, and osteocalcin, and improve intestinal flora.

[0005] Invention CN118985920A, entitled "Compositions Containing Bifidobacterium longum BORI for Improving, Preventing or Treating Osteoarthritis," discloses that Bifidobacterium longum BORI can be used to prepare compositions for improving, preventing or treating osteoarthritis. Mouse models have demonstrated that this bacterium can relieve pain, control joint tissue destruction, and reduce the expression of inflammatory markers IL-1β, IL-6, LTB4R, and cartilage destruction markers MMP9, MMP13, CTX-II, and PGE2.

[0006] The invention CN111902531A, entitled "Bifidobacterium longum RAPO strain and composition containing the same for improving, preventing or treating rheumatoid arthritis", discloses that the novel Bifidobacterium longum RAPO can inhibit the onset of rheumatoid arthritis and joint tissue destruction, reduce inflammation and osteochondral damage, inhibit intestinal damage and inflammatory cytokine IL-17, and has no ammonia production or hemolytic ability, and can safely and effectively improve, prevent or treat rheumatoid arthritis.

[0007] The aforementioned existing technologies all focus on the role of live bacterial preparations in rheumatoid arthritis or osteoarthritis, and do not involve the anti-rheumatoid arthritis efficacy and related mechanisms of heat-inactivated Bifidobacterium longum as a metabiotic. That is, once the aforementioned patents are prepared into the corresponding heat-inactivated Bifidobacterium longum, it will lead to a significant decrease or even loss of the corresponding performance.

[0008] Postbiotics are preparations of non-living microorganisms and / or their components that are beneficial to the host's health. Compared with live probiotics, postbiotics have significant advantages such as high stability, strong safety, ease of industrial production, and immunity to antibiotic interference. Studies have shown that postbiotics can alleviate immune-related diseases such as allergies and enteritis through multiple mechanisms, including regulating gut microbiota structure, repairing the intestinal mucosal barrier, regulating immune cell differentiation, and inhibiting inflammatory signaling pathways. Bifidobacterium longum, as a core beneficial bacterium in the human gut, has been shown to have immunomodulatory and anti-inflammatory activities in its derived postbiotics.

[0009] Invention CN113925885B, entitled "Use of a post-biotic extract for the prevention and / or treatment of arthritis and joint degeneration," discloses a method for preparing a post-biotic extract of *Bifidobacterium longum* CB108 as follows: *Bifidobacterium longum* CB108 is inoculated into MRS medium and cultured at 37°C for 16 hours. High-temperature short-time sterilization (HTST) is then performed, heating at 73±2°C for 15 seconds for heat inactivation. The heat-inactivated culture is centrifuged at 10,000 rpm for 15 minutes at 25°C. After discarding the supernatant, the precipitate is collected and spray-dried to obtain dried bacterial powder (including heat-inactivated bacterial cells and a small amount of insoluble matter and metabolic residue from the culture medium). Example 3 only demonstrates that this post-biotic extract can: significantly promote the proliferation of human primary central chondrosarcoma cell line (SW1353 cells) and significantly promote the secretion of transforming growth factor-β (TGF-β) by SW1353 cells.

[0010] It should be noted that substances that significantly promote SW1353 cell proliferation in in vitro experiments demonstrate their potential for anti-inflammatory effects, chondrocyte protection, and resistance to cartilage damage in rheumatoid arthritis (RA). However, this does not equate to their ability to alleviate rheumatoid arthritis. This is because SW1353 is a human chondrosarcoma cell line, and its gene expression profile has only limited similarity to that of primary human chondrocytes. The core pathological mechanism of rheumatoid arthritis is synovial inflammation and cartilage matrix degradation driven by pro-inflammatory factors such as TNF-α and IL-1β, rather than insufficient chondrocyte proliferation. Summary of the Invention

[0011] The technical problem to be solved by the present invention is to provide an application of heat-inactivated Bifidobacterium longum ZFML0012 in relieving rheumatoid arthritis.

[0012] To address the aforementioned problems, this invention provides the application of heat-inactivated Bifidobacterium longum ZFML0012 in relieving rheumatoid arthritis: used in the preparation of drugs to relieve rheumatoid arthritis, the heat-inactivated Bifidobacterium longum ZFML0012 bacterial solution can downregulate (significantly downregulate) the condition. TNF-α , iNOS , IL-1β The mRNA expression level can be upregulated (significantly upregulated). IL-10 The mRNA expression level; the taxonomic name of Bifidobacterium longum ZFML0012 is Bifidobacterium longum ( Bifidobacterium longum ZFML0012, its accession number is CCTCC NO: M 20252539.

[0013] That is, heat-inactivated Bifidobacterium longum ZFML0012 bacterial culture has anti-inflammatory activity against lipopolysaccharide (LPS)-induced inflammation (inhibiting LPS-induced expression of pro-inflammatory factors in RAW264.7 cells and promoting the secretion of anti-inflammatory factors).

[0014] As an improvement to the application of the present invention: heat-inactivated Bifidobacterium longum ZFML0012 can improve joint damage and immune imbalance (joint damage and immune imbalance in the CIA model).

[0015] As a further improvement to the application of this invention, the improvement of joint damage includes: reducing foot thickness, alleviating inflammatory infiltration, and having a bone-protective effect (inhibiting joint bone destruction and improving bone microstructure). It can also alleviate the trend of weight loss.

[0016] As a further improvement to the application of the present invention: improving immune imbalance includes correcting the imbalance of Th17 / Treg cells in the spleen (i.e., having a clear immunomodulatory effect).

[0017] As a further improvement to the application of the present invention: heat-inactivated Bifidobacterium longum ZFML0012 bacterial solution can regulate the intestinal flora structure (regulate the intestinal flora structure of collagen-induced arthritis model mice), reduce the Firmicutes / Bacteroidetes ratio, restore the abundance of beneficial bacteria, and increase the content of short-chain fatty acids.

[0018] As a further improvement to the application of the present invention: the seed culture of Bifidobacterium longum ZFML0012 was transferred to MRS selective liquid medium containing 0.05% L-cysteine ​​at an inoculation rate of 1.5~2.5% (volume%) and anaerobic cultured at 37±1℃ for 24±1h to obtain the fermentation broth. Collect the bacterial cells by centrifugation (3-5℃, 7000-9000×g for 15 min), wash with phosphate buffer (0.01 M, pH 7.3-7.5), resuspend, and adjust the concentration of the bacterial suspension to (0.5-1.5)×10⁻⁶. 9 CFU / mL, heat-inactivated (heated in a water bath at 95±3℃ for 20±2 min) to obtain heat-inactivated Bifidobacterium longum ZFML0012 bacterial suspension (stored at 4℃ for later use).

[0019] In this invention, heat-inactivated Bifidobacterium longum ZFML0012 has the following functions: It can inhibit the expression of pro-inflammatory cytokines in LPS-induced RAW264.7 cells and promote the expression of anti-inflammatory cytokines.

[0020] It can reduce arthritis scores, alleviate joint swelling, and inhibit bone erosion in collagen-induced arthritis model mice; it can also regulate the Th17 / Treg immune balance in the spleen.

[0021] It can regulate the gut microbiota structure in mice with collagen-induced arthritis, reduce the Firmicutes / Bacteroidetes ratio, restore the abundance of beneficial bacteria, and increase the content of short-chain fatty acids.

[0022] This invention has the following technical advantages: (1) The application protocol of heat-inactivated Bifidobacterium longum in RA intervention is provided for the first time: Existing patents mostly focus on the application of live Bifidobacterium in RA. This invention adopts a heat inactivation method, which retains anti-inflammatory activity while avoiding problems such as colonization dependence, antibiotic sensitivity, and poor storage stability that exist in live bacterial preparations (these are common problems of live bacterial preparations recognized in this industry), and has better industrial production potential and application safety.

[0023] (2) Achieving synergistic improvement across multiple pathways, from cellular anti-inflammatory effects to joint protection: Heat-inactivated Bifidobacterium longum ZFML0012 can inhibit the release of pro-inflammatory factors from macrophages in vitro, and reduce arthritis scores, alleviate joint swelling and bone erosion in CIA mice in vivo. At the same time, it can correct the Th17 / Treg imbalance, forming a systemic improvement effect from inflammation suppression to tissue protection.

[0024] (3) Clearly establish the regulatory pathway of "gut microbiota-short-chain fatty acids-immune balance": This invention confirms that heat-inactivated Bifidobacterium longum ZFML0012 can simultaneously regulate the intestinal flora structure of CIA mice (reducing the F / B ratio and increasing the abundance of Bacteroides) and the level of short-chain fatty acids (increasing the content of acetic acid and butyric acid), providing a complete chain of experimental evidence for improving RA through the gut-immune axis. This systemic regulatory mechanism has not been clearly reported in existing RA-related Bifidobacterium patents.

[0025] The beneficial effects of this invention are mainly reflected in: The heat-inactivated Bifidobacterium longum ZFML0012 described in this invention is a postbiotic preparation. While retaining the biological activity of Bifidobacterium longum, it has the characteristics of high postbiotic stability, good safety and no colonization dependence, providing a more promising option for long-term intervention of RA.

[0026] Compared with existing published patents, this invention provides the application of heat-inactivated Bifidobacterium longum ZFML0012 in alleviating rheumatoid arthritis. It can significantly inhibit joint inflammation, reduce bone erosion, correct Th17 / Treg immune imbalance, and exert its effects by regulating the structure of intestinal flora and the level of short-chain fatty acids. The above effects and mechanisms of action have not been disclosed in existing patents, which reflects the unique advantages and significant progress of this invention. Attached Figure Description

[0027] The specific embodiments of the present invention will be further described in detail below with reference to the accompanying drawings.

[0028] Figure 1 Image of Bifidobacterium longum ZFML0012 colony and Gram staining; Figure 1 middle: A is a ZFML0012 colony; B represents the Gram staining result of ZFML0012.

[0029] Figure 2 The graph shows the cell viability results under different multiplicity of infection (MOI).

[0030] Figure 3 The image shows the results of inflammatory factor secretion in RAW264.7 cells. Figure 3 middle: A is TNF-α Expression level; B is IL-1β Expression level; C is iNOS Expression level; D is IL-10 Expression level.

[0031] Figure 4 The results show the changes in mouse body weight, arthritis scores, and paw thickness. Figure 4 middle: A represents the rate of change in body weight; B represents the arthritis score (the sum of scores for the mouse's four limbs). C represents the thickness of the left hind limb foot. D represents the thickness of the right hind limb foot.

[0032] Figure 5 HE staining images of the knee and ankle joints of mice; Figure 5 middle: A shows HE staining of the mouse knee joint; B shows the HE staining image of the mouse ankle joint.

[0033] Figure 6 Histopathological scores were assigned to the knee and ankle joints of mice. Figure 6 middle: A represents the histopathological score of the mouse knee joint tissue; B represents the histopathological score of the mouse ankle joint.

[0034] Figure 7 Image showing the results of mouse joint bone microstructure (Micro-CT image of mouse hind paw).

[0035] Figure 8 Comparison of bone mineral density, bone volume fraction, and number of trabeculae in mouse joint bones; Figure 8 middle: A shows a comparison of bone mineral density; B represents a comparison of bone volume fraction; C represents a comparison of the number of trabeculae.

[0036] Figure 9 This is a graph showing the results of mouse immune cell assays. Figure 9 middle: A represents quantitative analysis of Th17 cells; B represents quantitative analysis of Treg cells; C represents the Th17 / Treg ratio.

[0037] Figure 10 This is a phylum-level stacking diagram of the mouse gut microbiota.

[0038] Figure 11 This represents the ratio of Firmicutes to Bacteroidetes in the mouse gut microbiota.

[0039] Figure 12 This is a stacked diagram of the genus level of the mouse gut microbiota.

[0040] Figure 13 A graph showing the genus-level abundance of the mouse gut microbiota; Figure 13 middle: A represents Bacteroides; B belongs to the genus *Parabacteroides*.

[0041] Figure 14 Figure showing the results of short-chain fatty acid analysis in the mouse intestine; Figure 14 middle: A represents the acetic acid content; B represents the butyric acid content. Detailed Implementation

[0042] The present invention will be further described below with reference to specific embodiments. However, it should be noted that the scope of protection of the present invention is not limited thereto: In the following embodiments, unless otherwise specified, the experimental operations used are all conventional methods in the art; the equipment used are all conventional instruments in the relevant field; and the reagents and materials involved, unless otherwise specified, are all commercially available conventional products or prepared by standard methods.

[0043] The MRS selective liquid medium (g / L) containing 0.05% L-cysteine ​​was prepared from the following ingredients: 10.0 g peptone, 8.0 g beef extract, 4.0 g yeast extract, 20.0 g glucose, 2.0 g dipotassium hydrogen phosphate, 2.0 g diammonium hydrogen citrate, 5.0 g sodium acetate, 0.2 g magnesium sulfate heptahydrate, 0.04 g manganese sulfate, 0.5 g L-cysteine, 1.0 mL Tween 80, and water was added to a final volume of 1 L. The pH was 6.2 ± 0.2.

[0044] Add 20.0 g of agar powder to the above to obtain MRS selective solid medium containing 0.05% L-cysteine.

[0045] Example 1: Isolation, culture and identification of Bifidobacterium longum ZFML0012 (1) Strains Isolation Infant fecal samples from Zhejiang Province were serially diluted 10-fold with phosphate buffer (0.01 M, pH 7.3–7.5). 100 μL of each sample was spread onto MRS selective solid medium containing 0.05% L-cysteine ​​and incubated anaerobicly at 37°C for 48 h. Single colonies were picked and streaked for purification to obtain pure cultures.

[0046] (2) Morphological characteristics After anaerobic culture for 48 h on MRS selective solid medium containing 0.05% L-cysteine, the colonies of this strain were milky white, round, smooth and moist, with regular edges and a raised center, and a diameter of approximately 1-2 mm. Gram staining was positive. Under a microscope, the bacteria were irregularly rod-shaped, often V-shaped, Y-shaped, or rod-shaped, sometimes showing typical bifid morphology (i.e., "bifidosis"), and without spores. These characteristics are consistent with the typical morphological features of *Bifidobacterium longum*.

[0047] Colony diagram and Gram staining diagram of Bifidobacterium longum ZFML0012 are as follows: Figure 1 As shown.

[0048] (3) Molecular biological identification Genomic DNA was extracted from the strain, and the 16S rDNA sequence was amplified. After sequencing, the sequence was compared with the GenBank database using BLAST. The identification results showed that the strain was *Bifidobacterium longum*. Bifidobacterium longum The sequence was named ZFML0012 and the obtained sequence is shown in SEQ ID NO:1.

[0049] (4) Preservation information The present invention contains Bifidobacterium longum ( Bifidobacterium longum The preservation information for ZFML0012 is as follows: Preservation name: Bifidobacterium longum ZFML0012 Bifidobacterium longum ZFML0012; Depository Institution: China Center for Type Culture Collection; Deposit Address: Wuhan University, Wuhan, China; Accession Number: CCTCC NO: M20252539; Deposit Date: November 12, 2025.

[0050] Example 2: Preparation of heat-inactivated Bifidobacterium longum ZFML0012 The *Bifidobacterium longum* ZFML0012 bacterial suspension from the preservation tube was streaked onto an MRS selective solid medium plate containing 0.05% L-cysteine ​​and activated by anaerobic culture at 37°C for 48 h. A single colony was picked and inoculated into 10 mL of MRS selective liquid medium containing 0.05% L-cysteine ​​and anaerobic cultured at 37°C for 24 h to obtain the seed culture. The seed culture was then transferred at a 2% (v / v) inoculation rate to the same liquid medium (MRS selective liquid medium containing 0.05% L-cysteine) and anaerobic cultured at 37°C for 24 h to obtain the fermentation broth. The fermentation broth was centrifuged at 8,000×g for 15 min at 4°C to collect the bacterial cells. The cells were washed twice with phosphate buffer (0.01 M, pH 7.3–7.5) and resuspended, and the concentration of the bacterial suspension was adjusted to 1×10⁻⁶. 9 The bacterial culture of *Bifidobacterium longum* ZFML0012 was obtained by heating at 95℃ for 20 min with CFU / mL and then storing at 4℃ for later use.

[0051] Example 3: Inhibitory effect of heat-inactivated Bifidobacterium longum ZFML0012 on LPS-induced inflammatory factors in RAW264.7 cells. 1. RAW264.7 mouse mononuclear macrophages were cultured and passaged in DMEM complete medium containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics at 37°C in a 5% CO2 incubator.

[0052] Macrophages are key effector cells in inflammatory diseases such as rheumatoid arthritis. Upon activation, they secrete large amounts of pro-inflammatory factors, exacerbating the inflammatory response. Cell viability is a fundamental indicator for evaluating the safety of biomaterials and the feasibility of subsequent experiments. Therefore, this invention first uses the CCK-8 assay to determine the effect of heat-inactivated Bifidobacterium longum ZFML0012 at different multiplicity of infection (MOI) on the viability of RAW264.7 cells to determine a safe treatment concentration for subsequent inflammatory model experiments.

[0053] Under culture conditions of 37℃ and 5% CO2, RAW264.7 cells were cultured at a density of 5 × 10⁶ cells per well. 3 The cells were inoculated at a density of 100 cells / well in 96-well plates and pre-cultured for 24 h.

[0054] The following groups were then set up: Blank group: Add 10 μL of DMEM medium to the blank wells; Control group: 10 μL of DMEM medium was added to the wells inoculated with RAW264.7 cells. Figure 2 MOI=0 was used as the baseline for cell viability without heat-inactivated bacterial treatment, and cell viability was defined as 100%. Sample group: 10 μL of heat-inactivated Bifidobacterium longum ZFML0012 bacterial suspension prepared in Example 2 was added to each well inoculated with RAW264.7 cells. The MOI gradients were set to 1, 10, 100, 200, 500, and 1000 (ratio of heat-inactivated bacteria to RAW264.7 cells). The values ​​of each gradient were logarithmic. 10 Logarithmic transformation processing.

[0055] After each group was treated under the above culture conditions (37℃, 5% CO2) for 16 h, 10 μL of CCK-8 (commercial ready-to-use reagent) was added to each well, and the mixture was incubated in an incubator under the above culture conditions for another 2 h. Finally, the absorbance value of each well was detected at a wavelength of 450 nm using an ELISA reader.

[0056] The formula for calculating cell viability is: Cell viability = [(sample group OD - blank group OD) / (control group OD - blank group OD)] × 100%.

[0057] The results are as follows Figure 2 As shown, when MOI ≤ 100, i.e., log 10 When MOI ≤ 2, heat-inactivated Bifidobacterium longum ZFML0012 showed no significant inhibitory effect on RAW264.7 cell viability. However, when MOI > 100 (log10(MOI) > 2), corresponding to MOI = 200, 500, and 1000, cell viability decreased significantly with increasing MOI, exhibiting obvious dose-dependent cytotoxicity. Therefore, MOI = 100 was selected as the safe treatment concentration for heat-inactivated Bifidobacterium longum ZFML0012 in subsequent inflammatory model construction.

[0058] 2. Lipopolysaccharide (LPS) can induce RAW264.7 macrophages to activate towards a pro-inflammatory phenotype and is a classic stimulant for constructing in vitro inflammation models. To investigate the regulatory role of heat-inactivated Bifidobacterium longum ZFML0012 on LPS-induced inflammatory responses, the mRNA expression levels of related inflammatory factors were detected using RT-qPCR.

[0059] RAW264.7 cells were spaced at 2 × 10⁶ cells per well. 5 Individual inoculum was seeded into 24-well plates and pre-cultured (37℃, 5% CO2) for 24 h. Subsequently, the following groups were established: Control group (CK): 1 mL of DMEM medium was added to the wells in which RAW264.7 cells were seeded; Control group (LPS): 1 mL of DMEM medium was added to the wells in which RAW264.7 cells were seeded; Sample group (HK-) B.longum): Add 1 mL of heat-inactivated Bifidobacterium longum ZFML0012 bacterial suspension (MOI=100) prepared in Example 2 to the wells inoculated with RAW264.7 cells.

[0060] After treatment under the above culture conditions (37℃, 5% CO2) for 12 h, the CK group was replaced with 1 mL of fresh DMEM medium, and the LPS group and HK- B.longum The cells were added to 1 mL of DMEM medium containing 100 ng / mL LPS and stimulated for another 4 h under the above conditions. The supernatant was then discarded, and 1 mL of TRIzol was added to each well to lyse the cells. Total RNA was extracted, and cDNA was synthesized via reverse transcription. GAPDH As an internal control, it was detected using a real-time quantitative PCR instrument. TNF-α , IL-1β , iNOS and IL-10 Relative expression level of mRNA (2) -ΔΔCt (Method), primer sequences are shown in Table 1.

[0061] Table 1 Primer Sequences

[0062] The results are as follows Figure 3 As shown, compared to the LPS group, HK- B.longum Processing group TNF-α , iNOS , IL-1β The mRNA expression levels were significantly downregulated. P < 0.05), and IL-10 The mRNA expression level was significantly upregulated ( P < 0.05). This indicates that the heat-inactivated bacteria can significantly inhibit the expression of pro-inflammatory factors induced by LPS in RAW264.7 cells and promote the secretion of anti-inflammatory factors, thus exhibiting significant anti-inflammatory activity.

[0063] Example 4: Effects of heat-inactivated Bifidobacterium longum ZFML0012 on joint damage and immune imbalance in CIA model mice (1) Construction of CIA animal model Collagen-induced arthritis (CIA) models share high similarities with human rheumatoid arthritis (RA) in terms of clinical manifestations, pathological features, and immune disorders, making them one of the internationally recognized classic animal models for studying RA mechanisms and evaluating intervention effects. To evaluate the intervention effect of heat-inactivated Bifidobacterium longum ZFML0012 on rheumatoid arthritis, this invention constructs a CIA mouse model for research.

[0064] DBA / 1 mice (male, 8 weeks old, 20-22 g) were purchased from Shanghai Yaokang Biotechnology Co., Ltd. They were acclimatized for one week under clean-grade conditions (12-hour light / dark cycle, temperature 23±2℃, humidity 55±5%). On day 0, an equal volume of 2 mg / mL bovine type II collagen was emulsified with complete Freund's adjuvant, and 100 μL of the emulsion was injected subcutaneously into the tail root of the mice for primary immunization. On day 21, a secondary immunization was performed using incomplete Freund's adjuvant in the same manner to obtain CIA model mice. Mice were randomly divided into 4 groups (6 mice in each group): Normal group (NC): Normal mice were administered 200 μL of 0.85% saline by gavage daily for 8 weeks.

[0065] Model group (MOD): CIA model mice were administered 200 μL of 0.85% saline by gavage daily for 8 weeks.

[0066] Methotrexate group (MTX): CIA model mice were administered 200 μL of 1 mg / kg methotrexate solution by gavage daily for 8 weeks.

[0067] Sample group (HK-) B.longum CIA model mice were administered 200 μL of 1×10⁻⁶ dextrose via gavage daily. 9 CFU / mL heat-inactivated Bifidobacterium longum ZFML0012 for 8 weeks.

[0068] During the intervention period, arthritis lesions in mice were scored every 7 days from weeks 1 to 4 (scoring criteria are shown in Table 2, which outlines the scoring criteria for a single limb in mice), and the thickness of the hind limb paw pads was measured. From weeks 5 to 8, arthritis lesions were scored every 3 days. All scoring and analysis were performed without the mice's knowledge of the grouping process. All procedures were conducted in accordance with animal ethics guidelines and were approved by the Animal Welfare and Ethics Committee of the Hangzhou Institute of Medical Sciences, Chinese Academy of Sciences (ethics number: AP2024-11-0314).

[0069] Table 2 Arthritis Scoring Criteria

[0070] The results are as follows Figure 4 As shown, compared with the NC group, the MOD group mice showed a sustained increase in arthritis scores and hind limb paw thickness one week after the second immunization, accompanied by a significant decrease in body weight. P (< 0.05), indicating that the CIA model was successfully constructed. (via HK-) B.longum After intervention, the trend of weight loss in mice was significantly alleviated, and arthritis scores and paw thickness were significantly reduced. P < 0.05), indicating that it can effectively improve joint inflammation and overall condition in CIA mice.

[0071] (2) Histopathological analysis of joint tissue Synovial hyperplasia, inflammatory cell infiltration, cartilage destruction, and bone erosion are the core pathological markers of RA joint injury, and histopathological observation is the gold standard for evaluating the effectiveness of joint protection.

[0072] At the experimental endpoint, mouse knee and ankle joints were collected, fixed in 4% paraformaldehyde, decalcified, and embedded in paraffin to prepare 5 μm sections. After HE staining, the sections were scanned in their entirety using a KF-PRX-040 fully automated slide scanner. Two researchers performed semi-quantitative scoring (0-3 points / item, total score 0-12 points) on four indicators—synovial hyperplasia, inflammatory cell infiltration, cartilage destruction, and bone erosion—under double-blind conditions. The scoring criteria are shown in Table 3.

[0073] Table 3 Scoring criteria for mouse joint tissue pathology

[0074] The results are as follows Figure 5 and Figure 6 As shown, the knee and ankle joints of mice in the NC group were structurally intact, with clear joint spaces and smooth cartilage surfaces; while the joint spaces of mice in the MOD group were narrowed, with severe synovial hyperplasia, extensive inflammatory cell infiltration, and significant erosion of bone and cartilage tissue. Compared with MOD, HK- B.longum The group showed a significant reduction in inflammatory infiltration, with the joint structure remaining largely intact, and a significant decrease in the pathological scores of the knee and ankle joints. P The value of < 0.05 indicates that it can effectively alleviate joint pathological damage in CIA mice and has a protective effect on joint structure.

[0075] (3) Micro-CT analysis Bone erosion and bone loss are key factors leading to joint deformities and functional impairments during the progression of rheumatoid arthritis (RA). Micro-CT can non-invasively and with high precision assess changes in bone microstructure and is an important means of evaluating the effectiveness of bone protection.

[0076] At the end of the experiment, hind limb samples were collected from mice and scanned using a Bruker SkyScan 1276 micro-CT imaging system. Specific scanning parameters were set as follows: scanning voltage 60 kV, scanning current 200 μA, 0.5 mm aluminum filter, resolution 13.06 μm, and reconstruction threshold 0-0.095. After scanning, 3D reconstruction was performed using CTVox software, and key bone microstructural parameters such as bone mineral density, bone volume fraction, and trabecular bone number were quantitatively analyzed using CTAN and DataViewer software.

[0077] The results are as follows Figure 7 and Figure 8As shown, the MOD group mice exhibited typical moth-eaten bone erosion at the edge of the paw joint, manifested as focal bone defects and discontinuity of the bone cortex; HK- B.longum The bone surface contours of the HK- group were clear and smooth, and the degree of bone erosion was significantly reduced. Compared with the MOD group, the HK- B.longum After treatment, bone density and bone volume fraction were significantly increased, and the number of trabeculae was significantly improved, indicating that heat-inactivated Bifidobacterium longum ZFML0012 can effectively inhibit joint bone destruction in CIA mice, improve bone microstructure, and has a clear osteoprotective effect.

[0078] (4) Flow cytometry analysis CD4 + Imbalance in T cell subsets is a core element of immune dysregulation in rheumatoid arthritis (RA). An elevated Th17 / Treg ratio is closely related to disease severity, and regulating the Th17 / Treg balance is an important indicator for evaluating the effectiveness of immune regulation in RA.

[0079] At the experimental endpoint, mouse spleens were used to prepare single-cell suspensions, which were then immunofluorescently labeled using KTH217 (Th17 cell staining) and KTR201 (Treg cell staining) kits, respectively. Th17 cells were stained using a combination of CD4-450 and IL-17-PE antibodies, while Treg cells were labeled using a three-color scheme of CD4-450, CD25-APC, and Foxp3-PE. After staining, flow cytometry was used for analysis, and the data were analyzed using FlowJo V10 software.

[0080] The results are as follows Figure 9 As shown, compared with the MOD group, after HK- B.longum After intervention, the proportion of Th17 cells in the spleen was significantly reduced. P < 0.05), the proportion of Treg cells was significantly increased ( P The Th17 / Treg ratio tended to normal (< 0.05), indicating that heat-inactivated Bifidobacterium longum ZFML0012 could effectively correct the imbalance of Th17 / Treg cells in the spleen of CIA mice and had a clear immunomodulatory effect.

[0081] Example 5: Regulatory effects of heat-inactivated Bifidobacterium longum ZFML0012 on intestinal flora and short-chain fatty acids in CIA mice. (1) 16S rRNA analysis Gut microbiota imbalance is closely related to the occurrence and development of rheumatoid arthritis (RA), and regulating the gut microbiota structure and restoring microbiota homeostasis are important ways to intervene in RA.

[0082] The experimental endpoint involved extracting microbial DNA from mouse colon contents using the FastPure Stool DNA Isolation Kit and amplifying the V3-V4 region of the 16S rRNA gene using barcoded primers 338F / 806R. The purified PCR products were used to construct libraries using the NEXTFLEX Rapid DNA-Seq Kit and sequenced on an Illumina NextSeq 2000 PE300 platform. The raw data underwent quality control, filtering, and assembly to obtain optimized sequences. Representative ASV sequences and abundance information were generated using DADA2 / Deblur noise reduction. Based on the ASV results, analyses were performed including species classification, diversity analysis, community comparison, differential species identification, correlation analysis, phylogenetic analysis, and functional prediction.

[0083] The results are as follows Figures 10-11 As shown, the gut microbiota of mice in each group mainly consisted of Firmicutes (Bacillota) and Bacteroidetes (Bacteroidota) at the phylum level, and the relative ratio of the two (F / B value) is an important indicator reflecting gut microbiota homeostasis. Compared with the NC group, the F / B value of the MOD group was significantly increased ( P < 0.05, indicating that CIA mice have typical gut microbiota imbalance, and intervention with heat-inactivated Bifidobacterium longum ZFML0012 significantly reduced the F / B ratio ( P (< 0.05), bringing it closer to normal. For example... Figures 12-13 As shown, at the genus level, intervention with heat-inactivated Bifidobacterium longum ZFML0012 significantly increased the abundance of beneficial bacteria such as Bacteroides and Parabacteroides. P < 0.05), indicating that the inactivated bacteria can effectively regulate the intestinal flora structure of CIA mice and restore flora homeostasis.

[0084] (2) Short-chain fatty acid analysis Short-chain fatty acids (SCFAs) are key metabolites produced by the fermentation of dietary fiber by gut microbiota. They have multiple physiological functions, such as anti-inflammation, maintaining the intestinal barrier, and regulating immune balance. They are also important mediators connecting gut microbiota with the systemic immune response.

[0085] The content of SCFAs in feces was determined by GC-MS. 25 mg of feces was added to 500 μL of 0.5% phosphoric acid aqueous solution, followed by freeze-grinding, ultrasonic extraction, and centrifugation (4℃, 13000×g, 15 min). The supernatant was extracted with n-butanol solution containing the internal standard 2-ethylbutyric acid, vortexed, sonicated at low temperature, and centrifuged again. The supernatant was then injected. The analytical instrument was an Agilent 8890B-5977BGC / MSD, with an HP-FFAP column (30 m × 0.25 mm × 0.25 μm). He was used as the carrier gas at a flow rate of 1.0 mL / min, with an injection temperature of 260℃, a split ratio of 10:1, and an injection volume of 1 μL. The temperature program was as follows: starting at 80℃, increasing to 120℃ at 40℃ / min, then to 200℃ at 10℃ / min, and finally holding at 230℃ for 3 min. Mass spectrometry conditions: EI source (70 eV), SIM mode acquisition. The data were processed using Masshunter software and quantified using the standard curve method.

[0086] The results are as follows Figure 14 As shown, after intervention with heat-inactivated Bifidobacterium longum ZFML0012, the levels of acetic acid and butyric acid in mouse feces were significantly increased ( P The value of < 0.05 indicates that the inactivated bacteria can effectively improve the metabolism of SCFAs in the intestine of CIA mice and promote the production of beneficial metabolites, which may be one of the important mechanisms by which it regulates the intestinal flora and improves systemic inflammation.

[0087] Comparative Example 1: Bifidobacterium longum CB108 was prepared into a heat-inactivated Bifidobacterium longum bacterial solution using the method described in this invention. Experiments were conducted according to the method described above, and the results were as follows: (1) Inhibiting LPS-induced pro-inflammatory factors in RAW264.7 cells ( TNF-α , IL-1β Regarding expression, the expression results of pro-inflammatory factors corresponding to heat-inactivated CB108 were only slightly lower than those of LPS, that is, there was no significant difference compared with the LPS group.

[0088] (2) In CIA model mice, the arthritis score of this invention was only 3 points on day 56 (e.g. Figure 4 The arthritis score on day 56 corresponding to heat-inactivated CB108 was approximately 5.5, which was not significantly different from the model group (MOD). Furthermore, the degree of bone erosion was not significantly different from the model group.

Claims

1. The application of heat-inactivated Bifidobacterium longum ZFML0012 in alleviating rheumatoid arthritis, characterized by: Used in the preparation of drugs to relieve rheumatoid arthritis, heat-inactivated Bifidobacterium longum ZFML0012 bacterial suspension can downregulate... TNF-α , iNOS , IL-1β The mRNA expression level can also be upregulated. IL-10 The mRNA expression level; the taxonomic name of Bifidobacterium longum ZFML0012 is Bifidobacterium longum ( Bifidobacterium longum ZFML0012, its accession number is CCTCC NO: M 20252539.

2. The application according to claim 1, characterized in that: Heat-inactivated Bifidobacterium longum ZFML0012 bacterial solution can improve joint damage and immune imbalance.

3. The application according to claim 2, characterized in that: Improvements to joint damage include: reducing foot thickness, alleviating inflammatory infiltration, and providing osteoprotection.

4. The application according to claim 3, characterized in that: Improving immune imbalance includes correcting the imbalance of Th17 / Treg cells in the spleen.

5. The application according to any one of claims 1 to 4, characterized in that: Heat-inactivated Bifidobacterium longum ZFML0012 bacterial solution can regulate the intestinal flora structure, reduce the Firmicutes / Bacteroidetes ratio, restore the abundance of beneficial bacteria, and increase the content of short-chain fatty acids.

6. The application according to claim 1, characterized in that: The seed culture of Bifidobacterium longum ZFML0012 was transferred to MRS selective liquid medium containing 0.05% L-cysteine ​​at an inoculation rate of 1.5~2.5% and anaerobic cultured at 37±1℃ for 24±1h to obtain the fermentation broth. The fermentation broth was centrifuged to collect the bacterial cells, washed with phosphate buffer, and resuspended. The concentration of the bacterial suspension was adjusted to (0.5~1.5)×10⁻⁶. 9 CFU / mL, heat inactivated, to obtain heat-inactivated Bifidobacterium longum ZFML0012 bacterial suspension.

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