Application of faecalis przewalskii FPS03 in preparation of medicine for preventing or treating inflammatory diseases
By using a drug prepared from Bacillus prednisolone FPS03, the aggregation of neutrophils and macrophages is inhibited, and the production of inflammatory factors is reduced, thus solving the problem of large side effects of existing drugs and achieving safe and effective treatment of inflammation.
Patent Information
- Application Number
- CN202310539282.1
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2023-05-15
- Publication Date
- 2026-04-14
AI Technical Summary
Existing medications for treating inflammatory diseases have significant side effects, necessitating a safe and effective microbial preparation to prevent or treat inflammation.
Drugs that inhibit the aggregation of neutrophils and macrophages were prepared using Bacillus prednisii FPS03, which also inhibited the production of inflammatory factors IL-1β, IL-6, and TNF-α.
FPS03 of *Bacillus prenanti* significantly inhibits the aggregation of neutrophils and macrophages and reduces the levels of inflammatory factors IL-1β, IL-6, and TNF-α, thus exhibiting good preventive or therapeutic effects against inflammatory diseases.
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Figure CN121846150A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of microbial technology, and more specifically to the use of *Proteus vulgaris* FPS03 in the preparation of drugs for the prevention or treatment of inflammatory diseases. Background Technology
[0002] Inflammation is a very common and important basic pathological process. External injuries and infections of the body surface, as well as most common and frequently occurring diseases of various organs (such as boils, carbuncles, pneumonia, hepatitis, nephritis, etc.), all fall under the category of inflammatory diseases. Inflammation is the defensive response of living tissues with vascular systems to damaging factors. During inflammation, on the one hand, damaging factors directly or indirectly cause damage to tissues and cells; on the other hand, inflammatory hyperemia and exudation dilute, kill, and surround the damaging factors. Simultaneously, the regeneration of parenchymal and interstitial cells allows damaged tissues to repair and heal. Therefore, inflammation can be considered a unified process of damage and resistance to damage. Existing drugs for treating inflammation have significant side effects. Probiotics, as a class of microorganisms beneficial to human health, can exert beneficial health effects by altering the intestinal microecology when a certain number of live probiotics colonize the host's intestines.
[0003] Therefore, providing the application of Prevotella f. p.O.3 in the preparation of drugs for the prevention or treatment of inflammatory diseases is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention
[0004] In view of this, the present invention provides the use of Prevotella f. p.O. 3 in the preparation of drugs for the prevention or treatment of inflammatory diseases.
[0005] Inflammation is a response of the immune system to tissue damage and infection, characterized by the aggregation of white blood cells (granulocytes and macrophages) around infected tissue. The immune system of zebrafish is very similar to that of mammals. When trauma occurs, neutrophils and macrophages respond to traumatic inflammation almost simultaneously, with macrophages and neutrophils being recruited to the site of injury.
[0006] The caudal fin of zebrafish was surgically severed to induce acute injury, prompting an immune response from the zebrafish's neutrophils. Using transgenic fluorescent neutrophil fish (green in color), a significant increase in the number of neutrophils at the caudal fin severance site was observed under a fluorescence microscope compared to normal zebrafish.
[0007] To achieve the above objectives, the present invention adopts the following technical solution:
[0008] The application of Faecalibacterium prausnitzii FPS03 in the preparation of drugs for the prevention or treatment of inflammatory diseases. The Faecalibacterium prausnitzii FPS03 has the accession number CGMCC No. 22513 and has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date was May 13, 2021, and the classification name is Faecalibacterium prausnitzii.
[0009] Furthermore, the application of *Proteus vulgaris* FPS03 in the preparation of a drug that inhibits the aggregation of neutrophils and macrophages to the inflamed area of the zebrafish tail fin.
[0010] Furthermore, the application of *Proteus vulgaris* FPS03 in the preparation of drugs that inhibit inflammatory factors IL-1β, IL-6, and TNF-α.
[0011] Furthermore, the *Proteus vulgaris* FPS03 is a bacterial suspension.
[0012] *Proteus vulgaris* FPS03 significantly inhibited the aggregation of neutrophils and macrophages to inflamed areas of the zebrafish tail fin in vivo, demonstrating good efficacy in preventing or treating inflammatory diseases. *Proteus vulgaris* FPS03 also significantly inhibited LPS and increased the levels of inflammatory factors IL-1β, IL-6, and TNF-α in zebrafish, showing potential for preventing or treating inflammatory diseases.
[0013] As can be seen from the above technical solution, compared with the prior art, the present invention discloses the application of *Bacillus predniseri* FPS03 in the preparation of drugs for the prevention or treatment of inflammatory diseases. In an in vivo inflammation model, *Bacillus predniseri* FPS03 can inhibit the aggregation of neutrophils and macrophages to the inflamed area of the zebrafish tail fin; *Bacillus predniseri* FPS03 can inhibit the increase in the levels of LPS-induced inflammatory factors IL-1β, IL-6, and TNF-α. This provides a theoretical reference and guiding basis for the development of drugs for the prevention or treatment of inflammatory diseases using *Bacillus predniseri* FPS03. Attached Figure Description
[0014] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.
[0015] Figure 1The attached figure shows the colony morphology of *Proteus vulgaris* FPS03 of the present invention on a PY+X agar plate;
[0016] Figure 2 The attached figure is a visual representation of the effect of *Bacillus prenanti* FPS03 of the present invention on the aggregation of neutrophils and macrophages at the site of inflammation in the caudal fin of zebrafish.
[0017] Wherein, A: normal group; B: model group; C: positive control group; D: 1×10 4 CFU / mL *Bacillus prednioides* FPS03; E: 1×10 5 CFU / mL *Bacillus prednioides* FPS03; F: 1×10 6 CFU / mL Prevotella f. p.O. 3;
[0018] Figure 3 The attached figure is a statistical graph showing the effect of the present invention, *Bacillus prenanti* FPS03, on the aggregation of neutrophils and macrophages to the site of inflammation in the caudal fin of zebrafish.
[0019] Figure 4 The attached figure shows the effect of *Bacillus prenanti* FPS03 of the present invention on LPS-induced increase in the content of the inflammatory factor IL-1β in zebrafish;
[0020] Figure 5 The attached figure shows the effect of *Bacillus prenanti* FPS03 of the present invention on LPS-induced increase in the level of the inflammatory factor IL-6 in zebrafish;
[0021] Figure 6 The attached figure shows the effect of *Bacillus prenanti* FPS03 of the present invention on LPS-induced increase in the content of the inflammatory factor TNF-α in zebrafish. Detailed Implementation
[0022] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0023] Zebrafish are classified into AB strain and Tg(corola:eGFP) strain.
[0024] Interleukin-6 (IL-6) ELISA kit, tumor necrosis factor-α (TNF-α) ELISA kit, and interleukin-1β (IL-1β) ELISA kit were all purchased from Wuhan Huamei Biotechnology Co., Ltd.; lipopolysaccharide (LPS) was purchased from Sigma-Aldrich; and tricaine was purchased from Beijing Huawiray Chemical Co., Ltd.
[0025] Example 1: Isolation, identification and preservation of *Bacillus prenanti* FPS03
[0026] (1) Separation:
[0027] 1) Dissolve the feces of the long-lived elderly (about 0.1g) in a 1.5mL centrifuge tube containing 1mL of sterile physiological saline, and mix thoroughly by pipetting with a 1mL sterile pipette tip.
[0028] 2) Add 900 μL of sterile physiological saline to each of six sterile 1.5 mL centrifuge tubes.
[0029] 3) Starting from the first one containing 10 -1 In the centrifuge tube containing the sample dilution, add 100 μL of the liquid to the second centrifuge tube (10 μL). -2 Dilute to 10 -2 ;
[0030] 4) From the second one containing 10 -2 In the centrifuge tube containing the sample dilution solution, add 100 μL of the liquid to the third centrifuge tube (10 μL). -3 Dilute to 10 -3 ;
[0031] 5) Repeat the previous step until the solution is diluted to 10. -4 10 -5 10 -6 10 -7 .
[0032] 6) From the container containing 10 -4 Pipe 100 μL of the sample dilution from centrifuge tubes and inoculate it onto PY+X solid medium (purchased from Shandong Top Biotechnology Co., Ltd.) and BS solid medium, respectively. Spread the 100 μL of bacterial culture evenly and dry it. Be careful to use a gentle and quick spreading technique, and ensure the operation is performed near an alcohol lamp flame. After spreading, label the side of the petri dish with information including name, sample number, medium name, incubation time, dilution gradient, and incubation conditions (anaerobic / aerobic).
[0033] 7) Repeat the previous step to complete 10. -5 10 -6 10 -7 Dilution gradient dilution coating.
[0034] 8) After coating, place the petri dishes in anaerobic conditions at 37°C and incubate for 48 hours. Observation and recording can be carried out after 48 hours.
[0035] 9) Use an inoculation loop to pick up a single colony from the plate and streak it onto PY+X solid medium. Incubate anaerobically at 37°C for 48 hours to obtain pure colonies.
[0036] 10) Inoculate the pure colonies on the plate into PY+X liquid medium, anaerobic culture at 37℃ for 12-16h, add 20% glycerol, and store in a -80℃ refrigerator.
[0037] (2) Molecular biological identification of the strain: Genomic DNA was extracted from the obtained strain, and the full-length 16S rDNA fragment was amplified by PCR using the universal 16S rDNA primers 27F and 1492R. Then, sequencing was performed to identify the species of the strain.
[0038] The primer sequences for universal primers 27F and 1492R are as follows:
[0039] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; SEQ ID NO.1;
[0040] 1492R: 5'-GGTTACCTTGTTACGACTT-3'; SEQ ID NO. 2.
[0041] Experimental results: The strains screened from the feces of centenarians in Jiaoling County, Meizhou City, Guangdong Province were identified as Bacillus prednisii by morphological observation and 16S rDNA identification. The 16S rDNA sequence of strain FPS03 is shown in SEQ ID NO.3.
[0042]
[0043] Single colonies of strain FPS03 were inoculated onto PY+X solid medium and grew well under anaerobic conditions at 37°C. The colonies were milky white, with regular edges, spherical shape, and smooth surface. Figure 1 The strain FPS03 has been deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date was May 13, 2021. The strain is classified as *Faecalibacterium prausnitzii*, and the accession number is CGMCC No. 22513.
[0044] Example 2: Preparation of *Bacillus prednisone* FPS03 bacterial suspension (cells)
[0045] After activation and culture, *Bacillus prenanti* FPS03 was inoculated into PY+X liquid medium and cultured at 37°C for 24 h. The culture was then centrifuged at 4°C, 6000 rpm for 10 min to obtain a bacterial pellet. The pellet was washed twice with PBS, and the cells were resuspended in PBS to adjust the cell concentration to 1 × 10⁻⁶. 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 A bacterial suspension (bacterial cells) was obtained at CFU / mL.
[0046] Example 3: Effect of *Bacillus prenanti* FPS03 on the aggregation of neutrophils and macrophages to the site of inflammation in the caudal fin of zebrafish.
[0047] Healthy zebrafish Tg(corola:eGFP) that have reached 3 days post-fertilization (dpf) were selected and placed in 6-well cell culture plates, 25 zebrafish / well. Both the normal group (without tail cutting) and the model group received PBS; the positive control group (indomethacin) received 0.4 μg / mL indomethacin. 1×10⁻⁶ cells / well were cultured. 4 CFU / mL *Bacillus prednioides* FPS03 intervention group added 1×10 4 CFU / mL *Bacillus prednioides* FPS03, 1×10 5 CFU / mL *Bacillus prednioides* FPS03 intervention group added 1×10 5 CFU / mL *Bacillus prednioides* FPS03, 1×10 6 CFU / mL *Bacillus prednioides* FPS03 intervention group added 1×10 6CFU / mL *Bacillus prenanti* FPS03 was added to each well at 5 mL and incubated at 28°C in a biochemical incubator, with the solution changed every 24 hours. After 72 hours of incubation, the zebrafish tail fins were surgically severed under a stereomicroscope and placed in 6-well cell culture plates at 20 zebrafish / well. PBS was added to both the normal group (without tail severance) and the model group, while 0.4 μg / mL indomethacin was added to the positive control group. 1×10⁻⁶ cells were cultured. 4 CFU / mL *Bacillus prednioides* FPS03 intervention group added 1×10 4 CFU / mL *Bacillus prednioides* FPS03, 1×10 5 CFU / mL *Bacillus prednioides* FPS03 intervention group added 1×10 5 CFU / mL *Bacillus prednioides* FPS03, 1×10 6 CFU / mL *Bacillus prednioides* FPS03 intervention group added 1×10 6 CFU / mL *Bacillus prenanti* FPS03 was added to each well (5 mL) and incubated at 28°C for 6 hours. Zebrafish were then anesthetized with tricaine, and the aggregation of neutrophils and macrophages at the caudal fin wound was observed and photographed under a fluorescence microscope. The number of neutrophils and macrophages was counted within a 150 μm radius of the incision site. Data were processed using SPSS 19.0 software. All experimental data are expressed as mean ± SEM value and analyzed using a t-test. Compared with the normal group: ### P<0.005, compared with the model group: &&& P < 0.005; using one-way ANOVA, compared with the model group: * P<0.05, *** P<0.005.
[0048] Depend on Figure 2 and Figure 3 It was found that there were almost no neutrophils and macrophages aggregated at the caudal fin of zebrafish in the normal group (0.65±0.17 cells). However, 6 hours after tail amputation, a large number of neutrophils and macrophages aggregated at the caudal fin wound of zebrafish in the model group; at the same time, the number of neutrophils and macrophages at the caudal fin wound of zebrafish in the model group was 19.75±1.20 cells, which was significantly different from that in the normal group (0.65±0.17 cells) (P<0.005), indicating that the zebrafish inflammation model was successfully established.
[0049] Depend on Figure 2 and Figure 3It was found that only a small number of neutrophils and macrophages aggregated at the caudal fin wound site of zebrafish in the positive control group (indomethacin). Furthermore, the number of neutrophils and macrophages at the caudal fin wound site of zebrafish in the positive control group was 8.40±0.57, which was significantly different from that in the model group (19.75±1.20) (P<0.005). Therefore, indomethacin has an anti-inflammatory effect, consistent with clinical results, indicating that this anti-inflammatory efficacy evaluation trial was effective. The concentration of *Bacillus prednisone* FPS03 was 1×10⁻⁶. 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 At CFU / mL, the number of neutrophils and macrophages at the zebrafish tail fin wound site were 15.75±1.25, 11.95±0.95, and 9.75±0.56, respectively, which were significantly different from those in the model group (19.75±1.20) (P<0.05). Therefore, the above results indicate that *Proteus vulgaris* FPS03 can significantly inhibit the aggregation of neutrophils and macrophages to the inflamed area of the zebrafish tail fin in vivo, demonstrating good efficacy in preventing or treating inflammatory diseases.
[0050] Example 4: Effect of *Bacillus prenanti* FPS03 on LPS-induced increases in levels of inflammatory cytokines IL-6, TNF-α, and IL-1β in zebrafish.
[0051] Healthy AB strain zebrafish that had reached 3 days post-fertilization (dpf) were selected and placed in 6-well cell culture plates, 40 zebrafish per well, with 6 replicates per group; PBS was added to the normal group; 5 μg / mL LPS solution was added to the model group; and indomethacin and 5 μg / mL LPS solution were added to the positive control group; 1×10 4 CFU / mL *Bacillus prednioides* FPS03 intervention group was added with a final concentration of 1×10⁻⁶. 4 CFU / mL *Bacillus prednioides* FPS03 and 5 μg / mL LPS solution, 1×10 5 CFU / mL *Bacillus prednioides* FPS03 intervention group was added with a final concentration of 1×10⁻⁶. 5 CFU / mL *Bacillus prednioides* FPS03 and 5 μg / mL LPS solution, 1×10 6 CFU / mL *Bacillus prednioides* FPS03 intervention group was added with a final concentration of 1×10⁻⁶. 6CFU / mL *Bacillus prenanti* FPS03 and 5 μg / mL LPS solution were mixed in 5 mL solutions per well and incubated at 28°C in a biochemical incubator, with the solution replaced every 24 h. After 72 h of incubation, zebrafish in each well were washed three times with PBS, and then collected into 1.5 mL centrifuge tubes (40 zebrafish per tube, 6 tubes per experimental group). The water in the centrifuge tubes was removed, and 100 μL of PBS was added. The zebrafish were homogenized using an S-18KS handheld micro-electric tissue homogenizer until no obvious tissue fragments remained. The homogenate was centrifuged at 12000 × g at 4°C for 10 min, and the supernatant was collected. The levels of IL-6, TNF-α, and IL-1β in the supernatant were detected using an enzyme-linked immunosorbent assay (ELISA) kit. Data were statistically processed using SPSS 19.0 software. Experimental data are expressed as mean ± SEM values, and t-tests were used for analysis. Compared with the normal group: ### P<0.005, compared with the model group: &&& P < 0.005; using one-way ANOVA, compared with the model group: * P<0.05, ** P<0.01, *** P<0.005.
[0052] Depend on Figure 4 It was found that compared with the normal group (66.01±3.10 pg / mL), the level of the inflammatory factor IL-1β in the zebrafish of the model group (126.58±7.48 pg / mL) was significantly increased (P<0.005), indicating that the LPS-induced inflammation model was successfully established. Figure 4 The positive control group (indomethacin) showed a significantly lower IL-1β level (76.81±5.43 pg / mL) in zebrafish compared to the model group (126.58±7.48 pg / mL) (P<0.005). Therefore, indomethacin has an anti-inflammatory effect, consistent with clinical results, indicating the effectiveness of this anti-inflammatory efficacy evaluation experiment. The concentration of *Bacillus prednisolone* FPS03 was 1×10⁻⁶. 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 At CFU / mL, the levels of the inflammatory factor IL-1β in zebrafish were 101.46±6.60 pg / mL, 84.21±5.66 pg / mL, and 78.59±2.94 pg / mL, respectively, which were significantly different from those in the model group (126.58±7.48 pg / mL) (P<0.05).
[0053] Depend on Figure 5It was found that compared with the normal group (10.38±1.22 pg / mL), the level of the inflammatory factor IL-6 in the zebrafish of the model group (29.52±2.84 pg / mL) was significantly increased (P<0.005), indicating that the LPS-induced inflammation model was successfully established. Figure 5 The positive control group (indomethacin) showed a significantly lower IL-6 level (12.69±1.26 pg / mL) in zebrafish compared to the model group (29.52±2.84 pg / mL) (P<0.005). Therefore, indomethacin has an anti-inflammatory effect, consistent with clinical results, indicating the effectiveness of this anti-inflammatory efficacy evaluation experiment. The concentration of *Bacillus prednisolone* FPS03 was 1×10⁻⁶. 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 At CFU / mL, the levels of the inflammatory factor IL-6 in zebrafish were 20.79±2.02 pg / mL, 16.82±1.20 pg / mL, and 15.55±1.02 pg / mL, respectively, which were significantly different from those in the model group (29.52±2.84 pg / mL) (P<0.05).
[0054] Depend on Figure 6 It was found that compared with the normal group (15.65±1.69 pg / mL), the level of the inflammatory factor TNF-α in the zebrafish of the model group (38.85±2.99 pg / mL) was significantly increased (P<0.005), indicating that the LPS-induced inflammation model was successfully established. Figure 6 The positive control group (indomethacin) showed a significantly lower TNF-α level (18.12±1.47 pg / mL) in zebrafish compared to the model group (38.85±2.99 pg / mL) (P<0.005). Therefore, indomethacin has an anti-inflammatory effect, consistent with clinical results, indicating the effectiveness of this anti-inflammatory efficacy evaluation. The concentration of *Bacillus prednisolone* FPS03 was 1×10⁻⁶. 4 CFU / mL, 1×10 5 CFU / mL, 1×10 6 At CFU / mL, the levels of the inflammatory cytokine TNF-α in zebrafish were 25.90±2.18 pg / mL, 21.93±2.28 pg / mL, and 19.33±1.18 pg / mL, respectively, which were significantly different from those in the model group (38.85±2.99 pg / mL) (P<0.01). Therefore, the above results indicate that *Proteus vulgaris* FPS03 can significantly inhibit the increase of LPS in the levels of inflammatory cytokines IL-1β, IL-6, and TNF-α in zebrafish, and has the potential to prevent or treat inflammatory diseases.
[0055] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims
1. The application of *Bacillus prenanti* FPS03 in the preparation of drugs for the prevention or treatment of inflammatory diseases, characterized in that, The preservation number of the *Proteus vulgaris* FPS03 is CGMCC No. 22513.
2. The application of *Proteus vulgaris* FPS03 according to claim 1 in the preparation of drugs for the prevention or treatment of inflammatory diseases, characterized in that, The *Proteus vulgaris* FPS03 was a bacterial suspension.
3. The use of *Proteus vulgaris* FPS03 as described in claim 1 in the preparation of a drug that inhibits the aggregation of neutrophils and macrophages to the inflamed area of the zebrafish tail fin.
4. The application of *Proteus vulgaris* FPS03 according to claim 3 in the preparation of a drug for inhibiting the aggregation of neutrophils and macrophages to the inflamed area of zebrafish tail fin, characterized in that... The *Proteus vulgaris* FPS03 was a bacterial suspension.
5. The use of *Proteus vulgaris* FPS03 as described in claim 1 in the preparation of drugs that inhibit inflammatory factors IL-1β, IL-6, and TNF-α.
6. The application of *Proteus vulgaris* FPS03 according to claim 5 in the preparation of drugs that inhibit inflammatory factors IL-1β, IL-6, and TNF-α, characterized in that... The *Proteus vulgaris* FPS03 was a bacterial suspension.