Application of clostridium butyricum LS05 in preparation of medicine for preventing or treating inflammatory diseases

Abstract

The invention discloses application of clostridium butyricum LS05 in preparation of drugs for preventing or treating inflammatory diseases, and belongs to the technical field of microorganisms. According to the application of the clostridium butyricum LS05 in preparing the medicine for preventing or treating the inflammatory diseases, disclosed by the invention, the clostridium butyricum LS05 can inhibit neutrophile granulocytes and macrophages from gathering towards the inflammation part of the tail fin of zebra fish in an in-vivo inflammation model; the clostridium butyricum LS05 can be used for inhibiting the increase of the content of LPS-induced inflammatory factors such as IL-1beta, IL-6 and TNF-alpha; theoretical reference and guidance basis are provided for developing medicines for preventing or treating inflammatory diseases by using the clostridium butyricum LS05.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and more specifically to the use of Clostridium butyricum LS05 in the preparation of drugs for the prevention or treatment of inflammatory diseases. Background Technology

[0002] Inflammation generally refers to localized inflammation caused by external injury. It can be caused by bacterial or viral infections, or by abnormalities in the autoimmune system. **Traumatic Inflammation:** If proper disinfection and care are not taken after an injury, local inflammation may occur. Applying anti-inflammatory medication topically is necessary to reduce inflammation and promote wound healing. **Bacterial or Viral Infections:** Bacterial or viral infections can affect bodily organs, leading to recurring local inflammation and discomfort. Treatment involves anti-infective, antiviral, and anti-inflammatory medications. **Autoimmune System Abnormalities:** Abnormalities may be caused by a weakened or compromised autoimmune system, potentially leading to inflammation. Immunotherapy or anti-inflammatory drugs are needed. However, the continuous use of these anti-inflammatory drugs has significant drawbacks and side effects. Probiotics are a type of microorganism that are beneficial to human health. When a certain number of live probiotics colonize the host's intestines, they can exert beneficial health effects by altering the intestinal microecology.

[0003] Therefore, providing the application of Clostridium butyricum LS05 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 Clostridium butyricum LS05 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 Clostridium butyricum LS05 in the preparation of drugs for the prevention or treatment of inflammatory diseases, wherein the preservation number of Clostridium butyricum LS05 is CGMCC No. 22095 (see patent number 202211458999.5).

[0009] Furthermore, the application of the aforementioned Clostridium butyricum LS05 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 the aforementioned Clostridium butyricum LS05 in the preparation of drugs that inhibit inflammatory factors IL-1β, IL-6, and TNF-α.

[0011] Furthermore, the Clostridium butyricum LS05 is a bacterial suspension.

[0012] At the same concentration, *Clostridium butyricum* LS05 showed stronger inhibitory effects than *Clostridium butyricum* 3627 on the aggregation of neutrophils and macrophages to the inflamed area of ​​the zebrafish tail fin in an in vivo inflammation model, demonstrating good efficacy in preventing or treating inflammatory diseases. At the same concentration, *Clostridium butyricum* LS05 also showed stronger inhibitory effects than *Clostridium butyricum* 3627 on the increase in levels of inflammatory factors IL-1β, IL-6, and TNF-α in zebrafish due to LPS, indicating potential for preventing or treating inflammatory diseases.

[0013] As can be seen from the above technical solutions, compared with the prior art, the present invention discloses the application of Clostridium butyricum LS05 in the preparation of drugs for the prevention or treatment of inflammatory diseases. In an in vivo inflammation model, Clostridium butyricum LS05 can inhibit the aggregation of neutrophils and macrophages to the inflammation site of zebrafish tail fin; Clostridium butyricum LS05 can inhibit the increase in the content 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 Clostridium butyricum LS05. 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 is a visual representation of the effect of the Clostridium butyricum LS05 of the present invention on the aggregation of neutrophils and macrophages to the site of inflammation in the caudal fin of zebrafish.

[0016] Among them, A: normal group; B: model group; C: positive control group; D: Clostridium butyricum 3627; E: Clostridium butyricum LS05;

[0017] Figure 2 The attached figure is a statistical graph showing the effect of the Clostridium butyricum LS05 of the present invention on the aggregation of neutrophils and macrophages to the inflamed area of ​​the caudal fin of zebrafish;

[0018] Figure 3 The attached figure shows the effect of Clostridium butyricum LS05 of the present invention on LPS-induced increase in the content of the inflammatory factor IL-1β in zebrafish;

[0019] Figure 4 The attached figure shows the effect of Clostridium butyricum LS05 of the present invention on LPS-induced increase in the content of the inflammatory factor IL-6 in zebrafish;

[0020] Figure 5 The attached figure shows the effect of Clostridium butyricum LS05 of the present invention on LPS to increase the content of the inflammatory factor TNF-α in zebrafish. Detailed Implementation

[0021] 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.

[0022] Zebrafish are classified into AB strain and Tg(corola:eGFP) strain.

[0023] 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; tricaine was purchased from Beijing Huawiray Chemical Co., Ltd.; and Clostridium butyricum 3627 (ATCC 3627) was purchased from Beijing Bio-Bio Biotechnology Co., Ltd.

[0024] Example 1: Preparation of Clostridium butyricum LS05 bacterial suspension (cells)

[0025] Clostridium butyricum LS05 was activated and cultured, then inoculated into BHI liquid medium and cultured at 37°C for 24 h. The culture was then centrifuged at 4°C and 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. 6 A bacterial suspension (bacterial cells) was obtained at CFU / mL.

[0026] Example 2: Preparation of Clostridium butyricum 3627 bacterial suspension (cells)

[0027] Clostridium butyricum 3627 was activated and inoculated into BHI liquid medium. After incubation at 37°C for 24 h, the culture was centrifuged at 4°C and 6000 r / min for 10 min to obtain a bacterial pellet. The bacterial pellet was washed twice with PBS, and the cells were resuspended in PBS to adjust the cell concentration to 1×10⁻⁶. 6 A bacterial suspension (bacterial cells) was obtained at CFU / mL.

[0028] Example 3: Effect of Clostridium butyricum LS05 on the aggregation of neutrophils and macrophages to the site of inflammation in the caudal fin of zebrafish.

[0029] Healthy zebrafish Tg(corola:eGFP) that had reached 3 days post-fertilization (dpf) were selected and placed in 6-well cell culture plates, 25 zebrafish / well. PBS was added to both the normal group (without tail cutting) and the model group; 0.4 μg / mL indomethacin was added to the positive control group; and 1 × 10⁻⁶ Clostridium butyricum 3627 intervention group was added. 6 Add 1×10 (CFU / mL) 6 CFU / mL Clostridium butyricum 3627; Clostridium butyricum LS05 intervention group (1×10 6 Add 1×10 (CFU / mL) 6 CFU / mL Clostridium butyricum LS05; 5 mL per well, incubated at 28℃ in a biochemical incubator, with fresh solution changed every 24 h; after 72 h of incubation, zebrafish tail fins were cut off under a stereomicroscope and placed in 6-well cell culture plates, 20 zebrafish / well. PBS was added to both the normal group (without tail cutting) and the model group, while 0.4 μg / mL indomethacin was added to the positive control group (indomethacin); Clostridium butyricum 3627 intervention group (1×10⁻⁶) 6 Add 1×10 (CFU / mL) 6 CFU / mL Clostridium butyricum 3627; Clostridium butyricum LS05 intervention group (1×10 6 Add 1×10 (CFU / mL) 6CFU / mL Clostridium butyricum LS05, 5 mL per well, was incubated at 28℃ in a biochemical incubator for 6 h. 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 counting range was defined as the area within 150 μm of the incision site, and the number of neutrophils and macrophages was counted. Data were statistically processed using SPSS 19.0 software. Experimental data are expressed as x±SEM data, and t-tests were used for analysis. Compared with the normal group: ### P<0.005, compared with the model group: *** P<0.005.

[0030] Depend on Figure 1 and Figure 2 It was found that there were almost no neutrophils and macrophages aggregated at the caudal fin of zebrafish in the normal group (0.85±0.18 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 21.60±1.65 cells, which was significantly different from that in the normal group (0.85±0.18 cells) (P<0.005), indicating that the zebrafish inflammation model was successfully established.

[0031] Depend on Figure 1 and Figure 2 It 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). Simultaneously, the number of neutrophils and macrophages at the caudal fin wound site of zebrafish in the positive control group was 10.85±1.04, which was significantly different from that in the model group (21.60±1.65) (P<0.005). Therefore, indomethacin has an anti-inflammatory effect, consistent with clinical results, indicating that this anti-inflammatory efficacy evaluation trial was effective. Clostridium butyricum 3627 intervention group (1×10⁻⁶) 6 The number of neutrophils and macrophages at the zebrafish tail fin wound (CFU / mL) was 19.80±1.46, which was not significantly different from that in the model group (21.60±1.65) (P>0.05). Additionally, the Clostridium butyricum LS05 intervention group (1×10⁻⁶ CFU / mL) showed no significant difference. 6 In zebrafish with CFU / mL caudal fin wounds, only a small number of neutrophils and macrophages aggregated, similar to the positive control group (indomethacin); Clostridium butyricum LS05 intervention group (1×10 6The number of neutrophils and macrophages at the zebrafish tail fin wound (CFU / mL) was 12.85±1.30, which was significantly different from that in the model group (21.60±1.65) (P<0.005). Therefore, these results indicate that at the same concentration, Clostridium butyricum LS05 has a stronger inhibitory effect on the aggregation of neutrophils and macrophages to the inflamed area of ​​the zebrafish tail fin than Clostridium butyricum 3627 in an in vivo inflammation model, demonstrating good efficacy in preventing or treating inflammatory diseases.

[0032] Example 4: Effect of Clostridium butyricum LS05 on LPS-induced increases in levels of inflammatory cytokines IL-6, TNF-α, and IL-1β in zebrafish.

[0033] 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; Clostridium butyricum 3627 intervention group (1×10 6 Add CFU / mL to a final concentration of 1×10⁻⁶. 6 CFU / mL Clostridium butyricum 3627 and 5 μg / mL LPS solution; Clostridium butyricum LS05 intervention group (1×10 6 Add CFU / mL to a final concentration of 1×10⁻⁶. 6 CFU / mL Clostridium butyricum LS05 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 changed every 24 h. After 72 h of incubation, zebrafish from 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). After removing the water from the centrifuge tubes, 100 μL of PBS was added, and 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<0.01, *** P<0.005.

[0034] Depend on Figure 3It was found that compared with the normal group (76.04±4.84 pg / mL), the level of the inflammatory factor IL-1β in the zebrafish of the model group (126.80±6.04 pg / mL) was significantly increased (P<0.005), indicating that the LPS-induced inflammation model was successfully established. Figure 3 The positive control group (indomethacin) showed that the IL-1β level in zebrafish was 88.04±4.20 pg / mL, which was significantly different from that in the model group (126.80±6.04 pg / mL) (P<0.005). Therefore, indomethacin has an anti-inflammatory effect, consistent with clinical results, indicating that this anti-inflammatory efficacy evaluation trial was effective. Clostridium butyricum 3627 intervention group (1×10⁻⁶ pg / mL) 6 The level of the inflammatory factor IL-1β in zebrafish (CFU / mL) was 120.80±4.63 pg / mL, which was not significantly different from that in the model group (126.80±6.04 pg / mL) (P>0.05). Additionally, the Clostridium butyricum LS05 intervention group (1×10⁻⁶ CFU / mL) showed a significantly lower level of IL-1β. 6 The level of the inflammatory factor IL-1β in zebrafish (CFU / mL) was 90.34±5.51 pg / mL, which was significantly different from that in the model group (126.80±6.04 pg / mL) (P<0.01).

[0035] Depend on Figure 4 It was found that compared with the normal group (10.34±1.19 pg / mL), the level of the inflammatory factor IL-6 in the zebrafish of the model group (24.38±2.21 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 that the IL-6 level in zebrafish was 12.09±1.31 pg / mL, which was significantly different from that in the model group (24.38±2.21 pg / mL) (P<0.005). Therefore, indomethacin has an anti-inflammatory effect, consistent with clinical results, indicating that this anti-inflammatory efficacy evaluation trial was effective. Clostridium butyricum 3627 intervention group (1×10⁻⁶ pg / mL) 6 The level of the inflammatory factor IL-6 in zebrafish (CFU / mL) was 22.05±1.56 pg / mL, which was not significantly different from that in the model group (24.38±2.21 pg / mL) (P>0.05). Additionally, the Clostridium butyricum LS05 intervention group (1×10⁻⁶ CFU / mL) showed a significantly lower level of IL-6. 6 The level of the inflammatory factor IL-6 in zebrafish (CFU / mL) was 14.19±1.42 pg / mL, which was significantly different from that in the model group (24.38±2.21 pg / mL) (P<0.01).

[0036] Depend on Figure 5It was found that compared with the normal group (16.56±1.39 pg / mL), the level of the inflammatory factor TNF-α in the zebrafish of the model group (35.56±2.11 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 TNF-α level (20.64±2.41 pg / mL) in zebrafish compared to the model group (35.56±2.11 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 trial. Clostridium butyricum 3627 intervention group (1×10⁻⁶ pg / mL) 6 The TNF-α level in zebrafish with CFU / mL was 28.53±3.16 pg / mL, which was not significantly different from that in the model group (35.56±2.11 pg / mL) (P>0.05). Additionally, the Clostridium butyricum LS05 intervention group (1×10⁻⁶ CFU / mL) showed a significantly lower CFU / mL level. 6 The TNF-α content in zebrafish (CFU / mL) was 23.53±2.26 pg / mL, which was significantly different from that in the model group (35.56±2.11 pg / mL) (P<0.01).

[0037] Therefore, the above results indicate that, at the same concentration, Clostridium butyricum LS05 has a stronger inhibitory effect on the levels of inflammatory factors IL-1β, IL-6, and TNF-α in zebrafish increased by LPS than Clostridium butyricum 3627, and has the potential to prevent or treat inflammatory diseases.

[0038] 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 Clostridium butyricum LS05 in the preparation of drugs for the prevention or treatment of inflammatory diseases, characterized in that, The preservation number of the Clostridium butyricum LS05 is CGMCC No. 22095.

2. The application of Clostridium butyricum LS05 according to claim 1 in the preparation of drugs for the prevention or treatment of inflammatory diseases, characterized in that, The Clostridium butyricum LS05 is a bacterial suspension.

3. The use of Clostridium butyricum LS05 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 Clostridium butyricum LS05 according to claim 3 in the preparation of a drug for inhibiting the accumulation of neutrophils and macrophages at the site of inflammation in the caudal fin of zebrafish, characterized in that, The Clostridium butyricum LS05 is a bacterial suspension.

5. The use of Clostridium butyricum LS05 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 Clostridium butyricum LS05 according to claim 5 in the preparation of drugs that inhibit inflammatory factors IL-1β, IL-6, and TNF-α, characterized in that, The Clostridium butyricum LS05 is a bacterial suspension.

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