Application of lactobacillus johnsonii or extracellular vesicles derived from lactobacillus johnsonii in preparation of medicine for treating and / or preventing adult degenerative scoliosis

Abstract

The invention discloses application of lactobacillus johnsonii or extracellular vesicles derived from the lactobacillus johnsonii in preparation of medicines for treating and / or preventing adult degenerative scoliosis. Experiments prove that the extracellular vesicles derived from lactobacillus johnsonii can promote synthesis of extracellular matrix (ECM) of nucleus pulposus cells derived from adult degenerative scoliosis (ADS), inhibit ECM degradation and effectively reverse nucleus pulposus ECM metabolism abnormality in the ADS progress process, and prompt that the extracellular vesicles can improve the ADS disease progress by regulating nucleus pulposus cell ECM metabolism.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to the use of Lactobacillus johnsonii or extracellular vesicles of its origin in the preparation of medicaments for the treatment and / or prevention of degenerative scoliosis in adults. Background Technology

[0002] Adult degenerative scoliosis (ADS) is a degenerative spinal disease characterized primarily by scoliosis, with a coronal Cobb angle greater than 10° as the diagnostic criterion. During the development of ADS, the spine experiences structural imbalance due to non-physiological load distribution, leading to deformity that gradually worsens. This not only impairs the functional integrity of the spine but also significantly negatively impacts the patient's overall health. Currently, clinical treatment for ADS falls into two main categories: conservative treatment and surgical treatment. Conservative treatment includes pain relief medication, exercise therapy, physical therapy, and bracing. When patients experience neurological deficits, spinal instability, or progressive deformities, surgical intervention becomes a necessary option. However, existing treatment options have significant limitations: conservative treatment has limited effectiveness and cannot achieve a fundamental cure; while surgery, although it can improve deformities, carries serious postoperative complications due to its highly invasive nature and significant blood loss risk. Therefore, there is an urgent need to develop new and effective drug interventions to slow the progression of ADS and ultimately treat the disease.

[0003] The pathogenesis of ADS is considered to be a combination of six etiologies, with the core mechanism being asymmetric degeneration of the intervertebral disc, the initiating cause of which is lesion of the nucleus pulposus. Anatomically, the intervertebral disc consists of three parts: the nucleus pulposus core, the outer annulus fibrosus, and the cartilaginous endplate. The nucleus pulposus maintains the integrity of the intervertebral disc by secreting extracellular matrix (ECM) components such as proteoglycans, type II collagen, and type X collagen. Decreased cell activity and a reduced number of cells in the nucleus pulposus tissue lead to abnormal ECM metabolism, cell proliferation, and cytokine secretion, resulting in disc degeneration, which is one of the main pathological features of degenerative disc diseases. Intervertebral disc degeneration appears to be an irreversible process; its avascular and ananeurysmal nature limits nutrient transport and cell density, making disc repair particularly difficult.

[0004] The gut microbiome (GM) has been identified as an important symbiotic partner in maintaining the health of animals and humans. Probiotics such as *Lactobacillus johnsonii* (…) Lactobacillus johnsonii , L. johnsonii As a potential beneficial bacterium, it is involved in regulating metabolic-related diseases, but whether it can alleviate ADS-related symptoms has not been reported.

[0005] Metabolites or extracellular vesicles (EVs) produced by gut microbiota have been identified as important mediators of communication between the gut microbiota and the host. Gut-derived EVs can easily diffuse across the mucosa or matrix and migrate directly to other tissues or immune cells, thereby participating in the regulation of host pathophysiology. Summary of the Invention

[0006] To address the aforementioned technical problems, this invention provides the use of Lactobacillus johnsonii or extracellular vesicles derived therefrom in the preparation of medicaments for the treatment and / or prevention of degenerative scoliosis in adults.

[0007] To achieve the above objectives, the technical solution adopted by the present invention is as follows: On one hand, the present invention provides the use of Lactobacillus johnsonii or extracellular vesicles of its origin in the preparation of medicaments for the treatment and / or prevention of degenerative scoliosis in adults.

[0008] Preferably, its use in the preparation of medicaments for treating and / or preventing intervertebral disc degeneration.

[0009] Preferably, its use in the preparation of drugs that promote the synthesis of extracellular matrix (ECM) in nucleus pulposus cells.

[0010] Preferably, its use in the preparation of drugs that inhibit the degradation of the extracellular matrix (ECM) of nucleus pulposus cells.

[0011] Preferably, its application in the preparation of drugs that upregulate the COL2A1 gene and the ACAN gene.

[0012] Preferably, its application in the preparation of drugs that downregulate the COL10A1 gene, MMP13 gene, and ADSMTS5 gene.

[0013] In another aspect, the present invention provides the use of Lactobacillus johnsonii or extracellular vesicles of its origin in the preparation of medicaments for the treatment and / or prevention of intervertebral disc degeneration.

[0014] In another aspect, the present invention provides the use of Lactobacillus johnsonii or extracellular vesicles derived therefrom in the preparation of a drug that regulates extracellular matrix (ECM) metabolism-related genes, wherein the regulation of extracellular matrix (ECM) metabolism-related genes includes upregulation of the COL2A1 gene, upregulation of the ACAN gene and downregulation of the COL10A1 gene, downregulation of the MMP13 gene and downregulation of the ADSMTS5 gene.

[0015] In another aspect, the present invention provides a pharmaceutical composition for treating and / or preventing degenerative scoliosis or intervertebral disc degeneration in adults, comprising extracellular vesicles of Lactobacillus johnsonii or derived therefrom as the active substance.

[0016] In another aspect, the present invention provides a pharmaceutical composition for regulating extracellular matrix (ECM) metabolism-related genes, comprising extracellular vesicles of Lactobacillus johnsonii or derived therefrom as active substances.

[0017] The above technical solution has the following advantages or beneficial effects: The extracellular vesicles in this invention are derived from the probiotic Lactobacillus johnsonii (Lactobacillus johnsonii). L. johnsonii Lactobacillus johnsonii, a Gram-positive bacterium, is an important medium for communication between the gut microbiota and the host. Extracellular vesicles readily diffuse through the mucosa or matrix, migrating directly to other tissues or immune cells, thereby participating in the regulation of host pathophysiology and avoiding intestinal discomfort caused by excessive live bacteria. This invention experimentally demonstrates that extracellular vesicles derived from Lactobacillus johnsonii can promote the synthesis of ECM in the nucleus pulposus derived from ADS and inhibit ECM degradation, effectively reversing ADS-induced abnormal ECM metabolism in the nucleus pulposus. This suggests that it can improve the disease progression of ADS by regulating ECM metabolism in nucleus pulposus cells. Attached Figure Description

[0018] Figure 1 This is a histological verification result of metabolic imbalance in the ECM of the ADS patient in Example 1 of the present invention.

[0019] Figure 2 This is a cellular-level verification result of metabolic imbalance in the ECM of the ADS patient in Example 2 of the present invention.

[0020] Figure 3 This is a diagram showing the experimental results of LJ-MVs promoting matrix synthesis and inhibiting matrix degradation in ADS-derived nucleus pulposus cells in Example 3 of this invention.

[0021] Figure 4 This is a graph showing the evaluation results of the therapeutic effect of LJ-MVs on ADS mice in Example 4 of this invention. Detailed Implementation

[0022] The following embodiments are merely some, not all, of the embodiments of the present invention. Therefore, the detailed descriptions of the embodiments provided below are not intended to limit the scope of the claimed invention, but merely to illustrate selected embodiments. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0023] In this invention, unless otherwise specified, all equipment and raw materials are commercially available or commonly used in the industry. The methods described in the following embodiments are conventional methods in the art, unless otherwise specified.

[0024] Preparation Example This invention isolates extracellular vesicles (LJ-MVs) from Lactobacillus johnsonii culture medium, and the specific process is as follows: (1) Purification: After centrifuging the Lactobacillus johnsonii culture at 4℃ and 6000 rpm for 20 min, the supernatant was filtered through a 0.22 μm filter membrane. The filtrate was then centrifuged at 4℃ and 120000 g for 2 h. 1 mL of the precipitate was retained in each centrifuge tube. (2) Resuspension: The precipitate was resuspended by adding the same volume of pre-cooled physiological saline (discarding the supernatant). The resuspension was centrifuged at 120000 g and 4℃ for 2 h. (3) Concentration: Approximately 1 mL of the precipitate was retained in each centrifuge tube and then placed into two centrifuge tubes. The tubes were then centrifuged at 120000 g and 4℃ for 2 h. (4) The supernatant was discarded. Approximately 1 mL of the precipitate was retained in each centrifuge tube and aliquoted for storage at -80℃ for later use.

[0025] Example 1 This embodiment verifies at the tissue level that ADS patients have abnormal ECM metabolism in the nucleus pulposus, as detailed below: In this embodiment, intervertebral disc tissues from ADS patients and HC patients (patients undergoing spinal fracture surgery, excluding scoliosis) were stained with Safranin O and Toluidine Blue. X-ray images of the intervertebral disc tissues from ADS and HC patients are shown below. Figure 1 As shown in Figure A. Figure 1 Figures B and D in the diagram show the results of Safranin O and Toluidine Blue staining and their statistical analysis, respectively. As can be seen from the figures, the Safranin O and Toluidine Blue staining of the intervertebral disc tissue in ADS patients is significantly lighter than that in HC patients, indicating that the intervertebral discs in ADS patients have undergone degeneration. Figure 1 Figures C and E in the diagram show the immunohistochemical (IHC) experiment and its statistical analysis, respectively. They reveal a significant increase in the number of cells positive for intervertebral disc matrix catabolism-related proteins (MMP13, ADAMTS5, COL10A1) from ADS patients, and a significant decrease in the number of cells positive for matrix synthesis-related proteins (ACAN, COL2A1). These results indicate increased ECM degradation and decreased ECM synthesis in ADS patients.

[0026] Example 2 This example verifies at the cellular level the presence of abnormal ECM metabolism in ADS patients, as detailed below: This embodiment describes the isolation and culture of nucleus pulposus cells from ADS and HC patients. The isolated nucleus pulposus cells were identified by detecting the relative-specific protein Ca3 in nucleus pulposus cells using immunofluorescence (IF) technology. The results are as follows: Figure 2 As shown in Figure A, blue represents the cell nucleus; red represents Ca3+, indicating that the isolated primary cells are nucleus pulposus cells. Subsequently, the isolated nucleus pulposus cells were stained with safranin O and toluidine blue, as shown... Figure 2As shown in Figure B, the staining of safranin O and toluidine blue in ADS-derived nucleus pulposus cells is significantly lighter than that in HC-derived nucleus pulposus cells, indicating that the matrix of ADS-derived nucleus pulposus cells has been degraded. Figure 2 Figures C and D in the figure show the results of the cell immunofluorescence assay (IF) and its statistical analysis, respectively. As can be seen from the figure, the expression of ECM degradation-related proteins (MMP13, ADAMTS5, COL10A1) from ADS patients is significantly increased, while the expression of ECM synthesis-related proteins (ACAN, COL2A1) is significantly decreased, which further confirms that the degradation of ECM in the nucleus pulposus of ADS patients is increased and the synthesis is reduced.

[0027] Example 3 This embodiment experimentally demonstrates that extracellular vesicles derived from Lactobacillus johnsonii (LJ-MVs) can improve the matrix metabolism level of nucleus pulposus cells derived from ADS patients, as detailed below: This embodiment experimentally verified that ADS-derived nucleus pulposus cells can internalize LJ-MVs. The internalization experiment used PKH67 fluorescent dye to label vesicles, such as... Figure 3 As shown in Figure A, LJ-MVs were observed to be extensively internalized in nucleus pulposus cells under a confocal fluorescence microscope. In Figure A, Hoechst (blue) stains the cell nucleus, WGA (red) stains the cell membrane, and PKH67 (green) stains the LJ-MVs. Figure 3 Figure B shows that the optimal concentration of LJ-MVs acting on nucleus pulposus cells was 64 µg / mL, as determined by the CCK8 assay. Figure 3 The CE diagram shows the staining results of the ECM of nucleus pulposus cells after LJ-MVs entered the nucleus pulposus cells using safranin O and toluidine blue staining, as well as the statistical analysis. As can be seen from the diagram, the staining deepens. Figure 3 Figures F and G in the figure show the Western blot results of metabolism-related proteins in the ECM of the nucleus pulposus and their statistical analysis. As can be seen from the figure, the expression levels of synthesis-related proteins ACAN and COL2A1 are significantly increased, while the expression levels of matrix degradation-related proteins MMP13, ADAMTS5, and COL10A1 are significantly decreased.

[0028] The above results indicate that LJ-MVs can promote ECM synthesis and inhibit ECM degradation in nucleus pulposus cells derived from ADS. Therefore, extracellular vesicles LJ-MVs derived from the probiotic Lactobacillus johnsonii can effectively reverse ADS-induced abnormal ECM metabolism in nucleus pulposus cells, suggesting that it can improve the disease progression of ADS by regulating ECM metabolism in nucleus pulposus cells.

[0029] Example 4 This embodiment demonstrates through experiments that LJ-MVs can effectively control the progression of scoliosis in ADS mice and improve metabolic imbalance in the ECM of the nucleus pulposus, as detailed below: likeFigure 4 As shown in Figure A, this embodiment uses 8-week-old female C57BL / 6J mice. An ADS mouse model was constructed by puncturing the intervertebral discs at the L2-L5 segments and dissecting the paraspinal muscles. The mice were divided into a sham-operated group, an ADS group, and an ADS+LJ-MVs treatment group (0.1 mL / mouse, 9.5 × 10⁻⁶ mmol / L). 10 (Particles / mL), all groups of mice stood for 8 hours a day to simulate human stress, and the experiment lasted for 10 weeks. Figure 4 Figures B and C show the X-ray images and Cobb angle statistics of the experimental mice at week 10 after modeling. The figures show that the progression of Cobb angle in the LJ-MVs treatment group was controlled, indicating that LJ-MVs significantly improved the progression of scoliosis in ADS mice. The structure of the intervertebral disc tissue in each group of mice at week 10 after modeling was observed using H&E, safranin-O, and toluidine blue staining. H&E staining showed that, compared to the ADS group, the intervertebral disc tissue structure of mice treated with LJ-MVs was restored to its original integrity. Safranin-O and toluidine blue staining showed an increase in ECM in the intervertebral disc tissue and a significantly higher score. Figure 4 Figures D and E in the diagram). Immunofluorescence assay was used to observe the ECM metabolism of intervertebral disc tissue in each group of mice at week 10 after the start of modeling. It was observed that the expression levels of COL2A1 and ACAN, which are related to ECM synthesis metabolism, increased in mice treated with LJ-MVs, while the expression levels of COL10A1, MMP13, and ADSMTS5, which are related to catabolic metabolism, decreased. This indicates that the imbalance of intervertebral disc ECM metabolism was improved and the pathological process of ADS was delayed. Figure 4 (F-plot and G-plot in the diagram).

[0030] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Use of Lactobacillus johnsonii or extracellular vesicles of its origin in the preparation of medicaments for the treatment and / or prevention of degenerative scoliosis in adults.

2. The application according to claim 1, characterized in that, Use in the preparation of drugs for the treatment and / or prevention of intervertebral disc degeneration.

3. The application according to claim 1, characterized in that, Application in the preparation of drugs that promote the synthesis of extracellular matrix in nucleus pulposus cells.

4. The application according to claim 1, characterized in that, Application in the preparation of drugs that inhibit the degradation of the extracellular matrix of nucleus pulposus cells.

5. The application according to claim 1, characterized in that, Application in the preparation of drugs that upregulate the COL2A1 gene and ACAN gene.

6. The application according to claim 1, characterized in that, Application in the preparation of drugs that downregulate the COL10A1 gene, MMP13 gene, and ADSMTS5 gene.

7. Use of Lactobacillus johnsonii or extracellular vesicles of its origin in the preparation of drugs for the treatment and / or prevention of intervertebral disc degeneration.

8. The use of *Lactobacillus johnsonii* or extracellular vesicles derived therefrom in the preparation of drugs that regulate genes related to extracellular matrix metabolism, characterized in that... The genes involved in regulating extracellular matrix metabolism include upregulating the COL2A1 gene, upregulating the ACAN gene, downregulating the COL10A1 gene, downregulating the MMP13 gene, and downregulating the ADSMTS5 gene.

9. A pharmaceutical composition for treating and / or preventing degenerative scoliosis or intervertebral disc degeneration in adults, characterized in that, Extracellular vesicles, including Lactobacillus johnsonii or its derivatives, are used as active substances.

10. A pharmaceutical composition for regulating genes related to extracellular matrix metabolism, characterized in that, Extracellular vesicles, including Lactobacillus johnsonii or its derivatives, are used as active substances.

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