Use of a secreted protein derived from akkermansia muciniphila in the preparation of a medicament for treating and / or repairing spinal cord injury

Abstract

The application belongs to the technical field of medicine, and specifically discloses application of a secretory protein derived from Akkermansia muciniphila in preparation of a medicine for treating and / or repairing spinal cord injury, so as to realize effective repair and / or treatment of spinal cord injury by the secretory protein derived from Akkermansia muciniphila (AmTARS). The medicine for treatment can effectively promote physiological and functional recovery after spinal cord injury, promote regeneration and germination of nerve axons, protect neurons from reduction after spinal cord injury, help to enhance structural integrity of nerve tissues, and thus improve overall repair efficiency. The medicine for treatment can be synthesized in large quantities by a biosynthetic method, and has a mature preparation process, stable quality, and good application prospect in treating and / or repairing spinal cord injury.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, specifically to the use of a secreted protein derived from Akkermansia myxophilus in the preparation of medicaments for treating and / or repairing spinal cord injuries. Background Technology

[0002] Spinal cord injury (SCI) refers to damage to the structure and function of the spinal cord caused by direct or indirect external factors. This results in various motor, sensory, and sphincter dysfunctions below the affected segment, accompanied by abnormal muscle tone and pathological reflexes. The process of SCI includes a direct stage caused by trauma, such as fractures and dislocations, leading to damage to axons and cell membranes; and a secondary stage caused by local hemorrhage, ischemia, hypoxia, ion imbalance, and the accumulation of oxidative stress, initiating cell death and neuroinflammation. In SCI intervention, reducing neuroinflammation and pro-inflammatory cell death in the secondary stage has always been key to promoting nerve repair. The high disability rate, the high costs of prolonged rehabilitation, and the loss of earning capacity of patients caused by SCI not only inflict a huge blow on individuals and their families but also place a heavy burden on society. Currently, apart from surgical and rehabilitation treatments, there is no effective treatment for repairing damaged spinal cords, making it a major clinical problem that urgently needs to be addressed by the medical community.

[0003] In recent years, the use of traditional Chinese medicine (TCM) to promote spinal cord injury repair via the gut-spinal axis has been increasingly applied in spinal cord injury treatment research, especially given the crucial role of small molecule secreted proteins from the gut microbiota. AmTARS, a secreted protein derived from Akkermansia myxophilus, is specifically secreted by this bacterium. Studies have confirmed that AmTARS can activate B cells by promoting IL-10 secretion from macrophages, thereby inducing anti-inflammatory immune homeostasis and exhibiting efficacy in preventing, alleviating, and treating inflammatory diseases. However, there are no reports of AmTARS being used to treat spinal cord injury. Therefore, it is urgent to clarify whether AmTARS is an effective component for treating spinal cord injury via the gut-spinal axis, and to synthesize large quantities of these functional monomers using methods such as protein expression with prokaryotic microorganisms for the treatment and / or repair of spinal cord injury. Summary of the Invention

[0004] In view of the shortcomings in the development of key effective components for promoting spinal cord injury repair in existing gut-spinal axis studies, and the limitations of the lack of clear key components in existing gut-spinal axis treatments for spinal cord injury, the present invention aims to provide the application of AmTARS, a protein secreted by beneficial intestinal bacteria, in the preparation of drugs for treating and / or repairing spinal cord injury, so as to achieve effective repair and / or treatment of spinal cord injury by AmTARS, thereby solving the problems mentioned in the background art.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] This invention provides the use of a secreted protein derived from *Ackermania myxotroph* in the preparation of medicaments for treating and / or repairing spinal cord injuries. The secreted protein from *Ackermania myxotroph* promotes the budding and growth of nerve axons and protects against neuronal reduction following spinal cord injury.

[0007] Furthermore, the secretory protein derived from Akkermansia myxophila includes the amino acid sequence shown in SEQ ID NO:1.

[0008] SEQ ID NO:1:

[0009] .

[0010] Furthermore, the method for preparing the secretory protein derived from Akkermansia myxophila includes the following steps: constructing the expression vector PET21a(+), transforming the PET21a(+) plasmid into competent Escherichia coli BL21, expressing AmTARS in large quantities after successful transformation, and obtaining purified AmTARS after purification by the polyhistidine affinity tag (HIS-tag).

[0011] The present invention also provides a pharmaceutical composition comprising a secretory protein derived from Akkermansia myxophilus as an active ingredient, said secretory protein derived from Akkermansia myxophilus comprising the amino acid sequence shown in SEQ ID NO:1.

[0012] Furthermore, the pharmaceutical composition also includes a pharmaceutically acceptable carrier.

[0013] The present invention also provides the use of the pharmaceutical composition described above in the preparation of a medicament for treating and / or repairing spinal cord injury.

[0014] Compared with the prior art, the beneficial effects of the present invention are:

[0015] 1. The therapeutic drug of the present invention has a significant therapeutic effect on spinal cord injury. The therapeutic drug of the present invention can effectively promote physiological and functional recovery after spinal cord injury, promote the germination and growth of nerve axons, and protect against the reduction of neurons after spinal cord injury.

[0016] 2. The therapeutic drug of the present invention can be expressed and synthesized in large quantities using biosynthesis methods, making it easy to obtain, safe, and economical, and has good application prospects in the treatment and / or repair of spinal cord injuries. Attached Figure Description

[0017] Figure 1 The BMS score was determined by administering AmTARS at 0.2 mg / kg immediately after spinal cord injury and 14 days later.

[0018] Figure 2 The changes in anterior horn syringe rate of spinal cord tissue 14 days after immediate administration of AmTARS at 0.2 mg / kg following spinal cord injury; where A shows HE staining of spinal cord tissue in the Sham group, SCI group, and SCI+AmTARS group; B shows the syringe rate results under 100× field of view.

[0019] Figure 3 The study aimed to assess the neuronal morphology of spinal cord tissue 14 days after immediate administration of AmTARS at 0.2 mg / kg following spinal cord injury. A shows Nissl staining of spinal cord tissue from the Sham, SCI, and SCI+AmTARS groups; B shows a comparison of neuronal numbers across the groups.

[0020] Figure 4Immunofluorescence images of spinal cord tissue 14 days after immediate administration of AmTARS at 0.2 mg / kg following spinal cord injury; where A shows the expression of NF-200 and NeuN in mouse spinal cord tissue detected by immunofluorescence staining, DAPI was used to stain cell nuclei, and Merge is the image after superimposing different channels; B shows the relative mean fluorescence intensity of NF-200; and C shows the relative mean fluorescence intensity of NeuN.

[0021] Figure 5 The expression of pathway proteins in spinal cord tissue 14 days after immediate administration of AmTARS at 0.2 mg / kg following spinal cord injury is shown. Among them, A is the band diagram of P-PI3K, P-AKT, PI3K, and AKT proteins; B is the statistical graph of PI3K protein expression; C is the statistical graph of AKT protein expression; D is the statistical graph of P-PI3K protein expression; and E is the statistical graph of P-AKT protein expression. 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] The embodiments provided below are intended as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way. Unless otherwise specified, the experimental methods in the following embodiments are conventional methods, performed according to the techniques or conditions described in the literature or according to the product instructions. Unless otherwise specified, the materials, reagents, etc., used in the following embodiments are commercially available.

[0024] This invention relates to the application of AmTARS, a protein secreted by beneficial intestinal bacteria, in the treatment and / or repair of spinal cord injury. The research method includes:

[0025] S1 and AmTARS in vitro recombinant protein expression: The target protein AmTARS was obtained by expressing and purifying the protein through gene recombination in E. coli.

[0026] S2. The effect of AmTARS on spinal cord injury repair in vivo: Monitor and record changes in nerve axons, neuronal indices, etc. in mouse spinal cord tissue after AmTARS intervention.

[0027] S3. Mechanism of AmTARS in promoting spinal cord injury repair in vivo: to verify the possible pathway of spinal cord nerve repair in mice after AmTARS intervention.

[0028] In one specific embodiment of the present invention, the specific process of step S1 is as follows: construct the expression vector PET21a(+), transform the plasmid into competent Escherichia coli BL21, express AmTARS in large quantities after successful transformation, and obtain purified AmTARS after HIS purification.

[0029] In one specific embodiment of the present invention, step S2 involves the following process: After constructing a mouse model of spinal cord injury, the mice are divided into a sham-operated group, an injury group, and an AmTARS group. The AmTARS group is administered AmTARS at 0.2 mg / kg once daily for 14 days. Motor function, neuronal function, and nerve axonal parameters are monitored in the mice.

[0030] Preferably, the indicators include BMS score, NF-200, NeuN, etc.

[0031] Example 1

[0032] In vitro recombinant protein expression of AmTARS:

[0033] (1) Obtain the target protein encoding gene SEQ ID NO:2: Akkermansia muciniphila,DNA Seq from the NCBI website.

[0034]

[0035] (2) Optimize the codons based on the required engineered bacteria - BL21(DE3) competent Escherichia coli, and design primers by adding restriction endonuclease sites BamHI and XhoI on both sides of the optimized gene sequence.

[0036] SEQ ID NO:3:

[0037] Upstream primer: FctagcatgactggtggacagcaaatgggtcgcGGATCCatgtccgaacacaaggaa;

[0038] SEQ ID NO:4:

[0039] Downstream primer RgcagccggatctcagtggtggtggtggtggtgCTCGAGaatcacgggagaaatatgacg.

[0040] (3) Obtain the gene sequence by PCR.

[0041] The PCR one-round reaction uses a 50 μL system: 2 μL each of primers, 2 μL template, 10 μL polymerase buffer, 1 μL 10mM dNTP, 1 μL Taq DNA polymerase (Global Gene China), and 32 μL ddH2O.

[0042] Cyclic parameters: 96℃ pre-denaturation for 5 min; 95℃ for 25 s, 58℃ for 25 s, 72℃ for 40 s, 23 cycles, 72℃ final extension for 1 min.

[0043] The PCR second-round reaction used a 100 μL system: 2 μL each of primers, 2 μL of the first-round synthesis product, 10 μL of polymerase buffer, 1 μL of 10 mM dNTPs, 1 μL of Taq DNA polymerase, and 82 μL of ddH2O.

[0044] Cyclic parameters: 96℃ pre-denaturation for 5 min; 95℃ for 25 s, 58℃ for 25 s, 72℃ for 40 s, 23 cycles, 72℃ final extension for 1 min 30 s.

[0045] (4) Cloning of fragments

[0046] The PCR-amplified fragment was assembled into the pET 21a vector. The ligation product was plated onto ampicillin-resistant plates and incubated overnight at 37°C. 10 μL of the ligation product was added to 100 μL of competent cells, gently agitated to ensure full contact between the product and cells, and incubated on ice for 10 minutes; then incubated in a 42°C water bath for 90 seconds without shaking; followed by an ice bath for 3 minutes; 500 μL of pre-warmed LB medium (37°C) was added to each tube, and the cells were incubated at 37°C with gentle shaking at 200 rpm for 40 minutes. After recovery, the cells were centrifuged at 6000 rpm for 2 minutes, and the supernatant was discarded. The remaining bacterial culture in the centrifuge tubes was thoroughly mixed and plated onto ampicillin-resistant agar plates, and incubated overnight at 37°C.

[0047] (5) Identification of cloned plasmids

[0048] The following day, the plates were retrieved and colony growth was observed and confirmed. Eight single-clone colonies were picked from the plates and cultured at 37℃ and 250 rpm / min while simultaneously performing colony PCR identification. The expected fragment length was 1902 bp. The colony PCR reaction used a 30 μL system: 1 μL each primer, 13 μL polymerase buffer, and 15 μL ddH2O. Cycling parameters: 96℃ pre-denaturation for 3 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 50 s, 23 cycles, with a final extension at 72℃ for 1 min. The amplified products were identified by electrophoresis. The obtained positive bacterial culture was cultured at 37℃ to extract plasmids, and sequencing confirmed that the plasmids were correct.

[0049] (6) Plasmid transformation

[0050] Dissolve 5 μg of plasmid in 50 μL of sterile water, add 1 μL to 100 μL of BL21(DE3), and incubate on ice for 30 min. Heat shock in a 42℃ water bath for 80 s, place on ice for 5 min, add 1 mL of antibiotic-free LB medium in a clean bench, and incubate on a shaker at 37℃ and 220 rpm for 1 h. Spread blank BL21(DE3) and transformed bacteria onto LB agar plates containing ampicillin (Amp), and incubate overnight in an inverted incubator at 37℃.

[0051] (7) Expression and purification

[0052] LB broth was used to induce expression by adding isopropyl-β-D-thiogalactoside (IPTG) overnight. After induction, bacteria were collected by centrifugation, and Hammer Super Lysis Buffer (BR0005-02, ACE) was added. The supernatant and precipitate were collected and purified using a NI-IDA gravity column. The purified AmTARS protein was identified by SDS-PAGE. The AmTARS protein includes the amino acid sequence shown in SEQ ID NO:1.

[0053] Example 2

[0054] AmTARS promotes spinal cord injury repair in vivo:

[0055] (1) Animal husbandry: The experimental animals used in this experiment were C57BL / 6N mice (female, 6-8 weeks old, weighing 18-22g), purchased from the Animal Experiment Center of Guangxi Medical University. All experimental procedures for the experimental animals complied with the relevant specifications of the "Guidelines for Ethical Review of Laboratory Animal Welfare" (GB / T 35892-2018). After obtaining approval for animal ethics review, the mice were housed in an animal biosafety laboratory with constant humidity and a temperature of 18-25℃. During the husbandry process, a 12 / 12h day-night cycle was adopted, and the mice were fed sterilized mouse feed and water.

[0056] (2) Animal grouping: Fifteen female C57BL / 6N mice were weighed and randomly divided into three groups of five each. The three groups were the sham operation group (Sham group), the spinal cord injury model group (SCI group), and the AmTARS treatment group (SCI+AmTARS group).

[0057] (3) Establishment of a spinal cord injury model: Before the surgical procedure, animals were intraperitoneally injected with 1% sodium pentobarbital at a dose of 50 mg / kg to ensure effective anesthesia. After anesthesia, a precise laminectomy was performed at the T9-T10 vertebral segment to expose the dorsal side of the spinal cord, while carefully avoiding damage to the dura mater. Subsequently, a modified Allen's percussion technique was used to induce contusion in the exposed spinal cord area. After contusion, the surgical incision was sutured layer by layer using 4-0 silk sutures. For animals assigned to the control group (i.e., the sham surgery group), laminectomy was performed only at the same vertebral segment without inducing any contusion. Postoperatively, the animals were placed in a temperature-controlled environment until their ability to regulate their body temperature was fully restored to ensure optimal recovery. In the days following the spinal cord injury, the animals were manually assisted three times a day to help empty their bladders until they regained normal urination function.

[0058] (4) Evaluation of mouse motor function: Hind limb motor function of each group of SCI mice was assessed using the Basso Mouse Scale (BMS) at 0, 1, 3, 7, and 14 days after modeling. After the mice were allowed to move freely on an open surface for 3 minutes, the hind limb motor function was scored based on joint range of motion, range of motion, weight-bearing, foot gait, and limb coordination. The highest score was 9 (normal activity), and the lowest was 0 (complete loss of motor function). The BMS scoring was performed by two researchers familiar with these guidelines using a randomized, double-blind method. The BMS results are as follows: Figure 1As shown, compared with the SCI group, the AmTARS treatment group showed a faster increase in BMS score (P<0.01). On days 7 and 14 post-injury, the scores of the SCI+AmTARS group were significantly higher than those of the SCI group (P<0.001). These results indicate that motor function in spinal cord injured mice recovered somewhat after AmTARS treatment. Note: Compared with Sham, the SCI group... P < 0.001: Comparison between SCI group and SCI+AmTARS, ## P < 0.002, ### P < 0.001.

[0059] (5) Changes in the syringe rate of the anterior horn of the spinal cord: Morphological changes of nerve cells in the anterior horn of the spinal cord were observed by HE staining. Hematoxylin staining solution was added to the prepared spinal cord tissue sections. After staining for 10 min, excess staining solution was washed away with distilled water. Differentiation was performed with 1% hydrochloric acid ethanol for a few seconds, followed by rinsing with double-distilled water. Eosin staining was performed for 3-5 min, followed by rinsing with double-distilled water, dehydration again, soaking in xylene, air drying, and mounting with neutral resin. The sections were observed and photographed under a fluorescence biological microscope. The syringe rate of the anterior horn of the spinal cord under a 10x objective lens was calculated using Image Pro Plus software. Figure 2 The results showed that, compared with the SCI group, the SCI+AmTARS group had a significantly lower syringomyelia rate, suggesting that AmTARS can improve the spinal cord tissue structure in mice with spinal cord injury. Note: P < 0.05, P < 0.01, P < 0.001. Scale: 50×: 200 μm, 100×: 100 μm, 200×: 50 μm.

[0060] (6) Observation of neuronal morphology in spinal cord tissue: The changes in the number and morphology of neurons in the spinal cord tissue of mice after intervention were observed using Nissl staining. Hematoxylin staining solution was added to the prepared spinal cord tissue sections, stained for 10 min, and then washed away with distilled water to remove excess staining solution. Differentiation was performed with 1% hydrochloric acid ethanol for a few seconds, followed by rinsing with double-distilled water, staining with Nissl staining solution for 10 min, rinsing with distilled water, dehydration, and mounting. Under a fluorescence biological microscope, the cells appeared mottled blue-purple. Semi-automatic counting of neurons in the anterior horn of the spinal cord was performed using ImageJ software under a 20x objective lens. Figure 3 The results showed that, compared with the SCI group, the SCI+AmTARS group had an increased number of Nissl bodies and a more orderly arrangement, suggesting that AmTARS can protect neurons in the spinal cord tissue of mice with spinal cord injury. Note: P < 0.05, P < 0.01, P < 0.001. Scale: 50×: 200 μm, 100×: 100 μm, 200×: 50 μm.

[0061] (7) Immunofluorescence staining of spinal cord tissue: Antigen retrieval: Microwave citric acid (pH 6.0) on medium heat for 8 min, turn off for 8 min, turn to medium-low heat for 7 min, and then cool naturally. Wash the slides three times in PBS, 3 min each time; Blocking: Block with 5% BSA for 30 min; Incubation with primary antibody: Mix neurofilament protein 200 (NF-200) and neuron-specific nuclear protein (NeuN) primary antibodies, add to paraffin sections, and incubate overnight at 4°C. The primary antibodies are all diluted 1:500. Add secondary antibody: Wash the slides three times in PBS, 3 min each time; After adding the fluorescent secondary antibody, incubate at room temperature in the dark for 1 h. The secondary antibody is all diluted 1:200; DAPI staining: Wash the slides three times in PBS, 3 min each time. Add DAPI staining solution and incubate at room temperature in the dark for 10 min. Wash the slides three times in PBS on a decolorizing shaker, 5 min each time. Add self-fluorescence quencher solution B for 5 min, rinse with running water for 10 min; mount with anti-fluorescence quenching mounting medium; then observe and acquire images under a fluorescence microscope. Figure 4 The results showed that, compared with the SCI group, the expression levels of NF-200 and NeuN were significantly increased in the SCI+AmTARS group. These results indicate that AmTARS can promote axonal growth and protect neurons. Note: NF-200 (green), NeuN (red), DAPI (blue), and Merge were co-stained. P<0.05, P<0.01, P<0.001; Scale bar: 200 μm.

[0062] Example 3

[0063] The mechanism by which AmTARS promotes spinal cord injury repair in vivo:

[0064] Western blotting analysis: Total protein was extracted from spinal cord tissue using RIPA lysis buffer (Beyotime, China) and PMSF (Beyotime, China). After centrifugation at 12000g, 4°C for 15 minutes, the supernatant was collected, and the total protein concentration was quantified using a BCA protein assay kit (Beyotime, China). Equal volumes of protein were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and then transferred to a PVDF membrane. The membrane was then blocked in TBST containing 5% BSA for 1 hour and incubated at 4°C for 12 hours with antibodies against P-AKT, AKT, P-PI3K, PI3K, and β-actin. After washing with TBST and incubation with the corresponding fluorescent secondary antibody (1:10000, Proteintech) for 1 hour, the bands were visualized using a near-infrared fluorescence imaging system, and the results were quantified using ImageJ software. The PI3K / AKT pathway plays a crucial role in neural repair, cell growth, and metabolic regulation, and its phosphorylation status is a marker of pathway activity. like Figure 5 The results showed that the phosphorylation levels of PI3K and AKT were significantly decreased in the SCI group, while the phosphorylation levels of PI3K and AKT were significantly restored in the AmTARS group, indicating that AmTARS can effectively activate the PI3K / AKT pathway and promote neural repair; Note: ns means not significant. P<0.05, P<0.02, P<0.001.

[0065] It should be noted that, in this document, relational terms such as "first" and "second" are used only to distinguish one entity or operation from another, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Furthermore, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such process, method, article, or apparatus.

[0066] It should be noted that the above content merely illustrates the technical concept of the present invention and should not be construed as limiting the scope of protection of the present invention. For those skilled in the art, various improvements and modifications can be made without departing from the principle of the present invention, and all such improvements and modifications fall within the scope of protection of the claims of the present invention.

Claims

1. The use of a secreted protein derived from *Akermansia myxophilus* in the preparation of medicaments for treating and / or repairing spinal cord injuries, characterized in that, The amino acid sequence of the secreted protein derived from Akkermansia myxophila is shown in SEQ ID NO:

1.

2. The use of a secreted protein derived from *Akkermansia myxophilus* according to claim 1 in the preparation of a medicament for treating and / or repairing spinal cord injury, characterized in that, The secreted protein derived from Akkermansia myxophila promotes the germination and growth of nerve axons and protects against neuronal reduction after spinal cord injury.

3. The use of a secreted protein derived from *Akermansia myxophilus* according to claim 1 in the preparation of a medicament for treating and / or repairing spinal cord injury, characterized in that... The method for preparing the secretory protein derived from Akkermansia myxophila includes the following steps: constructing the expression vector PET21a(+), transforming the PET21a(+) plasmid into competent Escherichia coli BL21, expressing the secretory protein in large quantities after successful transformation, and obtaining purified secretory protein derived from Akkermansia myxophila after HIS purification.

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