Use of piezo1 antagonist gsmtx-4 in the preparation of a drug for treating spinal cord injury

Abstract

The application discloses application of a PIEZO1 inhibitor molecule GsMTx-4 in preparation of a spinal cord injury treatment drug. GsMTx-4 plays a protective role in spinal cord injury. In vitro cell experiments show that GsMTx-4 can antagonize the inflammatory reaction process of microglia cells caused by mechanical injury. Meanwhile, an animal model shows that GsMTx-4 can reduce the occurrence of limb paralysis caused by spinal cord injury, improve the condition, and no obvious toxic side effects are found in long-term application, so that GsMTx-4 can be applied to the fields of spinal cord injury and the like, and has wide application value and market prospect.

Description

Technical Field

[0001] The present invention relates to the field of medicine, and in particular to application of a PIEZO1 antagonist GsMTx-4 in the preparation of a drug for treating spinal cord injury. Background Art

[0002] Currently, the treatment of spinal cord injury is a global problem and has a high disability rate. There is currently no specific drug for the repair process of spinal cord injury, and the clinically used drugs are ineffective. If the patient's spinal cord injury is severe, there is a high mortality rate [1]. Currently, surgical decompression of the spinal canal or conservative treatment strategies have associated risks and complications and their own limitations. With the increasing number of patients with spinal cord injury, the treatment of this disease has placed a huge burden on society and families.

[0003] Currently, most clinically used treatments target specific symptoms, while few drugs can protect or improve the spinal cord structure itself, leaving patients with limited options. Furthermore, currently used medications, including hormones and mannitol, have limited efficacy and are associated with side effects with long-term use, limiting their use to a certain extent.

[0004] The discovery of the PIEZO1 channel and its mechanism of action in pressure transduction were awarded the 2021 Nobel Prize in Medicine. Recent studies have suggested that the PIEZO1 antagonist GsMTx-4 has a potential protective function in the process of suppressing the inflammatory microenvironment of macrophages [2]. Through preliminary experiments, we have studied GsMTx-4 treatment and found that it has an ameliorative effect on the spinal cord injury process, which may provide a potential method for long-term use to reduce the disability and mortality rate of spinal cord injury.

[0005] [1]Kathrin J.Allen,Stephen W.Leslie;Autonomic Dysreflexia In:StatPearls[Internet].Treasure Island(FL):StatPearls Publishing;2022Jan.2022Feb 14.

[0006] [2]Yun Shen, Yongchu Pan, Shuyu Guo, Lian Sun, Chi Zhang, Lin Wang;The roles of mechanosensitive ion channels and associated downstream MAPK signaling pathways in PDLC mechanotransduction Mol Med Rep 2020 May;21(5):2113-2122. doi:10.3892 / mmr.2020.11006. Epub 2020 Feb 27. SUMMARY

[0007] The application aims to provide an application of a bone protective preparation of a PIEZO1 antagonist GsMTx-4 as an effective component in preparation of a spinal cord injury treatment drug.

[0008] The GsMTx-4 polypeptide is used as an anti-inflammatory reaction and cell regeneration promoting component, has the effects of improving cell metabolism and reducing the severity of spinal cord injury, and no cytotoxicity of GsMTx-4 is shown in an in-vitro experiment, and long-term use of the drug is safer.

[0009] The technical scheme of the application is:

[0010] The GsMTx-4 (purity 98.4%, CAS: 1209500-46-8, the molecular formula of which is C 185 H 273 N 49 O 45 S6) (Jier Biochemical Co., Ltd.) has a molecular weight of 4095.85.

[0011] The GsMTx-4 has good functions of maintaining cell metabolism stability and inhibiting cell inflammation, low local use dosage and low price, which are also the advantages of the GsMTx-4 compared with other spinal cord injury protective molecules. At present, the purity of the GsMTx-4 has reached more than 98%, and the purity meets the requirements of drug preparation.

[0012] The application provides an application of the GsMTx-4 polypeptide in preparation of a spinal cord injury treatment drug, in particular, as a cell metabolism protective factor and apoptosis antagonizing molecule, the application blocks the excessive activation of microglial cells and the function of inflammatory reaction.

[0013] In particular, the application is especially in preparation of a spinal cord injury protective drug.

[0014] Further, the application is especially in preparation of a treatment drug for spinal cord injury caused by trauma.

[0015] Furthermore, the invention is particularly used in the preparation of drugs for alleviating traumatic acute spinal cord injury.

[0016] Specifically, it includes the use of drugs for paralysis caused by spinal cord injury, lack of limb movement, and limb sensory impairment.

[0017] The drugs also include pharmaceutically acceptable molecular subtypes and analogs, salts of derivatives and the like of GsMTx-4 polypeptides.

[0018] The medicine of the present invention further includes a pharmaceutically acceptable carrier, adjuvant or diluent.

[0019] The preferred application form of the drug is intrathecal injection of the agent or local external application of nanoparticles.

[0020] Results from in vitro cell-based and in vivo animal model studies demonstrate that GsMTx-4 peptide therapy can antagonize microglial overactivation and inflammatory responses, exerting a protective effect in spinal cord injury. Its functional component, GsMTx-4, is a molecule that antagonizes cellular metabolic disorders and apoptosis and can be directly extracted and purified using existing technologies, thereby reducing costs. Furthermore, in animal models, long-term topical application of GsMTx-4 peptide has demonstrated no significant toxic side effects, suggesting its potential application in specific conditions associated with spinal cord injury.

[0021] Therefore, it has a foreseeable application in the preparation of spinal cord injury treatment drugs and thus has broad market prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 BMS statistical analysis of hindlimb muscle strength results in a mouse spinal cord injury model treated with GsMTx-4

[0023] Figure 2 This is an MRI image of the therapeutic effect of GsMTx-4 in a mouse spinal cord injury model.

[0024] Figure 3 HE staining images of GsMTx-4 treatment in a mouse spinal cord injury model

[0025] Figure 4 This is the immunohistochemical staining image of Iba-1 in the mouse spinal cord injury model treated with GsMTx-4

[0026] Figure 5 Western Blot images of inflammatory marker molecules in a mouse spinal cord injury model treated with GsMTx-4

[0027] Figure 6 The antagonistic effect of GsMTx-4 treatment on the excessive inflammatory response of microglia induced by excessive mechanical pressure. DETAILED DESCRIPTION

[0028] Sources of experimental animals, reagents, culture media, and buffers involved in the following examples:

[0029] PIEZO1 channel inhibitor GsMTx-4 (molecular weight: 4095.85) (Gill Biochemical Co., Ltd.)

[0030] C57 / BL6 wild-type mice (Animal Center of Shandong University)

[0031] Model 68100 Mouse Spinal Cord Percussion Device (Shenzhen RWD Technology Co., Ltd.)

[0032] Cell pressure culture system, (Wuxi Puhe Biotechnology Co., Ltd.)

[0033] PBS buffer (Biyuntian Bioreagent Company)

[0034] COX-2 antibody, iNOS antibody, Iba-1 antibody (Thermo Fisher, Pierce)

[0035] RIPA cell protein extraction and lysis buffer (Thermo Fisher, Pierce)

[0036] Protease inhibitors (Beijing Solebow Technology Co., Ltd.)

[0037] BCA protein quantification kit (Shanghai Yanxi Biotechnology Co., Ltd.)

[0038] Complete EDTA-Free (Roche Biopharmaceuticals)

[0039] Xylene (Sinopharm Chemical Reagent Co., Ltd.)

[0040] Neutral rubber (Shanghai Titan Technology Co., Ltd.)

[0041] Concentrated hydrochloric acid (Sinopharm Chemical Reagent Co., Ltd.)

[0042] Yi Hong (Shanghai Titan Technology Co., Ltd.)

[0043] Hematoxylin (Shanghai Titan Technology Co., Ltd.)

[0044] Methanol (Sinopharm Chemical Reagent Co., Ltd.)

[0045] Citrate buffer (0.01 M, pH = 6.0) (Shanghai Sangon Biotech Co., Ltd.)

[0046] 10% NGS (Shanghai Sangon Biotech Co., Ltd.)

[0047] Hydrogen peroxide (H2O2) (Sinopharm Chemical Reagent Co., Ltd.)

[0048] BSA (Shanghai Sangon Biotech Co., Ltd.)

[0049] Anhydrous ethanol (Sinopharm Chemical Reagent Co., Ltd.)

[0050] The cell counter was purchased from Thermo Fisher, USA

[0051] Microscope purchased from Shanghai Caikang Optical Instrument Co., Ltd.

[0052] Centrifuge purchased from Jinan Olaibo Medical Equipment Co., Ltd.

[0053] Electronic balance purchased from Jinan Oulaibo Medical Equipment Co., Ltd.

[0054] Enzyme-linked immunosorbent assay (ELISA) was purchased from Beijing Meihuayi Technology Co., Ltd.

[0055] Ice maker purchased from Jinan Olaibo Medical Equipment Co., Ltd.

[0056] The ultrapure water system was purchased from Jinan Olaibo Medical Equipment Co., Ltd.

[0057] Vortex mixer purchased from Jinan Olabo Medical Instrument Co., Ltd.

[0058] 1. Construction of Spinal Cord Injury Rat Model

[0059] A pneumatic percussion spinal cord injury model was established in 10-week-old wild-type C57 / BL6 mice (15 mice in total). The mice in the blank group (5 mice) did not receive any treatment. The remaining 10 mice underwent T10 thoracic laminectomy and used pneumatic percussion to create spinal cord injury. The positive control group received no treatment, and the experimental group received local subdural injection of the PIEZO1 inhibitor GsMTx-4 (280 μg / kg body weight) 3 times a week. On day 28, the spinal cord injury muscle strength recovery BMS score was used to compare the spinal cord function recovery of mice in different groups. 0 No ankle joint activity. 1 Slight ankle joint activity. 2 Extensive ankle joint activity. 3 Standing on the sole of the foot or without standing on the sole of the foot. 4 Frequent and continuous activity, occasionally standing on the sole of the foot. 5 Frequent and continuous standing on the sole of the foot, with some coordination. But not completely coordinated. The scores of both hind limbs were scored separately and then added together. After the experiment, all groups of mice underwent magnetic resonance imaging to determine the status of spinal cord injury and repair. Then, euthanasia was performed, and spinal cord specimens were collected for histological and molecular biological tests to analyze the inflammatory changes and regeneration process of the spinal cord.

[0060] Figure 1-2 The results of this part of the experiment are Figure 1 The BMS scores for each group were Figure 2The spinal cord magnetic resonance images after induction treatment (sagittal view shows the continuity of the spinal cord injury site, and axial view shows the integrity of the spinal cord structure) both showed that the spinal cord injury of mice was significantly alleviated under GsMTx-4 treatment.

[0061] 2. Tissue Slice Preparation

[0062] Spinal cord tissue from mice in all groups was fixed in 10% formalin for at least 72 hours at room temperature. Tissue was then dehydrated in 50% ethanol (60 minutes), 70% ethanol (60 minutes), 85% ethanol (60 minutes), 95% ethanol (60 minutes), 100% ethanol (30 minutes), and 100% ethanol (30 minutes). Tissue was then treated sequentially with ethanol and xylene (60 minutes), followed by xylene (60 minutes). Tissue was then cleared with xylene and paraffin (60 minutes), followed by paraffin (80 minutes). Tissue was placed in a cassette, filled with paraffin, and then placed on the cold stage of a paraffin embedding machine. The embedded paraffin block was placed on a microtome and sectioned to a thickness of approximately 4 μm. The paraffin slide containing the tissue was gently spread in 42°C water. After complete flattening, the sections were gently lifted with a clean glass slide. The sections were placed on slides, numbered, and baked in a 68°C oven for at least 6 hours.

[0063] 3. Hematoxylin / eosin staining

[0064] The sections were dewaxed with conventional fat-soluble solvents until water was obtained (xylene twice, 15 minutes each time; 100% alcohol for 5 minutes; 95% alcohol for 5 minutes; 75% alcohol for 5 minutes; 50% alcohol for 5 minutes), then stained with hematoxylin staining solution for 5 minutes. After rinsing with clean water, the sections were stained with eosin staining solution for 5 minutes. After rinsing with clean water, the sections were dehydrated (50% alcohol for 5 minutes; 75% alcohol for 5 minutes; 95% alcohol for 5 minutes; 100% alcohol for 5 minutes; xylene twice, 15 minutes each time). After the sections were dried, they were sealed with neutral gum and observed under an optical microscope.

[0065] Figure 3 This is the result of the experiment in this part. HE histological staining shows that GsMTx-4 treatment improved the regeneration process and structural continuity of spinal cord tissue in the spinal cord injury mouse model.

[0066] 4. Immunohistochemical Staining

[0067] The tissue sections were deparaffinated and hydrated. That is, 8 minutes of sequential xylene, 8 minutes of xylene, 8 minutes of xylene, 8 minutes of absolute ethanol, 8 minutes of absolute ethanol, 8 minutes of 95% ethanol, 8 minutes of 80% ethanol, and 8 minutes of 75% ethanol. After 8 minutes of 70% ethanol, the sections were rinsed with water 4 times for 5 minutes each; next, the deparaffinized and hydrated sections were placed in a 3% hydrogen peroxide solution and reacted for 20 minutes at 37°C to block endogenous peroxidase. The antigen was repaired with double distilled water 4 times for 5 minutes each. The citrate buffer was placed in a metal heater and brought to a boil. Boil for 15 minutes, turn off the power and let sit for 15 minutes. Cool naturally to room temperature; then wash in PBS 5 times for 5 minutes each, blot the surrounding tissue, add 5% goat serum to block non-specific antigens, react for 1 hour at room temperature, then add microglial Iba-1 antibody dropwise to the tissue sections and incubate overnight at 4°C in a humidified box; the next day, remove the sections, incubate for 1 hour in a 37°C incubator, rinse with PBS 5 times every 15 minutes, add enhanced horseradish peroxidase conjugated secondary antibody, incubate for 2 hours at room temperature. Wash excess secondary antibody with PBS (5 times for 5 minutes each); add dropwise freshly prepared DAB color solution, observe under light microscope, and present a yellowish brown color, and wash with PBS to stop the color development; next, perform hematoxylin counterstaining. Place the colored sections in modified hematoxylin stain and stain for 5 minutes. Observe the staining under light microscope. Then separate the sections with 0.2% hydrochloric acid and rinse the sections with running water. Finally, the sections are sequentially rinsed through 70% alcohol for 10 minutes, 75% alcohol for 8 minutes, 80% alcohol for 8 minutes, 90% alcohol for 8 minutes, absolute ethanol for 8 minutes, absolute ethanol for 8 minutes, xylene for 8 minutes, and xylene for 8 minutes. After 2 minutes of dehydration, blot the surrounding tissue of the tissue and blot, add dropwise neutral gel, place a coverslip under light microscope to observe.

[0068] Figure 4 For the results figures of this section of the experiment, it was shown by Iba-1 immunohistochemical staining specific to microglia that the use of GsMTx-4 reduced the aggregation process of inflammatory microglia in a mouse model of spinal cord injury.

[0069] 5. Rat microglial cell culture

[0070] Rat microglia were stable cell lines. MC3T3-E1 cells were cultured synchronously in DMEM / F-12 medium (Hyclone, Thermo Fisher Scientific) supplemented with 10% fetal bovine serum (FBS, Gibco, USA), 1% 100 u / ml penicillin, and 100 mg / ml streptomycin (Hyclone, USA) at pH 7.2 (95% air, 5% CO2, 37°C). The medium was changed every 3 days, and cells were passaged when they reached 80-90% confluence. Second- or third-passage cells were used for the indicated experiments. Microglia were stimulated with 900 kPa excessive mechanical stress and treated with either PBS (control group) or GsMTx-4 peptide (experimental group).

[0071] 6.Western Blot

[0072] After in vitro culture and stimulation, mouse spinal cord tissue and rat microglia from each group were placed on ice, treated, and washed with ice water. Proteins were extracted using lysis buffer (p0013c, Beyotime Biotechnology). Cultured nucleus pulposus cells were lysed in RIPA buffer (p0013c, Beyotime Biotechnology) and total protein was collected from each sample. Proteins in loading buffer were heated at 100°C for 10 minutes (ThermoFisher). Protein electrophoresis was performed using 10% SDS-PAGE gels (beyondtime Biotechnology) (30 g per lane) and transferred to nitrocellulose membranes. The membranes were blocked with Tween 20 (10 mM Tris-HCl, pH 8.0; 150 mM NaCl; 0.5% Tween 20) in 5% skim milk for 2 h. The membranes were incubated with specific primary antibodies (COX-2, iNOS, β-Tubulin) for 1 h at 37°C, washed three times with PBS, and incubated overnight at 4°C. A secondary antibody (dilution 1:2000) containing horseradish peroxidase was added and incubated for 1 h at room temperature. The membranes were removed with blunt forceps and rinsed at least three times with PBS. 1 ml of working buffer (p0018s, Biotime Biotechnology) was added to each membrane and detected (Amersham Life Sciences, Ellington, IL, USA). Statistical analysis of grayscale values ​​was performed using Image J software.

[0073] Figure 5-6 This is the experimental result. By using Western blot, we observed the abnormal levels of inflammatory markers in spinal cord tissue in vivo and microglia in vitro caused by mechanical injury, and GsMTx-4 alleviated the inflammatory response process under mechanical injury stimulation.

[0074] 7. Statistical analysis

[0075] All data were expressed as the mean ± standard deviation of at least three independent experiments. Statistical analysis of two groups of data was performed using paired t test, and statistical analysis of more than two groups of data was performed using one-way analysis of variance.

[0076] Through the above experiments, it is confirmed that the GsMTx-4 polypeptide can directly antagonize the spinal cord inflammatory microenvironment disorder mediated by spinal cord microglia cells, has a definite curative effect on an animal model of mechanical impact-induced spinal cord injury, and effectively promotes the homeostasis of the spinal cord internal environment. In addition, the GsMTx-4 polypeptide is convenient for microglial cell absorption and utilization, and fills the blank of the current domestic spinal cord injury prevention and treatment drugs.

[0077] In addition, the cell experiment shows that the GsMTx-4 polypeptide can reduce the inflammatory reaction process of microglial cells caused by excessive mechanical stress stimulation, and plays a protective role in the process of spinal cord injury.

[0078] More importantly, the GsMTx-4 polypeptide has a simple purification process and a mature preparation method, and can significantly reduce the cost, thereby reducing the economic burden of patients.

[0079] The above has carried out the detailed description to one embodiment of the application, but the content described is only the preferred embodiment of the application, and cannot be considered as used for limiting the implementation range of the application. Any equivalent changes and improvements made according to the application scope should still belong to the patent coverage range of the application.

Claims

1. Application of GsMTx-4 polypeptide in the preparation of a drug for treating spinal cord injury, wherein the GsMTx-4 polypeptide protects the spinal cord by antagonizing the microglial inflammatory response caused by mechanical injury, and the drug is administered by intrathecal injection.

2. Use of the GsMTx-4 polypeptide in the preparation of a drug for treating spinal cord injury caused by trauma, wherein the drug is administered by intrathecal injection.

3. Use of GsMTx-4 polypeptide in the preparation of a drug for alleviating traumatic acute spinal cord injury, wherein the drug is administered by intrathecal injection.

4. The use of GsMTx-4 polypeptide in the preparation of a drug for alleviating paralysis, limb weakness, and limb sensory impairment caused by spinal cord injury, wherein the drug is administered by intrathecal injection.

5. The use according to any one of claims 1 to 4, characterized in that: The GsMTx-4 polypeptide also includes pharmaceutically acceptable salts thereof.

6. The use according to any one of claims 1 to 4, characterized in that: The medicament includes pharmaceutically acceptable adjuvants.

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