Use of an esyt1-derived polypeptide in the preparation of a medicament for treating a central nervous system demyelination injury disease

Abstract

This invention discloses the application of an ESYT1-derived polypeptide in the preparation of drugs for treating central nervous system demyelinating diseases. The amino acid sequence of the ESYT1-derived polypeptide is shown in SEQ ID NO.1, specifically EEPELSSGGPPHITS. In vitro experiments confirmed that this polypeptide is non-cytotoxic and can inhibit LPS-induced reactive oxygen species production and the secretion of pro-inflammatory factors TNF-α and IL-1β in a concentration-dependent manner, significantly inhibiting microglial pro-inflammatory polarization. In vivo copper hydrazone-induced demyelinating mouse model experiments showed that intraventricular injection of this polypeptide can effectively inhibit abnormal glial activation, restore the expression of myelin proteins MAG and MOG, improve myelin ultrastructure, reduce the G-ratio, and increase axonal myelination efficiency. The ESYT1-derived polypeptide provided by this invention has the dual functions of inhibiting neuroinflammation and promoting myelin regeneration, has high safety, and can be used to prepare drugs for treating multiple sclerosis and other central nervous system demyelinating diseases.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an ESYT1-derived polypeptide and its application in the preparation of drugs for treating demyelinating diseases of the central nervous system. Background Technology

[0002] Central nervous system demyelinating diseases are a group of neurological disorders characterized primarily by damage and loss of the myelin sheath, including multiple sclerosis, neuromyelitis optica, and acute disseminated encephalomyelitis. Multiple sclerosis is the most common, with a global incidence of 5–300 per 100,000. It predominantly affects young adults and women, characterized by a prolonged course and recurrent episodes, leading to severe neurological deficits such as visual impairment, motor dysfunction, sensory abnormalities, and cognitive decline, significantly impacting patients' quality of life. Currently, disease-modifying therapies (DMTs) used clinically to treat multiple sclerosis primarily target the peripheral immune system, with an efficacy rate of only 29%–68%. Furthermore, they have adverse reactions such as bradycardia, infection, cardiac conduction block, and infusion reactions, making it difficult to meet clinical needs. More importantly, while these drugs can slow disease progression to some extent, they cannot effectively repair existing myelin damage or promote myelin regeneration and neurological function recovery.

[0003] In recent years, promoting myelin regeneration and repair has become an important research direction for the treatment of central demyelinating diseases. Existing studies have shown that promoting the differentiation of oligodendrocyte precursor cells (OPCs) into mature oligodendrocytes (OLs), thereby promoting myelin regeneration, is an effective strategy for treating demyelinating diseases. For example, Chinese patent application CN117442618A discloses compound LY2940094 as an opioid-associated nociceptor receptor 1 antagonist, which can promote the differentiation of OPCs into mature oligodendrocytes and has shown a promoting effect on myelin regeneration in a copper hydrazone (CPZ)-induced mouse demyelinating model. However, this compound only focuses on promoting OPC differentiation and does not involve the regulation of central nervous system inflammation. Neuroinflammation is a core driving factor in the occurrence and progression of demyelinating diseases. Abnormal activation of microglia and astrocytes can continuously release large amounts of inflammatory factors and reactive oxygen species, exacerbating myelin destruction and nerve damage, which is a key reason for the continuous progression of demyelinating lesions. Therefore, simply promoting OPC differentiation while neglecting the regulation of the inflammatory microenvironment may not achieve the desired repair effect.

[0004] On the other hand, Chinese patent application CN113846052A discloses the application of endothelial cells and their precursor cells in the treatment of demyelinating diseases. Studies have shown that these cells can secrete neurotrophic factors such as BDNF, promoting the proliferation, migration, and maturation of OPCs. Simultaneously, they can activate microglia / macrophages and induce their transformation from M1 to M2 types, thus exhibiting a dual effect of promoting myelin regeneration and regulating inflammation to a certain extent. However, this technical solution uses cell preparations as the active ingredient, which presents challenges such as complex cell preparation processes, difficulty in controlling quality consistency, stringent storage and transportation conditions, and high risks to in vivo activity and safety, thus limiting its clinical translation and widespread application.

[0005] Peptide drugs, with their advantages of small molecular weight, high specificity, low toxicity, good biocompatibility, and easy metabolism, are a hot research area in the development of new drugs for neurological diseases. However, no peptide drugs have yet been reported that can both effectively inhibit central nervous system inflammation and directly promote myelin regeneration and repair. Therefore, developing a peptide drug that combines anti-inflammatory and myelin repair functions, with high safety and good drug-like properties, has significant clinical significance and application value for the treatment of central demyelinating diseases. Summary of the Invention

[0006] In view of the limitations of existing drugs for treating central nervous system demyelinating diseases, such as limited efficacy, significant side effects, and the lack of dual-function drugs that can simultaneously inhibit neuroinflammation and promote myelin regeneration and repair, this invention provides an application of ESYT1-derived polypeptide in the preparation of drugs for treating central nervous system demyelinating injury and the resulting pharmaceutical composition.

[0007] This ESYT1-derived polypeptide can effectively inhibit pro-inflammatory polarization of microglia, reduce the release of reactive oxygen species and pro-inflammatory factors, and promote the proliferation of oligodendrocyte precursor cells, upregulate myelin protein expression, and repair myelin ultrastructure. It has dual activities of inhibiting central nervous system inflammation and promoting myelin regeneration and repair. Moreover, it has a small molecular weight, low toxicity, and good biocompatibility, and can effectively treat central demyelinating diseases such as multiple sclerosis, showing good prospects for clinical translation and application.

[0008] The technical solution adopted in this invention is as follows: The application of an ESYT1-derived polypeptide in the preparation of a drug for treating demyelinating injuries of the central nervous system, wherein the amino acid sequence of the ESYT1-derived polypeptide is shown in SEQ ID NO.1, specifically: Glu-Glu-Pro-Glu-Leu-Ser-Gly-Gly-Pro-Pro-His-Ile-Thr-Ser (EEPELSGGPPHITS).

[0009] Preferably, the central nervous system demyelinating injury disease is multiple sclerosis.

[0010] Preferably, the drug is a drug with activity that inhibits neuroinflammation of the central nervous system.

[0011] Preferably, the drug is a drug that has the activity of promoting myelin regeneration and repair in the central nervous system.

[0012] Furthermore, the activity of inhibiting central nervous system neuroinflammation includes inhibiting microglial cell activation, reducing reactive oxygen species production, and inhibiting the secretion of pro-inflammatory factors TNF-α and IL-1β.

[0013] Furthermore, the activities that promote myelin regeneration and repair in the central nervous system include increasing oligodendrocyte precursor cell density, upregulating the expression of myelin proteins MAG and MOG, improving myelin ultrastructure, reducing the myelin G-ratio, and improving axonal myelination efficiency.

[0014] The present invention also provides a pharmaceutical composition for treating demyelinating injuries of the central nervous system, the pharmaceutical composition comprising an effective dose of an ESYT1-derived polypeptide with an amino acid sequence as shown in SEQ ID NO.1, and a pharmaceutically acceptable carrier or excipient.

[0015] Preferably, the pharmaceutical composition is administered via intraventricular injection.

[0016] Preferably, the effective concentration of the ESYT1-derived polypeptide is 100 nM to 400 nM.

[0017] Preferably, the in vivo dose of the ESYT1-derived polypeptide is 0.2 μmol / 5 μL.

[0018] This invention also provides a method for preparing the above-mentioned ESYT1-derived polypeptide, comprising the following steps: (1) Preparation of solid-phase synthetic resin: Rink amide resin is used as solid-phase carrier. The resin is balanced with dichloromethane for 2 hours, washed and dried with DMF and then ready for use.

[0019] (2) Amino acid coupling: Fmoc-protected amino acids were activated using the HOBt, DIC, and DMAP system, and each amino acid residue was coupled sequentially from the C-terminus to the N-terminus according to the amino acid sequence shown in SEQ ID NO. 1. After each coupling step, the completeness of coupling was detected by the ninhydrin reaction, and then the Fmoc protecting group was removed.

[0020] (3) Peptide cleavage and precipitation: After all amino acids are coupled, the peptides are cut off from the resin using TFA / water / triisopropylsilane (volume ratio 95:2.5:2.5) cleavage buffer, the filtrate is collected by filtration, and the crude peptide is obtained by cold ether precipitation.

[0021] (4) Purification and freeze-drying: The crude peptide was purified by preparative HPLC (C18 reverse phase column), the target peak was collected, and the ESYT1 derivative peptide freeze-dried powder with a purity of ≥95% was obtained after freeze-drying.

[0022] Preferably, the coupling reaction in step (2) takes 1 to 2 hours and is carried out at room temperature; the pyrolysis reaction in step (3) takes 2 to 4 hours; and the HPLC mobile phase in step (4) is acetonitrile / water (containing 0.1% TFA) gradient elution.

[0023] The present invention also provides a method for preparing the above-mentioned pharmaceutical composition, comprising the following steps: The above-mentioned ESYT1-derived lyophilized peptide powder was dissolved in sterile phosphate buffer (PBS, pH 7.2–7.4) or physiological saline, and the peptide concentration was adjusted to 100 nM–400 nM. After sterilization by filtration through a 0.22 μm filter membrane, it was dispensed into sterile injection vials to obtain ESYT1 peptide injection solution.

[0024] Preferably, the pharmaceutical composition can also be prepared as a lyophilized powder for injection: a pharmaceutically acceptable lyophilization protectant is added to the ESYT1 polypeptide solution, wherein the lyophilization protectant is selected from one or more of mannitol, trehalose, and sucrose, and the mass-volume concentration of the lyophilization protectant is 2% to 10%; the mixed solution is dispensed into vials, and after freeze-drying (pre-freezing temperature -40℃ to -50℃, primary drying temperature -20℃ to 0℃, secondary drying temperature 20℃ to 30℃), the vials are sealed to obtain the ESYT1 polypeptide lyophilized powder for injection.

[0025] The present invention also provides a method for verifying the activity of the above-mentioned ESYT1-derived peptides, including in vitro activity verification and in vivo activity verification.

[0026] The in vitro activity verification includes the following steps: using the BV2 mouse microglia cell line, an inflammation model was induced with 100 ng / mL LPS, and the cells were treated with 100 nM, 200 nM, and 400 nM ESYT1 peptide for 24 h, respectively. Cytotoxicity was detected by CCK-8 assay, intracellular reactive oxygen species level was detected by DHE staining, and secretion levels of pro-inflammatory factors TNF-α and IL-1β were detected by ELISA.

[0027] The in vivo activity verification included the following steps: Six-week-old SPF-grade male C57BL / 6 mice were selected and fed a diet containing 0.2% copper hydrazone (CPZ) for 8 weeks to establish a central demyelinating model. From week 6 to week 8, 0.2 μmol / 5 μL of ESYT1 peptide was injected into the intracerebrospinal ventricle weekly. The repair effect of ESYT1 peptide on demyelinating injury was comprehensively evaluated by observing the ultrastructure of myelin sheath and G-ratio analysis by LFB myelin staining, MAG / MOG immunofluorescence staining, transmission electron microscopy, and detecting microglial activation by Iba1 immunofluorescence staining.

[0028] Compared with the prior art, the present invention has the following beneficial effects: (1) The central demyelination repair function of ESYT1-derived polypeptide was discovered for the first time. The ESYT1-derived polypeptide (SEQ ID NO.1) described in this invention is derived from amino acid residues 944 to 957 of human extended synapse binding protein 1. There are currently no reports on its related functions, which is original and innovative.

[0029] (2) It has the dual function of inhibiting neuroinflammation and promoting myelin regeneration and repair, overcoming the shortcomings of single-mechanism drugs. In the prior art, the compound LY2940094 disclosed in CN117442618A can only promote OPC differentiation and cannot regulate neuroinflammation; although the cell preparation used in CN113846052A can take into account some anti-inflammatory and repair-promoting effects, it has problems such as complicated preparation, difficult quality control, and high safety risks. The ESYT1 polypeptide of the present invention effectively inhibits central nervous system inflammation by inhibiting microglia activation, reducing ROS generation, and inhibiting TNF-α and IL-1β secretion; on the other hand, it significantly promotes myelin regeneration and repair by increasing oligodendrocyte precursor cell density, upregulating MAG and MOG expression, improving myelin ultrastructure, and reducing G-ratio, thus achieving a dual synergistic therapeutic effect of "anti-inflammatory + repair-promoting".

[0030] (3) High safety, good biocompatibility, and low cytotoxicity. CCK-8 assay confirmed that within the effective concentration range of 100 nM to 400 nM, the ESYT1 peptide had no significant effect on the survival rate of BV2 microglia for 24 h to 72 h (p>0.05). This peptide is an endogenous protein fragment, and its metabolites are amino acids. It has no toxic side effects and possesses good in vitro and in vivo safety.

[0031] (4) The drug has a clear in vivo efficacy and a significant effect on myelin repair. In a CPZ-induced demyelinating mouse model, after intraventricular injection of ESYT1 peptide, LFB staining showed a significant reduction in the area of ​​demyelination (p<0.01); immunofluorescence showed upregulated expression of MAG and MOG proteins (p<0.01); transmission electron microscopy showed that the myelin lamellar structure was dense and regular, the axonal morphology was normal, and the G-ratio was significantly reduced (p<0.01); Iba1 staining showed that microglial activation was significantly inhibited (p<0.01). The above multidimensional evidence fully confirms its strong ability to repair demyelinating damage.

[0032] (5) The preparation process is mature and suitable for clinical translation. The polypeptide of this invention is prepared by a mature solid-phase synthesis method, with high purity (≥95%) and good batch-to-batch consistency; the drug composition is simple to prepare, can be administered by intraventricular injection, with a clear dosage (0.2 μmol / 5 μL, once a week), and can be further prepared into lyophilized powder for injection to extend the shelf life, with good prospects for clinical translation and application.

[0033] In summary, this invention provides an ESYT1-derived polypeptide and its pharmaceutical composition that have dual functions of central nervous system inflammation suppression and myelin regeneration and repair. It can effectively treat central nervous system demyelinating diseases such as multiple sclerosis, filling the technical gap that existing drugs cannot simultaneously address persistent inflammation and insufficient myelin repair. It has extremely high clinical application value and commercialization potential. Attached Figure Description

[0034] Figure 1 The cytotoxicity of ESYT1 peptide against BV2 microglia was detected by treating BV2 cells with 100 nM, 200 nM, and 400 nM ESYT1 peptide for 24 h, 48 h, and 72 h, respectively, and then using the CCK-8 assay to detect cell viability. The results showed that ESYT1 peptide at each concentration did not significantly affect cell viability at any time point, confirming that ESYT1 peptide has no cytotoxicity within its effective concentration range. Figure 2 The in vitro anti-inflammatory activity of ESYT1 peptide was validated using a BV2 microglial cell inflammation model induced by 100 ng / mL LPS. Cells were treated with 100 nM, 200 nM, and 400 nM ESYT1 peptide for 24 h. (A) DHE staining was used to detect the level of intracellular reactive oxygen species (ROS). Statistical results showed that ESYT1 peptide concentration-dependently inhibited LPS-induced excessive ROS production. (B) ELISA was used to detect the secretion levels of pro-inflammatory factors IL-1β and TNF-α in the cell supernatant. Results showed that ESYT1 peptide significantly downregulated the levels of IL-1β and TNF-α. Data are expressed as mean ± standard deviation, ×p<0.05, ××p<0.01 vs. LPS model group. Figure 3 The effect of ESYT1 peptide on promoting the repair of demyelinating injury in vivo was verified. A central demyelinating model was established by feeding C57BL / 6 mice with 0.2% copper hydrazone (CPZ) diet for 8 weeks. From week 6, 0.2 μmol / 5 μL of ESYT1 peptide was injected into the intracerebrospinal fluid weekly for 2 weeks. (A) LFB myelin staining was used to detect myelin loss in the corpus callosum region of mice. The results showed that the area of ​​myelin loss was significantly reduced after ESYT1 peptide intervention (scale bar = 200 μm). (B) Immunofluorescence staining was used to detect the expression of myelin marker proteins MAG and MOG. The results showed that ESYT1 peptide significantly upregulated the expression levels of MAG and MOG (scale bar = 50 μm). μm; (C) Transmission electron microscopy observation of the ultrastructure of the myelin sheath of the corpus callosum and G-ratio analysis showed that after ESYT1 peptide intervention, the myelin sheath lamellar structure was dense and regular, the axon morphology was normal, and the myelin sheath G-ratio was significantly reduced; (D) Iba1 immunofluorescence staining to detect microglia activation showed that ESYT1 peptide could significantly inhibit the abnormal activation of microglia in the brain of model mice, scale bar = 50 μm; data are expressed as mean ± standard deviation, ×p<0.05, ××p<0.01 vs. CPZ model group or CPZ+Scr group. Detailed Implementation

[0035] The technical solution of the present invention will be clearly and completely described below through specific embodiments. Unless otherwise specified, the experimental operation methods used in the embodiments of the present invention are all conventional experimental techniques and standard operating procedures in the art, and the experimental conditions not specifically defined are all common conventional conditions in the art. Unless otherwise specified, the cells, reagents, consumables and instruments involved in the embodiments of the present invention are all commercially available products that are readily available in the market.

[0036] 1. Experimental cells and culture conditions The mouse microglia cell line BV2 used in this experiment was purchased from Shanghai Zhongqiao Xinzhou Biotechnology Co., Ltd. (Catalog No.: ZQ0397). Cells were cultured in high-glucose DMEM medium (Gibco, Catalog No.: 11965-092) supplemented with 10% fetal bovine serum (Gibco, Catalog No.: 10099141C) and 1% penicillin-streptomycin solution. Cells were cultured routinely at 37℃ in a 5% CO2 incubator, and cells in the logarithmic growth phase were used for subsequent experiments. A cellular inflammation model was constructed by treating BV2 cells with 100 ng / mL LPS (Sigma, Catalog No.: L2880) for 24 h.

[0037] 2. Peptide Synthesis and Preparation The ESYT1 polypeptide of this invention originates from amino acid residues 944-957 of human extended synapse-binding protein 1 (NCBI Reference Sequence: NP_001171725), with the amino acid sequence EEPELSGGPPHITS (SEQ ID NO.1); the disordered control polypeptide Scr sequence is SGPEHPLGITEPSG (SEQ ID NO.2). Both polypeptides were prepared using a solid-phase polypeptide synthesis method.

[0038] 3. Laboratory animals, model establishment, and drug administration regimen Six-week-old SPF-grade male C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. Mice were acclimatized for two weeks under an environmental temperature of 22℃, humidity of 40%–60%, and a 12-hour light-dark cycle. Eight-week-old mice were randomly divided into a blank control group, a CPZ model group, a CPZ+Scr control group, and a CPZ+ESYT1 experimental group. Except for the blank group, all other groups were fed a diet containing 0.2% copper hydrazone (CPZ) for eight consecutive weeks to establish a central demyelinating model. From week 6 to week 8, mice in the ESYT1 and Scr groups received weekly intraventricular injections of 0.2 μmol at a volume of 5 μL, while the blank and model groups received an equal volume of sterile saline.

[0039] 4. Cell viability assay (CCK-8 assay) BV2 cells were loaded at 5 × 10 4 Cells were seeded at a density of 100 cells / well in 96-well plates and cultured until adherent. Then, 100 nM, 200 nM, and 400 nM ESYT1 peptides were added for 24 h each. 10 μL of CCK-8 assay solution was added to each well, and the plates were incubated at 37°C in the dark for 1 h. The absorbance at 450 nm was measured using a microplate reader, and cell viability was calculated to assess the cytotoxicity of the ESYT1 peptide.

[0040] 5. Intracellular reactive oxygen species (ROS) detection Intracellular ROS levels were detected using a DHE reactive oxygen species (ROS) assay kit. After treatment of each cell group, 10 μM DHE probe was added, and the cells were incubated at 37°C in the dark for 30 min. Residual probes were removed by washing with PBS. Fluorescence images were acquired using a Leica SP8 laser confocal microscope, and fluorescence intensity was quantitatively analyzed using ImageJ software to evaluate the level of ROS generation in the cells.

[0041] 6. Immunofluorescence staining detection Mouse brain tissue was fixed with 4% paraformaldehyde for 24 h and dehydrated with 30% sucrose to prepare 30 μm frozen sections, which were then incubated with Iba1, MAG, and MOG primary antibodies, respectively. Subsequent staining and imaging methods were the same as for cell samples.

[0042] 7. Luxol Fast Blue (LFB) myelin staining Paraffin sections of mouse brain tissue were dewaxed with xylene and rehydrated with gradient ethanol. They were then stained in LFB myelin staining solution preheated to 65°C for 60 min in the dark. After rapid differentiation with differentiation solution and termination of the reaction with tap water, the differentiation was monitored under a microscope until the myelin sheath turned blue and the background was transparent. The sections were then dehydrated with gradient ethanol, cleared with xylene, mounted with neutral resin, photographed under a microscope, and quantitatively analyzed for the area of ​​myelin loss and the integrity of the myelin sheath.

[0043] 8. Transmission electron microscopy (TEM) ultrastructure detection of myelin sheath Mice were perfused with heparinized saline and 3% glutaraldehyde fixative via cardiac perfusion. Brain tissue from the corpus callosum was isolated and fixed at 4°C for 24 h. After fixation with 1% osmium tetroxide for 2 h, the tissue was dehydrated in gradient ethanol, embedded, and solidified to prepare ultrathin sections. The ultrastructure of myelin sheath was observed using a Tecnai G2 20Twin transmission electron microscope. The myelin sheath G-ratio (axon inner diameter / total myelin outer diameter) was measured and calculated using ImageJ to assess the level of myelination.

[0044] 9. ELISA inflammatory factor detection BV2 cell lysates from each group were collected, centrifuged at 12000 r / min for 20 min at 4℃, and the supernatant was collected. The mouse TNF-α and IL-1β specific ELISA kits were used according to the instructions, and the absorbance at 450 nm and 570 nm was measured using an enzyme-linked immunosorbent assay (ELISA) reader. The concentrations of inflammatory factors in each group were calculated based on the standard curve.

[0045] 10. Statistical Analysis All experimental data were statistically analyzed using GraphPad Prism 9.0 software. Independent samples t-tests were used to compare two groups of data, and one-way ANOVA combined with Tukey's test was used to compare multiple groups of data. p < 0.05 was considered statistically significant. All data are expressed as mean ± standard deviation (mean ± SD).

[0046] Example 1 Synthesis and preparation of ESYT1 polypeptide.

[0047] The amino acid sequence of the ESYT1 polypeptide described in this invention is EEPELSGGPPHITS (SEQ ID NO.1); the sequence of the disordered control polypeptide Scr is SGPEHPLGITEPSG (SEQ ID NO.2), both prepared by solid-phase synthesis.

[0048] Synthesis process: Rink amide resin was used as the solid-phase support. The resin was equilibrated with dichloromethane for 2 hours. After washing and drying with DMF, the amino acids were activated using the HOBt, DIC, and DMAP system. Each amino acid residue was coupled sequentially, and the completeness of coupling was detected by ninhydrin reaction. After each round of coupling, the Fmoc protecting group was removed. Finally, the peptide was cleaved using TFA lysis buffer, the crude product was precipitated with cold diethyl ether, purified by preparative HPLC, and lyophilized to obtain pure peptide powder.

[0049] Example 2 Cytotoxicity assay of ESYT1 peptide.

[0050] Toxicity experiments were conducted using the BV2 mouse microglia cell line. Cells were cultured in high-glucose DMEM medium (containing 10% FBS and 1% penicillin-dextrose antibody) at 37°C with 5% CO2. BV2 cells were seeded into 96-well plates at 5 × 10⁶ wells. 4 Cells were divided into control group, 100 nM ESYT1 group, 200 nM ESYT1 group, and 400 nM ESYT1 group. Cell viability was detected using a CCK-8 assay kit after 24 h, 48 h, and 72 h of treatment.

[0051] like Figure 1 The results showed that ESYT1 peptide at concentrations of 100 nM, 200 nM, and 400 nM had no significant effect on the survival rate of BV2 cells from 24 h to 72 h, confirming that the ESYT1 peptide described in this invention has no cytotoxicity within its effective concentration range.

[0052] Example 3 Validation of the in vitro anti-inflammatory activity of ESYT1 peptide.

[0053] A BV2 microglial inflammation model was induced using 100 ng / mL LPS. The model included a blank control group, an LPS model group, an LPS+400 nM Scr control group, and an LPS+100 / 200 / 400 nM ESYT1 experimental group. The treatment was continued for 24 h.

[0054] like Figure 2 As shown in Figure A, ESYT1 significantly reduced LPS-induced excessive ROS production in BV2 cells in a concentration-dependent manner, with the 400 nM concentration showing the best effect.

[0055] like Figure 2 As shown in Figure B, ELISA results showed that ESYT1 could significantly downregulate the expression and secretion of pro-inflammatory factors TNF-α and IL-1β, and effectively inhibit microglial cell inflammatory activation and pro-inflammatory polarization.

[0056] Example 4 Verification of the effect of ESYT1 peptide on promoting the repair of demyelinating injury in vivo.

[0057] Experimental animals: Eight-week-old male C57BL / 6 mice were randomly divided into a blank control group, a CPZ demyelination model group, a CPZ+Scr control group, and a CPZ+ESYT1 experimental group, with 15 mice in each group. Mice in the model group and drug treatment group were fed 0.2% copper hydrazone (CPZ) diet for 8 weeks to establish a demyelination model. From weeks 6 to 8, mice in the ESYT1 group received weekly intracerebroventricular injections of ESYT1 peptide (mice were anesthetized with isoflurane and fixed in a stereotaxic instrument; the skull was drilled 0.3 mm posterior to the anterior fontanelle and 1.0 mm to the right of the midline; a micro-injector was inserted vertically to a depth of 2.5 mm, and 5 μL of 0.2 μmol peptide was slowly injected at a rate of 0.5 μL / min; the needle was left in place for 3 minutes and then slowly withdrawn). The Scr group received an equal amount of disordered peptide. The intervention was continued for 2 weeks.

[0058] like Figure 3 A LFB myelin staining results showed that the corpus callosum of CPZ model mice had severe demyelination lesions. After ESYT1 intervention, the range of myelin loss was significantly reduced and the myelin damage was significantly repaired. like Figure 3 Results B show that ESYT1 can significantly upregulate the expression of myelin marker proteins MAG and MOG; like Figure 3 The results showed that CPZ model mice had pathological changes such as myelin sheath loosening, axonal swelling, and myelin sheath structure disorder. After ESYT1 intervention, the myelin sheath lamellar structure was dense and regular, the axonal morphology was normal, the myelin sheath G-ratio was significantly reduced, and the axonal myelination efficiency was significantly improved. like Figure 3 As shown in the results, Iba1 immunofluorescence staining confirmed that ESYT1 can significantly inhibit abnormal activation of microglia in the brain of model mice and reduce inflammatory damage to the central nervous system microenvironment.

[0059] As can be seen from Examples 1-4 above, this invention discloses an ESYT1-derived polypeptide EEPEL SGGPPHITS and its novel application in the preparation of drugs for central demyelinating injury repair and anti-neuroinflammatory purposes. The ESYT1 polypeptide of this invention exhibits good drug safety, showing no significant cytotoxicity to BV2 microglia within the effective concentration range of 100 nM, 200 nM, and 400 nM, thus providing a safe basis for in vitro drug use. In vitro inflammatory model experiments show that this polypeptide can significantly inhibit LPS-induced microglial overactivation in a concentration-dependent manner, effectively reduce abnormal intracellular ROS accumulation under inflammatory stress, and significantly downregulate the secretion and expression of key pro-inflammatory factors such as TNF-α and IL-1β, significantly improving the pro-inflammatory polarization state of central microglia and exerting excellent in vitro anti-inflammatory effects.

[0060] In vivo copper hydrazone-induced central demyelination model experiments further confirmed that the ESYT1 peptide of this invention possesses significant demyelination damage repair and neuroprotective capabilities. LFB myelin staining results confirmed that the ESYT1 peptide significantly improved CPZ-induced extensive demyelination lesions in the mouse corpus callosum, effectively reducing the area of ​​demyelination. Protein expression detection results showed that the ESYT1 peptide significantly upregulated the expression levels of myelin marker proteins MAG and MOG, promoting the functional recovery of myelin structure-related proteins. Myelin ultrastructure analysis confirmed that ESYT1 intervention effectively reversed typical pathological damage caused by CPZ, such as lamellar myelin sheath loosening, axonal swelling, and myelin sheath structural disorder, significantly reduced the myelin G-ratio, effectively improved axonal myelination efficiency, and promoted functional myelin regeneration. Simultaneously, glial cell immunofluorescence results confirmed that the ESYT1 peptide significantly inhibited the abnormal activation and excessive proliferation of microglia in the brains of demyelinating model mice, effectively improving the central inflammatory microenvironment and blocking secondary myelin damage mediated by persistent neuroinflammation.

[0061] In summary, the ESYT1 polypeptide described in this invention possesses both significant central nervous system inflammation-inhibiting activity and demyelinating injury repair function. It can simultaneously achieve multiple therapeutic effects, including inhibiting neuroinflammation, stabilizing myelin sheath structure, promoting myelin sheath regeneration and repair, and improving the central nervous system pathological microenvironment. It can serve as a novel active candidate molecule for the treatment of central nervous system demyelinating diseases, and can also be used to prepare drugs or functional preparations that inhibit central nervous system inflammation and promote myelin sheath repair. It has broad basic research value and clinical translational application prospects.

[0062] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the above embodiments do not limit the present invention in any way, and all technical solutions obtained by equivalent substitution or equivalent transformation fall within the protection scope of the present invention.

Claims

1. The use of an ESYT1-derived polypeptide in the preparation of a medicament for treating demyelinating injuries of the central nervous system, characterized in that, The amino acid sequence of the ESYT1-derived polypeptide is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The central nervous system demyelinating injury disease mentioned is multiple sclerosis.

3. The application according to claim 1, characterized in that, The drug is a drug with activity that inhibits neuroinflammation in the central nervous system.

4. The application according to claim 3, characterized in that, The activity of inhibiting central nervous system neuroinflammation includes one or more of the following: inhibiting microglia activation, reducing reactive oxygen species production, and inhibiting the secretion of pro-inflammatory factors TNF-α and IL-1β.

5. The application according to claim 1, characterized in that, The drug is a drug that has the activity of promoting myelin regeneration and repair in the central nervous system.

6. The application according to claim 5, characterized in that, The activities that promote myelin regeneration and repair in the central nervous system include one or more of the following: increasing oligodendrocyte precursor cell density, upregulating the expression of myelin proteins MAG and MOG, improving myelin ultrastructure, reducing the myelin G-ratio, and improving axonal myelination efficiency.

7. A pharmaceutical composition for treating demyelinating injuries of the central nervous system, characterized in that, The pharmaceutical composition comprises an effective dose of an ESYT1-derived polypeptide with an amino acid sequence as shown in SEQ ID NO.1, and optionally includes a pharmaceutically acceptable carrier or excipient.

8. The pharmaceutical composition according to claim 7, characterized in that, The drug composition is administered via intraventricular injection.

9. The pharmaceutical composition according to claim 7, characterized in that, The effective concentration of the ESYT1-derived polypeptide is 100 nM to 400 nM.

10. The pharmaceutical composition according to claim 7, characterized in that, The in vivo dosage of the ESYT1-derived polypeptide is 0.2 μmol / 5 μL.

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