Application of CLASP2 protein in preparation of medicine for promoting spinal cord injury repair

Abstract

The invention discloses an application of CLASP2 protein in preparation of a medicine for promoting spinal cord injury repair. According to the invention, a bioinformatics and phosphorylated proteomics technology combined method is utilized to screen out that the functional protein of curcumin for promoting spinal cord injury repair is CLASP2 protein. The molecular biological technique verifies that curcumin regulates and controls microtubule stability by increasing phosphorylation of CLASP2 protein to promote functional repair after spinal cord transection injury. Therefore, the curcumin can regulate and control microtubules by promoting the phosphorylation of the CLASP2 to dynamically promote the motor function repair after the spinal cord injury of the mouse. The method has an important application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of CLASP2 protein in preparing a medicine for promoting the repair of spinal cord injury. Background Art

[0002] Spinal cord injury is a central nervous system injury disease. The long-term rehabilitation treatment and loss of labor force of patients with spinal cord injury have a huge impact on their families and individuals. Previous research data show that the incidence of spinal cord injury is between 1.2 and 5.8 cases per 100,000 people per year. The recent surge in the number of patients with spinal cord injury has become an increasingly serious social and medical problem. Promoting the recovery of motor function in patients with spinal cord injury can not only reduce the patient's physical limitations and promote the patient's physical and mental health, but also reduce the family and socioeconomic burden.

[0003] Spinal cord injury is usually caused by trauma. Pathological processes such as inflammatory response and free radical generation lead to neuronal damage and destruction of the blood-spinal cord barrier. The regenerative capacity of the central nervous system is limited, and the persistent existence of inflammation leads to excessive neuronal clearance; the ischemic, hypoxic, and nutrient-deficient spinal cord environment after damage is the reason why neurons are difficult to repair after spinal cord injury.

[0004] The ability of neuronal axons to regenerate is one of the important factors in the recovery of the central nervous system after injury. Axon regeneration is affected by many factors, among which the correct reconstruction of the cytoskeleton is one of the important factors in axon regeneration. Neurons that have successfully regenerated and repaired form growth cones at the tips of damaged axons. The growth cones include microtubules and actin. The dynamic reorganization of the two forms lamellipodia and filopodia, which are necessary structures for axon elongation. Retraction balls will form at the injured part of the axons of damaged and unrepaired neurons, preventing axon regeneration. Retraction balls are composed of depolymerized or disordered microtubules, without the correct arrangement of actin and tubulin. The homeostatically unbalanced cytoskeleton structure causes the inability of spinal cord neuron axons to regenerate after injury.

[0005] Curcumin (Curcumin, molecular formula: C 21 H 20 O6, molecular weight: 368.37) is a polyphenol compound extracted from the rhizome of turmeric. It is a common natural food colorant and has been used in traditional medicine for more than 2,000 years as an active ingredient in a variety of natural plants. Numerous studies have shown that curcumin is a highly effective antibacterial agent and has been shown to be active against various chronic diseases, including various types of cancer, diabetes, obesity, cardiovascular, pulmonary, nervous system and autoimmune diseases. To date, curcumin has completed more than 100 different clinical trials involving more than 6,000 participants, and patients have tolerated it without adverse reactions. Summary of the invention

[0006] The purpose of the present invention is to select drugs that promote the repair of spinal cord injury.

[0007] The present invention firstly protects the use of a substance for detecting CLASP2 protein phosphorylation, a substance for increasing CLASP2 protein phosphorylation and / or a substance for detecting CLASP2 protein expression and / or activity in the preparation of a product for selecting a drug for promoting spinal cord injury repair.

[0008] In the above application, the drug may be curcumin.

[0009] The present invention also protects the use of CLASP2 protein or a substance capable of increasing the phosphorylation of CLASP2 protein in vivo in the preparation of a drug for promoting the repair of spinal cord injury.

[0010] In any of the above applications, the promotion of spinal cord injury repair may be manifested as at least one of A1)-A6):

[0011] A1) Promote functional recovery after spinal cord injury;

[0012] A2) Promote the recovery of motor function after spinal cord injury;

[0013] A3) Reduce fibrous scar formation after spinal cord injury;

[0014] A4) Promotes neuron formation after spinal cord injury;

[0015] A5) Stabilize microtubules;

[0016] A6) Maintains the structure of the Golgi apparatus.

[0017] In any of the above applications, the cells or tissues for repairing spinal cord injury may be neurons, oligodendrocytes and / or myelin sheaths.

[0018] In any of the above applications, the spinal cord injury may be a spinal cord transection injury.

[0019] In any of the above applications, the CLASP2 protein phosphorylation may specifically be serine phosphorylation at position 1025 of the CLASP2 protein.

[0020] The present invention also protects a pharmaceutical composition, which comprises CLASP2 protein or a substance capable of increasing the phosphorylation of CLASP2 protein in vivo.

[0021] The pharmaceutical composition may specifically consist of CLASP2 protein or a substance capable of increasing the phosphorylation of CLASP2 protein in vivo.

[0022] Any of the above-mentioned pharmaceutical compositions may further include curcumin and / or a pharmaceutically acceptable carrier.

[0023] Any of the above-mentioned pharmaceutical compositions may specifically consist of CLASP2 protein or a substance capable of increasing the phosphorylation of CLASP2 protein in vivo, curcumin and a pharmaceutically acceptable carrier.

[0024] Any of the above-mentioned pharmaceutical compositions may specifically consist of CLASP2 protein or a substance capable of increasing the phosphorylation of CLASP2 protein in vivo and curcumin.

[0025] Any of the above-mentioned pharmaceutical compositions may specifically consist of CLASP2 protein or a substance capable of increasing the phosphorylation of CLASP2 protein in vivo and a pharmaceutical carrier.

[0026] The present invention also protects the use of CLASP2 protein or phosphorylated CLASP2 protein, which may be at least one of A1)-A7):

[0027] A1) Promote functional recovery after spinal cord injury;

[0028] A2) Promote the recovery of motor function after spinal cord injury;

[0029] A3) Reduce fibrous scar formation after spinal cord injury;

[0030] A4) Promotes neuron formation after spinal cord injury;

[0031] A5) Stabilize microtubules;

[0032] A6) Maintain the structure of the Golgi apparatus;

[0033] A7) Promote the repair of spinal cord injury.

[0034] In the above application, the CLASP2 protein phosphorylation may specifically be the serine phosphorylation at position 1025 of the CLASP2 protein.

[0035] In the above application, the cells or tissues for repairing spinal cord injury may be neurons, oligodendrocytes and / or myelin sheaths.

[0036] In the above application, the spinal cord injury may be a spinal cord transection injury.

[0037] The Gene ID of any of the above-mentioned CLASP2 proteins may specifically be 76499.

[0038] Any of the above applications are used for non-disease diagnosis and treatment.

[0039] The inventors of the present application constructed a mouse model of complete spinal cord transection and verified the effectiveness of curcumin in promoting nerve repair after spinal cord injury. Using a method combining bioinformatics and phosphorylation proteomics technology, the protein that curcumin promotes spinal cord injury repair is CLASP2 protein. Molecular biology technology verified that curcumin promotes functional repair after spinal cord transection injury by increasing the phosphorylation of CLASP2 protein to regulate microtubule stability. It can be seen that curcumin promotes motor function repair after spinal cord injury in mice by promoting CLASP2 phosphorylation to regulate microtubule dynamics, that is, curcumin promotes spinal cord injury repair by targeting CLASP2 protein phosphorylation sites. CLASP2 protein or a substance that can increase CLASP2 protein phosphorylation in vivo can be used as a drug to promote spinal cord injury repair. The present invention has important application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 Curcumin effectively improves the recovery of motor function and reduces fibrous scar formation in the mouse model of complete spinal cord transection. A is a representative picture of the hind limb gait of mice in the control group, curcumin-medium dose group and curcumin-high dose group. B is the BMS score of the hind limb of mice in the control group, curcumin-medium dose group and curcumin-high dose group; * indicates P<0.05, ** indicates P<0.01, and *** indicates P<0.001. C is the deposition of fibrous scars in the injured area of ​​mice in the control group, curcumin-medium dose group and curcumin-high dose group, which were immunostained with chicken anti-GFAP (green) antibody and mouse anti-Lamini antibody (red), and the nuclei were stained with DAPI. D is the quantitative analysis of Lamini in C; * is P<0.05, ** is P<0.01, and ns is P>0.05. E is the quantitative analysis of GFAP in C; ns is P>0.05. F shows the production of MBP in the caudal area of ​​the mouse injury area in the control group, curcumin-medium dose group, and curcumin-high dose group. Immunostaining was performed using chicken anti-GFAP (green) antibody and mouse anti-MBP antibody (red), and the nuclei were stained with DAPI. G shows the quantitative analysis of F; * is P<0.05, ns is P>0.05. The scale bar is 100 μm, the scale bar of the small figure on the right is 20 μm, the error bar is the mean ± standard deviation, n=3, and the one-way analysis of variance (ANOVA) test was used.

[0041] Figure 2This is a phosphorylation proteomic analysis and enrichment analysis experiment of curcumin in promoting spinal cord injury repair. Among them, A is the normalized cluster analysis of phosphorylated peptides in the control group and curcumin group after 4 weeks of spinal cord injury. B is the volcano map of the differential phosphorylation sites in the curcumin group compared with the control group after 4 weeks of spinal cord injury, in which red is the phosphorylation-upregulated peptides and yellow is the phosphorylation-downregulated peptides. C is a Venn diagram used to represent the differential peptides upregulated by curcumin and Taxol. D is the normalized cluster analysis of phosphorylated peptides in the control group and Taxol group after 4 weeks of spinal cord injury. E is the volcano map of the phosphorylation differential sites in the Taxol group compared with the control group after 4 weeks of spinal cord injury, in which purple is the phosphorylation-upregulated peptides and pink is the phosphorylation-downregulated peptides. F is a Venn diagram used to represent the differential peptides downregulated by curcumin and Taxol. G is the enrichment analysis of the proteins to which the phosphorylated peptides of curcumin in the treatment of spinal cord injury belong based on cellular components, biological processes and molecular functions; the screening range is p<0.05. H is a chord diagram used to represent the correlation between the most frequently occurring phosphorylated regulatory proteins in GO and biological processes; in the figure, aj corresponds to the "negative regulation of microtubule depolymerization" to "negative regulation of microtubule polymerization or depolymerization" of BP in G. I is a Western blotting experiment verifying that curcumin upregulates CLASP2 phosphorylation. J is a quantitative analysis of the results of I; * is P<0.05, ns is P>0.05. Data are mean ± standard deviation, n=3, and one-way analysis of variance (ANOVA) test was used.

[0042] Figure 3Serine phosphorylation of CLASP2-1025 can regulate microtubule dynamics and maintain the normal structure of the Golgi apparatus. CLASP2-WT, CLASP2-S1025-A or CLASP2-S1025-D were transfected into Hela cells to obtain transfected cells. A shows that transfected cells were stained with rabbit anti-GFP antibody (green) and mouse anti-EB1 antibody (red), and the cell nuclei were stained with DAPI; the scale bar is 20 μm. B is a statistical analysis of the fluorescence intensity of BE1 at 5 μm from the cell edge in A, and the data are mean ± standard deviation; the number of cells in the control group (CLASP2-WT) is n=31, the number of cells in the phosphorylation group (CLASP2-S1025-D) is n=34, and the number of cells in the dephosphorylation group (CLASP2-S1025-A) is n=33; there are significant differences between the control group and the phosphorylation group and the dephosphorylation group; **** is P<0.0001, ns is P>0.05, and the one-way analysis of variance (ANOVA) test is used. C is a transfected cell stained with rabbit anti-GFP antibody (green) and mouse anti-Acetyl-Tubulin antibody (red), and the cell nucleus is stained with DAPI. The scale bar is 20 μm. D is the statistical analysis of the mean fluorescence intensity of Acetyl-Tubulin in C, and the data are mean ± standard deviation; the number of cells in the control group (CLASP2-WT) is n=39, the number of cells in the phosphorylation group (CLASP2-S1025-D) is n=46, and the number of cells in the dephosphorylation group (CLASP2-S1025-A) is n=46; the control group is different from the phosphorylation group and the dephosphorylation group, and there is no difference between the phosphorylation group and the dephosphorylation group; * is P<0.05, ns is P>0.05, and the one-way analysis of variance (ANOVA) test is used. E is the transfected cells stained with rabbit anti-GFP antibody (green) and mouse anti-GM130 antibody (red), and the cell nuclei are stained with DAPI. The scale bar is 20μm. F is the statistical analysis of the average fluorescence intensity of GM130 in E, and the data are presented as mean ± standard deviation; the number of cells in the control group (CLASP2-WT) is n=55, the number of cells in the phosphorylation group (CLASP2-S1025-D) is n=55, and the number of cells in the dephosphorylation group (CLASP2-S1025-A) is n=68; there are significant differences between the phosphorylation group and the control group and the dephosphorylation group; **** is P<0.0001, ns is P>0.05, and one-way analysis of variance (ANOVA) test was used.

[0043] Figure 4 Single-cell transcriptome sequencing was used to analyze the transcription level of the Clasp2 gene in 21 types of cells. DETAILED DESCRIPTION

[0044] The present invention is further described in detail below in conjunction with specific embodiments, and the examples provided are only for illustrating the present invention, rather than for limiting the scope of the present invention. The examples provided below can be used as a guide for further improvements by those of ordinary skill in the art, and do not constitute a limitation of the present invention in any way.

[0045] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0046] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0047] Avertin, paclitaxel, sodium vanadate, sodium pyrophosphate, β-glycerophosphate, phosphatase inhibitors, sodium deoxycholate, glutathione, trypsin, mounting medium, acetylated-tubulin antibody, GFP antibody, α-tubulin antibody and DAPI are all products of Sigma-Aldrich, with catalog numbers T48402-25G, P3456, S6508-10G, 7722-88-5, 154804-51-0, 524633, V900388-50G, 70-18-8, R00950, P36930-2, T6793, G1546, 28718-90-3 and T9026, respectively. The concentration of acetylated-tubulin antibody was 1:8000 for IF and 1:50000 for WB; the concentration of GFP antibody was 1:500 for IF; and the concentration of α-tubulin antibody was 1:10000.

[0048] Penicillin sodium is a product of FeiyuBIO, with the product number FY20311.

[0049] Curcumin, Tris, Tween 20 and 30% acrylamide are all products of Solarbio, with product numbers C7090, T8060, T8220 and A1010 respectively.

[0050] Sodium hydroxide, disodium hydrogen phosphate, sodium dihydrogen phosphate, sodium chloride, paraformaldehyde, sucrose, hydrogen chloride, EDTA, sodium fluoride, acetic acid, sodium acetate, zinc acetate, ammonium persulfate and methanol are all products of SCR Company, with the product numbers being 1310-73-2, 7558-79-4, 13472-35-0, 7647-14-5, 30525-89-4, 57-50-1, 7647-01-0, 60-00-4, 7681-49-4, 64-19-7, 127-09-3, 5970-45-6, 7727-54-0 and 67-56-1, respectively.

[0051] Triton-X-100 is a product of Aladdin, and its product number is T109026.

[0052] The OCT embedding medium is a product of Sakura Company, with the product number 4583.

[0053] NP-40 is a product of Thermo Scientific, with the catalog number being 28324.

[0054] The protease inhibitor is a product of Roche with the product number CO-RO.

[0055] Phos-tag agarose is a product of Wako Company, with the product number 302-93561.

[0056] Skimmed milk powder is a product of Beyotime Company, item number P0216.

[0057] The ECL kit is a product of PerkinElmer, with the product number being 122799-10.

[0058] TEMED and bovine serum albumin are products of AMRESCO, with catalog numbers 110-18-9 and 9048-46-8, respectively.

[0059] Fetal bovine serum is a product of VivaCell, with the product number C04001-050.

[0060] The penicillin-streptomycin double antibody mixture is a product of Pricella, with the product number PB180120.

[0061] Poly-lysine is a product of Yeasen Company, with the product number 60716ES08.

[0062] The exposure solution is a product of Millipore, with the product number 345789.

[0063] Laminin antibody, GFAP antibody and NFH antibody are all products of Abcam, with catalog numbers ab133645, ab7260 and ab207176.

[0064] The concentration of laminin antibody was 1:1000 for IF; the concentration of GFAP antibody was 1:1000 for IF; the concentration of NFH antibody was 1:1000 for IF.

[0065] The MBP antibody is a product of SANTACRUZ, with the catalog number sc-66064. The concentration of the MBP antibody is IF 1:100.

[0066] EB1 antibody and GM130 antibody are both products of BD, with catalog numbers 610535 and 610822 respectively. The concentration of EB1 antibody is IF 1:500, and the concentration of GM130 antibody is IF 1:500.

[0067] CLASP2 antibody is a product of CST, with the catalog number 14629s. The concentration of CLASP2 antibody used in WB is 1:1000.

[0068] goat HRP-conjugated anti-mouse IgG is a product of CWBIO, with the product number CW0102S. The concentration of goat HRP-conjugated anti-mouse IgG used in WB is 1:1000.

[0069] goatAlexa Fluor 555-conjugated anti-rabbit, goatAlexa Fluor 488-conjugated anti-mouse and goatAlexa Fluor 488-conjugated anti-chicken are all products of Invitrogen, with catalog numbers A27039, A11001 and A11039 respectively. The concentrations of goatAlexa Fluor 555-conjugated anti-rabbit, goatAlexa Fluor 488-conjugated anti-mouse and goatAlexaFluor 488-conjugated anti-chicken are all IF 1:500.

[0070] In the following examples, the Gene ID of the CLASP2 protein is 76499; the Genebank number of the Clasp2 gene is NM_001286602.

[0071] Example

[0072] 1. Experimental Materials

[0073] 1. Experimental Animals

[0074] C57BL / 6 mice were purchased from Sibeifu Biotechnology Co., Ltd. C57BL / 6 mice are referred to as mice.

[0075] C57BL / 6 mice were bred under SPF (specific pathogen free) conditions at the Experimental Animal Center of the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

[0076] 2. Construction of a mouse model of complete spinal cord transection

[0077] In order to detect whether curcumin can promote the regeneration of spinal axons, the inventors of the present application prepared a mouse model of complete spinal cord transection. The specific steps are as follows: after the mouse is anesthetized with Avertin (the dosage is 400 mg / kg), 1mm of the T7-T8 spinal cord is surgically removed; the mouse is then placed on a warm mat until it is fully awake, and penicillin is injected intramuscularly to prevent infection (the penicillin injection dose is 1100-2200U / kg); after the spinal cord injury surgery, penicillin is administered once a day for 3 days; during the experiment, the bladder is artificially emptied twice a day; and a mouse model of complete spinal cord transection is obtained.

[0078] 3. Cell culture and transfection

[0079] HeLa cells were cultured in DMEM medium containing 10% (v / v) fetal bovine serum and 1% (v / v) penicillin-streptomycin double antibody mixture at 37°C and 5% CO2.

[0080] All cells were frozen at -80°C in a mixture of 70% DMEM medium + 20% FBS + 10% DMSO and transferred to liquid nitrogen for storage.

[0081] When the cells grow to 60%-80% confluence, the cell medium is changed and transfection is performed. The plasmid is transfected according to the instructions of Lipofectamine 2000. Immunofluorescence staining is performed 24 hours after transfection.

[0082] 4. Construction of plasmid

[0083] (1) The small DNA fragment between the restriction endonucleases EcoRI and SalI in the pEGFP-C1 vector (purchased from Clontech, Catalog #6084-1) was replaced with the Clasp2 gene sequence to obtain the pEGFP-C1-CLASP2-WT plasmid (or CLASP2-WT). The pEGFP-C1-CLASP2-WT plasmid expresses the CLASP2 protein.

[0084] (2) Using the pEGFP-C1-CLASP2-WT plasmid as a template, the pEGFP-C1-CLASP2-S1025A plasmid (or CLASP2-S1025-A) and

[0085] pEGFP-C1-CLASP2-S1025D plasmid (or referred to as CLASP2-S1025-D).

[0086] The pEGFP-C1-CLASP2-S1025A plasmid expresses the CLASP2-S1025A protein (or CLASP2-S1025-A protein). Compared with the CLASP2 protein, the only difference of the CLASP2-S1025A protein is that the serine at position 1025 from the N-terminus is mutated to alanine.

[0087] The pEGFP-C1-CLASP2-S1025D plasmid expresses the CLASP2-S1025D protein (or CLASP2-S1025-D protein). Compared with the CLASP2 protein, the only difference of the CLASP2-S1025D protein is that the serine at position 1025 from the N-terminus is mutated to aspartic acid.

[0088] 2. Experimental Methods

[0089] The effectiveness of curcumin in treating spinal cord injury model mice was evaluated by measuring the contents of laminin (LN) and myelin basic protein (MBP) in the spinal cord complete transection mouse model through behavioral function scoring and immunofluorescence staining. Bioinformatics techniques such as phosphorylation proteomics, gene ontology (GO) and enrichment analysis were used to analyze and screen the proteins that curcumin promotes neural repair after spinal cord injury, and the results were verified by phosphorylation enrichment analysis experiments. Cell immunofluorescence staining experiments were used to verify the effect of CLASP2 phosphorylation on microtubule stability. Based on single-cell transcriptomics data, the transcription level of Clasp2 in cells related to spinal cord injury repair was analyzed, and the microtubule sedimentation experiment was further used to verify that curcumin promotes microtubule stability after spinal cord injury in mice.

[0090] The details are as follows:

[0091] 1. Behavioral analysis

[0092] The Basso Mouse Scale (BMS) (DM Basso, LC Fisher, AJ Anderson, LB Jakeman, DM Mctigue, PG Popovich. Basso Mouse Scale for locomotion detects differences in recovery after spinal cord injury in five common mouse strains. Journal of neurotrauma. 2006) was used to evaluate the recovery of motor function in the hind limbs of injured mice. Two researchers independently observed and scored each time, once a week, and the final score was averaged. The mice were placed in the center of a rectangular transparent box (60 cm long, 30 cm wide, and 10 cm high) with a non-slip mat to evaluate the motor function of the mouse trunk, tail, and hind limbs. Before each evaluation, check whether the mouse perineum, hind limbs, tail, and feet were infected.

[0093] 2. Mass spectrometry analysis

[0094] After spinal cord transection injury in mice, PBS (1×PBS, 300μL / day), curcumin (dose 4mg / day) or paclitaxel (256ng / day) were intraperitoneally injected for two weeks; samples from the injured part of the mice were then collected to determine the changes in phosphorylated modified proteins in the injured part of the spinal cord after treatment. The spinal cord was homogenized with 4% SDC solution (solvent pH7.5, 100mM Tris buffer) and the supernatant was obtained after centrifugation at 4°C and 12000rpm for 10 minutes. The supernatant protein concentration was 1.7mg / mL and was used for phosphorylation mass spectrometry analysis.

[0095] 3. Single-cell RNA-seq data analysis

[0096] The single-cell RNA sequencing data of spinal cord injury in mice was derived from the Gene Expression Omnibus (GEO) database. The data of the group with functional recovery of spinal cord injury in mice after 2 weeks of medication were selected from the above data set for reanalysis. The RNA single-cell sequencing data was filtered. The filtered data was normalized and standardized using the "NormalizeData" and "ScaleData" functions, and the highly variable genes were screened out for downstream analysis using the "FindVariableFeature" function. The standardized data was clustered using the "Umap" function. Combining the results of automatic annotation and manual annotation of the R package, the genes of the obtained cell cluster markers were verified according to the "FindAllMarkers" function, and each cell cluster was annotated based on the results of the two. The differential gene visualization applied the violin plot drawn by the "VlnPlot" function. The bubble chart of gene expression levels in different cell categories was drawn using the "ggplot2" function.

[0097] 4. Bioinformatics analysis

[0098] The identified phosphorylated peptides (including CLASP2-S1025) were included in the analysis when they were present in all three replicates and had an ion intensity score greater than zero. The ion intensity values ​​were globally normalized relative to the overall abundance of total protein in each replicate, and the normalized values ​​were log2 transformed for downstream bioinformatics analysis. Heat maps and chord diagrams were drawn by the bioinformatics platform (https: / / www.bioinformatics.com.cn / ) using gplots and chordDiagram. Volcano plots were drawn when p<0.05 and log2FC≥1. GO (Gene Ontology) analysis was drawn using the R package clusterProfile (version 3.14.3).

[0099] 5. Immunofluorescence staining

[0100] Hela cells were used for immunofluorescence staining experiments. The specific steps are as follows:

[0101] (1) HeLa cells were fixed in -20°C methanol for 5 minutes or in pre-warmed 4% PFA at 37°C for 15 minutes.

[0102] (2) Wash with PBST (0.1% TritonX-100 diluted in PBS buffer) three times, 5 minutes each time; then block with 1% BSA (3 g BSA diluted in 100 mL 1×PBST) for 30 minutes at room temperature;

[0103] (3) Incubate with primary antibody at room temperature for 1 hour and wash with PBST three times, 5 minutes each time;

[0104] (4) Add fluorescently labeled secondary antibody, incubate at room temperature in the dark for 1 hour, and wash three times with PBST for 5 minutes each time.

[0105] (5) Add 3 μL of anti-fluorescence quencher on the glass slide, place the glass disc upside down on the glass slide, leave it at room temperature for more than 30 minutes, and then observe and photograph it under a fluorescence microscope.

[0106] Use spinal cord tissue for immunofluorescence staining experiments. The specific steps are as follows:

[0107] (1) The spinal cord of mice was removed after perfusion with 4% PBS at 4°C. The spinal cord tissue was fixed with 4% PFA overnight, the PFA was discarded, and 30% sucrose was poured in for dehydration. After the spinal cord was immersed in the bottom of the sucrose, it was taken out and trimmed to a length of about 1 cm. It was placed in an embedding box with OCT compound added. The spinal cord tissue was adjusted to ensure that the dorsal side was upward and the ventral side was downward. The head and tail sides were marked. After embedding, it was placed in a -80°C freezer for 2 hours and sliced. The thickness of the spinal cord tissue slices was 10 μm. After slicing, it was stored at -20°C for immunofluorescence staining.

[0108] (2) Use an immunohistochemistry pen to draw a circle around the spinal cord tissue and wash the frozen sections with PBST three times, 5 minutes each time.

[0109] (3) Add an appropriate amount of 5% BSA for blocking and block at room temperature for 30 minutes in a light-proof humid box.

[0110] (4) Discard 5% BSA, add primary antibody, incubate at 4°C overnight, and wash three times with PBST, 5 minutes each time.

[0111] (5) Add secondary antibody, incubate at room temperature in the dark for 1 hour, and wash three times with PBST, 5 minutes each time.

[0112] (6) Add an appropriate amount of mounting medium onto the slide and seal it with a coverslip. Then observe and photograph using an inverted fluorescence microscope.

[0113] 6. Microtube sedimentation experiment

[0114] (1) Mice were perfused with PBS buffer at room temperature, and tissues were collected from the 2 mm area before and after the injury.

[0115] (2) After completing step (1), add microtubule stabilization buffer (solutes and their concentrations are 2M glycerol, 5mM MgCl2, 2mM EGTA, 100mM NaF, 0.1% NP-40 and 1% PIC, and the solvent is pH 6.8, 100mM PIPES) to the tissue at a ratio of 1% (w / v), grind at room temperature to obtain a total lysate.

[0116] (3) After completing step (2), add 2×sample buffer (solvent is pH 6.8, 50 mM Tris-HCl buffer, solute and its concentration are 100 mM DTT, 2% (w / v) SDS, 0.1% (w / v) bromodimethicone blue and 10% (v / v) glycerol) to 100 μL total lysate, boil for 15 minutes, centrifuge at 15000 rpm for 15 minutes, and collect the supernatant and precipitate.

[0117] (4) After completing step (3), add 2×sample buffer to the supernatant collected in step (3) and boil for 15 minutes. Add 100 μL of 10% SDS solution (solvent is water) to the precipitate collected in step (3) to resuspend, boil at 98°C for 30 minutes, and then add 2×sample buffer. The two together constitute the total protein lysate.

[0118] The sample volume was converted according to the concentration of the total protein lysate in each group so that the total amount of protein in each group was the same, and then detected by western blot.

[0119] 7. Curcumin regulates protein phosphate enrichment experiment

[0120] (1) The mouse spinal cord was added to phosphorylated protein lysis buffer (solvent was pH 6.8, 50 mM Tris-HCl buffer, solute and its concentration were 150 M m NaCl, 1% (v / v) NP-40, 0.25% (w / v) SDC, 1 mM EDTA, 1% PIC, 1 mM Na3VO4, 1 mM NaF and 10% (v / v) phosphatase inhibitor) at a ratio of 1:10 (w / v), and lysed for 30 minutes; then centrifuged at 12000 rpm for 20 minutes to obtain the mouse spinal cord supernatant.

[0121] (2) Use a 5 mm insulin needle to pierce the bottom of the 500 μL centrifuge tube to form a sleeve with the 1.5 mL centrifuge tube.

[0122] (3) After completing step (2), the phosphorylated enriched magnetic beads and the mouse spinal cord supernatant were thoroughly mixed, 30 μL was taken into a cannula, centrifuged at 4°C, 2000g for 1 minute, and the supernatant was discarded. 30 μL of Sol.C buffer (solute and its concentration were 20 μmol / L Zn(CH3COO)2, solvent was Sol.B buffer, pH value was 7.5) was added to the magnetic beads, incubated for 5 minutes, centrifuged at 4°C, 2000g for 1 minute, and the filtrate was discarded.

[0123] (4) Repeat the following steps three times, each time with the following steps: add 40 μL of Sol.B buffer (solute and its concentration are 0.10 mol / L Tris-CH3COOH and 1.0 mol / L CH3COONa, solvent is Sol.Abuffer, pH value is 7.5; Sol.Abuffer is prepared by dissolving 12.1 g of Tris in 70 mL of deionized water, then adjusting the pH value to 7.5 with 3 M acetic acid, and then diluting to 100 mL with deionized water) to the magnetic beads, wash for 5 minutes, centrifuge at 4°C and 2000 g for 1 minute, and discard the filtrate.

[0124] (5) After completing step (4), add 10 μL of protein lysate (the total amount of protein in the lysate is less than 15 μg) to the magnetic beads, incubate for 5 minutes, centrifuge at 4°C and 2000 g for 1 minute, and discard the filtrate. Then add 100 μL of Sol.B buffer to the magnetic beads, wash for 5 minutes, centrifuge at 4°C and 2000 g for 1 minute, and discard the filtrate.

[0125] (6) After completing step (5), add 500 μL of deionized water and transfer the magnetic beads to a 1.5 mL centrifuge tube. Centrifuge at 4°C and 2000 g for 1 minute and discard the supernatant. Add 10 μL of 6× loading buffer to the magnetic beads and boil at 98°C for 5 minutes to obtain the sample.

[0126] (7) After completing step (6), detect the samples by western blot.

[0127] 8. Immunoblotting

[0128] (1) Prepare SDS-PAGE gel for protein electrophoresis, place it in the electrophoresis tank, pour in electrophoresis buffer, and take an appropriate amount of the cooked sample for loading. Concentrate the protein bands at 80V and run the electrophoresis for 30 minutes; after the protein enters the separation gel, increase the voltage to 120V and run the electrophoresis for 1.5 hours to separate the protein bands.

[0129] (2) Take out the protein gel and soak it, the filter paper and the PVDF membrane in the semi-dry transfer buffer for more than 3 minutes. Use a semi-dry transfer instrument to transfer the membrane, placing the filter paper-protein gel-PVDF membrane-filter paper in order from the negative electrode to the positive electrode, without any bubbles between the layers. Cover the positive electrode and transfer the membrane at a constant current of 190mA for an appropriate time.

[0130] (3) After the transfer is completed, remove the PVDF membrane and soak it in TBST for 3-5 minutes to wash away the residual transfer solution.

[0131] (4) Place the membrane in a blocking solution containing 5% skimmed milk powder and block at room temperature for 1 hour.

[0132] (5) Dilute the primary antibody with blocking solution and incubate the primary antibody at room temperature for 1.5 hours. Keep the antibody incubation box moist, or place it in a cold room at 4°C for overnight incubation. Wash the membrane three times with TBST, 5 minutes each time.

[0133] (6) Dilute the secondary antibody with blocking solution and incubate in a wet box at room temperature for 1 hour. Wash the membrane three times with TBST, 5 minutes each time.

[0134] (7) Take equal volumes of ECL solution A and solution B, mix them, add them onto the membrane, let it sit for about 10 seconds, and scan and image using a chemiluminescence imaging system.

[0135] 9. Data Statistical Analysis

[0136] Data were analyzed using GraphPad Prism 8.0 software and expressed as mean ± standard deviation (SEM). Unpaired t-test was used for two groups, and one-way analysis of variance (ANOVA) was used for multiple groups. * indicates P < 0.05, ** indicates P < 0.01, and *** indicates P < 0.001, all of which were considered statistically significant.

[0137] 3. Experimental Results

[0138] 1. Curcumin promotes the recovery of motor function of the hind limbs of mice with spinal cord injury, reduces fibrous scars and promotes myelination

[0139] There are many types of animal models used in the study of spinal cord injury repair, including different models such as contusion, crush injury and chemical injury. Compared with the above-mentioned trauma models, the complete spinal cord transection injury model excludes the possibility of axonal sprouting and can truly reflect the regeneration of axons. In order to better study the effect of curcumin on the promotion of axonal regeneration after spinal cord injury, the inventors of the present application constructed a complete spinal cord transection mouse model. Mice were given 300 μL 1×PBS (control group, i.e., Control group), 100 mg curcumin / kg (curcumin-medium dose group, i.e., CM-M group) or 200 mg curcumin / kg (curcumin-high dose group, i.e., CM-H group) by intraperitoneal injection every day for 56 days (8 weeks). The results are as follows: The motor function score of mice after spinal cord injury (Basso Mouse Scale, BMS) was used to evaluate the recovery of motor function of the hind limbs of injured mice; compared with the control group, the motor function of the hind limbs of mice in the curcumin-medium dose group and the curcumin-high dose group was significantly restored ( Figure 1 A and B), the mice frequently planted their hind limbs, but the coordination was poor, and the joints rotated when the soles touched the ground and lifted up, that is, the hind limbs of the mice turned over, supported the ground and moved forward; compared with the control group, the main component of glial scar GFAP in the curcumin-medium dose group and curcumin-high dose group did not decrease ( Figure 1 C and E), the main component of fibrous scar LN decreased significantly ( Figure 1C and D), the myelin component MBP of mature central neurons increased significantly ( Figure 1 F and G); and the effects of motor function restoration and fibrous scar reduction were both better with the high dose than with the medium dose.

[0140] The above results indicate that curcumin can promote the recovery of motor function in the spinal cord complete transection mouse model, reduce the area of ​​fibrous scars in the spinal cord injury area, and promote the formation of myelin sheaths of mature neurons after spinal cord injury.

[0141] 2. Curcumin promotes spinal cord injury repair by increasing CLASP2 phosphorylation

[0142] Phosphoproteomics and bioinformatics techniques were used to analyze the proteins and biological processes related to curcumin promoting the repair of spinal cord injury. The details are as follows:

[0143] Hierarchical cluster analysis was performed on the phosphorylated differential peptide data. The results showed that the phosphorylated peptide modifications were significantly different between the spinal cord transection mouse model injury group, the spinal cord transection mouse model + curcumin group, and the spinal cord transection mouse model + paclitaxel group, but the differences within the groups were small ( Figure 2 A and D). This shows that the sample preparation is qualified.

[0144] Based on the results of phosphorylation mass spectrometry, a volcano plot was used to visualize the differentially phosphorylated peptides. The results showed that compared with the spinal cord transection mouse model injury group, the spinal cord transection mouse model + curcumin group regulated 46 phosphorylation up-regulated peptides and 31 phosphorylation down-regulated peptides ( Figure 2 Middle B); In the spinal cord complete transection mouse model + paclitaxel group, 90 peptides were upregulated by phosphorylation, and 15 peptides were downregulated by phosphorylation ( Figure 2 Middle E); For the intersection of the phosphorylated modified proteins of curcumin and paclitaxel in the treatment of spinal cord injury, it was found that there were 13 common up-regulated peptides ( Figure 2 Middle C), there are 3 down-regulated peptides ( Figure 2 (F) Therefore, there are 16 common peptides regulated by curcumin and paclitaxel in the treatment of spinal cord injury, belonging to 12 proteins, as shown in Table 1.

[0145] Table 1

[0146]

[0147]

[0148] GO enrichment analysis was further performed on the phosphorylated peptides regulated by curcumin. The results showed that the biological processes (BP) regulated by curcumin included regulation of microtubule polymerization or depolymerization (GO:0031110), negative regulation of cytoskeleton organization (GO:0051494), etc.; the involved cellular components (CC) included microtubules (GO:0005874), main axons (GO:0044304), etc.; molecular functions (MF) included tubulin binding (GO:0015631), microtubule plus end binding (GO:0051010), etc.; GO biological processes mainly regulated proteins such as CLASP1, CLASP2 and SPECC1L; mainly cytoskeleton regulation, of which the core was the regulation of microtubule stability ( Figure 2 G and H) and Table 2.

[0149] Taxol, a microtubule stabilizing drug, was selected as a positive control drug for spinal cord injury, and a hierarchical clustering analysis was performed on the differential peptides regulated by Taxol. The results showed that compared with the control group, Taxol regulated 90 up-regulated phosphorylation peptides and 15 down-regulated phosphorylation peptides; GO biological processes mainly regulated proteins such as CAMSAP2, CLASP1, CLASP2, MAP1A, MAP2, and SPECC1L. The proteins co-regulated by curcumin and Taxol were intersected with the proteins co-regulated by the GO biological processes of the two, and CLASP2 was found to be a common regulatory protein.

[0150] Table 2. GO enrichment analysis results of phosphorylated differentially expressed proteins in curcumin-induced spinal cord injury repair

[0151]

[0152]

[0153] The results of phosphorylated protein enrichment showed that curcumin could upregulate the phosphorylation modification of CLASP2, that is, curcumin could promote the phosphorylation of CLASP2 ( Figure 2 I and J), which is consistent with the trend of CLASP2 phosphorylation data in Table 1.

[0154] 3. CLASP2 phosphorylation can regulate microtubule homeostasis and maintain the normal morphology of the Golgi apparatus

[0155] In order to study the effect of serine phosphorylation at the CLASP2-S1025 site on microtubule dynamics, plasmids for serine phosphorylation and dephosphorylation at this site were constructed and transfected into Hela cells for immunofluorescence staining to verify the effect of CLASP2 phosphorylation on microtubule stability. Given that CLASP2 interacts with EB1 to regulate microtubule dynamics, the amount of EB1 can reflect the dynamics of microtubules, thereby affecting functions such as cell polarity and migration ability. Therefore, EB1 is used to characterize microtubule stability. The results showed that the phosphomimetic mutant of CLASP2 (CLASP2-S1025D) can reduce the aggregation of EB1 at the cell edge compared to the dephosphorylated mutant (CLASP2-S1025A). Figure 3 A and B), but phosphorylation did not affect the number of acetylated microtubules ( Figure 3 C and D).

[0156] By observing the results of immunofluorescence staining, it was found that phosphorylation mutations would lead to changes in the Golgi structure, and GM130 was selected to characterize the Golgi structure. The results showed that the dephosphorylation mutant (S1025A) had a decreased ability to maintain the Golgi structure compared with the pseudophosphorylation mutant (S1025D). Figure 3 E and F).

[0157] It can be seen that curcumin-regulated CLASP2-S1025 site phosphorylation can regulate microtubule dynamics and maintain the normal morphology of the Golgi apparatus.

[0158] 4. The main cell types involved in spinal cord injury repair by CLASP2 are neurons and oligodendrocytes

[0159] Single-cell RNA-Seq can be used to explore the transcriptional level of a single cell. By analyzing a single cell, it solves the problem that traditional omics technology can only obtain cell mean information. Therefore, it can reflect the transcriptional differences between cells and characterize the disease according to the characteristics of different types of cells. Therefore, applying single-cell sequencing technology to spinal cord injury can reveal the specific cell types in which CLASP2 is involved in the repair of spinal cord injury.

[0160] The cell types involved in spinal cord injury repair were obtained from the spinal cord injury single cell dataset. The cascade map was derived using the UMAP method, and 21 major cell clusters were finally identified. The gene transcription level of Clasp2 gene in the above 21 types of cells was further analyzed. The results showed (see Figure 4 ), CLASP2 is highly expressed in neurons, oligodendrocytes and oligodendrocyte precursor cells. Single-cell sequencing results show that the main cells involved in the repair of spinal cord injury by CLASP2 are neurons and oligodendrocytes; curcumin can play a repairing role in neurons and myelin sheaths of spinal cord injury by regulating the phosphorylation of CLASP2.

[0161] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, and without the need to carry out unnecessary experimental conditions, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principles of the present invention, the application is intended to include any changes, uses or improvements to the present invention, including departure from the disclosed scope in the application, and changes made with conventional techniques known in the art.

Claims

1. Use of a substance for detecting CLASP2 protein phosphorylation, a substance for increasing CLASP2 protein phosphorylation, and / or a substance for detecting CLASP2 protein expression and / or activity in the preparation of a product for selecting a drug for promoting spinal cord injury repair.

2. The use according to claim 1, characterized in that: The drug is curcumin.

3. Use of CLASP2 protein or a substance capable of increasing the phosphorylation of CLASP2 protein in vivo in the preparation of a drug for promoting the repair of spinal cord injury.

4. The use according to any one of claims 1 to 3, characterized in that: The promotion of spinal cord injury repair is manifested as at least one of A1)-A6): A1) Promote functional recovery after spinal cord injury; A2) Promote the recovery of motor function after spinal cord injury; A3) Reduce fibrous scar formation after spinal cord injury; A4) Promotes neuron formation after spinal cord injury; A5) Stabilize microtubules; A6) Maintains the structure of the Golgi apparatus.

5. The use according to any one of claims 1 to 3, characterized in that: The cells or tissues for repairing spinal cord injury are neurons, oligodendrocytes and / or myelin sheaths.

6. The use according to any one of claims 1 to 3, characterized in that: The spinal cord injury is a spinal cord transection injury.

7. The use according to any one of claims 1 to 3, characterized in that: The CLASP2 protein phosphorylation is the serine phosphorylation at position 1025 of the CLASP2 protein.

8. A pharmaceutical composition comprising CLASP2 protein or a substance capable of increasing the phosphorylation of CLASP2 protein in vivo.

9. The pharmaceutical composition according to claim 8, characterized in that: The pharmaceutical composition further comprises curcumin and / or a pharmaceutically acceptable carrier.

10. Use of CLASP2 protein or phosphorylated CLASP2 protein, which is at least one of A1) to A7): A1) Promote functional recovery after spinal cord injury; A2) Promote the recovery of motor function after spinal cord injury; A3) Reduce fibrous scar formation after spinal cord injury; A4) Promotes neuron formation after spinal cord injury; A5) Stabilize microtubules; A6) Maintain the structure of the Golgi apparatus; A7) Promote the repair of spinal cord injury.

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