Application of agents related to expression of glutaminase 1

Abstract

The invention belongs to the technical field of biological medicine, particularly relates to application of a reagent related to expression of glutaminase 1, and provides application of a reagent for detecting the expression quantity of GLS1 in preparation of a diagnostic product for intervertebral disc degeneration due to the fact that the expression level of GLS1 protein is found to be remarkably reduced in degenerated nucleus pulposus tissue. According to the present invention, the GLS1 regulates and controls the iron ion homeostasis by promoting the expression of cysteine desulfatase so as to antagonize ferroptosis and senescence of nucleus pulposus cells and further delay the degenerative change of the intervertebral disc, such that the GLS1 is adopted as the treatment target, and the GLS1 is overexpressed by using the overexpression reagent provided by the invention so as to achieve the intervertebral disc degeneration regulation and control effect of the GLS1; therefore, the invention provides application of a reagent for overexpressing GLS1 in preparation of a product for treating intervertebral disc degeneration.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of a reagent related to expression of glutaminase 1. BACKGROUND

[0002] Low back pain has become a major cause of disability worldwide, and intervertebral disc degeneration is considered to be the most important pathophysiological mechanism of low back pain, but its pathogenesis is not fully clear. Studies have shown that metabolic diseases are closely related to intervertebral disc degeneration, such as obesity and diabetes, which are considered to be the main risk factors for intervertebral disc degeneration.

[0003] At present, the main methods for treating intervertebral disc degeneration in clinical practice are conservative treatment and fusion surgery, which are difficult to fundamentally repair the progression of intervertebral disc degeneration. Glutaminase 1 (GLS1) is a key enzyme in glutamine metabolism, which can convert glutamine into glutamic acid. Although current research has revealed that glucose metabolism and lipid metabolism are crucial for the nucleus pulposus cells of the intervertebral disc, there is still a long way to go to fully understand the nutritional metabolism mechanism of the nucleus pulposus cells, which also limits the exploration of the potential mechanism of intervertebral disc degeneration, especially the lack of exploration of the role of amino acid metabolism in intervertebral disc degeneration. Therefore, it is necessary to clarify the relationship between amino acid metabolism and intervertebral disc degeneration. SUMMARY

[0004] To solve the above problems, the application provides application of a reagent related to expression of glutaminase 1.

[0005] Application of the reagent for detecting expression amount of glutaminase 1 in preparation of a diagnostic product for intervertebral disc degeneration.

[0006] Preferably, the reagent for detecting expression amount of glutaminase 1 includes any one of a reagent used in an immunohistochemical staining method and a reagent used in an immunofluorescent staining method.

[0007] Preferably, the reagent used in the immunohistochemical staining method or the immunofluorescent staining method includes a GLS1 antibody, and the GLS1 antibody is produced by Proteintech, and the item number is 12855-1-AP.

[0008] Preferably, the reagent used in the immunohistochemical staining method or the immunofluorescent staining method further includes a goat anti-mouse / rabbit IgG.

[0009] A kit includes the reagent for detecting expression amount of glutaminase 1.

[0010] Application of a reagent for overexpressing glutaminase 1 in preparation of a therapeutic product for intervertebral disc degeneration.

[0011] Preferably, the reagent for overexpressing the glutaminase 1 comprises any one of primers shown as SEQ ID NO. 1-2.

[0012] Preferably, the therapeutic product maintains the integrity of the intervertebral disc.

[0013] Preferably, the therapeutic product reduces the degeneration of the intervertebral disc.

[0014] Compared with the prior art, the present application has the advantages of:

[0015] The present application finds that the expression level of GLS1 protein is significantly decreased in degenerated nucleus pulposus tissue, and therefore proposes the use of a reagent for detecting the expression amount of glutaminase 1 in the preparation of a diagnostic product for intervertebral disc degeneration.

[0016] The present application finds that GLS1 antagonizes the iron death and aging of nucleus pulposus cells by regulating iron homeostasis through promoting the expression of cysteine desulfurase (Nitrogen fixation 1, NFS1), thereby delaying the degenerative changes of the intervertebral disc, and therefore proposes to use GLS1 as a therapeutic target, overexpress GLS1 using the overexpression reagent provided by the present application, achieve the effect of GLS1 regulating intervertebral disc degeneration, and therefore proposes the use of a reagent for overexpressing glutaminase 1 in the preparation of a therapeutic product for intervertebral disc degeneration. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 The relationship between glutaminase GLS1 and intervertebral disc degeneration is shown, wherein A is a representative MRI image of T2 weighted sequence evaluated using the Pfirrmann grading system, B is a verification of key genes in the GSE176205 dataset, C is a representative image of GLS1 immunohistochemical staining in degenerated human nucleus pulposus tissue (first row scale = 100 pm; second row scale = 50 pm), D is the quantification of GLS1 positive cells in severely degenerated nucleus pulposus tissue (n = 10 per group), E is a representative image of H&E and SafraninO / fast green stained samples (scale = 200 pm), F is the analysis of GLS1 expression in rat nucleus pulposus tissue by immunofluorescence staining (first row scale = 100 pm; second row scale = 50 pm), statistical significance: *p < 0.05, **p < 0.01.

[0018] Figure 2Figure 6 shows GLS1 modulates ferroptosis, senescence in intervertebral disc nucleus pulposus cells, wherein A is Western blot analysis and quantification of GPX4 and SLC7A11 protein levels under GLS1 overexpression or non-overexpression conditions after TBHP (50 mM) treatment for 24 hours, B is flow cytometry analysis for detection of ROS levels in nucleus pulposus cells under GLS1 overexpression or non-overexpression conditions after TBHP (50 mM) treatment for 24 hours, C is transmission electron microscopy (TEM) images of nucleus pulposus cells treated with different treatments (scale bar = 5 pm in the first row; scale bar = 1 pm in the second row; red triangle indicates mitochondria), D is representative images of C11-BODIPY staining (scale bar = 20 pm), E is representative images of MitoSOX and MitoTracker staining in nucleus pulposus cells (scale bar = 20 pm), F is Western blot analysis for detection of p16, p21 and p53 protein levels under GLS1 overexpression or non-overexpression conditions after TBHP (50 mM) treatment for 24 hours, G is representative images of beta-galactosidase staining in nucleus pulposus cells *p < 0.05, **p < 0.01.

[0019] Figure 3 Figure 7 shows GLS1 regulates iron homeostasis by enhancing NFS1 expression, wherein A is a volcano plot displaying RNA sequencing results of rat nucleus pulposus cells after TBHP (50 mM) treatment and GLS1 overexpression, B is representative immunostaining images showing NFS1 expression in degenerated human nucleus pulposus tissue (scale bar = 100 pm in the first row; scale bar = 50 pm in the second row), C is quantification analysis of NFS1 positive cells in severely degenerated nucleus pulposus tissue (n = 10 per group), D is single-factor linear regression analysis of GLS1 and NFS1 positive cells in the same patient’s nucleus pulposus tissue, E is Western blot analysis and quantification of NFS1 protein levels under GLS1 overexpression or non-overexpression conditions after TBHP (50 mM) treatment for 24 hours, F shows representative images of GLS1 and NFS1 immunofluorescence staining in nucleus pulposus cells (scale bar = 20 pm), statistical significance is represented as *p < 0.05 and **p < 0.01.

[0020] Figure 4NFS1 and GLS1 activation slows down intervertebral disc degeneration in rats, where A is a schematic illustration of the surgical procedure, B is a representative magnetic resonance imaging image of the tail vertebrae of rats four weeks after surgery (n=5), green triangles indicate non-punctured intervertebral discs; red triangles indicate punctured intervertebral discs treated with AAV-NC; blue triangles mark punctured intervertebral discs treated with AAV-GLS1; yellow triangles highlight punctured intervertebral discs treated with AAV-NFS1, C is the Pfirrmann grading score of the tail vertebrae of rats assessed according to MRI, D is the histological images stained with H&E and Safranin O / fast green (scale bar = 200 pm), E is the quantitative histological score (n=5), F is the immunofluorescence analysis of GLS1 and NFS1 expression levels in the nucleus pulposus (NP) tissue of rat intervertebral discs, G is the quantification of F, data are expressed as mean ± standard deviation, statistical significance is indicated by *p<0.05 and **p<0.01. DETAILED DESCRIPTION

[0021] The specific embodiments of the present application are described in detail below, but it should be understood that the scope of the present application is not limited to the specific embodiments. Based on the examples in the present application, all other examples obtained by those of ordinary skill in the art without making creative efforts fall within the scope of the present application. The experimental methods described in the embodiments of the present application are conventional methods unless otherwise specified.

[0022]

[0023] 1. Clinical sample collection

[0024] The present application has collected a total of 20 clinical specimens of nucleus pulposus tissue from patients undergoing lumbar surgery in the Department of Spinal Surgery, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, with a time span from 2021 to 2023. Prior to surgery, all patients have signed the informed consent for the use of clinical specimens and clinical images. After entering the patient information, including 13 female patients and 7 male patients. According to the Pfirrmann classification of the intervertebral disc at the surgical site on the preoperative lumbar MRI sagittal T2 image, patients with Pfirrmann classification ≤ III were classified as mild degeneration group, while patients with Pfirrmann classification ≥ IV were classified as severe degeneration group. After the specimen was removed during the operation, part of it was soaked in 4% paraformaldehyde for at least 24 hours, then paraffin-embedded for H&E staining and immunohistochemical staining. Another part was taken out and placed in liquid nitrogen, then transferred to -80°C for storage for subsequent RNA extraction and detection. This part of the study has been reviewed and approved by the Ethics Committee of Sun Yat-sen Memorial Hospital of Sun Yat-sen University, with approval number SYSKY-2022-217-0121.

[0025] 2. H&E staining

[0026] (1) After fixation and decalcification, human and SD rat intervertebral disc specimens were entrusted to Wuhan Saiver Company for paraffin embedding and sectioning.

[0027] (2) Place the paraffin sections evenly on the slide rack and place them in a 60°C environment for 2 hours.

[0028] (3) After baking, the paraffin sections are immersed in three histological staining tanks containing xylene solution, each for 10 minutes.

[0029] (4) Place the sections in anhydrous ethanol, 95% alcohol, 85% alcohol, 75% alcohol, and distilled water, respectively, for 5 minutes each time.

[0030] (5) Immerse the sections in Mayer's hematoxylin solution for 1 minute, then rinse with tap water for 10 minutes.

[0031] (6) Place the sections in the eosin staining solution for 2 minutes, then rinse with distilled water for 1 minute.

[0032] (7) After natural air drying, use neutral resin solution to mount the sections and cover with a glass slide.

[0033] (8) Observe the sections under a microscope and record the experimental results, and take photos at the same time.

[0034] 3. Immunohistochemical staining

[0035] (1) Preparation of paraffin-embedded sections The conventional dewaxing and hydration steps were used, with the same details as in the "H&E staining" procedure.

[0036] (2) Antigen retrieval with pepsin The steps for antigen retrieval with pepsin were as follows: the range of the nucleus pulposus tissue was marked on the section using an immunohistochemical pen, and then pepsin digestion solution was added to ensure complete coverage of the nucleus pulposus tissue. After incubation at 37°C for 30 min, the section was washed 3 times with PBS solution for 5 min each time.

[0037] (3) Treatment step for neutralizing endogenous peroxidase: 50 μL of endogenous peroxidase blocking agent was added, and incubation was performed at room temperature for 20 min, followed by immersion in PBS solution for 5 min, repeated 3 times.

[0038] (4) Blocking step: 50 μL of goat serum was added, and blocking was performed at room temperature for 30 min.

[0039] (5) Incubation step of primary antibody: the goat serum blocking solution was removed, 50 μL of primary antibody solution was added, and then the section was placed in a wet box for incubation at 4°C overnight. The source and concentration of the primary antibody solution are shown in Table 1.

[0040] Table 1. Antibody information

[0041] Antibody name Catalogue number and company Concentration GLS1 12855-1-AP, Proteintech 1:200

[0042] (6) The next day, the primary antibody solution was recovered, and the section was washed 3 times with PBS solution for 5 min each time. Then, 50 μL of biotin-labeled goat anti-mouse / rabbit IgG (SP-9001, Zhongshanjingqiao) polymer solution was added, and incubation was performed at room temperature for 20 min. The section was again immersed in PBS solution for 5 min, repeated 3 times.

[0043] (7) 50 μL of horseradish enzyme-labeled streptavidin working solution was added at room temperature, and incubation was performed for 20 min. Then, the section was immersed in PBS solution for washing, each time for 5 min, repeated 3 times.

[0044] (8) DAB color development: 50 μL of DAB color developing solution was added, and the staining was observed under a microscope. After color development, the section was immersed in pure water to terminate the staining. Then, the section was immersed in PBS solution for 5 min, repeated 3 times.

[0045] (9) Counterstaining step: the section was immersed in modified Mayer's hematoxylin staining solution for 1 min, and then distilled water was continuously washed for 10 min.

[0046] (10) Mounting: neutral resin and a cover glass were used for mounting, and then the resin gel was naturally air-dried.

[0047] (11)Photographing and statistics: human nucleus pulposus tissue sections were photographed under a microscope, and at least 5 high-power fields were photographed. According to the ratio of the number of positive cells to the total number of cells in the high-power field, the positive cell rate of the section was obtained, and further statistical analysis was carried out. At the same time, rat nucleus pulposus tissue sections were photographed under a microscope, and at least 5 high-power fields were photographed. The cumulative optical density value (IOD) of each section was calculated using Image J software for further statistical analysis.

[0048] 4, Extraction and culture of rat primary nucleus pulposus cells

[0049] (1) Male SD rats weighing about 300 g were selected, and they were killed by excessive injection of sodium pentobarbital solution.

[0050] (2) After disinfection with conventional iodine and alcohol, the mouse tail was cut off at the root of the tail, about 6 / 7 segments of the intervertebral space of the tail vertebrae. The mouse tail skin and muscle tissue were carefully separated. Then, the intervertebral disc outer annulus fibrosus was cut open, and the nucleus pulposus tissue was exposed and quickly placed in an EP tube using DMEM medium containing 10% fetal bovine serum. Eight nucleus pulposus tissues of the intervertebral disc of each rat were obtained.

[0051] (3) The nucleus pulposus tissue in the EP tube was centrifuged at 1000 rpm for 5 min, and the precipitate was obtained and the supernatant was discarded. Then, 500 μL of 0.25% collagenase type II was added to the EP tube, and the digestion was carried out in a 37°C incubator for 2 h. After digestion, 500 μL of medium containing 10% fetal bovine serum was added, and the centrifugation was carried out again at 1000 rpm for 5 min, and the supernatant was discarded. Finally, the tissue was washed three times with PBS solution.

[0052] (4) The nucleus pulposus tissue was carefully added to the DMEM medium containing 1% anti-double antibody solution and 10% fetal bovine serum, and cultured in a constant temperature cell incubator at 37°C and 5% carbon dioxide.

[0053] 5, Construction and transfection of GLS1 overexpression plasmid

[0054] (1) The expression plasmid used in the present application was constructed and prepared by Wuhan Jin Kai Rui Company, and the specific sequence is shown in Table 2. When performing the transfection step, first add 200 μL of JetPRIME buffer to the EP tube, then add 2 μg of plasmid, shake for 10 s, then quickly centrifuge to deposit the liquid in the tube at the bottom.

[0055] (2) Then add 4 μL of JetPRIME reaction reagent, vortex for 1 s, then quickly centrifuge again, and incubate at room temperature for about 10 min.

[0056] (3) Add the mixture to the nucleus cell culture medium which has been grown for 80% area.

[0057] (4) After incubation for at least 24 hours, detect the transfection efficiency.

[0058] "OE" means overexpression.

[0059] Table 2 Plasmid sequences

[0060]

[0061] 6. Cell protein extraction

[0062] (1) Discard the cell culture medium and replace it with PBS solution to rinse the cells three times. Then, add the lysis solution mixed by RIPA lysis solution, protease inhibitor and phosphatase inhibitor at a ratio of 100:1:1. Shake the lysis solution to ensure that the cells are fully covered, and then place the sample on ice to lyse the cells for 30 min.

[0063] (2) Quickly scrape off the entire protein layer and transfer the protein suspension to a 1.5 mL EP tube. Centrifuge at 14000 g / min for 30 min at 4°C, transfer the protein-containing supernatant to a new EP tube, and place it on ice. Record the volume of the supernatant, and be careful not to touch the cell debris and sediment.

[0064] (3) Determine the protein concentration using the BCA kit.

[0065] (4) Add 1 / 4 of the recorded data volume of loading buffer to the EP tube and vortex well.

[0066] (5) Boil the sample at 100°C for 10 min, and then quickly transfer it to -80°C for storage.

[0067] 7. Western blotting

[0068] (1) Gel preparation: According to the instructions of the Yease gel preparation kit, prepare the lower gel solution and the upper gel solution according to Table 3 and Table 4.

[0069] Table 3 Required components and volumes for each piece of separation gel

[0070] Component Volume Lower separation gel buffer 4 mL Lower separation gel solution 4 mL Modified ammonium persulfate solution 100 μL

[0071] Table 4 Required components and volumes for each piece of concentrated gel

[0072] Component Volume Upper color concentrated gel buffer 1 mL Upper concentrated gel solution 1 mL Modified ammonium persulfate solution 50 μL

[0073] (2) Loading: Prepare fresh electrophoresis solution according to Table 5, add it to the inner and outer electrophoresis tanks, calculate the protein sample loading amount according to the protein concentration, and add 2.5 μL of protein marker to each end of the loading well.

[0074] Table 5 Electrophoresis liquid composition and dosage

[0075] Component Amount SWE rapid high-resolution electrophoresis buffer 1 package Deionized water 1000 mL

[0076] (3) Electrophoresis: After pressing for 10 min at 70 V, adjust to 150 V to separate proteins, about 0.5 h or so until the proteins are electrophoresed to the bottom of the gel.

[0077] (4) Electrotransfer: Wrap the electrophoresis gel with a "sandwich" made of cotton pad, filter paper and PVDF membrane to perform electrotransfer. Pay attention to avoid bubbles during the transfer process. Use 400 mA constant current for 40 min. The electrotransfer liquid is shown in Table 6.

[0078] Table 6 Formula of rapid electrotransfer liquid

[0079] Component Amount Ice-free rapid transfer membrane buffer 1 package Deionized water 800 mL Anhydrous ethanol 200 mL

[0080] (5) Blocking: After electrotransfer, transfer the PVDF membrane to 5% BSA solution and block at room temperature for 1 h.

[0081] (6) Membrane cutting: Wash the blocking solution in the PVDF membrane in TBST solution, and then cut the PVDF membrane according to the molecular weight position shown by the protein marker.

[0082] (7) Incubation: Place the strip in the primary antibody solution, and incubate overnight at 4°C. The primary antibody solution is shown in Table 7.

[0083] Table 7 Source and concentration of primary antibody solution

[0084] Antibody name Catalogue number and company Concentration GLS1 12855-1-AP, Proteintech 1:5000 NFS1 67021-1-Ig, Proteintech 1:5000 GPX4 ab125066, Abeam 1:1000 SLC7A11 26864-1-AP, Proteintech 1:1000 COL2A ab34712, Abeam 1:500 Aggrecan ab36861, Abeam 1:500 MMP3 A1202ABclonal 1:500 ADAMTS5 A2836, ABclonal 1:1000 P53 60283-2-Ig, Proteintech 1:1000 P21 ab109199, Abeam 1:1000 P16 ab51243, Abeam 1:1000 β-actin AC006, ABclonal 1:1000

[0085] (8) Incubation of secondary antibody: Take out the strip from the primary antibody dilution every other day, soak in TBST solution, and wash three times for 10 min each time. Add goat anti-rabbit secondary antibody at a concentration of 1:5000, and incubate at room temperature for 1 h. After incubation, soak in TBST solution again, and wash three times for 10 min each time.

[0086] (9) Exposure and imaging: Add ECL luminescent liquid to the strip, and take a photo in the exposure and imaging system.

[0087] (10) Statistical analysis: Perform gray value semi-quantitative analysis on the protein strip imaging picture using ImageJ.

[0088] 8. Cell senescence β-galactosidase staining

[0089] (1) Discard the cell culture solution, wash with PBS for 3 times, and then add 1 mL of fixing solution to cover the entire cell surface, and fix at room temperature for 20 min.

[0090] (2) Mix the staining solution in advance according to the following ratio: 10 μL of staining solution A + 10 μL of staining solution B + 930 μL of staining solution C + 50 μL of X-Gal solution, to obtain 1 mL of staining solution. Ensure uniform mixing of the components by rotating and shaking thoroughly.

[0091] (3) Discard the fixing solution and rinse with PBS for 3 times. Cover the cell surface with 1 mL of fresh staining solution prepared in advance, and then incubate at 37°C overnight.

[0092] (4) Discard the staining solution, rinse with PBS for 3 times, and use an optical microscope to take pictures of the sample and obtain images.

[0093] 9. Transmission electron microscopy

[0094] (1) Uniformly spread the nucleus pulposus cell suspension in a 10 cm diameter culture dish, and then add various stimulating substances after one day.

[0095] (2) Add trypsin to the culture dish, and after successful cell digestion, add an equal volume of basic medium containing 10% fetal bovine serum by volume to terminate the digestion reaction. Then, centrifuge at 1000 rpm for 5 min.

[0096] (3) Discard the supernatant, add 1 mL of electron microscope fixing solution, and fix at room temperature in the dark for 2 h, then store at 4°C, continuing to store in the dark.

[0097] (4) Transport the sample to Wuhan Saiver Biological Company at 4°C to prepare ultrathin sections.

[0098] (5) Use a transmission electron microscope to observe and image, and save the pictures

[0099] 10. Flow cytometry

[0100] (1) Uniformly spread the nucleus pulposus cell suspension in a six-well plate, and then apply various stimuli after one day.

[0101] (2) After the stimulation is completed, discard the culture medium, wash with PBS, and then add 1 mL of serum-free medium containing a concentration of 10 μm / L of reactive oxygen species probe to each well, and incubate in a 37°C cell incubator for 0.5 h.

[0102] (3) Discard the supernatant containing the reactive oxygen species probe, and wash the cells with serum-free medium 3 times to thoroughly wash the probe.

[0103] (4) Add 1 mL of trypsin to each well, and after successful cell digestion, add an equal volume of basic medium containing 10% fetal bovine serum to terminate the digestion. Then, centrifuge at 1000 rpm for 5 min.

[0104] (5) Add PBS to the cell pellet, resuspend using a vortex device, and then transfer to the flow machine for analysis. Use an excitation wavelength of 488 nm and observe the emission wavelength at 525 nm.

[0105] 11. Lipid Peroxidation Probe Staining

[0106] (1) Distribute the nucleus pulposus cell suspension evenly in the 24-well plate, and after 1 day, introduce different stimulation conditions.

[0107] (2) After the stimulation is complete, discard the culture medium, wash with PBS, and then add 1 mL of serum-free medium containing a lipid peroxidation probe at a concentration of 10 μm / L to each well. Incubate in a cell culture incubator at 37°C for 30 min.

[0108] (3) Discard the supernatant containing the active oxygen probe, and wash the cells with serum-free medium 3 times to thoroughly remove the probe. Then, observe and take images under a fluorescence microscope, and save the records.

[0109] 12. Mitochondrial Probe Staining

[0110] (1) Distribute the nucleus pulposus cell suspension evenly in the 24-well plate, and then after 1 day of culture, apply different stimulation conditions.

[0111] (2) After the stimulation is complete, discard the culture medium, wash with PBS, and then add 1 mL of serum-free medium containing a mitochondrial superoxide red fluorescent probe at a concentration of 5 μm / L to each well. Incubate in a cell culture incubator at 37°C for 30 min.

[0112] (3) Discard the supernatant containing the mitochondrial superoxide red fluorescent probe, and wash the cells with serum-free medium 3 times to thoroughly remove the probe. Then, add 1 mL of serum-free medium containing a mitochondrial green fluorescent probe at a concentration of 0.5 μm / L to each well, and incubate in a cell culture incubator at 37°C for 30 min.

[0113] (4) Discard the supernatant containing the mitochondrial green fluorescent probe, and wash the cells with serum-free medium 3 times to thoroughly remove the probe. Then, observe and take images under a fluorescence microscope.

[0114] 13. Cell Immunofluorescence Staining

[0115] (1) Distribute the evenly blown nucleus pulposus cell suspension into the 24-well plate, and after 1 day of culture, perform various types of stimulation.

[0116] (2) After the stimulation is over, the culture medium is discarded, and three washes of 3 min each are performed using a PBS solution. Subsequently, 4% paraformaldehyde is dropped at room temperature, the cells are covered and incubated for 30 min.

[0117] (3) The 4% paraformaldehyde solution is discarded, and three washes of 3 min each are performed using a PBS solution again. Next, 0.5% Triton-100 is dropped, and incubation is performed for 5 min.

[0118] (4) The 0.5% Triton-100 solution is discarded, and three washes of 3 min each are performed using a PBS solution again. Next, 10% goat serum is dropped, and blocking is performed for 30 min.

[0119] (5) The 10% goat serum is discarded, 200 μL of the primary antibody diluent is added per well, and incubation is performed overnight at 4°C.

[0120] (6) The primary antibody diluent is recovered, three washes of 3 min each are performed using a PBS solution. Then, a fluorescent secondary antibody solution at a concentration of 1:100 is added, and incubation is performed for 1 h at room temperature in the dark.

[0121] (7) The secondary antibody solution is discarded, three washes of 3 min each are performed using a PBS solution again. Next, a DAPI solution is added, incubation is performed for 10 min at room temperature, and three washes of 3 min each are performed using a PBS solution.

[0122] (8) The photographs are taken under an inverted fluorescence microscope for observation, and the imaging pictures are saved.

[0123] 14. Establishment of an SD rat needle puncture intervertebral disc degeneration model

[0124] (1) Animal source and ethical approval: All SD rats used in the present application are from the Animal Experiment Center of Sun Yat-sen University, and have obtained the ethical review approval of the Sun Yat-sen University Experimental Animal Management and Use Committee (ethical number: SYSU-IACUC-2023-000704).

[0125] (2) Experimental grouping: 10 male rats with a body weight of about 300 g and an age of about 12 weeks are selected in the present experiment. Among them, 5 rats are subjected to needle puncture to manufacture a model, and the other 5 rats are subjected to sham operation as a control group. In all experiments, the segments subjected to needle puncture are selected as the caudal vertebrae 7 / 8.

[0126] (3) Anesthesia: The rats are subjected to fasting for 12 h before anesthesia, and then general anesthesia is performed by intraperitoneal injection of 2% sodium pentobarbital (0.3 mL / 100 g).

[0127] (4) Preoperative preparation: The location of the disc between the 7th and 8th coccygeal vertebrae was confirmed by palpation of the sacrum, transverse processes and spinous processes of the coccygeal vertebrae and marked with a marker pen. Subsequently, the skin of the tail was disinfected with iodophor and 75% alcohol.

[0128] (5) Needle insertion.

[0129] (6) Postoperative care: Routine rewarming was performed after the operation to prevent hypothermia. Fasting was prohibited within 8 hours after the operation to prevent aspiration. Routine disinfection care was performed for the surgical wound.

[0130] (7) Specimen preparation: After 4 weeks of needle insertion, the disc and the adjacent vertebral bodies were retained and the paravertebral muscles were separated. After careful separation and trimming, the specimen was fixed in 4% paraformaldehyde solution and then subjected to room temperature decalcification treatment in EDTA decalcification solution for 8 weeks.

[0131] 15. Histological scoring criteria

[0132] The scoring criteria were based on five degeneration categories, with a total score ranging from 0 for a normal disc to 15 for a severely degenerated disc, and each category accounted for 3 points to evaluate the degree of disc degeneration in rats.

[0133] (1) Morphological score for nucleus pulposus tissue

[0134] Score 0: Nucleus pulposus tissue accounted for more than 75% of the disc area; Score 1: Nucleus pulposus tissue was round and accounted for 50-75% of the disc area; Score 2: Nucleus pulposus tissue accounted for 25-50% of the disc area; Score 3: Nucleus pulposus tissue accounted for less than 25% of the disc area.

[0135] (2) Cytological score for nucleus pulposus tissue

[0136] Score 0: Mainly star-shaped cells, proteoglycan matrix uniformly distributed in the periphery; Score 1: Partial star-shaped and partial round cells, with more star-shaped cells; Score 2: Mostly round cells separated by dense areas of proteoglycan matrix; Score 3: Mainly round cells separated by dense areas of proteoglycan matrix.

[0137] (3) Morphological score for annulus fibrosus

[0138] Score 0: Collagen layer is intact and no rupture occurs; Score 1: Inward protrusion of the annulus fibrosus, with ruptured fibers constituting less than 25% of the area; Score 2: Inward protrusion of the annulus fibrosus, with ruptured fibers constituting 25-50% of the area; Score 3: Inward protrusion of the annulus fibrosus, with ruptured fibers constituting more than 50% of the area.

[0139] (4) Cytological score for annulus fibrosus

[0140] Score 0: fibroblast cell ratio > 90%; Score 1: fibroblast cell ratio > 75-90%; Score 2: fibroblast cell ratio 50%-75%; Score 3: cartilage cell ratio > 75%.

[0141] (5) Score for the junction of the nucleus pulposus tissue and the annulus fibrosus

[0142] Score 0: normal, no interruption; Score 1: mild interruption; Score 2: moderate interruption; Score 3: severe interruption.

[0143] 16. Immunohistochemical staining

[0144] (1) Paraffin-embedded section, section conventional deparaffinization and hydration, and use pepsin to repair antigen, and the specific details are the same as those in the step of "immunohistochemical staining".

[0145] (2) Blocking: drop 50 μL of goat serum and block at room temperature for 30 min.

[0146] (3) Incubation of primary antibody: shake off the goat serum blocking solution, drop 50 μL of primary antibody solution, and put into a wet box for overnight incubation at 4°C. The primary antibody solution is derived from and has a concentration of: GLS1 (12855-1-AP, Proteintech, 1:50).

[0147] (4) Incubation of secondary antibody: the next day, recover the primary antibody solution, wash with PBS solution for three times, each time for 5 min. Drop 50 μL of goat anti-rabbit fluorescent secondary antibody (ZF-0516, ZSbio) under light shielding conditions, and incubate at room temperature for 2 h. Wash with PBS solution for 5 min repeatedly for three times.

[0148] (5) Counterstaining: drop DAPI solution under light shielding conditions, and incubate at room temperature for 10 min. Wash with PBS solution for 5 min repeatedly for three times.

[0149] (6) Mounting: use neutral resin and cover glass for mounting, and pay attention to avoid air bubbles.

[0150] (7) Photography and statistics: take pictures of the rat nucleus pulposus tissue section under a fluorescence microscope, at least three high-power fields, and use Image J to calculate the fluorescence intensity of the section for statistical analysis.

[0151] Results

[0152] The present application first collects magnetic resonance imaging data of patients receiving spinal surgery before surgery and nucleus pulposus tissue with different degrees of degeneration removed during surgery, classifies the degree of intervertebral disc degeneration through the Pfirrmann classification system Figure 1 A).

[0153] Subsequently, the present application performs transcriptomic sequencing on the collected specimens, and the results show that the expression of GLS1 is significantly reduced in severely degenerated intervertebral disc nucleus pulposus tissue Figure 1 B).

[0154] To further verify the relationship between GLS1 and intervertebral disc degeneration, the present application performs immunohistochemical staining on human nucleus pulposus tissue specimens, and the results suggest that the expression level of GLS1 protein is significantly reduced in degenerated nucleus pulposus tissue Figure 1 C-D).

[0155] The present application subsequently establishes an intervertebral disc degeneration model in rats by needle puncture induction, and the staining results verify the intervertebral disc degeneration changes in the intervertebral disc degeneration group Figure 1 E) Immunofluorescence staining results show that the protein expression of GLS1 is significantly reduced in the intervertebral disc degeneration group Figure 1 F).

[0156] To clarify the regulatory role of GLS1 in intervertebral disc degeneration, the present application overexpresses GLS1 in nucleus pulposus cells. Under oxidative stress stimulation, the expression of iron death markers glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) is significantly reduced, and these effects can be reversed by overexpression of GLS1 Figure 2 A). Flow cytometry experiments confirm the protective effect of GLS1 against oxidative stress, manifested as a decrease in reactive oxygen species levels Figure 2 B). Transmission electron microscopy shows that GLS1 alleviates the damage to nucleus pulposus cell mitochondria under oxidative stress conditions Figure 2 C). Confocal fluorescence microscopy results show that GLS1 overexpression can alleviate lipid peroxidation Figure 2 D) and maintain mitochondrial stability, eliminating the generation of reactive oxygen species within mitochondria Figure 2 E). In addition, overexpression of GLS1 effectively alleviates the aging of nucleus pulposus cells, as evidenced by a decrease in the expression level of aging markers Figure 2 F) and a decrease in the positive rate of beta-galactosidase staining Figure 2 G).

[0157] Meanwhile, to clarify the specific mechanism by which GLS1 regulates intervertebral disc degeneration, the present application performs transcriptomic sequencing analysis on nucleus pulposus cells overexpressing GLS1, and the results show that the expression of NFS1, a key enzyme for stable iron ion homeostasis, is significantly increased Figure 3 A). Immunohistochemical results on degenerated human nucleus pulposus tissue show that the NFS1 protein level is significantly reduced Figure 3B, C).

[0158] In addition, linear regression analysis showed a positive correlation between GLS1 and NFS1 levels, indicating a functional interaction between the two proteins Figure 3 D) Overexpression of GLS1 effectively counteracted the decrease in NFS1 protein expression under oxidative stress Figure 3 E) Confocal microscopy showed that GLS1 and NFS1 co-localized after GLS1 overexpression Figure 3 F).

[0159] In addition, the present application investigated the in vivo significance of GLS1 and NFS1 in a rat model of degenerative disc disease. After induction of disc degeneration by needle puncture, rats were injected with adeno-associated viral vectors overexpressing GLS1 and NFS1 into the intervertebral disc to overexpress these genes Figure 4 A) MRI analysis showed that overexpression of GLS1 and NFS1 was able to maintain disc integrity Figure 4 B, C) Histological evaluation by H&E staining and SO&FG staining showed that disc degeneration was significantly reduced in rats receiving GLS1 and NFS1 overexpression therapy Figure 4 D, E) In addition, the decrease in GLS1 and NFS1 levels induced by needle puncture was alleviated by overexpression of GLS1 and NFS1 Figure 4 F, G).

[0160] The present application in-depth research on the role of amino acid metabolism, especially glutamine metabolism in intervertebral disc degeneration, helps to comprehensively evaluate the role of glucose, lipid and amino acid in maintaining the normal physiological function of nucleus pulposus cells, and provides a basis for prevention and treatment strategies for intervertebral disc degeneration. Glutaminase 1 (GLS1) is a key enzyme in glutamine metabolism, which can convert glutamine to glutamic acid. In view of the key role of GLS1 in glutamine metabolism, a new target is provided for the treatment of intervertebral disc degeneration, thereby providing an innovative strategy for the treatment of low back pain.

[0161] It should be noted that in the claims of the present application, when a numerical range is involved, both endpoints of each numerical range and any number between the two endpoints can be selected. In order to prevent repetition, the present application describes preferred embodiments.

[0162] Although preferred embodiments of the present application have been described, those skilled in the art can make further changes and modifications to these embodiments once they know the basic inventive concept. Therefore, the appended claims are intended to include the preferred embodiments and all changes and modifications falling within the scope of the present application.

[0163] Obviously, many modifications and variations of the present application are possible in light of the above teachings. It is, therefore, to be understood that within the scope of the appended claims and their equivalents, the application can be practiced otherwise than as specifically described.

Claims

1. Use of a reagent for detecting expression amount of glutaminase 1 in preparation of a diagnostic product for intervertebral disc degeneration.

2. Use according to claim 1, characterized in that, The reagent for detecting expression amount of glutaminase 1 includes any one of reagents used in an immunohistochemical staining method and reagents used in an immunohistochemical fluorescent staining method.

3. Use according to claim 2, characterized in that, The reagent used in the immunohistochemical staining method or the immunohistochemical fluorescent staining method includes a GLS1 antibody.

4. Use according to claim 3, characterized in that, The reagent used in the immunohistochemical staining method or the immunohistochemical fluorescent staining method further includes a goat anti-mouse / rabbit IgG.

5. A kit characterized in that, The kit includes the reagent for detecting expression amount of glutaminase 1 as claimed in claim 1. 6.Use of a reagent for overexpressing glutaminase 1 as claimed in claim 1 in preparation of a therapeutic product for intervertebral disc degeneration.

7. Use according to claim 6, characterized in that, The reagent for overexpressing the glutaminase 1 includes any one of primers as shown in SEQ ID NO. 1-2.

8. Use according to claim 6, characterized in that, The therapeutic product maintains the integrity of the intervertebral disc.

9. Use according to claim 6, characterized in that, The therapeutic product alleviates the degeneration of the intervertebral disc.

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