Application of apolipoprotein D in the preparation of medicine for treating intervertebral disc degeneration

Abstract

The present invention discloses the use of apolipoprotein D in the preparation of drugs for treating intervertebral disc degeneration, and relates to the field of biotechnology. APOD undergoes S-palmitoylation, which competitively inhibits its ubiquitination and degradation process mediated by GNB2. S-palmitoylated APOD changes extracellular matrix metabolism and regulates cellular oxidative stress by inhibiting the GATA4-P65 signaling pathway, thereby inhibiting the aging of nucleus pulposus cells and delaying the progression of intervertebral disc degeneration. In the results of the examples of the present application, the role of S-palmitoylation in the pathogenesis of intervertebral disc degeneration is elucidated for the first time, and the in vivo supplementation is verified. Etc. The degenerative phenotype of the intervertebral disc can be significantly reversed. This application provides a new intervention target for the treatment and prognosis of intervertebral disc degeneration, and is expected to provide a potential new strategy for clinical patients with intervertebral disc degeneration to restore joint homeostasis.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to application of apolipoprotein D in preparing a medicine for treating intervertebral disc degeneration. Background Art

[0002] Intervertebral Disc Degeneration (IVDD) is an important disease that affects human health. The low back pain caused by it is one of the most common disabling diseases in the world in the past 30 years. With the aging of the population and the change of lifestyle, the incidence of IVDD has been increasing year by year, bringing heavy economic burden and life pressure to society and individuals. Although the diagnosis and treatment methods of intervertebral disc degeneration are constantly improving, its prevention and treatment are still major problems faced by clinicians and researchers. Therefore, actively exploring and clarifying the pathogenesis of low back pain and finding effective biological targets to delay the progression of intervertebral disc degeneration are of great practical significance for the development of society and the country.

[0003] As a key mechanism for regulating the functional diversity of proteins, protein post-translational modifications (PTMs) have received increasing attention in recent years for their role in the pathophysiological processes of various diseases. Among them, lipid modification, as an important type of PTMs, changes the lipid solubility of proteins, thereby affecting their localization, stability and interactions, thus playing a core role in cell signaling, metabolic regulation and disease progression.

[0004] S-palmitoylation is the most common protein lipid modification, also known as protein palmitoylation, which is catalyzed by the S-palmitoyltransferase DHHC (Asp-His-His-Cys) family. This modification process connects palmitic acid to the cysteine ​​residue of the protein through a sulfhydryl bond, thereby changing the localization, stability and activity of the protein and participating in basic biological processes such as cell adhesion, growth and division. More and more studies have shown that abnormal S-palmitoylation is closely related to the progression of various diseases, such as cancer, metabolic diseases, inflammatory diseases and neurodegenerative diseases. However, there are few reports on the pathogenesis of S-palmitoylation modification in intervertebral disc degeneration.

[0005] Apolipoprotein D (APOD), as a highly conserved soluble lipophilic molecular carrier protein, is mainly responsible for the transport and metabolism of lipids in the body, and also exhibits important functions in neuroprotection and anti-oxidative stress. APOD can regulate cell stress response and aging process by reducing lipid peroxidation and maintaining extracellular matrix (ECM) homeostasis, which provides a theoretical basis for exploring its potential therapeutic effects in aging-related diseases, especially disc degeneration. Although the role of APOD in various pathological and physiological processes has been gradually revealed, there are still no reports on its post-translational modification. At the same time, the specific role of S-palmitoylation in the pathogenesis of disc degeneration has not yet been elucidated.

[0006] Therefore, studying the specific pathophysiological role and molecular mechanism of APOD in the occurrence and development of intervertebral disc degeneration will provide a new direction and strategy with great development potential for the development of drugs related to intervertebral disc degeneration. Summary of the invention

[0007] Based on the deficiencies in the prior art, the present invention provides the use of apolipoprotein D in the preparation of a drug for treating intervertebral disc degeneration. S-palmitoylated apolipoprotein APOD exerts pathophysiological effects by inhibiting the GATA4-P65 signaling pathway, and competitively inhibits the ubiquitination and degradation of APOD mediated by GNB2, thereby supplementing the body with Apod It can significantly reverse the phenotype of intervertebral disc degeneration.

[0008] In one aspect, the present invention provides the use of apolipoprotein D in the preparation of a medicament for treating intervertebral disc degeneration.

[0009] Preferably, the drug regulates apolipoprotein D overexpression.

[0010] After APOD overexpression, the expression of ECM anabolism-related genes (COL2A1, SOX9) increased, the expression of ECM catabolism-related genes (ADAMTS4) decreased, the expression of aging-related secretory SASPs markers (MMP3, IL-6) decreased, and the expression of cell senescence-related genes (P53, P21) decreased. In addition, after H2O2 treatment, the positive area of ​​SA-β-gal staining in human nucleus pulposus cells with APOD overexpression decreased, indicating that Apod Overexpression of the gene (Gene ID: 347) inhibits senescence of nucleus pulposus cells.

[0011] Preferably, when used, the drug regulates the overexpression of apolipoprotein D, and apolipoprotein D acts by S-palmitoylation. APOD undergoes S-palmitoylation, competitively inhibiting the ubiquitination and degradation of APOD mediated by GNB2. S-palmitoylated APOD participates in the metabolism of the extracellular matrix and the oxidative stress process of cells by inhibiting the GATA4-P65 signaling pathway, thereby inhibiting the aging of nucleus pulposus cells and delaying the progression of intervertebral disc degeneration.

[0012] After APOD was overexpressed, the expression of S-palmitoyltransferase increased significantly. After H2O2 treatment, the level of APOD S-palmitoylation was significantly reduced. Treatment with the palmitoylase inhibitor 2BP (50 μM) for 24 hours significantly inhibited the S-palmitoylation of APOD. These evidences indicate that apolipoprotein APOD regulates its anti-aging effect of nucleus pulposus cells by S-palmitoylation.

[0013] Preferably, the active ingredient of the drug includes a gene or mRNA encoding apolipoprotein D, which is introduced into the nucleus pulposus via an adenovirus vector.

[0014] Further preferably, the adenovirus vector is an adeno-associated virus. Apod The expression level of glial cell carcinoma was significantly increased, which could significantly improve the pathological phenotype of intervertebral disc degeneration.

[0015] Preferably, the drug is in the form of tablets, capsules, granules, oral liquid, pills, aerosol or injection.

[0016] On the other hand, the present invention also provides a drug for treating intervertebral disc degeneration, wherein the active ingredient of the drug contains apolipoprotein D.

[0017] The medicine for treating intervertebral disc degeneration of the present invention further comprises a pharmaceutically acceptable excipient.

[0018] Preferably, the drug is in the form of tablets, capsules, granules, oral liquid, pills, aerosol or injection.

[0019] Compared with the prior art, the embodiments of the present application have at least the following advantages or beneficial effects:

[0020] The present invention clarifies for the first time the role of S-palmitoylation in the pathogenesis of intervertebral disc degeneration. S-palmitoylated APOD has the effects of changing extracellular matrix metabolism, regulating cellular oxidative stress, and inhibiting senescence of nucleus pulposus cells in the occurrence and development of intervertebral disc degeneration. The implementation of this application provides a new intervention target for the treatment and prognosis of intervertebral disc degeneration, and is expected to provide a potential new strategy for reestablishing homeostasis in clinical patients with intervertebral disc degeneration.

[0021] At the mechanistic level, the present invention first discovered that S-palmitoylated apolipoprotein APOD exerts pathophysiological effects by inhibiting the GATA4-P65 signaling pathway, while competitively inhibiting the ubiquitination and degradation of APOD mediated by GNB2. Apod It can significantly reverse the phenotype of intervertebral disc degeneration. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The immunohistochemical staining results of nucleus pulposus tissue of patients with intervertebral disc degeneration with different degrees of lesions, where A is immunohistochemical staining, B is the statistics of APOD-positive cell rate, and the scattered points represent the APOD-positive cell rate of a sample respectively; the scale is 100 μm; "***" means P < 0.001.

[0023] Figure 2 Figure 3 is the functional verification of APOD knockdown in vitro, where A is the expression results of real-time fluorescence quantitative PCR detection of genes related to anabolism (COL2A1, SOX9), catabolism (ADAMTS4), senescence-associated secretory phenotype (SASPs) markers (MMP3, IL-6) and cell senescence (P53, P21), and B is the Western blot protein expression detection of genes related to anabolism (COL2A1, SOX9), catabolism (ADAMTS4), SASPs markers (MMP3) and cell senescence (P53, P21); * represents the statistical difference between Si-NC group and Si-APOD#1 group, # represents the statistical difference between Si-NC group and Si-APOD#2 group, "**" indicates P < 0.01, "***" indicates P < 0.001, "##" indicates P < 0.01, and "###" indicates P < 0.001.

[0024] Figure 3 Figure 3 is the functional verification of APOD overexpression in vitro, where A is the expression results of real-time fluorescence quantitative PCR detection of genes related to anabolism (COL2A1, SOX9), catabolism (ADAMTS4), SASPs markers (MMP3, IL-6) and cell senescence (P53, P21), and B is the Western blot protein expression detection of genes related to anabolism (COL2A1, SOX9), catabolism (ADAMTS4), SASPs markers (MMP3) and cell senescence (P53, P21); "*" indicates P < 0.05, "**" indicates P < 0.01, and "***" indicates P < 0.001.

[0025] Figure 4Micro-CT analysis of the changes in intervertebral disc height in mice, where A is the Micro-CT images of wild-type sham-operated group, IVDD model group and Apod AAV-treated group mice, and B is the quantitative analysis result of the changes in intervertebral disc height in each group of mice, where the scattered points represent the changes in intervertebral disc height of one sample respectively; "***" indicates P < 0.001.

[0026] Figure 5 The results of HE staining and Alcian blue staining of mouse intervertebral disc tissue, where A is a representative image, B is the statistical results of histological scores of each group, and the scattered points represent the histological score of a sample respectively; the scale is 50 μm, "**" indicates P < 0.01, and "***" indicates P < 0.001.

[0027] Figure 6 Figure 3 shows the S-palmitoylation of APOD in human nucleus pulposus cells, where A is the sample processing process of co-immunoprecipitation mass spectrometry (Co-IP / MS), B is the result of ABE palmitoylation experiment under simulated excessive oxidative stress environment, and C is the result of ABE method detection of palmitoylase inhibitor 2BP inhibiting APOD S-palmitoylation.

[0028] Figure 7 The co-immunoprecipitation assay (Co-IP) was used to detect the interaction between APOD and S-palmitoyltransferase (ZDHHC11) in human nucleus pulposus cells.

[0029] Figure 8 Western blot protein expression was used to detect the expression of APOD in human nucleus pulposus cells at different 2BP treatment times (A) and doses (B).

[0030] Fig. 9 Western blot was used to detect the degradation of APOD in human nucleus pulposus cells after adding 2BP and proteasome inhibitor (Borz) or lysosome inhibitor (CHQ) based on the addition of protein synthesis inhibitor CHX (cycloheximide).

[0031] Fig.10 Co-IP / MS experiment and UbiBrowser bioinformatics analysis (referred to as bioinformatics prediction) and the Venn diagram of the E3 ubiquitin ligase GNB2 that interacts with APOD.

[0032] Fig.11 Co-IP experiments were performed to verify the interaction between E3 ubiquitin ligase GNB2 and APOD.

[0033] Fig.12The results of Co-IP experiments, A shows that overexpression of GNB2 increases the ubiquitination level of APOD, B shows the effect of downregulating the expression of S-palmitoyltransferase on the ubiquitination level of APOD, and C shows the immunofluorescence results of MMP13, COL2A1, and P16 in human nucleus pulposus cells; the scale bar is 20 μm.

[0034] Fig.13 Volcano plot results of transcriptome sequencing analysis of APOD knockdown.

[0035] Fig.14 These are the results of real-time fluorescence quantitative PCR (RT-qPCR) detection. A shows the up-regulation of GATA4 expression after APOD knockdown, and B shows the decrease of GATA4 expression after APOD overexpression. The scattered points represent the GATA4 expression level of a sample respectively. “***” indicates P < 0.001.

[0036] Fig.15 The effects of APOD or GATA4 knockdown on P65 phosphorylation and nuclear localization. A is the Western blot detection result, B is the immunofluorescence image, and the scale bar is 100 μm. DETAILED DESCRIPTION

[0037] The following is a clear and detailed description of the examples of the present application in conjunction with the specific embodiments. If no specific conditions are specified in the examples, the conditions are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0038] It should be understood that these embodiments are only used to illustrate the present application and they should not be construed as limiting the scope of protection of the present application.

[0039] Example 1

[0040] This example is the research method and experimental means in this application

[0041] 1. Acquisition and processing of human tissue specimens

[0042] After informed consent from the patients and approval from the ethics committee (approval number: 20230756), intervertebral disc samples with different degrees of lesions were collected from patients with intervertebral disc degeneration. The obtained intervertebral disc specimens were used to collect nucleus pulposus cells and histological experiments.

[0043] 1. Immunohistochemistry

[0044] Human intervertebral disc samples were fixed with 4% paraformaldehyde solution for 2 days and subsequently decalcified with 12.5% ​​ethylenediaminetetraacetic acid (EDTA) for 10 days. The tissues were then dehydrated in different concentrations of ethanol and embedded in melted paraffin, and all tissues were cut into 5-μm-thick sections under a microscope. Paraffin sections were dewaxed with xylene and rehydrated in graded alcohols, and the alcohol was washed off with PBS. They were then treated with 0.1% trypsin without EDTA at 37°C for 1 hour and then washed three times with PBS. Endogenous peroxidase activity was eliminated with 3% hydrogen peroxide for 10 minutes and then washed three times with PBS. Next, the sections were blocked with 5% goat serum at 37°C for 1 hour and incubated with primary antibodies overnight at 4°C. After the initial incubation, the sections were washed three times with PBS and then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit secondary antibodies (Boster, USA) at 37°C for 1 hour and then washed three times with PBS. Finally, the sections were incubated with DAB colorimetric solution (Boster, USA) for 1 min and finally stained with hematoxylin solution (Boster, USA) for 2 min at room temperature. Tissue images were captured by light microscopy. Quantitative analysis was performed in a blinded manner, and the proportion of positive cells was evaluated using Image J software (version 1.5.3), and the significance of the data was evaluated using GraphPad Prism (version 8.0.1).

[0045] 2. Pfirrmann classification system

[0046] According to the MRI manifestations, it is divided into grade I: the intervertebral disc is uniform, with bright high-signal white intensity, and the intervertebral disc height is normal; grade II intervertebral disc is uneven, but maintains a high-intensity white signal, the cell nucleus and cell annulus are clearly distinguished, a gray horizontal band may appear, and the intervertebral disc height is normal; grade III intervertebral disc is uneven, with intermittent gray signal intensity, the distinction between the nucleus and the annulus is unclear, and the intervertebral disc height is normal or slightly decreased; grade IV intervertebral disc is uneven, with a dark gray low signal, there is no longer a distinction between the nucleus and the annulus, and the intervertebral disc height is slightly or moderately reduced; grade V: the intervertebral disc is uneven, with a low signal black, there is no more distinction between the nucleus and the annulus, and the intervertebral disc space is collapsed.

[0047] 2. Cell and Molecular Experiments

[0048] 1. Primary nucleus pulposus cell culture

[0049] Under sterile conditions, the human nucleus pulposus samples were cut into pieces and washed three times with PBS. Then they were digested in 0.2% type II collagenase (Sigma, USA) at 37°C overnight. The supernatant was then taken and filtered with a 100μm cell strainer (Biosharp, China) and centrifuged at 1000rpm for 5 minutes. Then they were washed three times with PBS and finally cultured in DMEM high-glucose medium supplemented with 10% fetal bovine serum (FBS) at 37°C in a 5% CO2 incubator.

[0050] 2. Cell transfection

[0051] Customized siRNA sequences were synthesized by Shanghai Genema Gene to knock down specific mRNAs. The siRNA sequences are shown in Table 1. The two bases at the 3′ end of the sequence were replaced with protruding TT, which can enhance the stability of the siRNA double-stranded complex and thus increase the knockdown efficiency of siRNA. Lipofectamine RNAi MAX transfection reagent (Invitrogen, USA) was used at 1 μl / 10 5 The cells were transfected into human nucleus pulposus cells, the medium was changed 8 hours after transfection, and the cell RNA and protein were extracted 48 hours later.

[0052] Table 1

[0053]

[0054] 3. Viral infection

[0055] The APOD overexpression plasmid was provided by Beijing Qingke Biotechnology, and the G protein β2 (GNB2) overexpression plasmid was provided by Wuhan Miaoling Biotechnology. One day before transfection, HEK293 cells were seeded in a 10 cm cell culture dish with surface pretreatment to a density of 70% to 80%. The medium was changed 1 hour in advance and the medium was discarded. After plasmid extraction, the auxiliary plasmids (pMD2.G and psPAX2) were co-transfected into HEK293 cells using Opti-MEM culture medium containing Lipofectamine 3000 (Invitrogen, USA). After incubation for 6 hours, the medium was discarded and the medium was changed. 24 hours after the medium change, the virus was collected and the medium was replaced. 48 hours later, the virus was collected again and stored at 4°C. The supernatant was centrifuged at 4°C and 3000 rpm for 15 minutes, and the medium was purified by 0.45 μm filter (Millipore, Billerica, MA, USA). Well-growing human primary nucleus pulposus cells were infected with a virus containing polybrene, and the final concentration of polybrene was 10 μg / mL. After mixing, they were placed in an incubator and cultured for 24 hours, and then the medium was changed. The overexpression efficiency was verified by RT-qPCR.

[0056] 4. RNA extraction and real-time quantitative PCR (RT-qPCR)

[0057] Total RNA was extracted from cells using the AG RNAex Pro kit (Acrobio), and reverse transcribed into cDNA using the PrimeScript RT kit (Acrobio). qRT-PCR was performed using the SYBR Green PCR kit (Yisheng Bio) on an ABI 7500 sequencing system (Applied Biosystems, USA). -△△CT Methods: β-Actin gene was used as an internal reference to calculate mRNA expression levels, and GraphPad Prism (version 8.0.1) was used to evaluate the significance of the data.

[0058] 5. Transcriptome sequencing (RNA-seq)

[0059] Human nucleus pulposus cells from the control group and Si-APOD (n=3 patients) were lysed with 1 ml Trizol and sent to a third-party organization (Megi Biotech) for HiSeq RNA-Seq detection. Then, according to the polyA selection method, oligo(dT) magnetic beads were used to isolate mRNA and then fragmented. Then, the SuperScript double-stranded cDNA synthesis kit (Invitrogen, USA) was used to synthesize double-stranded cDNA using the fragmented mRNA as a template and random hexamer primers (Illumina). The synthesized cDNA was then end-repaired, phosphorylated, and 3'-terminal bases were added according to the Illumina library construction protocol. On a 2% low-range super agarose gel, a cDNA target fragment of 300 bp was selected, and the selected cDNA fragment was PCR amplified using Phusion DNA polymerase (NEB) for 15 cycles. After quantification using Qubit 4.0, the prepared libraries were sequenced with a NovaSeq 6000 sequencer (2 × 150 bp read length). Genes with P < 0.05 and an absolute value of log2 (fold changes) > 1 were considered differentially expressed genes.

[0060] 6. Western blot

[0061] The cultured cells were placed on ice, gently scraped with a cell scraper, and lysed with RIPA lysis buffer at 4°C for 1 hour. The total protein was quantified by the BCA method, and SDS loading buffer was added and boiled at 100°C for 5 minutes to denature the protein. The total protein sample of the cell was separated by polyacrylamide gel electrophoresis (SDS-PAGE), then transferred to a PVDF membrane, blocked with 5% skim milk at room temperature, and then incubated with the corresponding primary antibody at 4°C overnight. The next day, TBST was washed three times, and then incubated with HRP-conjugated secondary antibody at room temperature for 1 hour. Finally, the binding of the antibody was detected using chemiluminescent substrate (ECL) and chemiluminescent gel imaging system (Bio-Rad, USA), and the bands were exposed and colored.

[0062] 7. β-galactosidase activity staining method (SA-β-gal)

[0063] The SA-β-gal staining kit (Biyuntian Biotechnology) was used according to the manufacturer's instructions to evaluate the level of senescence-related β-galactosidase in cells. The treated cells were fixed at room temperature for 30 minutes and then washed three times with PBS. Subsequently, the PBS was removed, and SA-β-gal staining solution (pH 6.0) was added and incubated at 37°C overnight, and the images were observed and captured using a normal optical microscope.

[0064] 8. Immunofluorescence staining

[0065] Nucleus pulposus cells cultured at 37°C were fixed in 4% paraformaldehyde for 15 min and washed three times with PBS. Then, the cells were permeabilized with 0.1% Triton X-100 for 15 min and washed three times with PBS. The cells were then blocked with 5% goat serum for 1 hour at room temperature, and then incubated with the corresponding primary antibody at 4°C overnight. The next day, they were washed three times with PBS and incubated with fluorescently conjugated goat anti-rabbit IgG secondary antibody (Fude Bio) at room temperature for 1 hour. The cell nuclei were counterstained with DAPI for 5 minutes and then sealed. All images were taken under a fluorescent inverted microscope.

[0066] 11. Co-immunoprecipitation experiment (Co-IP)

[0067] Experiments were performed using rProtein A / G MagBeads (Yisheng Bio) according to the instructions provided by the manufacturer. The culture medium of human nucleus pulposus cells was discarded, washed twice with PBS, and the cells were scraped off with a cell scraper. The treated cells were collected and mixed with RIPA buffer, and lysis buffer containing protease inhibitors was added. After mixing, the supernatant was collected by centrifugation (4°C, 14000g, 10 minutes) and placed on ice for later use. After magnetic bead pretreatment, antibody adsorption was performed, and then the sample containing antigen was added, and the antigen and magnetic bead-antibody complex were evenly dispersed and placed on a flip mixer at 4°C overnight. The magnetic bead-antibody-antigen complex was then magnetically separated and the supernatant was collected. Washing solution was added to the centrifuge tube, and the complex was evenly dispersed by pipetting, and magnetic separation was performed again and the supernatant was discarded. After repeated washing twice, the IP protein in the complex was released by protein thermal denaturation method, and detected by Western blot or mass spectrometry.

[0068] 12. Acyl-biotin exchange (ABE) palmitoylation experiment

[0069] The chondrocytes were mixed with lysis buffer for 30 minutes. The lysate was then sonicated and centrifuged. The supernatant was then blocked, cut, and biotin was added, and finally incubated with streptavidin magnetic beads. After incubation, the palmitoylation level of the protein was detected by Western blot. The specific operation steps are detailed in this reference: Yao H et al. InhibitingPD-L1 palmitoylation enhances T-cell immune responses against tumours. Nat Biomed Eng . 2019 Apr;3(4):306-317.

[0070] 13. Drug treatment methods

[0071] Drug treatment method of nucleus pulposus cells in in vitro experiments: H2O2 (200μM) was cultured for 2 days to simulate excessive oxidative stress conditions of cells, proteasome inhibitor Borz (250 nM), lysosome inhibitor CHQ (50μM), protein synthesis inhibitor CHX (10μg / mL), palmitoylase inhibitor 2BP (50μM).

[0072] 3. In vivo experiments

[0073] 1. Construction of mouse intervertebral disc degeneration model

[0074] Male C57BL / 6 mice were randomly divided into four groups: sham control group (WT-Sham and cko-Sham), mouse intervertebral disc degeneration group (IVDD) and Apod AAV treatment group (IVDD+Apod AAV), with 6 mice in each group. The operation steps are briefly described as follows: 3-month-old male C57BL / 6J mice were approved by the Experimental Animal Ethics Committee of the Run Run Shaw Hospital of Zhejiang University. The tail disc (Co 7 / 8) was determined by palpation and confirmed by X-ray counting of vertebral bodies in the sacral region. The annulus fibrosus was punctured with a 27G needle, and the adeno-associated virus (AAV) with a specific sequence was subsequently injected into the nucleus pulposus. After surgery, the health status of the rats was monitored daily, and they were allowed to move without restriction, fed regularly, and had free access to water. After surgery, they were placed under an infrared warming lamp until they were fully awakened from anesthesia and returned to their cages. Ampicillin was injected subcutaneously at 20 mg / kg according to body weight to prevent infection, and buprenorphine was injected subcutaneously at 0.05 mg / kg according to body weight for analgesia at 0 and 4 hours after surgery.

[0075] 2. Apod AAV therapy trial

[0076] Apod AAV was constructed by Shanghai Hanheng Biotechnology. In the mouse adeno-associated virus (AAV) treatment experiment, mice in the Apod AAV treatment group (IVDD+Apod AAV) received intradiscal injections of 5μL two weeks after IVDD modeling.

[0077] 3. Micro-CT and quantitative analysis

[0078] Mouse intervertebral discs were fixed in 4% paraformaldehyde for 48 h, then washed and stored in 70% ethanol. The samples were then scanned using high-resolution μCT (Skyscan 1072, Belgium) in a 17 mm scanning tube with a scanning volume of 11 mm3, scanning conditions of 180 mA, 50,000 V, and acquisition time of 5 min. After acquiring CT images, three-dimensional imaging reconstruction was performed by Skyscan software (VGStudio MAX, Germany) to observe the changes in the height of the mouse intervertebral disc. The quantitative analysis of the changes in the height of the intervertebral discs of each group of mice was performed by one-way analysis of variance, and GraphPad Prism (version 8.0.1) was used to evaluate the significance of the data.

[0079] 4. Alcian blue staining

[0080] After the mouse intervertebral disc was scanned with Micro-CT, it was decalcified with 12.5% ​​ethylenediaminetetraacetic acid (EDTA) for one week, and then the tissue was dehydrated in ethanol and embedded in melted paraffin. All intervertebral disc tissues were cut into 5-micron thick sections under a microscope. The paraffin sections were dewaxed with xylene and rehydrated in gradient alcohol, and the alcohol was washed off with PBS. The sections were then stained with Alcian blue for 30 minutes and then stained with nuclear fast red solution for 5 minutes. After gradient alcohol dehydration, the sections were sealed with neutral resin, and images of intervertebral disc tissue were taken under a microscope.

[0081] 5. Histological scoring

[0082] After staining with Alcian blue, the disc sections were histologically scored, ranging from 0 to 15 for severe degeneration (3 points for each category).

[0083] For the morphology of the nucleus pulposus, 0 points: round, the nucleus pulposus cells (NP) occupy 75% of the disc area; 1 point: round, the NP occupies 50-75% of the disc area; 2 points: round, the NP occupies 25-50% of the disc area; 3 points: round, the NP occupies less than 25% of the disc area.

[0084] For the cellularity of the nucleus pulposus, 0 points: astrocytes, surrounded by proteoglycan matrix, uniformly distributed; 1 point: partially astrocytes and partially round cells, more astrocyte than round; 2 points: mostly large and round cells separated by dense areas of proteoglycan matrix; 3 points: large and round cells separated by dense areas of proteoglycan matrix.

[0085] For annular fibrosis morphology, 0 points: well-organized collagen sheets without ruptures; 1 point: inward bulging, ruptures, or serpentine fibers occupy less than 25% of the annular fibrosis; 2 points: inward bulging, ruptures, or serpentine fibers occupy 25%-50% of the annular fibrosis; 3 points: inward bulging, ruptures, or serpentine fibers occupy 50% of the annular fibrosis.

[0086] Regarding the cellular structure of the annulus fibrosus, 0 point: the proportion of fibroblasts in the total number of all cells in the annulus fibrosus is >90%; 1 point: the proportion of fibroblasts in the total number of all cells in the annulus fibrosus is >75%-90%; 2 points: the proportion of fibroblasts in the total number of all cells in the annulus fibrosus is 50%-75%; 3 points: the proportion of chondrocytes in the total number of all cells in the annulus fibrosus is >75%.

[0087] For the boundary between the nucleus pulposus and the annulus fibrosus, 0 points: normal, without any interruption; 1 point: minimal interruption; 2 points: moderate interruption; 3 points: severe interruption.

[0088] Each sample was evaluated by three independent researchers who were blinded to the experimental design, and GraphPadPrism (version 8.0.1) was used to assess data significance.

[0089] IV. Results

[0090] 1. APOD expression is downregulated in aging nucleus pulposus cells, and the APOD level in tissues is negatively correlated with the severity of intervertebral disc degeneration

[0091] Immunohistochemical staining of nucleus pulposus tissue of patients with different degrees of intervertebral disc degeneration. Figure 1 As shown, the proportion of APOD-positive cells in the nucleus pulposus tissue of patients with mild intervertebral disc degeneration is higher.

[0092] 2. APOD can promote ECM anabolism, control ROS generation, and inhibit nucleus pulposus cell aging

[0093] To investigate the effect of APOD on the progression of aging-related intervertebral disc degeneration, APOD siRNA was transfected into primary human nucleus pulposus cells to verify the function of APOD knockdown in vitro. Figure 2 As shown, compared with the control group, after APOD knockdown, the expression of ECM anabolism-related genes (COL2A1, SOX9) decreased, the expression of ECM catabolism-related genes (ADAMTS4) increased, the expression of SASPs markers (MMP3, IL-6) increased, and the expression of cell senescence-related genes (P53, P21) increased. In addition, the positive area of ​​SA-β-gal staining in human nucleus pulposus cells increased after APOD knockdown, indicating that the knockdown of the APOD gene can significantly induce cell senescence. The fluorescence intensity was then evaluated by staining with fluorescent dyes DHE and BODIPY581 / 591 C11. The results showed that the fluorescence intensity of the Si-APOD group was significantly higher than that of the control group, indicating that the knockdown of APOD increased the level of cellular ROS and promoted the lipid peroxidation process.

[0094] To verify the above results, the function of APOD overexpression in vitro was verified, as shown in Figure 3 As shown. Compared with the control group, after APOD overexpression, the expression of ECM anabolism-related genes (COL2A1, SOX9) increased, the expression of ECM catabolism-related genes (ADAMTS4) decreased, the expression of SASPs markers (MMP3, IL-6) decreased, and the expression of cell senescence-related genes (P53, P21) decreased. In addition, after H2O2 treatment, the positive area of ​​SA-β-gal staining of human nucleus pulposus cells with APOD overexpression decreased, indicating that the overexpression of the APOD gene inhibited the senescence of nucleus pulposus cells. The fluorescence intensity was then evaluated by staining with fluorescent dyes DHE and BODIPY581 / 591 C11. The results showed that the overexpression of APOD reduced the cellular ROS level and inhibited the lipid peroxidation process.

[0095] 3. Apod can improve the pathological phenotype of intervertebral disc degeneration in gene knockout mice

[0096] Three-month-old C57BL / 6J male mice underwent IVDD surgery, and Apod AAV was injected into the nucleus pulposus two weeks after surgery (divided into 4 groups, n=6). The animals were killed eight weeks after surgery to collect histological samples. Micro-CT analysis of the changes in the intervertebral disc height of mice Figure 4 The results showed that compared with the sham operation group, the intervertebral disc height of mice in the IVDD model group was significantly decreased, while the supplementation of Apod effectively delayed the occurrence of degeneration and significantly increased the intervertebral disc height.

[0097] like Figure 5 As shown in A, the intervertebral disc tissues of the above four groups of mice were stained with hematoxylin and eosin and alcian blue. The results showed that the nucleus pulposus of the mice in the IVDD model group was significantly damaged. After the injection of Apod AAV, the degree of damage to the nucleus pulposus tissue was significantly reduced, which significantly alleviated the related pathological characteristics of intervertebral disc degeneration.

[0098] like Figure 5 As shown in B, the above four groups of mice were scored for synovitis quantification, and the results showed that the relevant scores of mice treated with Apod AAV were lower than those of the IVDD group, suggesting that Apod reduced the severity of the disease in the mouse intervertebral disc degeneration model.

[0099] 4. APOD plays an anti-aging role in nucleus pulposus cells through S-palmitoylation

[0100] like Figure 6 As shown, in order to clarify the potential mechanism by which APOD exerts its anti-aging effect on nucleus pulposus cells in vivo, normal nucleus pulposus cells were infected with APOD-overexpressing lentivirus, and Co-IP experiments were performed and used for mass spectrometry (MS) analysis. The ABE method was then used to detect the level of S-palmitoylated APOD (Palm-APOD) after 48 hours of H2O2 (200 μM) treatment. The results showed that after H2O2 treatment, the level of S-palmitoylation of APOD was significantly reduced. Treatment with the palmitoylase inhibitor 2BP (50 μM) for 24 hours significantly inhibited the S-palmitoylation of APOD. These evidences indicate that apolipoprotein APOD may regulate its anti-aging effect on nucleus pulposus cells by undergoing S-palmitoylation. Co-IP experiments confirmed the interaction between S-palmitoyltransferase and APOD (such as Figure 7 as shown).

[0101] 5. S-palmitoylation of APOD competitively inhibits its ubiquitination and degradation mediated by GNB2

[0102] like Figure 8As shown, normal human nucleus pulposus cells were treated with different concentrations (0, 12.5, 25, 50, 100 μM) of 2BP, or treated with a specified concentration of 2BP for different time periods (0, 3, 6, 12, 24, 48 hours), and the expression of APOD decreased in a time- and dose-dependent manner. This may indicate that the de-palmitoylated APOD protein is degraded, and S-palmitoylation may keep the APOD protein in a stable state.

[0103] Protein degradation in eukaryotes occurs mainly through the proteasome system, lysosome-mediated autophagy, and other pathways. To further explore the protein degradation pathway of APOD depalmitoylation, Fig. 9 As shown in the figure, after adding 2BP and Borz on the basis of adding protein synthesis inhibitor CHX, the degradation of APOD in human nucleus pulposus cells was significantly inhibited, while after adding 2BP and CHQ, the degradation of APOD in human nucleus pulposus cells did not improve significantly. Since the ubiquitin-proteasome system (UPS) is the main pathway for intracellular protein degradation, it is involved in the degradation of more than 80% of intracellular proteins. The experimental results suggest that the degradation of depalmitoylated APOD in vivo may occur through the ubiquitin-proteasome system, and the potential E3 ubiquitin ligase involved in regulating the stability of APOD protein needs to be further identified.

[0104] Through IP-MS experiments and UbiBrowser bioinformatics analysis, we explored the E3 ubiquitin ligases that interact with APOD. Fig.10 GNB2 is the only E3 ubiquitin ligase involved in APOD degradation. Fig.11 It was further shown that there is an interaction between GNB2 and APOD.

[0105] The effect of GNB2 overexpression on APOD ubiquitination was verified by Co-IP experiments. Fig.12 As shown in the results, overexpression of GNB2 increased the ubiquitination level of APOD; meanwhile, downregulating the expression of S-palmitoyltransferase increased the ubiquitination level of APOD. These results indicate that S-palmitoylation of APOD competitively inhibits its ubiquitination mediated by GNB2 and its subsequent protein degradation.

[0106] The immunofluorescence results of MMP13, COL2A1, and P16 in C28 / I2 cells showed that inhibition of S-palmitoylation could block the protective effect of APOD on nucleus pulposus cells, while downregulation of GNB2 could significantly reverse this phenomenon.

[0107] 6. S-palmitoylated APOD can alleviate the progression of intervertebral disc degeneration by inhibiting the GATA4-P65 signaling pathway

[0108] Transcriptome sequencing was performed on normal human nucleus pulposus cells after APOD knockdown, and differential gene expression and KEGG enrichment analysis were performed. The results are as follows Fig.13 As shown, in nucleus pulposus cells with decreased APOD expression, the cellular senescence pathway was significantly activated, and the transcription factor GATA binding protein 4 (GATA4) was the most expressed aging-related gene in nucleus pulposus cells with APOD knockdown.

[0109] To explore whether GATA4 is a downstream regulatory factor of APOD, the RT-qPCR test results are as follows Fig.14 As shown (n=9), when APOD was knocked down in human nucleus pulposus cells (Si-APOD#1 and Si-APOD#2), the expression level of GATA4 was significantly increased; while when APOD was overexpressed (OE-APOD), the expression level of GATA4 was significantly decreased.

[0110] To further explore the effects of APOD or GATA4 knockdown on P65 phosphorylation and nuclear localization, APOD siRNA or GATA4 siRNA was transfected into human nucleus pulposus cells. Fig.15 Western blot results showed that downregulating APOD could promote the phosphorylation of P65, while silencing GATA4 could inhibit this effect; immunofluorescence results also verified this conclusion.

Claims

1. Application of apolipoprotein D in the preparation of drugs for treating intervertebral disc degeneration.

2. The use according to claim 1, characterized in that: The dosage form of the medicine is tablet, capsule, granule, oral liquid, dripping pill, aerosol or injection.

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