Application of YAP protein in preparation of product for preventing and treating intervertebral disc nucleus pulposus cell aging

Abstract

The invention discloses application of YAP protein in preparation of a product for preventing and treating intervertebral disc nucleus pulposus cell aging. The YAP protein is selected from an amino acid sequence as shown in SEQ ID NO: 1. Experiments prove that the aging of the nucleus pulposus cells can be effectively inhibited by overexpressing the YAP in the aged nucleus pulposus cells, and the discovery provides a new thought for the treatment of the intervertebral disc degeneration. More importantly, the invention provides a strategy of inhibiting mitochondrial division by overexpression of YAP for the first time to inhibit aging of nucleus pulposus cells. The innovative viewpoint not only is fully proved through experiments, but also shows a very high application value. By regulating and controlling the expression level of YAP, a brand new and effective treatment method is expected to be provided for intervertebral disc degeneration patients, so that the pain of the intervertebral disc degeneration patients is relieved, and the life quality of the intervertebral disc degeneration patients is improved. In addition, the discovery may also provide new enlightenment and reference for treatment of other diseases related to cell aging.

Description

Technical Field

[0001] The present invention belongs to the technical field of preventing and treating aging of intervertebral disc nucleus pulposus cells, and specifically relates to the application of YAP protein in preparing products for preventing and treating aging of intervertebral disc nucleus pulposus cells. Background Art

[0002] Low back pain is a common complaint seen in orthopedic clinics, affecting people of all ages. Approximately 23% of patients develop chronic low back pain, a condition closely associated with intervertebral disc degeneration. Intervertebral disc degeneration is a complex process involving biological degeneration of disc tissue under the combined influence of multiple factors, leading to changes in the mechanical and structural properties of the disc. During this process, the disc undergoes component absorption and metabolic disorders. Combined with the accumulation of multiple confounding factors, the disc gradually loses its normal protein and water content, replacing it with fibrous and cartilage components. This ultimately leads to symptoms such as pain, radiculopathy, and muscle strain. In severe cases, it can even lead to loss of ability to work or even disability.

[0003] The nucleus pulposus, the core structure of the intervertebral disc, plays a crucial role in the degeneration of the intervertebral disc due to its intrinsic cellular aging. Therefore, in-depth research on the specific mechanisms of cellular aging in the nucleus pulposus during intervertebral disc degeneration is of great significance for the treatment and prevention of intervertebral disc degeneration.

[0004] In related research, the Hippo signaling pathway has been shown to be involved in the regeneration of multiple organs following injury, including the heart, nervous system, and intestines. The activity of the YAP protein has a significant impact on physiological and pathological events such as cell differentiation, proliferation, and cell death. Mitochondria, as complex organelles within cells, are not only energy "workstations" but also play a key role in regulating cell aging, apoptosis, and homeostasis.

[0005] Compared to existing research, this study specifically explores the molecular biological mechanisms of YAP protein in preventing and treating aging in intervertebral disc nucleus pulposus cells from a novel perspective, focusing on mitochondrial homeostasis. By deeply studying how YAP protein plays a role in regulating mitochondrial homeostasis, this study aims to provide a new theoretical basis for intervertebral disc degeneration and uncover potential therapeutic targets. This research direction is expected to not only bring new breakthroughs in the treatment of intervertebral disc degeneration, but also provide new ideas and methods for the prevention and treatment of related diseases. Summary of the Invention

[0006] Technical problem to be solved: The purpose of the present invention is to provide a method for inhibiting the aging of intervertebral disc nucleus pulposus cells to solve the problems of intervertebral disc degeneration and the resulting low back pain and lumbar disc herniation.

[0007] Technical solution: A use of a YAP protein in the preparation of a drug for delaying or treating aging of intervertebral disc nucleus pulposus cells, wherein the YAP protein is selected from: a) the amino acid sequence shown in SEQ ID NO: 1; b) a variant having at least 80% homology with SEQ ID NO: 1 and retaining YAP functional activity.

[0008] A use of a nucleic acid molecule encoding a YAP protein in the preparation of a drug for delaying or treating aging of intervertebral disc nucleus pulposus cells, wherein the nucleic acid molecule is selected from: a) a nucleotide sequence shown in SEQ ID NO: 2; b) a variant having at least 80% homology with SEQ ID NO: 2 and encoding a functional YAP protein.

[0009] A pharmaceutical composition for delaying the aging of intervertebral disc nucleus pulposus cells comprises a therapeutically effective amount of YAP protein and a pharmaceutically acceptable carrier, wherein the YAP protein is as described above.

[0010] A gene therapy product for delaying the aging of intervertebral disc nucleus pulposus cells comprises an expression vector and the nucleic acid molecule described above, wherein the expression vector is selected from a viral vector or a non-viral vector.

[0011] The above viral vector is an adeno-associated virus (AAV) vector, which contains a tissue-specific promoter.

[0012] A method for delaying aging of nucleus pulposus cells comprises administering a therapeutically effective amount of a YAP protein activator to a subject in need thereof, wherein the activator is selected from: i) an expression vector encoding a YAP protein; ii) YAP protein mRNA; iii) a small molecule YAP agonist.

[0013] Preferably, the above expression vector is delivered to the intervertebral disc tissue by local injection.

[0014] The above product is used in the preparation of a drug for treating intervertebral disc degenerative diseases, including but not limited to intervertebral disc herniation, spinal canal stenosis or degenerative scoliosis.

[0015] Beneficial effects: The present invention has demonstrated through experiments that in the process of intervertebral disc degeneration, the aging of nucleus pulposus cells is accompanied by a decrease in the expression level of YAP, while the mitochondrial fission in the cells increases. These changes will further promote the aging process of nucleus pulposus cells. Overexpression of YAP in aged nucleus pulposus cells can effectively inhibit the aging of nucleus pulposus cells. This discovery provides a new approach for the treatment of intervertebral disc degeneration. More importantly, the present invention proposes for the first time the strategy of inhibiting mitochondrial fission by overexpressing YAP to inhibit the aging of nucleus pulposus cells. This innovative viewpoint has not only been fully demonstrated through experiments, but also has shown strong application value. By regulating the expression level of YAP, we hope to provide patients with intervertebral disc degeneration with a new and effective treatment method, thereby alleviating their pain and improving their quality of life. In addition, this discovery may also provide new inspiration and reference for the treatment of other diseases related to cell aging. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is a comparison of the gross observation, growth curve and aging rate of normal and aged nucleus pulposus cells.

[0017] Figure 2 This is a graph of YAP gene and protein expression in P3 nucleus pulposus cells transfected with YAP-overexpressing lentivirus.

[0018] Figure 3 The gross observation, growth curve and aging rate of P10 nucleus pulposus cells transfected with YAP-overexpressing lentivirus were compared with those of P10 nucleus pulposus cells.

[0019] Figure 4 The changes in mitochondrial membrane potential of P10 nucleus pulposus cells transfected with YAP-overexpressing lentivirus and P10 nucleus pulposus cells were observed under a laser confocal microscope.

[0020] Figure 5 The differences in mitochondrial morphology between P10 nucleus pulposus cells transfected with YAP-overexpressing lentivirus and P10 nucleus pulposus cells were observed under a laser confocal microscope.

[0021] Figure 6 The difference in ROS expression between P10 nucleus pulposus cells transfected with YAP-overexpressing lentivirus and P10 nucleus pulposus cells was observed under a fluorescence microscope. DETAILED DESCRIPTION

[0022] The present invention will be described in detail below with reference to the accompanying drawings and specific examples, but they should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0023] The YAP-overexpressing agents described herein preferably include other cytokines and chemical molecules that indirectly increase YAP expression in intervertebral disc nucleus pulposus cells; other vectors carrying the YAP gene, such as plasmids, lentiviruses, adenoviruses, or adeno-associated viruses, or other microorganisms containing these vectors. Any agent that artificially increases intracellular YAP levels in nucleus pulposus cells to modify nucleus pulposus cytological characteristics and delay cell aging is protected by this invention.

[0024] Example 1:

[0025] Extraction, culture, passage and replicative aging of nucleus pulposus cells

[0026] 1. Collect normal human nucleus pulposus tissue, mince the tissue as much as possible and place it in a 15 mL centrifuge tube. Add 5 mL of type II collagenase and digest at 37°C in a shaking incubator for 5 hours. Remove the centrifuge tube every 60 minutes and pipette the digestion solution and nucleus pulposus tissue mixture. Stop digestion when no obvious clumps of nucleus pulposus tissue are visible. Centrifuge at 1000 rpm for 5 minutes and aspirate the digestion solution.

[0027] 2. Add 3 mL of PBS to wash the cells, centrifuge again at 1000 rpm for 5 minutes, and remove the PBS;

[0028] 3. Add 5 mL of DMEM / F12 culture medium containing 10% FBS and double antibody, pipette evenly, and transfer to a 25 cm 2 Culture the cells in a culture flask at 5% carbon dioxide and 37°C in a cell culture incubator, changing the medium every 3 days.

[0029] 4. When the nucleus pulposus cells grow to 80% confluence, use cell digestion enzymes to digest and passage them;

[0030] 5. During the digestion process, the DMEM / F12 medium was used to stop the digestion. The cells were pipetted and aspirated into a 15 mL centrifuge tube. The tube was centrifuged at 1000 rpm for 5 min, and the digestion liquid was aspirated. Fresh medium was added and the cells were subcultured at a ratio of 1:3.

[0031] 6. A replicative aging nucleus pulposus cell model can be induced by continuous passage to the 10th generation.

[0032] Example 2:

[0033] Identification of a replicative aging nucleus pulposus cell model

[0034] Experimental groups: P3 group: the third generation of nucleus pulposus cells, P10 group: the tenth generation of nucleus pulposus cells.

[0035] 1. Cell Morphology Observation

[0036] like Figure 1 : A and Figure 1: B shows that under an inverted microscope, NPCs in the P3 group grew at a uniform density, with regular morphology and a long spindle-shaped appearance. NPCs in the P10 group were induced by continuous replicative aging through cell passage, with increased cell volume and irregular shapes, such as triangles or polygons. The cells were flattened and spread out. Under the microscope, an increase in cytoplasmic particles, increased metabolic debris products, and increased nucleus and nucleolus volumes were observed.

[0037] 2. Growth Curve Determination

[0038] NPCs at P3 and P10 were digested and counted, and the cell density was adjusted to 1×10 4 / mL, open the 96-well cell culture plate, add 100 microliters of cell suspension to each well, about 2000 cells, set up 6 replicates for each group, add PBS buffer around it to reduce the impact of volatilization, after 12 hours, add 10 microliters of CCK-8 solution to each well, continue to incubate in a 37℃ incubator with 5% carbon dioxide for 4 hours, and detect with a microplate reader at a wavelength of 450nm, and continue to detect for 7 days. Figure 1 : C, The growth curves of the two groups were roughly "S" shaped, entering the exponential growth phase on day 2-3, and the proliferation rate gradually slowed down as time went on, entering the growth plateau phase on day 5-7. Compared with the P3 group, the cell proliferation ability of the P10 group was significantly reduced.

[0039] 3. Cell Senescence β-Galactosidase Staining

[0040] We purchased the Senescence β-galactosidase Staining Kit (SA-β-gal) from Biyuntian Biotechnology Co., Ltd. P10 aging NPCs produce their own specific β-galactosidase, which catalyzes the X-gal substrate to produce a dark blue product, visualizing the aging phenotype. Blue-stained cells expressing β-galactosidase can be observed under an ordinary microscope.

[0041] NPCs at P3 and P10 were digested and counted, and the cell density was adjusted to 1×10 6 / mL, open a 6-well cell culture plate and add 1mL of cell suspension to each well. Set up three replicates per group and incubate for 12 hours. Remove the cultured cells from the 6-well plate, remove the culture medium with a pipette, and wash three times with PBS buffer for 1 minute each. Add 1mL of the β-galactosidase staining fixative provided in the kit to each well of the 6-well plate and fix for 20-30 minutes. Remove the cell fixative with a pipette, wash three times with PBS buffer for 1 minute each, and blot any remaining liquid with absorbent paper. Prepare the senescent β-galactosidase staining solution, add 1mL to each well, seal with sealing film, and incubate in a standard incubator at 37°C overnight. Observe the number and proportion of blue-stained cells under a microscope. If it is not possible to observe and count them in time, remove the staining solution with a pipette, add PBS buffer, and store in a refrigerator at 4°C. The 6-well cell culture plate was placed under an ordinary inverted microscope for observation. The number of positively stained cells was counted. Six random fields of view were read continuously in each well (100× field of view). The aging rate = number of positive cells / total number of cells × 100%. Compared with the P3 group, the average positive rate of SA-β-gal in the P10 group was significantly increased ( Figure 1 :DF).

[0042] Example 3:

[0043] Determination of transfection multiplicity of YAP overexpression lentiviral vector and overexpression detection

[0044] 1. Lentiviral packaging Using the GV248-hU6-MCS-Ubiquitin-EGFP-IRES-puromycin vector (Shanghai Jikai Biotechnology Co., Ltd.), the YAP overexpression lentiviral vector sequence was designed. In the present invention, the nucleotide sequence of the human YAP gene is shown in SEQ ID NO.1.

[0045] 2. Use TCID50 to determine the titer and calculate the virus titer according to the above formula T = 1.0 × 10 10 PFU / mL. Lentiviral YAP overexpression vectors were transfected into P3 and P10 NPCs at different MOIs (0, 10, 50, and 100). After 48 hours, transfection efficiency was observed under an inverted fluorescence microscope. The appropriate MOI was selected for subsequent experiments, which included calculating GFP protein expression under different fields of view. The results showed that the transfection efficiency gradually increased with increasing MOI of the YAP overexpression vector. At an MOI of 100, some cells were rounded and a small number had detached and died. At an MOI of 10, the transfection efficiency reached 50%, and at an MOI of 50, the transfection efficiency exceeded 90%. An MOI of 50 was selected as the optimal transfection efficiency. P3 and P10 NPCs were transfected with the negative control and YAP overexpression vectors.

[0046] 3. Grouping: P3 group, P3-LV-YAP group, P10 group and P10-LV-YAP group. Figure 2 As shown in Figure 3A, the YAP gene expression in the P3-LV-YAP group was significantly higher than that in the P3 group, and the difference between the two groups was statistically significant. Figure 2 : As shown in BC, the expression of YAP protein in the P3-LV-YAP group was significantly increased compared with the P3 group; compared with the P10 group and P10-LV-YAP, the YAP protein also increased after LV-YAP transfection in P10 cells, and the difference was statistically significant.

[0047] Example 4:

[0048] Detection of the inhibition of nucleus pulposus cell aging by lentiviral overexpression vector LV-YAP

[0049] 1. Select P10 cells and divide them into two groups: P10 group and P10-LV-YAP group.

[0050] 2. The cell morphology observation, growth curve determination, and cell senescence β-galactosidase staining detection methods are as described in Examples 1 and 2.

[0051] 3. Such as Figure 3 : AB shows that compared with the P10 group, the irregular morphology of the nucleus pulposus cells in the P10-LV-YAP group was reduced, and they were spread out flatly. Under the microscope, the intracytoplasmic particles were relatively reduced, and the metabolic debris products in the cells were reduced; Figure 3 : As shown in C, the cell growth rate increased, and as time went on, the proliferation rate slowed down after 3-5 days, but was still higher than that of the P10 group; Figure 3 As shown in Figures DF, the positive rate of senescent cells was significantly reduced by β-galactosidase staining. According to the results of Example 4, YAP protein can slow down the aging process of nucleus pulposus cells and increase cell growth rate.

[0052] Example 5:

[0053] Mitochondrial membrane potential, mitochondrial morphology and ROS detection

[0054] 1. Select P10 cells and divide them into two groups: P10 group and P10-LV-YAP group.

[0055] 2. Digest and count P10 NPCs and adjust the cell density to 1×10 6 / mL, open the 6-well cell culture plate, add 1mL of cell suspension to each well, set up 3 replicates for each group, culture for 12h, remove the cultured cells in the 6-well plate, remove the culture medium with a pipette, and wash 3 times with PBS buffer, each time for 1min. Add 1mL of cell culture medium to the cells in the 6-well plate, add 1mL of JC-1 staining working solution, mix well, incubate at 37℃ in a cell culture incubator for 20 minutes, observe the changes in membrane potential under a laser confocal microscope, and analyze the differences between the two groups. Figure 4As shown in the experimental results, it can be seen that the membrane potential of the P10-LV-YAP group is higher than that of the P10 group.

[0056] 3. Take another 6-well plate of cells, remove the cell culture medium, add Mito-Tracker Green staining working solution and incubate at 37℃ for 30 minutes. Remove the Mito-Tracker Green staining working solution, add fresh cell culture medium, and observe under a laser confocal microscope. At this time, mitochondria can be observed to be brightly fluorescent. Analyze the morphology and length of mitochondria in nucleus pulposus cells under different conditions. Figure 5 As shown in the figure, the experimental results show that the mitochondria in the P10-LV-YAP group are longer than those in the P10 group, and their morphology is relatively regular and orderly.

[0057] 4. Take another 6-well plate of cells, remove the cell culture medium, prepare DCFH-DA to a final concentration of 10 micromol / L, add 1 ml of diluted DCFH-DA to each well, incubate in a 37°C cell culture incubator for 20 minutes, wash the cells three times with serum-free cell culture medium to fully remove the DCFH-DA that has not entered the cells, observe under a laser confocal microscope, and DCF fluorescence can be seen showing cells expressing ROS. Figure 6 As shown in the experimental results, the DCF fluorescence positivity rate in the P10-LV-YAP group was higher than that in the P10 group.

[0058] 5. According to the results of Example 5, YAP protein can slow down the aging process of nucleus pulposus cells. The cellular molecular mechanism is that YAP inhibits mitochondrial fission and reduces ROS release.

Claims

1. A use of a YAP protein in the preparation of a medicament for delaying or treating aging of intervertebral disc nucleus pulposus cells, wherein the YAP protein is selected from: a) the amino acid sequence shown in SEQ ID NO: 1; b) a variant having at least 80% homology to SEQ ID NO: 1 and retaining YAP functional activity.

2. Use of a nucleic acid molecule encoding a YAP protein in the preparation of a medicament for delaying or treating aging of intervertebral disc nucleus pulposus cells, wherein the nucleic acid molecule is selected from: a) the nucleotide sequence shown in SEQ ID NO: 2; b) a variant having at least 80% homology to SEQ ID NO: 2 and encoding a functional YAP protein.

3. A pharmaceutical composition for delaying the aging of intervertebral disc nucleus pulposus cells, characterized in that: Comprising a therapeutically effective amount of a YAP protein and a pharmaceutically acceptable carrier, wherein the YAP protein is as defined in claim 1.

4. A gene therapy product for delaying the aging of intervertebral disc nucleus pulposus cells, characterized in that: The method comprises an expression vector and the nucleic acid molecule according to claim 2, wherein the expression vector is selected from a viral vector or a non-viral vector.

5. The product according to claim 4, characterized in that The viral vector is an adeno-associated virus (AAV) vector, which contains a tissue-specific promoter.

6. A method for delaying aging of nucleus pulposus cells, characterized in that: The method comprises administering a therapeutically effective amount of a YAP protein activator to a subject in need thereof, wherein the activator is selected from: i) an expression vector encoding a YAP protein; ii) a YAP protein mRNA; iii) a small molecule YAP agonist.

7. The method according to claim 6, characterized in that The expression vector is delivered to the intervertebral disc tissue by local injection.

8. Use of the product according to claim 3 or 4 in the preparation of a medicament for treating intervertebral disc degenerative diseases, wherein the diseases include but are not limited to herniated disc, spinal stenosis or degenerative scoliosis.

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