Application of HSP90 beta and / or APOB as target spot in preparation of AIS treatment medicine

Abstract

The invention provides application of HSP90 beta and / or APOB as a target spot in preparation of a medicine for treating AIS, and belongs to the technical field of biological medicine. The invention provides application of HSP90 beta and / or APOB as a target spot in preparation of a medicine for treating AIS and a medicine composition for treating AIS, and the medicine composition comprises an HSP90 beta inhibitor and / or an APOB inhibitor and pharmaceutically acceptable auxiliary materials. The target spot HSP90beta and APOB protected by the invention are key molecules for driving AIS development, and all means for inhibiting the target spot HSP90beta and APOB can effectively delay AIS development. The medicine protected by the invention takes a key molecule for driving AIS progress as a target spot, and has better targeting and specificity than the existing physical treatment of AIS.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the use of HSP90β and / or APOB as targets in the preparation of drugs for treating scoliosis. Background Art

[0002] Adolescent Idiopathic Scoliosis (AIS) is the most common form of pediatric scoliosis. The main clinical manifestation is a three-dimensional spinal deformity with a Cobb angle ≥ 10°, which can affect 1-4% of adolescents globally, causing pain, appearance deformity, and physical and psychological disabilities as the curve progresses. A large data study spanning more than 40 years and covering more than two million adolescents shows that the incidence of AIS in mainland China is 1.18%. Among them, only 4.1% of severe patients (Cobb angle > 40°) who need surgical treatment, while the proportion of mild and moderate (Cobb angle = 10° - 39°) patients is as high as 95.9%. This indicates that non-surgical treatment is crucial for the prevention and control of AIS. However, due to the still unclear specific causes of the onset and progression of AIS, there is a lack of drugs for scoliosis-modifying in clinical practice. Currently, only physical means such as long-term follow-up and long-term wearing of orthopedic braces are used to delay the progression of AIS, which only treats the symptoms but not the root cause, and causes a financial burden of 7-27%. Therefore, it is urgent to explore drug treatments targeting the key molecules driving the onset and progression of AIS, and a comprehensive etiological study is the only way to determine the key molecules driving the occurrence and development of the disease.

[0003] Corylin is an active ingredient from the traditional Chinese medicine Psoralea corylifolia, which can bind to HSP90β and reduce cholesterol synthesis. Avasimibe, as a cholesterol acyltransferase inhibitor, can enhance the intracellular degradation of APOB.

[0004] The present invention focuses on the significantly diseased bone and cartilage tissues in the spine, and determines that the key exosomal protein HSP90β (Heat Shock Protein HSP 90-beta) or APOB (Apolipoprotein B-100) is an effective target for clinically delaying the progression of AIS by studying the progression of Cobb angle in experimental AIS mouse models when Corylin and Avasimibe are used alone or in combination. Summary of the Invention

[0005] Aiming at the problems existing in the above-mentioned prior art, the purpose of the present invention is that HSP90β and / or APOB can be used as drug targets for treating the progression of adolescent idiopathic scoliosis.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] On the one hand, the present invention provides the use of HSP90β and / or APOB as targets in the preparation of drugs for treating scoliosis.

[0008] On the second hand, the present invention provides the use of HSP90β and / or APOB as targets in the preparation of drugs for treating AIS.

[0009] In the above use, the HSP90β is an exosome protein derived from chondrocytes.

[0010] In the above use, the HSP90β weakens the osteogenic differentiation and mineralization ability of AIS osteoblasts by promoting cholesterol biosynthesis, and at the same time excretes a large amount of cholesterol into the microenvironment.

[0011] In the above use, the APOB is an exosome protein derived from abnormally differentiated osteoblasts.

[0012] In the above use, the APOB promotes cholesterol influx into AIS intervertebral facet joint chondrocytes, and the increased intracellular cholesterol promotes the degradation of AIS cartilage matrix and maintains the activity of mTORC1 to accelerate the senescence of AIS chondrocytes.

[0013] On the third hand, the present invention provides the use of HSP90β inhibitors and / or APOB inhibitors in the preparation of drugs for treating AIS.

[0014] In the above use, the HSP90β inhibitor is Corylin.

[0015] In the above use, the APOB inhibitor is Avasimibe.

[0016] On the fourth hand, the present invention provides a pharmaceutical composition for treating AIS, which comprises an HSP90β inhibitor and / or an APOB inhibitor, and a pharmaceutically acceptable excipient.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] The target HSP90β and APOB protected by the present invention are key molecules driving the progression of AIS, and all means of inhibiting them can effectively delay the progression of AIS. The drugs protected by the present invention are drugs for scoliosis-modifying, and these drugs target the key molecules driving the progression of AIS, and are more targeted and specific than the existing physical therapies for AIS. Description of the Drawings

[0019] Figure 1In AIS, there are phenomena of cellular senescence and matrix degradation in the intervertebral facet joint cartilage, and the osteogenic differentiation and mineralization ability of the vertebrae at the corresponding anatomical positions are weakened; among them, (A) Schematic diagram of the sampling positions of cartilage and bone tissues, CC: concave side; CV: convex side; Up: upper vertebra; Apex: apical vertebra; Down: lower vertebra; (B) Results of the isolation and identification of primary cells, CH: chondrocytes; OB: osteoblasts; (C) Effect diagrams of SA-β-Gal and safranin O-fast green staining (SF) of chondrocytes; (D) Effect diagrams of ALP and ARS staining of osteoblasts;

[0020] Figure 2 For the results of the conditioned medium experiment, among them, (A) The supernatant of the intervertebral facet joint chondrocytes of the upper and lower vertebrae in AIS was used as the co-culture conditioned medium, and ALP and ARS staining were performed after co-incubation with normal osteoblasts; (B) Statistics based on A; (C) Western blot was used to verify the osteogenesis and bone mineralization markers Runx2 and Ocn of osteoblasts after co-incubation with the supernatant of the intervertebral facet joint chondrocytes of the upper and lower vertebrae in AIS as the co-culture conditioned medium; (D) The supernatant of the vertebral osteoblasts of the upper and lower vertebrae in AIS was used as the co-culture conditioned medium, and β-galactosidase staining and safranin O staining were performed after co-incubation with normal osteoblasts; (E) Statistics of the number of β-galactosidase staining-positive cells in D; (F) Western blot was used to detect the senescence markers P16 and P21 of chondrocytes after co-incubation with the supernatant of the vertebral osteoblasts of the upper and lower vertebrae in AIS as the co-culture conditioned medium and normal chondrocytes;

[0021] Figure 3Results of exosome research, where (A) Transmission electron microscopy images of chondrocyte exosomes and osteoblast exosomes; (B) Dynamic light scattering analysis of the particle sizes of chondrocyte exosomes and osteoblast exosomes, with peaks around 112.5 nm and 118.6 nm respectively; (C) Western blot verification of exosome markers HSP70 and CD81; (D) Laser confocal scanning microscopy observation of the internalization of chondrocyte exosomes and osteoblast exosomes by AIS osteoblasts and chondrocytes respectively; (E) Incubation of normal osteoblasts with conditioned medium from co-culture of normal chondrocytes (CH(Con)-CM), conditioned medium from co-culture of AIS upper vertebral chondrocytes (CH(CV-UP)-CM), exosomes derived from AIS upper vertebral chondrocytes (CH(CV-UP)-EVs), and exosome-free supernatant from AIS upper vertebral chondrocytes (CH(CV-UP)-CM(non-EVs)), followed by ALP staining and statistics after incubation; (L) Incubation of normal chondrocytes with conditioned medium from co-culture of normal osteoblasts (OB(Con)-CM), conditioned medium from co-culture of AIS upper vertebral osteoblasts (OB(CV-UP)-CM), exosomes derived from AIS upper vertebral osteoblasts (OB(CV-UP)-EVs), and exosome-free supernatant from AIS upper vertebral osteoblasts (OB(CV-UP)-CM(non-EVs)), followed by β-galactosidase staining, safranin O staining, and statistics of the number of β-galactosidase-positive cells after incubation;

[0022] Figure 4HSP90β in chondrocyte extracellular vesicles (CH-EVs) inhibits osteogenesis by promoting cholesterol synthesis while increasing cholesterol efflux in vertebral osteoblasts. Among them, (A) Bioinformatics analysis of the clustering heatmap of significant difference analysis of exosomes derived from intervertebral facet joint chondrocytes of the upper and lower vertebrae on the concave side of AIS; (B) Bioinformatics analysis of the clustering heatmap of significant difference analysis of exosomes derived from intervertebral facet joint chondrocytes of the upper and lower vertebrae on the convex side of AIS; (C) Bioinformatics analysis of the Venn diagram of differentially expressed genes in exosomes of intervertebral facet joint chondrocytes of the upper and lower vertebrae on both the concave and convex sides of AIS; (D) Western blot assay to detect the upregulation of HSP90β in chondrocyte exosomes CH-EVs; (E) ALP and ARS staining after osteoblasts were supplemented with human recombinant HSP90β; (F) Statistics based on E; (G) Western blot detection of bone formation and bone mineralization markers Runx2 and Ocn after overexpression of HSP90β and statistics; (H) Fluorometric analysis of total cholesterol content in AIS vertebral osteoblasts; (I) Western blot verification of cholesterol synthesis marker HMGCR and efflux markers ABCA1 and APOA1 in AIS vertebral osteoblasts; (J) Western blot verification of the promotion of cholesterol efflux (ABCA1, APOA1) by HSP90β in osteoblasts; (K) Western blot verification that HSP90β can inhibit osteoblast differentiation and mineralization (Rux2 and Ocn) by promoting cholesterol biosynthesis (HMGCR);

[0023] Figure 5For APOB in OB-EVs to exacerbate chondrocyte senescence and matrix degradation, (A) Bioinformatics analysis of the volcano plot of the significant difference analysis of AIS osteoblast exosomes; (B) Bioinformatics analysis of the cluster heat map of the significant difference analysis of AIS osteoblast exosomes; (C) Bioinformatics analysis of the scatter plot of the significant difference analysis of AIS osteoblast exosomes; (D) Verification of the upregulation of APOB in AIS vertebral osteoblast exosomes by Western blotting; (E) Results of fluorescence analysis of the cholesterol ester content in AIS chondrocytes; (F) Verification of the cholesterol synthesis marker HMGCR and the influx marker OLR1 in AIS chondrocytes by Western blotting; (G) Statistics based on F; (H) Western blotting verification of the mTORC1 activity (phosphorylated S6K), senescence markers (P16, P21), cholesterol metabolism markers CH25H, CYP7B1, RORA, and matrix degradation markers (ADAMTS5, MMP13) in AIS facet joint chondrocytes; (I) Statistics of the mTORC1 activity (phosphorylated S6K) and senescence markers (P16, P21) in AIS facet joint chondrocytes in H; (J) Statistics of the cholesterol metabolism markers CH25H, CYP7B1, RORA, and matrix degradation markers (ADAMTS5, MMP13) in AIS facet joint chondrocytes in H; (K) Verification of the expression of senescence markers P16 and P21 after rapamycin Rapa inhibits mTORC1 activity by Western blotting; (L) Statistics based on K; (M) Western blotting knockdown of CYP7B1 to inhibit the cholesterol metabolism pathway and observe the expression of chondrocyte matrix degradation markers ADAMTS5 and MMP13.

[0024] Figure 6 For the high-cholesterol microenvironment between AIS osteocytes and chondrocytes to drive the progression of idiopathic scoliosis in mice, HSP90β and APOB can be used as effective drug treatment targets. Among them, (A) X-ray scans of the mouse spine and Cobb angle measurement after single or combined treatment; (B) Determination of the optimal drug concentration of the HSP90β activity inhibitor Corylin on osteoblasts by CCK8; Verification of Corylin's ability to reverse the low osteogenesis caused by chondrocyte exosomes by Western blotting of bone formation and mineralization markers Runx2 and Ocn; Determination of the optimal drug concentration of the APOB degradation enhancer Avasimibe on chondrocytes by CCK8; Verification of Avasimibe's ability to reverse the senescence and matrix degradation caused by osteoblast exosomes by Western blotting of chondrocyte senescence markers P16, P21 and matrix degradation markers ADAMTS5, MMP13. Specific implementation manners

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments.

[0026] Example 1:

[0027] To verify whether cellular senescence occurs in the facet joint cartilage during the progression of AIS, the following experimental studies were conducted:

[0028] 1. Isolated and extracted the facet joint cartilage cells of AIS patients during spinal osteotomy orthopedic internal fixation fusion surgery, and performed primary culture and corresponding senescence phenotype and matrix degradation detection.

[0029] The specific process is as follows:

[0030] (1) In this experiment, cell cultures were performed on the facet joint cartilage tissue and vertebral bone tissue separated during spinal osteotomy orthopedic internal fixation fusion surgery. For the facet joint cartilage tissue, first rinse it 3 times with sterile 0.01M PBS buffer in a laminar flow hood, cut it into about 1mm 3 granules, then rinse with PBS, and then treat it successively with 0.25% trypsin solution and collagenase P. After each treatment, centrifuge and rinse with PBS. Finally, resuspend it with DMEM / F12 complete medium containing 10% fetal bovine serum, and place it in a 37°C, 5% CO2 incubator for 2 weeks to observe whether the typical polygonal morphology of chondrocytes appears; for the vertebral bone tissue, rinse it 3 times with sterile PBS buffer in a laminar flow hood, cut it into about 1mm 3 granules, rinse with PBS, and then resuspend it with DMEM / F12 complete medium containing 0.5 ng / mL D-valine and 10% fetal bovine serum, and place it in the same culture conditions for 1 week to observe whether the typical morphology of osteoblasts appears.

[0031] (2) β-galactosidase staining: Seed the primary chondrocytes at 3×10 4 cells / well in a 24-well plate and culture at 37°C / 5% CO2 for 48 hours; after washing with pre-cooled PBS, add β-Gal fixing solution at pH 6.0 and fix at room temperature for 15 minutes; wash with gradient PBS to remove residual fixing solution, add fresh staining reaction solution and incubate at 37°C in the dark for 16 - 18 hours; after washing with PBS, observe the blue-stained cells (β-Gal positive cells) under an optical microscope.

[0032] (3) Tissue safranin-fast green staining: After fixing the vertebral bone samples with 4% paraformaldehyde (4°C, 24 - 48 hours), decalcify them with EDTA (0.5M, pH 7.4, 25°C, 8 weeks), dehydrate them with gradient ethanol, clear them with xylene, impregnate them with paraffin and embed them. Prepare 4μm serial sections and bake them; after dewaxing and rehydrating, perform hematoxylin (nuclear staining), fast green (bone tissue) and safranin O (cartilage tissue) staining successively. Retain the staining specificity through differentiation with hydrochloric acid ethanol and dehydration with gradient ethanol, and finally mount with neutral balsam for observation. The cartilage appears red (safranin O), the bone tissue appears green (fast green), and the depth of red reflects the degree of cartilage loss.

[0033] The results are as Figure 1 shown in A, B, and C below. Compared with the normal control, obvious senescence and matrix degradation were observed in the facet joint cartilage of AIS. Moreover, regardless of the concave or convex side, the degree of chondrocyte senescence in the upper vertebra and apical vertebra of the main curve was much higher than that in the lower vertebra of the main curve.

[0034] 2. Detect the osteogenic differentiation and mineralization ability of osteoblasts at the corresponding positions.

[0035] Alkaline phosphatase (ALP) and alizarin red (ARS) staining: Seed 3×10 4 cells into a 24-well plate. After routine culture for 48 hours, change the medium to osteogenic induction medium (containing DMEM / F12, 10% FBS, 1% double antibody, 10 mM β-glycerophosphate, 50 μM ascorbic acid phosphate, and 0.1 μM dexamethasone), and change the medium every 3 days. For the ALP staining group, after 3 days of induction, wash with PBS, fix with 4% paraformaldehyde, stain with ALP working solution at room temperature in the dark for 3 hours, and observe under the microscope after terminating the reaction; for the ARS staining group, continuously induce for 14 days, fix, stain with 1% alizarin red S (pH 4.2) for 10 minutes, and observe the formation of calcium nodules under the microscope after washing with PBS.

[0036] The results are as Figure 1 shown in D below. Compared with normal osteoblasts, the ALP (alkaline phosphatase) staining and ARS (alizarin red) staining of osteoblasts from AIS patients incubated with osteogenic induction medium were significantly weakened. Moreover, regardless of the concave or convex side, the osteogenic differentiation ability of osteoblasts from the upper vertebra and apical vertebra of the main curve was significantly weaker than that of osteoblasts from the lower vertebra of the main curve.

[0037] In summary, the senescence and matrix degradation of AIS facet joint chondrocytes were aggravated, and the osteogenic differentiation and mineralization ability of osteoblasts in the corresponding vertebrae were weakened. The consistency of the pathological changes between the two suggests that there may be an interaction between them. In addition, the degree of pathological changes in the upper vertebra and apical vertebra of the main curve was similar, and there were significant differences compared with the pathological changes in the lower vertebra.

[0038] Example 2:

[0039] In this invention, through conditioned medium experiments and exosome research, the interaction between AIS facet joint chondrocytes and vertebral osteoblasts was verified. The specific experimental process is as follows:

[0040] 1. Experimental process:

[0041] (1) Co - culture of AIS intervertebral facet joint chondrocyte conditioned medium and normal osteoblasts: Select normal osteoblasts with good growth status and a density of 80% - 90%, and seed 4×10 4 cells per well into a 12 - well plate, and culture at 37℃ and 5% CO2 for 48 hours; at the same time, select well - grown AIS chondrocytes, wash them 3 times with PBS, and then culture them with DMEM / F12 complete medium for 48 hours. Aspirate the supernatant as the conditioned medium; after the normal osteoblasts are cultured for 48 hours, wash them with PBS, add 1 mL of the conditioned medium to each well, co - culture for 48 hours, and then perform subsequent detections.

[0042] (2) Co - culture of AIS vertebral osteoblast conditioned medium and normal chondrocytes: Select normal chondrocytes with good growth status and a density of 80% - 90%, and seed 4×10 4 cells per well into a 12 - well plate, and culture at 37℃ and 5% CO2 for 48 hours; select well - grown AIS osteoblasts, wash them 3 times with PBS, and then culture them with DMEM / F12 complete medium for 48 hours. Aspirate the supernatant as the conditioned medium; after the normal chondrocytes are cultured for 48 hours, wash them with PBS, add 1 mL of the conditioned medium to each well, co - culture for 48 hours, and then perform subsequent detections.

[0043] (3) Western blot detection of proteins: Use lysis buffer to fully lyse cells or exosomes, collect the samples in EP tubes, and treat them in a 100℃ metal bath thermostat for 10 minutes to denature the proteins; load the prepared samples for 70 - minute SDS - PAGE gel electrophoresis; after electrophoresis, transfer the membrane using a 0.45μm NC membrane; after membrane transfer, place the NC membrane in 5% skim milk and block it on a shaker at room temperature for 1 hour; after blocking, wash the membrane with TBST to remove the blocking solution; then immerse the NC membrane in the dilution containing the primary antibody and incubate it on a shaker at 4℃ overnight; recover the primary antibody, wash the membrane 3 times with TBST, 10 minutes each time; then prepare a secondary antibody with an appropriate concentration using the antibody dilution solution and incubate the secondary antibody at room temperature for 1 hour; then wash the membrane on a shaker with TBST 3 times, 10 minutes each time, and finally add chemiluminescent reagent and expose the bands using a chemiluminescent imager.

[0044] (4) Exosome isolation: After collecting 60 mL of cell supernatant cultured with exosome - free serum, perform gradient centrifugation in sequence: centrifuge at 800g for 10 minutes at 4℃ to remove suspended cells, and centrifuge at 2000g for 30 minutes to remove sub - cellular debris; filter the supernatant through a 0.22μm PVDF membrane, and then sediment exosomes by ultra - centrifugation (120000g, 4℃, 2 hours). Discard the supernatant, resuspend the precipitate with pre - cooled PBS and repeat ultra - centrifugation three times (parameters are the same above) to remove impurities; then resuspend it with pre - cooled PBS with the same volume as the initial culture medium and perform three - cycle ultra - centrifugation washes again. Finally, gently resuspend the precipitate with 1 mL of PBS.

[0045] (5) Identification of exosomes: Western blotting was used to verify the exosome markers HSP70 and CD81. The exosomes were diluted into three concentration gradients by a multiple of 10. 10 μL of each solution was added to the carbon-free aromatic membrane. After adsorption for 90 seconds, excess water was absorbed with filter paper and then allowed to dry naturally in the air. Then, the staining step was performed. 10 μL of phosphotungstic acid dye solution was added to the carbon-free aromatic membrane and placed in a cool place away from light for 5 minutes. Water was absorbed with filter paper and dried under a heat source lamp. Finally, the prepared sample was photographed to capture the exosomes as cup-shaped vesicles. Then, the exosomes were quantitatively characterized based on the light scattering characteristics of the nanoparticle tracking analysis (NTA) technology.

[0046] (6) Internalization of exosomes by primary cells: 100 μg of exosomes were mixed with 50 μL of PKH67 green fluorescent dye in the dark, vortexed for 60 seconds and then allowed to stand at room temperature for 10 minutes to construct a tracing system; the labeled exosomes were then co-cultured with primary cells at 37°C and 5% CO2 for 4-6 hours, the supernatant was discarded and washed three times with PBS; then the cells were co-incubated with DMEM / F12 complete medium containing 1:200 WGA555 in the dark for 10 minutes, and then washed again with PBS; finally, Hoechst33258 dye was added for 10 minutes of cell nucleus staining, and observation under a laser confocal fluorescence microscope showed that the green labeled exosomes were taken up by the cells. The whole process was carried out in the dark, and the key steps were to remove the unbound reagent by washing with PBS.

[0047] (7) Co-culture of AIS chondrocyte conditioned medium, exosomes and normal osteoblasts: Normal osteoblasts with good growth status were cultured at 4×10 4 The cells were seeded in 12-well plates at a density of 10 cells / mL and pre-cultured at 37°C and 5% CO2 for 48 hours. At the same time, the conditioned medium of normal chondrocytes and AIS upper vertebrae / apical vertebrae chondrocytes after washing with PBS three times and culturing with DMEM / F12 complete medium for 48 hours was collected, and the supernatant of AIS upper vertebrae / apical vertebrae chondrocytes after culturing with exosome-free serum medium for 48 hours was collected to separate the exosomes and retain the supernatant without exosomes. Subsequently, the original culture medium in the 12-well plate was discarded, washed with PBS three times, and the above-mentioned conditioned medium, exosome suspension and supernatant without exosomes were added respectively, and the co-culture was continued for 48 hours. Finally, the regulatory effect of chondrocyte-derived exosomes on osteoblast differentiation was analyzed by staining.

[0048] (8) Co-culture of AIS osteoblast-conditioned medium, exosomes and normal chondrocytes: Normal chondrocytes with good growth status were cultured at 4×10 4Cells were seeded in 12-well plates at a density of [number of cells] and cultured at 37°C and 5% CO2 for 48 hours. Meanwhile, conditioned media from normal osteoblasts and osteoblasts from the upper vertebra / apical vertebra of AIS after being washed 3 times with PBS and cultured in DMEM / F12 complete medium for 48 hours were collected. Additionally, the supernatant of osteoblasts from the upper vertebra / apical vertebra of AIS after being cultured in exosome-free serum medium for 48 hours was taken, and exosomes were isolated while retaining the exosome-free supernatant. Subsequently, the original culture medium in the 12-well plates was discarded, and after being washed 3 times with PBS, the above-mentioned conditioned media, exosome suspension, and exosome-free supernatant were added respectively, and co-culture was continued for 48 hours. Finally, the regulatory effect of osteoblast-derived exosomes on chondrocyte senescence was analyzed by detection.

[0049] The results are as Figure 2 shown in Figure 3, demonstrating that exosomes are the material basis for the interaction between the intervertebral facet cartilage and osteoblasts in AIS. Figure 2 A-C in it illustrate that the culture supernatant of senescent chondrocytes can inhibit osteoblast bone formation and mineralization. Figure 2 D-F in it illustrate that the culture supernatant of abnormally differentiated osteoblasts can accelerate chondrocyte senescence. Figure 3 A-D in it are for exosome identification. Figure 3 E and F in it illustrate that the material basis for the interaction between AIS cartilage and bone tissue is exosomes.

[0050] 2. In order to find the exosomal proteins that are highly circulated between prematurely senescent chondrocytes and functionally abnormal osteoblasts and serve as key molecules driving the progression of scoliosis, the following experimental tests were conducted:

[0051] Taking exosomes from the intervertebral facet cartilage or osteoblasts of the lower end vertebra (control group) of the main curve of the same patient as the control, the proteins enriched in exosomes from the corresponding upper vertebra and apical vertebra (lesion group) were analyzed by 4D / 480 label-free proteomics. The mass spectrometry data was compared with the "uniport_homo_20231008_20427_9606_swiss_prot" database. Proteins with a protein expression fold change greater than 2 or less than 0.5 and a p-value (Student’s t-test) less than 0.05 were considered differentially expressed proteins.

[0052] The results are as Figure 4 shown in A-D in it. Bioinformatics analysis found that exosomes secreted by overly senescent chondrocytes are rich in HSP90β (Heat Shock Protein HSP 90-beta), verifying the upregulation of HSP90β in CH-EVs. As Figure 5As shown in A-D, a large amount of APOB (Apolipoprotein B-100) is loaded in the exosomes secreted by abnormally differentiated osteoblasts, verifying that cholesterol synthesis in AIS chondrocytes is reduced and the influx is increased. Subsequent studies will focus on these two functional molecules.

[0053] 3. Experimental procedure:

[0054] (1) Human recombinant HSP90β at 700 ng / mL was added to osteoblasts and cultured at 37 °C under 5% CO2 for 48 hours, followed by ALP staining and ARS staining. After treatment under the same conditions, the cells were lysed thoroughly with lysis buffer, and the samples were collected for protein denaturation treatment. Western blot was used to detect the bone formation and bone mineralization markers Runx2 and Ocn, as well as the efflux markers ABCA1 and APOA1.

[0055] (2) Fluorometric assay for total cholesterol content in AIS vertebral osteoblasts: First, osteoblasts were seeded in 12-well plates at a density of 3×10 4 cells per well and cultured at 37 °C under 5% CO2 for 48 hours. After aspirating the culture medium, the cells were washed 1-2 times with PBS, and 100 μL of pre-cooled BeyoLysis TM Buffer A lysis buffer (operated on ice throughout the process) was added to each well. After gentle pipetting, the mixture was transferred to a 1.5 mL centrifuge tube. According to the Amplex Red kit instructions, the working solutions for total cholesterol and free cholesterol detection were prepared respectively, and a cholesterol concentration standard curve was established. After mixing the lysed samples with the working solutions, the mixture was incubated at 37 °C in the dark for 30 minutes, and the absorbance was measured at a wavelength of 570 nm. The total cholesterol content was calculated through the standard curve.

[0056] (3) Osteoblasts from the concave and convex sides of AIS vertebrae were isolated and cultured at 37 °C under 5% CO2 for 48 hours. Western blot was used to detect the cholesterol synthesis marker HMGCR and the cholesterol efflux markers ABCA1 and APOA1. Under the same culture conditions, 250 nM Lovastain was added. After culturing for 48 hours, the cells were collected, lysed thoroughly with lysis buffer, and the samples were collected for protein denaturation treatment. Western blot was used to detect the cholesterol synthesis marker HMGCR and the bone formation and bone mineralization markers Runx2 and Ocn.

[0057] The results are as Figure 4As shown in E-K, exosomes secreted by AIS-overaged chondrocytes transport HSP90β to osteoblasts, leading to an increase in its concentration by promoting intracellular cholesterol biosynthesis. Administration of the cholesterol synthesis inhibitor Lovastatin demonstrated that HSP90β can weaken osteoblast differentiation and mineralization ability by promoting cholesterol biosynthesis. In addition, the expression levels of cholesterol efflux-related proteins APOA1 and ABCA1 were upregulated in AIS primary osteoblasts, indicating an increase in cholesterol efflux in abnormally differentiated osteoblasts. It can be seen that the exosomal protein HSP90β derived from over-aged chondrocytes weakens the osteogenic differentiation and mineralization ability of AIS osteoblasts by promoting cholesterol biosynthesis, while a large amount of cholesterol is excreted into the microenvironment.

[0058] Example 3:

[0059] By detecting the key proteins of cholesterol concentration, synthesis (HMGCR) and influx (APOB, OLR1) in AIS zygapophyseal joint chondrocytes, it was verified whether cholesterol in the microenvironment enters chondrocytes.

[0060] The specific process is as follows:

[0061] (1) Detection of total cholesterol content in AIS zygapophyseal joint chondrocytes by fluorescence method: First, chondrocytes were seeded in a 12-well plate at a density of 3×10 4 cells per well and cultured at 37 °C and 5% CO2 for 48 hours; after aspirating the culture medium, the cells were washed 1-2 times with PBS, and 100 μL of pre-cooled BeyoLysis TM Buffer A lysis buffer (operated on ice throughout the process) was added to each well, gently pipetted, and then transferred to a 1.5 mL centrifuge tube; according to the Amplex Red kit instructions, the working solutions for detecting total cholesterol and free cholesterol were prepared respectively, and a cholesterol concentration standard curve was established; after the lysed samples were mixed with the working solutions, they were incubated at 37 °C in the dark for 30 minutes, and the absorbance was measured at a wavelength of 570 nm; the total cholesterol content was calculated through the standard curve

[0062] (2) Isolate and extract AIS zygapophyseal joint chondrocytes on both the concave and convex sides and culture them at 37 °C and 5% CO2 for 48 hours. Protein immunoblotting was used to detect the cholesterol synthesis marker HMGCR, the cholesterol influx markers APOB and OLR1, the mTORC1 activity marker P-S6K and the senescence markers P16 and P21, the cholesterol metabolism markers CH25H, CYP7B1, RORA, and the matrix degradation markers ADAMTS5 and MMP13.

[0063] (3) Isolate and extract primary mouse chondrocytes: Select neonatal mice aged 1 - 7 days. After decapitation and 5 - minute disinfection with 75% alcohol, transfer them to a laminar flow hood and aseptically isolate the rib cage and remove membranous tissues; Cut the rib cage into 1 mm 3 granules, and wash them 3 times with PBS (centrifugation at 1000 rpm for 5 min); Add 2 volumes of collagenase P (prepared with basal medium), and digest at 37°C on a shaker for 4 - 6 hours; Filter through a 120 - mesh sieve and then centrifuge (1000 rpm for 5 min). After discarding the supernatant, rinse twice with PBS, resuspend the cells in DMEM / F12 complete medium containing 10% fetal bovine serum, seed them in a 6 - well plate, and culture at 37°C and 5% CO2 for 48 hours to observe the morphology of typical polygonal chondrocytes.

[0064] (4) After culturing primary mouse chondrocytes for 48 hours, add 50 μM cholesterol to simulate the chondrocyte state in AIS, add 100 nM Rapa to inhibit the activity of mTORC1, culture the cells at 37°C and 5% CO2 for 48 hours, then collect the cells, lyse the samples and denature them, and use Western blot to detect the mTORC1 activity marker P - S6K and the senescence markers P16 and P21.

[0065] (5) Twenty - four hours before transfection, seed primary mouse chondrocytes into a 24 - well plate at 500 μL / well (medium without antibiotics) to achieve a density of 30% - 50% at the time of transfection; When preparing the transfection complex, dilute siRNA - CYP7B1 (siRNA concentration 20 μM) and 2 μL of Lipo2000 separately with 50 μL of serum - free basal medium, mix the two after standing at room temperature for 5 minutes, and continue to incubate for 20 minutes to form the complex; Add 100 μL of the complex to the wells, gently shake and mix, and culture at 37°C and 5% CO2 for 4 - 6 hours, then replace with complete medium. As above, construct a high - cholesterol chondrocyte model, continue to culture for 48 hours, collect the cells, and use Western blot to detect the matrix degradation markers ADAMTS5 and MMP13.

[0066] The results are as Figure 5As shown in E-M, the cholesterol level in chondrocytes increases, but its synthesis decreases while the influx increases, indicating that the cholesterol excreted by osteoblasts enters chondrocytes. High concentrations of cholesterol can drive an increase in catabolism of the OA phenotype, such as promoting OA matrix degradation through the cholesterol metabolic pathway CH25H-CYP7B1-RORα axis and accelerating cell senescence by maintaining the activity of mTORC1. The exosomal protein APOB derived from abnormally differentiated osteoblasts promotes cholesterol influx into chondrocytes of the AIS intervertebral facet joints to increase intracellular cholesterol concentration, and the increased cholesterol promotes AIS cartilage matrix degradation through the CH25H–CYP7B1–RORα axis and maintains the activity of mTORC1 to accelerate the senescence of AIS chondrocytes.

[0067] Since, in the spine, in addition to the intervertebral facet joint cartilage, there are also intervertebral discs and vertebral growth plate cartilage that transmit 70%-80% of the load, and they have a high degree of consistency in the pathological changes of OA, it is considered that the above-mentioned positive feedback loop mediated by exosomes characterized by high cholesterol flux exists in all adjacent bones and cartilages of the AIS spine.

[0068] Example 4:

[0069] As confirmed above, the positive feedback regulation between bone and cartilage in the spine mainly involves exosome-mediated cholesterol synthesis, excretion, and influx. To verify the important role of high cholesterol flux in this microenvironment in the progression and aggravation of AIS, we constructed a recombinant adeno-associated virus rAAV9-RUNX2-HMGCR, and the construction process is as follows:

[0070] rAAV9 can successfully target and infect osteoblasts, and the RUNX2 promoter further ensures the expression of the key cholesterol synthesis gene HMGCR in osteoblasts to achieve increased cholesterol synthesis in osteoblasts. In theory, increased cholesterol synthesis in osteoblasts can lead to abnormal osteogenic differentiation ability, and the exosomes rich in APOB secreted by them can accelerate cholesterol influx into chondrocytes, thereby inducing OA-like changes in cartilage. The cartilage with aggravated OA-like changes secretes exosomes rich in HSP90β and upregulates the synthesis and excretion of cholesterol in osteoblasts, and the finally formed positive feedback loop accelerates the progression of AIS.

[0071] Furthermore, after injecting the recombinant adeno-associated virus rAAV9-RUNX2-HMGCR into C57BL / 6J mice and supplementing with double hindlimb standing to simulate the human vertical stress pattern, an experimental AIS mouse model was established. The therapeutic effects of Corylin or Avasimibe were verified in the established experimental AIS mouse model and human AIS primary cells.

[0072] The specific process is as follows:

[0073] (1) Establishment of an experimental AIS mouse model: C57BL / 6J mice at 2 weeks of age were selected for modeling. Adeno-associated virus AAV9-runx2-HMGCR overexpressing HMGCR and control virus (n = 5) were intraperitoneally injected respectively. Injection dose: 7.5×10 13 vg / kg (AAV9-runx2-NC), 7.5×10 13 vg / kg (AAV9-runx2-HMGCR), 200 μL per mouse. From the 4th week, the mice stood on their two hind legs in water (8 hours per day) to simulate the vertical stress pattern of humans, and the Cobb angle changes were observed by X-ray scanning every two weeks.

[0074] (2) Pharmacodynamic verification of Corylin (Cor) and Avasimibe (Ava): C57BL / 6J mice at 2 weeks of age were selected. A model was constructed by intraperitoneally injecting adeno-associated virus AAV9-runx2-HMGCR overexpressing HMGCR, and the double hind limb standing model was combined for intervention 2 weeks after injection. The mice were divided into five groups (n = 5 / group): normal saline control, AAV model group, AAV+Cor (30 mg / kg), AAV+Ava (15 mg / kg), AAV+Cor+Ava combination administration group, and gavaged once every two days (200 μL / mouse). The mice started standing at the 4th week, and the Cobb angle was observed by X-ray scanning every two weeks to evaluate the therapeutic effect of the drug on skeletal abnormalities induced by HMGCR overexpression.

[0075] The results are as Figure 6 shown in A and B below. The positive feedback regulation mediated by exosomes between cartilage and bone tissues in the spine, characterized by high cholesterol flux, is a key factor in the progression and aggravation of AIS. Targeting the key exosome proteins HSP90β or APOB as drug treatment targets can effectively delay the progression of AIS. Figure 6 (A) X-ray scanning and Cobb angle measurement of the spine of mice intraperitoneally injected with rAAV9-RUNX2-HMGCR and gavaged with Avasimibe (15 mg / kg), Corylin (30 mg / kg), alone or in combination. (B) The drug concentration of Corylin was determined by CCK8 to be 30 μM; Western blot verified that Corylin could reverse the low bone mass caused by HSP90β in CH-EVs; the drug concentration of Avasimibe was determined by CCK8 to be 2 μM; Western blot verified that Avasimibe could reverse the effect of APOB on chondrocyte senescence and matrix degradation in OB-EVs.

[0076] In summary, the exosome-mediated positive feedback regulation between bone and cartilage in AIS spine may be a key factor promoting the progression of AIS. Targeting the key exosomal proteins HSP90β and APOB breaks this positive feedback loop of pathological exacerbation and delays the progression of AIS.

Claims

1. Use of HSP90β and / or APOB as targets in the preparation of a medicament for treating scoliosis.

2. Use of HSP90β and / or APOB as targets in the preparation of a medicament for treating AIS.

3. The application according to claim 2, characterized in that, The HSP90β is an exosome protein derived from chondrocytes.

4. The application according to claim 2, characterized in that The HSP90β weakens the osteogenic differentiation and mineralization ability of AIS osteoblasts by promoting cholesterol biosynthesis, and at the same time excretes a large amount of cholesterol into the microenvironment.

5. The application according to claim 2, characterized in that, The APOB is an exosome protein derived from abnormally differentiated osteoblasts.

6. The application according to claim 2, wherein The APOB promotes the influx of cholesterol in the microenvironment into chondrocytes, and the increased cholesterol in the cells promotes the degradation of the AIS cartilage matrix and maintains the activity of mTORC1 to accelerate the senescence of AIS chondrocytes.

7. Use of an HSP90β inhibitor and / or an APOB inhibitor in the preparation of a medicament for treating AIS.

8. The application according to claim 7, characterized in that, The HSP90β inhibitor is Corylin.

9. The application according to claim 7, characterized in that, The APOB inhibitor is Avasimibe.

10. A pharmaceutical composition for treating AIS, characterized in that, Comprising an HSP90β inhibitor and / or an APOB inhibitor, and a pharmaceutically acceptable excipient.

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