Application of AGP2 in preparation of medicine for promoting osteogenesis and relieving osteoporosis

Abstract

The invention belongs to the technical field of medicine research and development. The invention finds that overexpression of AGP2 in the liver can promote osteogenesis and relieve the progress of osteoporosis. Therefore, the invention provides the application of the AGP2 and the substance capable of over-expressing the AGP2 in the liver in the aspect of preparing the medicine for promoting osteogenesis and relieving the osteoporosis, and the application range of the AGP2 and the range of the medicine capable of treating the osteoporosis are expanded.

Description

Technical Field

[0001] The present invention belongs to the technical field of drug research and development, and specifically relates to the application of AGP2 in the preparation of drugs for promoting osteogenesis and alleviating osteoporosis. Background Art

[0002] Osteoporosis is a systemic bone disease caused by various reasons, mainly characterized by decreased bone density and bone mass, damaged bone microstructure, increased bone fragility, and a state prone to fractures. Common drugs for treating osteoporosis include alendronate sodium, zoledronic acid, risedronate ammonium, etidronate disodium, etc.

[0003] In addition to developing new drugs, progress has also been made in screening substances with activity against osteoporosis within the range of known substances. For example, Guo Donggui et al. provided the application of Mulberrin in the preparation of anti-osteoporosis drugs in Chinese patent application CN110051659A; Wang Huijuan et al. provided the application of Cyclomorusin in the preparation of anti-osteoporosis drugs in Chinese patent CN115381811B.

[0004] However, although a variety of substances that can be used to treat osteoporosis have been discovered, these discoveries are still not sufficient for people to summarize a reliable path for discovering new active substances, making it difficult to discover substances that can be used to treat osteoporosis among existing substances. Summary of the Invention

[0005] Aiming at the deficiencies of the prior art, the purpose of the present invention is to provide new substances for promoting osteogenesis and alleviating osteoporosis and their application in the preparation of drugs for promoting osteogenesis and alleviating osteoporosis.

[0006] To achieve this technical purpose, the present invention discovers through research that AGP2 can promote osteogenesis and alleviate osteoporosis. Thus, the technical solutions provided by the present invention are as follows:

[0007] The application of AGP2 in the preparation of drugs for promoting osteogenesis and alleviating osteoporosis.

[0008] Optionally, the AGP2 is applied in the form of any pharmaceutically acceptable preparation.

[0009] Preferably, the preparation is an injection.

[0010] The application of a substance capable of overexpressing AGP2 in the liver in the preparation of drugs for promoting osteogenesis and alleviating osteoporosis, wherein the AGP2 is α1-acid glycoprotein 2.

[0011] Optionally, the substance capable of overexpressing AGP2 in the liver is applied in the form of any pharmaceutically acceptable preparation.

[0012] Preferably, the preparation is an injection.

[0013] AGP2 is the abbreviation of α1-acid glycoprotein 2, and α1-acid glycoprotein 2 is also known as orosomucoid 2 (ORM2).

[0014] Currently, there are many studies on the function of α1-acid glycoprotein (or ORM 1), such as Lei Hong [1] found that ORM 1 can act as an endogenous anti-fatigue protein and may also serve as a diagnostic marker for chronic fatigue syndrome; He Zhengxin et al. [2] found that α1-acid glycoprotein can be used for the diagnosis of rheumatoid arthritis; Adila Sulitan et al. [3] found that detecting the changes in blood lipids and α1-acid glycoprotein can be an important means to predict the treatment effect of MM patients;

[0015] AGP2 (or α1-acid glycoprotein 2, ORM2) is different from α1-acid glycoprotein (or ORM 1) and is another substance. Regarding the research on the new medical uses of this substance, as far as the inventors know, there are relatively few existing studies, and sporadic reports can be found in Chinese patents CN109490556B, CN113358870B, CN114075600B and the paper reported by Wang Yupeng et al. [4]. However, these studies all focus on AGP2 in the preparation of disease diagnosis products and lack the aspect of preparing drugs for treating related diseases.

[0016] Advantages of the present invention:

[0017] The present invention discovers that overexpressing AGP2 in the liver can promote osteogenesis and relieve the progression of osteoporosis. Therefore, the present invention provides the application of AGP2 and the substance capable of overexpressing AGP2 in the liver in the preparation of drugs for promoting osteogenesis and relieving osteoporosis, expanding the scope of uses of AGP2 and the scope of drugs for treating osteoporosis.

[0018] The prior art cited in this part:

[0019] [1] Lei Hong. New Discoveries of Alpha-1 Acid Glycoprotein in Fatigue [D]. Second Military Medical University, 2011. DOI: 10.7666 / d.d151620.

[0020] [2] He Zhengxin, Chen Xing, Wang Wei, et al. Value of α1-acid glycoprotein in the diagnosis of rheumatoid arthritis [J]. Hebei Medical Journal, 2013, 35(003): 404-405. DOI: 10.3969 / j.issn.1002-7386.2013.03.047.

[0021] [3] Adila Sultana, Liao Hongli. Significance of detecting blood lipid and serum α1-acid glycoprotein in patients with multiple myeloma [J]. Journal of Modern Laboratory Medicine, 2015(5): 2. DOI: 10.3969 / j.issn.1671-7414.2015.05.054.

[0022] [4] Wang Yupeng, Song Kai, Sun Yuansong, Jiang Datong, Li He. Predictive value of serum AGP2, LBP and PCT for the severity of patients with acute pancreatitis [J]. Journal of Hepatobiliary Surgery, 2023, 31(5): 349-355. Brief Description of the Drawings

[0023] Figure 1 It is a diagram of the experimental results of the effect of liver-specific knockout of MCT1 on bone formation;

[0024] Figure 2 It is a diagram of the experimental results of proteomics and Cut&Tag sequencing identification of the present invention;

[0025] Figure 3 It is a diagram of the experimental results of the effect of H3K14 lactylation in hepatocytes on AGP2 expression;

[0026] Figure 4 It is a diagram of the experimental results of overexpressing AGP2 in the liver on osteogenesis and in the treatment of osteoporosis;

[0027] Figure 5 It is a diagram of the results of the expression of AGP2 within 24 hours after exercise;

[0028] Figure 6 It is a diagram of the experimental research results of the mechanism of AGP2 in promoting osteogenesis. Detailed Implementation Modes

[0029] The present invention will be specifically described below through examples. It is necessary to point out here that the following examples are only used to further illustrate the present invention and cannot be understood as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art according to the above invention content still fall within the protection scope of the present invention.

[0030] Example 1

[0031] I. Experimental Methods

[0032] 1. HIIT training for mice:

[0033] (1) One week before the experiment, endurance training for mice was carried out. The experimental mice were placed on a treadmill and trained starting from a speed of 5 m / min. The speed was adjusted every 5 minutes and gradually increased to the maximum speed of 30 m / min, which was used as the HIIT training speed.

[0034] (2) The mice were randomly divided into an experimental group and a control group, with 5 mice in each group. The experimental group underwent HITT training. Before each experiment, they were warmed up at a speed of 5 m / min. There were 10 cycles of HIIT (running for 4 minutes and resting for 2 minutes), continuously for 4 weeks.

[0035] (3) Orbital blood of mice was extracted using a blood sampling puncture needle. Approximately 500 μl of blood was taken from each mouse. After blood sampling, it was immediately centrifuged at 2000 rpm / min. After centrifugation, the upper serum was extracted for lactic acid concentration determination and Elisab determination; Sodium pentobarbital at 5% (w / v) was diluted 10-fold (note that it was filtered and sterilized after dilution), and all the mice in the experiment were anesthetized at a dose of 0.1 ml / 10 g; (6) After the mice fell asleep, they were quickly sacrificed, and the brains, hearts, lungs, livers, spleens, kidneys, muscles, femurs, and tibias of the mice were removed for subsequent experiments.

[0036] 2. Preparation of homogenates of various tissues of mice

[0037] First, the tissue blocks were transferred into a glass homogenizer, and 5 - 10 ml of pre-cooled PBS (the mass-volume ratio of tissue to PBS was 1:5) was added and thoroughly ground using a tissue grinder. This process was carried out on ice; the obtained homogenate was then ultrasonically disrupted. The prepared homogenate was centrifuged at 5000×g for 5 minutes, and the supernatant was retained for detection.

[0038] 3. Determination of the content of AGP2 in serum by Elisa

[0039] (1) Take out the ELISA test kit (from the enzyme immunoassay company, mouse AGP2, product number MM-4681M1) from the refrigerator; (2) Equilibrate all reagents in the kit to room temperature (to increase the experimental accuracy); (2) Prepare according to the instructions: a. 330-fold concentrated washing solution; b. enzyme-labeled reagent; c. enzyme-coated plate; d. sample diluent; e. chromogenic agent A solution; chromogenic agent B solution; f. termination solution; g. standard product (3200 μg / L); h. standard product diluent, and mix all reagents thoroughly (no bubbles are allowed); (3) Dilute the serum to be tested according to the instructions (it was found in the preliminary experiment that a 1:50 dilution is just right); (4) Add samples: Set up blank wells (no samples and enzyme-labeled reagents are added to the blank control wells, and the same operations are performed in the following steps), standard wells, and wells for samples to be tested. Accurately add 50 μl of the standard product to the standard wells on the enzyme-coated plate. First, add 40 μL of the sample diluent to the wells for samples to be tested, and then add 10 μl of the sample to be tested (the final dilution factor of the sample is 5 times). When adding the sample, add it to the bottom of the wells on the enzyme plate, try not to touch the well walls, and gently shake to mix evenly. (5) Incubate: Seal the plate with a sealing film and incubate at 37°C for 30 minutes. (6) Prepare the solution: Dilute the 30-fold concentrated washing solution 30-fold with distilled water and set aside for later use. (7) Wash: Carefully remove the sealing film, discard the liquid, shake dry, fill each well with the washing solution, let it stand for 30 seconds and then discard, repeat this 5 times, and pat dry. (8) Add enzyme: Add 50 μl of the enzyme-labeled reagent to each well except the blank well. (9) Incubate: The operation is the same as in step 3. (10) Wash: The operation is the same as in (7). (11) Develop color: First add 50 μl of chromogenic agent A to each well, then add 50 μl of chromogenic agent B, gently shake and mix evenly, and develop color at 37°C in the dark for 10 minutes. (12) Terminate: Add 50 μl of the termination solution to each well to terminate the reaction (at this time, the blue immediately turns yellow). (13) Measure: Zero with the blank well, and sequentially measure the absorbance (OD value) of each well at a wavelength of 450 nm. The measurement should be carried out within 15 minutes after adding the termination solution. (14) Use the concentration of the standard substance as the abscissa and the OD value as the ordinate, draw a standard curve on the coordinate paper, find the corresponding concentration from the standard curve according to the OD value of the sample; then multiply by the dilution factor; or calculate the linear regression equation of the standard curve with the concentration of the standard substance and the OD value, substitute the OD value of the sample into the equation, calculate the sample concentration, and then multiply by the dilution factor to obtain the actual concentration of the sample.

[0040] 4. Perform Micro-CT scanning on the tibia of the mouse.

[0041] 5. Perform a three-point bending stress experiment on the femur of the mouse.

[0042] 6. HE staining:

[0043] Rinse the obtained bone tissue twice in PBS. (1) Use clean ophthalmic scissors to clean the soft tissue on the surface of the bone tissue, and immerse it in 10% paraformaldehyde (4% formalin can also be used) solution for 24 hours; (2) Decalcify with EDTA decalcifying solution for 7 days. Mark the decalcified bone tissue and put it into a disposable wax box, and operate according to the following steps: 70% Ethanol for 60 minutes, 80% Ethanol for 60 minutes, 90% Ethanol for 60 minutes, 95% Ethanol for 60 minutes, Ethanol for 40 minutes, Ethanol for 40 minutes, Xylene for 40 minutes, Xylene for 40 minutes, Paraffin for 60 minutes, Paraffin for 60 minutes, Paraffin for 60 minutes, and embed after impregnation with wax; (3) Cut 5-15 μm sections from the embedded tissue wax block and dry the sections (1 hour is enough); (4) If baking the sections, place the sections in an oven at 65 °C for 50 minutes and operate according to the following procedure: Xylene for 10 minutes, Xylene II for 8 minutes, 95% Ethanol for 1 minute (repeat 95% Ethanol for 1 minute again), 80% Ethanol for 1 minute, rinse with tap water for 5 minutes; (5) Stain briefly with hematoxylin (about 5-10 seconds); (6) Rinse the sections with double-distilled water (until colorless to the naked eye); (7) Differentiate with 1% ethanol hydrochloride for 1 second; (8) Wash with double-distilled water for 1 minute and stain with 1% eosin for 1 minute; (9) Wash with double-distilled water for 1 minute, 95% Ethanol for 1 minute, 95% Ethanol for 1 minute, Ethanol for 1 minute, Ethanol for 1 minute, Xylene for 1 minute, Xylene for 1 minute, and after draining, seal the sections with neutral gum overnight and observe.

[0044] 7. IF staining of liver tissue (AGP2 and Pan-kla)

[0045] (1) Dewax the paraffin sections to water: Place the sections in xylene-115min, xylene-215min, 100% anhydrous ethanol for 5min, 95% anhydrous ethanol for 5min, 85% alcohol for 5min, 75% alcohol for 5min, and wash with PBS for 5min. (2) Antigen repair: Place the tissue sections in a repair box filled with EDTA antigen repair buffer (pH=8.0) in a microwave oven for antigen repair, high heat for 4min, medium heat for 15min. During this process, prevent the buffer from evaporating excessively and do not dry the sections. After natural cooling, place the slides in PBS and shake on a decolorizing shaker for 3 times, 5min each time. (The repair solution and repair conditions are determined according to the tissue). (3) Draw a circle around the tissue with a tissue pen to prevent the antibody from flowing away. Add autofluorescence quencher A solution in the circle for 30min, rinse with pure water for 5min, B solution for 5min, and rinse with running water for 3min. (4) Serum blocking: Add BSA to the circle for 30min. (5) Adding primary antibody: shake off the blocking solution, add PBS to the slices according to a certain ratio of primary antibody, place the slices flat in a humidified box, and incubate at 4°C overnight. (Add a small amount of water in the humidified box to prevent the antibody from evaporating). (6) Adding secondary antibody: place the slices in PBS (PH7.4) and shake on a decolorizing shaker to wash 3 times for 5 minutes each time. After the slices are slightly dried, add the secondary antibody of the same species as the primary antibody to cover the tissue in the circle, and incubate at room temperature in the dark for 50 minutes. (7) DAPI counterstaining of cell nuclei: place the slides in PBS and shake on a decolorizing shaker to wash 3 times for 5 minutes each time. (8) After the slices are shaken dry, add DAPI staining solution in the circle, and incubate at room temperature in the dark for 10 minutes. (9) Sealing: place the slides in PBS (PH7.4) and shake on a decolorizing shaker to wash 3 times for 5 minutes each time. After the slices are slightly dried, seal them with anti-fluorescence quenching sealing agent. (10) Microscopic examination and photography: observe the slices under a fluorescence microscope and collect images.

[0046] 8. Liver proteomics analysis

[0047] The samples were sent to Shanghai Ouyi Biomedical Technology Co., Ltd. for determination.

[0048] 9. Liver Tissue Protein Extraction

[0049] Operate according to the instruction manual of the Biyuntian Tissue Lysis Kit: (1) Wash the freshly cut liver twice with PBS. Use clean ophthalmic scissors to cut 50 mg of liver tissue and cut it into small pieces. (2) Thaw the RIPA lysis buffer, mix well. Take an appropriate amount of the lysis buffer and add PMSF within a few minutes before use to make the final concentration of PMSF 1 mM, or add the appropriate above protease phosphatase inhibitor mixture according to experimental needs. (3) Add the lysis buffer according to the ratio of adding 150 - 250 μl of lysis buffer per 20 mg of tissue. (If the lysis is insufficient, more lysis buffer can be added appropriately. If a high-concentration protein sample is required, the amount of lysis buffer can be reduced appropriately.) (4) Grind with a tissue grinder until fully lysed. The tissue sample can also be frozen and ground in liquid nitrogen. After sufficient grinding, add the lysis buffer for lysis. (5) After sufficient lysis, centrifuge at 10,000 - 14,000 g for 3 - 5 minutes, take the supernatant, and subsequent PAGE, Western, and immunoprecipitation operations can be carried out; the protein concentration of the supernatant obtained by lysing 20 mg of frozen mouse liver tissue with 200 μl of this lysis buffer is approximately 15 - 25 mg / ml, which varies for different tissues in different states. (6) Dissolve the SDS-PAGE loading buffer (5X) in a water bath at room temperature or not exceeding 37°C. After water bath dissolution, store at room temperature immediately, and try to avoid placing it in the water bath for a long time. (7) Mix the protein sample and the protein loading buffer (5X) according to the ratio of adding 1 μl of the protein loading buffer (5X) per 4 μl of the protein sample. (8) Heat at 100°C or in a boiling water bath for 3 - 5 minutes to fully denature the protein.

[0050] 10. Detect the expression of the ORM2 gene in tissues or cells by fluorescence real-time quantitative PCR technology:

[0051] (1) Extraction of total RNA from tissues and cells. It is necessary to obtain cells in good growth condition, which should be in the logarithmic growth phase; (2) Digest the cells with trypsin and finally collect them into a 1.5 ml centrifuge tube; (3) Wash 3 times with PBS (completely remove the serum) and add 1 ml of TRIzol reagent; (4) Repeatedly aspirate with a 1 ml pipette tip (RNase-free) until no cell precipitate can be seen; (5) Use a vortex oscillator to shake vigorously until it turns white, and let it stand at room temperature for 10 minutes; (6) If tissue specimens are used, add 1 ml of Trizol reagent according to the ratio of 50 mg of tissue per 1 ml, and the rest of the operations are the same as above; (7) Add 0.2 ml of chloroform to every 1 ml of Trizol (extract RNA), and use a vortex oscillator to shake vigorously for 15 seconds; (8) Let it stand in a small tube (at room temperature) for 15 minutes; (9) Centrifuge at 12,000 rpm at 2 - 8°C for 20 minutes; (10) Carefully transfer the upper aqueous phase (about 0.5 ml) to another treated EP tube; (11) Add an equal volume (about 0.5 ml) of isopropanol, shake vigorously at room temperature, and let it stand for 10 minutes; (12) Centrifuge at 12,000 rpm at 2 - 8°C for 15 minutes; (13) Discard the supernatant (RNA adheres to the inclined plane of the tube wall), add 1 ml of 75% ethanol (prepared with DEPC water) to wash the white flocculent precipitate; (14) Centrifuge at 7,500 rpm at 2 - 8°C for 2 minutes; (15) Carefully suck dry the supernatant, open the lid and expose it to the air, and dry for about 5 - 10 minutes; (16) Add 40 μl of DEPC water to dissolve the precipitated RNA (the dissolved water can be preheated to 65°C in advance); (17) Measure the RNA concentration; (18) Prepare an agarose gel (concentration of 2%), and use nucleic acid electrophoresis to detect the quality of the RNA sample; (19) Use deionized water to zero, measure the absorbance of the sample, and calculate the concentration of total RNA; (20) If the absorbance A260 / 280 ratio is between 1.8 - 2.0, the sample is judged to be qualified; (21) The RNA running gel band has 3 bands (sometimes 2 bands) without smear is qualified; (22) Aliquot the samples and make good marks; (23) Can be stored in a -80°C refrigerator for a long time; if stored at -20°C, use it as soon as possible for subsequent experiments.

[0052] 11. Reverse transcription of total mRNA into cDNA

[0053] Strictly follow the reagent instructions (MCE, RT Master Mix for qPCR II (gDNA digesterplus, HY- K0511A) Perform the following operations: (1) Remove the residual DNA molecules in the total RNA and prepare the experimental reaction system: a. 3 μl of 5× gDNA digester Mix; b. 50 pg - 5 μg / 50 pg - 500 ng of Total RNA / mRNA; c. Supplement with RNase-Free H2O to 15 μl. The operation of preparing the reaction solution should be carried out on ice. (2) Gently pipette and mix well, and incubate at 42 °C for 2 mins. (3) Prepare the reverse transcription reaction system (20 μL system), directly add 4× Super RT Mix to the reaction tube in step 1, and gently pipette and mix well μl; (4) Set the program: a. 25 °C, 15 mins; b. 55 °C, 15 mins; c. 85 °C for 2 min; (5) Dilute the reverse transcription product 4 - 5 times with deionized water and store at -20 °C for later use or use immediately.

[0054] 12. Operation of real-time quantitative fluorescence PCR (qRT-PCR)

[0055] (1) Prepare under light protection: a. 10 μl of SYBR-Green qPCR Mix; b. 0.4 μl of Forward Primer; c. 0.4 μl of Reverse Primer; d. 2 μl of cDNA; e. 7.2 μl of deionized water, totaling 20 μl; (3) Add 3 replicate wells for each sample and perform the operation on the machine; (4) Reaction program: a. 95 °C, pre-denaturation for 5 minutes; b. 95 °C, denaturation for 10 seconds; c. 57.5 °C, annealing; d. 72 °C, extension, for 20 seconds; e. The process of b - d is repeated for 39 cycles; f. 95 °C, denaturation for 10 seconds; g. 65 °C for 5 s to 95 °C for incremental reading; h; (5) Statistical analysis method: Use the 2-ΔΔCt method to convert the gene expression level into a fold change, and perform data sorting and analysis for easy result observation; (6) Ct value, the value of the number of cycles experienced by the fluorescence signal that the instrument can detect (this fluorescence signal must reach the set threshold); (7) ΔCt is the Ct value of each sample minus the Ct value of the internal reference (i.e., ΔCt = Ct of the target gene - Ct of the internal reference); (8) ΔΔCt = Ct of the experimental group - Ct of the internal reference group; (9) Calculated by 2-ΔΔCt, the obtained value is the relative fold change of the expression of a certain gene in the experimental group compared to the control group, which is called the relative gene expression level; (6) Fluorescence PCR is very sensitive. For each sample in the experiment, not only three replicate wells should be made, but the ΔCt values between the replicate wells should not exceed 0.5.

[0056] 13. Detection of the expression of ORM2 protein by Western Blot technology:

[0057] (1) Wear rubber gloves and a mask, fix the glass plate holder (it can be washed with tap water), and continue to add deionized water to rinse 3 times (drain). (2) Fill the glass plate with deionized water and observe for 10 minutes. (3) The subsequent experiment can only be carried out when there is no liquid leakage. (4) Tilt the glass plate and pour out the deionized water, making sure to pour it out completely (it can also be blotted dry with filter paper for the water remaining on the glass plate). (5) Prepare the corresponding concentration of separating gel freshly according to the method described above (in this experiment, 10% separating gel can be used for ORM2). (6) Inject about 7 ml of separating gel, leaving the gel about 1 cm from the edge of the plate. (7) Slowly add deionized water to the glass plate (this is called pressing the gel, and the purpose is to keep the gel surface level). (8) Let it stand at room temperature for 30 minutes and observe the formation of folding lines. (9) Only after the separating gel has polymerized can the deionized water be poured out, and the remaining deionized water be blotted dry with filter paper. (10) Prepare the stacking gel according to the above formula (pink dye can be added), immediately insert a 10-well comb after injecting it into the gel plate, and let it stand at room temperature until it solidifies. (11) Check the electrophoresis apparatus and assemble it. (12) Pour in freshly diluted electrophoresis buffer (it can be reused). (13) Vertically place the gel plate into the electrophoresis tank and then slowly pull out the comb (to avoid bending the gel). (14) Load 30 μg of protein into each sample well (add 5 μl of protein marker to the well next to the sample well as an indicator). (15) Add 1x protein denaturing buffer to the extra empty wells (to make the bands neat and reduce the protein stacking force). (16) Fill the electrophoresis tank with freshly prepared electrophoresis solution. (17) Connect the power supply with attention to electrical safety. (18) First, use a constant voltage electrophoresis method of 80 V (until the blue sample line reaches the junction of the stacking gel and the separating gel). (19) Then adjust the voltage to 120 V (until the bromophenol blue runs to the bottom of the separating gel). (20) Take 2 thick sponges, 2 thick filter papers, and 1 PVDF membrane (the membrane requires pretreatment: cut the membrane into a suitable size and then soak it in methanol for 1 - 2 minutes), put them into the transfer buffer, and equilibrate for a few minutes (it can be operated when the electrophoresis is almost over). (21) After the electrophoresis is over, according to the principle of black gel and white membrane, place them in order: a. sponge; b. filter paper; c. PVDF membrane; d. gel; e. filter paper; f.Sponge, without air bubbles; (22) Place the "sandwich" electrotransfer clip into the electrotransfer tank, fill it with transfer buffer, and start electrotransfer; (23) Place the electrotransfer tank in ice-water mixture in advance. The electrotransfer conditions are: constant current of 250 mA and time of 100 minutes; (24) Cut the PVDF membrane according to the protein marker (separate the membrane with the target protein band and the internal reference protein); (25) Place the cut membrane (protein side up) into TBST, rinse it 3 times, 1 minute each time; (26) Place the rinsed PVDF membrane into 5% milk (without small clots), and incubate it at room temperature for 1 hour; (27) After blocking, place the PVDF membrane into the primary antibody (SANTA CRUZ, catalog number sc-515724, 1:200) dilution solution, and incubate it overnight in the cold room for at least 16 hours; (28) Take out the PVDF membrane from the cold room, place it into TBST, and rinse it 3 times on a shaker (40 revolutions per minute), 10 minutes each time; (29) After rinsing, place the NC membrane into the secondary antibody (ABclonal, catalog number AS014, 1:5000) dilution solution, and incubate it at room temperature on a shaker (20 revolutions per minute) for 60 minutes; (30) Place the PVDF membrane into TBST, and rinse it 3 times on a shaker (40 revolutions per minute), 5 minutes each time; (31) Mix the ECL luminescent solution (solution A and solution B are fully mixed) in a 1:1 ratio; (32) Perform detection in a luminescence instrument (pay attention to controlling the exposure time), and take a photo for preservation; (33) Analyze the gray value of the protein using Image J software.

[0058] 14. Primary osteoblast extraction

[0059] (1) Primary BMSC extraction: Culture medium: Alpha-MEM, 20% fetal bovine serum (FBS) 100 μl penicillin, 100 μg / ml Steptomycin. Anesthetize the mouse with CO2, dissect the femur and tibia, remove as much muscle as possible, cut the epiphyseal end of each bone, place the cut end of the bone downward into a 0.6 ml tube, and transfer the 0.6 ml to a 1.6 ml tube. Use a tabletop centrifuge to spin quickly until reaching 10,000×g, then turn off the centrifuge to precipitate the bone marrow plasma into the 1.6 ml tube. Then transfer the bone marrow stromal cells to a 15 ml tube containing 5 ml of culture medium. Centrifugation: 1000 rpm, 4 minutes. Remove the culture medium, add 1 ml of red blood cell lysis buffer, and incubate at room temperature for 5 minutes. Add 5 ml of culture medium; Centrifugation: 1000 rpm, 4 minutes; Resuspend in 10 ml of culture medium. Filter through a 70 μm sterile cell filter, and plate the cells on a 100 mm tissue culture dish (one mouse can obtain 30×10 6cells). The cells were cultured for 3 days, and half of the medium (5 ml) was replaced; the cells were cultured for another 3 days, and the medium was completely replaced. On the 7th or 8th day, the adherent cells were trypsinized and re-seeded at a concentration of 0.5 - 1×10 5 cells / cm 2 .

[0060] (2) Osteogenic medium induction: Osteogenic medium: aMEM with 10% fetal bovine serum (FBS), 1% P / S containing 50 μg / ml ascorbic acid, 10 mM β-glycerophosphate; 2. The medium was changed every 3 days. 3. ALP staining or RNA analysis was usually performed after culturing in osteogenic medium for 7 days.

[0061] 15. After 7 days of intervention of AGP2 recombinant protein on primary osteoblasts, alkaline phosphatase (ALP) staining, WB and qPCR were used to detect osteogenic markers, and alizarin red (ARS) staining was performed at 21.

[0062] 16. WB was used to detect the protein expression levels of osteogenic markers RUNX2, Osterix, OPN, and OCN, and the method was the same as in step 13.

[0063] 17. qPCR was used to detect the gene expression levels of osteogenic markers RUNX2, Osterix, OPN, and OCN, and the method was the same as in step 12.

[0064] 18. ALP staining of osteoblasts

[0065] It was carried out strictly according to the instructions of the BCIP / NBT alkaline phosphatase color development kit (product number C3206 of Beyotime): a. For tissue or cell samples for detecting endogenous alkaline phosphatase, the cells were fixed with 4% paraformaldehyde for 10 minutes, and then washed 3 - 5 times with an appropriate washing solution for 3 - 5 minutes each time. b. The following solutions were added in sequence and mixed to prepare the BCIP / NBT staining working solution: 3 ml of alkaline phosphatase color development buffer + 10 μl of BCIP solution (300X) + NBT solution (150X). c. After the last washing, the washing solution was removed, and an appropriate amount of BCIP / NBT staining working solution was added to ensure that the sample was fully covered. d. Incubate at room temperature in the dark for 5 - 30 minutes or longer (up to 24 hours) until the color development reaches the expected depth. e. Remove the BCIP / NBT staining working solution and wash 1 - 2 times with distilled water to terminate the color development reaction. f. For tissue sections or cell samples, after the color development reaction is terminated, if necessary, it can be stained with neutral red staining solution for easy observation. For membranes, after the color development reaction is terminated, it can be air-dried at room temperature and stored in the dark. Then it can be observed and photographed under a microscope.

[0066] 19. Osteoblast Alizarin Red S (ARS) staining:

[0067] Strictly follow the Osteoblast Mineralized Nodule Staining Kit (Alizarin Red S method) (product number C0148S from Beyotime): a. When using for the first time, add 40 ml of absolute ethanol to the fixing solution and mix well for later use. b. Sample treatment: (1) After cell induction and differentiation, remove the culture medium and wash once with PBS (C0221A); (2) Fix with the fixing solution for 20 minutes; (3) Wash 3 times with PBS. c. Stain with Alizarin Red S staining solution. d. Add an appropriate amount of Alizarin Red S staining solution to evenly cover the cells and stain at room temperature for 30 minutes. Note: Please adjust the staining time according to the actual staining effect. e. Wash thoroughly with distilled water, and then observe and take pictures under the microscope.

[0068] 20. Establishment of mouse ovariectomy (OVX) osteoporosis model:

[0069] a. Anesthetize the mouse by intraperitoneal injection of sodium pentobarbital; b. After the anesthesia takes effect, place the mouse in a supine position on the surgical board and depilate the back; c. Make a skin incision about 0.5 - 1 cm long along the midline of the back with surgical scissors and bluntly separate the skin and muscle; d. The muscle layer can be seen from the incision. Cut the psoas muscle 1 cm below the rib away from the spine, and immediately the adipose tissue surrounding the ovary and the ovary closely connected can be seen; e. Gently clamp the adipose tissue and pull it out of the incision. At this time, the cauliflower-like structure visible is the ovary. Ligate at the upper and lower parts of the uterine horn and the fallopian tube; f. Peel off the adipose tissue surrounding the ovary; g. Cut short the uterine horn with surgical scissors and remove the ovary; h. Push the adipose tissue back into the abdominal cavity, suture the peritoneum and muscle layer together, and then suture the skin.

[0070] 21. Intravenous injection of AAV8-TBG-AGP2 into the mouse tail vein:

[0071] a. Fix the mouse with a special holder to fully expose the tail; b. Wipe the tail with an alcohol cotton ball to make the blood vessels dilate; c. Fix the tail with the thumb and index finger; d. Hold the syringe in the right hand and insert the needle at the posterior 1 / 4 of the mouse tail along the direction parallel to the vein.

[0072] 22. Extract the liver tissue protein after adeno-associated virus intervention and detect the expression of AGP2 by gene.

[0073] 23. Micro-CT scan the change of mouse tibia bone mass after adeno-associated virus intervention.

[0074] 24. Perform calcein detection on hard tissue sections:

[0075] a. Twenty-four weeks after tail vein injection of AAV8-TBG-AGP into mice, calcein (10 μg / g) was intraperitoneally injected 10 days and 3 days before sacrificing the mice. b. After anesthetizing the mice, they were sacrificed by cervical dislocation, and the femurs and tibias were dissected and fixed in PLP for 24 hours in the dark. c. Hard tissue embedding and ultra-thin sectioning were performed in the dark, and photographs were taken immediately.

[0076] II. Experimental Results

[0077] 1. Liver-specific knockout of MCT1 can inhibit exercise-induced bone formation

[0078] After 1 month of running exercise, we measured the changes in lactate levels in different organs of mice ( Figure 1 Part A in it), and found that the changes in muscle, serum and liver were the most significant, and the lactate level in the liver increased to (14.11 ± 3.538 mmol / L) ( Figure 1 Part B in it). To determine whether lactate mediates the bone formation effect in the liver, we constructed an adeno-associated virus (AAV) to specifically knockout the lactate transporter MCT1 in the liver ( Figure 1 Part C in it). Two weeks after tail vein injection, the mice started a one-month exercise protocol (4 minutes of running, 2 minutes of rest, 10 cycles / day). Consistent with previous studies using the same high-intensity interval exercise model [References], our micro-CT results showed that in the control group ( Figure 1 Parts D-J in it), exercise significantly increased the femoral cortical bone thickness, as well as the trabecular bone volume, number and thickness, while reducing the trabecular bone spacing. Notably, in the exercise group with mct1 gene knockdown, bone mass did not increase significantly, and bone loss was not observed in the control group of mice with liver-specific mct1 gene knockdown. Compared with the control group of mice, there was a significant improvement in their bone mechanical properties after exercise. We did not detect any mechanical changes after exercise in the mice with liver-specific mct1 gene knockdown ( Figure 1 Parts K-N in it). Therefore, we found that knocking out MCT1 in the liver can inhibit the bone-promoting effect of exercise by HE staining.

[0079] 2. Identification of H3K14 lactylation-promoted liver-derived factors by proteomics and Cut&Tag sequencing

[0080] We further tested the changes in pan-lactylation modification in the livers of mice after running exercise and found that the number of cells with pan-lactylation modification in the liver tissue increased significantly ( Figure 2 Parts A-B in it). In addition, when primary hepatocytes were treated with different concentrations of lactate, we observed a dose-dependent increase in the overall lactylation modification ( Figure 2Part C). Among the modification sites, H3K14 is the most significantly modified compared to other sites ( Figure 2 Part D). Immunofluorescence staining of cells also showed that the fluorescence signal at the H3K14la site increased significantly under 10 nM lactate stimulation ( Figure 2 Part E). To study the changes in the expression of downstream genes after H3K14 lactylation in hepatocytes, we performed Cut&Tag analysis, and the results showed that lactylation at the H3K14 site might bind to the promoter regions of 1,567 downstream genes ( Figure 2 Parts G - I). Meanwhile, we screened the liver proteome of exercised mice and identified 151 differential proteins ( Figure 2 Parts J - K). Through comprehensive analysis of the proteomics and CUT&TAG results, 8 key factors (AGP2, LECT2, ST3GAL4, QSOX1, GPC6, ATP2B2, ADGRL2, CEP85) were found ( Figure 2 Part L). After lactate induction, the peak in the promoter region of AGP2 also increased significantly ( Figure 2 Part M). Then, we treated osteoblasts with recombinant proteins and found that compared with the control group, ST3GAL4, QSOX1, and AGP2 promoted the expression of OPN. However, only AGP2 also promoted the expression of osteocalcin, RUNX2, and OSX ( Figure 2 Parts N - Q). Therefore, through our screening, it was determined that AGP2 is a potential liver-derived factor promoting bone formation.

[0081] 3. Lactylation of H3K14 in hepatocytes promotes the expression of the liver-derived factor AGP2

[0082] We further detected the expression of AGP2 in the livers of exercised mice. Compared with the control group, the expression of AGP2 increased significantly ( Figure 3 Parts A - E). ELISA detection also showed an increase in the expression of serum AGP2 in exercised mice ( Figure 3 In primary hepatocytes in vitro, exogenous addition of lactate led to a dose-dependent increase in the expression of AGP2 ( Figure 3 According to the existing research results in this field, HDAC3 has a strong ability to remove lactylation modifications. In our experiment, we found that HDAC3 could eliminate overall lactylation and H3K14 lactylation. Meanwhile, as H3K14 lactylation was removed, the expression of AGP2 decreased accordingly ( Figure 3 Parts K - L).

[0083] 4. Overexpression of AGP2 in the liver can promote osteogenesis and alleviate the progression of osteoporosis

[0084] Then, we constructed an AAV8-TBG to specifically overexpress AGP2 in the liver and intervened for one month in OVX model mice ( Figure 4 Part A in Figure 4 ). First, in in vivo optical imaging, we confirmed that AGP2 could be transported from the liver to bone. As shown in Figure 4 Part B, a small amount of AGP2 signal was detected in the liver, bone, and kidney, and the luciferase-labeled AGP2 signal was detected. Next, micro-CT results showed that the cortical bone thickness, trabecular bone volume, number, and thickness of mice with overexpressed AGP2 in the liver were significantly increased, while the trabecular bone spacing was decreased ( Figure 4 Part C in Figure 4 ). Notably, in OVX mice, overexpression of AGP2 in the liver could also reverse bone loss. Compared with the control group, after overexpressing AGP2 in the liver, the bone mechanical properties were significantly improved, and similar mechanical property changes were also observed in OVX mice after exercise ( Figure 4 Parts D-I in Figure 4 ). The three-point bending stress experiment showed that the maximum load and stiffness of the AGP2 and OVX+AGP2 groups increased ( Figure 4 Parts J-M in

[0085] ). HE staining of mouse tibia showed that the number of osteoblasts in the AGP2 and OVX+AGP2 groups increased (

[0086] Parts -O in Figure 5 ). Serum ELISA detection showed that compared with the OVX group, the cell survival rate β-CTX of osteoclasts in the OVX+AGP2 group was significantly decreased ( Figure 5 Part P in Figure 5 ). The markers of osteoblast collagen synthesis, P1NP, were increased in both the AGP2 group and the OVX+AGP2 group (

[0087] Part Q in ). In summary, these findings suggest that the hepatic factor AGP2 can be transported to bone, where it promotes bone growth and inhibits osteoclast activation.

[0085] 5. An increase in AGP2 expression was observed within 24 hours after exercise in a population study

[0086] A total of 21 volunteers were recruited in this study, and each participated in 15 minutes of high-intensity interval exercise ( Figure 5 Part A in Figure 5 ). The demographic characteristics of the subjects were as follows: average age (26.26 ± 3.18 years), height (170.68 ± 8.82 cm), weight (65.01 ± 11.91 kg), exercise frequency (3 times / week), BMI (22.2 ± 2.85) ( Figure 5 Part B in

[0087] ). None of the volunteers had a history of hypertension, hyperlipidemia, or diabetes. The levels of AGP2 in serum at different time points were detected using ELISA ( Figure 5 Part C in

[0087] ). The results showed that the level of AGP2 began to increase 24 hours after exercise.

[0087] 6. AGP2 promotes osteogenesis through the activation of the lrp5-mediated Wnt / β-catenin signaling pathway

[0088] To evaluate the effect of AGP2 on osteoblast mineralization, MC3T3-E1 osteoblasts were cultured in osteogenic medium at 37 °C for 14 days. AGP2 treatment significantly enhanced alkaline phosphatase (ALP) activity and mineral deposition, as confirmed by alizarin red staining ( Figure 6 parts A-D in Figure 6 ). In addition, AGP2 significantly upregulated the expression of key osteogenic markers, including RUNX2, OPN, Osterix (Osx), and osteocalcin ( Figure 6 parts E-G in Figure 6 ). To investigate the potential mechanism of AGP2, we performed mRNA sequencing (mRNA-seq) and found that 646 genes were upregulated and 152 genes were downregulated after AGP2 treatment (

[0089] part H in Figure 6 ). Gene ontology (GO) enrichment analysis showed that these differentially expressed genes were mainly related to collagen biosynthesis and extracellular matrix organization. Pathway analysis further confirmed that these genes were significantly involved in the Wnt signaling pathway and osteoblast proliferation ( Figure 6 parts I-K in

[0090] ). Figure 6 In terms of downstream signaling, phosphorylation of β-catenin at Ser33 and Ser37 promotes its degradation, while phosphorylation at Ser552 enhances its nuclear translocation. Immunofluorescence staining showed that AGP2 treatment promoted the nuclear accumulation of p-β-catenin (Ser552) ( Figure 6 part N in

[0091] At the protein level, AGP2 treatment induced phosphorylation of β-catenin at Ser552, upregulating the expression of downstream Wnt / β-catenin target genes Axin2, DKK1, and CyclinD1 ( Figure 6 part of P-T in). Taken together, these results indicate that AGP2 activates the Wnt / β-catenin signaling pathway by interacting with LRP5, thereby promoting osteogenesis.

Claims

1. The application of AGP2 in the preparation of drugs for promoting osteogenesis and alleviating osteoporosis, characterized in that, The AGP2 is α1-acid glycoprotein 2.

2. The application according to claim 1, characterized in that, The AGP2 is applied in the form of any pharmaceutically acceptable preparation.

3. The application according to claim 2, characterized in that, The preparation is an injection.

4. Use of a substance capable of overexpressing AGP2 in the liver in the preparation of a drug for promoting osteogenesis and relieving osteoporosis, characterized in that, The AGP2 is α1-acid glycoprotein 2.

5. The application according to claim 4, characterized in that, The substance capable of overexpressing AGP2 in the liver is applied in the form of any pharmaceutically acceptable preparation.

6. The application according to claim 5, wherein The preparation is an injection.

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