Regulation network for regulating intracellular GLUT1 expression level and plasma membrane localization and application thereof

Abstract

The invention relates to a regulation and control network for regulating and controlling intracellular GLUT1 expression level and plasma membrane localization and application of the regulation and control network. The regulation and control network is a regulation and control network of lactic acid modification enzyme P300-SDCCAG3 lactic acid modification-GLUT1, or a regulation and control network of lactic acid demodification enzyme HDAC3-SDCCAG3 lactic acid demodification-GLUT1, or a regulation and control network of lactic acid demodification enzyme HDAC3-SDCCAG3 lactic acid demodification-GLUT1; the NCBI (National Center of Biotechnology Information) ID (Identity) of the SDCCAG3 is 306322, the NCBI ID of the lactic acid modification enzyme P300 is 170915, the NCBI ID of the GLUT1 is 24778, and the NCBI ID of the lactic acid demodification enzyme HDAC3 is 84578. According to the invention, from the level of transport and bioenergy science, a lactic acid modification enzyme P300 and a lactic acid de-modification enzyme HDAC3 are illuminated to carry out lactic acid modification and lactic acid de-modification on SDCCAG3, so that the expression of GLUT1 is influenced, and the GLUT1 is transported to plasma membrane to be repositioned, thereby regulating and controlling the osteogenic differentiation of BMSCs in a high-fat environment and the bone combination and bone formation mechanism of a hyperlipemia rat implant. The invention provides a new clinical treatment target for treating hyperlipemia bone diseases and realizing bone regeneration, and has important significance in treating hyperlipemia rat bone formation and implant osseointegration.

Description

Technical Field

[0001] The invention relates to a regulatory network for regulating the expression level and plasma membrane localization of GLUT1 in cells and application thereof, belonging to the technical field of genetic engineering. Background Art

[0002] Hyperlipidemia is a metabolic disease characterized by elevated lipid levels in the blood. Its elevated blood lipid levels inhibit osteoblast differentiation, leading to bone metabolism disorders, poor bone formation, reduced bone density, volume and strength, and increased fracture healing. Clinical studies have also found that hyperlipidemia is often associated with osteoporosis, and about 63% of osteoporosis patients suffer from hyperlipidemia. Oral implant surgery is currently a common procedure for patients with edentulous and missing teeth to restore chewing function and aesthetics. Among them, bone integration is the biological basis for the success and long-term stability of implant dentures. The physiological process of bone integration is similar to fracture healing. Therefore, the good or bad condition of the host bone directly determines the quality of bone integration. Clinical studies have shown that the failure rate of implant dentures in hyperlipidemia patients undergoing bone augmentation surgery is 7.48 times higher than that of normal patients, which is one of the risk factors affecting implant bone integration. At the same time, this research group and related experimental studies have confirmed that hyperlipidemia can lead to poor implant bone integration. Therefore, studying the mechanism of hyperlipidemia affecting implant bone integration and bone metabolism is of great significance for improving the success rate of implant dentures and preventing and treating bone metabolic diseases such as osteoporosis.

[0003] The diversity of bone functions is achieved by the interaction of osteoblasts, chondrocytes, adipocytes and stromal cells, all of which are generated by skeletal stem cells. Different skeletal cells have unique metabolic requirements related to specific functions and directly determine the fate of skeletal stem cell lineage differentiation. Studies have shown that the energy demand for osteogenic differentiation of bone marrow stromal cells (BMSCs) to lineages mainly comes from glucose, and they are highly glycolytic, efficiently producing ATP and carbon sources to supply energy needs and produce biomass. Glucose transporters 1 (GLUT1) is a switch for glucose to enter cells, and its accurate localization on the plasma membrane and recycling relocalization are crucial for glucose metabolism. At the same time, in experimental mice induced by a high-fat diet, bone glucose uptake and distribution were significantly reduced. However, little is known about the key molecular mechanisms that affect BMSCs metabolism and bioenergetics under a high-fat environment.

[0004] Proteins are the main executors of biological functions, and post-translational modifications (PTMs) can increase the diversity of proteome functions. Therefore, studying key proteins and their post-translational modifications is of great significance for more fully revealing the specific mechanisms of osteogenic differentiation and poor bone formation in hyperlipidemic rats and providing clinical therapeutic targets. Among them, lactylation modification, as a new type of PTM, was first reported in Nature magazine in 2019. As a switch of glycolysis epigenetics, it plays different roles in different biological behaviors such as tumor development, macrophage polarization, inflammation and tissue damage, and myocardial infarction repair, but its research in bone metabolism is still blank; and current research is limited to lactylation modification of histones, and lactylation modification of non-histones is rarely reported. At the same time, lactylation modification affects protein conformation, protein-protein interaction, and subcellular localization of proteins.

[0005] Serologically defined colon cancer antigen-3 (SDCCAG3), as an endosomal protein, plays an important role in endosomal-plasma membrane protein sorting. Technical studies have shown that knocking out SDCCAG3 in Hela cells leads to the aggregation of GLUT1 in the lysosomal cavity and the reduction of GLUT1 levels on the plasma membrane. In Glut1 knockout mice, osteoblast differentiation is delayed and bone formation is impaired. However, the specific mechanism by which SDCCAG3 and its lactylated modification regulate hyperlipidemia bone metabolism and poor bone integration by affecting the expression and plasma membrane relocalization of GLUT1 from the perspective of bioenergetics is still unclear. Summary of the invention

[0006] In view of the deficiencies of the prior art, the present invention provides a regulatory network for regulating the expression level and plasma membrane localization of GLUT1 in cells and its application.

[0007] The technical solution of the present invention is as follows:

[0008] A regulatory network for regulating the expression level and plasma membrane localization of GLUT1 in cells, wherein the regulatory network is a lactylation modification enzyme P300-SDCCAG3 lactylation modification-GLUT1 regulatory network, or a lactylation demodification enzyme HDAC3-SDCCAG3 lactylation demodification-GLUT1 regulatory network;

[0009] Among them, the NCBI ID of the SDCCAG3 is 306322, the NCBI ID of the lactylation modification enzyme P300 is 170915, the NCBI ID of the GLUT1 is 24778, and the NCBI ID of the lactylation demodification enzyme HDAC3 is 84578.

[0010] The present invention has found through research that at the protein level, lactylation modification enzyme P300 will lactate SDCCAG3, and SDCCAG3 lactylation modification will inhibit the expression of GLUT1 and transport plasma membrane relocation, cascade inhibition of glycolysis, inhibit the osteogenic differentiation of BMSCs in a high-fat environment, and lead to bone formation in hyperlipidemia rats and poor bone integration around implants. Therefore, lactylation modification enzyme P300 and lactylation demodification enzyme HDAC3 play a key role in the osteogenic differentiation of BMSCs in a high-fat environment and bone integration and bone formation in hyperlipidemia rats through the SDCCAG3 lactylation modification-GLUT1 transport relocation pathway.

[0011] That is, in high-fat environment BMSCs and hyperlipidemic rats, the expression of lactylation modification enzyme P300 inhibits the osteogenic differentiation of high-fat environment BMSCs and leads to poor bone binding and bone formation in hyperlipidemic rats. The expression of lactylation demodification enzyme HDAC3 promotes the osteogenic differentiation of high-fat environment BMSCs and bone binding and bone formation in hyperlipidemic rats.

[0012] The application of the above-mentioned lactylation modification enzyme P300-SDCCAG3 lactylation modification-GLUT1 regulatory network, or the lactylation demodification enzyme HDAC3-SDCCAG3 lactylation demodification-GLUT1 regulatory network in the preparation of drugs that promote osteogenic differentiation and bone formation in hyperlipidemia rats is characterized in that the application is the application of the regulatory factors of the lactylation modification enzyme P300-SDCCAG3 lactylation modification-GLUT1 regulatory network, or the lactylation demodification enzyme HDAC3-SDCCAG3 lactylation demodification-GLUT1 regulatory network in the preparation of drugs that promote bone formation and peri-implant bone integration in hyperlipidemia rats.

[0013] Preferably according to the present invention, the drug inhibits the expression of lactate modification enzyme P300 and / or promotes the expression of lactate demodification enzyme HDAC3 in hyperlipidemic rats.

[0014] Preferably according to the present invention, the regulatory factors of the lactylation modification enzyme P300-SDCCAG3 lactylation modification-GLUT1 regulatory network, or the lactylation demodification enzyme HDAC3-SDCCAG3 lactylation demodification-GLUT1 regulatory network include but are not limited to: nucleic acid molecules, carbohydrates, lipids, polypeptides, proteins or lentiviruses.

[0015] Further preferably, the nucleic acid molecule is siRNA-3239, siRNA-2131 or siRNA-3604 that inhibits the expression of lactylation modifying enzyme P300;

[0016] The sequences of the siRNA-3239, siRNA-2131 or siRNA-3604 are as follows:

[0017] siRNA-3239: 5'-GGAGAGAGGCCCUGAGUUATTUAACUCAGGGCCUCUCUCCTT-3';

[0018] siRNA-2131: 5'-AGAAAGAACUAGAAGAGAATTUUCUCUUCUAGUUCUUUCUTT-3';

[0019] siRNA-3604: 5'-CUUCAAGGGUAUACAAAUATTUAUUUGUAUACCCUUGAAGTT-3'.

[0020] Preferably according to the present invention, the drug contains an effective dose of the regulatory factor of the above regulatory network.

[0021] Preferably according to the present invention, the drug contains a pharmaceutically acceptable carrier.

[0022] The beneficial effects of the present invention are:

[0023] 1. The present invention explains from the perspective of transport and bioenergetics that lactylation modification enzyme P300 and lactylation demodification enzyme HDAC3 affect the expression and transport of GLUT1 to the plasma membrane for relocation by lactylation modification and lactylation demodification of SDCCAG3, thereby regulating the osteogenic differentiation of BMSCs in a high-fat environment and the bone binding and bone formation of hyperlipidemic rats, and provides a regulatory network of lactylation modification enzyme P300-SDCCAG3 lactylation modification-GLUT1 or lactylation demodification enzyme HDAC3-SDCCAG3 lactylation demodification-GLUT1. In high-fat environment BMSCs and hyperlipidemic rats, the expression of lactylation modification enzyme P300 inhibits the osteogenic differentiation of high-fat environment BMSCs and leads to poor bone formation in hyperlipidemic rats. The expression of lactylation demodification enzyme HDAC3 promotes the osteogenic differentiation of high-fat environment BMSCs and bone formation in hyperlipidemic rats.

[0024] 2. The present invention also provides siRNA-3239, siRNA-2131 or siRNA-3604 for inhibiting the expression of lactylation modifying enzyme P300. Experiments have shown that inhibiting the expression of lactylation modifying enzyme P300 can promote the osteogenic differentiation of BMSCs in a high-fat environment and bone formation in hyperlipidemic rats, which provides a new clinical treatment target for the treatment of hyperlipidemic bone disease and the realization of bone regeneration, and is of great significance in the treatment of bone formation in hyperlipidemic rats and implant bone integration. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 Liquid chromatography-tandem mass spectrometry was used to identify SDCCAG3 lactylation and its K278 site.

[0026] Figure 2 To characterize the lactylation of SDCCAG3 in bone tissue and BMSCs of hyperlipidemic rats;

[0027] In the figure, NC is normal condition and HF is high-fat condition;

[0028] a After immunoprecipitation of SDCCAG3 in BMSCs, western blotting of lactic acid pan-antibody (PTM-140RM) was used to characterize the expression of SDCCAG3-Kla in BMSCs;

[0029] b After immunoprecipitation of SDCCAG3 in the bone tissue of hyperlipidemic rats, western blotting of lactic acid pan-antibody (PTM-140RM) was used to characterize the expression of SDCCAG3-Kla in the bone tissue of hyperlipidemic rats;

[0030] c is the Co-IP verification of the binding of SDCCAG3 with the lactylation modification enzyme P300;

[0031] d is the Co-IP verification of the binding of SDCCAG3 with the lactate demodification enzyme HDAC3;

[0032] Figure 3 Lactylation of SDCCAG3 inhibits the osteogenic differentiation of BMSCs under high-fat environment.

[0033] In the figure, a is the verification of the efficiency of simulating SDCCAG3 lactation and non-lactation in BMSCs under high-fat environment;

[0034] b shows the effects of SDCCAG3 lactylation and non-lactylation on the expression of osteogenesis-related factors;

[0035] c: The effects of SDCCAG3 lactation and non-lactation on ALP and ARS staining of BMSCs.

[0036] Figure 4 SDCCAG3 lactylation inhibits GLUT1 expression and plasma membrane relocalization;

[0037] In the figure, a shows the total protein expression level of GLUT1 in BMSCs under high-fat environment promoted by overexpression of SDCCAG3;

[0038] b is the efficiency verification of successful overexpression of SDCCAG3;

[0039] c shows that overexpression of SDCCAG3 promotes the expression level of plasma membrane GLUT1 protein in BMSCs under high-fat environment;

[0040] d is Co-IP demonstrating the interaction between SDCCAG3 and GLUT1;

[0041] e and f are the transfection of Sdccag3(WT)-3×Flag, KQ is Sdccag3(K278Q)-3×Flag, and KR is Sdccag3(K278R)-3×Flag in BMSCs in a high-fat environment to verify the binding of SDCCAG3-K278la to GLUT1;

[0042] g and h are the results of stably transfecting sh-Sdccag3 in BMSCs in a high-fat environment to eliminate the interference of endogenous SDCCAG3, and then transfecting Sdccag3(WT)-3×Flag, KQ is Sdccag3(K278Q)-3×Flag, and KR is Sdccag3(K278R)-3×Flag to verify the binding of SDCCAG3-K278la to GLUT1;

[0043] i and j show BMSCs transfected with Sdccag3(WT)-3×Flag, KQ is Sdccag3(K278Q)-3×Flag, and KR is Sdccag3(K278R)-3×Flag in a high-fat environment. Plasma membrane proteins were extracted and western blotting was used to detect the effect of SDCCAG3-3-K278la on the expression of total GLUT1 protein and plasma membrane protein.

[0044] Figure 5 Lactylation of SDCCAG3 inhibits the expression of key glycolytic enzymes.

[0045] Figure 6 Characterization of SDCCAG3 lactylation for glycolysis stress experiments to inhibit glycolysis.

[0046] Figure 7 Stable isotope labeled U- 13 C6 Characterization of glucose metabolic flux SDCCAG3 lactylation inhibits glycolytic flux in BMSCs under high-fat environment.

[0047] Figure 8 Lactylation of SDCCAG3 inhibits bone formation in hyperlipidemic rats;

[0048] In the figure, a is the validation of the simulation efficiency of lactic acid and non-lactic acid of SDCCAG3 in hyperlipidemic rats;

[0049] b shows that after simulating SDCCAG3-K278 lactation and non-lactation in hyperlipidemic rats, SDCCAG3 lactation inhibits the expression of key glycolytic enzymes;

[0050] c shows that after simulating SDCCAG3-K278 lactation and non-lactation in hyperlipidemic rats, SDCCAG3 lactation inhibits the expression of osteogenic-related factors;

[0051] d and e are the results of simulating SDCCAG3-K278 lactation and non-lactation in hyperlipidemic rats. Micro-CT showed that SDCCAG3 lactation inhibited new bone formation in the skull defect.

[0052] f and g show the BMD and BV / TV index of the SDCCAG3 lactate group decreased after simulating SDCCAG3-K278 lactate and non-lactate in hyperlipidemic rats;

[0053] h is the simulation of SDCCAG3-K278 lactic acid and non-lactic acid in hyperlipidemic rats. HE staining shows that SDCCAG3 lactic acid inhibits new bone formation in the skull defect.

[0054] i: After simulating the lactic acid and non-lactic acid of SDCCAG3-K278 in hyperlipidemia rats, Masson's staining showed that SDCCAG3 lactic acid inhibited new bone formation in the skull defect.

[0055] Fig. 9 Lactylation of SDCCAG3 inhibits peri-implant bone formation and bone integration in hyperlipidemic rats;

[0056] In the figure, a and b are the simulated lactic acidification and non-lactic acidification of SDCCAG3-K278 in hyperlipidemic rats. Micro-CT shows that lactic acidification of SDCCAG3 inhibits new bone formation around implants;

[0057] c~h show the effects of SDCCAG3 lactation on bone metrology parameters after simulating SDCCAG3-K278 lactation and non-lactation in hyperlipidemic rats: BV / TV, trabecular thickness, and trabecular number were all decreased (cde), while trabecular spacing, bone surface area / bone volume, and trabecular pattern factor were increased (fgh);

[0058] i After simulating SDCCAG3-K278 lactation and non-lactation in hyperlipidemic rats in vivo, HE staining of hard tissue sections showed that SDCCAG3 lactation inhibited implant bone integration.

[0059] Fig.10 To investigate the regulation of SDCCAG3 lactylation by knockdown of P300 or HDAC3

[0060] In the figure, a is the result of immunoprecipitation of SDCCAG3 and western blotting of pan-lactylating antibody after transfection of siRNA to knock down the lactylation modifying enzyme P300: knocking down P300 reduces the lactylation of SDCCAG3;

[0061] b is the result of immunoprecipitation of SDCCAG3 and western blotting with pan-lactylase antibody after transfection of siRNA to knock down the lactylation demodification enzyme HDAC3: knocking down HDAC3 increased the lactylation of SDCCAG3;

[0062] c is a western blotting experiment after transfection of siRNA knockdown of lactic acid modification enzyme P300 and siRNA knockdown of lactic acid demodification enzyme HDAC3, showing that knockdown of P300 promoted the expression of osteoblast-related factors, while knockdown of HDAC3 inhibited the expression of osteoblast-related factors;

[0063] d: After transfection with siRNA to knock down the lactylation modification enzyme P300, glycolysis, glycolytic capacity and reserve capacity were all increased; after transfection with siRNA to knock down the lactylation demodification enzyme HDAC3, glycolysis, glycolytic capacity and reserve capacity were all decreased. DETAILED DESCRIPTION

[0064] The present invention is further described below in conjunction with the examples, but the protection scope of the present invention is not limited thereto. The experimental operations involved in the examples are all carried out according to conventional operations unless otherwise specified; the drugs and reagents involved are all common commercially available products unless otherwise specified. Unless otherwise specified, the percentages in the examples are all mass percentages.

[0065] The culture medium and other materials required to be prepared in the examples are as follows.

[0066] Complete culture medium containing 10% FBS: prepared at a volume ratio of FBS:α-MEM=1:9 and stored at 4°C before use.

[0067] High-fat / normal osteogenic induction medium containing 5% FBS:

[0068] ① Separately weigh 3.93 mg of dexamethasone (dissolved in 2 mL of anhydrous ethanol), 306 mg of sodium β-glycerophosphate (containing 5H2O), and 5 mg of vitamin C in a dark place and store in a dark place;

[0069] ② Add 3 mL of 5% FBS complete medium to 2 mL of anhydrous ethanol containing 3.93 mg of dexamethasone, filter with a 0.22 μm filter to sterilize, and obtain 2×10 -3 mol / L dexamethasone stock solution a; take 5 μL of stock solution a to 5 mL of 5% FBS complete medium to obtain 2×10 -6 mol / L dexamethasone stock solution b, stored at 4℃ and protected from light;

[0070] ③ Dissolve 306 mg of sodium β-glycerophosphate (containing 5H2O) and 5 mg of vitamin C in 5 mL of 5% FBS complete medium and filter with a 0.22 μm filter for sterilization;

[0071] ④ Take 250 μL of dexamethasone stock solution b and 2.5 mL of solution ③ and add them to 47.25 mL of 5% FBS high-fat / normal culture medium to obtain dexamethasone 10 -8 mol / L, β-phosphoglycerate sodium (containing 5H2O) 10mmol / L, 5mg vitamin C 50mg / L high-fat / normal osteogenic induction solution, stored at 4℃ away from light before use.

[0072] 10% ammonium persulfate substitute (APS): weigh 10 mg APS and fully dissolve it in 10 mL triple-distilled water.

[0073] Electrophoresis fluid (1×): Add 1 pack of finished electrophoresis powder to 1L of triple-distilled water and shake until fully dissolved.

[0074] Transfer solution (1×): Add 1 package of finished transfer powder to 800 mL of triple distilled water and add 200 mL of methanol.

[0075] 0.1% TBST buffer: 1 pack of finished TBST powder is completely dissolved in 2L triple distilled water and 2mL Tween-20 is added.

[0076] 5% skim milk powder: weigh 2 g skim milk powder into 40 mL TBST buffer and shake until fully dissolved.

[0077] Western blotting secondary antibody: add 4uL horseradish enzyme-labeled goat anti-rabbit / mouse IgG (H+L) (affinity purified) to every 40mL 5% skim milk solution, i.e. the secondary antibody dilution ratio is 1:1w.

[0078] PBS buffer: 1 pack of PBS powder is completely dissolved in 2L of triple distilled water and placed at room temperature for use;

[0079] 12.5% ​​EDTA solution (2L system): Dissolve 250g EDTA powder, 27.5g NaOH powder, and 1 pack of PBS powder in 2L triple distilled water and stir thoroughly until completely dissolved.

[0080] High-fat feed formula: basic feed 49.25%, lard 16%, sucrose 10%, whole milk powder 10%, casein 9%, experimental animal premix 2%, calcium hydrogen phosphate 2%, cholesterol 1.25%, sodium cholate 0.5%.

[0081] Example 1: Isolation, culture and identification of primary rat BMSCs

[0082] 1. Isolation of primary rat BMSCs

[0083] Whole bone marrow adherence method, the specific method is as follows:

[0084] ①, 3-week-old Wistar male rats were killed by cervical dislocation and then immersed in 75% ethanol for 5 minutes for disinfection;

[0085] ②In a biosafety cabinet, cut the skin at the hind leg area of ​​the rat, peel off the soft tissue and muscle, expose the bone surface, remove the bilateral femurs and tibiae under sterile conditions, and wash them three times with sterile PBS solution containing 1% triple antibody;

[0086] ③. Cut off the ends of the femur and tibia with ophthalmic scissors to expose the bone marrow cavity. Use a syringe to repeatedly flush the bone marrow cavity with 20% FBS α-MEM complete medium into the culture dish until the bone marrow cavity turns white; ④ Place the culture dish in a 37°C, 5% CO2 cell culture incubator for culture; After 3 days, replace the medium with 10% FBS α-MEM complete medium until passage. The experiment used BMSCs of P2-3 generations.

[0087] 2. Culture of primary rat BMSCs

[0088] ①. Cell passaging: Cells in the culture dish are passaged when the confluence reaches 80% or more. Aspirate the old culture medium in the dish, wash the cells with PBS, add 2mL of trypsin for digestion, closely observe the cell morphology, and when the cells become round and float away from the bottom of the dish in a suspended state, add 4mL of complete culture medium to terminate digestion, gently blow repeatedly until the cells fall off, collect the cell suspension into a 15mL centrifuge tube, centrifuge (1000r, 5min), discard the supernatant, resuspend the cells with 10% FBSα-MEM culture medium, and evenly inoculate them into the cell culture dish, and culture them in a 37°C, 5% CO2 cell culture incubator. Depending on the cell status, the ratio of 1:2 or 1:3 is generally used for passaging.

[0089] ②. Cell freezing: Aspirate the old culture medium in the dish, wash the cells with PBS, digest with trypsin, collect the cell suspension and centrifuge, discard the supernatant, resuspend the cells with the finished product of Cardiomycetin serum-free freezing solution, transfer to 2 mL sterile cryopreservation tubes, and store at -80℃.

[0090] ③. Cell recovery: quickly put the cryopreservation tube into a 37℃ constant temperature water bath to thaw, transfer to a sterile biosafety cabinet, aspirate the cell suspension in the cryopreservation tube into a 15mL centrifuge tube, add culture medium, centrifuge (1000r, 5min), discard the supernatant, resuspend the cells with 10% FBSα-MEM culture medium, and evenly inoculate into the cell culture dish, and culture in a 37℃, 5% CO2 cell culture incubator.

[0091] 3. Identification of primary rat BMSCs

[0092] ① Flow cytometry identification of rat BMSCs surface markers: P3 generation BMSCs were collected, and the cells were counted. 5×10 5 Place the cells in a 1.5mL EP tube and centrifuge to remove the supernatant; wash the cells twice with PBS and centrifuge to remove the supernatant; add 100ul PBS to each 1.5mL EP tube, and add CD44, CD45, CD90 antibodies and isotype negative control respectively, and incubate at room temperature for 30min. Wash the cells 3 times with PBS to remove unbound antibodies, and resuspend each tube with 500ul PBS after the last centrifugation, and detect on the machine according to the operating requirements of the flow cytometer.

[0093] ②Alkaline phosphatase staining: P3 generation BMSCs were inoculated in a 12-well plate. After 14 days of osteogenic induction, the cells were incubated in culture medium, washed twice with PBS, fixed with 4% paraformaldehyde for 30 min, and washed twice with PBS. The cells were stained according to the instructions of the alkaline phosphatase staining kit (Biyuntian, China), photographed under an inverted microscope, and the entire cell plate was scanned.

[0094] ③. Alizarin red staining: P3 generation BMSCs were inoculated into 12-well plates. After 28 days of osteogenic induction of BMSCs, the old culture medium was discarded, the cells were washed twice with PBS, the cells were fixed with 4% paraformaldehyde for 30 min, washed twice with PBS, covered with 1% Alizarin red staining solution (PH4.2) for 30 min, the staining solution was removed, and the cells were washed twice with PBS. A small amount of liquid was left at the bottom of the cell plate. Pictures were taken under an inverted microscope, and the cell plate was scanned in its entirety.

[0095] ④. Oil Red O staining: P3 generation BMSCs were inoculated in a 12-well plate. After 28 days of osteogenic induction, the cells were incubated in culture medium and washed twice with PBS. The cells were fixed with 4% paraformaldehyde for 30 min and washed twice with PBS. The cells were stained according to the instructions of the cell-specific Oil Red O staining kit (Solebo, G1262, China). The cells were photographed under an Olympus inverted microscope and the entire cell plate was scanned.

[0096] Example 2: Osteogenesis induction of rat BMSCs

[0097] Normal / high-fat osteogenic induction solution was prepared, and P2 or P3 generation BMSCs were taken for normal / high-fat osteogenesis (normal group: NC, high-fat group: HF), and osteogenic induction was performed according to the requirements of subsequent experiments.

[0098] Example 3: Establishment of hyperlipidemia rat model

[0099] 60 4-week-old Wistar male rats (180-220 g) were fed in the animal experimental platform purchased from the Shandong Provincial Key Laboratory of Oral Tissue Regeneration & Shandong Provincial Oral Biomaterials and Tissue Regeneration Engineering Laboratory. All experiments have been approved and recognized by the Ethics Committee of Shandong University Stomatological Hospital (No. 20210509). Because the simulation is similar to hyperlipidemia caused by changes in human dietary structure, this study used a high-fat diet to induce. The high-fat diet formula is as follows: 49.25% basic feed, 16% lard, 10% sucrose, 10% whole milk powder, 9% casein, 2% experimental animal premix, 2% calcium hydrogen phosphate, 1.25% cholesterol, and 0.5% sodium cholate. Among them, 20 were randomly divided into a high-fat group (HF) and a normal group (Normal), and were given a high-fat diet and a normal diet, respectively; the remaining 40 were given a high-fat diet. After 8 weeks, all rats fasted for 12 hours, and 0.5 ml of whole blood was collected from the capillary orbital medial angular vein and placed in a 1.5 mL EP tube. The blood was centrifuged (4 ° C, 4000r, 30min) to obtain serum in a new 1.5 mL EP tube and sent to the Laboratory Department of Qilu Hospital of Shandong University to test the levels of total cholesterol (TC), triglyceride (TG), high-density lipoprotein (HDL), and low-density lipoprotein (LDL). At the same time, blood was collected from the tail vein and peripheral blood glucose was tested using a blood glucose meter to exclude the influence of high blood sugar on this experimental study. The blood lipid levels of rats in the normal group and the high-fat group are shown in Table 1, which are statistically different (p < 0.05).

[0100] Table 1: Blood lipid levels of rats in the normal group and high-fat group (mean ± SD, mmol / L)

[0101]

[0102] As shown in Table 1, the levels of TC, TG, LDL, and HDL in the high-fat group rats were significantly higher than those in the normal group rats, which means that the hyperlipidemia rats were successfully modeled, and subsequent experiments were conducted and high-fat diet was continued.

[0103] Example 4: Lentivirus packaging and synthesis

[0104] According to the changes in the charge properties of amino acids after protein modification, 3×Flag-tagged lentiviral vectors were packaged: Sdccag3(WT)-3×Flag, Sdccag3(K278Q)-3×Flag (simulating lactic acid state), Sdccag3(K278R)-3×Flag (simulating non-lactated state), and negative control group LV17-nc. The above lentiviral vectors were synthesized by Shanghai Jima Pharmaceutical Technology Co., Ltd. Specifically, Sdccag3(WT), Sdccag3(K278Q) and Sdccag3(K278R) were used as templates to clone their coding frames into LV17(EF-1a / Luciferase17&Puro) lentiviral plasmid by PCR, and then packaged into lentiviral plasmid.

[0105] The Sdccag3 (WT) is the wild-type SDCCAG3, and its Genbank ID is 306322; the Sdccag3 (K278Q) is the wild-type SDCCAG3 in which the 278th lysine is mutated to glutamine, which is used to simulate the SDCCAG3 in the lactic acid state; the Sdccag3 (K278R) is the wild-type SDCCAG3 in which the 278th lysine is mutated to arginine, which is used to simulate the SDCCAG3 in the non-lactated state.

[0106] Example 5: Implantation in hyperlipidemic rats

[0107] Normally fed and high-fat fed rats were selected as normal group and high-fat group, with 8 rats in each group. All surgical instruments were sterilized by high-pressure steam sterilization. 10% chloral hydrate was injected intraperitoneally at a dose of 100g / 0.3ml. After the rats were anesthetized, they were fixed on the surgical board in supine position, the femoral epiphysis was prepared, disinfected, and covered with a towel. A No. 11 scalpel was used to make an incision of about 2cm in the skin on the anterior medial side of the bilateral femur, and the muscle layer and periosteum were bluntly separated to fully expose the femoral epiphysis. Under continuous irrigation with normal saline, a simple implanter was used to prepare a cavity with a diameter of 1.2mm and a depth of about 2.5mm at 5mm from the distal end of the bilateral femoral epiphysis, and the implant was manually screwed into the implant cavity. The muscles and skin were sutured in layers with 4-0 sutures, and 100,000 units / rat of cefazolin sodium were injected intramuscularly. 100,000 units / rat of cefazolin sodium were continued intramuscularly for 3 days after surgery to prevent postoperative infection.

[0108] Example 6: Establishment of a unilateral skull defect model in hyperlipidemia rats

[0109] After implant placement and injection of LV17-nc, Sdccag3(WT)-3×Flag, Sdccag3(K278Q)-3×Flag (simulating lactated state), and Sdccag3(K278R)-3×Flag (simulating non-lactated state) lentiviral vectors in hyperlipidemia rats, the rats were turned over and fixed on the operating board in a prone position. The skull area was prepared, disinfected, and covered with a towel. The skin was incised at the midline of the skull, the periosteum was separated, and the skull was exposed. A 5-mm diameter defect was prepared on the left side of the midline of the skull. The defects were rinsed with saline and sutured. The wounds were disinfected with iodine and cefazolin sodium was injected intramuscularly 3 days after surgery to prevent infection.

[0110] Example 7: Lentivirus transfection of BMSCs and intramuscular injection into hyperlipidemia rat model

[0111] P2 BMSCs in good condition were taken and transferred into Sdccag3(WT)-3×Flag, Sdccag3(K278Q)-3×Flag, Sdccag3(K278R)-3×Flag, and the negative control group LV17-nc lentiviral vector at MOI=100 and 5μg / mL transfer agent Polybrene. The medium was changed according to the cell status. If there was no abnormality, the medium was changed 3 days after transfection, and 5μg / mL puromycin was added for selection for 48h. The medium was changed or passaged according to the cell status to obtain stably transfected cells for subsequent experiments.

[0112] Thirty-two successfully constructed hyperlipidemia rats were randomly divided into LV17-nc, SDCCAG3-WT, SDCCAG3-KQ, and SDCCAG3-KR groups, with 8 rats in each group. All surgical instruments were sterilized by high-pressure steam sterilization. Three days before surgery, the corresponding lentiviral expression vectors (injected virus number: 1×10) were injected intramuscularly 5 mm from the distal end of the bilateral femoral epiphysis in the LV17-nc, SDCCAG3-WT, SDCCAG3-KQ, and SDCCAG3-KR groups. 8 Titer / mouse), and the same dose and the same lentivirus were injected intramuscularly again with a microsyringe during the operation.

[0113] Example 8: Identification of SDCCAG3 lactylation and its modification sites by SDCCAG3 immunoprecipitation combined with liquid chromatography-tandem mass spectrometry

[0114] (1) After enriching SDCCAG3 antigen through immunoprecipitation experiment, western blotting gel electrophoresis was performed to prepare gel strips (the protein was required to run into the separation gel for about 1.5 cm in length and the gel cutting area was about 1.0 cm) 2 ), the strips were placed in sterile triple-distilled water and sent to Hangzhou Jingjie Biotechnology Co., Ltd. for SDCCAG3 lactic acid modification and site identification. The specific method is as follows.

[0115] (2) Liquid chromatography-tandem mass spectrometry

[0116] This part of the experiment was mainly completed by Hangzhou Jingjie Biotechnology Co., Ltd.

[0117] ① In-gel enzymatic hydrolysis

[0118] Cut the strips into pieces and decolorize the gel blocks with 50% acetonitrile containing 50mM ammonium bicarbonate. Dehydrate the gel blocks by incubating with 100% acetonitrile for 5 minutes, then remove the liquid phase in the system, add dithiothreitol solution with a final concentration of 10mM, and incubate at 37°C for 60 minutes. Dehydrate again with 100% acetonitrile, remove the liquid phase, add iodoacetamide with a final concentration of 55mM, and incubate at room temperature in the dark for 45 minutes. Then wash with ammonium bicarbonate with a final concentration of 50mM, and dehydrate again with 100% acetonitrile. Finally, resuspend the gel blocks with 50mM ammonium bicarbonate containing 10ng / μl trypsin and incubate on ice for 1 hour. After removing the excess solution in the sample, the gel blocks were enzymatically hydrolyzed at 37°C overnight. The enzymatic peptides were extracted from the gel blocks using 50% acetonitrile / 5% formic acid and 100% acetonitrile in turn, and the peptide solution was frozen and spin-dried for use.

[0119] ②Mass spectrometry parameter setting

[0120] The peptides were dissolved in liquid chromatography mobile phase A and separated using the EASY-nLC 1000 ultra-high performance liquid phase system. Mobile phase A was an aqueous solution containing 0.1% formic acid and 2% acetonitrile; mobile phase B was an aqueous solution containing 0.1% formic acid and 90% acetonitrile. The liquid phase gradient was set as follows: 0-22 min, 6% to 35% B phase; 22-26 min, 35% to 80% B phase; 26-30 min, 80% B phase, and the flow rate was maintained at 550 nL / min.

[0121] The peptides were separated by ultra-high performance liquid chromatography (UPLC) and injected into the NSI ion source for ionization. TMQ Exactive Plus mass spectrometry was used for analysis. The ion source voltage was set to 2.2 kV, and the peptide parent ions and their secondary fragments were detected and analyzed using a high-resolution Orbitrap. The primary mass spectrometry scan range was set to 350-1800 m / z, and the scan resolution was set to 70,000; the secondary scan resolution was set to 17,500. The data acquisition mode used a data-dependent scan (DDA) program, that is, after the primary scan, the top 20 peptide parent ions with the highest signal intensity were selected to enter the HCD collision cell in turn and use 28% fragmentation energy for fragmentation, and the secondary mass spectrometry analysis was also performed in turn. In order to improve the effective utilization of the mass spectrometry, the automatic gain control (AGC) was set to 5e4, the signal threshold was set to 5e3 ions / s, the maximum injection time was set to 200 ms, and the dynamic exclusion time of the tandem mass spectrometry scan was set to 15.0 s to reduce repeated scanning of the parent ion.

[0122] ③Data search and analysis

[0123] The secondary mass spectrometry data were searched using Proteome Discoverer 2.1. The search parameters were set as follows: the database was set to the target protein sequence; the enzyme cleavage method was set to Trypsin / P; the missed cleavage site was set to 2; the primary parent ion mass error tolerance was set to 10ppm; the secondary fragment ion mass error tolerance was set to 0.02Da; the fixed modification was set to cysteine ​​alkylation; the variable modification was set to methionine oxidation, protein N-terminal acetylation; serine, threonine, and tyrosine phosphorylation; lysine acetylation, crotonylation, dihydroxyisobutyrylation, ubiquitination, succinylation, malonylation, benzoylation, propionylation, butyrylation, trihydroxybutyrylation, N-glycosylation, glutarylation, lactylation, monomethylation, dimethylation, and trimethylation; arginine monomethylation and dimethylation; tyrosine nitration. The peptide ion score was required to be higher than 20, and the identification result peptide confidence was set to High. The results are as follows: Figure 1 shown.

[0124] Depend on Figure 1 It can be seen that the SDCCAG3 protein is lactylated and occurs at the lysine K278 site. The lactylated SDCCAG3 protein is called SDCCAG3-Kla.

[0125] Example 10: SDCCAG3 immunoprecipitation combined with pan-lactylating antibody to verify SDCCAG3 lactylation

[0126] Through immunoprecipitation experiments, SDCCAG3 antigens were enriched in high-fat osteogenesis-induced BMSCs and bone tissues of hyperlipidemic rats, and western blotting was performed with lactic acid pan antibody (PTM-140RM). The results are as follows Figure 2 a and Figure 2 As shown in b.

[0127] Depend on Figure 2 a and Figure 2 b It can be seen that the expression of SDCCAG3-Kla was increased in the bone tissue and BMSCs of hyperlipidemic rats, indicating that the lactylation level of SDCCAG3 was increased both in vivo and in vitro under a high-fat environment.

[0128] Example 11: Co-IP experiment to detect the binding of SDCCAG3 with lactylation modification enzyme P300 and lactylation demodification enzyme HDAC3

[0129] Through immunoprecipitation experiments, SDCCAG3, P300, and HDAC3 antigens were enriched, followed by protein denaturation and gel electrophoresis. Western blotting was then performed using SDCCAG3, P300, and HDAC3 antibodies to detect the binding of SDCCAG3 to P300 and HDAC3. The results are as follows: Figure 2 c and Figure 2 As shown in d.

[0130] Depend on Figure 2 a and Figure 2 b It can be seen that SDCCAG3 protein can bind to the lactylation modification enzyme P300 and the lactylation demodification enzyme HDAC3, respectively, which further shows that SDCCAG3 can undergo lactylation modification and lactylation demodification.

[0131] Example 12: Lactation of SDCCAG3 inhibits osteogenic differentiation of BMSCs under high-fat environment

[0132] 1. Sdccag3(WT)-3×Flag(WT group), Sdccag3(K278Q)-3×Flag(KQ group), Sdccag3(K278R)-3×Flag(KR group), and negative control group LV17-nc(NC group) lentiviral vector were transferred into high-fat osteogenic induced BMSCs, total protein was extracted, Flag was immunoprecipitated, and western blotting was performed using lactylated pan antibody (PTM-140RM). The results are shown in Figure 1. Figure 3 As shown in a.

[0133] Depend on Figure 3 a It can be seen that compared with other groups, the lactation level of the KQ group was significantly increased, indicating that the simulation of SDCCAG3 lactation in BMSCs was successful.

[0134] 2. According to the same method as above, western blotting was performed using antibodies against osteogenic regulatory factors ALP, RUNX2, and type I collagen. The results are as follows: Figure 3 As shown in b.

[0135] Depend on Figure 3 b It can be seen that compared with other groups, the KQ group inhibited the expression of osteogenic factors, indicating that SDCCAG3 lactylation would inhibit the expression of osteogenic factors in a high-fat environment.

[0136] 3. According to the same method as above, high-fat osteogenic BMSCs transferred with lentiviral vector were obtained, and then ALP and Alizarin Red (ARS) staining experiments were performed. The results were as follows Figure 3 As shown in c.

[0137] Depend on Figure 3 c It can be seen that compared with other groups, the KQ group had fewer densely stained osteoblasts and fewer red mineralized nodules, indicating that SDCCAG3 lactylation inhibited the osteogenic differentiation of BMSCs under a high-fat environment.

[0138] Example 13: SDCCAG3 lactylation inhibits GLUT1 expression and plasma membrane relocalization

[0139] 1. The lentiviral vectors overexpressing Sdccag3(WT)-3×Flag and the negative control group LV17-nc were transferred into high-fat osteogenic induced BMSCs, and the regulation of SDCCAG3 on the expression of GLUT1 protein and plasma membrane relocalization was verified by plasma membrane protein separation. The results are as follows Figure 4 a. Figure 4 b and Figure 4 As shown in c.

[0140] Depend on Figure 4 a. Figure 4 b and Figure 4 c It can be seen that after overexpression of Sdccag3(WT)-3×Flag, the total protein level of GLUT1 increased ( Figure 4 a and b), and the expression level of GLUT1 on the plasma membrane was also increased ( Figure 4 c).

[0141] 2. After enriching SDCCAG3 and GLUT1 antigens through immunoprecipitation experiments, protein denaturation and gel electrophoresis were performed, and then western blotting was performed with SDCCAG3 and GLUT1 antibodies to detect the binding of SDCCAG3 and GLUT1. The results are as follows: Figure 4 As shown in d.

[0142] Depend on Figure 4 d It can be seen that SDCCAG3 protein can bind to GLUT1.

[0143] 3. Sdccag3(WT)-3×Flag(WT group), Sdccag3(K278Q)-3×Flag(KQ group), Sdccag3(K278R)-3×Flag(KR group), and negative control group LV17-nc(NC group) lentiviral vectors were transferred into high-fat osteogenic induced BMSCs, and then Co-IP experiments were performed to verify the effect of SDCCAG3 lactylation modification on GLUT1 binding. The results are shown in Figure 4 e and Figure 4 f.

[0144] Depend on Figure 4 e and Figure 4 f It can be seen that SDCCAG3 lactylation inhibits the binding of SDCCAG3 to GLUT1.

[0145] 4. In order to eliminate the interference of endogenous SDCCAG3, SDCCAG3 was knocked out by sh-Sdccag, and then the lentiviral vectors of Sdccag3(WT)-3×Flag(WT group), Sdccag3(K278Q)-3×Flag(KQ group), Sdccag3(K278R)-3×Flag(KR group), and negative control group LV17-nc(NC group) were transferred into BMSCs induced by high-fat osteogenic, and then Co-IP experiments were performed to verify the effect of SDCCAG3 lactylation modification on GLUT1 binding. Figure 4 g and Figure 4 As shown in h.

[0146] The sequence of the sh-Sdccag3 is:

[0147] 5'-CGACAGAAUUACUGAAGUUTTAACUUCAGUAAUUCUGUCGTT-3'.

[0148] Depend on Figure 4 g and Figure 4 h shows that SDCCAG3 lactylation inhibits the binding of SDCCAG3 to GLUT1. This result is consistent with Figure 4 e Figure 4 f is consistent with the results shown.

[0149] 5. The lentiviral vectors of Sdccag3(WT)-3×Flag(WT group), Sdccag3(K278Q)-3×Flag(KQ group), Sdccag3(K278R)-3×Flag(KR group), and the negative control group LV17-nc(NC group) were transferred into BMSCs induced by high-fat osteogenic stimulation, and the plasma membrane proteins were extracted to verify the effect of SDCCAG3 on the plasma membrane relocalization of GLUT1. The results are as follows: Figure 4 i and Figure 4 j shown.

[0150] Depend on Figure 4 i and Figure 4 j It can be seen that after SDCCAG3 lactylation, the total protein expression level of GLUT1 decreased and its plasma membrane localization decreased, indicating that SDCCAG3 lactylation inhibits the total protein expression and plasma membrane relocalization of GLUT1.

[0151] Example 14: SDCCAG3 lactylation inhibits the expression of key glycolytic enzymes

[0152] The lentiviral vectors of Sdccag3(WT)-3×Flag(WT group), Sdccag3(K278Q)-3×Flag(KQ group), Sdccag3(K278R)-3×Flag(KR group), and the negative control group LV17-nc(NC group) were transferred into BMSCs induced by high-fat osteogenic stimulation. After protein extraction, western blotting experiments were performed to verify the effect of SDCCAG3 on key enzymes of glycolysis. The results are shown in Figure 5 shown.

[0153] Depend on Figure 5 It can be seen that SDCCAG3 lactylation inhibits the expression of key glycolytic enzymes HK1, HK2, PKM, LDHA and LDHB, indicating that SDCCAG3 lactylation will inhibit intracellular glycolysis, and glycolysis is closely related to the osteogenic differentiation of BMSCs in a high-fat environment and the bone formation in hyperlipidemia rats.

[0154] Example 15: Glycolysis stress experiment characterizes that SDCCAG3 lactic acidification inhibits glycolysis

[0155] 1. The day before the experiment - preparation

[0156] ①. Turn on and preheat the detection system: turn on the Seahorse / XFe analyzer and software Wave, connect them successfully, and heat them to 37℃ to keep them stable;

[0157] ②. Hydration probe plate - Step 1: Take out the probe plate assembly (including the hydration plate at the bottom, the green probe plate and the lid), remove the lid and the probe plate and invert it on the laboratory bench, add 200μL / well Seahorse XF Calibrant (pH 7.4) to the hydration plate; align the lid and the probe plate with each well of the hydration plate and put them back on the hydration plate so that the probe plate Sensor is immersed in the XFCalibrant, and place it in a 37℃ CO2-free cell culture incubator for overnight incubation. At the same time, add at least 20mL / plate of XFCalibrant to a 50mL centrifuge tube, place it in a 37℃ CO2-free cell culture incubator for overnight incubation for the next day's experiment. Note: Make sure to place a water box in the incubator to ensure the humidity in the incubator;

[0158] ③. Inoculation of cells: Open the XF96 V3 PS cell culture microplate in a biosafety cabinet, add 80 μL / well cell growth medium to the background correction wells (A1, A12, H1, H12) of the cell culture plate, add 1w osteogenic-induced BMSCs to each of the remaining wells, and place the cell culture plate in the biosafety cabinet for 1 hour to allow the cells to settle naturally to maintain uniform cell distribution and reduce edge effects; then transfer to a 37°C, CO2 cell culture incubator overnight to allow the cells to adhere.

[0159] 2. The day before the experiment - computer test

[0160] ①, Hydration probe plate - Step 2: Take out the incubated XF Calibrant and probe plate device from the 37℃ CO2-free cell culture incubator, remove the lid and probe plate and invert it on the laboratory bench, discard the XF Calibrant in the hydration plate, add 200μL / well of XF Calibrant incubated in the 37℃ CO2-free cell culture incubator again, align the lid and probe plate with each well of the hydration plate and put it back on the hydration plate so that the probe plate Sensor is immersed in the XF Calibrant, put it in the 37℃ CO2-free cell culture incubator and incubate it for 60 minutes, and wait for the drug to be dispensed;

[0161] ②. Prepare the drugs and add them to the dosing wells of the probe plate: Take out oligomycin (light blue cover), 1 vial of grape (blue cover) and 1 vial of 2-DG (green cover) from the foil bag of the kit, add 720μL, 3000μL and 3000μL of detection solution respectively, so that the concentrations are 100μmol / L for oligomycin, 100mmol / L for glucose and 500mmol / L for 2-DG; then use the fixed concentration dosing method (see Table 2 below) to add drugs to the dosing wells with the assistance of the dosing auxiliary plate.

[0162] Table 2

[0163]

[0164]

[0165] ③. Prepare the detection solution: add 500 μL of Seahorse XF glutamine solution (200 mmol / L) to 49.5 mL of Seahorse XF DMEM medium (pH 7.4) and mix well to obtain a glutamine solution with a final concentration of 2 mmol / L as the detection solution;

[0166] ④. Cell washing: remove the cell plate, observe the cell status and confluence (80-90%) under a microscope, aspirate 60 μL of the growth medium in each well but leave 20uL; add 200 μL of detection solution to each well, then aspirate and discard 200 μL, repeat this step once; add 160 μL of detection solution to all wells for the last time, making the final volume 180 μL, place the cell culture plate in a 37°C CO2-free cell culture incubator for 60 minutes and wait for the machine test.

[0167] ⑤. Run the experiment on the machine: open the glycolysis pressure detection template in the Wave software interface, and complete the group definition according to the experimental design, click Start Run, and a dialog box will pop up to select the save location of the experimental data, and then the instrument will pop up the plate tray. According to the software prompts, remove the probe plate cover and place the probe plate and hydration plate combination on the tray, click I'm ready, the tray enters the instrument, and calibration begins (about 20 minutes); after the calibration is completed, take out the cell culture plate from the 37℃ CO2-free cell culture incubator, click Open Tray on the software pop-up interface, the tray pops up and removes the hydration plate and places the cell culture plate on the tray, click Load Cell Plate; after the cell plate enters the instrument, it enters the detection stage, after the measurement is completed, the software pops up the interface and clicks Eject, the tray pops up and removes the probe plate and cell culture plate, click OK, and the tray enters the instrument.

[0168] 3. Data analysis: Install Seahorse XF report generator and Wave software to perform data analysis. The results are as follows: Figure 6 shown.

[0169] Depend on Figure 6 It can be seen that the results of the Seahorse experimental data analysis show that SDCCAG3 lactylation inhibits the glycolysis, glycolytic energy and glycolytic reserve capacity of BMSCs under high-fat conditions. SDCCAG3 lactylation will cascade inhibit glycolysis, thereby inhibiting the osteogenic differentiation of BMSCs in a high-fat environment and leading to poor bone formation in hyperlipidemic rats.

[0170] Example 16, Stable Isotope Labeled U- 13 C6 Glucose Metabolic Flux Assay to Characterize the Inhibition of Glycolytic Metabolic Flux by SDCCAG3 Lactation

[0171] (1) Prepare a stable isotope labeled U- 13 C6 glucose solution: weigh 10.02 mg of stable isotope labeled U- 13 C6 glucose, completely dissolved in 10 mL Seahorse XF DMEM medium (pH 7.4), and 10% FBS was added;

[0172] (2) After the treatment and induction of BMSCs, one dish of cells from each sample was cultured in a 10 cm diameter dish, the culture medium was discarded, the cells were washed once with PBS buffer, and 8 mL / dish of 5.56 mM stable isotope labeled U- 13 C6 glucose solution, cultured in a cell culture incubator at 37°C and 5% CO2 for 48 h;

[0173] (3) The cells were washed once with PBS buffer, and 2 mL / dish of trypsin was added for digestion and collection of the cells. The cells were frozen in liquid nitrogen and transported on dry ice to Shanghai Puling Biotechnology Co., Ltd. for metabolic flux experiments. The details are as follows:

[0174] ①. Take the cell sample frozen and stored in 80% ice-cold methanol solvent system, place it in an ice water bath, and process it with "ultrasound 1min-stop 1min" for 5 cycles. Place it at -20℃ for 30min; centrifuge it at 1500r for 15min (4℃); take 1mL of supernatant extract, dry it under warm nitrogen; redissolve the dried material in 50 microliters of 50% acetonitrile aqueous solution (1:1, volume ratio) for UHPLC-HRMS testing;

[0175] ②. The samples were chromatographed using the Thermo Fisher Ultimate 3000 liquid chromatography system and the Waters BEH Amide column (2.1 mm × 100 mm, 1.7 μm). The injection volume was 5 μL. The flow rate was 0.35 mL / min. The mobile phase consisted of (A) ultrapure water containing 15 mM ammonium acetate (pH = 8.5) and (B) acetonitrile / water (90:10, volume ratio). The metabolites were eluted and separated using a linear gradient mode, with the following gradient: starting at 90% B and maintained for 2 min, linearly decreasing to 75% B at 14 min, linearly decreasing to 65% B at 15 min, linearly decreasing to 50% B at 15.2 min and maintained until 16.9 min, returning to the starting gradient at 17 min and balancing to 20 min.

[0176] ③. The metabolites were ionized and mass spectrometry data were collected using the Q Exactive quadrupole-orbital field mass spectrometer (QE) from Thermo Fisher Scientific. Metabolites were ionized in negative ion mode (HESI-) using a heated electrospray (HESI) ion source; the main parameters of the ion source were set as follows: the spray voltage was 3500 V (HESI-), the capillary temperature and the needle heater temperature were 250 °C and 300 °C, respectively; the sheath gas flow rate was 35 (arbitrary units, the same below), the auxiliary gas flow rate was 10 (arbitrary units); the S-Lens RF was set to 50 (arbitrary units); the primary mass spectrometry scanning range was mass-to-charge ratio (m / z) 70-1050, the resolution was 70000 (mass-to-charge ratio 200), and the AGC threshold was 1×10 6 ;

[0177] ④. The spectra were viewed using the Thermo Fisher analytical software Xcalibur (version 4.0.27.19), and the target metabolites were integrated. Manual inspection was also performed to ensure the accuracy of the integrated area of ​​each target. The data were exported to obtain the original integrated area. At the same time, the original data were corrected for natural isotopes using reference methods. The results are as follows: Figure 7 shown.

[0178] Depend on Figure 7 It can be seen that the glycolysis flow in the SDCCAG3 lactate group is specifically manifested as reduced glucose (m+6) uptake, G6P (m+6), F6P (m+6), F1,6P (m+6), 2-BP (m+3), PEP (m+3), pyruvic acid (m+3), and lactic acid (m+3), which further indicates that SDCCAG3 lactate inhibits the glycolysis of BMSCs under high-fat environment, glycolytic energy, and glycolytic reserve capacity. SDCCAG3 lactate will cascade inhibit glycolysis, thereby inhibiting the osteogenic differentiation of BMSCs in a high-fat environment and leading to poor bone bonding and bone formation in hyperlipidemic rats.

[0179] Example 15: Lactylation of SDCCAG3 inhibits bone formation in hyperlipidemic rats

[0180] 1. Sdccag3(WT)-3×Flag(WT group), Sdccag3(K278Q)-3×Flag(KQ group), Sdccag3(K278R)-3×Flag(KR group), and negative control group LV17-nc(NC group) lentiviral vectors were injected into hyperlipidemic rats, and bone tissue proteins in the skull defect area were extracted. Western blotting was used to verify the lactylation level of SDCCAG3 in hyperlipidemic rats. The results are as follows: Figure 8 As shown in a.

[0181] Depend on Figure 8 a It can be seen that compared with other groups, the lactic acidification of the KQ group was significantly increased, indicating that the lactic acidification of SDCCAG3 was successfully simulated in hyperlipidemic rats.

[0182] 2. After the lentiviral vectors of Sdccag3(WT)-3×Flag(WT group), Sdccag3(K278Q)-3×Flag(KQ group), Sdccag3(K278R)-3×Flag(KR group), and the negative control group LV17-nc(NC group) were injected into the hyperlipidemic rats, the bone tissue proteins in the skull defect area were extracted, and the western blotting experiment was used to verify the effect of SDCCAG3 lactylation on glycolytic enzymes in hyperlipidemic rats. The results are as follows Figure 8 b and Figure 8 As shown in c.

[0183] Depend on Figure 8 b and Figure 8 c It can be seen that SDCCAG3 lactylation inhibits the expression of glycolytic enzymes HK1, HK2, PKM, LDHA, and LDHB in hyperlipidemic rats ( Figure 8 b), while inhibiting the expression of osteogenic factors ( Figure 8 c), further indicating that SDCCAG3 lactylation would cascade inhibit glycolysis, leading to poor bone formation in hyperlipidemic rats.

[0184] 3. Micro-CT detection of new bone formation in rat skull defects

[0185] ① Sample placement: Place the sample vertically in the sample container of the Inveon MM CT machine (SIEMENS, Germany) with the skull defect area as the center and fix it with foam around it;

[0186] ② Sample scanning: Set parameters and use Inveon Acquisition Workplace (SIEMENS, Germany) software for scanning. Skull defect tissue scanning parameters: effective pixel 9.08 μm, scanning time 360 ​​s, exposure time 1000 ms, rotation angle 360°, voltage 80 kV, current 500 μA.

[0187] ③. Data analysis: The maximum height of new bone in the skull defect sample (0.923 mm) was selected as the region of interest (ROI), and bone morphometric analysis was performed using Inveon Research Workplace (SIEMENS, Germany) software.

[0188] ④. Three-dimensional reconstruction: Use COBRA_Exxim (EXXIM Computing Corp., CA) software to visualize the new bone formation in the ROI area and observe the new bone formation in the skull defect area. The results are as follows: Figure 8 d. Figure 8 f and Figure 8 As shown in g.

[0189] Depend on Figure 8 d. Figure 8 f and Figure 8 g It can be seen that the new bone formation in the defect area of ​​the KQ group was significantly lower than that of the KR and WT groups ( Figure 8 d); BMD and BV / TV in the KQ group were lower than those in the KR and WT groups. In addition, in the two-dimensional interface, there was continuous new bone formation of uneven thickness in the defect area in the KR and WT groups, which was not visible in the KQ group. In conclusion, SDCCAG3 lactic acidification inhibits bone formation in hyperlipidemic rats ( Figure 8 f and Figure 8 g).

[0190] 4. Morphological staining of decalcified bone tissue

[0191] The skulls of rats in the normal group and the high-fat group were separated and fixed, decalcified, dehydrated, transparent, wax-impregnated, paraffin-embedded, and sectioned before HE and Masson's staining to detect new bone formation in the skull defect area. Figure 8 h and Figure 8 As shown in i.

[0192] Depend on Figure 8 h and Figure 8 i It can be seen that continuous new bone formation was observed in the defect area of ​​the KR and WT groups, while new bone formation was interrupted in the KQ group, indicating that SDCCAG3 lactylation inhibits bone formation in hyperlipidemic rats.

[0193] Example 16: Lactation of SDCCAG3 inhibits implant osseointegration in hyperlipidemic rats

[0194] 1. Micro-CT detection of new bone formation around implants in rats

[0195] Scanning was performed using Inveon Acquisition Workplace (SIEMENS, Germany) software. Scanning parameters for the femur with implants were as follows: effective pixel 8.89 μm, scanning time 965 s, exposure time 2000 ms, rotation angle 360°, voltage 80 kV, current 300 μA; 0.5 mm bone tissue around the implant was selected as the region of interest (ROI), and bone morphometric analysis was performed using Inveon Research Workplace (SIEMENS, Germany) software. The results are shown in the figure. Fig. 9 shown.

[0196] Depend on Fig. 9 It can be seen that the new bone formation around the implants increased in the KR group and the WT group ( Fig. 9 a), New bone formation around implants decreased in the KQ group ( Fig. 9 b); BV / TV, trabecular thickness, and trabecular number were all decreased in the KQ group ( Fig. 9 c. Fig. 9 d and Fig. 9 e), while trabecular spacing, bone surface area / bone volume, and trabecular pattern factor increased ( Fig. 9 f. Fig. 9 g and Fig. 9 h). The above results all indicate that SDCCAG3 lactylation inhibits new bone formation around implants in hyperlipidemic rats.

[0197] 2. HE staining of hard tissue sections to detect implant-bone integration in rats

[0198] ① Dehydration and infiltration: After Micro-CT scanning and analysis, the samples were dehydrated with 75%, 80%, 85%, 95%, and 100% ethanol gradients, with each gradient dehydration for 24 hours, for a total of 5 days; infiltration with 50%, 70% 7200 resin stock solution, 7200 resin stock solution I, and II, for 5 to 7 days at each level, for a total of 25 days;

[0199] ②, Resin embedding: Use polymethacrylate resin to embed the sample along the slice axis, remove air bubbles, and cure the resin overnight in a light curing machine;

[0200] ③, Slicing: Assemble the diamond blade to the hard tissue slicer, cut the embedded sample along the long axis of the implant, and obtain 70μm thick hard tissue slices;

[0201] ④. Grinding and polishing: Use sandpaper with 500, 800, 1200, 2500, 4000 grit for grinding and polishing;

[0202] ⑤. Perform HE staining according to the instructions of the hematoxylin and eosin (HE) staining kit and seal the slides with neutral resin;

[0203] ⑥ Observe the new bone formation and trabecular arrangement at the implant-bone interface under an Olympus upright microscope. Fig. 9 As shown in i.

[0204] Depend on Fig. 9 i It can be seen that the new bone formation around the implants in the KR group and the WT group increased and was closely integrated with the implants; the new bone formation around the implants in the KQ group decreased and was tightly and loosely integrated with the implants, which indicates that SDCCAG3 lactylation inhibits implant bone integration in hyperlipidemic rats.

[0205] Example 17 Effects of knocking down lactylation modifying enzyme P300 and lactylation demodifying enzyme HDAC3 on SDCCAG3 lactylation, glycolysis and osteogenesis

[0206] 1. siRNA transfection experiment: Lipofectamine TM 2000 transfected with 20μM siRNA in 6-well plate as an example

[0207] ① After receiving the freeze-dried powdered siRNA, centrifuge it instantly, dissolve it in RNase-free H2O to a 20μM storage solution, and store it at -20℃;

[0208] Among them, the siRNAs are siRNA-3239, siRNA-2131 and siRNA-3604 for knocking down the lactylation modification enzyme P300, and siRNA-741, siRNA-408 and siRNA-288 for knocking down the lactylation demodification enzyme HDAC3. The specific sequences are as follows:

[0209] siRNA-3239: 5'-GGAGAGAGGCCCUGAGUUATTUAACUCAGGGCCUCUCUCCTT-3';

[0210] siRNA-2131: 5'-AGAAAGAACUAGAAGAGAATTUUCUCUUCUAGUUCUUUCUTT-3';

[0211] siRNA-3604: 5'-CUUCAAGGGUAUACAAAUATTUAUUUGUAUACCCUUGAAGTT-3';

[0212] siRNA-741: 5'-GCAUUGAUGACCAGAGUUATTUAACUCUGGUCAUCAAUGCTT-3';

[0213] siRNA-408: 5'-CAACACAGCUAAACAACAATTUUGUUGUUUAGCUGUGUUGTT-3';

[0214] siRNA-288: 5'-CCACCAAUAUGCAGGGUUUTTAAACCCUGCAUAUUGGUGGTT-3'.

[0215] ②Inoculation of cells: 1×10 5 BMSCs were seeded in 6-well plates / well and transfected when the cell confluence reached 80-90%;

[0216] ③、Transfection of siRNA:

[0217] a. Discard the old culture medium and rinse once with PBS;

[0218] b. 5 μL / well siRNA dissolved in 250 μL / well Opti-MEM medium , let stand at room temperature for 5 minutes;

[0219] c. 5μL / well Lipofectamine TM 2000 dissolved in 250 μL / well Opti-MEM medium , let stand at room temperature for 5 minutes;

[0220] d. Mix b and c to form a transfection complex and let it stand at room temperature for 30 minutes;

[0221] e. Add 500 μL of transfection complex to each well, fill up to 2 mL of liquid in each well with original culture medium, and culture in a cell culture incubator at 37°C and 5% CO2;

[0222] f. Closely observe the cell status and decide whether to change the medium based on the cell status. Collect samples 48 hours after transfection and perform western blotting experiments to verify the transfection efficiency and related follow-up experiments.

[0223] After transfection of siRNA to knock down the lactylation modifying enzyme P300, SDCCAG3 was immunoprecipitated, and then western blotting was performed with a lactylation pan antibody (PTM-140RM). The results are shown in Fig.10 shown.

[0224] Depend on Fig.10 It can be seen that after knocking down the lactylation modification enzyme P300, the lactylation level of SDCCAG3 was significantly reduced ( Fig.10 a). After knocking down the lactylation demodification enzyme HDAC3, the lactylation level of SDCCAG3 was significantly increased ( Fig.10 b). Knockdown of lactate modification enzyme P300 promoted the expression of osteogenic factors, while knockdown of lactate demodification enzyme HDAC3 reduced the expression of osteogenic factors ( Fig.10 c); Saehorse experiments showed that knocking down the lactate modification enzyme P300 increased glycolysis, glycolytic capacity, and reserve capacity; while knocking down the lactate demodification enzyme HDAC3 reduced glycolysis, glycolytic capacity, and reserve capacity ( Fig.10 d). The above results indicate that siRNA knocking down the lactate modification enzyme P300 can inhibit SDCCAG3 lactate modification and promote glycolysis, thereby promoting osteogenesis. Alternatively, overexpression of the lactate demodification enzyme HDAC3 can promote glycolysis, thereby promoting osteogenesis.

Claims

1. A regulatory network for regulating the expression level and plasma membrane localization of GLUT1 in cells, characterized in that: The regulatory network is a lactylation modification enzyme P300-SDCCAG3 lactylation modification-GLUT1 regulatory network, or a lactylation demodification enzyme HDAC3-SDCCAG3 lactylation demodification-GLUT1 regulatory network; Among them, the NCBI ID of the SDCCAG3 is 306322, the NCBI ID of the lactylation modification enzyme P300 is 170915, the NCBI ID of the GLUT1 is 24778, and the NCBI ID of the lactylation demodification enzyme HDAC3 is 84578.

2. The use of the lactylation modification enzyme P300-SDCCAG3 lactylation modification-GLUT1 regulatory network or the lactylation demodification enzyme HDAC3-SDCCAG3 lactylation demodification-GLUT1 regulatory network according to claim 1 in the preparation of a drug for promoting osteogenic differentiation and bone formation in hyperlipidemia rats, characterized in that: The application is the use of a lactylation modification enzyme P300-SDCCAG3 lactylation modification-GLUT1 regulatory network, or a regulatory factor of a lactylation demodification enzyme HDAC3-SDCCAG3 lactylation demodification-GLUT1 regulatory network in the preparation of a drug for promoting bone formation and peri-implant bone integration in hyperlipidemia rats.

3. The use according to claim 2, characterized in that The drug inhibits the expression of lactate modification enzyme P300 and / or promotes the expression of lactate demodification enzyme HDAC3 in hyperlipidemic rats.

4. The use according to claim 2, characterized in that The regulatory factors of the lactylation modification enzyme P300-SDCCAG3 lactylation modification-GLUT1 regulatory network or the lactylation demodification enzyme HDAC3-SDCCAG3 lactylation demodification-GLUT1 regulatory network include: nucleic acid molecules, carbohydrates, lipids, polypeptides, proteins or lentiviruses.

5. The use according to claim 4, characterized in that The nucleic acid molecule is siRNA-3239, siRNA-2131 or siRNA-3604 that inhibits the expression of lactylation modification enzyme P300; The sequences of the siRNA-3239, siRNA-2131 or siRNA-3604 are as follows: siRNA-3239: 5'-GGAGAGAGGCCCUGAGUUATTUAACUCAGGGCCUCUCUCCTT-3'; siRNA-2131: 5'-AGAAAGAACUAGAAGAGAATTUUCUCUUCUAGUUCUUUCUTT-3'; siRNA-3604: 5'-CUUCAAGGGUAUACAAAUATTUAUUUGUAUACCCUUGAAGTT-3'.

6. The use according to claim 2, characterized in that The medicine contains an effective dose of the regulatory factor of the regulatory network according to claim 4.

7. The use according to claim 2, characterized in that The drug contains a pharmaceutically acceptable carrier.

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