Application of AMPK activator or PPAR gamma inhibitor in prevention or treatment of diabetic osteoporosis

Abstract

The invention discloses an application of an AMPK (adenosine monophosphate kinase) activator or a PPAR (peroxisome proliferator activated receptor) gamma inhibitor in preparation of a medicine for preventing or treating diabetic osteoporosis. The invention discloses a key effect of a brand new signal channel, namely an AMPK-PPAR gamma-ERS axis, in osteogenic differentiation disorder of diabetes mellitus, and provides a method for preventing and treating diabetic osteoporosis by regulating and controlling the signal axis by taking metformin as a core. The invention provides new guidance for research and development of medicines for treating osteoporosis, and has potential clinical application and mechanism research values.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to the application of an AMPK activator or PPARγ inhibitor in the prevention or treatment of diabetic osteoporosis. Background Technology

[0002] Diabetic osteoporosis (DOP) is a common chronic complication of diabetes, characterized by decreased bone mineral density, bone microarchitectural deterioration, and a significantly increased risk of fractures. Hyperglycemia disrupts bone metabolic homeostasis through multiple mechanisms, particularly inhibiting osteoblast differentiation, leading to reduced bone formation. Current treatments for diabetic osteoporosis are limited, and most fail to address its unique molecular mechanisms.

[0003] Currently, the following bottlenecks exist in the clinical medication and mechanism research of diabetic osteoporosis: 1. Currently, conventional drugs (such as bisphosphonates) or hypoglycemic agents (such as thiazolidinediones) used clinically to treat osteoporosis have the following limitations in treating diabetic osteoporosis (DOP): Bisphosphonates (such as alendronate sodium) mainly reduce bone resorption by inhibiting osteoclast activity, but they cannot effectively promote osteoblast differentiation and cannot reverse the core pathological mechanism of osteogenic dysfunction in diabetes. Thiazolidinediones (TZDs) can improve insulin sensitivity, but by activating PPARγ, they inhibit osteogenic differentiation and promote adipogenic differentiation, exacerbating bone loss, and are not suitable for diabetic patients with pre-existing osteoporosis. 2. Currently, there are no drugs specifically targeting the key mechanism by which high glucose inhibits osteogenic differentiation, and existing treatment strategies have failed to achieve the dual goals of lowering blood glucose and promoting bone formation simultaneously. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the purpose of this invention is to provide an application of an AMPK activator or PPARγ inhibitor in the prevention or treatment of diabetic osteoporosis.

[0005] The objective of this invention is achieved through the following technical solution: In a first aspect, the present invention provides the use of an AMPK activator or PPARγ inhibitor in the preparation of a medicament for the prevention or treatment of diabetic osteoporosis.

[0006] As a preferred embodiment, the AMPK activator includes at least one of metformin, 5-aminoimidazole-4-carboxamide nucleoside, and MT63-78.

[0007] As a preferred embodiment, the PPARγ inhibitor includes at least one of metformin, GW9662, and T0070907.

[0008] Secondly, the present invention provides the use of metformin in the preparation of a medicament for the prevention or treatment of diabetic osteoporosis.

[0009] Metformin improves osteoblast differentiation by regulating the AMPK-PPARγ-ERS signaling axis.

[0010] As a preferred embodiment, the effective dose of metformin in the drug for preventing diabetic osteoporosis is 400-600 mg / day.

[0011] As a preferred embodiment, the effective dose of metformin in the drug for treating diabetic osteoporosis is 1000-3000 mg / day.

[0012] As a preferred embodiment, the drug also includes a carrier that promotes bone integration. Combining metformin with a carrier that promotes bone integration can create bone-targeted sustained-release microspheres or local implants. These can be administered to the site of bone injury via local injection, achieving high local concentrations and long-lasting release of the drug, significantly improving its bioavailability in bone tissue while reducing side effects from systemic exposure (such as gastrointestinal reactions). This approach is particularly suitable for the precision treatment of diabetic patients undergoing fracture repair or with localized bone defects.

[0013] As a preferred embodiment, the carrier includes at least one of calcium phosphate and hydroxyapatite.

[0014] Compared with the prior art, the present invention has the following beneficial effects: 1) This invention is the first to verify that metformin can improve diabetic osteoporosis and reveals the key role of a novel signaling pathway—the AMPK-PPARγ-ERS axis—in diabetic osteogenic differentiation disorder. It provides a method for preventing and treating diabetic osteoporosis by regulating this signaling axis with metformin as the core.

[0015] 2) This invention provides new guidance for the development of drugs for the treatment of osteoporosis and has potential clinical application and mechanism research value. Attached Figure Description

[0016] Other features, objects, and advantages of the present invention will become more apparent from the following detailed description of non-limiting embodiments with reference to the accompanying drawings: Figure 1 The fasting blood glucose, insulin content, and HbA1c results of each group of SD rats in Example 1; Figure 2 The results of bone tissue and bone metabolism analysis of SD rats in each group in Example 1 are shown; among them, Figure 2 A represents the whole-body bone mineral density result; Figure 2B represents the bone mineral density results for the upper limbs, lower limbs, and spine; Figure 2 C1 is an image of the L4 lumbar vertebra obtained by Micro-CT. Figure 2 C2 represents the results of bone mineral density (BMD), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N). Figure 2 D represents the HE staining result; Figure 2 E represents the serum ALP expression level; Figure 2 F represents the expression level of OCN in serum; Figure 2 G represents the expression level of TRAP5b in serum; Figure 3 The results of protein analysis in the bone tissue of SD rats in each group in Example 1 are shown; among them, Figure 3 A1 shows the SDS-PAGE electrophoresis analysis results of BMP-2, PPAR-γ, p-AMPK, and AMPK proteins; Figure 3 A2 shows the expression levels of BMP-2, PPAR-γ, p-AMPK, and AMPK proteins; Figure 3 B1 shows the SDS-PAGE electrophoresis results of PERK, ATF4, and CHOP proteins; B represents the SDS-PAGE electrophoresis results. Figure 3 B2 shows the expression levels of PERK, ATF4, and CHOP proteins; Figure 4 The results of ARS staining, BMP-2 and PPAR-γ protein analysis for each group of cells in Example 2 are shown; among them, Figure 4 A represents the ARS staining result; Figure 4 B represents the SDS-PAGE electrophoresis analysis results of BMP-2 and PPAR-γ proteins; Figure 4 C represents the relative amount of mineralized nodules; Figure 4 D represents the expression levels of BMP-2 and PPAR-γ proteins; Figure 5 The results of AMPK and p-AMPK protein analysis in each group of cells in Example 2 are shown; among them, Figure 5 A represents the results of SDS-PAGE electrophoresis analysis; Figure 5 B represents the expression level result; Figure 6 The results of PERK, ATF4, and CHOP protein analysis in each group of cells from Examples 2 and 4 are shown; among them, Figure 6 A1 shows the SDS-PAGE electrophoresis analysis results of each group of cells in Example 2; Figure 6 A2 represents the expression levels of cells in each group in Example 2; Figure 6 B1 shows the SDS-PAGE electrophoresis analysis results of each group of cells in Example 4; Figure 6B2 represents the expression levels of cells in each group in Example 4; Figure 7 The results show the cell viability and ALP and OCN protein analysis of each group of cells in Example 3; among them, Figure 7 A represents the cell viability result; Figure 7 B represents the ALP analysis results; Figure 7 C represents the OCN protein analysis result; Figure 8 The results show the cell viability, ARS staining, and protein analysis of each group of cells in Example 4; among them, Figure 8 A1 shows the SDS-PAGE electrophoresis analysis results of AMPK and p-AMPK proteins in cells from groups BC and Compound C. Figure 8 A2 shows the expression levels of AMPK and p-AMPK proteins in cells of group BC and Compound C. Figure 8 B represents the cell viability results for the HG group, HG+MF group, HG+CompoundC group, and HG+MF+CompoundC group. Figure 8 C represents the relative amounts of ARS staining and mineralized nodules in the HG group, HG+MF group, HG+Compound C group, and HG+MF+Compound C group. Figure 8 D and Figure 8 E represents the expression levels of ALP and OCN proteins in the HG group, HG+MF group, HG+Compound C group, and HG+MF+Compound C group, respectively. Figure 9 The results of BMP-2, PPAR-γ, AMPK, and p-AMPK protein analysis in the HG group, HG+MF group, HG+Compound C group, and HG+MF+Compound C group in Example 4 are shown; among them, Figure 9 A represents the results of SDS-PAGE electrophoresis analysis; Figure 9 B represents the expression level result; Figure 10 The results of PERK, ATF4, and CHOP protein analysis in the HG group, HG+MF group, HG+Compound C group, and HG+MF+Compound C group in Example 4 are shown; among them, Figure 10 A represents the results of SDS-PAGE electrophoresis analysis; Figure 10 B represents the expression level result. Detailed Implementation

[0017] The present invention will now be described in detail with reference to specific embodiments. These embodiments will help those skilled in the art to further understand the present invention, but do not limit the invention in any way. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention. These all fall within the scope of protection of the present invention.

[0018] The sources of the main reagents used in the following examples are shown in Table 1, and the main experimental instruments used are shown in Table 2.

[0019] Table 1 Main Reagents Table 2 Experimental Instruments Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Other materials and reagents used in the following examples are commercially available unless otherwise specified.

[0020] Example 1 This embodiment investigates the effect of metformin on osteoporosis in diabetic rats by treating them with metformin. The specific steps are as follows: 1. Animal grouping and intervention Male SD rats aged 5-6 weeks (purchased from Cyagen Biosciences) were selected. After a one-week acclimatization period, their body weight was measured, and they were divided into two groups: a normal control group (BC group, n = 20) and a high-sugar, high-fat diet group (HGHF group, n = 20). Rats in the HGHF group were fed a high-sugar, high-fat diet for 16 weeks to induce obesity. After fasting for 16 hours (with water access), their body weight was measured. A 1% STZ solution was prepared as described above, and streptozotocin (STZ) injection (30 mg / kg) was administered intraperitoneally for 3 days, calculated based on the rats' daily body weight. Tail venous blood was collected by tail clipping at 5, 7, and 14 days post-injection, and fasting blood glucose was measured using a glucometer (all rats were fasted but had access to water for 16 hours before measurement). Rats with a fasting blood glucose ≥ 16.7 mmol / L were considered successful type 2 diabetes models. The control group rats were injected with an equal volume of physiological saline. After successful modeling, 12 rats in the type 2 diabetes model group and 12 rats in the control group were randomly divided into two groups using a random number table. One group of type 2 diabetes model rats was treated with metformin (900 mg / kg / d for 16 weeks) as the diabetes + metformin group (DM+MF group, n=6). The other group of type 2 diabetes model rats was administered an equal volume of physiological saline by gavage as the diabetes group. One group of control rats was treated with metformin (900 mg / kg / d for 16 weeks) as the metformin group (MF group, n=6). The other group of control rats was administered an equal volume of physiological saline by gavage as the control group (BC group, n=6).

[0021] 2. Bone mineral density measurement After the intervention period, the SD rats in each group were fasted for 12 hours, and their fasting blood glucose, insulin levels, and HbA1c were measured. Under anesthesia with intraperitoneal injection of 10% chloral hydrate (3ml / kg), the bone mineral density of the upper limbs, lower limbs, spine, and whole body of the SD rats in each group was measured in the bone mineral density laboratory of our unit using dual-energy X-ray absorptiometry (DEXA).

[0022] 3. ELISA was used to determine the expression of serum ALP, OCN, and TRAP5b in rats of each group. 3.1 Sample Preparation SD rat serum samples: Blood was collected from the abdominal aorta of SD rats under anesthesia in each group. After centrifugation at 3000 rpm for 5 min, the serum was collected and stored at -20 ℃.

[0023] 3.2 Reagent Preparation Rat ELISA kits were selected. The operation steps and concentration units for detecting different indicators varied. The operation was carried out in accordance with the instructions of different ELISA kits.

[0024] 1) Rat ELISA Standard Working Solution: Centrifuge the standard at 10000 rpm for 1 min, add 1.0 ml of standard & sample diluent to the lyophilized standard, tighten the cap, let stand for 10 min, invert several times to ensure complete dissolution, then gently mix to prepare the standard diluent. Then, perform serial dilutions as needed. Serial dilution method: Take 7 EP tubes, add 500 μl of standard & diluent to each tube, and transfer 500 μl of the standard working solution to one of the EP tubes to dilute the standard working solution (S1). Repeat this process for subsequent tubes to perform serial dilutions (Table 3).

[0025] Table 3. Preparation of working solutions for rat ELISA standards using stepwise concentrations. 2) Washing solution: Dilute the concentrated washing solution with double-distilled water (1:25).

[0026] 3) Biotinylated antibody working solution: Dilute 100× concentrated biotinylated antibody to 1× working concentration using biotinylated antibody diluent.

[0027] 4) Enzyme conjugate working solution: Dilute 100× concentrated HRP enzyme conjugate to 1× working concentration with enzyme conjugate diluent.

[0028] 3.3 Rat ELISA kit operation steps: 1) Sample addition: Take the 96-well plate from the kit and set up standard wells and test sample wells. 3 replicates for each concentration. Add 100 μl of standard or test sample to each well, placing the sample at the bottom of the plate, avoiding contact with the well walls, and gently shake to mix.

[0029] 2) Incubation: Cover with a plate and incubate at 37 ℃ for 90 min. Discard the liquid, spin dry, and do not wash.

[0030] 3) Labeled antibody incubation: Add 100 μl of biotinylated antibody working solution to each well, mix well, cover with a new plate, and incubate at 37°C for 60 min.

[0031] 4) Washing: Shake off the liquid in the wells, add 350 μl of washing solution to each well, soak for 1-2 minutes, shake off the liquid in the plate, and pat dry on thick absorbent paper. Repeat 3 times.

[0032] 5) Enzyme incubation: Add 100 μl of horseradish peroxidase-labeled avidin working solution to each well, cover with a plate, and incubate at 37°C for 30 min.

[0033] 6) Washing: Shake off the liquid in the wells, add 350 μl of washing solution to each well, soak for 1-2 min, shake off the liquid in the plate, and pat dry on thick absorbent paper. Repeat 5 times.

[0034] 7) Color development: Add 90 μl of substrate solution (TMB) to each well, cover with a plate, and incubate at 37°C in the dark for 15 min.

[0035] 8) Termination of reaction: Add 50 μl of stop solution to each well to terminate the reaction.

[0036] 9) Detection: Zero the blank well and measure the optical density (OD value) of each well at a wavelength of 450 nm within 5 min after the reaction ends.

[0037] 4. Micro-CT Detection of Microstructural Indicators of the L4 Lumbar Vertebra in Rats 4.1 Collect bone tissue from the fourth lumbar vertebra, femur, tibia and fibula of rats, remove soft tissues such as muscles, cartilage and surrounding connective tissue, rinse with physiological saline and store in liquid nitrogen.

[0038] 4.2 The L4 lumbar vertebrae were scanned along the longitudinal axis of the specimen using a SkyScan 1076 microcomputation scanner with a resolution of 9 µm, followed by analysis using guidelines for evaluating rodent skeletal microstructure using microcomputed tomography. Scanning parameters were set to 40 kV X-ray power and 0.25 mA tube current. Three images were averaged at each angle (0.9º) to generate the final image, and trabeculae were determined by a fixed threshold. These images were reconstructed using NRecon software (Micro-Photonics Inc., Allentown, PA, USA). Lumbar vertebral microstructural parameters, such as volumetric bone mineral density (vBMD), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N), were analyzed.

[0039] 5. Total protein in bone tissue 5.1 Extraction of total protein from bone tissue 1) Preparation of extraction solution: According to the number of samples, add 2 μl of protease inhibitor and 2 μl of protein stabilizer to every 500 μl of cold bone tissue protein extract solution, mix well and set at 2-8 ℃ for later use.

[0040] 2) Take fresh bone tissue samples and soak them thoroughly in PBS buffer or physiological saline at 4 ℃ and pH 7.4. Replace with fresh physiological saline or PBS and soak thoroughly again, then rinse with pure water to remove blood and red blood cells.

[0041] 3) Cut the bone tissue into small pieces, weigh them, and put them into a mortar containing liquid nitrogen. Grind the bone tissue into powder while ensuring that the liquid nitrogen does not completely evaporate.

[0042] 4) Add bone tissue powder to a centrifuge tube and add 500 μl of protein extraction solution for every 200 mg of bone tissue.

[0043] 5) After mixing, shake at 4 ℃ for 30 minutes. Use a low shaking speed; the extract should only be able to swirl slightly.

[0044] 6) 10 ultrasound sessions in an ice bath under conditions of 80 W, 10 s ultrasound / 10 s interval.

[0045] 7) Centrifuge at 4 ℃ and 12000 rpm for 10 minutes.

[0046] 8) Aspirate the supernatant into another pre-cooled clean centrifuge tube to obtain total protein from bone tissue.

[0047] 9) The above protein extracts can be used directly in downstream experiments or packaged and stored at -80 ℃ for later use.

[0048] 5.2 Protein concentration was determined by BCA method.

[0049] 1) Diluting BSA standards: Dilute BSA standards with the same diluent as the protein sample to be tested, as shown in Table 4 below.

[0050] Table 4 Dilution Table of BSA Standards 2) Calculate the total volume of BCA working fluid. Total BCA working fluid volume = (Number of BSA standards + Number of unknown samples) × Number of replicates × BCA working fluid volume per sample

[0051] 3) Based on the calculated total amount of BCA working solution, prepare BCA working solution by mixing BCA Solution A and BCA Solution B at a volume ratio of 50:1 and mix thoroughly.

[0052] 4) Quantitative detection i. According to the table above, add 25 μl each of the diluted BSA standard and the protein sample to be tested to the labeled 96-well plate. It is recommended to perform 2-3 parallel reactions for each sample (the sample can be diluted 5- or 10-fold in advance depending on the concentration). ii. Add 200 μl of BCA working solution to each well, mix thoroughly, cover the 96-well plate, incubate at 37°C for 30 min, cool to room temperature, and complete the detection within 3 min. iii. Use a microplate reader to measure the absorbance value of each sample and BSA standard at 562 nm, and record the results.

[0053] iv. Plot a standard curve and calculate the protein concentration in the sample.

[0054] 5.3 SDS-PAGE Electrophoresis 1) Prepare 15 ml of 8% separating gel (5 ml distilled water, 4.0 ml 30% Acr-Bis (29:1), 5.7 ml 1M Tris (pH 8.8), 150 μl 10% SDS, 150 μl 10% gel polymerization catalyst, and 9 μl TEMED) according to the ratio, quickly pour it into a glass plate, cover it with water seal gel, and polymerize for 30-60 min; then use filter paper to absorb the water, prepare 4 ml of 5% stacking gel (2.7 ml distilled water, 670 μl 30% Acr-Bis (29:1), 500 μl 1M Tris (pH 8.8), 40 μl 10% SDS, 40 μl 10% gel polymerization catalyst, and 4 μl TEMED), quickly pour it into a glass plate, insert a comb, and polymerize for about 30 min.

[0055] 2) Based on the measured concentration, calculate 40 μg of total protein sample per well and an equal proportion of loading buffer, shake and mix well, boil in a water bath at 95°C for 10 min, place in an ice bath for 5 min, and then start loading the sample.

[0056] 3) Fix the prepared SDS-PAGE gel vertically on the gel plate, place it in the electrophoresis tank, add 1× electrophoresis buffer, and remove the comb.

[0057] 4) Add protein marker (5 μl / well) and protein sample (40 μg / well).

[0058] 5) First, use 80V constant voltage electrophoresis. When bromophenol blue enters the separating gel, switch to 120V constant voltage electrophoresis. Stop electrophoresis when the bromophenol blue moves to 1cm from the front edge of the gel plate.

[0059] 6) Transfer: After electrophoresis, cut the desired area from the gel according to the marker bands and completely immerse it in 1× transfer buffer. Cut an appropriate NC membrane according to the size of the gel and completely immerse it in 1× transfer buffer. Assemble the sandwich in the following order: "negative electrode → sponge pad → thick filter paper → gel → NC membrane → thick filter paper → sponge pad → positive electrode". Ensure there are no air bubbles between the gel and the membrane, and between the gel and the filter paper. Add pre-cooled 1× transfer buffer and transfer at a constant voltage of 100V for 100 minutes. Note that the transfer must be performed in an ice bath to avoid severe overheating.

[0060] 7) Sealing: Place the transferred NC membrane (with the sharp corners marked) into ddH2O and rinse for 1-2 minutes to remove the transfer solution. Place the rinsed NC membrane into a 5% skim milk powder sealing solution and place it on a horizontal shaker for 1 hour at room temperature.

[0061] 8) Primary antibody incubation: Refer to the primary antibody instructions and dilute the primary antibodies with the appropriate dilution buffer according to the following ratios: β-actin antibody (1:1000), BMP2 antibody (1:1000), PPARγ antibody (1:1000), AMPK antibody (1:1000), p-AMPK (1:1000), ATF4 antibody (1:1000), CHOP antibody (1:1000), PERK antibody (1:1000).

[0062] The blocked NC membrane was incubated with primary antibody overnight at 4°C on a shaker. Then the NC membrane was removed from the primary antibody, 1× TBST was added, and the membrane was placed on a horizontal shaker and washed at room temperature for 10 min. This process was repeated 3 times.

[0063] 9) Secondary antibody incubation: Dilute the secondary antibody with 5% skim milk blocking buffer (HRP-labeled goat anti-rabbit secondary antibody diluted 1:3000). Incubate the NC membrane with the secondary antibody on a horizontal shaker at room temperature for 1 hour. Remove the NC membrane from the secondary antibody, add 1× TBST, and wash on a horizontal shaker at room temperature for 10 minutes. Repeat 3 times.

[0064] 10) Mix ECL chemiluminescence reagent A and B in equal volumes at a ratio of 1:1 to prepare ECL luminescence working solution.

[0065] 11) The ECL luminescent working solution is evenly dropped onto the NC membrane and placed in a gel imaging system for exposure and imaging.

[0066] 12) The gray values ​​of each band were measured using ImageJ software and compared with the results of the internal reference β-actin. Statistical analysis was performed based on the ratio.

[0067] 6. Bone tissue decalcification fixation and embedding Rat femur, lumbar vertebrae, and tibia / fibula tissues were harvested, and the attached muscle and connective tissue were quickly separated. The tissues were then immersed in a decalcification solution, which was changed every 3 days for 3 consecutive months. After decalcification, the tissues were fixed in neutral paraformaldehyde, dehydrated, and embedded in paraffin blocks.

[0068] 1) Dehydration: Remove the bone tissue from the formaldehyde fixative and trim the target area with a scalpel in a fume hood. Place the trimmed tissue in a dehydration box and label it. Place the dehydration box in a basket and dehydrate it sequentially with alcohol in a dehydrator. 75% alcohol 4h - 85% alcohol 2h - 90% alcohol 2h - 95% alcohol 1h - anhydrous ethanol I 30min - anhydrous ethanol II 30min - benzene 10min - xylene I 10min - xylene II 10min - wax I 1h - wax II 1h - wax III 1h.

[0069] 2) Embedding: The paraffin-impregnated tissue is embedded in an embedding machine. First, the molten paraffin is placed into the embedding frame. Before the paraffin solidifies, the tissue is removed from the dehydration box and placed into the embedding frame according to the embedding surface requirements, and the corresponding label is attached. The tissue is then cooled on a -20°C freezing stage. After the paraffin solidifies, the paraffin block is removed from the embedding frame and trimmed.

[0070] 7. Hematoxylin and Hematoxylin (HE) staining Paraffin sections of femoral tissue from each group of SD rats were stained with hematoxylin and eosin (HE) to observe the differences in the morphology of femoral trabeculae between the normal group and the diabetic group.

[0071] 1) Sectioning: Place the trimmed wax block on a paraffin microtome and section it to a thickness of 4 μm. Float the sections on 40°C warm water in a slide spreader to flatten the tissue. Use a glass slide to lift the tissue and place it in a 60°C oven to bake. After the water has dried and the wax has melted, remove it and store it at room temperature for later use.

[0072] 2) Dewaxing paraffin sections: The sections were placed in xylene I for 20 min - xylene II for 20 min - anhydrous ethanol I for 10 min - anhydrous ethanol II for 10 min - 95% ethanol for 5 min - 90% ethanol for 5 min - 80% ethanol for 5 min - 70% ethanol for 5 min - and then washed 3 times with distilled water.

[0073] 3) Hematoxylin staining of cell nuclei: Slices were stained with Harris hematoxylin for 8 min, washed with water, differentiated with 1% hydrochloric acid for a few seconds, washed with water, and then blued with 0.6% ammonia water, and washed with water.

[0074] 4) Eosin staining of cytoplasm: Immerse the section in eosin staining solution for 3 min.

[0075] 5) Dehydration and mounting: Place the sections in 95% ethanol I for 5 min - 95% ethanol II for 5 min - anhydrous ethanol I for 5 min - anhydrous ethanol II for 5 min - xylene I for 5 min - xylene II for 5 min to dehydrate and clear them. Remove the sections from the xylene and let them dry slightly before mounting them with neutral resin.

[0076] 6) Microscopic examination and image acquisition and analysis. Staining results: cell nuclei are blue, cytoplasm is red.

[0077] 8. Statistical Analysis SPSS 19.0 software was used for statistical analysis. Quantitative data that followed a normal distribution and had homogeneous population variance were expressed as mean ± standard deviation. (mean ± standard deviation) indicates the statistical significance of data. One-way ANOVA was used for comparisons among multiple groups. LSD-t tests were then used for pairwise comparisons between groups, with a two-tailed P < 0.05 indicating statistical significance. For continuous data that did not meet the requirements of normality or homogeneity of variance, the median was used, and the rank-sum test was used for comparisons between groups. All statistical analyses were performed using R software (https: / / www.r-project.org).

[0078] 9. Results 9.1 The fasting blood glucose, insulin levels, and HbA1c results of each group of SD rats are as follows: Figure 1 As shown. By Figure 1 It is evident that, compared to the DM group, the DM+MF group significantly reduced fasting blood glucose and HbA1c in rats and significantly increased insulin levels.

[0079] 9.2 Whole-body bone mineral density results of SD rats in each group are as follows: Figure 2 As shown in Figure A, the measured bone mineral density results for the upper limb, lower limb, and spine are as follows: Figure 2 As shown in B. Figure 2 A and Figure 2 The results of B showed that, compared with the DM group, the DM+MF group significantly increased the whole-body bone mineral density of rats, specifically significantly increasing the bone mineral density of the upper limbs and spine.

[0080] 9.3 Images of the L4 lumbar vertebrae in each group of SD rats were obtained using Micro-CT. Figure 2 As shown in C1, the volumetric bone mineral density (L4 BMD), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), and trabecular number (Tb.N) were analyzed as follows: Figure 2 As shown in C2. Figure 2 C1 and Figure 2 The results of C2 showed that, compared with the DM group, the DM+MF group significantly increased vBMD, BV / TV, Tb.Th and Tb.N in the lumbar spine of rats.

[0081] 9.4 HE staining results of SD rats in each group are as follows: Figure 2 As shown in D.

[0082] 9.5 The expression levels of ALP, OCN, and TRAP5b in the serum of SD rats in each group were measured by ELISA. Figure 2 E, Figure 2 F and Figure 2 As shown in G. The results show that, compared with the DM group, the DM+MF group significantly increased the expression levels of ALP, OCN, and TRAP5b.

[0083] 9.6 Protein analysis results in bone tissue of SD rats in each group are as follows: Figure 3 As shown. Figure 3 A1 and Figure 3 B1 shows the SDS-PAGE electrophoresis analysis results of each protein. Figure 3 A2 and Figure 3 B2 shows the expression levels of each protein. Figure 3 The results showed that, compared with the DM group, the DM+MF group significantly increased the expression levels of BMP-2 and p-AMPK, and significantly decreased the expression levels of PPAR-γ, PERK, ATF4, and CHOP.

[0084] The results above show that metformin can significantly improve bone metabolism disorders in diabetic rats, and this effect is related to the expression of PPARγ and endoplasmic reticulum stress.

[0085] Example 2 This embodiment uses metformin to intervene in osteoblasts and studies the effect of metformin on glycemic osteoblasts. The specific steps are as follows: 1. Experimental Materials 1.1 Cell line: Mouse pre-osteoblast precursor MC3T3-E1 cells were used, obtained from the Shanghai Cell Bank of the Chinese Academy of Sciences.

[0086] 1.2 Preparation of main reagents 1.2.1 Preparation of complete culture medium: 10% fetal bovine serum + 1% penicillin and streptomycin antibiotics + 89% α-MEM medium.

[0087] 1.2.2 Preparation of complete culture medium containing osteogenic induction solution: Measure the required doses of sodium β-glycerophosphate powder and ascorbic acid powder, and dilute them with α-MEM medium to concentrations of 10 mM and 50 mg / L, respectively. Take 1 ml of 50 mg / L ascorbic acid solution and add it to 9 ml of 10 mM sodium β-glycerophosphate solution to prepare an osteogenic induction solution concentrated 100 times. After sterilization by filtration through a 0.22 μm CA filter, dispense the solution, store it at -20℃ protected from light, and dilute it with complete culture medium at a ratio of 1:100 before use.

[0088] 1.2.3 Preparation of high-glucose solution: Measure the required amount of glucose powder, dilute it with α-MEM medium to 25 mmol / L (including the sugar content of the medium), filter it through a 0.22 μm CA filter for sterilization, and then dispense it at 4℃ for later use.

[0089] 1.2.4 Preparation of metformin intervention solution: Weigh the required dose of metformin powder, dilute it with complete culture medium containing osteogenic induction solution to 25, 50, and 100 μmol / L, filter it through a 0.22 μm CA filter for sterilization, and dispense it at 4℃ for later use.

[0090] 1.2.5 Preparation of Reagents for Western Blot 1) 10% ammonium persulfate (APS): Dissolve 0.5g ammonium persulfate powder in 5 ml of deionized water, shake well, store away from light, and use within 2 months.

[0091] 2) 10× Electrophoresis Buffer: Dissolve 30.3g Tris, 188g glycine and 10g SDS in 800 ml of deionized water, bring the volume to 1L with deionized water, store at room temperature, and dilute to 1× before use.

[0092] 3) 10× Electroporation Buffer: Dissolve 30.3g Tris and 144g glycine in 800 ml of deionized water, bring the volume to 1L with deionized water, store at 4℃, and dilute to 1× before use.

[0093] 4) TBS buffer: 8g NaCl, 0.2g KCl, 3g Tris, add water to 800 ml, adjust pH to 7.4 with hydrochloric acid, and add deionized water to bring the volume to 1L.

[0094] 5) TBST buffer: Add 1 ml of Tween-2000 to 1 L of TBS, mix well, and store at room temperature.

[0095] 6) Blocking solution: Dissolve 5g skim milk powder in 100ml TBST, mix thoroughly, and store at 4℃.

[0096] 7) Acrylamide and N,N'-methylenebisacrylamide should be prepared as a stock solution containing 29% (w / v) acrylamide and 1% (w / v) N,N'-methylenebisacrylamide in warm (to facilitate the dissolution of bisacrylamide) deionized water. Add 29 g of acrylamide and 1 g of N,N'-methylenebisacrylamide to 100 ml of water and store in a brown bottle at 4°C protected from light. Strictly verify that the pH does not exceed 7.0, as a deamination reaction can occur, which is photocatalyzed or alkali-catalyzed. The stock solution should not be used for more than two months; it must be prepared fresh every few months. If precipitation occurs, it can be filtered.

[0097] 8) Sodium dodecyl sulfate SDS solution: 0.1 g SDS (10% w / v) prepared with 1 ml H2O deionized water, stored at room temperature.

[0098] 9) Separating gel buffer: 1.5 mmol / L Tris-HCl (pH 8.8): Mix 18.15 g Tris and 48 ml 1 mol / L HCl, and dilute with water to a final volume of 100 ml. Filter and store at 40°C.

[0099] 10) Stacking gel buffer: 0.5 mmol / L Tris-HCl (pH 6.8): Dissolve 6.05 g Tris in 40 ml H2O, adjust the pH to 6.8 with approximately 48 ml of 1 mol / L HCl, and dilute with water to a final volume of 100 ml. Filter and store at 40°C. Both buffers must be prepared using Tris base and then adjusted to pH with HCl.

[0100] 11) TEMED stock solution: N,N,N'N' tetramethylethylenediamine catalyzes the formation of free radicals from ammonium persulfate, accelerating the polymerization of the two acrylamides. The polymerization reaction is inhibited at too low a pH. 10% (w / v) ammonium persulfate solution. Provides the free radicals necessary for the polymerization of the two acrylamides. Prepare a few ml with deionized water, immediately before use.

[0101] 12) SDS-PAGE loading buffer: pH 6.8 0.5 mol / L Tris buffer 8 ml, glycerol 6.4 ml, 10% SDS 12.8 ml, mercaptoethanol 3.2 ml, 0.05% bromophenol blue 1.6 ml, H2O 32 ml. Mix well and set aside. Mix with protein sample at a 1:1 or 1:2 ratio, boil in boiling water for 10 min to mix well, and then load the sample. Generally, the volume is 20-25 μl, with a total protein content of 100 μg.

[0102] 2. Cell treatment 2.1 Cell resuscitation and culture 1) Preparation before the experiment: The electric thermostatic water bath was preheated to 37℃, the ultra-clean bench in the cell operation room was sterilized with ultraviolet light for 30 minutes, and the relevant experimental materials were prepared.

[0103] 2) Remove the cell cryopreservation tubes from the liquid nitrogen tank and quickly place them in a 37°C water bath to thaw. Shake to ensure complete thawing. The thawing time should be controlled within 1-2 minutes to avoid cell damage.

[0104] 3) Use a pipette to transfer the cell suspension from the cryopreservation tube into a 15 ml centrifuge tube, quickly add complete culture medium (about 10 times the volume of the cell suspension), mix well, centrifuge at 1000 rpm for 5 min, and discard the supernatant.

[0105] 4) Add an appropriate amount of complete culture medium, gently pipette to mix the cell suspension, transfer it to a cell culture flask, and label the culture flask with information such as cell passage number, recovery date, cell type and name.

[0106] 5) Incubate in an incubator and observe the cell condition under an inverted microscope the next day. Since the metabolic products of dead cells may affect living cells, the medium needs to be changed when there are too many dead cells. After changing the medium, continue incubating in the incubator. Change the medium every 2-3 days depending on cell growth and changes in the culture medium.

[0107] 2.2 Cell passage 1) Prepare the materials and perform ultraviolet irradiation in the clean bench before the experiment. Remove the cell culture flask from the incubator and observe the cell density under a microscope. When the cell density reaches about 80%, perform cell passage.

[0108] 2) Discard the old culture medium in the culture flask and rinse the cells twice with 1×PBS.

[0109] 3) Add about 1 ml of trypsin digestion solution containing EDTA, gently shake the culture flask from side to side to fill the bottom of the culture flask with the digestion solution, place it in a 37°C incubator for 2 minutes, remove the culture flask and gently tap it with your palm, place the culture flask under a microscope and observe that most of the intercellular spaces have increased, and the cells have shrunk and become rounded. Immediately add 3 ml of complete culture medium to stop the trypsin action.

[0110] 4) If local digestion is inadequate, gently blow on the bottle wall with a pipette to allow the cells to detach completely from the bottle wall.

[0111] 5) Transfer the cell suspension to a 15 ml centrifuge tube and centrifuge at 1000 rpm for 5 min. The cells will settle to the bottom of the tube. Carefully aspirate the supernatant. Add an appropriate amount of complete culture medium, gently pipette to mix the cell suspension, adjust the cell density, and passage at a 1:3 ratio.

[0112] 6) Label the cells, including cell type, cell passage number, and operation date, and place them in an incubator for culture.

[0113] 2.3 Cell cryopreservation 1) Preparation before the experiment: Prepare the necessary items for cell cryopreservation, such as cryopreservation tubes, serum-free cell cryopreservation solution, etc.

[0114] 2) Select cells in the logarithmic growth phase, digest adherent cells with trypsin containing 0.25% EDTA, transfer cells to centrifuge tubes and centrifuge (1000 rpm, 5 min).

[0115] 3) After centrifugation, discard the supernatant, add serum-free cell cryopreservation solution, and gently pipette the cells repeatedly to adjust the cell concentration to approximately 1×10⁻⁶. 6 per ml.

[0116] 4) Transfer the cell suspension to cryovials, 1 ml per tube, seal the cryovials and label them with information such as cell name, cryopreservation time, and cell passage number.

[0117] 5) Control the cooling rate: Place the cell cryopreservation tubes at -80℃ for 24 hours, and then transfer the cryopreservation tubes to a liquid nitrogen tank for long-term storage.

[0118] 3. Cell grouping BC: Control group (only supplemented with complete culture medium containing osteogenic induction solution); MF: Metformin treatment group (with 100 mmol / L metformin intervention solution); HG: High glucose intervention group (with high glucose solution); HG+25MF: High glucose + 25 mmol / L metformin intervention group (with both high glucose and 25 mmol / L metformin intervention solution); HG+50MF: High glucose + 50 mmol / L metformin intervention group (with both high glucose and 50 mmol / L metformin intervention solution); HG+100MF: High glucose + 100 mmol / L metformin intervention group (with both high glucose and 100 mmol / L metformin intervention solution); Cells in each group were supplemented with complete culture medium containing osteogenic induction solution and treated at 37 ℃ in a 5% CO2 incubator for 14 or 21 days, with the medium changed every 2-3 days.

[0119] 4. Detection and Analysis 4.1 Alizarin Red (ARS) Staining 4.1.1 Experimental Principle: Alizarin Red S is an anthraquinone derivative and is the sodium salt of alizarin sulfonate. It can chelate with calcium salts in calcium carbonate or calcium phosphate to form an orange-red complex. Alizarin Red S can reliably stain small amounts of sediment.

[0120] 4.1.2 Operating steps: 1) Washing: Discard the cell culture medium and rinse the adherent cells twice with 1×PBS.

[0121] 2) Fixation: Add 4% paraformaldehyde solution to fix for 10 min, remove the fixative, and wash 1-2 times with double-distilled water.

[0122] 3) Staining: Add alizarin red staining solution at pH 4.2 and stain for 20 min. Remove the alizarin red staining solution, wash 3 times with double-distilled water, and observe and photograph under a microscope.

[0123] 4.2 Western Blot Experiment 4.2.1 Extraction of total cellular protein 1) Remove the cells to be tested from the incubator, discard the cell culture medium, wash the cells twice with pre-cooled 1×PBS, and place them on ice.

[0124] 2) Add cell lysis buffer (10 μl PMSF per 1 ml of cell lysis buffer) and lyse thoroughly. Shake on ice for 15 min.

[0125] 3) Gently scrape the lysate with a cell scraper and collect it into a 1.5 ml EP tube. Incubate at 4°C on a shaker for complete lysis. If necessary, sonication can be used to assist lysis: sonicate on ice for 3 min at 80 W with 5 s sonication / 5 s intervals.

[0126] 4) Centrifuge at 12000 rpm for 20 min, transfer the supernatant to another 1.5 ml EP tube, aliquot, and store at -80℃.

[0127] 4.2.2 Detection of total protein concentration by BCA method The protein concentration of each sample was calculated by measuring the OD value using the new SEMET BCA protein concentration assay kit and microplate reader method.

[0128] 1) Preparation of working solution: Based on the number of standards and samples, prepare BCA working solution by adding 1 volume of Cu reagent to 50 volumes of BCA reagent (50:1) and mix thoroughly. Total BCA working solution = (number of BSA standards + number of unknown samples) × number of replicates × volume of BCA working solution per sample.

[0129] 2) Dilution of Standards: Dilute 10 μl of BSA standard to 100 μl with PBS to a final concentration of 0.5 mg / ml. Add 0, 2, 4, 6, 8, 12, 16, and 20 μl of the standard to the protein standard wells of a 96-well plate, and then add PBS to bring the total to 20 μl, resulting in concentrations of 0, 0.05, 0.1, 0.15, 0.2, 0.3, 0.4, and 0.5 (mg / ml). Plot a standard curve by measuring the OD values ​​of each concentration well.

[0130] 3) Add samples to each group: Take 2 μl of sample (dilute the sample 10 times according to the preliminary experimental results), add PBS to make up to 20 μl, and add it to the sample well of the 96-well plate. Since the pipette has a large error when taking small amounts of sample, the point before the standard line may not be very accurate, so try to make the sample point fall within 1 / 2 of the standard line.

[0131] 4) Add 200 μl of BCA working solution to each well and incubate at 37°C for 30 minutes. Measure the OD value of each standard and sample well at A562 nm using a microplate reader, and calculate the protein concentration based on the standard curve. When using an incubator, seal the plate in a sealed bag to prevent moisture evaporation from affecting the detection results.

[0132] 4.2.3 SDS-PAGE electrophoresis The steps are the same as step 5.3 in Example 1.

[0133] 5. Results 5.1 ARS staining results of cells in each group are as follows: Figure 4 As shown in Figure A, the relative amounts of mineralized nodules are as follows: Figure 4 As shown in C, by Figure 4 The results of A and 4C show that metformin can increase the mineralization nodules of glycotoxic osteoblasts.

[0134] 5.2 The results of BMP-2 and PPAR-γ protein analysis in each group of cells are as follows: Figure 4 B and Figure 4 As shown in D. Figure 4 B represents the results of SDS-PAGE electrophoresis analysis. Figure 4 D represents the protein expression level. (From...) Figure 4 B and Figure 4 The results from D showed that metformin increased BMP2 expression and inhibited PPARγ, and this effect was concentration-dependent. This suggests that metformin's improvement of glucosamined osteoblast differentiation function is associated with the inhibition of PPARγ expression.

[0135] 5.3 Results of AMPK and p-AMPK protein analysis in each group of cells are as follows: Figure 5 As shown. Figure 5 A represents the results of SDS-PAGE electrophoresis analysis. Figure 5 B represents the protein expression level. (From...) Figure 5 The results showed that, compared with the high glucose intervention group, the metformin + high glucose group had increased cellular pAMPK levels in a concentration-dependent manner. These results suggest that metformin can improve the inhibitory effect of glucose toxicity on osteogenic differentiation, and this improvement may be related to AMPK activation.

[0136] 5.4 Results of PERK, ATF4, and CHOP protein analysis in each group of cells are as follows: Figure 6 As shown. Figure 6 A1 shows the results of SDS-PAGE electrophoresis analysis. Figure 6 A2 represents the protein expression level. (From...) Figure 6 The results showed that the expression of endoplasmic reticulum stress (ERS)-related proteins PERK, ATF4, and CHOP was increased in osteoblasts, suggesting that high glucose may inhibit osteogenic differentiation by activating ERS stress. Metformin, on the other hand, reduced the expression of ERS-related proteins PERK, ATF4, and CHOP in glucotoxic osteoblasts in a concentration-dependent manner, suggesting that metformin may improve the inhibitory effect of high glucose on osteogenic differentiation by inhibiting ERS.

[0137] The results of this embodiment demonstrate that metformin can significantly improve glucotoxicity-induced osteogenic differentiation disorder, and this is achieved by inhibiting glucotoxicity-induced endoplasmic reticulum stress in osteoblasts through AMPK.

[0138] Example 3 This embodiment uses PPARγ activators / inhibitors to intervene in osteoblasts, and studies the effect of regulating PPARγ expression levels or activity on metformin intervention in glycotoxic osteoblasts, in order to verify the regulatory relationship between metformin and the effector protein PPARγ.

[0139] 1. Experimental Materials GW9662 and PIO were both purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The cells, other experimental reagents, and main instruments used were the same as in Example 2.

[0140] 2. PPARγ activation and inhibition 2.1 Preparation of PPARγ inhibitors The PPARγ inhibitor GW9662 was dissolved in DMSO to prepare a 5 mmol / L solution according to the instructions and stored at -20°C. Based on the CCK-8 assay results, the intervention concentration of GW9662 used was 10 μmol / L.

[0141] 2.2 Preparation of PPARγ agonists Following the instructions, the PPARγ agonist Pioglitazone (PIO) was dissolved in DMSO to prepare a 5 mmol / L solution and stored at -20°C. Based on the results of the CCK-8 assay, the intervention concentration of PIO we used was 1 μmol / L.

[0142] 3. Cell grouping BC: Control group (only supplemented with complete culture medium containing osteogenic induction solution); HG: High glucose intervention group (supplemented with high glucose solution); G+GW9662: High glucose + PPARγ inhibitor intervention group (supplemented with high glucose solution and GW9662); HG+PIO: High glucose + PPARγ activator intervention group (supplemented with high glucose solution and PIO); HG+MF: High glucose + metformin intervention group (supplemented with high glucose solution and metformin intervention solution); HG+MF+GW9662: High glucose + metformin + PPARγ inhibitor intervention group (supplemented with high glucose solution, metformin intervention solution and GW9662); HG+MF+PIO: High glucose + metformin + PPARγ activator intervention group (supplemented with high glucose solution, metformin intervention solution and PIO). Cells in each group were supplemented with complete culture medium containing osteogenic induction solution and incubated at 37°C under 5% CO2 for 14 or 21 days, with the medium changed every 2-3 days.

[0143] 4. Detection and Analysis 4.1 Cell proliferation and cytotoxicity detection - CCK8 assay (Cell Counting Kit-8) 4.1.1 Experimental Principle CCK-8 reagent contains WST-8 (chemical name: 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfonic acid benzene)-2H-tetrazole monosodium salt), which, in the presence of the electron coupling reagent 1-methoxy-5-methylphenazineonium sulfate dimethyl ester (1-Methoxy PMS), can be reduced by mitochondrial dehydrogenases to the highly water-soluble orange-yellow formazan product, Formazan. The amount of Formazan produced is directly proportional to the number of viable cells. Therefore, this property can be used to directly analyze cell proliferation and cytotoxicity; the more and faster the cell proliferation, the darker the color; the greater the cytotoxicity, the lighter the color.

[0144] 4.1.2 Experimental Procedure 1) Digest cells in culture flasks or wells to prepare cell suspensions: cell counting.

[0145] 2) Seeding into 96-well plates: Based on the appropriate cell number (5000-7000 cells / well), use approximately 100 μl of cell suspension per well, setting up 3-5 replicate wells. The plate should include 3-5 replicates of a control group (untreated, normally cultured cells) and 3 replicates of a blank control group (no cells seeded). Do not seed cells in the outer ring; add 100 μl of PBS buffer to each well to prevent evaporation of the culture medium in the inner ring.

[0146] 3) Culture in a 37℃ incubator: After cell seeding, it takes about 2-4 hours to adhere the cells.

[0147] 4) After the cells adhere, replace the culture medium with different concentrations of drugs, 100 μl per well.

[0148] 5) Incubation at 37℃: The culture time for adding the test drug is selected based on the drug properties and cell sensitivity. The commonly used time points are 6 h, 12 h, 24 h, 36 h, 48 h, and 72 h (using one 96-well plate for each time point).

[0149] 6) When the incubation time is up, remove the drug-containing culture medium, wash twice with PBS, and slowly add 100 μl of culture medium containing 10% CCK-8 to each well (prepare the total amount according to the number of wells to be tested). Be careful not to generate air bubbles in the wells, as this may affect the absorbance detection.

[0150] 7) Incubate in the incubator for 0.5-4 hours: The incubation time can be determined based on the absorbance (OD value) of the normal control group (around 1.0 is optimal). If the color development is insufficient, continue incubation to confirm the optimal conditions.

[0151] 8) Place the cells in an ELISA reader and measure the absorbance at 450 nm. Calculate cell viability based on the absorbance value.

[0152] 9) The calculation formula is as follows: Cell viability = [(As - Ab) / (Ac - Ab)] × 100%; Inhibition rate = [(Ac - As) / (Ac - Ab)] × 100%; Wherein, As: absorbance of the experimental well (containing cell culture medium, CCK-8, and the drug to be tested); Ac: Absorbance of control wells (containing cell culture medium, CCK-8, and no test drug); Ab: Absorbance of blank wells (culture medium and CCK-8 containing no cells and test drug).

[0153] 4.2 Alizarin Red (ARS) Staining Same as step 4.1 in Example 2.

[0154] 4.3 ELISA assay for ALP and OCN expression in osteoblasts 1) Protein dilution: Dilute with TBST, 50ul per well.

[0155] Diluted 0 times Dilute 2 times: 50ul protein + 50ul dilution Dilute 4 times: 25ul protein + 75ul dilution Dilute 8 times: 12.5ul protein + 87.5ul dilution 2) Dilution of standard Human IgG (50 ug / uL): ① Take 1 Human IgG, added to 499 Protein dilution buffer, mix thoroughly by pipetting -- 0.1 ug / ul ② Dilute to 50 pg / ul: Take four 1.5 ml EP tubes A. 500ul① + 500ul protein diluent = 50 B. Take 100ul A + 900ul protein diluent = 5 C. Take 100ul B + 900ul protein diluent = 500 D. Take 100ul C + 900ul protein diluent = 50 Protein dilution buffer: PBS containing 5% glycerol (42.75 ml PBS + 2.25 ml glycerol) Take D and dilute it sequentially with 100 μL of standard D and 100 μL of D + 100 μL of diluent.

[0156] 3) Antigen coating: Add 50 mg of antigen to each well. The corresponding protein was incubated overnight at 4°C.

[0157] 4) Washing: Discard the liquid in the wells, wash each well once with PBST, and pat clean.

[0158] 5) Sealing: Add 100 to each hole Incubate with 3% BSA in TBST at room temperature for 1 h.

[0159] 6) Washing: Discard the liquid in the wells, wash each well once with PBST, discard the liquid in the wells, and pat clean.

[0160] Sample loading: (standard curve + PBST) ① Preparation of serum diluent: PBST 45.05 mL, 10% BSA 4.5 mL, PBS 0.15 mL. ② Serum treatment: 12000 rpm, 4℃, 20 min. ③ Serum dilution factors: 25, 50, 100, 200. ④ Add 150 mL of PBS to the corresponding well. The diluted serum.

[0161] Dilution calculation: 650 per person (4 wells) Diluted 25 times: 26 +624 diluent Diluted 50 times: 13 +637 diluent Diluted 100 times: 6.5 +643.5 diluent Diluted 200 times: 3.25 +646.75 diluent 7) Incubation: Incubate at 37℃ for 1.5 h.

[0162] 8) Washing: Discard the liquid in the well, wash 5 times with PBST, and pat clean.

[0163] 9) Enzyme-labeled antibody incubation: Add 100 μL of enzyme-labeled antibody to each well. Dilute the enzyme-labeled antibody (RabbitHRP-Anti Human IgG antibody) in PBST at a ratio of 1:10000 and incubate at 37°C for 35 minutes.

[0164] 10) Washing: Pour out the liquid from the holes and add 200 ml of water to each hole. Wash 5 times with PBST and pat clean.

[0165] 11) Developing color: Add 100g of TMB developing substrate to each well. Develop color at 37℃ in the dark for 15 minutes (observe every 5 minutes).

[0166] 12) Termination: Add 100 to each hole The reaction was terminated with 1M H₂SO₄. (In sequence) 13) Microplate reader reading: Measure the optical density (OD450) of each well at a wavelength of 450 nm using a single-channel microplate reader.

[0167] 14) Data processing.

[0168] 5. Results 5.1 The results of cell viability tests in each group are as follows: Figure 7 As shown in A, by Figure 7 The results of A showed that, compared with the metformin intervention group, siRNA-PPARγ could increase the protective effect of metformin against glycotoxic osteoblasts, manifested as increased osteoblast activity.

[0169] 5. The ALP and OCN content of cells in each group are as follows: Figure 7 B and Figure 7 As shown in C, by Figure 7 B and Figure 7 The results of C showed that, compared with the metformin intervention group, siRNA-PPARγ could promote the secretion of ALP and OCN.

[0170] The results of this embodiment demonstrate that metformin improves bone metabolism disorders in diabetic rats in association with PPARγ protein.

[0171] Example 4 This embodiment uses a glycotoxic component differentiation cell model to investigate the mechanism by which metformin antagonizes glycotoxic osteoblast differentiation disorder through the AMPK inhibitor Compound C to inhibit AMPK and β-Thapsigargin to induce endoplasmic reticulum stress intervention.

[0172] 1. Experimental Materials Dorsomorphin (Compound C) and β-thapsigargin (TG) were both purchased from MCE.

[0173] The cells, other experimental reagents, and main instruments used were the same as in Example 2.

[0174] 2. Formulation of AMPK pathway inhibitors Following the instructions, the AMPK pathway inhibitor Dorsomorphin (Compound C) was dissolved in DMSO to prepare a 5 mmol / L solution and stored at -20°C. Based on the results of the CCK-8 assay, the intervention concentration of Compound C we used was 10 μmol / L.

[0175] 3. Preparation of endoplasmic reticulum stress activators Following the instructions, the endoplasmic reticulum stress activator β-thapsigargin (TG) was dissolved in DMSO to prepare a 5 mmol / L solution and stored at -20℃. Based on the results of the CCK-8 experiment, the intervention concentration of β-thapsigargin (TG) we used was 100 nmol / L.

[0176] 4. Cell grouping BC: Control group (complete culture medium containing osteogenic induction solution only); Compound C: AMPK inhibitor group (AMPK inhibitor added); HG: High glucose group (high glucose solution added); MF: Metformin intervention group (100 μmol / L metformin intervention solution added); TG: Endoplasmic reticulum stress activator intervention group (endoplasmic reticulum stress activator added); HG+ Compound C: High glucose + AMPK inhibitor group (high glucose solution and AMPK inhibitor added); HG+MF: High glucose + metformin group (high glucose solution and 100 μmol / L metformin intervention solution added); HG+MF+ Compound C: High glucose + metformin group + AMPK inhibitor group (high glucose solution, 100 μmol / L metformin intervention solution and AMPK inhibitor added); TG +MF: β-Thapsigargin + metformin intervention group (endoplasmic reticulum stress activator and 100 μmol / L metformin intervention solution added). Cells in each group were added to complete culture medium containing osteogenic induction solution and treated for 14 or 21 days in a 37°C incubator with 5% CO2, with the medium changed every 2-3 days.

[0177] 5. Detection and Analysis 5.1 Cell proliferation and cytotoxicity detection - CCK8 assay (Cell Counting Kit-8) Same as step 4.1 in Example 3.

[0178] 5.2 Alizarin Red (ARS) Staining Same as step 4.1 in Example 2.

[0179] 5.3 ELISA assay for ALP and OCN expression in osteoblasts The same as step 4.3 in Example 3.

[0180] 5.4 Western Blot Experiment The same as step 4.2 in Example 2.

[0181] 6. Results 6.1 Results of AMPK and p-AMPK protein analysis in BC group and Compound C group cells are as follows: Figure 8 A1 and Figure 8 As shown in A2, by Figure 8 A1 and Figure 8 The results of A2 showed that Compound C could inhibit the expression of p-AMPK protein in osteoblasts. Furthermore, after Compound C inhibited p-AMPK protein expression, we found that metformin's protective effect against glucocorticoid-induced osteogenic differentiation damage was weakened: compared with the metformin intervention group, the osteoblast viability was decreased in the metformin + Compound C intervention group.

[0182] 6.2 The cell viability test results of HG group, HG+MF group, HG+Compound C group, and HG+MF+Compound C group are as follows: Figure 8 As shown in B, the relative amounts of ARS staining and mineralized nodules are as follows: Figure 8 As shown in Figure C, the expression levels of ALP and OCN proteins are as follows: Figure 8 D and Figure 8 As shown in E, by Figure 8 B- Figure 8 The results from E showed a reduction in mineralized nodules and decreased secretion of OCN and ALP, suggesting that metformin's effect in improving glucosamine-induced osteoblast differentiation depends on AMPK activation.

[0183] 6.3 The results of BMP-2, PPAR-γ, AMPK, and p-AMPK protein analysis in the HG group, HG+MF group, HG+Compound C group, and HG+MF+Compound C group are as follows: Figure 9 As shown. Figure 9 A represents the results of SDS-PAGE electrophoresis analysis. Figure 9 B represents the protein expression level. (From...) Figure 9 The results showed that, compared with the metformin intervention group, the expression level of BMP2 in osteoblasts of the metformin + Compound C group decreased and the expression level of PPARγ protein increased, suggesting that metformin's regulation of PPARγ expression depends on AMPK activity.

[0184] 6.4 The results of PERK, ATF4, and CHOP protein analysis in the HG group, HG+MF group, HG+Compound C group, and HG+MF+Compound C group are as follows: Figure 10 As shown. Figure 10A represents the results of SDS-PAGE electrophoresis analysis. Figure 10 B represents the protein expression level. (From...) Figure 10 The results showed that, compared with the high glucose + metformin intervention group, the high glucose + metformin + Compound C group had increased levels of osteoblast ERS-related proteins (PERK, CHOP and ATF4), confirming that metformin's inhibition of glucosinolate endoplasmic reticulum stress depends on AMPK activity.

[0185] 6.5 The results of PERK, ATF4, and CHOP protein analysis in the BC group, MF group, TG group, and TG+MF group are as follows: Figure 6 As shown. Figure 6 B1 shows the results of SDS-PAGE electrophoresis analysis. Figure 6 B2 represents the protein expression level. (From...) Figure 6 The results showed that, compared with the ERS group (TG), metformin inhibited the expression of β-Thapsigargin-induced PERK, CHOP, and ATF4 proteins. This confirms that metformin can improve glucotoxicity-induced osteoblast ERS.

[0186] The results of this embodiment demonstrate that metformin can significantly improve glucotoxicity-induced osteogenic differentiation disorder, and this is achieved by inhibiting glucotoxicity-induced endoplasmic reticulum stress in osteoblasts through AMPK.

[0187] This invention has many specific applications, and the above description is only a preferred embodiment. It should be noted that the above embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention. For those skilled in the art, several improvements can be made without departing from the principle of this invention, and these improvements should also be considered within the scope of protection of this invention.

Claims

1. The use of an AMPK activator or PPARγ inhibitor in the preparation of a medicament for the prevention or treatment of diabetic osteoporosis.

2. The application according to claim 1, characterized in that, The AMPK activator includes at least one of metformin, 5-aminoimidazole-4-carboxamide nucleoside, and MT 63-78.

3. The application according to claim 1, characterized in that, The PPARγ inhibitors include at least one of metformin, GW9662, and T0070907.

4. The use of metformin in the preparation of a drug for the prevention or treatment of osteoporosis in diabetes.

5. The application according to claim 4, characterized in that, Among the drugs mentioned for preventing osteoporosis in diabetes, the effective dose of metformin is 400-600 mg / day.

6. The application according to claim 4, characterized in that, Among the medications mentioned for treating diabetic osteoporosis, the effective dose of metformin is 1000-3000 mg / day.

7. The application according to claim 1 or 4, characterized in that, The drug also includes a carrier that can promote bone integration.

8. The application according to claim 7, characterized in that, The carrier includes at least one of calcium phosphate and hydroxyapatite.

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