Use of small molecule drug AN698 / 40746067 in treating hyperlipidemia and osteoporosis

Abstract

The present application relates to the application of small molecule drug AN698 / 40746067 in treating hyperlipidemia and osteoporosis. The small molecule drug AN698 / 40746067 targets SDCCAG3 protein, reduces serum lipid level by inhibiting adipogenic differentiation of BMSCs, and is used for preparing a drug for treating hyperlipidemia and a drug for treating osteoporosis. The present application first discovers that the small molecule drug AN698 / 40746067 has obvious therapeutic effect on hyperlipidemia and osteoporosis, and in vitro and in vivo verification experiments show that the small molecule drug AN698 / 40746067 can significantly reduce serum lipid level, thereby treating hyperlipidemia, obesity caused or accompanied by hyperlipidemia and osteoporosis. The present application first screens a small molecule drug for treating hyperlipidemia and osteoporosis by taking SDCCAG3 protein as a target, and the research results first show that the small molecule drug AN698 / 40746067 reduces serum lipid level by inhibiting adipogenic differentiation of BMSCs, thereby achieving the effect of treating hyperlipidemia, obesity caused or accompanied by hyperlipidemia and osteoporosis.

Description

TECHNICAL FIELD

[0001] The application relates to application of a small molecule drug AN698 / 40746067 in treatment of hyperlipidemia and osteoporosis and belongs to the technical field of biological medicine. BACKGROUND

[0002] Hyperlipidemia (HL) is a kind of lipid metabolism disorder disease, which is characterized by increase of triglyceride, total cholesterol and low-density lipoprotein in serum or decrease of high-density lipoprotein, and is closely related to metabolic diseases such as atherosclerosis, obesity and fatty liver. The clinical characteristics of osteoporosis (OP) are reduction of bone mass, destruction of bone microstructure, increase of bone fragility and high incidence of bone fracture. Investigation shows that 63% of osteoporosis patients have hyperlipidemia, and there is obvious correlation between blood lipid disorder and osteoporosis.

[0003] Bone marrow is the only region where fat tissue and bone tissue are in close contact in the body. Both adipocytes and osteoblasts in bone marrow originate from bone marrow mesenchymal stem cells (BMSCs). BMSCs have good proliferation capacity, and also have growth potential and multipotential differentiation stemness. BMSCs can be differentiated into preosteoblasts and finally develop into mature osteoblasts. Osteoblasts can synthesize bone matrix and finally complete the osteogenesis process. In the initial stage of differentiation of BMSCs into fat, multipotential mesenchymal stem cells participate in the formation of adipocyte lineage, and in the final differentiation stage, preadipocytes become adipocytes and exert new functions, including lipid synthesis and storage, and production of adipocyte-specific proteins. Bone marrow adipose tissue (MAT) occupies 70% of the space of the bone marrow cavity in adults and plays an important role in bone microenvironment metabolism. Long-term high-fat diet can cause excessive expansion of MAT, cause bone marrow adiposity, inhibit osteogenic differentiation of BMSCs in the bone marrow cavity, enhance adipogenic differentiation, cause bone lipid imbalance and increase the incidence of osteoporosis.

[0004] Small molecule drugs are a class of synthetic drugs with molecular weight less than 1000, which often target intracellular proteins to exert biological functions, and have the advantages of small side effects, stable performance, and easy synthesis. Compared with biological macromolecules, small molecule drugs have good drug performance and pharmacokinetic properties, and are more likely to penetrate the cell membrane and act on the intracellular with high efficiency. Using small molecule ligands and their key proteins as therapeutic targets has become a research hotspot. Currently, drugs for the treatment of osteoporosis include small molecules and protein antibody macromolecules, such as bisphosphonates and denosumab. In the field of hyperlipidemia treatment, chemical small molecule drugs still occupy a dominant position, such as statins and fibrates. In 1999, Mundy et al. first discovered that the commonly used clinical cholesterol-lowering drug, statins, had a strong effect on promoting bone formation. It is currently the only drug that has a "double-barreled" effect on hyperlipidemia and osteoporosis, but the specific mechanism has not been clearly defined. Recent studies have found that statins can inhibit osteoporosis-induced bone marrow obesity and alleviate the progression of bone metastasis, suggesting that statins may target MAT. This suggests that targeting the adipogenic differentiation of BMSCs and inhibiting the excessive growth of MAT may be the key to treating hyperlipidemia with osteoporosis.

[0005] Serologically defined colon cancer antigen-3 (Sdccag3), also known as Endosome associated trafficking regulator 1 (Entr1), was first discovered in the serum of colon cancer patients and is involved in various life activities in cells, including cell cycle, cell transport, flagellum formation, and apoptosis. Our previous studies have found that regulating the expression of Sdccag3 can promote the osseointegration of implants in the femur of hyperlipidemic rats, suggesting the possibility of Sdccag3 as a target for treating hyperlipidemia with osteoporosis by regulating the osteogenic / adipogenic differentiation of BMSCs.

[0006] Drug screening is the first step of discovering new drugs. In the past, animal models are used to screen new drugs, which is high cost, low efficiency, large sample size, and is not conducive to drug research and development. With the rapid development of computer technology, high-throughput screening (HTS) has entered the field of drug research and development since the 1990s. The HTS technology system integrates pharmacology, molecular and cellular biology, computer and automation multidisciplinary theory and technology, and realizes rapid, efficient, automated and large-scale drug screening. Virtual screening (VS) is an active compound screening based on a small molecule database. Based on the theory of drug design, with the help of computer technology and professional application software, the molecular docking operation between small molecule compounds and drug targets is used to quickly select active lead compounds with drug properties from tens to millions of molecules, greatly reducing the number of experimental screening compounds, shortening the research cycle and reducing the cost of drug research and development. It is reported that the positive rate of virtual screening is 5% to 20%, and the number of cases of successful drug design assisted by virtual screening increases year by year. Virtual screening has become the most potential drug development tool. SUMMARY

[0007] In view of the deficiencies of the prior art, the application provides an application of a small molecule drug AN698 / 40746067 in treatment of hyperlipidemia and osteoporosis.

[0008] The technical scheme of the application is as follows:

[0009] The application of the small molecule drug AN698 / 40746067 in preparation of a drug for treating hyperlipidemia, wherein the small molecule drug AN698 / 40746067 has the following structural formula:

[0010]

[0011] According to the application, the small molecule drug AN698 / 40746067 targets SDCCAG3 protein, and reduces serum lipid level by inhibiting adipogenic differentiation of BMSCs.

[0012] According to the application, the drug for treating hyperlipidemia is composed of the small molecule drug AN698 / 40746067 and pharmaceutically acceptable adjuvants.

[0013] According to the application, the concentration of the small molecule drug AN698 / 40746067 is 0.5-1.5 mg / kg.

[0014] The application of the small molecule drug AN698 / 40746067 in preparation of a drug for treating osteoporosis, wherein the small molecule drug AN698 / 40746067 has the following structural formula:

[0015]

[0016] According to the application, the osteoporosis is caused by hyperlipidemia or accompanied by osteoporosis.

[0017] According to the application, the osteoporosis treatment drug is composed of the small molecule drug AN698 / 40746067 and pharmaceutically acceptable excipients.

[0018] According to the application, the concentration of the small molecule drug AN698 / 40746067 is 0.5-1.5 mg / kg.

[0019] The application has the following beneficial effects:

[0020] 1. The application first discovers that the small molecule drug AN698 / 40746067 has obvious therapeutic effect on hyperlipidemia and osteoporosis, and the in vitro and in vivo verification experiments show that the small molecule drug AN698 / 40746067 can significantly reduce the serum lipid level, thereby treating hyperlipidemia, obesity caused or accompanied by hyperlipidemia and osteoporosis.

[0021] 2. The application first screens the small molecule drug for treating hyperlipidemia and osteoporosis by taking the SDCCAG3 protein as a target, and the research result first shows that the small molecule drug AN698 / 40746067 can reduce the serum lipid level by inhibiting the adipogenic differentiation of BMSCs, thereby treating hyperlipidemia, obesity caused or accompanied by hyperlipidemia and osteoporosis.

[0022] 3. The use of the small molecule drug AN698 / 40746067 targeting the SDCCAG3 protein in the preparation of a drug for treating hyperlipidemia and osteoporosis is first disclosed, which provides a new method and new idea for the clinical treatment of hyperlipidemia and osteoporosis, can improve the hydrophilicity and solubility of the drug through drug structure optimization, improve the in vivo conversion rate, and explore the action mechanism and active metabolites in vivo metabolism, thereby providing a new vision for clinical treatment. BRIEF DESCRIPTION OF DRAWINGS

[0023] Figure 1 The names and structural formulas of the first 30 small molecule compounds selected according to the affinity of the SDCCAG3 protein.

[0024] Figure 2 The results of qPCR detection of the mRNA levels of PPAR-γ and FABP4 after 16 small molecule compounds and rat BMSCs are co-cultured in complete high-fat medium for 7 days.

[0025] Figure 3 Oil red O staining (upper) and quantitative analysis (lower) results of small molecule compounds AF-399 / 42018065, AN698 / 40746067 and AN-465 / 42897535 co-cultured with BMSCs in complete high-fat medium for 14 days.

[0026] Figure 4 AN698 / 40746067 binding to target protein SDCCAG3 was verified by intracellular thermal shift assay.

[0027] Figure: The left graph is the western blot result, and the right graph is the western blot result quantitative line graph. Control is the control group, and AN698 / 40746067 is the experimental group with small molecule drugs added.

[0028] Figure 5 Effect of AN698 / 40746067 on the expression level of SDCCAG3 protein.

[0029] Figure: The left graph is the qRT-PCR result, and the right graph is the western blot result. Control is the control group, and AN698 / 40746067 is the experimental group with small molecule drugs added.

[0030] Figure 6 CCK8 experiment results of BMSCs after 1, 5 and 7 days of adding small molecule drug AN698 / 40746067.

[0031] Figure 7 Fat-related index gene expression level (upper) and fat-related index protein expression level (lower) of the experimental group with AN698 / 40746067 added under high-fat environment and the high-fat group.

[0032] Figure 8 Oil red O staining (left) and quantitative experiment (right) results after 28 days of culture of the normal group, high-fat group and experimental group.

[0033] Figure 9 Fat-related index gene expression level (upper) and fat-related index protein expression (lower) level of the experimental group and the fat group.

[0034] Figure 10 Oil red O staining (left) and quantitative experiment (right) results after 28 days of culture of the normal group, high-fat group and experimental group.

[0035] Figure 11 Inflammatory-related factor gene expression level after 7 days of culture of the normal group, high-fat group and experimental group.

[0036] Figure 12The results of alizarin red staining (left) and quantitative experiment (right) for the control group and experimental group.

[0037] Figure 13 The serum lipid levels of the common group (P group) and high-fat diet group (G group) after 10 weeks of feeding.

[0038] Figure 14 The body size comparison (left) and the calculation results of obesity index Lee's index (right) of the three groups of rats at the end of the experiment.

[0039] Figure 15 The change in body weight of rats before and after injection of small molecule drug AN698 / 40746067.

[0040] Figure 16 The HE staining results of the perirenal white adipose tissue of rats in the P, G, and G+ groups.

[0041] Figure 17 The serum lipid level detection results of rats in the P, G, and G+ groups after 16 weeks of feeding.

[0042] Figure 18 The photos of important organs (heart, liver, spleen, lung, kidney) of rats in the P, G, and G+ groups after 16 weeks of feeding.

[0043] Figure 19 The net weight of important tissues and organs of rats in the P, G, and G+ groups after 16 weeks of feeding.

[0044] Figure 20 The organ index of important tissues and organs of rats in the P, G, and G+ groups after 16 weeks of feeding.

[0045] Figure 21 The HE staining results of liver sections of rats in the P, G, and G+ groups after 16 weeks of feeding.

[0046] Figure 22 The HE staining results of heart, spleen, lung, and kidney tissue sections of rats in the P, G, and G+ groups after 16 weeks of feeding.

[0047] Figure 23 The expression level of SDCCAG3 protein in bone tissue of rats in the P, G, and G+ groups after 16 weeks of feeding.

[0048] Figure 24 The expression level of fat-related factors PPARγ and C / EBPα and lipid metabolism-related factor LDLR protein in bone tissue of rats in the P, G, and G+ groups after 16 weeks of feeding.

[0049] Figure 25qPCR detection of the gene expression levels of fat-related factors (Pparγ, C / ebpα) and lipid metabolism-related factors (Fabp4, Ldlr, Adipoq) in the proximal tibial bone marrow of P, G, G+ groups of rats after 16 weeks of feeding.

[0050] Figure 26 qPCR detection of the gene expression levels of inflammatory factors Il-1β, Il-6 in the bone tissue of P, G, G+ groups of rats after 16 weeks of feeding.

[0051] Figure 27 HE staining results of the distal femur and proximal tibia bone marrow of P, G, G+ groups of rats after 16 weeks of feeding.

[0052] Figure 28 Immunohistochemical detection of RUNX2 expression results of the distal femur and proximal tibia of P, G, G+ groups of rats after 16 weeks of feeding.

[0053] Figure 29 Typical images (top) and bone parameter measurement results (bottom) of the distal femur Micro-CT of P, G, G+ groups of rats after 16 weeks of feeding. DETAILED DESCRIPTION

[0054] The technical solutions of the present application will be further described below in combination with examples and drawings, but the protection scope of the present application is not limited to this. The experimental operations involved in the examples are all carried out according to the conventional operations if not otherwise specified; the involved drugs and reagents are all ordinary commercially available products if not otherwise specified. The percentages in the examples are all mass percentages if not otherwise specified.

[0055] The small molecule drugs including AN698 / 40746067 in the examples are all purchased from Specs company (https: / / www.specs.net / ).

[0056] The high-fat feed is purchased from Jinan Coolset Biotechnology Co., Ltd.

[0057] Example 1, small molecule drug screening

[0058] 1. First, download the protein sequence of SDCCAG3 from the NCBI database (NCBI ID: 195540228), perform homology modeling using Swiss-model, remove other small molecules, water molecules, etc. in the model, set the docking pocket, and dock with small molecule compounds in the Specs small molecule library, and select the top 30 small molecule drugs according to the affinity for subsequent screening. The names and structural formulas of the 30 small molecule compounds are as shown in Table 1. Figure 1

[0059] ​2. Excluding 11 poorly soluble and 3 unobtainable small molecule compounds, the remaining 16 small molecule compounds (AK968 / 1157309, AF399 / 42019019, AN806 / 41205070, AK918 / 11939016, AF-399 / 42017853, AF-399 / 42018065, AN698 / 40746067, AF-399 / 13567362, AN-989 / 15133074, AN-989 / 15133060, AN-329 / 4) were included. 3385681, AN-465 / 41587276, AK-968 / 41924930, AF-399 / 40634403, AN-758 / 15106208, AN-465 / 42897535 (available at Specs) and rat BMSCs were cultured in a completely high-fat medium (Pythonbio, catalog number AAPR156-d500) for 7 days. RNA was then extracted and qRT-PCR was performed to detect the mRNA levels of adipogenesis-related markers peroxisome proliferator-activated receptor-γ (Pparγ) and fatty acid binding protein 4 (Fabp4). The results are as follows: Figure 2 As shown.

[0060] Depend on Figure 2 It is known that small molecule compounds AF-399 / 42018065, AN698 / 40746067 and AN-465 / 42897535 can lead to a decrease in the mRNA levels of screening indicators Pparγ and Fapp4.

[0061] 3. Small molecule compounds AF-399 / 42018065, AN698 / 40746067, and AN-465 / 42897535 were mixed at a concentration of 10... - 5 DMSO at a concentration of mol / L was dissolved in the medium and co-cultured with BMSCs in a completely high-fat medium. BMSCs cultured under high-fat conditions with an equal concentration of DMSO served as a control. After 14 days, Oil Red O staining was performed, and lipid droplets were dissolved in 60% isopropanol for quantitative absorbance analysis. The results are as follows: Figure 3 As shown.

[0062] Depend on Figure 3 It is known that the small molecule compound AN698 / 40746067 has the potential to inhibit the adipogenic differentiation of BMSCs and can be used as a small molecule drug.

[0063] Example 2: Intracellular thermal migration experiment

[0064] Take well-grown BMSCs and measure 6 × 10⁶ cells using a cell counting chamber. 6 After thoroughly mixing, the cells were evenly seeded into two new culture dishes. After 24 hours of cell attachment, AN698 / 40746067 (experimental group) and DMSO (control group) were added at a concentration of 10... -6 Incubate the cells in M's ordinary culture medium (BI, catalog number C3060-0500) in an incubator for 4 hours; discard the culture medium, wash with PBS buffer, digest with trypsin for 2 minutes, collect the suspension containing cells, centrifuge at 1000 r / min for 5 minutes, discard the supernatant, resuspend the cells in pre-chilled PBS buffer, centrifuge again and discard the supernatant to thoroughly remove residual culture medium; add 6 ml of pre-chilled PBS buffer containing 1×cocktail protease inhibitor to resuspend the cells, and add 100 μl / tube to ep tubes. The Mastercycler Nexus GSX1 gradient PCR instrument was pre-set with six temperature gradients (range 30–55°C). Cells from both the experimental and control groups were heated for 5 minutes at each temperature, then cooled to 4°C for 3 minutes before removal. Cells were lysed three times using liquid nitrogen freezing followed by a room temperature water bath. After centrifugation at 12000 rpm and 4°C for 30 minutes, the supernatant was transferred to a 1.5 ml EP tube, which was the obtained protein sample. The concentration was then determined using Western blot to detect the Sdccag3 protein content (β-actin as an internal control). The results are as follows: Figure 4 As shown.

[0065] Depend on Figure 4 It is known that the small molecule drug AN698 / 40746067 can bind to the SDCCAG3 protein in cells and improve the thermal stability of the SDCCAG3 protein.

[0066] Experiment 3: Effect of AN698 / 40746067 on SDCCAG3 protein expression level

[0067] BMSCs in the experimental group and control group were obtained according to the method described in Example 2. They were then cultured in a completely high-fat medium for 7 days. RNA was extracted for qRT-PCR, and proteins were extracted for Western blot experiments according to the method described in Example 2. The results are as follows: Figure 5 As shown.

[0068] Depend on Figure 5 It is known that the small molecule drug AN698 / 40746067 does not affect the expression level of Sdccag3 mRNA transcription, but it can inhibit and reduce the expression of SDCCAG3 protein in BMSCs.

[0069] Experimental Example 4, Effect of AN698 / 40746067 on BMSCs cell proliferation

[0070] Take well-grown BMSCs, inoculate 5000 cells per well into a 96-well culture plate, and place the culture plate in a 37°C, 5% carbon dioxide incubator for 24 hours of pre-culture. Discard the old culture medium, and add different concentrations (0, 0.01, 10 -3 , 10 -4 , 10 -5 mol / L) of small molecule AN698 / 40746067 dissolved in ordinary culture medium to the culture plate, with 5 replicates per group. After 1, 5, and 7 days of culture, discard the old culture medium, gently rinse 1-2 times with PBS buffer, and add 100 μL of CCK-8 solution (CCK-8 stock solution to culture medium at a volume ratio of 1:10) to each well. Place in a 37°C incubator for 30 minutes of incubation in the dark, and use a microplate reader to measure the absorbance at 450 nm. The results are shown in Figure 6 .

[0071] As can be seen from Figure 6 , the small molecule drug AN698 / 40746067 does not significantly affect BMSCs cell proliferation.

[0072] Experimental Example 5, Effect of AN698 / 40746067 on BMSCs adipogenesis

[0073] 1. Take well-grown BMSCs, inoculate 8x10 4 cells per well into a six-well culture plate, and after the cells grow to 80%, divide into an ordinary group, an experimental group, and a high-fat group. Add 2 ml of α-MEM culture medium containing 10% FBS to each well of the ordinary group, and add 2 ml of complete high-fat culture medium to each well of the experimental group and the high-fat group. Add small molecule AN698 / 40746067 dissolved in DMSO to the experimental group to a final concentration of 10 -5 mol / l, and add an equal amount of DMSO to the high-fat group.

[0074] Culture the experimental group and the high-fat group for 7 days, and then extract RNA for qRT-PCR to detect the levels of adipogenesis-related indicators Pparγ, Fabp4, CCAAT Enhancer Binding Protein α (C / ebpα), Adiponectin (Adipoq), and Low Density Lipoprotein Receptor (Ldlr). Extract total cell protein for Western Blot experiments to detect the levels of adipogenesis-related indicators PPARγ, C / EBPα, and LDLR according to the method described in Example 2. The results are shown inFigure 7 As shown in Fig. 2, the expression levels of the lipogenic related index genes Pparγ, Fabp4, C / ebpα, Adipoq and Ldlr in the experimental group were significantly lower than those in the high-fat group.

[0075] As shown in Fig. 2, the expression levels of the lipogenic related index genes Pparγ, Fabp4, C / ebpα, Adipoq and Ldlr in the experimental group were significantly lower than those in the high-fat group. Figure 7 As shown in Fig. 2, the expression levels of the lipogenic related index genes Pparγ, Fabp4, C / ebpα, Adipoq and Ldlr in the experimental group were significantly lower than those in the high-fat group.

[0076] 2, grouping same as the experiment 1 of this embodiment, then the ordinary group, the experimental group, the high-fat group BMSCs in complete high-fat medium culture 28 days, discard old culture medium to carry out oil red O staining, and after staining with 60% isopropanol to dissolve lipid droplet, carry out absorbance quantitative analysis, the results are shown in Fig. 3. Figure 8

[0077] As shown in Fig. 3, the lipid droplet formation of the high-fat group BMSCs was more obvious than that of the ordinary group, and the lipid droplet formation of the experimental group BMSCs was weaker than that of the high-fat group. Figure 8 As shown in Fig. 3, the lipid droplet formation of the high-fat group BMSCs was more obvious than that of the ordinary group, and the lipid droplet formation of the experimental group BMSCs was weaker than that of the high-fat group.

[0078] 3, take 25mg insulin powder dissolved in 12.5ml dilute hydrochloric acid, obtain 57.3μg / ml concentration of insulin solution; take 25mg dexamethasone powder dissolved in 25ml anhydrous ethanol, obtain 1mg / ml concentration of dexamethasone solution; take 35.8mg indomethacin powder dissolved in 0.5ml DMSO, obtain 0.2mol / l concentration of indomethacin solution; take 100mg IBMX powder dissolved in 2.7ml DMSO, obtain 0.17mol / l concentration of IBMX solution. Liquid is placed in a biological safety cabinet, pipette 1.4325ml insulin solution, 50μl dexamethasone solution, 50μl indomethacin solution, 150μl IBMX solution dissolved in 20ml of α-MEM medium, add 10μl of 1mol / l concentration of sodium hydroxide solution, adjust the pH value to 7.0, filter with a bacteria filter, add 5ml of FBS, 0.25ml of penicillin-streptomycin mixture and 25ml of α-MEM medium, obtain 50ml of lipid induction A liquid, store at 4℃ in the dark; pipette 1.4325ml insulin solution, 50μl dexamethasone solution dissolved in 20ml of α-MEM medium, add 10μl of 1mol / l concentration of sodium hydroxide solution, adjust the pH value to 7.0, filter with a bacteria filter, add 5ml of FBS, 0.25ml of penicillin-streptomycin mixture and 25ml of α-MEM medium, obtain 50ml of lipid induction B liquid. Take well grown BMSCs, according to 8×10 4 ​The cells were seeded at a density of 1 x 104 / cm2 in six-well plates, and after the cells grew to 80%, they were divided into a normal group, an experimental group, and an adipogenic group. The old culture medium was discarded, 2 ml of a-MEM culture medium containing 10% FBS was added to each well of the normal group, 2 ml of adipogenic induction A liquid was added to each well of the experimental group and the adipogenic group, 3 days later, the adipogenic induction B liquid was changed, 1 day later, the A liquid was changed back, and the experimental group was added with DMSO-dissolved small molecules AN698 / 40746067 to a final concentration of 10 -5 mol / l, and the adipogenic group was simultaneously added with an equal amount of DMSO.

[0079] The experimental group and the adipogenic group were subjected to 14-day adipogenic induction, and then RNA was extracted for qRT-PCR to detect the levels of adipogenic-related indicators Pparγ, Fabp4, C / ebpα, Adipoq, and Ldlr, and total cell protein was extracted for Western Blot experiments to detect the levels of adipogenic-related indicators PPARγ, C / EBPα, and LDLR according to the method described in Example 2, and the results are shown in Figure 9 .

[0080] It can be known from Figure 9 that the expression levels of adipogenic-related indicator genes Pparγ, Fabp4, C / ebpα, Adipoq, and Ldlr in the experimental group were significantly lower than those in the adipogenic group; and the expression levels of adipogenic-related indicator proteins PPARγ, C / EBPα, and LDLR in the experimental group were significantly lower than those in the adipogenic group.

[0081] 4、The grouping was the same as in Example 3 of the present embodiment. After the normal group, the experimental group, and the high-fat group were subjected to adipogenic induction for 14 days, the old culture medium was discarded, and oil red O staining was performed. After staining, the lipid droplets were dissolved with 60% isopropanol, and absorbance quantitative analysis was performed, and the results are shown in Figure 10 .

[0082] It can be known from Figure 10 that oil red O staining showed that the lipid droplet formation of the BMSCs in the adipogenic group was more obvious than that in the normal group, and the lipid droplet formation of the BMSCs in the experimental group was weaker than that in the adipogenic group. Quantitative analysis showed that the lipid level of the BMSCs in the adipogenic group was significantly higher than that in the normal group, and the lipid level of the BMSCs in the experimental group was significantly lower than that in the adipogenic group.

[0083] Example 6, Effect of AN698 / 40746067 on the Inflammatory Response of BMSCs in a High-fat Environment

[0084] Following Experiment 1 in Example 5, the cells were divided into groups. The normal group and the high-fat group were cultured in a completely high-fat medium for 7 days. RNA was then extracted and qRT-PCR was performed to detect the gene expression levels of inflammation-related factors tumor necrosis factor α (Tnf-α), transforming growth factor β (Tgf-β), interleukin 1β (Il-1β), and interleukin 6 (Il-6) in the high-fat group. Similarly, BMSCs from both the experimental group and the high-fat group were cultured in a completely high-fat medium for 7 days. RNA was then extracted and qRT-PCR was performed to detect the gene expression levels of inflammation-related factors Tnf-α, Tgf-β, Il-1β, and Il-6 in the high-fat group. The results are as follows: Figure 11 As shown.

[0085] Depend on Figure 11 It was found that after culturing BMSCs in the normal group, high-fat group and experimental group for 7 days, the gene expression levels of inflammation-related factors Tnf-α, Tgf-β, Il-1β and Il-6 in the high-fat group were significantly higher than those in the normal group, while the gene expression levels of inflammation-related factors Tnf-α, Tgf-β, Il-1β and Il-6 in the experimental group were significantly lower than those in the high-fat group.

[0086] Experimental Example 7: The effect of AN698 / 40746067 on osteogenic activity of BMSCs

[0087] Weigh 0.0039 g of dexamethasone into a centrifuge tube under light-protected conditions, dissolve in 2 ml of anhydrous ethanol, add 3 ml of ordinary culture medium, filter through a bacterial filter to obtain a 2*10^-3 mol / L dexamethasone solution. Take 5 μl of this solution and dissolve in 5 ml of ordinary culture medium to dilute to 2*10^-6 mol / L. Weigh 0.206 g of sodium β-glycerophosphate and 0.005 g of vitamin C into a centrifuge tube, dissolve in 5 ml of ordinary culture medium, filter through a bacterial filter, take 2.5 ml of this solution and 0.25 ml of the dexamethasone solution, add to 47.25 ml of ordinary culture medium to prepare 50 ml of osteogenic induction solution. The ordinary culture medium used here is supplemented with 10% fetal bovine serum.

[0088] Take well-grown BMSCs and process them at a ratio of 8 × 10⁻⁶. 4 Cells were seeded at a density of 1 cell / well in six-well plates. After the cells reached 80% growth, they were divided into experimental and control groups. The old culture medium was discarded, and 2 ml of osteogenic induction solution was added to each well. For the experimental group, small molecule AN698 / 40746067 dissolved in DMSO was added to bring the final concentration to 10. -5mol / L, and the control group was added with the same amount of DMSO. After 28 days of osteogenic induction of the BMSCs in the experimental group and the control group, the old culture medium was discarded, and alizarin red staining was performed on the cell mineralization nodules. After the staining, 10% cetylpyridinium chloride was used to dissolve the alizarin red nodules, and the absorbance was quantitatively analyzed, and the results are shown in Figure 12

[0089] As can be seen from Figure 12 , alizarin red staining of the cell mineralization nodules showed that obvious mineralization nodules were formed in both the experimental group and the control group, but the mineralization nodules in the experimental group treated with AN698 / 40746067 were enhanced compared with the control group, and the staining degrees were different. The quantitative analysis results showed that there was a statistical difference between the experimental group and the control group, and the formation of mineralization nodules in the experimental group was stronger than that in the control group.

[0090] Example 8, Establishment of Hyperlipidemia Animal Model, Drug Injection, and Specimen Collection and Processing

[0091] 1. Fifteen 10-week-old male Wistar rats were randomly divided into three groups, namely, a normal group (P group), a high-fat diet group (G group), and a high-fat diet plus small molecule drug treatment group (G+AN698 / 40746067 group, referred to as G+ group), with 5 rats in each group. The P group was given a normal diet, and the G and G+ groups were given a high-fat diet during the establishment of the hyperlipidemia rat model. The body weight was recorded every week, and after 10 weeks of feeding, the rats were fasted and deprived of water for 16 hours, and blood was taken from the medial canthus vein. The serum lipid levels were detected using a full-automatic biochemical analyzer, and the results are shown in Figure 13 and Table 1.

[0092] Table 1, Serum Lipid Detection Results of Rats in the P and G Groups after 10 Weeks of Feeding (mean ± SD, unit: mmol / L)

[0093]

[0094]

[0095] As can be seen from Figure 13 and Table 1, the serum total cholesterol (T-CHO), low-density lipoprotein cholesterol (LDL-C), and triglyceride (TG) levels in the Wistar rats in the high-fat diet group (G group) were significantly higher than those in the Wistar rats in the normal group (P group), and the difference was statistically significant (P<0.05), indicating that the hyperlipidemia model of Wistar rats was successfully constructed.

[0096] ​2. After successfully establishing the hyperlipidemic rat model, the control group (P group) and the high-fat diet group (G group) were raised as usual until 16 weeks. The high-fat diet plus drug group (G+ group) was given drug injection, specifically: the high-fat diet plus drug group (G+ group) was given 1 mg / kg of small molecule drug AN698 / 40746067 via intraperitoneal injection, once every 3 days, and samples were collected after 6 weeks. The small molecule drug AN698 / 40746067 was fully dissolved in DMSO to prepare a 10 mg / ml injection stock solution, which was diluted with physiological saline before injection to ensure that the final DMSO concentration was less than 1%.

[0097] During tissue collection, Wistar rats in groups P, G, and G+ were intraperitoneally injected with an excessive amount of sodium pentobarbital to confirm cardiac arrest. The length from the tip of the nose to the anus was then measured. The skin, subcutaneous tissue, and muscles were cut open from below the xiphoid process upwards and downwards. The thoracic and abdominal cavities were opened, and the pericardial adipose tissue surrounding the heart, liver, spleen, lungs, kidneys, and mesentery was bluntly dissected. The dissected organs and tissues were rinsed with physiological saline, blotted dry with absorbent paper, placed in clean petri dishes, weighed, and photographed. The corresponding organ index was subsequently calculated based on body weight (calculation method: organ index = net weight of organ (g) / body weight (g) * 100%). The leg skin and muscles were then cut open sequentially to dissect the femur and tibia, carefully removing the surrounding muscles. The dissected organs and tissues were fixed in test tubes containing 4% paraformaldehyde for subsequent experiments. The remaining half of the dissected femur and tibia were rapidly frozen in liquid nitrogen for subsequent RNA and protein experiments.

[0098] Example 9: Effect of AN698 / 40746067 treatment on obesity signs in hyperlipidemic rats

[0099] 1. After 16 weeks of feeding according to the method described in Example 8, compared with group P, the body weight of rats in groups G and G+ increased significantly, the body size difference among the three groups was significant, and the Lee's index of rats in groups G and G+ was significantly higher than that in group P. Figure 14 The formula for calculating Lee's index is: Lee's index = weight (g)^1 / 3 * 10 / body length (cm). Monitoring of body weight showed that after 11 weeks of injection of the small molecule drug AN698 / 40746067, the weight gain trend in the G+ group decreased. Figure 15 The G+ group, after treatment with the small molecule drug AN698 / 40746067, showed a significant decrease in Lee's index compared to the G group, with a statistically significant difference. Figure 14 (P < 0.05).

[0100] 2. HE staining was performed on the white adipose tissue surrounding the rat mesentery obtained in Example 8, and the results are as follows: Figure 16 As shown.

[0101] Depend onFigure 16 It can be seen that, compared with group P, the diameter of white adipocytes around the mesentery of rats in groups G and G+ was increased. However, after treatment with the small molecule drug AN698 / 40746067, the diameter of adipocytes in group G+ was reduced compared with group G, and was closer to the adipocyte morphology (400×) of group P. This indicates that the small molecule drug AN698 / 40746067 alleviated the degree of obesity in rats.

[0102] 3. Serum lipid levels in rats of the P, G, and G+ groups after 16 weeks of feeding were as follows: Figure 17 As shown.

[0103] Depend on Figure 17 It was found that after treatment with the small molecule drug AN698 / 40746067, the serum T-CHO and LDL-C levels in the G+ group rats were significantly lower than those in the G group (P < 0.05), while the TG level showed a decreasing trend but no significant statistical difference (P > 0.05). The HDL-C level increased, with a statistically significant difference (P < 0.05). This indicates that the small molecule drug AN698 / 40746067 can effectively improve the elevated serum lipid levels induced by a high-fat diet, and its effect is more pronounced on lipids mainly composed of cholesterol.

[0104] 4. The important organs (heart, liver, spleen, lung, and kidney) taken in Example 8 were weighed, measured, and their soft tissue sections were stained with hematoxylin and eosin (HE). The results are as follows: Figures 18-20 As shown.

[0105] HE staining of soft tissue sections: Soft tissue samples from various organs obtained in Example 8 were fixed in 4% paraformaldehyde for 12–24 h, rinsed, dehydrated with a gradient of alcohols, cleared with xylene, embedded in paraffin at 65°C, and then sectioned to a thickness of 5 μm. After obtaining the paraffin sections of each soft tissue, HE staining was performed using the Solarbio HE staining kit (catalog number: G1120) according to the manufacturer's instructions. The specific steps are as follows: Bake the sections of heart, liver, spleen, lung, kidney, and adipose tissue that need staining on a slide baking machine for 1-2 hours to melt the paraffin; place the sections in a fume hood and dewax them in xylene (xylene I for 10 minutes → xylene II for 10 minutes); rehydrate them in a gradient of 100%, 95%, 85%, and 75% alcohols, soaking for 3 minutes each; soak in distilled water for 2 minutes; stain with hematoxylin for 3 minutes, then rinse off the staining solution with distilled water; differentiate with differentiation solution for 3 minutes, then gently rinse twice with distilled water for 2 minutes each time; stain with eosin for 30 seconds to 1 minute, then quickly rinse off the staining solution with distilled water; rinse with 75%, 85%, 95%, and 100% alcohol for 2-3 seconds each; soak in 100% alcohol II for 1 minute; clear with xylene I and xylene II for 1 minute each; remove the sections, allow them to dry slightly, and then mount them with neutral resin. The results are shown in Figures 21-22.

[0106] Depend on Figures 18-22 It was found that neither a high-fat diet nor treatment with the small molecule drug AN698 / 40746067 had any adverse effects on the morphology of tissue cells or the weight of organs such as the heart, spleen, lungs, and kidneys, indicating that the small molecule drug AN698 / 40746067 did not have significant acute toxicity to important organs in rats. Group G rats exhibited pathological changes of fatty liver, specifically diffuse enlargement of the liver, a grayish-yellow color, and HE staining of liver sections showing hepatocytes filled with lipid droplets of varying sizes and vacuoles, with loss of normal lobular morphology. In contrast, the fatty liver symptoms in Group G+ were alleviated after treatment with the small molecule drug AN698 / 40746067.

[0107] Example 11: Effects of AN698 / 40746067 treatment on the expression levels of adipogenic and lipid metabolism-related factors and inflammatory factors RNA and protein in bone tissue.

[0108] 1. Total protein was extracted from rat bone tissue using the Invent Bone Tissue Total Protein Extraction Kit (catalog number: SA-02-BT). The steps were as follows: 50-100 mg of proximal tibial bone tissue was placed in a pre-chilled mortar, and liquid nitrogen was added in small amounts several times to grind it into powder. The bone tissue powder was transferred to a collection tube containing a filter column. 50-80 mg of protein extraction powder and 100 μl of lysis buffer were added, and the mixture was incubated at room temperature for 5-10 minutes. The mixture was gently ground with a plastic rod for 2-3 minutes, and then 100 μl of lysis buffer was added again and ground for 30 seconds to 1 minute. The mixture was centrifuged at 14000×g for 1 minute at room temperature, the filter column was discarded, and the supernatant was transferred to a new 1.5 ml ep tube, which was the obtained total protein from the bone tissue. The protein concentration was determined using BCA, and the expression levels of SDCCAG3, PPARγ, C / EBPα, and LDLR in the bone tissue were detected by Western blotting. The results are shown below. Figures 23-24 As shown.

[0109] Depend on Figure 23 It can be seen that, compared with the G group, the G+ group treated with the small molecule drug AN698 / 40746067 had significantly lower SDCCAG3 protein levels (P<0.05), indicating that the small molecule drug AN698 / 40746067 can effectively inhibit the expression of SDCCAG3 in rats.

[0110] Depend on Figure 24It can be seen that the expression levels of PPARγ and C / EBPα proteins in group G were increased, and the difference was statistically significant (P<0.05), indicating that the adipogenic differentiation of BMSCs was enhanced by high-fat diet. After treatment with small molecule drug AN698 / 40746067, the expression levels of PPARγ and C / EBPα proteins in group G+ were significantly decreased compared with group G (P<0.05), while the expression level of LDLR showed an increasing trend in group G and a decreasing trend in group G+, but the difference was not statistically significant (P>0.05). The above results showed that small molecule drug AN698 / 40746067 can significantly inhibit the adipogenic differentiation of bone marrow in hyperlipidemic rats.

[0111] 2、According to the Vazyme Fast The RNA of the bone tissue of rats in groups P, G and G+ was extracted according to the EACell / Tissue Total RNAIsolation Kit (product number: RC101) kit instructions. Specifically, 20 mg of proximal tibial bone tissue was taken and immediately ground into powder in a pre-cooled mortar with liquid nitrogen. 500 μl of Buffer RL1 (β-mercaptoethanol was added to a final concentration of 1% in advance) was added, and the pipette was blown thoroughly. Centrifugation was performed at room temperature at 12000 rpm for 3 minutes. The supernatant was transferred to gDNA-Filter Columns (which had been placed in a collection tube), and centrifugation was performed at room temperature at 12000 rpm for 2 minutes. The gDNA-Filter Columns were discarded, and the liquid in the collection tube was retained. 800 μl of Buffer RL2 (which had been added with a specified volume of anhydrous ethanol) was added to the collection tube, and the mixture was gently mixed. The mixture was transferred to RNAPure Columns (which had been placed in a collection tube), and centrifugation was performed at room temperature at 12000 rpm for 1 minute, and the waste liquid was discarded. 500 μl of Buffer RW1 was added to the RNAPure Columns, and centrifugation was performed at room temperature at 12000 rpm for 1 minute, and the waste liquid was discarded. 700 μl of Buffer RW2 was added to the RNAPure Columns, and centrifugation was performed at room temperature at 12000 rpm for 1 minute, and the waste liquid was discarded. The above step was repeated once. The RNAPure Columns were placed back into the collection tube, and the empty tube was centrifuged at room temperature at 12000 rpm for 2 minutes. The RNAPure Columns were transferred to a new RNase-free 1.5 ml ep tube, and 100 μl of enzyme-free double distilled water was added to the center of the adsorption column. It was placed at room temperature for 2 minutes, and then centrifuged at 1200 rpm for 1 minute to elute the RNA. The extracted RNA was subjected to reverse transcription for PCR experiment or stored at -80°C.

[0112] qRT-PCR experiment was used to detect the expression levels of Pparγ, C / ebpα, Fabp4, Adipoq, Ldlr, Il-1β and Il-6 in the bone tissues of P, G and G+ groups, and Actin was used as a control.2 -ΔΔct The relative expression levels were calculated, and the results are shown in Figure 25

[0113] As can be seen from Figure 25 , compared with the G group, the levels of Pparγ, C / ebpα, Fabp4 and Ldlr in the proximal tibial bone tissues of the G+ group were significantly reduced (P<0.05), and the expression of Adipoq showed a downward trend, indicating that the small molecule drug AN698 / 40746067 can effectively reduce the expression of adipogenic related factors in bone marrow.

[0114] qRT-PCR experiment was used to detect the expression levels of Il-1β and Il-6 in the bone tissues of P, G and G+ groups, and the results are shown in Figure 26

[0115] As can be seen from Figure 26 , high-fat diet can increase the expression of Il-1β and Il-6 in the bone marrow of mice, and the expression of inflammatory factors in the G+ group after treatment with the small molecule drug AN698 / 40746067 is significantly reduced, indicating that the small molecule drug AN698 / 40746067 can effectively alleviate the bone marrow inflammation induced by high-fat diet.

[0116] Example 12, Effect of AN698 / 40746067 treatment on bone marrow obesity in hyperlipidemic rats

[0117] The bone tissues of P, G and G+ groups of rats were subjected to decalcification section HE and RUNX2 immunohistochemical staining, and the specific method was as follows.

[0118] Preparation of bone tissue decalcification section: the bone tissue was fixed, washed and decalcified with 10% EDTA for 30 days, followed by dehydration, transparency, wax immersion, embedding, sectioning and HE staining, and the results are shown in Figure 27

[0119] ​​​The steps of RUNX2 immunohistochemical staining are as follows: after the bone tissue paraffin section is baked on the section warmer for 2 hours, it is sequentially placed in xylene I and II for 5 minutes; gradient alcohol dehydration: 100% alcohol I→100% alcohol II→95% alcohol→85% alcohol→75% alcohol, each for 5 minutes, PBS buffer gentle soaking and washing for 3 times, each for 5 minutes; 0.1% trypsin digestion solution is added dropwise to cover the tissue, and wet box incubation is performed at 37°C for 15 minutes, PBS washing for 3 times, each for 5 minutes; 3% hydrogen peroxide is added dropwise to cover the tissue, and wet box incubation is performed at 37°C for 10 minutes, PBS washing for 3 times, each for 5 minutes; goat serum is added dropwise to cover the tissue, and wet box incubation is performed at 37°C for 2 minutes, and the old liquid is discarded; the diluted RUNX2 antibody is added dropwise, and wet box incubation is performed at 4°C overnight; recovery of the RUNX2 primary antibody, PBS gentle washing for 3 times, each for 5 minutes; the diluted secondary antibody (ratio 1:100) is added dropwise, and wet box incubation is performed at 37°C for 20 minutes, the secondary antibody is poured into the wet box, and PBS washing is performed for 3 times, each for 5 minutes; the excess PBS buffer is absorbed by the water-absorbing paper, the streptavidin-POD working solution is added dropwise, wet box incubation is performed at 37°C for 15 minutes, PBS buffer washing is performed for 3 times, each for 5 minutes; the DAB working solution is added dropwise and observed under a microscope, and when the appropriate staining degree is reached, the staining solution is discarded, and PBS is washed; hematoxylin staining is performed for 1-2 seconds, and water washing is performed for 4 minutes, gradient alcohol 85%→95%→100% I→100% II each for 5 minutes for rehydration; xylene I→xylene II each for 5 minutes for transparency, and neutral resin is used for section sealing, and the results are as shown in Figure 28 .

[0120] It can be known from Figures 27-28 that the increase of adipocytes in the bone marrow cavity of the rats in group G shows bone marrow obesity, and the number and diameter of adipocytes in group G+ treated by the small molecule drug AN698 / 40746067 are reduced. The immunohistochemical results show that the treatment of the small molecule drug AN698 / 40746067 can relieve the decrease of the growth plate width and the decrease of the length of the lower trabecula, and the expression of RUNX2 is increased.

[0121] Example 13, effect of AN698 / 40746067 treatment on osteoporosis of hyperlipidemic rats

[0122] The distal femur of the rats in groups P, G and G+ is subjected to Micro-CT detection and analysis, and the specific method is as follows.

[0123] The femur and tibia of three groups of rats were scanned by Quantum GX2 small animal live Micro-CT imaging system to obtain the information of cancellous bone, compact bone and microstructure. The obtained femur and tibia were fixed in 4% paraformaldehyde for 24-48 hours, then taken out and placed in a 50 ml centrifuge tube containing 100% ethanol. When Micro-CT detection was performed, the soft tissues such as muscle and ligament around the bone tissue were removed as much as possible, wrapped with sealing film and fixed on the sample bed for scanning. The specific scanning conditions were set as follows: source voltage 90 kV, current 88 μA, scanning time 14 minutes, scanning type High resolution. The three-dimensional reconstruction was performed by the data workstation (Shandong University Higher Medical Research Institute), the ROI was set as 2.0 mm range near the distal metaphysis of the femur, and the bone mineral density (Ct.BMD), bone volume percentage (Ct.BV / TV) and trabecular bone thickness (Tb.N) were measured. The bone trabecula and bone stem in different cross sections (sagittal, coronal and horizontal) of the bone tissue were observed by Data Viewer software, and the results are shown in Figure 29 .

[0124] As can be seen from Figure 29 , the cortical bone bone mineral density (Ct.BMD) and bone volume percentage (Ct.BV / TV) of the distal femur of the hyperlipidemia rats were reduced, the trabecular bone thickness (Tb.Th) was reduced, and the above indexes were recovered after AN698 / 40746067 treatment. Combined with the results of immunohistochemical staining, it is indicated that AN698 / 40746067 can relieve the osteoporosis of the hyperlipidemia rats.

Claims

1. The use of a small molecule drug AN698 / 40746067 in the preparation of a medicament for treating hyperlipidemia, the small molecule drug AN698 / 40746067 has the following structural formula:

2. Use according to claim 1, wherein The small molecule drug AN698 / 40746067 targets SDCCAG3 protein, and reduces serum lipid levels by inhibiting the adipogenic differentiation of BMSCs.

3. The use according to claim 1, wherein The medicament for treating hyperlipidemia is composed of the small molecule drug AN698 / 40746067 and pharmaceutically acceptable adjuvants.

4. The use according to claim 1, wherein The use concentration of the small molecule drug AN698 / 40746067 is 0.5-1.5 mg / kg.

5. The use of a small molecule drug AN698 / 40746067 in the preparation of a medicament for treating osteoporosis, the small molecule drug AN698 / 40746067 has the following structural formula:

6. The use according to claim 5, wherein the compound is ###0002### The osteoporosis is an osteoporosis condition caused or accompanied by hyperlipidemia.

7. The use according to claim 5, wherein the compound is ###0002### The medicament for treating osteoporosis is composed of the small molecule drug AN698 / 40746067 and pharmaceutically acceptable adjuvants.

8. The use according to claim 5, wherein the compound is ###0002### The use concentration of the small molecule drug AN698 / 40746067 is 0.5-1.5 mg / kg.

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