Application of a celastrol based on a trem2 target point in preparation of an anti-osteosarcoma drug

Abstract

The application provides an application of tripterine based on a TREM2 target in preparation of an anti-osteosarcoma drug and belongs to the technical field of biological medicines.Tripterine screened with the TREM2 as a target can be specifically combined with the TREM2 protein and promote the expression of the TREM2 protein, and then plays an anti-osteosarcoma role.The application first finds that tripterine can be used as an activator of the TREM2, effectively inhibits the proliferation and migration and invasion of osteosarcoma cells by activating the expression of the TREM2.This provides a potential therapeutic drug of a therapeutic target for the treatment of osteosarcoma and has important clinical application value.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of triptolide based on the TREM2 target in the preparation of anti-osteosarcoma drugs. Background Technology

[0002] Osteosarcoma is the most common malignant bone tumor in adolescents and children, with 75% of patients developing the disease between the ages of 10 and 20. The incidence is slightly higher in males than females, and it commonly occurs in the metaphysis of long bones such as the distal femur and proximal tibia. Its etiology remains unclear. Clinically, it is characterized by localized pain and a mass. Late-stage osteosarcoma is prone to lung metastasis, seriously threatening the patient's life. Currently, the clinical treatment model combines surgery with radiotherapy and chemotherapy, which has increased the 5-year survival rate to 60%–80%. However, the long-term survival rate for metastatic or recurrent cases remains below 30%, and long-term chemotherapy easily leads to tumor cell resistance and significant toxic side effects. Traditional Chinese medicine (TCM) has advantages such as multi-target therapy, fewer side effects, and the ability to reverse tumor drug resistance, making it a focus of attention in cancer prevention and treatment. Given the limitations of modern medical treatment for osteosarcoma, in-depth exploration of the anti-tumor potential of TCM, investigating its mechanisms of action in inhibiting osteosarcoma cell proliferation, invasion, and metastasis, and finding safe and effective TCM formulas and compound prescriptions have become important directions in current osteosarcoma treatment research, providing new ideas and support for improving patient prognosis and treatment outcomes.

[0003] Tripterygium wilfordii is a natural active ingredient extracted from Tripterygium wilfordii. Studies have shown that it has inhibitory effects on various tumors, such as breast cancer and liver cancer, but its specific mechanism of action in osteosarcoma has not been fully understood.

[0004] TREM2 protein belongs to the immunoglobulin superfamily. Recent studies have confirmed its low expression in osteosarcoma tissues, and overexpression of TREM2 can significantly inhibit osteosarcoma cell proliferation and metastasis, suggesting it as a potential tumor suppressor target. However, there are currently no reports on triptolide exerting anti-osteosarcoma effects by targeting TREM2 protein, and there is also a lack of traditional Chinese medicine drugs targeting TREM2 for osteosarcoma treatment. Therefore, this invention provides an application of triptolide-activated TREM2 in the preparation of drugs to inhibit osteosarcoma. Summary of the Invention

[0005] The purpose of this invention is to provide the application of triptolide based on the TREM2 target in the preparation of anti-osteosarcoma drugs.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of triptolide based on TREM2 target in the preparation of anti-osteosarcoma drugs. The triptolide obtained by screening with TREM2 as the target can specifically bind to TREM2 protein and promote TREM2 protein expression, thereby exerting an anti-osteosarcoma effect.

[0007] Preferably, the anti-osteosarcoma effect is to inhibit the growth and / or proliferation of osteosarcoma cells.

[0008] This invention provides an anti-osteosarcoma drug comprising triptolide and excipients.

[0009] Preferably, the concentration of triptolide in the drug is 200-400 nM.

[0010] Compared with the prior art, the present invention has the following beneficial effects: This invention is the first to discover that triptolide can act as an activator of TREM2, effectively inhibiting the proliferation, migration, and invasion of osteosarcoma cells by activating TREM2 expression. This provides a potential therapeutic target for osteosarcoma treatment and has significant clinical application value. Attached Figure Description

[0011] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0012] Figure 1 In Example 1, triptolide was initially screened as a potential agonist of TREM2 and its effects on the proliferation, colony formation, migration and invasion of 143B osteosarcoma cells were explored. Figure 2 The effect of triptolide on TREM2 protein levels in Example 2; Figure 3 This describes the effect of triptolide on the inhibitory effect on osteosarcoma after TREM2 knockdown in Example 3. Detailed Implementation

[0013] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0014] The 143B cell line used in the following examples was purchased from the Cell Bank of the Chinese Academy of Sciences (Shanghai Institute of Cell Biology). 143B cells were cultured in MEM medium supplemented with 10% fetal bovine serum, 100 U / mL penicillin, and 0.1 mg / mL streptomycin at 37 °C and 5% CO2.

[0015] Example 1

[0016] This embodiment preliminarily screens triptolide as a potential agonist of TREM2 and explores its effects on the proliferation, colony formation, migration, and invasion of 143B osteosarcoma cells.

[0017] 1. Based on the 3D protein structure of TREM2 (5ELI), docking pockets of the protein receptor were identified using the CB-Dock algorithm on the DrugRep platform, and compounds that could bind to the TREM2 target were screened from a traditional Chinese medicine database using AutoDock Vina software. A total of 173 traditional Chinese medicine compounds were screened and ranked according to their virtual molecular docking scores. The top 5 traditional Chinese medicine compounds with the lowest binding energies were obtained: triptolide, icariin II, baicalin, naringin, and psoralen, among which triptolide had the lowest binding energy (Table 1).

[0018] The process of screening traditional Chinese medicine compounds based on TREM2 as a target is as follows: Figure 1 As shown in Figure A, triptolide has the following chemical formula: Figure 1 As shown in B.

[0019] Table 1. Top 5 active compounds of traditional Chinese medicine that are potentially associated with TREM2

[0020] 2. CCK-8 Experiment

[0021] 143B cells were added at a concentration of 2 × 10⁶ cells per well (100 μL per well). 3 Cells were seeded at a density of 1000 mcg / well in 96-well plates and treated with different concentrations of triptolide (four replicates per concentration). The cells were then incubated at 37 ℃ in a 5% CO2 incubator for 48 h. Afterward, 10 μL of CCK-8 reagent and 90 μL of MEM medium were added to each well, and the cells were incubated for another 1 h. The absorbance was then measured at 450 nm using a microplate reader, and the half-maximal inhibitory concentration (IC50) was calculated using GraphPad Prism 8 software. 50 value.

[0022] The results are as follows Figure 1 As shown in Figure C, triptolide can significantly inhibit the proliferation of osteosarcoma cells and has an effect on the IC50 of 143B cells. 50 The value is 381.1 nM.

[0023] 3. Cloning experiment

[0024] 143B cells were seeded into 6-well plates at a density of 500 cells / well and treated with 0, 200, 300, and 400 nM triptolide for 48 h, respectively. During this period, the cell spheroidization ability was observed and the culture medium was replaced with fresh medium. After 10 days, the culture medium was removed, and the cells were washed twice with 1 mL PBS. The cells were then fixed with 4% paraformaldehyde at room temperature for 10 min. After removing the paraformaldehyde, the cells were stained with 0.1% crystal violet for 10 min. The cells were then washed three times with 1 mL PBS. The cells were photographed and counted using image software.

[0025] The results are as follows Figure 1 As shown in Figure D, triptolide can significantly inhibit the colony formation of 143B cells in a dose-dependent manner.

[0026] 4. Scratch test

[0027] 143B cells were spaced at 5 × 10⁶ cells per well. 5 Cells were seeded at a density of 100 μL into 6-well plates and cultured for 24 h. Vertical scratches were made using a 200 μL pipette tip and photographed to obtain the initial scratch area at 0 h. Different concentrations of triptolide (0, 200, 300, 400 nM) were added and the cells were cultured in a cell culture incubator. After 48 h, the scratch area was photographed and recorded again. ImageJ software was used to quantify the area and calculate the number of cells that migrated.

[0028] The results are as follows Figure 1 As shown in Figure E, triptolide can significantly inhibit the migration of 143B cells in a dose-dependent manner.

[0029] 5. Transwell migration and invasion experiments

[0030] 143B cells were fed at a rate of 2 × 10 4 Cells were seeded at a density of 100 cells / well in 6-well plates and pre-cultured for 24 h, followed by treatment with medium containing different concentrations of triptolide (0, 200, 300, 400 nM) for 48 h. Cells were then treated with trypsin and cultured in MEM medium at a concentration of 2 × 10⁻⁶ cells / well. 4Cells / well were seeded into the upper chamber of a Transwell chamber, and 600 μL of MEM medium containing 10% FBS was added to the lower chamber. For the invasion assay, 40 μL of 1:5 diluted Matrix gel was added to the upper chamber before adding cells, and the mixture was incubated for 30 min. Cell suspension was then added. After 24 h, the chamber was removed and the medium was discarded. Cells were fixed with 4% paraformaldehyde at room temperature for 10 min, the 4% paraformaldehyde was removed, and the cells were stained with 0.1% crystal violet for 10 min. The chamber was then washed three times with 1 mL PBS. The bottom inside of the chamber was gently wiped with a cotton swab to remove cells from the upper chamber. Cells were then photographed using an optical microscope, and their counts were quantified using Image software.

[0031] The results are as follows Figure 1 As shown in Figure F, triptolide can significantly inhibit the migration and invasion of 143B cells in a dose-dependent manner.

[0032] As shown above, triptolide can significantly inhibit the proliferation, colony formation, migration and invasion of osteosarcoma cells in a dose-dependent manner.

[0033] Example 2

[0034] This example investigated the effect of triptolide on TREM2 protein levels.

[0035] 1. Tripterygium wilfordii is an agonist of TREM2.

[0036] Download the PDB file of the TREM2 protein from PDB and the MOL2 file of triptolide from PubChem. Upload them to CB-DOCK2 and construct the Celastrol-TREM2 complex structure using CB-DOCK2, showing the pocket where the drug triptolide (Celastrol) binds to TREM2, with the lowest binding energy (-10.5 kcal / mol). Figure 2 As shown in Figure A.

[0037] Root mean square deviation (RMSD) is a good indicator of the conformational stability of proteins and ligands, and also measures the degree of deviation of atomic positions from their initial positions. The smaller the deviation, the better the conformational stability. From 0 to 50 ns: RMSD increases rapidly, from 0 Å to approximately 8 Å, indicating that the protein is adjusting from its initial conformation to a more stable state; from 50 to 100 ns: RMSD stabilizes at around 8 to 9 Å with smaller fluctuations, indicating that the Celastrol-TREM2 system structure tends to be stable. Figure 2 As shown in B.

[0038] The radius of gyration (Rg) measures the average distance from a protein atom to its center of mass, reflecting the protein's compactness and overall folding state. The system reached a stable folded state after 20 ns, without significant unfolding, indicating that the Celastrol-TREM2 complex did not undergo significant expansion and contraction during motion. Figure 2 As shown in C.

[0039] Solvent-accessible surface area (SASA) measures the surface area of ​​a protein, and the simulation calculated the solvent-accessible surface area between the target protein and the small molecule. Throughout the 100 ns simulation, the SASA remained stable at around 22,000 Ų, with only minor fluctuations, indicating that the surface exposure of the protein remained stable throughout the simulation. This suggests that the hydrophobic core of the Celastrol-TREM2 receptor was not disrupted after ligand binding, and its structural integrity remained good. Figure 2 As shown in D.

[0040] The hydrogen bond network remained dynamically stable throughout the simulation, without large-scale breakage, indicating the overall stability of the structure. Figure 2 As shown in E.

[0041] The core domains of proteins are stable, while the terminal or loop regions are highly dynamic; this is a common characteristic in the regulation of protein function, such as... Figure 2 As shown in F.

[0042] Kaplan-Meier survival curve analysis of osteosarcoma patients with high / low TREM2 expression based on the TARGET database showed that high TREM2 expression can prolong the survival of osteosarcoma patients. Figure 2 As shown in G.

[0043] 2. Effect of triptolide on TREM2 protein stability as determined by CETSA cell thermal displacement assay

[0044] Osteosarcoma 143B cells were treated with 400 nM triptolide for 1 h, while the control group was treated with DMSO for the same time. After cell collection, the cells were washed with PBS and resuspended in PBS containing 1 mM PMSF protease inhibitor. Cells were aliquoted into PCR tubes and subjected to a temperature gradient heating process (45°C–65°C, 3 min at each temperature). After heating, the PCR tubes were quickly transferred to liquid nitrogen. The tubes underwent a first freeze-thaw cycle: immersion in liquid nitrogen for 20 seconds; thawing: brief transfer to room temperature (or a 37°C water bath); and a second freeze-thaw cycle: immersion in liquid nitrogen again for 20 seconds (to enhance cell lysis efficiency). Lysis buffer (containing 1 mM PMSF protease inhibitor) was added, and the cells were lysed on ice for 30 min. Cells were centrifuged at high speed (12000 r / min, 20 min, 4°C), followed by denaturation with SDS-PAGE protein loading buffer (5×) at 95°C for 10 min. Electrophoresis was then performed, followed by membrane transfer; blocking buffer was applied at room temperature for 1 h. Add specific rabbit anti-human TREM2 monoclonal antibody (Wuhan Sanying, 1:2000) and incubate overnight at 4 °C. Wash three times with 1×TBST, then incubate with the corresponding HRP-labeled goat anti-rabbit IgG and HRP-labeled goat anti-mouse IgG (BBI, 1:10000) secondary antibodies at room temperature for 1.5 h; develop color with ECL high-sensitivity chemiluminescence reagent.

[0045] The results are as follows Figure 2 As shown in Figure H, triptolide can enhance the protein stability of TREM2.

[0046] 3. Western blot analysis of the effect of triptolide on TREM2 protein expression levels

[0047] To assess whether triptolide affects the protein level of TREM2, TREM2 expression was detected by Western blot.

[0048] The results are as follows Figure 2 As shown in Figure I, the protein expression level of TREM2 was upregulated in a dose-dependent manner with increasing triptolide concentration.

[0049] Example 3

[0050] To investigate whether triptolide inhibits the proliferation and metastasis of osteosarcoma cells by regulating TREM2, triptolide was added to TREM2-knockdown 143B cells for a rescue experiment.

[0051] In 143B cells, siNC and siTREM2 cells were transfected using the LIPO2000 transfection reagent. After 24 h, DMSO and triptolide were added, respectively, for rescue experiments. The effect of triptolide on the cell viability of TREM2 knockdown cells was detected by CCK-8 assay. The change in the number of TREM2 knockdown cells was detected by Transwell migration and invasion assays. The changes in related proteins after TREM2 knockdown were detected by Western blot, using the same methods as in Example 1.

[0052] The results are as follows Figure 3 As shown, knocking down TREM2 can reverse the inhibitory effect of triptolide on 143B cells. Figure 3 (A), blocking the inhibitory effect of triptolide on cell migration and invasion ( Figure 3 (C, D); Western blot results showed that knocking down TREM2 blocked the regulatory effect of triptolide on β-catenin and c-myc. Figure 3 (See Figure B). It is evident that triptolide can target and activate TREM2 to inhibit Wnt / β-catenin and suppress osteosarcoma progression.

[0053] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The application of triptolide based on the TREM2 target in the preparation of anti-osteosarcoma drugs, characterized in that, Tripterygium wilfordii, obtained by screening with TREM2 as the target, can specifically bind to TREM2 protein and promote TREM2 protein expression, thereby exerting an anti-osteosarcoma effect.

2. The application according to claim 1, characterized in that, The anti-osteosarcoma treatment aims to inhibit the growth and / or proliferation of osteosarcoma cells.

3. A drug for treating osteosarcoma, characterized in that, Including triptolide and excipients.

4. The drug according to claim 3, characterized in that, The concentration of triptolide in the drug is 200-400 nM.

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