Application of THBS1 as drug target for screening and promoting or inhibiting osteoclast generation

Abstract

The invention relates to the field of biological medicine application, in particular to application of THBS1 as a drug target for screening and promoting or inhibiting osteoclast generation. The invention also relates to application of the THBS1 inhibitor or agonist in preparation of drugs for inhibiting or promoting osteoclast generation.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical applications and relates to the use of THBS1 as a drug target for screening drugs that promote or inhibit osteoclastogenesis. Background Art

[0002] THBS1 (Thrombospondin-1) is a secretory matricellular protein and a 450kDa monomeric homodimeric glycoprotein. THBS1 can regulate cell proliferation and apoptosis by binding to cell surface receptors such as CD36, CD47, and LRP1. In summary, THBS1 is a multifunctional matricellular protein that plays an important role in cell adhesion, proliferation, angiogenesis, and immune regulation, and has complex pathophysiological functions in various diseases. Further research and clinical application development of THBS1 is expected to provide new ideas and methods for drug screening for the treatment of various diseases.

[0003] In the existing technology, for example, patent CN119932192A discloses the use of THBS1 in the preparation of pancreatic cancer diagnosis and treatment products; for another example, CN113041350B discloses the use of Thbs1 and its inhibitors in the preparation of anti-tumor drugs. However, these studies on THBS1 have not yet been able to go deep into all aspects and need further exploration.

[0004] Studies have shown that postmenopausal osteoporosis (PMOP) in women is caused by an imbalance in the interactions between osteoclasts, osteoblasts, and immune cells in the bone marrow microenvironment. Research results show that increased expression of cytokines and chemokines in specific cell subsets (including hematopoietic stem cells, B cells, T cells, dendritic cells, neutrophils, granulocyte-macrophage progenitor cells, and erythrocytes) may help promote osteoclastogenesis. Specifically, enhanced monocyte-to-osteoclast differentiation is associated with enhanced B cell communication.

[0005] Further research was conducted on the correlation between the above three monocyte subtypes and osteoclasts. The results of correlation analysis showed that the proportion of MONO1 / 4 / 5 subtypes in the bone marrow was significantly positively correlated with the number of osteoclasts. Their common feature is the high expression of THBS1. Knocking out THBS1 can inhibit menopausal-induced bone loss and osteoclast formation. The above research results provide the possibility of using THBS1 as a drug target for screening to promote or inhibit osteoclast formation.

[0006] The information disclosed in this background technology section is only intended to enhance understanding of the overall background of the invention and should not be regarded as an admission or any form of suggestion that the information constitutes the prior art already known to a person skilled in the art. Summary of the Invention

[0007] In view of the shortcomings of the prior art, the present invention provides the use of THBS1 as a drug target for screening drugs that promote or inhibit osteoclastogenesis.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] THBS1 is used as a target for screening drugs that promote or inhibit osteoclastogenesis. When the drug can promote THBS1 expression, the drug can promote osteoclastogenesis; when the drug can inhibit THBS1 expression, the drug can inhibit osteoclastogenesis.

[0010] Preferably, the drug for promoting or inhibiting osteoclastogenesis is not used for preventing or treating tumors.

[0011] The present invention also provides a use of a THBS1 inhibitor in preparing a drug for inhibiting osteoclastogenesis.

[0012] Preferably, the drug for inhibiting osteoclastogenesis is not used for preventing or treating tumors.

[0013] The present invention also provides a use of a THBS1 activator in preparing a drug for promoting osteoclastogenesis.

[0014] Preferably, the drug for promoting osteoclastogenesis is not used for preventing or treating tumors.

[0015] The present invention also provides a use of the THBS1 gene as a drug target in screening drugs that promote or inhibit osteoclastogenesis. When the drug can promote THBS1 gene expression, the drug can promote osteoclastogenesis; when the drug can inhibit THBS1 gene expression, the drug can inhibit osteoclastogenesis.

[0016] Preferably, the drug for promoting or inhibiting osteoclastogenesis is not used for preventing or treating tumors.

[0017] Beneficial effects of the present invention:

[0018] This invention simulates and discloses the detailed characteristics of bone marrow cell heterogeneity in postmenopausal women, providing insights into potential therapeutic strategies for PMOP in the future. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Schematic diagram of the nine subpopulations of monocytes that are most likely to differentiate into osteoclasts;

[0020] Figure 2Schematic diagram of three subpopulations that widely express genes associated with osteoclast differentiation: (B) Heat map of marker genes in each monocyte subpopulation; (C) Feature map showing the normalized expression levels of marker genes in MONO 1, MONO 4, and MONO 5 groups; (D) Bubble plot of gene expression levels associated with osteoclast differentiation in each cell subpopulation; (E) KEGG analysis of specific genes based on MONO 1, MONO 4, and MONO 5 groups;

[0021] Figure 3 Shown is a volcano plot of differential gene expression analysis in MONO 1, MONO 4, and MONO 5 groups;

[0022] Figure 4 Schematic diagram: (G) Representative immunofluorescence images of CD14 and JUND in cancellous bone sections of healthy controls and PMOP patients (left) and quantification of the proportion of positive cells (right); (H) Representative immunofluorescence images of CD11b and CXCL8 in cancellous bone sections of healthy controls and PMOP patients (left) and quantification of the proportion of positive cells (right);

[0023] Figure 5 Schematic diagram: (I) representative images of distal femur sections of female mice based on TRAP staining, (J) representative images of distal femur sections of female mice based on immunofluorescence staining (CD14 and CCL20, LPR1 and C1QA are used as markers of MONO 1, MONO 4 and MONO 5, respectively (marked by red arrows));

[0024] Figure 6 Figure 3 is the correlation curve fitting graph between the number of TRAP-positive cells (osteoclasts) and the number of MONO 1, MONO 4, and MONO 5 cells based on Pearson correlation and Spearman rank correlation analysis (n = 30);

[0025] Figure 7 Schematic diagram of the t-distributed stochastic neighbor embedding (t-SNE) plot of monocytes, showing two clusters, one of which contains the MONO1, MONO4, and MONO5 subpopulations;

[0026] Figure 8 Schematic diagram showing the comparison of the proportions of MONO1 / 4 / 5 subpopulations in the control group and PMOP group;

[0027] Figure 9 Heat maps showing gene expression profiles of the two monocyte populations;

[0028] Figure 10 Figure 1 shows KEGG enrichment analysis of upregulated genes in the MONO1 / 4 / 5 subgroup, highlighting significantly enriched pathways.

[0029] Figure 11 Schematic diagram of TRAP staining of cells on days 2, 4, and 6;

[0030] Figure 12 Schematic diagram of the quantification of TRAP-positive cells;

[0031] Figure 13 Schematic diagram of animal experiments;

[0032] Figure 14 Representative flow cytometric images of monocyte populations;

[0033] Figure 15 Schematic diagram of flow cytometric analysis of MONO1, MONO4, and MONO5 subsets in mouse bone marrow;

[0034] Figure 16 Representative micro-CT images of trabecular bone in the proximal femoral metaphysis and schematic diagrams of quantification of bone mineral density and trabecular thickness;

[0035] Figure 17 Representative TRAP staining images of osteoclasts and schematic diagram of the quantification of the number of osteoclasts per trabecular bone surface;

[0036] Figure 18 Figure 2 shows the results of qPCR analysis of monocytes. DETAILED DESCRIPTION

[0037] The present invention is further described below by specific examples. In order to make the invention purpose, technical solution and beneficial technical effect of the present invention clearer, the present invention is further described in detail below with reference to the examples. It should be understood that the embodiments described in this specification are only for explaining the present invention and are not intended to limit the present invention.

[0038] Unless otherwise stated, all films and reagents used in the examples are commercially available or synthesized according to conventional methods and can be used directly without further treatment. The instruments used in the examples are also commercially available.

[0039] The above-mentioned features of the present invention or the features described in the embodiments may be combined in any combination. All features disclosed in this patent specification may be used in any combination, and each feature disclosed in the specification may be replaced by any alternative feature that can provide the same, equal, or similar purpose. Therefore, unless otherwise specified, the features disclosed are only general examples of equal or similar features.

[0040] Example 1: Screening the cell subpopulation most likely to differentiate into osteoclasts by single-cell sequencing

[0041] 1.1 Experimental Materials

[0042] Bone marrow cells (93,867 cells) from 10 PMOP patients and 10 controls were provided by volunteers;

[0043] 1.2 Experimental methods

[0044] Single-cell RNA sequencing was performed on both groups of bone marrow cells, supplemented by histological validation and in vitro modulation of key pathways;

[0045] 1.3 Experimental Results

[0046] Monocytes with the greatest potential to differentiate into osteoclasts were identified as nine subpopulations (MONO 1 to 9; Figure 1 The results showed that specific cell subsets—including hematopoietic stem cells, B cells, T cells, dendritic cells, neutrophils, granulocyte-macrophage progenitor cells, and erythrocytes—had elevated expression of cytokines and chemokines, which may help promote osteoclastogenesis.

[0047] Further verification showed that the enhancement of monocyte differentiation into osteoclasts was associated with the enhancement of B cell communication. + )、MONO 4(LRP1 + ) and MONO 5 (C1QA) + The above three cell subpopulations widely express genes related to osteoclast differentiation, see Appendix Figure 2 .

[0048] Example 2: Verification of the characteristics of the mononuclear cell population susceptible to osteoclast differentiation

[0049] KEGG enrichment analysis of the samples showed that the MONO 1, MONO 4 and MONO 5 subgroups were significantly enriched. Compared with the control group, more genes were expressed in these three subgroups in the PMOP group. For details, please refer to the attached Figure 2-3 Immunofluorescence detection was used to further verify the enrichment, and the results are shown in the attached Figure 4 .

[0050] The three monocyte populations identified above as susceptible to osteoclast differentiation were combined to generate related monocyte subpopulations (MONO1, MONO4, and MONO5) to study their common effects on osteoclast differentiation. Single-cell RNA sequencing analysis of bone marrow mononuclear cells revealed two distinct clusters, one of which was composed of the MONO1, MONO4, and MONO5 subpopulations (see

[15] ). Figure 7 These subpopulations were more prevalent in the postmenopausal osteoporosis (PMOP) group compared to controls, as detailed in Figure 8Heat map analysis showed that the expression level of THBS1 gene was higher in MONO1 / 4 / 5 subgroups. Figure 9 KEGG enrichment analysis showed that the upregulated genes in the MONO1 / 4 / 5 subgroup were significantly enriched in the osteoclast differentiation pathway. Figure 10 .

[0051] Example 3: Further verification of the correlation between the three screened monocyte subtypes and osteoclasts

[0052] Further immunostaining was performed on the femoral sections of mice. TRAP staining was used to mark osteoclasts, and MONO 1 (CD14 CCL20 ++ )、MONO 4(CD14 LRP1 ++ ) and MONO 5(CD14 C1QA ++ ) and perform cell counting. Figure 5 The results of correlation analysis showed that the amount of MONO 1, MONO 4 and MONO 5 was significantly positively correlated with the number of osteoclasts in the cancellous bone marrow of the proximal femur of mice ( Figure 6 ).

[0053] The above examples are about the identification of monocytes with osteoclast differentiation tendency and metabolic pathway analysis.

[0054] Example 4: Verification of the inhibitory effect of inhibitors on osteoclast differentiation

[0055] Immunofluorescence staining, ELISA, and qPCR were performed on days 2, 4, and 6 of osteoclast differentiation. Quantitative analysis showed that the inhibitors GSK2795039, MCC950 sodium, and NF-κB-IN-4 significantly reduced the fluorescence intensity and IL-1β concentration (an indicator of osteoclast differentiation activity) in the supernatant on day 6 compared with NAT treatment. Correlation analysis further showed a significant positive correlation between the fluorescence intensity of CTSK (a marker of osteoclast maturation) and the marker IL-1β / nuclear NF-κB on day 6. In addition, qPCR results showed that the inhibitors significantly suppressed the expression of key osteoclast differentiation markers, including Trap (Acp5), Ctsk, and Nfatc1, further confirming their inhibitory effects on osteoclastogenesis.

[0056] Example 5: Verification of the inhibitory effect of conditional deletion of THBS1 in monocytes on osteoclast differentiation

[0057] siRNA-mediated gene knockdown was performed for the following monocyte subset marker genes: MONO1 (CCL20), MONO4 (LRP1), MONO5 (C1QA), and the pan-subpopulation marker gene THBS1. Figure 11 TRAP staining was performed on days 2, 4, and 6. Scale bar = 20 μm. (F) Quantification of TRAP-positive cells. ****P < 0.0001, based on Student's t-test. + Comparisons of the RANKL group) are indicated by black and red asterisks, respectively, to quantify osteoclast formation. The results showed that NAT significantly promoted osteoclast differentiation, while GSK2795039 and siRNA-mediated knockdown significantly inhibited osteoclast formation before day 6. Notably, knockdown of THBS1, representing all three subpopulations, led to a more significant inhibition of osteoclast differentiation than knockdown of MONO1, MONO4, or MONO5 alone, suggesting that targeting THBS1 can more effectively inhibit osteoclastogenesis. Figure 11-12 .

[0058] Example 6: Inducing differentiation in vitro and verifying the promoting effect of activators on differentiation

[0059] Primary bone marrow mononuclear cells were isolated and induced to differentiate into osteoclasts using macrophage colony-stimulating factor (M-CSF), receptor activator of nuclear factor kappa-B ligand (RANKL), and pathway activator (NAT) or inhibitor (GSK2795039); NAT significantly promoted osteoclast differentiation.

[0060] Example 7: The effects of GSK2795039 and conditional deletion of THBS1 in monocytes (PMOP_THBS1fl / fl) were further investigated in an in vivo PMOP model.

[0061] Osteoporosis was induced by ovariectomy followed by administration of GSK2795039 or conditional knockout of THBS1 in monocytes, Figure 13 . Specifically: One week after surgery, THBS1 conditional knockout or GSK2795039 (10 mg / kg per day) was intraperitoneally injected for 40 days. Bone marrow mononuclear cells were collected, and the proportions of MONO1, MONO4, and MONO5 subpopulations (marked by CCL20, LRP1, and C1QA) were analyzed by flow cytometry. The expression of genes related to osteoclast differentiation (AKT, BLNK, RANKL, and NFATc1) and the NOD-like receptor pathway (TNF-α, IL-1β, NLRP3, NOX2) were quantified by qPCR. Flow cytometric analysis of bone marrow mononuclear cells showed that GSK2795039 or THBS1 gene deletion treatment significantly reduced the proportions of MONO1, MONO4, and MONO5 compared with untreated PMOP mice. Figure 14-15Bone marrow immunofluorescence analysis further confirmed that GSK2795039 significantly reduced the number of monocytes associated with osteoclast differentiation (MONO1, MONO4, and MONO5), as indicated by CD14 and CCL20, LRP1, and C1QA markers, respectively.

[0062] Micro-CT imaging showed that GSK2795039 treatment and conditional THBS1 knockout improved trabecular morphology in the proximal femur compared with the PMOP control group, as evidenced by increased bone mineral density and trabecular thickness. Figure 16 TRAP staining of bone sections also showed that both treatments could significantly reduce the number of osteoclasts on each bone surface. Figure 17 qPCR analysis of bone marrow mononuclear cells, *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, based on Student's t-test (n≥4 mice per group), showed that genes related to osteoclast differentiation (AKT, BLNK, RANKL, NFITc1) and genes (TNF-α, IL-1β, NLRP3, NOX2) were significantly upregulated in the PMOP group, while GSK2795039 treatment or THBS1 gene deletion could effectively reverse these upregulations, refer to Figure 18 .

[0063] The above examples show that conditional deletion of THBS1 in monocytes can inhibit osteoclast differentiation, thereby improving osteoporosis.

[0064] The various aspects of the present invention have been described above. However, it should be understood that, without departing from the spirit of the present invention, those skilled in the art may make equivalent changes and modifications thereto, which also fall within the scope of the appended claims.

Claims

1. Use of THBS1 as a drug target for screening for promoting or inhibiting osteoclastogenesis, characterized in that: When the drug can promote the expression of THBS1, the drug can promote osteoclast formation; when the drug can inhibit the expression of THBS1, the drug can inhibit osteoclast formation.

2. The use according to claim 1, wherein the drug for promoting or inhibiting osteoclastogenesis is not used for preventing or treating tumors.

3. Use of THBS1 inhibitors in the preparation of drugs for inhibiting osteoclastogenesis.

4. The use according to claim 3, wherein the drug for inhibiting osteoclastogenesis is not used for preventing or treating tumors.

5. Use of THBS1 activator in the preparation of drugs for promoting osteoclastogenesis.

6. The use according to claim 5, wherein the drug for promoting osteoclastogenesis is not used for preventing or treating tumors.

7. Use of the THBS1 gene as a drug target in screening drugs that promote or inhibit osteoclastogenesis, characterized in that: When the drug can promote the expression of THBS1 gene, the drug can promote osteoclast formation; when the drug can inhibit the expression of THBS1 gene, the drug can inhibit osteoclast formation.

8. The use according to claim 7, wherein the drug for promoting or inhibiting osteoclastogenesis is not used for preventing or treating tumors.

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