Application of S100A9 in preparation of osteoporosis diagnostic reagent and therapeutic drug
By using S100A9 protein as a diagnostic reagent and therapeutic drug, the multi-target targeting problem of existing osteoporosis treatment drugs has been solved, enabling early diagnosis and effective treatment, reducing adverse reactions and costs, and improving treatment outcomes.
Patent Information
- Application Number
- CN202511242765.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-02
- Publication Date
- 2025-11-21
AI Technical Summary
Existing osteoporosis treatments are unable to target multiple pathogenic targets simultaneously, resulting in limited efficacy and problems such as adverse drug reactions, high cost, complex synthesis, and low availability.
The concentration of S100A9 protein in blood was detected using a diagnostic reagent and diagnosed by enzyme-linked immunosorbent assay (ELISA). It was also administered locally into the bone marrow cavity as a therapeutic drug to promote osteogenic differentiation of bone marrow mesenchymal stem cells and inhibit osteoclast differentiation. The drug was prepared into an injection form for use.
It enables early diagnosis of osteoporosis, significantly improves bone density, reduces adverse drug reactions, lowers costs, and improves treatment efficacy, especially showing significant effects on bone loss caused by estrogen deficiency after oophorectomy.
Smart Images
Figure CN120992962A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of medicine, in particular to the application of S100A9 in the preparation of osteoporosis diagnostic reagents and therapeutic drugs. BACKGROUND
[0002] Osteoporosis (OP) is a chronic metabolic bone disease characterized by decreased bone mass, damaged bone microstructure, and increased bone fragility. It can be divided into primary and secondary according to the cause. Primary osteoporosis can be divided into postmenopausal osteoporosis and senile osteoporosis. Secondary osteoporosis has a clear cause, which is often due to endocrine metabolic diseases and drug effects, etc. Each year, there are 8.9 million cases of fractures worldwide due to OP, which seriously affects the physical and mental health and quality of life of patients and brings a heavy burden to the medical and health services. Due to the complexity of the pathogenesis of OP, the current treatment methods aim to promote bone formation and inhibit bone resorption to prevent fractures and improve the quality of life of patients.
[0003] Currently, the drugs for treating osteoporosis are mainly divided into two categories: one is bone formation promoters, such as parathyroid hormone-related peptide, sclerostin monoclonal antibody, etc.; the other is bone resorption inhibitors, such as cathepsin K inhibitors, RANKL monoclonal antibodies, etc. However, the occurrence and development of osteoporosis is the result of the interaction of multiple factors, and the current treatment drugs are difficult to target multiple pathogenic targets at the same time to exert more comprehensive efficacy. In addition, osteoporosis requires long-term drug treatment, which is prone to drug adverse reactions such as liver and kidney toxicity, gastrointestinal discomfort, drug resistance, etc., and these drugs have high preparation cost, complex synthesis process, limited acquisition rate, and high storage requirement. SUMMARY
[0004] The purpose of the present application is to provide the application of calcium-binding protein S100A9 in the preparation of osteoporosis diagnostic reagents and therapeutic drugs.
[0005] The application of S100A9 in the preparation of osteoporosis diagnostic reagents.
[0006] Preferably, the reagent is a reagent for detecting the concentration of S100A9 in blood; the concentration of S100A9 is positively correlated with the bone density of the patient.
[0007] Preferably, the reagent detects the concentration of S100A9 by enzyme-linked immunosorbent assay (ELISA), and the detection sample includes peripheral blood and bone marrow samples of clinical patients.
[0008] The application of S100A9 in the preparation of osteoporosis therapeutic drugs.
[0009] Preferably, the drug is an anti-osteoporosis drug that promotes osteogenic differentiation of bone marrow mesenchymal stem cells and inhibits osteoclast differentiation.
[0010] Preferably, the drug is an anti-osteoporosis drug that improves bone loss caused by estrogen deficiency after ovariectomy.
[0011] Preferably, the drug comprises S100A9, and the administration method is local administration in the bone marrow cavity.
[0012] Preferably, the drug is an injection liquid preparation of S100A9 recombinant protein dissolved in water for injection.
[0013] Preferably, the water for injection is PBS.
[0014] Preferably, the drug up-regulates the expression levels of osteogenic genes Col1a1, Alp, Runx2, Spp1, Bglap, and Sp7.
[0015] Preferably, the drug increases the expression amounts of osteogenic proteins Col1, Alp, and Runx2.
[0016] The S100 protein family is the largest subclass of calcium-binding protein families, which transduces signals by binding to calcium ions and plays an important role in cell proliferation, differentiation, and apoptosis. As a common member of the S100 protein family, S100A9 has been gradually revealed to have potential in regulating bone metabolism in recent years, involving malnutrition calcification in atherosclerosis, inhibition of peripheral blood-derived monocyte osteoclast differentiation, and the like. In addition, due to the advantages of S100A9, such as wide source, mature extraction technology, good biological activity and biological safety, and the like, it has considerable application prospects in the preparation of anti-osteoporosis drugs.
[0017] The present application first discovers that S100A9 has significant effects in the diagnosis and treatment of osteoporosis. In the present application, S100A9 has the effect of promoting bone formation, which can induce osteogenic differentiation of bone marrow mesenchymal stem cells by increasing early alkaline phosphatase activity and late osteogenic mineralization capacity, and can significantly inhibit osteoclast differentiation, thereby ultimately improving bone loss caused by estrogen deficiency after ovariectomy. Therefore, S100A9 as an effective ingredient has important significance and clinical application value in the preparation of drugs for treating osteoporosis.
[0018] The present application provides the administration dose of S100A9 as an anti-osteoporosis drug, which is injected into the bone marrow cavity by injection preparation, and plays its maximum efficacy by locally supplementing S100A9.
[0019] Advantages: Compared with the prior art, the present application has the following remarkable advantages:
[0020] The present application first discovers that S100A9 and the bone density of patients have a significant positive correlation, can be used for early diagnosis of OP; and S100A9 has a significant effect in promoting osteogenic differentiation of bone marrow mesenchymal stem cells, inhibiting osteoclast differentiation and improving estrogen deficiency osteoporosis, and has important social and economic significance in preparing drugs for treating osteoporosis. S100A9 used in the present application has been widely used in animal models of diabetes, and has high biological safety. At the same time, the synthesis method of S100A9 is mature, and intensive production has been realized, and the storage and transportation conditions are not high, which greatly reduces the cost of S100A9 as a drug. BRIEF DESCRIPTION OF DRAWINGS
[0021] Figure 1 For detection of S100A9 concentration in peripheral blood and bone marrow of clinical patients.
[0022] Figure 2 For detecting the relationship graph of the effect of S100A9 on osteogenic differentiation genes of bone marrow mesenchymal stem cells.
[0023] Figure 3 For the relationship graph of the effect of S100A9 on protein expression of osteogenic differentiation of bone marrow mesenchymal stem cells.
[0024] Figure 4 For the relationship graph of the effect of S100A9 on early ALP activity and late osteogenic mineralization ability of bone marrow mesenchymal stem cells.
[0025] Figure 5 For the relationship graph of the effect of S100A9 on osteoclast differentiation.
[0026] Figure 6 For the relationship graph of the therapeutic effect of S100A9 on ovariectomy-induced osteoporosis animal model by bone marrow cavity injection. DETAILED DESCRIPTION
[0027] The technical solutions of the present application will be further described below in combination with the drawings and examples.
[0028] Example 1
[0029] Analysis of the relationship between S100A9 and human bone density
[0030] In the bone marrow and peripheral blood samples of clinical patients, centrifuge at 1500g for 15 minutes at 4°C, obtain the supernatant, detect the concentration of S100A9 therein by ELISA, and detect the bone density of these patients at the same time, perform linear correlation analysis, evaluate the correlation between the concentration of S100A9 and the bone density, see Figure 1 , which shows that the concentration of S100A9 in human bone marrow (BM) and peripheral blood (PB) and bone density are significantly positively correlated.
[0031] Example 2
[0032] (1) Detection of the effect of S100A9 on the expression of osteogenic differentiation genes of bone marrow mesenchymal stem cells
[0033] Primary rat bone marrow mesenchymal stem cells were extracted and passaged to P1 generation and placed in a six-well plate. When the cell density reached 70%, 0, 20 ng / ml, and 100 ng / ml of S100A9 were added to the culture medium, respectively. After 24 hours, the cell culture solution was removed, and the RNA of each group of cells was extracted and reverse transcribed into cDNA using a kit. The results of fluorescence quantitative PCR detection showed that the expression levels of osteogenic genes (Col1a1, Alp, Runx2, Spp1, Bglap, Sp7) were significantly up-regulated after S100A9 treatment, as shown in Figure 2 .
[0034] (2) Detection of the effect of S100A9 on the expression of osteogenic differentiation proteins of bone marrow mesenchymal stem cells.
[0035] Primary rat bone marrow mesenchymal stem cells were extracted and passaged to P1 generation and placed in a six-well plate. When the cell density reached 70%, 0, 20 ng / ml, and 100 ng / ml of S100A9 were added to the culture medium, respectively. After 48 hours, the cell culture solution was removed, and the protein of each group of cells was extracted with RIPA lysis buffer. Western Blot detection showed that the expression of osteogenic proteins (Col1, Alp, Runx2) was significantly increased after S100A9 treatment, as shown in Figure 3 .
[0036] (3) Detection of the effect of S100A9 on the early alkaline phosphatase activity and late osteogenic mineralization capacity of bone marrow mesenchymal stem cells
[0037] Primary rat bone marrow mesenchymal stem cells were placed in a six-well plate and a twelve-well plate, respectively. When the cell density reached 70%, 0, 20 ng / ml, and 100 ng / ml of S100A9 were added to the culture medium, respectively. After 7 days, ALP staining and ALP activity determination were performed; after the culture of calcium nodules, alizarin red staining was performed, and after elution with 10% hexadecylpyridinium chloride, the liquid was transferred to a 96-well plate, and the OD value was determined at 562 nm. The results showed that S100A9 could significantly improve the early ALP activity and late osteogenic mineralization capacity of bone marrow mesenchymal stem cells, as shown in Figure 4 .
[0038] Example 3
[0039] Evaluation of the inhibitory effect of S100A9 on osteoclast differentiation.
[0040] Primary bone marrow-derived monocytes of mice were placed in a twelve-well plate, and 0, 20 ng / ml, 100 ng / ml of S100A9 was added to the culture medium, respectively. After 7 days, tartrate-resistant acid phosphatase (TRAP) detection and cytoskeleton staining were performed to evaluate the degree of osteoclast differentiation, as shown in Figure 5 , indicating that S100A9 inhibits osteoclast differentiation.
[0041] Example 4
[0042] Preparation of S100A9 injection:
[0043] Each mouse was injected with recombinant S100A9 protein through the bone marrow cavity at a concentration of 500 nM. The injection composition system included S100A9 recombinant protein and PBS, which were mixed uniformly, filtered through a 0.22 μm filter, and then injected into the OP mouse model through the bone marrow cavity to locally supplement S100A9 protein. The injection dose was 10 μL each time, and the injection was performed twice a week for a total of 4 weeks.
[0044] Example 5
[0045] Establishment of an osteoporosis model induced by ovariectomy (OVX) in mice
[0046] Ten-week-old BALB / c female mice were selected for the experiment. After anesthesia by intraperitoneal injection of 5% chloral hydrate, the back of both sides was routinely prepared and disinfected, a longitudinal incision (less than 1 cm) was made on one side of the back, then the surrounding tissue was bluntly separated after the skin was cut, and the reddish-brown cauliflower-like ovaries were found, cut off at the oviduct, and the ovaries were removed. Finally, the incision was closed layer by layer by suturing the muscle and skin. The same method was used on the other side. Penicillin was injected intramuscularly daily for 3 consecutive days. After 3 days, the mice were observed for infection, and the non-infected mice continued to the next step of the experiment.
[0047] Example 6
[0048] The mice were divided into two groups: group 1, OVX group + intramedullary injection of sterile PBS, and group 2, OVX group + intramedullary injection of S100A9 recombinant protein. The one-time dose of S100A9 injection was 500 nM, 10 μL, and the injection was performed twice a week for a total of 4 weeks. At the end of the experiment, the experimental animals were sacrificed, and the femur specimens were obtained for micro-CT scanning to evaluate the bone condition, as shown in Figure 6 , indicating that intramedullary injection of S100a9 can effectively inhibit bone loss in OP animal models. Bone mineral density (BMD), bone volume (BV), bone volume tissue volume ratio (BV / TV), trabecular bone number (Tb.N), trabecular bone thickness (Tb.Th), trabecular bone separation (Tb.Sp), and cortical bone thickness (Cortex.Th).
Claims
1. Use of S100A9 in the preparation of a diagnostic reagent for osteoporosis.
2. The use of S100A9 according to claim 1 for the preparation of a diagnostic agent for osteoporosis, characterized in that, The reagent is a reagent for detecting the concentration of S100A9 in blood.
3. Use of S100A9 in the preparation of a therapeutic drug for osteoporosis.
4. The use of S100A9 according to claim 3 for the preparation of a medicament for the treatment of osteoporosis, characterized in that, The drug is an anti-osteoporosis drug that promotes osteogenic differentiation of bone marrow mesenchymal stem cells and inhibits osteoclast differentiation.
5. The use of S100A9 according to claim 3 for the preparation of a medicament for the treatment of osteoporosis, characterized in that, The drug is an anti-osteoporosis drug that improves bone loss caused by estrogen deficiency after ovariectomy.
6. The use of S100A9 according to claim 3 for the preparation of a medicament for the treatment of osteoporosis, characterized in that, The drug comprises S100A9, and the administration method is local administration in the bone marrow cavity.
7. The use of S100A9 according to claim 3 for the preparation of a medicament for the treatment of osteoporosis, characterized in that, The drug is an injection liquid preparation composed of S100A9 recombinant protein dissolved in water for injection.
8. The use of S100A9 according to claim 7 for the preparation of a medicament for the treatment of osteoporosis, characterized in that, The water for injection is PBS.
9. The use of S100A9 according to claim 3 for the preparation of a medicament for the treatment of osteoporosis, characterized in that, The drug up-regulates the expression levels of osteogenic genes Col1a1, Alp, Runx2, Spp1, Bglap, and Sp7.
10. The use of S100A9 according to claim 3 for the preparation of a medicament for the treatment of osteoporosis, characterized in that, The drug increases the expression levels of osteogenic proteins Col1, Alp, and Runx2.