WO2020192232A1 - Application of andrographolide in inhibiting formation and activation of osteoclasts - Google Patents

Abstract

Provided is application of an osteoclast formation and/or activation inhibitor agent and medication, and further provided are a medication containing an effective dose of andrographolide, a method for inhibiting formation of osteoclasts, and a method for inhibiting the activity of a GLS promoter induced by ERRa/PGC1-β. The andrographolide can inhibit formation and activation of osteoclasts, reduce bone mass loss, and finally achieve the effect of preventing and treating bone loss diseases such as osteoporosis. Moreover, the andrographolide is a natural substance, and thus has a good therapeutic effect and small side effects.

Description

Application of andrographolide in inhibiting the formation and activation of osteoclasts Technical field

The invention belongs to the technical field of biomedicine, and specifically relates to an application of andrographolide in inhibiting the formation and activation of osteoclasts.

Background technique

Bone supports the body's muscles or organs and protects them from external shocks by surrounding the internal organs. Bone is an important part of the human body, which not only stores calcium in the body, but also stores necessary inorganic substances such as phosphorus or magnesium. The old bone matrix of the adult is removed and replaced with a new bone matrix. Through repeated resorption and destruction processes, that is, bone remodeling, the balance of bone production is maintained.

According to the existing literature, there are two types of cells involved in bone remodeling. One is osteoblasts responsible for building bone, and the other is osteoclasts responsible for destroying bone. That is, under normal conditions, the formation and maintenance of bone homeostasis depends on the dynamic balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. In the life process of an individual, this dynamic balance is affected by genetics, immunity, endocrine, metabolism and nutrition at the overall level; at the tissue cell level, it is affected by the functions of stem cells, osteoblasts, osteoclasts, and bone cells. State regulation; at the molecular level, it is regulated by key signaling molecular pathways. Under physiological conditions, all levels and links are interconnected and regulated to maintain bone homeostasis together. Any problem in any link may cause bone instability, decrease in bone density, and ultimately lead to osteoporosis.

At present, the clinical treatment of osteoporosis is mainly divided into two types: promoting bone formation and inhibiting bone resorption. The drugs that promote bone formation mainly include parathyroid hormone and its similar compounds, vitamin D and its derivatives, strontium salts, etc.; drugs that inhibit bone resorption include estrogen, calcitonin, and bisphosphonates. However, clinical studies have found that the current clinical treatment of osteoporosis is not particularly effective and has many side effects. The main side effects currently found include venous thrombosis, hypocalcemia, hyperthyroidism, esophageal and digestive tract side effects. The treatment brought great pain to the patient both physically and mentally. Because traditional Chinese medicine has the advantages of overall treatment, good curative effect, and small side effects, researchers are currently exploring how to use modern technology to screen high-efficiency and low-toxic traditional Chinese medicine to treat osteoporosis. Commonly used Chinese medicine mainly includes epimedium, psoralen, angelica, drynaria and so on.

Andrographis paniculata (Burm.f.) Nees is a dry aerial part of the Acanthaceae plant. It is a commonly used traditional Chinese medicine. It has the effects of clearing heat and detoxification, cooling blood and reducing swelling. It is clinically used for upper respiratory tract infections, acute dysentery, and gastrointestinal Treatment of inflammation, cold and fever. Andrographolide is one of the main chemical components of Andrographis paniculata. However, there are no reports about the use of andrographolide in the prevention and treatment of bone loss diseases.

technical problem

The purpose of the present invention is to overcome the above-mentioned shortcomings of the prior art, and to provide an application of andrographolide as an inhibitor of osteoclast formation and/or activation and preparation of drugs for the prevention/treatment of bone diseases to solve At present, Andrographis paniculata is often used to clear away heat and detoxify, cool blood and reduce swelling, and the effects of existing drugs for treating osteoporosis are not particularly satisfactory, and there are many technical problems with side effects.

Technical solutions

In order to achieve the above-mentioned object of the invention, one aspect of the present invention provides an application method of andrographolide. Application of the andrographolide as an inhibitor of osteoclast formation and/or activation.

Another aspect of the present invention provides the application of andrographolide in the preparation of drugs for the prevention/treatment of bone diseases.

In another aspect of the present invention, a medicine for preventing/treating bone diseases is provided. The medicine for preventing/treating bone diseases includes andrographolide in effective dose.

In another aspect of the present invention, a method for inhibiting the formation of osteoclasts is provided. The method for inhibiting the formation of osteoclasts includes the step of contacting an effective dose of andrographolide or the drug for preventing/treating bone diseases of the present invention with osteoclast precursor cells.

At the same time, it also provides a method to inhibit the activity of GLS promoter induced by ERRa/PGC1-β. The method for inhibiting the activity of the GLS promoter induced by ERRa/PGC1-β includes the step of contacting cells with an effective amount of andrographolide or the drug for preventing/treating bone diseases of the present invention.

Beneficial effect

Compared with the prior art, the application of andrographolide of the present invention has the following beneficial effects:

Experiments have proved that the andrographolide has a relatively strong function of inhibiting at least one of osteoclast formation and activation. Therefore, after the andrographolide is used as an inhibitor of osteoclast formation and/or activation, it can It can effectively inhibit the differentiation, formation and activation of osteoclasts.

After the andrographolide of the present invention is used in the preparation of drugs for the prevention/treatment of bone diseases, and the drugs for prevention/treatment of bone diseases prepared based on its application can be used to inhibit the differentiation and maturation of osteoclasts, and can significantly reduce The expression of genes related to osteoclast differentiation can improve bone loss, and ultimately play a role in preventing bone loss diseases such as osteoporosis. In addition, because andrographolide is a natural substance, it has good curative effect and small side effects.

Description of the drawings

The present invention will be further described below in conjunction with the accompanying drawings and embodiments. In the accompanying drawings:

Figure 1 shows the stained photos and histograms of osteoclast numbers after different concentrations of andrographolide inhibited the osteoclast differentiation of mouse bone marrow monocytes. Figure 1-A is the negative control group after TRAP staining on the 5th day of induction. (Control group), positive control group (RANKL group), andrographolide 0.5μM, 1μM, 2μM and 5μM group photos; Figure 1-B is a histogram of the number of osteoclasts on the 5th day of induction; and Figure 1-B ***P<0.001vs Control, ##P<0.01vs RANKL;

Figure 2 is a graph showing the level of andrographolide inhibiting the expression of genes related to osteoclast differentiation. Figure 2-A is a bar graph showing the expression levels of osteoclast ERRa genes in the negative control group, positive control group and 0.2μm andrographolide group. Figure 2-B is a histogram of the expression levels of osteoclast PGC-1β gene in the negative control group, positive control group and 0.2μm andrographolide group; Figure 2-C is the negative control group, positive control group and 0.2μm andrographolide The histogram of the expression level of osteoclast GLS gene in the lactone group; Figure 2-D is the histogram of the expression level of osteoclast TRAP gene in the negative control group, the positive control group and the 0.2 μm andrographolide group; Figure E is negative The histogram of the control group, the positive control group and the 0.2μm andrographolide group on the expression level of osteoclast CTSK gene; and *P<0.05, **P<0.01, ***P<0.001vs in Figure 2-AE Con,#P<0.05,##P<0.01vs RANKL;

Figure 3 is a bar graph of andrographolide inhibiting ERRa/PGC1-β-induced GLS promoter activity, where ***P<0.001vs Con, #P<0.05,##P<0.01,## #P<0.001vs DMSO;

Figure 4 shows the enzyme activity of andrographolide on osteoporotic bone loss, bone density, and serum type 1 collagen carboxy-terminal peptide in male mice induced by high-fat diet; Figure 4-A is the normal group (CD) , High-fat group (HFD) and high-fat plus andrographolide group (HFD+AP) mouse bone photos; Figure 4-B is the normal group (CD), high-fat group (HFD) and high-fat plus andrographolide group (HFD+AP) Bone density, bone volume fraction, number of bone trabeculae and separation of trabecular bone in mice; Figure 4-C is the normal group (CD), high fat group (HFD) and high fat plus andrographis The enzyme activity diagram of type 1 collagen carboxyl terminal peptide in the serum of ester group (HFD+AP) mice; and in Figure 4-AC (*P<0.05, **P<0.01 vs CD, #P<0.05 vs HFD)

Figure 5 is a graph showing the effects of andrographolide on ovarian removal-induced osteoporotic bone loss, bone density, and enzyme activity of type 1 collagen carboxy-terminal peptide in serum; Figure 4-A shows the sham operation group (sham) , Ovarian removal group (OVX) and ovarian removal plus andrographolide group (OVX+AP) mouse bone photos; Figure 4-B is the sham operation group (sham), ovarian removal group (OVX) and ovarian removal plus andrographolide Group (OVX+AP) mice bone density, bone volume fraction, bone trabecular bone number and bone trabecular separation diagram; Figure 4-C is the sham operation group (sham), ovarian removal group (OVX) and ovarian removal plus Enzyme activity diagram of type 1 collagen carboxyl terminal peptide in serum of andrographolide group (OVX+AP) mice; and (**P<0.01,***P<0.001vs sham,#P< in Figure 5-AC 0.05vsOVX).

Embodiments of the invention

In order to make the technical problems, technical solutions, and beneficial effects to be solved by the present invention clearer and clearer, the present invention will be further described in detail below in conjunction with embodiments and attached tables. It should be understood that the specific embodiments described here are only used to explain the present invention, but not to limit the present invention.

Explanation of the special name:

Osteoclast (also known as bone-resorbing cells, OC): is a kind of bone-resorbing cells, derived from the mononuclear macrophage lineage, is a multinucleated giant cell with bone resorption function, and is the only bone in the human body Absorb cells to perform the function of bone resorption. Insufficiency or increase in its quantity and activity can lead to osteosclerosis, osteoporosis and other osteolytic diseases, respectively. Highly expressed tartrate resistant acid phosphatase and cathepsin K are the main signs of osteoclasts.

ERRα: It belongs to estrogen-related receptors, a type of orphan nuclear hormone receptor with continuous activation transcription activity.

Glutaminase (GLS): Located in the cytoplasm, mitochondrial matrix and mitochondrial membrane, it is the first catalytic enzyme in the main metabolic pathway of glutamine. It requires inorganic phosphate and ammonium ions as activators to catalyze the hydrolysis of glutamine It becomes glutamic acid and ammonia, and then is converted into α-ketoglutarate to enter the tricarboxylic acid cycle. It is completely oxidized to generate a large amount of ATP, which becomes the other required substrates and the carbon source of fat in the TCA cycle.

The inventors of the present invention have discovered based on a large number of studies that andrographolide has the ability to inhibit the formation and activation of osteoclasts. Based on this, the embodiments of the present invention provide applications of andrographolide in the following related aspects.

In one aspect, the embodiments of the present invention provide the use of andrographolide as an inhibitor of osteoclast formation and/or activation. According to related experiments constructed by the inventor, as an active ingredient, the andrographolide can effectively inhibit the formation and activation of osteoclasts.

As in one example, the andrographolide has the function of inhibiting the differentiation and maturation of osteoclasts. Therefore, the andrographolide can be used as an inhibitor of osteoclast formation, and further can be used for Preparation of related drugs to inhibit the formation of osteoclasts. This effectively inhibits the differentiation and maturation of osteoclasts, thereby balancing osteoclasts and osteocytes, thereby improving corresponding bone diseases.

In another embodiment, the andrographolide has the function of inhibiting the expression of genes related to osteoclast differentiation. Therefore, the andrographolide can be used for osteoclast differentiation genes TRAP, CTSK, ERRa, PGC-1β, Gls expression inhibitor, and further can be used to prepare drugs for inhibiting the expression of osteoclast differentiation genes TRAP, CTSK, ERRa, PGC-1β, and Gls. This effectively inhibits the differentiation of osteoclasts, thereby balancing osteoclasts and bone cells, thereby improving the corresponding bone diseases.

In another embodiment, the andrographolide has the function of inhibiting the activity of the GLS promoter induced by ERRa/PGC1-β. Among them, ERRα is currently considered to mainly interact with peroxisome proliferator receptor γ coactivator 1α, 1β (PGC-1α, PGC-1β) to jointly regulate the transcription of genes in the oxidative phosphorylation pathway, thereby Regulates the balance of energy metabolism. Recent studies have also shown that ERRα may have an important regulatory role in the process of osteoclast formation and bone resorption. Glutaminase (GLS) can not only catalyze the hydrolysis of glutamine into glutamate and ammonia, and then convert it into α-ketoglutarate to enter the tricarboxylic acid cycle, complete oxidation to generate a large amount of ATP, and become other requirements in the TCA cycle As the carbon source of substrate and fat, the metabolites produced in the process of glutamine metabolism can be used as an important material basis for cell proliferation and differentiation. There have been reports that ERRa can regulate bone by regulating the expression of GLS. However, the role of GLS in the process of osteoclast differentiation is not yet known. Therefore, the inventors, through the corresponding experiments constructed, effectively confirmed that andrographolide as described above has the function of inhibiting the GLS promoter activity signal induced by ERRa/PGC1-β, specifically the clinical experimental model constructed in Example 3 below, It is effectively confirmed that andrographolide can effectively inhibit the GLS promoter activity mediated by ERRa/PGC-1β, and it is also confirmed that the GLS signal pathway mediated by ERRa/PGC-1β is involved in the process of osteoclast differentiation. Therefore, It is further confirmed that andrographolide has the function of inhibiting osteoclast differentiation. Thus, the andrographolide can be used as an inhibitor of ERRa/PGC1-β-induced GLS promoter activity signal pathway, and further can be used to prepare related drugs that inhibit osteoclast formation. This effectively inhibits the differentiation and maturation of osteoclasts, thereby balancing osteoclasts and osteocytes, thereby improving corresponding bone diseases.

In another embodiment, the andrographolide has the function of inhibiting the activation of osteoclasts. Therefore, the andrographolide can be used to inhibit osteoclast activation inhibitors, and further can be used to prepare related drugs that inhibit osteoclast activation. This effectively inhibits the activation of osteoclasts, thereby inhibiting the bone absorption effect of osteoclasts, thereby improving the corresponding bone diseases.

Based on the function and effect of andrographolide in the field of inhibiting osteoclasts as described above, andrographolide can be used in the preparation of drugs for the prevention/treatment of bone diseases. In this way, in the prepared drugs for the prevention/treatment of bone diseases, the andrographolide is an effective ingredient that inhibits the formation or/and activation of osteoclasts, which can inhibit the formation of osteoclasts, including differentiation and maturation, thereby achieving Balance between bone cells and osteoclasts, thereby improving the corresponding bone diseases; or directly inhibit the activation of osteoclasts, thereby inhibiting the bone resorption of osteoclasts, thereby improving the corresponding bone diseases.

In addition, in one embodiment, the bone diseases described above include primary osteoporosis, secondary osteoporosis, rheumatoid arthritis, multiple myeloma, Paget's disease, and hypercalcemia of malignant tumors. At least one of osteogenesis imperfecta and alveolar bone loss.

Secondly, the andrographolide in the above embodiments can be extracted from the original medicinal material of Andrographis paniculata according to the existing conventional methods. Of course, a new extraction method can also be designed to extract from the original medicinal material of Andrographis paniculata.

On the other hand, based on the function of andrographolide described above in inhibiting osteoclasts, embodiments of the present invention also provide a medicine for preventing/treating bone diseases. The medicine includes an effective amount of active ingredients for preventing/treating bone diseases. Wherein, the active ingredient includes andrographolide. Of course, the active ingredient may also include other active ingredients that can effectively inhibit osteoclast-related properties. The "effective" mentioned here refers to the prevention or treatment of bone diseases alone. The clinically effective component can also be a component that can improve andrographolide to prevent or treat bone diseases after being compounded with andrographolide. The "effective dose" refers to an effective amount capable of preventing or treating bone diseases, and refers to an amount of andrographolide sufficient to show benefits or clinical significance to an individual. Those skilled in the art will understand that the actual amount or dose administered and the time course of administration will depend on the nature and severity of the disease to be prevented or treated, the age and general condition of the subject to be prevented or treated, and the administration. Medicine method etc. As in one embodiment, the effective dose of andrographolide is 30 mg/kg-100 mg/kg, specifically, the effective dose for mouse clinical experiments is 30 mg/kg-100 mg/kg.

In addition, the drug for preventing/treating bone diseases may further include a pharmaceutically acceptable carrier component of andrographolide. The carrier component of the pharmaceutically acceptable andrographolide may be a corresponding carrier of a corresponding dosage form prepared according to the administration mode of the drug for treating tumors. As in one embodiment, the carrier includes but not only corn oil. As long as it is a carrier that can support the andrographolide and facilitate its stability and absorption, it is within the scope of the disclosure of the present invention. Therefore, according to the choice of the carrier, the dosage form of the drug may be at least one of an oral dosage form, an injection dosage form and an external dosage form.

Therefore, since the drug for preventing/treating bone diseases contains the above andrographolide, the drug can effectively inhibit the formation and activation of osteoclasts. Specifically as described above, the andrographolide can significantly inhibit the differentiation and maturation of osteoclasts; inhibit the expression of osteoclast differentiation genes TRAP, CTSK, ERRa, PGC-1β, and Gls; inhibit ERRa/PGC1-β Induced GLS promoter activity; In addition, it can directly inhibit the activation of osteoclasts, thereby inhibiting the bone resorption of osteoclasts, improving bone loss, and ultimately preventing osteoporosis. In addition, because andrographolide is a natural substance, it has good curative effect, small side effects and safety.

In another aspect, based on the application of andrographolide described above and the drug containing the andrographolide, in one embodiment, the embodiment of the present invention provides a method for inhibiting the formation of osteoclasts. The method includes the step of contacting an effective dose of the andrographolide or the drug for preventing/treating bone diseases described above with osteoclast precursor cells. The method can effectively inhibit the differentiation and maturation of osteoclast precursor cells into osteoclasts. Wherein, the osteoclast precursor cells can be but not only monocytes.

In another embodiment, the embodiment of the present invention provides a method for inhibiting the activity of GLS promoter induced by ERRa/PGC1-β. The method includes the step of contacting cells with an effective dose of andrographolide or the drug for preventing/treating bone diseases described above. The method can effectively inhibit the expression of genes related to osteoclast differentiation, thereby inhibiting the formation of osteoclasts.

In conjunction with specific examples, the application of andrographolide for inhibiting the formation and activation of osteoclasts will be described in further detail.

Example 1: The effect of andrographolide on the osteoclast differentiation of mouse bone marrow monocytes

The experimental method is as follows:

1) Take C57BL\6 mice, remove their necks and put them to death, soak them in 75% alcohol for 5 minutes;

2) Separate the long bones of the hind limbs and the humerus of the forelimbs under aseptic conditions;

3) Remove the metaphysis from the ultra-clean table, and use a sterile syringe to draw a-MEM medium and gently wash the bone marrow cavity repeatedly until the bone marrow cavity turns white;

4) Centrifuge at 1200r/min for 5min after filtration with 100um cell filter;

5) Discard the supernatant, add 10 times the volume of sterile red blood cell lysate, mix by pipetting, lyse on ice for 5 min, centrifuge at 1000 r/min for 5 min, discard the red supernatant to remove red blood cells;

6) Resuspend the pellet in serum-free a-MEM medium and wash it twice;

7) Resuspend the cells in a-MEM medium containing 10% FBS and 1% PS, inoculate them in a 25 cm ² culture flask, and place them in a 37°C, 5% CO ₂ incubator overnight;

8) Collect the supernatant, wash the culture flask twice with serum-free a-MEM medium, centrifuge at 1200r/min for 5min, and resuspend in a-MEM medium containing 10% FBS, 1% PS, 30ng/mL M-CSF After the cells were seeded in 96-well plates at 3×10 ⁴ /mL.

9) The experiment was divided into the negative control group (Control group) with only 40ng/mL M-CSF, the positive control group (RANKL group) and the drug group both added 40ng/mL M-CSF and 100ng/mL RANKL, and the drug group added Andrographis at the same time Lactone 0.5μM, 1μM, 2μM and 5μM, 2 replicates in each group, change the medium every 3 days, TRAP staining on the 5th day of induction, observe and take pictures under a microscope, and count as TRAP positive and more than 3 nuclei .

The results are shown in Figure 1. Figure 1-A shows the negative control group (Control group), positive control group (RANKL group), andrographolide 0.5μM, 1μM, 2μM and 5μM after TRAP staining on the 5th day of induction. Group photomicrograph; Figure 1-B is a histogram of the number of osteoclasts on the fifth day of induction. It can be seen from Figure 1 that within the effective dose concentration, andrographolide concentration-dependently inhibits the differentiation of monocytes into osteoclasts.

Example 2: Effect of Andrographolide on the expression of genes related to osteoclast differentiation

The experimental method is as follows:

According to the operation in Example 1, mouse primary monocytes were extracted for osteoclast differentiation, and the cells were seeded in a 24-well plate at 4×10 ⁵ /mL. The experiment was grouped as follows: the negative control group (Control group) only added 40ng /mL M-CSF, 40ng/mL M-CSF and 100ng/mL RANKL were added to the positive control group (RANKL group) and the drug group, and 2μM andrographolide was added to the drug group at the same time. Each group had 3 replicate holes. After induction for 3 days , Discard the medium, wash with PBS, add Trizol to lyse the cells, extract the total RNA in the cells, and prepare cDNA by reverse transcription. The system is shown in the following expression 1:

Table 1

Reverse transcription PCR conditions:

a. Reverse transcription reaction: 42℃ 60min

b. Reverse transcriptase inactivation: 70℃ 5min

4°C

Realtime PCR uses a 20μl reaction system, and prepare the PCR reaction solution according to the components in Table 2 (the reaction solution is prepared on ice). The primer sequence is shown in Table 3 below:

Table 2

table 3

Real time PCR reaction conditions are as follows:

a. Reverse transcription reaction: 95°C 30s

b. PCR reaction: 40cycles 95°C 5s

At 60°C 34s

c. Dissolution curve 95°C 15s

60°C 1min

95°C 15s

The results are shown in Figure 2. It can be seen from Figure 2 that 2μM andrographolide can significantly inhibit the expression of osteoclast-related genes TRAP, CTSK, ERRa, and PGC-1β.

Example 3. Detecting the effect of andrographolide on the activity of GLS promoter induced by ERRa/PGC1-β

The experimental method is as follows:

Inoculate 293T cells into a 96-well plate at 2×10 ⁵ cells/mL. After the cells are attached, the pGL3 reporter plasmid containing the GLS gene promoter and the ERRa and PGC-1β overexpression plasmids are co-transfected into the cells. The phRL-TK plasmid was transfected as the internal reference plasmid, and andrographolide of different drug concentrations was added 6 hours after transfection, and detection was performed 18 hours later. The detection method is as follows:

1) Discard the culture medium, add 100μl PBS to the wells of the 96-well plate for cell inoculation to wash once, operate gently, and aspirate the PBS as much as possible;

2) After washing, add 50μl 1×PLB, at room temperature, shake on a shaker for 20 minutes, observe under the microscope to ensure that the cells are completely lysed;

3) Turn on the platform computer and GloMax 96 microplate luminescence detector, open the software, and set the program;

4) Aspirate 20μl of lysate from each hole of the cell well to the 96-well detection whiteboard, add 30μl of luciferase substrate to the whiteboard holes in the order of detection, and put the whiteboard into the GloMax 96-well plate luminescence detector for testing Luciferase activity;

5) After the test, take out the white board, add 30μl of 1×Stop&Glo substrate sequentially, and put it back into the instrument to measure the internal reference phRL-TK Renilla luciferase activity.

The results are shown in Figure 3. It can be seen from Figure 3 that andrographolide can effectively inhibit the GLS promoter activity mediated by ERRa/PGC-1β, indicating that this signaling pathway is involved in the process of osteoclast differentiation.

Example 4. Detecting the effect of andrographolide on the osteoporosis model of male mice induced by high-fat diet

The experimental method is as follows:

Six-week-old male C57BL/6 mice were divided into 3 groups, ordinary group (CD), high-fat group (HFD) and high-fat plus andrographolide group (HFD+AP). The general group was given regular feeding, and the high-fat group and the high-fat drug group were given a high-fat diet. After 7 weeks, the high-fat medicated group was given a high-fat diet and andrographolide 50 mg/kg was administered daily. After 18 weeks, the mice were sacrificed, and blood and bone samples were collected for testing. The imaging characteristics of the mice were analyzed by MicroCT, and the results are shown in Figure 4. It can be seen from Figure 4 that andrographolide can reverse the bone loss of osteoporosis induced by high-fat diet, as shown in Figure 4-A. Through the analysis of bone-related parameters, the protective effect of andrographolide on bone loss is mainly reflected in increasing the bone density, bone volume fraction, bone trabecular number and reducing the separation of trabecular bone in mice, as shown in Figure 4-B Show. Andrographolide can also significantly reduce the enzyme activity of the carboxyl terminal peptide of type 1 collagen in the serum and reduce the osteoclast absorptive capacity, as shown in Figure 4-C.

Example 5. Testing the effect of andrographolide on the osteoporosis model of female mice induced by bilateral ovarian removal

The experimental method is as follows:

Seven-week-old female C57BL/6 mice were randomly divided into 3 groups, sham operation group (sham), ovarian removal group (OVX) and ovarian removal plus andrographolide group (OVX+AP). After bilateral incisions were made on the back of the mice, the ovarian removal group and the ovarian removal plus medicine group had their ovaries removed, and the sham operation group only had bilateral incisions. Two weeks after the operation, the ovarian removal plus medicine group was given andrographolide 30 mg/kg per day. Three weeks later, the mice were sacrificed, and blood and bone samples were collected for testing. The imaging characteristics of the mice were analyzed by MicroCT, and the results are shown in Figure 5. It can be seen from Figure 5 that andrographolide reduces bone loss in mice induced by ovarian removal, as shown in Figure 5-A. Through the analysis of bone-related parameters, the protective effect of andrographolide on bone loss is mainly reflected in increasing the bone density, bone volume fraction, number of bone trabeculae and reducing the degree of trabecular bone separation, as shown in Figure 5-B. Andrographolide can also significantly reduce the enzyme activity of the bone resorption marker enzyme type 1 collagen carboxyl terminal peptide, as shown in Figure 5-C.

The above-mentioned embodiments are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in this Within the scope of protection of the invention.

Claims (10)

1. The use of andrographolide as an inhibitor of osteoclast formation and/or activation.
2. The application according to claim 1, wherein the andrographolide is used as an inhibitor of osteoclast differentiation and maturation.
3. The application according to claim 1, wherein the andrographolide is used as an inhibitor of the expression of osteoclast differentiation genes TRAP, CTSK, ERRa, PGC-1β, and Gls.
4. The application according to claim 1, wherein the andrographolide is used as an inhibitor of GLS promoter activity induced by ERRa/PGC1-β.
5. Application of andrographolide in the preparation of medicines for preventing/treating bone diseases.
6. The application according to claim 5, characterized in that: the bone diseases include primary osteoporosis, secondary osteoporosis, rheumatoid arthritis, multiple myeloma, Paget's disease, and malignant tumors. At least one of calcemia, osteogenesis imperfecta, and alveolar bone loss.
7. A medicine for preventing/treating bone diseases, which is characterized in that it includes andrographolide in an effective dose.
8. The medicine according to claim 7, wherein the dosage form of the medicine is at least one of oral dosage form, injection dosage form and external dosage form.
9. A method for inhibiting the formation of osteoclasts, which is characterized in that it comprises the step of contacting an effective dose of andrographolide or the drug of any one of claims 7-8 with osteoclast precursor cells.
10. A method for inhibiting the activity of GLS promoter induced by ERRa/PGC1-β, which is characterized in that it comprises the step of contacting cells with an effective amount of andrographolide or the drug of any one of claims 7-8.

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