Application of protostemonine in preparation of medicine for inhibiting osteoclast activity and / or osteoclast formation

Abstract

The invention discloses application of protostemonine in preparation of a medicine for inhibiting osteoclast activity and / or osteoclast formation, and belongs to the technical field of biological medicine. The invention particularly discloses application of protostemonine in preparation of medicines for inhibiting osteoclast activity, inhibiting osteoclast generation, treating bone destruction diseases and the like. Research finds that protostemonine has an obvious inhibiting effect on proliferation and differentiation activity of osteoclasts, the IC50 value of protostemonine is 6.83 mg / mL, and protostemonine is expected to be applied to preparation of drugs for treating and / or preventing osteoporosis and other bone destruction diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and in particular relates to a use of protostemonine in the preparation of a drug for inhibiting osteoclast activity and / or osteoclast formation. Background Art

[0002] Osteoclasts (OCs) are special cells formed by the fusion of monocyte / macrophage hematopoietic lineage precursor cells. They are the only cells in the human body that have bone resorption function. The stability of bone metabolism requires a dynamic balance and coupling between bone resorption mediated by osteoclasts (OCs) and bone formation mediated by osteoblasts (OBs). In healthy individuals, the two maintain a functional balance, and once the balance is broken, bone diseases may occur.

[0003] Osteoclasts are the only cells in the body responsible for bone absorption. Since differentiated and mature osteoclasts can absorb bone, their overactivation can cause bone loss, osteoporosis, fracture pain, secondary reactive bone hyperplasia, and even disability. There are many types of bone destruction diseases related to osteoclast overactivation, and the causes are also very diverse. Even a disease can have multiple pathogenic factors, and some of them are refractory diseases with unclear or diversified causes. However, drugs that directly target osteoclasts and inhibit osteoclast differentiation or bone absorption function can bypass the cause of the disease, directly improve the symptoms of bone destruction in related diseases, and effectively reduce the patient's fractures, pain and other related suffering. Therefore, osteoclast differentiation inhibitors have become a class of drugs in recent years, which can be used for various osteoclast overactivation-related bone destruction diseases such as postmenopausal osteoporosis. Summary of the invention

[0004] In view of the above-mentioned deficiencies in the prior art, the present invention provides a use of protostemonine in the preparation of a drug for inhibiting osteoclast activity and / or osteoclast formation, which can effectively inhibit osteoclast activity and avoid its over-activation.

[0005] To achieve the above purpose, the technical solution adopted by the present invention to solve the technical problem is:

[0006] The purpose of the present invention is to provide the use of protostemonine in preparing a medicine for inhibiting osteoclast activity.

[0007] Another object of the present invention is to provide the use of protostemonine in the preparation of a drug for inhibiting osteoclastogenesis.

[0008] Another object of the present invention is to provide the use of protostemonine in preparing medicines for treating bone destruction diseases.

[0009] Further, bone destructive diseases include osteoporosis, Paget's disease, osteolysis, hypercalcemia of malignancy, osteogenesis imperfecta, osteoarthritis and alveolar bone loss.

[0010] Another object of the present invention is to provide a drug combination for treating bone destruction diseases, which contains stemonae as an active ingredient.

[0011] Furthermore, protostemonine comes from Echinops cyanidis.

[0012] Further, bone destructive diseases include osteoporosis, Paget's disease, osteolysis, hypercalcemia of malignancy, osteogenesis imperfecta, osteoarthritis and alveolar bone loss.

[0013] Another object of the present invention is to provide a drug for preventing and / or treating osteoporosis, which contains stemonae as an active ingredient.

[0014] Furthermore, the medicine is an injection, tablet or capsule.

[0015] Beneficial effects of the present invention:

[0016] The present invention found that protostemonine has a significant inhibitory effect on the proliferation and differentiation activity of osteoclasts. 50 The value is 6.83 mg / mL, and it is expected to be applied to the preparation of drugs for the treatment and / or prevention of bone-destructive diseases such as osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 IC of protostemonine 50 Value detection;

[0018] Figure 2 This is the TRAP staining picture;

[0019] Figure 3 The effect of prostemonine on the proliferation activity of human osteoclasts;

[0020] Figure 4 This is the effect of protostemonine on the expression of OPG, TRACP-5b and OCN in human osteoclasts. DETAILED DESCRIPTION

[0021] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0022] The original Stemonine used in the present invention comes from the Mongolian medicine Echinops cylindrica and is purchased from Chengdu Gelipu Biotechnology Co., Ltd.

[0023] Example 1

[0024] 1. Osteoclast IC 50 and IC 20 Detection

[0025] (1) Human osteoclasts in the logarithmic growth phase of each experimental group were trypsinized, resuspended in complete medium into a cell suspension and counted.

[0026] (2) Determine the cell density for plating according to the cell growth rate (mostly 2000 cells / well), 100 μl per well, 3-5 wells per group, and the number of plates to be plated is determined according to the experimental design (e.g., if the test is for 5 days, 5 96-well plates should be plated)

[0027] (3) After uniform plating, wait for the cells to completely settle down, and observe the cell density of each experimental group under a microscope. If the density is uneven, fix one group, fine-tune the amount of cells in other groups and plate them again (for example: if it is found that there are more cells in the Con group, reduce the amount of cells and plate them again), and culture them in a cell culture incubator.

[0028] (4) Starting from the second day after plating, add 10 μL of CCK-8 reagent to each well 2 to 4 hours before the end of culture. No need to change the medium.

[0029] (5) After 4 hours, the 96-well plate was placed on an oscillator for 2-5 minutes and the OD value was measured at 450 nm using a microplate reader.

[0030] (6) Data analysis.

[0031] according to Figure 1 The results of the test showed that the half inhibitory concentration (IC 50 ) is 6.83 mg / mL, and its IC 20 It is 5.28mg / mL.

[0032] 2. TRAP staining

[0033] The staining experiment was carried out using a tartaric acid-resistant acid phosphate staining kit, with protostemonine, vitexin and privet glycoside as the experimental group and NC as the control. The results are shown in Figure 2 .

[0034] like Figure 2As shown in the figure, compared with the control group, no obvious specific staining was observed after treatment with vitexin and privet glycosides, indicating that vitexin and privet glycosides did not change the performance of cells in tartaric acid-resistant acid phosphatase staining, while after treatment with prostemonine, obvious purple granular staining could be seen in the cells, which may be a manifestation of tartrate-resistant acid phosphatase (TRAP) activity. This indicates that prostemonine may have an effect on certain functions of cells, resulting in changes in TRAP activity.

[0035] Example 2

[0036] 1. Effect of Stemonine on Cell Proliferation

[0037] The effect of protostemonine on the proliferation of human osteoclasts was detected by CCK8 method. Figure 3 , the horizontal axis unit is day, Figure 3 Vitexin is human osteoclasts + vitexin-IC 20 ;Protostemonine is human osteoclasts + protostemonine-IC 20 ;Qligustroside is human osteoclasts + Ligustrum lucidum-IC 20 .

[0038] like Figure 3 As shown, after adding the three groups of drugs for 48 hours, each group of drugs could inhibit the proliferation of human osteoclasts and had a significant effect on their proliferation level, among which protostemonine had the most significant effect on the proliferation activity of human osteoclasts.

[0039] 2. WB detection

[0040] Western blotting was used to detect the expression of OCN (osteocalcin), OPG (osteoprotegerin) and TRACP-5b (tartrate-resistant acid phosphatase) in human osteoclasts treated with protostemonine, vitexin and ligusticum glycosides. The results are shown in Figure 4 .

[0041] like Figure 4 As shown in the results, among the treatments of protostemonine, vitexin and ligusticum glycoside, protostemonine had the most significant inhibitory effect on OCN and the most significant increase in the expression of OPG and TRACP-5b, indicating that it can reduce bone resorption, maintain and increase bone quality, and further reverse osteoporosis and improve bone health.

[0042] Finally, it should be noted that the above specific implementation methods are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to examples, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solution of the present invention, which should be included in the scope of the claims of the present invention.

Claims

1. Use of protostemonine in the preparation of drugs for inhibiting osteoclast activity.

2. Use of protostemonine in the preparation of drugs for inhibiting osteoclastogenesis.

3. Use of protostemonine in the preparation of medicines for treating bone destruction diseases.

4. The use according to claim 3, characterized in that Bone-destructive diseases include osteoporosis, Paget's disease, osteolysis, hypercalcemia of malignancy, osteogenesis imperfecta, osteoarthritis, and alveolar bone loss.

5. A drug combination for treating bone destruction diseases, characterized in that: The active ingredient is stemonae.

6. The pharmaceutical combination according to claim 5, characterized in that The protostemonine is derived from Echinops cyanidis.

7. The pharmaceutical combination according to claim 5, characterized in that Bone-destructive diseases include osteoporosis, Paget's disease, osteolysis, hypercalcemia of malignancy, osteogenesis imperfecta, osteoarthritis, and alveolar bone loss.

8. A drug for preventing and / or treating osteoporosis, characterized in that: The active ingredient is stemonae.

9. The drug according to claim 8, characterized in that The medicine is in the form of injection, tablet or capsule.

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