Application of Wip1 inhibitor in preparation of medicine for treating osteoporosis

Abstract

The invention belongs to the technical field of biological medicine, and discloses an application of a Wip1 inhibitor in preparation of a medicine for treating osteoporosis, the Wip1 inhibitor comprises one of GSK2830371, CCT007093 and SL-176, and the Wip1 inhibitor realizes treatment of osteoporosis by inhibiting osteoclast differentiation and inhibiting osteoclast bone resorption function.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of a Wip1 inhibitor in preparation of a drug for treating osteoporosis. BACKGROUND

[0002] Osteoporosis is a systemic bone metabolic disease characterized by bone mass loss, bone tissue microstructure destruction and increased risk of fracture, and is particularly common in postmenopausal women. Excessive differentiation and activation of osteoclasts are considered to be one of the important pathological mechanisms leading to osteoporosis. Existing anti-osteoporosis drugs are mostly focused on inhibiting osteoclast absorption or promoting osteogenesis, but still have problems such as limited efficacy and large side effects, and new targets and intervention means are urgently needed.

[0003] Wip1 (wild-type p53-induced phosphatase 1), also known as PPM1D, is a phosphatase involved in cell cycle, DNA damage response and inflammation regulation, and its role in bone metabolism is not clear. Currently, there is no literature reporting the exact function of Wip1 in osteoclast differentiation and osteoporosis occurrence. SUMMARY

[0004] The purpose of the present application is to provide application of a Wip1 inhibitor in preparation of a drug for treating osteoporosis. The Wip1 inhibitor inhibits osteoclast differentiation and osteoclast bone absorption function, thereby achieving treatment of osteoporosis.

[0005] In order to achieve the above-mentioned purpose, the present application adopts the following technical solutions: In a first aspect, the present application provides application of a Wip1 inhibitor in preparation of a drug for treating osteoporosis.

[0006] In the above technical solution, the Wip1 inhibitor includes one of GSK2830371, CCT007093 and SL-176.

[0007] The chemical structural formula of GSK2830371 is as follows:

[0008] The chemical structural formula of CCT007093 is as follows:

[0009] The chemical structural formula of SL-176 is as follows:

[0010] In the above technical solution, the Wip1 inhibitor inhibits osteoclast differentiation and osteoclast bone absorption function, thereby achieving treatment of osteoporosis.

[0011] In a second aspect, the present invention provides a drug for treating osteoporosis, which is composed of a Wipl inhibitor at an effective dose. The drug can be a single component or a composition comprising an effective dose of the Wipl inhibitor. The composition can include pharmaceutically acceptable excipients.

[0012] In the above technical solution, the auxiliary materials include at least one of excipients, fillers, adhesives, wetting agents, absorption promoters, surfactants, lubricants, stabilizers, flavoring agents, sweeteners, and pigments.

[0013] The present invention has the following beneficial effects: It has discovered for the first time the key regulatory role of Wip1 in osteoclast differentiation, providing a new molecular target for studying the pathogenesis of osteoporosis. It has also been shown for the first time that Wip1 inhibitors can be used to inhibit osteoclast function and treat osteoporosis, revealing a new application for the treatment of bone metabolic diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 This is an experimental diagram of the changes in Wip1 mRNA and protein levels during osteoclast differentiation detected by qRT-PCR and WB; Figure 2 This is an experimental diagram showing the toxic effects of GSK2830371 on RAW264.7 cell lines and BMM cells within the effective concentration range; Figure 3 This is an experimental diagram of GSK2830371 inhibiting osteoclast differentiation in vitro; Figure 4 This is an experimental diagram showing the inhibition of osteoclast bone resorption by GSK2830371 in vitro; Figure 5 This is a micro-CT result showing that GSK2830371 reduces bone loss in OVX mice; Figure 6 The results of TRAP staining of femoral tissue sections of OVX mice show that GSK2830371 reduces the number of osteoclasts in the distal femur and alleviates bone loss in OVX mice. DETAILED DESCRIPTION

[0015] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. The present invention can be implemented in many different forms and should not be construed as limited to the embodiments set forth herein. On the contrary, these embodiments are provided so that this disclosure will be thorough and complete and will fully convey the concept of the present invention to those skilled in the art. The present invention will be limited only by the claims.

[0016] This invention discovered for the first time that Wip1 (wild-type p53-induced phosphatase 1) plays an important positive regulatory role in osteoclast differentiation, and further revealed the potential of Wip1 small molecule inhibitors in anti-osteoporosis treatment.

[0017] Example 1: Wip1 is upregulated during osteoclast differentiation BMM cell isolation (1) Six-week-old C57BL / 6 mice were euthanized after anesthesia and then soaked in 70% ethanol for 20 minutes.

[0018] (2) Separate the bilateral tibia and femur.

[0019] (5) Use a 10 ml syringe and a 20G needle to flush the bone marrow cavity with complete culture medium and collect the bone marrow cells into a culture dish.

[0020] (6) Mix the bone marrow cells thoroughly and culture them in a 37°C incubator for 16 hours. Transfer the supernatant to a new culture dish and supplement with complete culture medium and 30 ng / mL M-CSF.

[0021] (7) After 24 hours, the adherent BMM cells were used for further experiments.

[0022] Osteoclast differentiation induction Take a 6-well plate as an example: (1) Inoculate 2.5 x 105 BMM cells per well and culture in a 37°C incubator overnight.

[0023] (2) After the cells adhered, the culture medium was replaced and M-CSF (30 ng / mL) and RANKL (100 ng / ml) were added. The culture medium was replaced and M-CSF and RANKL were supplemented every day.

[0024] (3) After 5 days, irregular multinucleated cells, namely osteoclasts, can be observed under the microscope. After collecting the cells, RNA and protein are extracted for qRT-PCR and WB detection.

[0025] like Figure 1 As shown in the results, the mRNA and protein expressions of Wip1 were significantly increased during the differentiation of mouse bone marrow-derived mononuclear cells (BMM). Figure 1 AB), suggesting that it may play a regulatory role in osteoclast formation.

[0026] Example 2: The Wip1 small molecule inhibitor GSK2830371 has no obvious toxicity to BMM cells within the effective concentration range RAW264.7 cells (2.5 x 10 3cells / well) or BMM cells (4x10 3 cells / well) were seeded in 96-well plates and cultured with complete medium. RANKL (100 ng / mL) was added to induce osteoclast differentiation. Different concentrations of GSK2830371 (0 μmol / L, 2.5 μmol / L, 5 μmol / L, and 10 μmol / L) were added, and cell proliferation was detected using a CCK-8 kit (Beyotime, C0038). Figure 2 As shown in the figure, CCK-8 assay revealed that GSK2830371 had no significant inhibitory effect on the proliferation of RAW264.7 cells and BMM cells within multiple concentration ranges, demonstrating that the drug has good safety in in vitro experiments.

[0027] Example 3: GSK2830371 inhibits osteoclast differentiation in a concentration-dependent manner RAW264.7 cells (0.8x104 cells / well) or BMM cells (1.5x104 cells / well) were seeded in 96-well plates and cultured with complete medium. RANKL (100 ng / mL) was used to induce osteoclast differentiation, and different concentrations of GSK2830371 (0 μmol / L, 2.5 μmol / L, 5 μmol / L, and 10 μmol / L) were added. After 7 days of culture, the cells were fixed and stained for TRAP (Sigma, 387A). The results showed that the number of TRAP-positive osteoclasts was significantly reduced under the action of GSK2830371, and the relationship was concentration-dependent (e.g., Figure 3 shown).

[0028] Example 4: GSK2830371 inhibits osteoclast bone resorption RAW264.7 cells were induced to differentiate into osteoclasts in 6-well plates using the method described in Example 3. The cells were digested with trypsin-EDTA (0.25%) (Gibico, 25200056) and plated onto a Corning Osteo Assay Surface. The cells were cultured in the presence of RANKL (100 ng / mL) and various concentrations of GSK2830371 (0 μmol / L, 2.5 μmol / L, 5 μmol / L, and 10 μmol / L). After 3 days, the cells were treated with 10% SDS for 5 minutes and rinsed twice with PBS. After air drying, the cells were observed under a microscope, photographed, and the area of ​​bone resorption was calculated. Figure 4 As shown, the results showed that the area of ​​bone resorption pits was significantly reduced after GSK2830371 treatment, further verifying that the inhibitor not only inhibited the differentiation process but also affected the bone resorption function of mature osteoclasts.

[0029] Example 5: GSK2830371 alleviates osteoporosis in an ovariectomized (OVX) mouse model An OVX (ovariectomized) model was established using 8-week-old female C57BL / 6 mice. The experimental group was given GSK2830371 (intraperitoneal injection) 0.25 mg / time, 5 times a week, and the control group was given the corresponding solvent. After 6 weeks, the mice were euthanized, and the femurs of the mice were evaluated using micro-CT. The results showed that the femoral bone mass of the experimental group mice was significantly higher than that of the control group, and the reduction of bone volume / tissue volume (BV / TV) and trabecular number (Tb.N) in OVX mice was significantly alleviated. (As shown in Figure 2) Figure 5 shown) In addition, we performed TRAP staining on femoral sections. The number and size of osteoclasts in OVX mice were significantly higher than those in sham-operated mice. After GSK2830371 treatment, the activity of osteoclasts in ovariectomized OVX mice was significantly reduced (e.g., Figure 6 shown).

[0030] It should be noted that other Wip1 inhibitors also have the same effect as GSK2830371, such as CCT007093 and SL-176.

[0031] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. Application of Wip1 inhibitors in the preparation of drugs for the treatment of osteoporosis.

2. The application according to claim 1, characterized in that: The Wipl inhibitor includes one of GSK2830371, CCT007093, and SL-176.

3. The application according to claim 1, characterized in that: The Wip1 inhibitor achieves the treatment of osteoporosis by inhibiting osteoclast differentiation and inhibiting osteoclast bone resorption function.

4. A drug for treating osteoporosis, characterized in that: The composition is composed of a Wipl inhibitor at an effective dose, and can be a single component or a composition comprising an effective dose of the Wipl inhibitor. The composition can include pharmaceutically acceptable excipients.

5. The drug according to claim 4, characterized in that: The auxiliary materials include at least one of excipients, fillers, adhesives, wetting agents, absorption promoters, surfactants, lubricants, stabilizers, flavoring agents, sweeteners, and pigments.

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