Application of LLDT-8 in preparation of medicine for preventing and / or treating osteoporosis

Abstract

The invention discloses an application of LLDT-8 in preparation of a medicine for preventing and / or treating osteoporosis. According to the application disclosed by the invention, the LLDT-8 blocks signal transduction by inhibiting TRAF6, so that downstream NF-kappa B and MAPK signal channels are influenced, the activation of the downstream NF-kappa B and MAPK signal channels on NFATc1 is weakened, and osteoclast differentiation is inhibited. In addition, the invention also discloses that the LLDT-8 can obviously improve bone loss of OVX mice. Therefore, the LLDT-8 provided by the invention provides a new direction for research and development of medicines for preventing and treating osteoporosis.

Description

Technical Field

[0001] The invention belongs to the field of new uses of traditional Chinese medicine, and specifically relates to a new use of LLDT-8 in preventing osteoporosis. Background Art

[0002] Osteoporosis (OP) is a systemic bone disease characterized by decreased bone mass, destruction of bone tissue microstructure, increased bone brittleness, and susceptibility to fractures. It can occur at any age, but is more common in the elderly. It is one of the main causes of disability and death in elderly patients and has attracted widespread attention from the international community. According to the World Health Organization (WHO), by 2050, the total number of OP patients will reach 221 million, and the risk of subsequent fractures and premature death in patients with fractures will increase, bringing a huge economic burden to society. Although the bisphosphonates, PTH and other drugs currently used internationally are effective to a certain extent, they also have many side effects. Therefore, there is an urgent need to deeply understand the mechanism of bone development and regeneration and develop more effective anti-osteoporosis drugs without side effects.

[0003] Osteoblasts (OB) and osteoclasts (OC) are important components of bone tissue. The dynamic balance between bone formation and bone resorption mediated by OB and OC respectively plays an important role in bone growth, development, repair and reconstruction. Once this balance is broken, the bone erosion of OC is enhanced, which will cause bone diseases such as OP. Therefore, inhibiting the differentiation of OC has become one of the strategies for the prevention and treatment of OP (Zhou Jianlie, Liu Zhonghou. Progress in global clinical guidelines for calcium and vitamin D supplementation for the prevention and treatment of osteoporosis [J]. Chinese Journal of Osteoporosis, 2017, 23(03): 371-80.).

[0004] Tripterygium wilfordii is one of the traditional Chinese herbal medicines in my country. It has the effects of dispelling wind and dampness, promoting blood circulation and unblocking collaterals, reducing swelling and relieving pain, killing insects and detoxifying. It is clinically used to treat immune rejection after organ transplantation, skin diseases, rheumatoid arthritis (RA) and kidney disease. Triptolide (TP) is one of the effective monomer components. Triptolide (TP) has anti-inflammatory effects and can inhibit the secretion of inflammatory factors by regulating the NF-κB pathway. Studies have found that the NF-κB signaling pathway plays a very important role in regulating osteoclast differentiation. Osteoporosis is often accompanied by osteoclast-regulated bone resorption greater than osteoblast-regulated bone formation. Therefore, we speculate that TP may have the potential to treat osteoporosis. LLDT-8 ((5R)-5-hydroxytriptolid Triptolide, LLDT-8) is a derivative of TP, which can be used in the treatment of immune-related diseases such as RA and nephritis, and has lower toxicity (RU ZHOU, WEI TANG, PEI-LAN HE, et al. (5R)-5-hydroxytriptolide inhibits the immune response of human peripheral blood mononuclear cells[J]. Int Immunopharmacol, 2009, 9(1): 63-9.). However, the effect and mechanism of LLDT-8 on osteoporosis are still unclear. Studies have found that LLDT-8 reduces the production of nitric oxide and tumor necrosis factor α (TNF-α) in the macrophage cell line Raw264.7 stimulated by lipopolysaccharide (LPS) by regulating the activity of the NF-κB pathway (RU ZHOU, SHEN-XIZHENG, WEI TANG, et al. Inhibition of inducible nitric-oxide synthase expression by (5R)-5-hydroxytriptolide in interferon-gamma-and bacterial lipopolysaccharide-stimulated macrophages[J]. J Pharmacol Exp Ther, 2006, 316(1):121-8.), and the NF-κB pathway is crucial for the differentiation of osteoclasts.In addition, LLDT-8 can also exert anti-tumor effects such as inhibiting cell proliferation and promoting tumor cell apoptosis by targeting transcription factors (TF), kinases or inhibiting ubiquitin / proteasomes (Zhang Xiaohui, Yang Encai, Wan Chunping. Research progress on the pharmacological effects of triptolide and its derivatives [J]. Yunnan Journal of Traditional Chinese Medicine, 2013, 34(8): 64-6.). In short, it is still unclear what effect LLDT-8 has on osteoclast differentiation and osteolytic diseases such as osteoporosis. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of LLDT-8.

[0006] LLDT-8 ((5R)-5-hydroxytriptolid Triptolide, LLDT-8) is a derivative of TP, and its specific structure is shown below:

[0007]

[0008] The new uses described in the present invention include:

[0009] Application of LLDT-8 and pharmaceutically acceptable salts thereof in the preparation of drugs for preventing and / or treating osteoporosis.

[0010] Furthermore, the LLDT-8 inhibits the differentiation of osteoclasts and the expression of genes TRAP, c-Src, NFATc1, and TRAF6 related to osteoclast bone absorption function.

[0011] Furthermore: the LLDT-8 inhibits the expression of TRAF6 in osteoclasts induced by RANKL, blocks signal transduction, affects its activation of downstream NF-κB and MAPK signaling pathways, and then weakens the regulatory effect of the above signaling pathways on NFATc1, the master transcription factor for osteoclast differentiation, ultimately inhibiting osteoclast differentiation and improving bone loss.

[0012] Furthermore, the LLDT-8 inhibits bone loss and increases the number of trabeculae and bone density.

[0013] The present invention also provides a pharmaceutical composition comprising LLDT-8 and a pharmaceutically acceptable salt thereof.

[0014] The present invention also provides the use of the pharmaceutical composition in preparing a drug for preventing and / or treating osteoporosis.

[0015] Furthermore, the LLDT-8 inhibits the differentiation of osteoclasts and the expression of genes TRAP, c-Src, NFATc1, and TRAF6 related to osteoclast bone absorption function.

[0016] Furthermore: the LLDT-8 inhibits the expression of TRAF6 in osteoclasts induced by RANKL, blocks signal transduction, affects its activation of downstream NF-κB and MAPK signaling pathways, and then weakens the regulatory effect of the above signaling pathways on NFATc1, the master transcription factor for osteoclast differentiation, ultimately inhibiting osteoclast differentiation and improving bone loss.

[0017] Furthermore, the LLDT-8 inhibits bone loss and increases the number of trabeculae and bone density.

[0018] Beneficial effects of the present invention: The research results of the present invention show that LLDT-8 blocks signal transduction by inhibiting TRAF6, thereby affecting the downstream NF-κB and MAPK signaling pathways, weakening their activation of NFATc1, thereby inhibiting osteoclast differentiation and improving bone loss. The present invention proposes that LLDT-8 is a potential candidate drug for preventing and treating osteolytic diseases such as osteoporosis. The present invention provides a new direction for the research and development of drugs for preventing and / or treating osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Figure 1 is the result of LLDT-8 inhibiting the differentiation of osteoclasts induced by RANKL; Figure A is the result of TRAP staining of osteoclasts induced by BMMs under the conditions of different concentrations of LLDT-8 treatment; Figure B is the statistical result of TRAP staining of osteoclasts induced by BMMs under the conditions of different concentrations of LLDT-8 treatment;

[0020] Figure 2 The results are for the survival rate of BMMs cells after 48 h of LLDT-8 treatment;

[0021] Figure 3 The results show that LLDT-8 inhibits the mRNA expression of osteoclast-related active genes;

[0022] Figure 4 The results of LLDT-8 inhibiting the expression of TRAF6 and NFATc1 proteins in osteoclasts; Figure A is the Western blot analysis of the expression of TRAF6 and NFATc1 proteins in BMMs of each group at different time points; Figure B is the quantitative analysis of the expression of TRAF6 and NFATc1 proteins in BMMs of each group at different time points;

[0023] Figure 5The results show that LLDT-8 inhibits the activation of NF-κB and MAPK signaling pathways induced by RANKL; Figure A shows the Western blot analysis of the expression of proteins related to NF-κB and MAPK signaling pathways under different concentrations of LLDT-8 treatment; Figure B shows the statistical analysis of the relative expression of p-p65 protein under different concentrations of LLDT-8 treatment; Figure C shows the statistical analysis of the relative expression of p-ERK protein under different concentrations of LLDT-8 treatment;

[0024] Figure 6 The results show the effect of LLDT-8 on the body weight of ovariectomized mice;

[0025] Figure 7 The results of LLDT-8 improving bone loss in ovariectomized mice; Figure A is a representative Micro-CT image of 4-month-old mice in each group; Figure B is a statistical analysis of the relative bone volume and bone density of mice in each group. DETAILED DESCRIPTION

[0026] The following is a clear and complete description of the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings in the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Example 1

[0028] LLDT-8 has an inhibitory effect on osteoclast differentiation in vitro

[0029] Experimental Materials:

[0030] Twenty specific pathogen-free experimental animals (SPF animals) male mice, C57BL / 6 strain, 6-8 weeks old, weighing (20±5) g, were purchased from the Minhang Experimental Animal Center of East China Normal University and raised according to SPF animal breeding conditions; LLDT-8 was provided by Shanghai Guanghua Hospital of Integrated Traditional Chinese and Western Medicine.

[0031] Experimental methods:

[0032] 1. Isolation, culture and differentiation of mouse bone marrow-derived macrophages (BMMs) into OC

[0033] 1) Kill 6-8 week old male wild-type mice by cervical dislocation; 2) Under sterile conditions, peel off the entire hind leg of the mouse; 3) Remove the muscles and tissues on the leg, and cut off the ends of the femur and tibia respectively; 4) Use a 1 mL syringe to draw PBS to rinse the cells in the bone into a centrifuge tube until the bone marrow cavity turns white; 5) Soak the collected cells in new culture medium and transfer them to a new culture dish, and culture them in a 37°C, 5% CO2 incubator; 6) After 24 hours, take the supernatant into a 15 mL centrifuge tube, centrifuge and discard the supernatant; 7) Resuspend with PBS and centrifuge and discard the supernatant; 8) Resuspend the precipitate with full culture medium containing 50 ng / mL M-CSF and 50 ng / mL RANKL, and transfer it to a new culture dish for continued culture. The adherent cells are BMMs; 9) Change the culture medium every other day, and change the medium every other day. Obvious osteoclasts (OC) can be obtained after about 6 days of induction.

[0034] 2. Tartrate-resistant acid phosphatase (TRAP) staining of osteoclasts

[0035] The mature osteoclasts were fixed with 4% paraformaldehyde solution at room temperature for 15 minutes, and the residual paraformaldehyde was washed off with PBS. The cells were permeabilized with 0.1% TritonX-100 at room temperature for 15 minutes, washed with PBS, and then incubated with PBST at room temperature for 20 minutes. The incubated cells were subjected to TRAP staining using the TRAP staining kit of Sigma under light-proof conditions, and the cells were stained in a 37°C incubator in the dark for 60 minutes. Then they were washed with PBS three times, and fresh PBS was added to infiltrate the cells for observation and photography.

[0036] 3. CCK-8 detection of cell activity

[0037] According to the experimental principle and steps of the CCK-8 kit No. C0037, the cell activity was detected, and 4 groups of parallel experiments were performed. The OD value at 450nm was read with an enzyme marker at 48 hours.

[0038] 4. Real-time fluorescence quantitative PCR (qRT-PCR) detection of mRNA expression

[0039] According to the principle and steps of RNA extraction kit of Thermo Fisher Scientific, the total RNA of each group of cells was extracted, and the extracted RNA system was reverse transcribed according to the following system and procedure, and the obtained cDNA was temporarily stored at -20℃. Reverse transcription system (20μL): 5× reverse transcriptase mixture, 4μL; template, 1μg; DEPC water, make up to 20μL. The reaction procedure of reverse transcription PCR is: 55℃, 15min; 85℃, 5s. The obtained cDNA was then subjected to qRT-PCR, and three duplicate wells were set for each sample. The obtained product was verified for its specificity using Gel-base. The qRT-PCR system (total volume 20μl) is: 2×SYBR Green Master Mix, 10μl; primer mix, 1μl; DEPC water, 8μl; template, 1μl. The reaction program of qRT-PCR is as follows: 1) pre-denaturation condition 95℃, 30s, 1 cycle; 2) PCR reaction program setting 95℃, 10s; 60℃, 30s, 40 cycles; 3) melting curve program 95℃, 15s; 60℃, 60s; 95℃, 15s. The primer sequences are shown in Table 1.

[0040] Table 1 Primer sequences of each gene

[0041]

[0042] 5. Western Blot Detection of Protein Expression

[0043] Osteoclasts were treated with RANKL (50 ng / mL) and RANKL+LLDT-8 (0.1, 0.5, 1 μM), and the culture medium was replaced every other day for 5 days. The total cell protein was extracted, and the protein was quantified (BCA method). Then the protein was denatured, and finally the conventional Western Blot experimental operation steps were followed: gel preparation (separation gel containing 10% polyacrylamide, stacking gel containing 4% polyacrylamide) - sample loading (the sample loading amount was determined according to the BCA quantitative results, generally within 30 μL) - electrophoresis (stacked gel 80V 30 minutes, separation gel 130V until it runs to the bottom) - transfer membrane (constant voltage 80V, 1KD / min) - 7% skim milk powder blocking for 1h - TBS washing 3 times (5 minutes / time - primary antibody incubation (β-actin, NFATc1, TRAF6, p65, p-p65, p38, p-p38, JNK, p-JNK, ERK, p-ERK; 3% BSA 1:1000 / 2000 diluted antibody, 4°C overnight) - TBST washing 3 times (5 minutes / time) - secondary antibody incubation (1:10000, 4°C, 1 hour) - TBST washing 3 times (5 minutes / time) - Odyssey Clx software read the results - data analysis.

[0044] 6. Statistical methods

[0045] Data were processed using Prism software 8.0 (GraphPad Software, San Diego, California, USA). ImageJ software was used to analyze the intensity of Western blot. Data were expressed as mean ± standard deviation (x-±s), and the means of two samples were compared using independent sample t-test. The difference was considered statistically significant when P < 0.05.

[0046] Experimental results:

[0047] 1.LLDT-8 inhibits osteoclast differentiation

[0048] After being induced by RANKL, mouse bone marrow macrophages will form a large number of TRAP-positive osteoclasts. In order to study the effect of LLDT-8 (LLDT-8) on osteoclast differentiation, the present invention induced BMMs with RANKL and treated them with 0.1, 0.5, and 1 μM LLDT-8 respectively. Figure 1 TRAP staining results showed that the number of osteoclasts showed a significant downward trend with the increase of LLDT-8 concentration. This indicates that LLDT-8 has an inhibitory effect on the differentiation of osteoclasts in vitro, and this effect is dose-dependent. ***P<0.001, ****P<0.0001.

[0049] 2. Cytotoxicity of LLDT-8

[0050] In order to study the toxic effect of LLDT-8 (LLDT-8) on cells, the present invention treated the mouse bone marrow-derived macrophages with the drug for 48 hours and detected the cell activity using the CCK8 method. Figure 2 is the survival rate of BMMs after treatment with different concentrations of LLDT-8. Figure 2 As shown, compared with the control group, there was no difference in the cell survival rate in the 0.1 μM treatment group, and as the concentration of LLDT-8 increased, the cell survival rate gradually decreased. ****P<0.0001.

[0051] 3.LLDT-8 inhibits the mRNA expression of osteoclast-related active genes

[0052] To further verify the inhibitory effect of LLDT-8 (LLDT-8) on osteoclast differentiation, the present invention used qRT-PCR to detect the mRNA expression level of osteoclast differentiation marker genes. Figure 3The results showed that compared with the control group, the expression of osteoclast differentiation marker genes NFATc1, TRAF6, TRAP and c-Src increased significantly after RANKL induction, indicating that the osteoclast differentiation signaling pathway was significantly activated. In the RANKL+0.5uM LLDT-8 group, NFATc1, TRAF6, TRAP and c-Src all decreased significantly, further indicating that LLDT-8 significantly inhibited the differentiation of osteoclasts induced by RANKL. **P<0.01, ***P<0.001, ****P<0.0001.

[0053] 4.LLDT-8 inhibited the expression of TRAF6 and NFATc1 proteins in osteoclasts

[0054] TRAF6, as a signal transduction protein, plays an important role in the differentiation of osteoclasts, and NFATc1 is a transcription factor necessary for osteoclast differentiation. To further explore the molecular mechanism of LLDT-8 (LLDT-8) inhibiting osteoclast differentiation, the present invention used Western Blot to detect the expression changes of TRAF6 and NFATc1 protein levels. Figure 4 A control group and a 0.5μM LLDT-8 treatment group were set up. At the same time, in order to observe the changes in the levels of TRAF6 and NFATc proteins in each group within 48 hours of RANKL induction and drug treatment, four time treatment groups were set up in each group, namely 0h, 8h, 24h, and 48h. The results showed that during the differentiation of BMMs cells into osteoclasts under RANKL induction, the expression of TRAF6 and NFATc1 increased with the extension of time, while under the same time gradient, the expression of TRAF6 and NFATc1 in the 0.5μM LLDT-8 treatment group was significantly inhibited, which further verified the above qRT-PCR results. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.

[0055] 5.LLDT-8 inhibited the activation of NF-κB and MAPK signaling pathways induced by RANKL

[0056] In order to further explore the potential mechanism of LLDT-8 (LLDT-8) inhibiting osteoclast differentiation, the present invention conducted Western Blot analysis on the classic signaling pathway of osteoclast differentiation. The present invention set up a BMMs cell group, a RANKL induction group, and a RANKL+LLDT-8 (0.1, 0.5, 1 μM) group. The results are as follows Figure 5The results showed that after LLDT-8 treatment, the expression of P-JNK and P-p38 did not change significantly, while P-ERK was significantly inhibited. Therefore, it was inferred that LLDT-8 affected the MAPK signaling pathway through ERK. The levels of proteins related to the NF-κB signaling pathway were detected, and the results showed that after RANKL stimulation, the expression of phosphorylated p65 increased significantly, while after treatment with different concentrations of LLDT-8, the expression of phosphorylated p65 decreased significantly, indicating that LLDT-8 downregulated the expression of phosphorylated p65, thereby preventing p65 nuclear translocation from regulating the transcription of target genes. ***P<0.001, ****P<0.0001.

[0057] Example 2

[0058] LLDT-8 improves bone loss in ovariectomized mice.

[0059] Experimental Materials:

[0060] Twenty specific pathogen-free experimental animals (SPF animals) female mice, C57 strain, 3 months old, weighing (25±2) g, were purchased from the Minhang Experimental Animal Center of East China Normal University and raised according to SPF animal breeding conditions; LLDT-8 was provided by Shanghai Guanghua Hospital of Integrated Traditional Chinese and Western Medicine.

[0061] Experimental methods:

[0062] 1. Modeling and grouping and drug administration

[0063] The mice were randomly divided into four groups, namely CTRL group, OVX group, PBS group, and LLDT-8 group, with 5 mice in each group. The CTRL group did not receive any treatment. The other three groups of mice underwent bilateral ovarian removal surgery to simulate osteoporosis. One week after the wounds of the mice healed, medication was started. The LLDT-8 group was given an intraperitoneal injection of LLDT-8 1 mg / kg, and the PBS group was injected with the same volume of PBS as a control. The injections were given every other day for one month, and the body weights of the four groups of mice were measured and recorded before each medication.

[0064] 2. Micro-CT Scanning of Mouse Tibia

[0065] After the mouse tibia was stripped and fixed, Micro-CT scanning was performed to detect bone mineral density (BMD), bone volume fraction (BV / TV) and other related indicators, and the 3D structure of the bone was reconstructed using NRcon and CTVOX software.

[0066] 3. Statistical methods

[0067] The statistical method is the same as that of “Example 1 of the present invention. LLDT-8 has an inhibitory effect on osteoclast differentiation in vitro”.

[0068] Experimental results:

[0069] 1.LLDT-8 has no significant effect on the body weight of ovariectomized mice

[0070] To determine whether LLDT-8 (LLDT-8) treatment would affect the body weight of OVX-induced osteoporotic mice, the body weights of mice in each group were statistically analyzed. Figure 6 The results showed that after four weeks of treatment, the weight of mice in the LLDT-8 group was slightly lower than that of mice in the PBS group, but there was no significant difference between the two groups, which indicates that LLDT-8 treatment will not have a significant effect on the weight of mice.

[0071] 2.LLDT-8 improves bone loss in ovariectomized mice

[0072] Micro-CT detects various indicators and reconstructs the three-dimensional structure of bones. The results are as follows Figure 7 , Figure 7 A is the three-dimensional image of the bones of mice in each group. Figure 7 B is the statistical analysis of bone mineral density (BMD) and bone volume fraction (BV / TV). Compared with the CTRL group, the number of trabeculae in the OVX group decreased significantly, resulting in bone loss, while the number of trabeculae in the LLDT-8 group increased compared with the OVX group and the PBS group ( Figure 7 A). Further statistical analysis of bone mineral density (BMD) and bone volume fraction (BV / TV) showed that the BMD and BV / TV of the LLDT-8 group were significantly higher than those of the OVX group, and were generally close to those of the CTRL group ( Figure 7 B), which indicates that LLDT-8 can improve bone loss and increase bone mass in ovariectomized mice.

[0073] Excessive activation of osteoclasts can lead to enhanced bone erosion, thus causing related osteolytic diseases. Inhibiting the differentiation and function of osteoclasts has become one of the strategies for preventing and treating such diseases. After binding to their respective receptors c-Fms and RANK, the two major cytokines activate downstream MAPK, NF-κB, Ca 2+Classic signaling pathways such as RANKL promote osteoclast differentiation. Many studies have shown that inhibiting the conduction of the NF-κB signaling pathway induced by RANKL can affect osteoclast differentiation. In the NF-κB signaling pathway, p65 NF-κB is inhibited by binding to IKBα. When NF-κB is activated, IKBα is degraded by the proteasome through phosphorylation and ubiquitination, promoting the nuclear translocation of p65 NF-κB. Protein immunoblotting results showed that after LLDT-8 treatment, the phosphorylation level of p65 decreased, the activity of p65 weakened, and the nuclear translocation was blocked, thereby affecting its regulation of target gene transcription. The MAPK signaling pathway is critical in the process of RANKL-induced differentiation of precursor cells into osteoclasts. Protein immunoblotting showed that LLDT-8 downregulated the phosphorylation level of ERK in the MAPK signaling pathway, thereby weakening the regulation of downstream target genes by this signaling pathway.

[0074] TNF receptor-associated factor 6 (TRAF6) also plays an important role in the differentiation of osteoclasts. As a signal transduction protein, it is recruited to the cytoplasmic binding domain after RANK receives the signal, transmits the signal to the downstream signaling pathway, and then regulates the expression of the NFATc1 gene. Nuclear factor of activated T cells 1 (NFATc1) is a transcription factor required for osteoclast differentiation, and directly regulates many genes important for osteoclast differentiation and bone resorption function, including TRAP, cathepsin K, etc. LLDT-8 reduces the expression of NFATc1, TRAF6, and TRAP in RANKL-induced osteoclasts, which further verifies the inhibitory effect of LLDT-8 on osteoclast differentiation.

[0075] In addition, experiments on the OVX animal model found that osteoporosis simulated after bilateral ovariectomy in mice was significantly alleviated by LLDT-8, and the bone density of OVX mice was significantly improved.

[0076] In summary, the present study showed that LLDT-8 affects the downstream NF-κB and MAPK signaling pathways by downregulating the expression of TRAF6, inhibiting p-p65 and p-ERK, thereby affecting their activation of NFATc1, and ultimately inhibiting the differentiation of osteoclasts and improving bone loss in OVX mice. Therefore, LLDT-8 is expected to be used in the treatment of osteoporosis and other related osteolytic diseases.

[0077] This specific embodiment is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make modifications to the present embodiment without any creative contribution as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. Application of LLDT-8 in the preparation of a drug for preventing and / or treating osteoporosis, characterized in that: The structure of the LLDT-8 is shown below:

2. The use according to claim 1, characterized in that: The LLDT-8 downregulates the protein levels of intracellular p-ERK and p-p65, and inhibits the activation of NF-κB and MAPK osteoclast differentiation-related signaling pathways.

3. The use according to claim 1, characterized in that: The LLDT-8 blocks signal transduction by inhibiting TRAF6, thereby affecting the downstream NF-κB and MAPK signaling pathways, weakening their activation of the osteoclast differentiation master transcription factor NFATc1, inhibiting osteoclast differentiation in vitro and improving bone loss in vivo.

4. The use according to claim 1, characterized in that: The LLDT-8 inhibits bone loss and increases the number of trabeculae and bone density.

5. A pharmaceutical composition, characterized in that It comprises LLDT-8 and a pharmaceutically acceptable salt thereof, and the structure of the LLDT-8 is shown below:

6. Use of the pharmaceutical composition according to claim 5 in the preparation of a medicament for preventing and / or treating osteoporosis.

7. The use according to claim 6, characterized in that: The pharmaceutical composition down-regulates the protein levels of p-ERK and p-p65 in cells, and inhibits the activation of NF-κB and MAPK osteoclast differentiation-related signal pathways.

8. The use according to claim 6, characterized in that: The pharmaceutical composition blocks signal transduction by inhibiting TRAF6, thereby affecting the downstream NF-κB and MAPK signaling pathways, weakening their activation of the osteoclast differentiation master transcription factor NFATc1, inhibiting osteoclast differentiation in vitro and improving bone loss in vivo.

9. The use according to claim 6, characterized in that: The pharmaceutical composition inhibits bone loss and increases the number of trabeculae and bone density.

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