CN113616631B - Application of DUSP6 inhibitor BCI in preparation of osteoporosis drugs - Google Patents

Abstract

The invention discloses application of DUSP6 inhibitor BCI in the preparation of medicaments for preventing and treating osteoporosis. In an effective dose, BCI has no toxic effect on mononuclear macrophages and does not influence the cell cycle and apoptosis of the mononuclear macrophages, remarkably inhibits the differentiation and maturation of RANKL-induced RAW264.7 cells and mouse primary mononuclear macrophages (BMMs) to osteoclasts, effectively inhibits the fusion and bone-feeding capability of RANKL-induced mature osteoclasts, remarkably reduces the expression of specific osteoclast differentiation genes such as NFATc1 and c-Fos, strongly inhibits STAT3 and NF-kB-NFATc 1 signal pathways in the osteoclast differentiation process, and inhibits the reduction of bone density and other related bone parameters in an osteoporosis in-vivo model.

Description

Application of DUSP6 inhibitor BCI in preparation of osteoporosis drugs

Technical Field

The invention belongs to the technical field of medicines, and particularly relates to an application of a DUSP6 inhibitor, in particular to a compound BCI in preparation of medicines for preventing and treating osteoporosis.

Background

Osteoporosis becomes an important health problem for people over 50 years old in China, and the osteoporosis problem of middle-aged and old women is particularly serious. 19 months and 10 months in 2018, the Chinese osteoporosis epidemiological survey result released by the national Weijian Commission for the first time shows that more than half of women over 65 years old in China have osteoporosis; the nearly half of the population over 50 years of age shows low bone mass and density, and is a high risk population for osteoporosis, and the residents have general insufficient cognition on osteoporosis. Osteoporosis is the most common bone disease affecting the health of residents, the early stage of the disease usually has no obvious clinical manifestation, if no attention is paid, the conditions of pain, spinal deformation, fracture and the like can be caused along with the progress of the disease, the disability fatality rate is high, the life quality of patients is seriously affected, and the huge medical treatment and nursing cost is also caused.

At present, bisphosphonates for osteoporosis, calcitonin, selective estrogen receptor modulators, estrogens and active vitamin D drugs are treated. Despite the great progress made in anti-osteoporosis therapy, the long-term effects thereof are still unsatisfactory due to the harmful side effects of the above-mentioned conventional drugs.

Osteoporosis is a common and frequent skeletal disease, the cause of which is an imbalance in bone remodeling, associated with increased osteoclastic bone resorption activity and insufficient osteoblastic osteogenic capacity. Osteoclast precursors are derived from the monocyte/macrophage lineage of hematopoietic stem cells and gradually differentiate into mature osteoclasts in the presence of macrophage colony stimulating factor (M-CSF) and nuclear factor kappa B receptor activator (NF-kappa B) ligand (RANKL). During the formation of osteoclast, the combination of M-CSF and CSF-1 receptor ensures the proliferation of cells, and the action of RANKL and its receptor RANK promotes the maturation and absorption of osteoclast. The interaction of osteoclast precursor surface RANKL-RANK results in the activation of a series of downstream signals, including activation of protein kinases (MAPKs) with NF- κ B and mitogen. These signals initiate osteoclast activation and differentiation. In addition, signal sensors and activator of transcription 3(STAT3) play important roles in osteoclastogenesis by regulating the NF-. kappa.B pathway. The above various signaling pathways synergistically activate key transcription factors in osteoclast formation, such as c-Fos and NFATc 1. Therefore, inhibition of these signaling pathways may be helpful in the treatment of osteoporosis. Recently, new drugs (such as bisphosphonates, calcitonin, and dinoselled, etc.) directed to osteoclastic bone resorption activity or osteoblastic bone formation have been widely developed and explored and used for the prevention and treatment of osteoporosis. However, most therapeutic drugs cause severe side effects, preventing their long-term use and good patient compliance. Therefore, there is an urgent need to develop alternative methods for preventing and treating osteoporosis that can improve the therapeutic effects and reduce the toxicity.

Bispecific phosphatase 6(DUSP6) is a mitogen-activated protein kinase phosphatase and is closely associated with a variety of cellular functions, including cell proliferation and differentiation. The current research direction on DUSP6 is mainly focused on the treatment of tumors and immunity, however its role in osteoclast differentiation-related diseases is not clear. The invention finds that the transcription and translation levels of DUSP6 are remarkably increased in the process of osteoclast formation and differentiation in research, and the knockout of DUSP6 remarkably inhibits the formation of osteoclasts. In addition, BCI hydrochloride (BCI) as a selective DUSP6 inhibitor attenuated RANKL-mediated osteoclast differentiation in vitro, alleviated bone loss in bilateral ovariectomy-induced osteoporotic mice without significant hepatorenal toxicity. Specifically, BCI disrupts osteoclast fusion and bone-phagocytic activity in a dose-dependent manner during osteoclast differentiation and reduces osteoclast-specific gene mRNA and protein levels, and GSEA, KEGG enrichment assays and western blot results based on transcriptome sequencing indicate that BCI inhibits RANKL-induced osteoclastogenesis by inhibiting STAT3 and NF- κ B signaling pathways and attenuating the interaction of NF- κ B/p65 with NFATc 1. These results suggest that inhibition of DUSP6 can prevent postmenopausal osteoporosis and is expected to be a novel therapeutic method for osteoporosis, and the present invention has been completed for this purpose.

Disclosure of Invention

The invention aims to provide application of DUSP6 inhibitor BCI in preparation of a medicament for preventing and treating osteoporosis. Provides a new potential treatment means for osteoporosis patients.

The present invention provides the following embodiments:

in one embodiment, use of a DUSP6 inhibitor that inhibits RANKL-induced osteoclast differentiation in the manufacture of a medicament for the prevention and treatment of osteoporosis.

Preferably, the DUSP6 inhibitor is capable of inhibiting osteoclast formation, fusion and bone resorption activity for the use according to the invention. The DUSP6 inhibitor is compound BCI or its cis-trans isomer and its pharmaceutically acceptable salt.

In another embodiment, the present invention provides the use of the compound BCI or its cis-trans isomers (e.g., E or Z configuration) and pharmaceutically acceptable salts thereof in the manufacture of a medicament for the prevention and treatment of osteoporosis.

The chemical structural formula of compound BCI is as follows:

bispecific phosphatase 6(DUSP6) is a mitogen-activated protein kinase phosphatase and is closely associated with a variety of cellular functions, including cell proliferation and differentiation. The current research direction on DUSP6 is mainly focused on the treatment of tumors and immunity, however its role in osteoclast differentiation-related diseases is not clear. The invention finds that the transcription and translation levels of DUSP6 are remarkably increased in the process of osteoclast formation and differentiation in research, and the knockout of DUSP6 remarkably inhibits the formation of osteoclasts. In addition, BCI hydrochloride (BCI) as a selective DUSP6 inhibitor attenuated RANKL-mediated osteoclast differentiation in vitro, alleviated bone loss in bilateral ovariectomy-induced osteoporotic mice without significant hepatorenal toxicity. Specifically, BCI disrupts actin loop formation and bone-phagocytic activity in a dose-dependent manner during osteoclast differentiation, and reduces osteoclast-specific gene and protein levels. KEGG, GSEA analysis and western blot results based on transcriptome sequencing indicate that BCI inhibits RANKL-induced osteoclastogenesis by inhibiting STAT3 and NF- κ B signaling pathways and attenuating the interaction of NF- κ B/p65 with NFATc 1. These results indicate that inhibition of DUSP6 can prevent postmenopausal osteoporosis and may be an effective method for treating osteoporosis.

The invention researches the effect of DUSP6 in regulation of bone homeostasis, researches the inhibition of RANKL-induced osteoclast differentiation by BCI and the regulation mechanism thereof, and proves that DUSP6 inhibits the formation, fusion and bone resorption activity of osteoclasts in a concentration-dependent manner. In addition, it was found that BCI-mediated inhibition of DUSP6 ameliorated bone loss in a mouse Ovariectomy (OVX) -induced osteoporosis model. These findings suggest that targeting DUSP6 may be a novel therapeutic strategy for osteoporosis.

Drawings

Figure 1 is the results of experiments on the role of DUSP6 in RANKL-induced osteoclast differentiation;

FIG. 2 shows the effect of BCI compound on the activity, apoptosis, cycle and osteoclast formation and differentiation of BMMs and RAW264.7 cells;

FIG. 3 shows that compound BCI inhibits RANKL-mediated nuclear translocation of NFATc1 and osteoclast differentiation marker gene expression;

figure 4 effect of inhibition of DUSP6 on osteoclast fusion and bone resorption activity using compound BCI experimental results;

FIG. 5 results of transcriptome change analysis of RAW264.7 cells treated with compound BCI during osteoclast differentiation;

FIG. 6 shows that BCI compound inhibits RANKL-induced osteoclast differentiation by reducing STAT3 and NF-kB-NFATc 1 signals;

FIG. 7 shows the effect of BCI compound on the level of p-p38, p-JNK, p-ERK during osteoclast differentiation;

FIG. 8 shows that compound BCI at a specific concentration does not affect osteoblast activity and differentiation assay results;

figure 9 compound BCI ameliorates the results of the OVX-induced bone loss experiment in a mouse model of osteoporosis in vivo;

fig. 10 schematic of the mechanism by which BCI of the compound affects osteoclast differentiation, in which: BCI negatively regulates the expression of MMP9 and TRAP by inhibiting STAT3 and NF-kB-NFATc 1 signals, and further has the effect of inhibiting osteoclast differentiation.

Detailed Description

The following examples are intended to illustrate the invention in detail to aid in understanding the spirit of the invention.

The invention adopts the following medicines and reagents:

BCI hydrochloride (abbreviated as BCI, also called (E) -BCI) was purchased from MCE corporation (Shanghai, China) and dissolved in DMSO. DMEM, alpha-MEM and Fetal Bovine Serum (FBS) were purchased from Hyclone (USA). Recombinant mouse RANKL and M-CSF were purchased from R & D, Inc., USA. The primary antibody to DUSP6 was from Abcam (uk). Anti- β -tubulin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), phospho-NF-. kappa.B/p 65, NF-. kappa.B/p 65, p-I.kappa.B.alpha., p38, p-ERK, CTSK, MMP9, and c-Fos antibodies were purchased from Protetech corporation (Wuhan, China). p-JNK, JNK and p-p38 specific primary antibodies were purchased from Affinity Biosciences, Inc. (Zhenjiang, China). anti-p-STAT 3 and STAT3 antibodies were purchased from Nanjing biosphere. anti-NFATc 1 antibody was from Santa Cruz (usa). Enzyme-labeled secondary antibodies against rabbit and mouse were purchased from Shanghai Biyuntian.

Example 1

Cell culture and osteoclast differentiation

Mouse macrophage RAW264.7 was derived from ATCC cell bank (Shanghai, China) and cultured in DMEM (10% fetal bovine serum and 1% P/S). Bone marrow mononuclear macrophages (BMMs) were isolated from mouse femurs and tibias and cultured in α -MEM (10% fetal bovine serum, 1% penicillin/streptomycin diabody and 50ng/mL M-CSF) for 48 hours. All cells were in 5% CO₂The culture was carried out at 37 ℃ below zero. DUSP 6-specific small interfering RNA (siRNA) oligonucleotide (40 nM; 5'-CGAUGCUUACGACAUUGUUAATTUUAACAAUGUCGUAAGCAUCGTT-3') was transfected 5h before osteoclast induction by Lipofectamine 3000 (Invitrogen) (Shanghai Production). Both cell types were differentiated using M-CSF (50ng/mL) and RANKL (100 ng/mL). The extent of osteoclast differentiation of RAW264.7 cells and BMMs was determined on induction days 3 and 5, respectively. MC3T3-E1 cells (pre-osteoblast line) were grown in alpha-MEM (10% fetal bovine serum and 1% penicillin/streptomycin diabody), and osteoblasts were induced using an osteogenic induction solution containing ascorbic acid (60. mu.g/mL) and beta-glycerophosphate (6 mM).

Cytotoxicity assays

The cytotoxicity of BCI was detected by using a cell counting kit-8 (CCK-8) to find an appropriate concentration. RAW264.7 cells and BMMs were seeded on a 96-well plate, and the viability of the cells was evaluated after 1 to 3 days of culture in BCI at a dose of 0 to 8 μm. The cells were washed with PBS and cultured in CCK-8 solution for 1.5 to 2 hours. The absorbance was read at 450nm using a spectrophotometer (Bio-Tek, USA).

And detecting the apoptosis condition and the cell cycle condition by adopting flow cytometry. 1.5X 10⁶Each RAW264.7 was cultured for 3 days on a differentiation medium with or without BCI (2 to 4 μm). Cells were washed with PBS and fixed in 70% ethanol, then stained with Propidium Iodide (PI) according to standard procedures and using a FACStar flow cytometer (Becton Dickinson, usa)Country) for the test. For detecting the apoptosis rate, cells are stained by Annexin V/PI and detected by a flow cytometer.

TRAP staining

RAW264.7 cells and BMMs were seeded in 96-well plates as described previously for growth and differentiation towards osteoclasts. TRAP staining was performed after multinucleated osteoclast formation was observed (TRAP staining kit, Sigma, usa). Cells were counted using light microscopy and TRAP + cells containing 3 or more nuclei were considered mature osteoclasts.

FAK staining

RAW264.7 cells in 96-well plates were washed with PBS, fixed with 4% paraformaldehyde at 37 ℃ for 30 minutes, then washed twice with wash buffer, and then disrupted with Triton X-100. Subsequently, the cells were washed again and blocked with 2% BSA for 20 min. After staining with tetramethylrhodamine-conjugated phalloidin for 1.5 hours, washing was performed 2 times with the washing solution. Counterstaining was performed with DAPI. The fluorescence image is displayed by a fluorescence microscope.

Bone pit formation experiment

BMMs were inoculated on bovine cortical bone sections and induced according to experimental requirements. After 8 days of induction, the bone chips were rinsed with sodium hypochlorite, incubated in toluidine blue for 5 minutes, and then observed microscopically for bone pit formation, and the relative areas of resorption were quantified by ImageJ software.

Alkaline phosphatase and alizarin Red S staining

MC3T3-E1 cells were cultured in 24-well plates and induced in osteogenic medium. After successful induction, cells were fixed in 70% ethanol for 30 min and washed 2 times with deionized water. ALP activity was assessed using the NBT/BCIP substrate system. When alizarin red S is stained, cells are prepared as described above, and then stained in alizarin red staining solution. Images were acquired using an inverted microscope.

RNA extraction and qRT-PCR

RNA was obtained using TRIzol buffer and concentration was measured using a NanoDrop ND-1000 microplate reader (Thermo, UK). Using PrimeScript^TMReverse transcriptase (Takara, Japan) synthesized cDNA. mu.L of cDNA was then ligated with SYBR Green super mix and corresponding primersAnd (4) mixing. The expression of DUSP6, CTSK, c-Fos, MMP9, NFATc1, CD9, OSCAR, PU.1, ATP6V0d2, RUNX2, COL1 alpha 1, ALPL was detected using GAPDH as a reference. All primer sequences are listed in table 1.

TABLE 1 qRT-PCR primer sequences

Transcriptome sequencing and bioinformatics analysis

RNA-seq was used to detect gene expression of RAW264.7 cells after 3 days of induction in differentiation medium with or without 2 μm BCI. Sequencing was performed by Shanghai Producer using HiseqTM 2500 system (Illumina). Data analysis was performed using the DEGseq R software package with a threshold for Differentially Expressed Genes (DEGs) of | logFC | >1, p < 0.05. Enrichment analysis was performed using clusterProfiler R package for Gene Ontology (GO) annotation and kyoto gene and genome encyclopedia (KEGG). Gene Set Enrichment Analysis (GSEA) was performed using the h.all.v. 7.2. symbol. p <0.05 and FDR <0.25 are considered statistically significant.

Immunoblotting

The induced cells were washed in pre-cooled PBS and lysed on ice for 30 minutes using cell lysates containing protease inhibitors and phosphatase inhibitors. SDS-PAGE gel electrophoresis was performed using at least 40g of protein, followed by transfer to PVDF membrane. Blocking was performed with 4% bovine serum albumin in TBST for 2 hours, followed by overnight incubation with specific primary antibody (1: 1000). The next day, after washing 3 times with TBST, incubation with enzyme-labeled secondary antibody was performed for 1.5 hours. After washing the secondary antibody 3 times with TBST, images were collected under a Bio-Rad imaging system (CA, USA) using ECL luminophores. Nuclear translocation of p65 and NFATc1

Observing the effect of BCI on NFATc1 nuclear translocation and NF-. kappa.B/p 65 during osteoclast differentiation, RAW264.7 cells were treated with medium with or without 2 μm BCI for 2 hours and then induced with 100ng/mL RANKL solution for 20 minutes. Followed by fixation in 4% paraformaldehyde and blocking for 1.5 hours with Triton X-100 medium permeable membrane and 4% BSA. The primary antibody was then incubated overnight in a shaker at 4 ℃ and finally a secondary antibody labeled with Alexa fluorite 647 (Proteintech). Nuclear counterstaining was performed with DAPI for 5 min. Fluorescence images were observed using a confocal microscope (Leica Microsystems) and the fluorescence intensity was analyzed using ImageJ.

Co-IP experiments

RAW264.7 cells were induced in differentiation medium with or without 2 μm BCI for 3 days, then lysed with lysis buffer on ice for 1 hour followed by centrifugation at 12000 g. An anti-NF-. kappa.B/p 65 antibody (NFATc1 antibody) (1:150) was added to the cell lysate, and incubated overnight at 4 ℃ to thereby form an immune complex. IP experiments with NF-. kappa.B/p 65(NFATc1) were performed using protein A/G magnetic beads (Thermo, USA) according to the protocol. Incubation with magnetic beads for 60 min at room temperature gave IP complexes. The magnetic beads were collected on a magnetic support, washed 3 times in IP buffer, the immune complex washed with eluent and finally detected with western blot.

Establishment of OVX mouse osteoporosis model

Female C57BL/6 mice aged 6 weeks were randomly divided into Sham group, OVX + low dose (15mg/kg) BCI group, and OVX + high dose (30mg/kg) BCI group. After 5% chloral hydrate is adopted for intraperitoneal injection and anesthesia, bilateral ovariectomy is carried out on the mice to establish an osteoporosis model. Sham mice were subjected to only incision and suture procedures. 3 days after surgery, each group of mice was injected intraperitoneally with BCI (15 or 30mg/kg) or saline for 7 weeks, respectively. All mice were housed in the laboratory animal center at army-military medical university. The experiment was approved by the ethical committee of the army medical university.

Micro-computerized tomography (micro-CT) and histological evaluation

After each group of mice died at the neck-broken site, micro-CT scanning was performed after fixing the femurs of the mice with 4% paraformaldehyde. All samples were then decalcified with 10% EDTA solution for 10 days. H & E, Masson and TRAP staining was performed on the mouse femurs after decalcification. Liver and kidney tissues of each group of mice were morphologically evaluated by H & E staining. The sections were observed using an inverted microscope.

Statistical analysis

Data are expressed as mean ± standard deviation and statistically analyzed using GraphPad Prism. Student's t-test is adopted for two groups of comparison, and one-factor analysis of variance is adopted for 3 or more than 3 groups of comparison. p <0.05 is defined as having statistical differences.

Results

Effect of DUSP6 on RANKL-mediated osteoclast formation and differentiation

To explore the role of DUSP6 in RANKL-mediated osteoclast differentiation, we (i.e. the inventors) induced RAW264.7 cells and BMMs with M-CSF and RANKL for 1, 3 and 5 days, respectively. The results showed that the mRNA level of DUSP6 gradually increased during osteoclast differentiation (fig. 1A). Following induction of RAW264.7 cells and BMMs with different doses of RANKL, expression of DUSP6 increased significantly in a RANKL dose-dependent manner (fig. 1B). Furthermore, we observed that DUSP6 protein expression was upregulated during osteoclast differentiation along with increased expression of osteoclast specific genes (MMP9, CTSK, NFATc1, and C-Fos) (fig. 1C). To better understand the effect of DUSP6 on osteoclastogenesis, we used siRNA to knock down the expression of DUSP6 and western blot results demonstrated that DUSP6 siRNA effectively reduced DUSP6 expression in RAW264.7 cells and BMMs (fig. 1D). Furthermore, TRAP staining and western blot showed that DUSP6 knockdown significantly reduced the number of RANKL-induced TRAP + cells and levels of osteoclastogenesis-associated proteins (fig. 1E-F), suggesting that DUSP6 plays an important role in modulating RANKL-mediated osteoclastogenesis.

Effect of BCI on cell cycle, viability and apoptosis

First, we evaluated BCI cytotoxicity on RAW264.7 cells and BMMs, and the results showed that low doses of BCI (≦ 2 μm and ≦ 4 μm) showed no toxicity on RAW264.7 cells and BMMs (FIGS. 2B and 2C). To further assess the effect of BCI on apoptosis and cell cycle progression, we performed flow cytometry. The results show that BCI (. ltoreq.4 μm) had no significant effect on the cell cycle progression (FIG. 2D) and apoptosis (FIG. 2E) of BMMs. We subsequently demonstrated that BCI inhibited DUSP6 protein expression in RAW264.7 cells and BMMs during RANKL-mediated osteoclast differentiation (fig. 2F). These results indicate that BCI is effective in inhibiting expression of DUSP6 protein without affecting cell cycle and apoptosis.

DUSP6 inhibitor BCI inhibits RANKL-mediated osteoclast formation

To determine whether BCI-mediated inhibition of DUSP6 would inhibit RANKL-mediated osteoclast differentiation, we performed TRAP staining on RAW264.7 cells and BMMs. Figure 2G shows that BCI inhibits osteoclast generation in a concentration-dependent manner. Current research suggests that NFATc1 translocates from the cytoplasm to the nucleus during osteoclast activation and promotes transcription of downstream osteoclast-specific genes. To evaluate the inhibitory effect of DUSP6 on NFATc1 nuclear translocation, we induced RAW264.7 cells for 1 day in differentiation medium with or without BCI. Cellular immunofluorescence results indicated that BCI treatment inhibited RANKL-mediated nuclear translocation of NFATc1 (fig. 3A). Furthermore, the evaluation of BCI-specific effects by qRT-PCR (CTSK, MMP9, NFATc1 and c-Fos) showed that mRNA levels of RAW264.7 cells after BCI treatment (fig. 3B-E) and BMMs (fig. 3F-I) were significantly down-regulated in concentration-dependence. Similar conclusions were drawn from the Western blot results (FIGS. 3J and K).

BCI attenuates RANKL-mediated osteoclast fusion and resorption

To further assess the effect of DUSP6 inhibition on osteoclast fusion, we performed FAK staining of RAW264.7 cells induced by different doses of BCI. FAK staining showed a significant reduction in the number of F-act in loops after BCI treatment (FIG. 4A). Furthermore, qRT-PCR results showed that BCI treatment reduced the mRNA levels of the osteoclast fusion genes (ATP6V0d2, pu.1, OSCAR, and CD9) (fig. 4B-E), consistent with FAK staining results. In the bone pit formation experiment, BMMs were induced in differentiation media with or without BCI for 7 days, and quantitative results showed a significant reduction in the number of bone pits under BCI treatment (fig. 4F).

Transcriptome sequencing and bioinformatics analysis

To elucidate the mechanism associated with BCI-mediated inhibition of osteoclastogenesis, we determined by RNA-seq analysis whether RANKL-induced RAW264.7 cells undergo BCI-treated transcriptome. As a result, 177 genes were down-regulated and 45 were up-regulated after BCI treatment. Fig. 5A shows the distribution of deg using volcano plots. In addition, the RNA-seq analysis results showed a significant decrease in the expression of osteoclast differentiation key genes after BCI treatment (fig. 5B), which is consistent with qRT-PCR results. We next performed GO and KEGG enrichment analyses predicting the primary function represented by the first 50 most significant DEGs after BCI treatment. The biological processes of these genes are mainly concentrated in receptor ligand activity, chemokine activity and osteoclast differentiation regulation; molecular functions were mainly related to response to lipopolysaccharide, neutrophil migration and neutrophil chemotaxis (fig. 5C). The results of the KEGG pathway enrichment show several major pathways including cytokine-cytokine receptor interaction, osteoclast differentiation, rheumatoid arthritis, NF- κ B and TNF signaling related pathways (fig. 5D). The above results demonstrate that BCI inhibits osteoclast differentiation and suggests a downstream signaling pathway.

BCI inhibits RANKL-induced STAT3 and NF- κ B-NFATc1 signals

Based on the results of the KEGG pathway analysis (fig. 5D), we next performed GSEA. The results show that TNF- α signaling by NF- κ B was significantly enriched in RANKL-induced group, but less enriched in BCI-treated group (fig. 6A). In addition, immunofluorescence staining experiments with nuclear translocation of NF-. kappa.B/p 65 showed that significant nuclear translocation of NF-. kappa.B/p 65 occurred in the RANKL-induced group, while this process was significantly inhibited in the BCI group (FIG. 6B). STAT3 induces NF-. kappa.B activity, which in turn activates transcriptional regulation of downstream osteoclast-associated genes (e.g., CTSK and TRAP). We treated RAW264.7 cells with and without BCI (2 μm) differentiation medium for 5, 15, 30 and 60 minutes respectively and then tested STAT3 and NF-. kappa.B signaling activity by western blot. The results in fig. 6C show that the BCI treated group significantly attenuated phosphorylation of STAT3 at Ser727 and Tyr705 sites at 5 min, 15 min and 30 min relative to RANKL group. In addition, BCI treatment group inhibited phosphorylation of p65 subunit and I κ B α at 15 min and 30 min relative to RANKL group (fig. 6C-D). NFATc1 is an important regulator of osteoclast differentiation-related genes. Our previous results in figure 4 show that NFATc1 protein expression gradually decreased with increasing BCI concentration and treatment time. In addition, the protein interaction between NF-KB/p65 and NFATc1 was found to be significantly inhibited by BCI by Co-IP experiments (FIG. 6E). Finally, we verified whether BCI inhibited osteoclast differentiation by MAPK signaling. Western blot results showed no significant change in the expression levels of p-p38, p-JNK and p-ERK after BCI treatment (FIGS. 7A and 7B). The above results indicate that BCI inhibits RANKL-mediated osteoclast differentiation by attenuating STAT3 and NF- κ B signals, and that this process is independent of MAPK signals.

BCI does not affect osteoblast differentiation or mineralization

In addition to osteoclasts, osteoblasts are also key regulatory cells of bone that regulate homeostasis. First, we confirmed by CCK-8 experiments that BCI was not toxic to MC3T3-E1 cells, and the data showed no significant change (≦ 4 μm) in MC3T3-E1 cell viability after 48 and 96h BCI treatment (FIG. 8A). Next, we evaluated the effect of BCI on mRNA levels of key osteogenic differentiation genes (RUNX2, COL1 α 1 and ALPL). The qRT-PCR results showed that BCI (. ltoreq.4 μm) did not significantly alter the expression of these osteoblast specific genes (FIG. 8B). Furthermore, we assessed the osteogenesis and mineralization of MC3T3-E1 cells under BCI treatment by ALP and alizarin Red S staining. As expected, the results showed that osteogenesis and mineralization of MC3T3-E1 cells did not significantly change between the control and BCI treated groups (fig. 8C and D). The above results indicate that BCI effectively inhibits osteoclast differentiation without affecting osteogenic differentiation.

BCI ameliorates OVX-induced bone loss

After establishing the OVX-induced osteoporosis mouse model, we subsequently evaluated the role of BCI in vivo. Low or high concentration (15mg/kg or 30mg/kg) BCI was injected intraperitoneally for 8 weeks and bone loss was assessed using micro-CT. We observed that bone loss was prevented in both the low and high concentration BCI groups (fig. 9A). Furthermore, the quantitative results showed a significant increase in bone volume/total tissue volume (BV/TV), trabecular number (tb.n), bone density (BMD) and bone surface density (BS/TV) for the two BCI treated groups compared to the OVX group (fig. 9B). Consistent with the micro-CT results, H & E and Masson staining showed significant bone destruction in the OVX group samples, however, this was rarely observed in the BCI treated group samples (fig. 9C). In addition, TRAP staining detected a high number of TRAP + cells in OVX group samples relative to Sham group, and both concentrations of BCI treatment reduced the number of TRAP + osteoclasts relative to OVX group (fig. 9C). To assess hepatorenal toxicity under BCI treatment, we performed histological analysis of the liver and kidney. Liver structures of mice in Sham and OVX groups were normal, portal triple structures were normal, central veins were intact, and liver structures of mice in BCI group were not significantly changed pathologically (fig. 9D). Also, no significant pathological changes were observed in the renal structure of BCI treated animals (fig. 9D). These results indicate that BCI has a protective effect on OVX-induced osteoporosis by inhibiting osteoclast activation, without significant hepatorenal toxicity. As shown in FIG. 10, the STAT3 and NF-. kappa.B-NFATc 1 signal pathway inhibit osteoclast differentiation under the intervention of compound BCI, so as to achieve the effect of protecting osteoporosis bone loss.

Discussion of the related Art

The present inventors intensively studied and studied the effect of DUSP6 on osteoclastogenesis, found that DUSP6 protein expression is significantly increased during osteoclastogenesis, and that down-regulation of DUSP6 inhibits osteoclastogenesis. In addition, the inhibitor BCI of DUSP6 inhibits osteoclast differentiation in a concentration-dependent manner. Bioinformatic analysis and experimental validation suggest that BCI inhibits osteoclastogenesis by altering STAT3 and NF- κ B-NFATc1 signaling pathways. In addition, in vivo experimental results show that BCI treatment can reduce bone loss in OVX-induced osteoporosis mouse models.

Assessment of DUSP6 expression during osteoclast differentiation showed that expression of DUSP6 gradually increased as osteoclasts formed and matured. Notably, siRNA transfected DUSP6 in BMMs and RAW264.7 cells significantly inhibited TRAP + osteoclast formation. Subsequently, pharmacological inhibition of DUSP6 by BCI was shown to significantly attenuate osteoclast formation, fusion and bone resorption by TRAP staining, FAK staining and bone pit formation experimental results.

Activation of NF-. kappa.B requires I.kappa.B kinase to phosphorylate I.kappa.B alpha, while I.kappa.B kinase promotes translocation of NF-. kappa.B/p 65 to the nucleus of the cell, thereby promoting the transcriptional activity of factors such as the osteoclast-specific gene NFATc 1. The inventors found that BCI treatment reversed RANKL-induced nuclear translocation of NF-. kappa.B/p 65 by inhibiting phosphorylation of I.kappa.B.alpha.. In addition, Co-IP assays indicate that BCI can attenuate the interaction of NF-. kappa.B/p 65 with NFATc 1. Taken together, we speculate that BCI inhibits the expression of NFATc1 by inhibiting the activation and nuclear translocation of NF- κ B/p65, thereby inhibiting the formation of osteoclasts. MAPK signaling plays a key role in osteoclastogenesis. However, western blot results in the present invention show that BCI inhibition of osteoclast differentiation is independent of MAPK signaling pathway. Furthermore, we found that BCI treatment attenuated the activity of RANKL-mediated STAT3 signaling in osteoclastogenesis, suggesting that BCI may inhibit c-Fos and NFATc1 expression in osteoclastogenesis by inhibiting the activity of NF- κ B and STAT 3.

The in vivo effect of BCI on osteoporosis was assessed in a mouse model. Micro-CT data show that BCI treatment can increase bone mass and bone density by increasing OVX osteoporosis mice BS/TV, BV/TV, tb.n and BMD. In addition, TRAP staining of bone tissue sections of mice showed a significant reduction in the number of TRAP + mature osteoclasts in distal trabecular tissue of femurs after BCI treatment, and no significant hepatotoxicity and nephrotoxicity was observed in mice of the BCI treated group.

Claims (1)

1. Use of a compound BCI, said compound BCI being in the E configuration, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the prevention and treatment of osteoporosis.

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