Application of Visomitin in preparation of medicine for reducing bone mass loss

Abstract

The invention discloses an application of Visomittin in preparation of a medicine for reducing bone mass loss, and through in-vitro and in-vivo experiments, it is revealed for the first time that Visomittin can inhibit differentiation of bone marrow-derived macrophages (BMMs), further inhibit generation of osteoclasts (OC) and reduce bone resorption, and the application of the Visomittin in preparation of the medicine for reducing bone mass loss in preparation of the medicine for reducing bone mass loss in preparation of the medicine for reducing bone mass loss in preparation of the medicine for reducing bone mass loss in preparation of the medicine for reducing bone mass loss in preparation of the medicine for reducing bone mass loss in preparation of the medicine for reducing bone mass loss in preparation of the medicine for reducing bone mass loss in preparation of the medicine for reducing bone mass loss. Visomittin directly interacts with a signal transduction and transcriptional activator 3 (STAT3) to inhibit phosphorylation and nuclear transport of the STAT3, so that expression of lactic dehydrogenase B (LDHB) is inhibited, energy generation is reduced, osteoclast differentiation is inhibited, and the effect of reducing bone mass loss is achieved. The invention provides a theoretical basis and experimental support for developing a novel bone mass loss treatment medicine and a novel STAT3 / LDHB pathway inhibitor.

Description

Technical Field

[0001] The present invention belongs to the field of biomedical technology, and particularly relates to the use of Visomitin in preparing a drug for reducing bone loss, and the use of STAT3 / LDHB as a target for treating osteoporosis. Background Art

[0002] Osteoporosis is a systemic metabolic disease whose pathogenesis is characterized by bone resorption exceeding bone formation, leading to bone loss, microarchitectural disruption, and increased bone fragility. Osteoclasts (OCs), the primary effector cells of bone resorption, require substantial energy for differentiation and rely on RANKL signaling to enhance glycolysis and oxidative phosphorylation. OC differentiation and activity are meticulously regulated by multiple cytokines and signaling pathways. Recent studies have revealed that mitochondrial dysfunction is closely associated with abnormal bone metabolism. Mitochondria serve as the cell's energy source and regulator of redox homeostasis. Mitochondria dysfunction can lead to excessive production of reactive oxygen species (ROS), which in turn impair the normal physiological function of bone cells, promote OC differentiation and activity, and inhibit the function of osteoblasts (OBs).

[0003] Visomitin, also known as SkQ1, is a mitochondrial-targeted antioxidant with CAS number 934826-68-3. Its chemical structure and molecular 3D model can be seen Figure 1 Visomitin can localize to mitochondria, scavenge mitochondrial ROS, and improve mitochondrial metabolism. Therefore, it has potential uses in the treatment of various ocular diseases, including dry eye disease (DED). However, the role of visomitin in bone metabolism has not been fully studied, and its application in other fields is still under further exploration. Summary of the Invention

[0004] The purpose of the present invention is to address the deficiencies of the existing technology and provide the application value of Visomitin in reducing bone loss.

[0005] In order to achieve the above object, the present invention adopts the following technical solutions: In a first aspect, the present invention provides use of Visomitin in preparing a drug for inhibiting bone loss.

[0006] Preferably, in in vitro experiments, Visomitin can reduce ROS accumulation in bone marrow-derived macrophages (BMMs) without affecting cell activity and inhibit the differentiation of BMMs into OCs.

[0007] As a preference, in in vivo experiments, Visomitin can alleviate bone loss in mouse osteolysis models and estrogen deficiency osteoporosis models, and reduce tissue peroxide levels; at the same time, it does not affect OB differentiation and bone formation.

[0008] Preferably, Visomitin acts on signal transducer and activator of transcription 3 (STAT3), inhibiting the phosphorylation and nuclear translocation of STAT3 during OC differentiation, thereby inhibiting the expression of lactate dehydrogenase B (LDHB), reducing energy production, inhibiting OC differentiation, and reducing bone resorption.

[0009] In a second aspect, the present invention provides use of Visomitin in preparing a cell differentiation inhibitor.

[0010] Preferably, the inhibitor is used to inhibit the differentiation of BMMs into OCs.

[0011] Preferably, the inhibitor reduces ROS accumulation in BMMs.

[0012] In a third aspect, the present invention provides use of Visomitin in preparing an LDHB expression inhibitor.

[0013] In a fourth aspect, the present invention provides a non-therapeutic method for inhibiting the differentiation of BMMs into OCs, wherein the method comprises placing BMMs in a Visomitin administration environment.

[0014] Compared with the prior art, the present invention has the following beneficial effects: By constructing mouse models of osteolysis and estrogen deficiency osteoporosis and conducting multiple in vitro cellular and molecular experiments, the present invention demonstrates for the first time that visomitin inhibits OC production and reduces bone resorption both in vivo and in vitro, thereby preventing the development and progression of osteoporosis. Furthermore, the present invention demonstrates that visomitin can directly bind to STAT3 and inhibit its activity, thereby reducing LDHB expression, lowering cellular glycolysis and energy production, and thereby inhibiting OC differentiation and delaying the development and progression of osteoporosis. Furthermore, the present invention provides insights into the role of visomitin in inhibiting OC differentiation, LDHB expression, and STAT3 activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the chemical structural formula of Visomitin.

[0016] Figure 2 Visomitin can reduce ROS accumulation in BMMs without affecting cell activity; A is ROS staining, B is quantitative analysis of A, C is mitochondrial superoxide staining, and D is quantitative analysis of C, ***: p<0.001, **: p<0.01.

[0017] Figure 3 Visomitin can inhibit OC differentiation and reduce bone loss in the mouse osteolysis model and estrogen deficiency osteoporosis model; A is tartrate-resistant acid phosphatase (TRAP) staining after co-treatment of BMMs with different concentrations of Visomitin and RANKL, B is quantitative analysis of A, C is TRAP staining after co-treatment of BMMs with 300 nm Visomitin and RANKL for different time periods, D is quantitative analysis of C, E is quantitative analysis of Micro-CT scanning results of osteolysis model, F is quantitative analysis of Micro-CT scanning results of osteoporosis model, ***: p<0.001, **: p<0.01, *: p<0.05.

[0018] Figure 4 Visomitin does not affect OB differentiation and bone mineralization. A is alkaline phosphatase (ALP) staining, and B is alizarin red (ARS) staining.

[0019] Figure 5Visomitin directly interacts with STAT3, inhibiting its phosphorylation and nuclear translocation during OC formation; A is a three-dimensional predicted simulation diagram of the interaction between Visomitin and STAT3, B is the Western Blot results after BMMs were co-treated with different concentrations of Visomitin and RANKL, C is STAT3 immunofluorescence staining, and D is the quantitative analysis of C, where ***: p < 0.001, *: p < 0.05.

[0020] Figure 6 LDHB overexpression can rescue the inhibition of OC differentiation by Visomitin, but does not affect normal OC differentiation; A shows whether LDHB is overexpressed in BMMs and TRAP staining after RANKL treatment; B shows the quantitative analysis of A, where *: p<0.05; C shows the Western Blot results.

[0021] Figure 7 Visomitin can reduce LDHB expression, lower cellular glycolysis levels, and reduce ATP production. A shows the LDHB RNA level in BMMs with and without co-treatment of STAT3 activator with Visomitin and RANKL. B shows the WB results. C shows the JC-1 mitochondrial membrane potential measurement results after co-treatment of BMMs with Visomitin and RANKL. D shows the ATP content measurement results. E and F show the Seahorse experiment results. **: p<0.01, *: p<0.05. DETAILED DESCRIPTION

[0022] The technical solution of the present invention is further explained below in conjunction with the accompanying drawings and embodiments, but the experimental conditions and setting parameters therein should not be regarded as limiting the basic technical solution of the present invention.

[0023] Example 1: Visomitin can reduce ROS accumulation in BMMs 1. Bone Marrow Cell Isolation and OC Differentiation: Bone marrow cells were harvested from the tibia and femur of 6-week-old C57BL / 6 mice, followed by flushing the bone marrow cavity with a 1 mL syringe. The harvested cells were lysed for erythrocytes and then cultured for 4 days in α-MEM medium supplemented with 25 ng / mL mouse M-CSF. Bone marrow cells were plated at an optimal density and stimulated with OC differentiation medium prepared in α-MEM supplemented with 25 ng / mL M-CSF and 50 ng / mL RANKL until mature multinucleated osteoclasts were formed.

[0024] 2. ROS determination: ROS levels were measured using a ROS detection kit (S0033S, Beyotime, China). BMMs were plated at appropriate densities and exposed to different treatment conditions. The culture medium was replaced with 10 μmol / L DCFH-DA working solution and the cells were cultured. Subsequently, the cells were rinsed with PBS to remove residual DCFH-DA. Fluorescence images were captured using a fluorescence microscope, and the relative mean fluorescence intensity was quantified using ImageJ software for significance analysis. Figure 2 As shown in Figures A and B, after RANKL treatment, the ROS level in BMMs increased, while ROS in BMMs pretreated with Visomitin was significantly reduced.

[0025] 3. Mitochondrial superoxide assay: The assay was performed using the MitoSOX detection kit (S0061S, Beyotime, China). Pretreated BMMs were incubated with 5 μM MitoSOX Red staining solution for 30 minutes. The cells were then rinsed with PBS to ensure complete removal of any excess stain. Fluorescence images were captured using a fluorescence microscope, and the relative mean fluorescence intensity was quantified using ImageJ software for significance analysis. Figure 2 As shown in Figures C and D, after RANKL treatment, the level of mitochondrial superoxide in BMMs increased, while mitochondrial superoxide in BMMs pretreated with Visomitin was significantly reduced.

[0026] Example 2: Visomitin can inhibit OC differentiation and reduce bone loss 1. TRAP staining: Fix fully differentiated osteoclasts with 4% paraformaldehyde (PFA). Then, discard the PFA and wash the cells three times with PBS buffer. Then, stain with the TRAP kit (G1492, Solarbio, China). Figure 3 As shown in Figures AD, Visomitin dose-dependently inhibited OC differentiation of BMMs.

[0027] 2. Mouse skull osteolysis model: 12-month-old male mice were randomly divided into groups and anesthetized with an intraperitoneal injection of 30 mg / kg pentobarbital. Subsequently, a skull osteolysis model was established with a subcutaneous injection of 25 mg / kg LPS. A sham-operated group underwent the same surgical procedure, except that an equal volume of saline was used instead of LPS. PBS or varying concentrations of Visomitin were administered orally starting two days after surgery. Seven days after surgery, skull bones were harvested for subsequent experiments.

[0028] 3. Mouse model of estrogen deficiency osteoporosis: 12-month-old female mice were randomly divided into groups and anesthetized with an intraperitoneal injection of 30 mg / kg pentobarbital. In the prone position, an incision was made at the midpoint of the iliac crest line. The pelvic cavity was accessed bilaterally, the ovaries were removed, and hemostasis was achieved with hemostats. The incisions were then disinfected and sutured. The sham surgery group underwent only skin and pelvic incisions, but no ovariectomy. Four weeks after surgery, mice were administered PBS or various concentrations of Visomitin. Four weeks later, the mice were euthanized, and bone tissue was collected for subsequent experiments.

[0029] Micro-CT Scanning: Mouse skull and femur specimens were fixed in 4% paraformaldehyde for 48 hours, then deveined of overlying muscle tissue and immersed in EP tubes containing 75% ethanol. CT scanning was performed using a SkyScan 1275 (rukermicroCT, Kontich, Belgium) with X-ray energy of 60 kV and intensity of 60 mA. Data were analyzed using DataViewer software (version 1.5.6.2) and CTan software (version 1.20.8.0) to quantitatively assess bone morphological parameters. Finally, representative three-dimensional reconstructions were created using CTvox software (version 3.3).

[0030] like Figure 3 As shown in EF, the bone mass of the sham group model mice was significantly reduced, and Visomitin rescued the bone mass reduction of the model mice in a dose-dependent manner.

[0031] Example 3: Visomitin does not affect OB differentiation and bone mineralization Osteoprogenitor cell extraction and OB differentiation: The skull was collected from 3-day-old C57BL / 6 mice, and the excess soft tissue and periosteum were removed. After cutting into small pieces, the cells were digested with type II collagenase overnight. The next day, the cell suspension obtained by digestion was filtered and inoculated into α-MEM medium, and then cultured in a sterile incubator at 37°C and 5% CO2. The obtained osteoprogenitor cells were plated at an optimal density and cultured for 7 days or 21 days in α-MEM medium containing 50µM ascorbic acid, 10 mM β-glycerophosphate and 100 nM dexamethasone. Subsequently, alkaline phosphatase (ALP) and Alizarin Red S (ARS) were used to evaluate the degree of OB differentiation and mineralization. As Figure 4 As shown in the results, Visomitin treatment did not affect OB differentiation and bone mineralization.

[0032] Example 4: Visomitin can directly interact with STAT3, inhibiting its phosphorylation and nuclear translocation during OC formation 1. Molecular modeling and molecular docking: The 3D structure of visomitin was retrieved from the PubChem database and then imported into Chem 3D v20.0 software for further structural refinement. PyMOL v2.5.4 was used to examine the amino acid residues involved in the hydrogen bonding between STAT3 and visomitin to create a three-dimensional interaction map: the orange double helix represents DNA, the yellow stick model depicts visomitin; the brown sphere represents Br - ions; the blue cartoon shows chain A of STAT3, while the purple cartoon shows chain B. Figure 5 As shown in A.

[0033] 2. Western Blot: After pretreatment, the cells were decellularized, washed three times with PBS, and lysed at 4°C for 30 minutes using RIPA lysis buffer (FD009, FDBIO, China) supplemented with a phosphatase inhibitor cocktail (FD1002, FDBIOs, China), 100 mM phenylmethanesulfonyl fluoride (FD0100, FDBIO, China), and a protease inhibitor cocktail (FD1001, FDBIOS, China). Lysates were then collected and centrifuged at 12,000 rpm for 10 minutes at 4°C. The supernatant was collected and protein quantified using a BCA protein assay kit (P0012, Beyotime, China). The extracted proteins were separated by SDS-PAGE gel electrophoresis and transferred to a PVDF membrane using the eBlot™ L1 Fast Wet Transfer System (L00686C, Genscript, China). The membrane was then blocked with 5% skim milk for 1 hour at room temperature. The membrane was then incubated with the specific primary antibody overnight at 4°C. On the second day, the PVDF membrane was incubated with the corresponding secondary antibody at room temperature for 1 hour. After each step, the membrane was rinsed three times with TBST buffer. Finally, the results were visualized using an eBlot Touch Imager™ (e-BLOT XLI, Genscript, China). Figure 5 As shown in Figure B, after RANKL treatment, nuclear STAT3 and phosphorylated STAT3 increased, and Visomitin dose-dependently reduced nuclear STAT3 and STAT3 phosphorylation.

[0034] 3. Immunofluorescence staining: Plate the cells at an appropriate density on sterile cell culture slides. After experimental treatment, remove the culture medium and fix with 4% paraformaldehyde; then permeabilize with 1% Triton X-100; block with 5% bovine serum albumin (BSA), and incubate with primary antibody at 4°C overnight; the next day, incubate with fluorescent-labeled secondary antibody at room temperature for 1 hour; then use DAPI dye for nuclear staining; finally, seal the slide with anti-fluorescence quenching sealing fluid. Rinse with PBS buffer 3 times after each of the above steps. Finally, use a fluorescence or confocal microscope to capture images and conduct detailed analysis of the slides. Figure 5 As shown in Figures C and D, RANKL treatment increased the nuclear localization of STAT3, and Visomitin dose-dependently reduced the nuclear localization of STAT3.

[0035] Example 5: Visomitim inhibits OC differentiation and can be partially rescued by LDHB overexpression Lentiviral infection: The LDHB adenovirus used in this study was purchased from Genechem. For adenoviral infection, BMMs were initially seeded and cultured at an appropriate density. Subsequently, a mixture of adenovirus and polyisoprene (H8761, Solarbio, China) was added at an appropriate multiplicity of infection (MOI) for infection. After an 8-hour infection period, the culture medium was replaced with fresh medium.

[0036] like Figure 6 As shown in Figures A and B, Visomitim treatment inhibited OC differentiation, which could be partially rescued by overexpression of LDHB; overexpression of LDHB alone had no significant effect on OC differentiation.

[0037] Example 6: Visomitin can reduce the generation of LDHB in BMMs, lower the level of cellular glycolysis, and reduce ATP production.

[0038] 1. RT-qPCR: RNA was extracted from cells using the Ultrapure RNA Kit (#CW0581, CWBIO, China). The extracted mRNA was then reverse transcribed into cDNA using the Evo M-MLV RT Kit (AG11705, Accurate Biology, China). For RT-qPCR analysis, cDNA was amplified by mixing it with a forward primer, a reverse primer, and SYBR Green Master Mix (11201ES08, Yeasen, China) in optimized ratios. Quantification was performed on an ABI Prism 7500 system (Applied Biosystems, USA). Actb (β-actin) was used as a reference gene, and the 2-ΔΔCt method was used for internal normalization.

[0039] like Figure 6 As shown in A and 6B, LDHB expression was reduced at both the mRNA and protein levels after treatment with Visomitin, and LDHB expression recovered after co-treatment with Colivelin (a STAT3 agonist).

[0040] 2. Mitochondrial membrane potential (MMP) assay: MMP assay was performed using the JC-1 fluorescent probe (C2006, Beyotime, China). Specifically, BMMs were cultured in confocal culture dishes and subjected to osteoclast differentiation with or without Visomitin. Subsequently, 1× JC-1 staining solution was added and the cells were incubated at 37°C for 20 minutes. Then, after carefully removing the unbound JC-1 probe by washing with PBS, imaging was performed using a Nikon A1-Ti confocal microscope. The relative mean fluorescence intensity was analyzed using ImageJ software to determine the ratio of aggregates to monomers. Figure 7 As shown in Figure C, MMP increased after RANKL treatment, and decreased after co-treatment with Visomitin.

[0041] 3. ATP assay: ATP assay kit (S0027, Beyotime, China) was used to evaluate BMMs treated under different conditions. Figure 7 As shown in D, ATP levels increased after RANKL treatment, while they decreased after co-treatment with Visomitin.

[0042] 4. Seahorse experiment: The oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured using the Seahorse XF Cell Mitochondrial Stress Kit (103015-100, Agilent, USA) and the Seahorse XF Cell Glycolysis Stress Test Kit (103020-100, Agilent, USA). Figure 7 As shown in Figures E and H, RANKL increased basal OCR, maximal OCR, ATP production, glycolysis level, and maximal glycolytic capacity; co-treatment with Visomitin reduced these levels.

[0043] The above are only some preferred embodiments of the present invention, and the present invention is not limited to the above embodiments. Those skilled in the art who, based on their understanding of the technical solution of the present invention, make modifications or equivalent substitutions without departing from the spirit or scope of the technical solution of the present invention shall be included in the scope of protection of the present invention.

Claims

1. Application of Visomitin in the preparation of drugs for inhibiting bone loss.

2. The use according to claim 1, characterized in that Visomitin reduces the accumulation of reactive oxygen species in bone marrow-derived macrophages without affecting cell activity and inhibits the differentiation of bone marrow-derived macrophages into osteoclasts.

3. The use according to claim 2, characterized in that Visomitin acts on the STAT3 transcription factor, inhibiting the phosphorylation and nuclear translocation of signal transduction and activator of transcription factor 3 during osteoclast differentiation, thereby inhibiting the expression of lactate dehydrogenase B, reducing energy production, inhibiting osteoclast differentiation, and reducing bone resorption.

4. The use according to claim 1, characterized in that Visomitin reduces tissue peroxide levels without affecting osteoblast differentiation and bone formation.

5. Application of Visomitin in the preparation of osteoclast differentiation inhibitors.

6. The use according to claim 5, characterized in that The inhibitor is used for inhibiting the differentiation of bone marrow-derived macrophages into osteoclasts.

7. The use according to claim 6, characterized in that The inhibitor reduces the accumulation of reactive oxygen species in bone marrow-derived macrophages.

8. Application of Visomitin in the preparation of lactate dehydrogenase B expression inhibitors.

9. A method for inhibiting the differentiation of bone marrow-derived macrophages into osteoclasts for non-therapeutic purposes, characterized in that: The following steps are involved: Bone marrow-derived macrophages were placed in a Visomitin administration environment.

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