Application of MMA-CMA pathway as target spot in preparation of medicine for preventing or treating bone loss

Abstract

The invention belongs to the technical field of medicines, and particularly discloses application of an MMA-CMA pathway as a target spot in preparation of a medicine for preventing or treating bone loss. The invention discloses the effect and mechanism that MMA (methylmalonic acid) inhibits osteogenic differentiation of mesenchymal stem cells by inhibiting a molecular chaperone-mediated autophagy (CMA) function and finally causes bone loss for the first time; meanwhile, in-vivo effective treatment of bone loss is carried out by utilizing a CMA activator. Compared with an existing bone loss treatment medicine, the medicine has the advantage of multi-target regulation and control, not only can aim at the pathological effect of MMA, but also can promote bone formation by activating CMA, and a brand new intervention thought and a medicine development target are provided for treatment of bone loss related diseases such as osteoporosis.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to the use of an MMA-CMA pathway as a target in the preparation of a drug for preventing or treating bone loss. Background Art

[0002] Bone loss refers to a pathological process characterized by a decrease in bone mass or destruction of bone microarchitecture. Its core mechanism is a disruption in the dynamic balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption. When bone resorption exceeds bone formation, it can lead to conditions such as osteoporosis, increased fracture risk, and osteoarthritis. Studies have shown that impaired osteoblast differentiation (such as blocked osteoblast-to-osteoblast differentiation from mesenchymal stem cells) is a key factor in bone loss. This process is regulated by multiple pathological conditions, including metabolic abnormalities, inflammation, and oxidative stress.

[0003] Currently, strategies for the prevention and treatment of bone loss primarily focus on inhibiting osteoclast activity (e.g., bisphosphonates, RANKL inhibitors) or promoting osteoblast function (e.g., PTH analogs, Wnt pathway activators). These drugs have single targets, making it difficult to comprehensively regulate bone metabolic imbalances: most drugs target only a single link in bone resorption or formation and are unable to simultaneously correct the "osteogenesis-osteoclast" imbalance. Long-term use may lead to side effects (e.g., mandibular osteonecrosis, cardiovascular risks). The mechanisms of bone loss are complex, involving the cross-talk of multiple factors, including metabolism, inflammation, and oxidative stress, and existing therapies do not fully integrate these regulatory networks. Therefore, current treatment strategies for bone loss still have limitations, and exploring new regulatory targets is of great significance.

[0004] Methylmalonic acid (MMA) is a byproduct of the methylmalonyl-CoA metabolic pathway. Its accumulation is closely associated with various systemic diseases, particularly vitamin B12 deficiency, mitochondrial dysfunction, and aging. Recent studies have found that MMA is significantly elevated in metabolic disorders such as diabetes and cardiovascular disease and can induce oxidative stress and cellular dysfunction. However, whether MMA directly regulates bone loss, particularly through its effects on osteoblast differentiation or bone metabolic homeostasis, remains unclear. Chaperone-mediated autophagy (CMA) is a key pathway for selective degradation of intracellular proteins, relying on the molecular chaperone Hsc70 to recognize substrate proteins and transport them through the lysosomal membrane protein LAMP-2A. Dysfunction of CMA is closely associated with neurodegenerative diseases, metabolic syndrome, and age-related diseases. In the field of bone metabolism, recent studies have suggested that CMA may influence bone homeostasis by regulating osteoblast differentiation or osteoclast activity. However, the specific role of CMA in bone loss and its interaction with MMA remain largely unexplored.

[0005] In summary, whether MMA participates in bone loss by inhibiting osteoblast differentiation, whether CMA plays a protective or aggravating role in MMA-induced bone loss, and whether targeted CMA regulation can become a new strategy to intervene in MMA-related bone loss, have not yet been studied. Summary of the Invention

[0006] The purpose of the present invention is to select methylmalonic acid as a research object, explore its role in regulating osteoblast differentiation and the key mechanism involved in CMA, and explore the efficacy of methylmalonic acid-CMA intervention in treating and slowing bone loss, and provide an application of methylmalonic acid-CMA intervention to develop a new strategy for treating bone loss. This application aims to solve the technical problem that drug treatment in the prior art is not effective in preventing or treating bone loss.

[0007] In order to achieve the above object, the specific technical solutions adopted by the present invention are as follows:

[0008] In a first aspect, the present invention provides a use of methylmalonic acid (MMA) in preparing a preparation for inhibiting chaperone-mediated autophagy (CMA) function in bone loss.

[0009] This invention proposes for the first time the hypothesis that MMA inhibits osteogenic differentiation by inhibiting CMA function, and explores its application value as a target for the treatment of bone loss. Experiments confirm the role and mechanism of MMA in inhibiting CMA and thereby inhibiting the osteogenic differentiation of mesenchymal stem cells, ultimately leading to bone loss, revealing the molecular relationship between MMA and CMA.

[0010] In a second aspect, the present invention provides the use of the MMA-CMA pathway as a target in the preparation of a drug for preventing or treating bone loss.

[0011] By targeting the MMA-CMA pathway and activating CMA function, this approach promotes bone formation while reversing MMA's inhibitory effect on osteoblastic differentiation, thereby alleviating symptoms of bone loss. By analyzing the molecular axis of MMA-CMA-osteoblastic differentiation and targeting bone loss, this study provides new intervention strategies and drug development targets for osteoporosis and aging-related bone diseases.

[0012] In a third aspect, the present invention provides use of a CMA activator in the preparation of a medicament for preventing or treating bone loss.

[0013] Furthermore, the CMA activator is CA77.1. CA77.1 acts on the MMA-CMA pathway, activates CMA function to promote bone formation, and reverses the inhibitory effect of MMA on osteogenic differentiation, thereby alleviating MMA-induced bone loss.

[0014] Furthermore, the drug contains CA77.1 as an active ingredient.

[0015] In a fourth aspect, the present invention provides a pharmaceutical composition for treating, preventing, or treating bone loss, comprising an active ingredient, wherein the active ingredient is a CMA activator, and a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means that the carrier does not produce adverse, allergic, or other untoward reactions when properly administered to an animal or human. The pharmaceutically acceptable carrier may be selected from one or more of a solvent, a diluent, a filler, a surfactant, an absorption enhancer, a disintegrant, a wetting agent, a dispersant, and the like.

[0016] Furthermore, the CMA activator is CA77.1.

[0017] The present invention has the following beneficial effects:

[0018] This invention reveals for the first time the molecular mechanism by which methylmalonic acid (MMA) inhibits the chaperone-mediated autophagy (CMA) function, thereby hindering the osteogenic differentiation of mesenchymal stem cells and ultimately leading to bone loss. Experiments have confirmed that MMA treatment can significantly reduce the expression level of LAMP2A, a key CMA marker, while inhibiting the osteogenic differentiation ability of bone marrow mesenchymal stem cells (BMMSCs), as manifested by reduced calcium nodule formation, decreased alkaline phosphatase (ALP) activity, and decreased expression of osteogenic marker genes. In animal models, MMA administration resulted in a significant decrease in bone mineral density (BMD) and bone volume fraction (BV / TV) in both young and old mice, confirming that MMA can induce bone loss both in vivo and in vitro.

[0019] More importantly, the present invention utilizes a CMA activator to effectively treat bone loss in vivo. This study found that activating CMA function with the CMA activator CA77.1 can effectively reverse the inhibitory effects of MMA on osteoblastic differentiation. In vitro experiments demonstrated that CA77.1 treatment significantly improved the osteoblastic differentiation phenotype inhibited by MMA. In an aged mouse model, CA77.1 administration effectively alleviated MMA-induced bone loss.

[0020] These results not only elucidate the mechanism of action of the novel regulatory axis of MMA-CMA-osteogenic differentiation in bone loss but also innovatively propose a novel therapeutic strategy for targeted intervention in this pathway. Compared to existing bone loss treatments, this invention offers the advantage of multi-target regulation, addressing both the pathological effects of MMA and promoting bone formation through CMA activation. This provides a novel intervention strategy and drug development target for the treatment of osteoporosis and other bone loss-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1Methylmalonic acid (MMA) promotes femoral bone loss in young and aged mice. From left to right, micro-CT analysis of cancellous bone, bone mineral density (BMD), and bone volume percentage (BV / TV) was performed in young (2-month-old) and aged (16-month-old) mice after vehicle administration and MMA administration. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01.

[0022] Figure 2 MMA inhibits osteogenic differentiation of mesenchymal stem cells in vitro. (A) Rat MSCs were induced to differentiate into osteoblasts in vitro and stimulated with different concentrations of MMA. ARS staining was performed 10 days after induction and ALP staining was performed 5 days after induction. (B) Quantitative analysis of calcium nodules and ALP activity. (C) Q-PCR analysis of the expression of osteogenic differentiation-related genes Sp7, Alp, Runx2, and Bglap using RNA collected 5 days after induction. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

[0023] Figure 3 MMA inhibits CMA and CMA activators inhibit MMA-induced bone loss. (A) Bone marrow mesenchymal stem cells isolated from SD rats were induced to differentiate into osteoblasts in vitro. MMA was added to the differentiation system, and expression of LAMP2a, a key CMA protein, was assessed by Western blot. (B) Bone marrow mesenchymal stem cells isolated from SD rats were induced to differentiate into osteoblasts in vitro. MMA and the CMA activator CA77.1 were added to the differentiation system for 10 days, and ARS staining and calcium nodule quantification were performed. (C) Three-dimensional imaging of cancellous bone, bone mineral density (BMD), and bone volume percentage (BV / TV) were analyzed by microCT analysis in aged (16-month-old) mice after intraperitoneal administration of a control (vehicle) and methylmalonic acid (MMA), along with CA77.1 and a control vehicle. Data are expressed as mean ± SD. *p < 0.05, **p < 0.01. DETAILED DESCRIPTION

[0024] The existing technologies have problems such as the unclear mechanism of action of methylmalonic acid (MMA) in bone loss and the lack of regulation of molecular chaperone-mediated autophagy (CMA), as follows: (1) The mechanism of action of MMA in bone metabolism is not yet clear: Although MMA accumulates in aging and metabolic diseases, there is currently a lack of direct evidence to prove whether it is directly involved in bone loss and how it affects osteoblast differentiation and CMA function. Previous studies have focused on the neurotoxicity or metabolic toxicity of MMA, while ignoring its effects on bone tissue; at the same time, there are insufficient in vitro and in vivo models of the interaction between MMA and osteocytes. (2) There is a lack of intervention strategies targeting the MMA-CMA axis: The molecular association between MMA and CMA has not been revealed, and the application of CMA activators / inhibitors in bone diseases has not been explored.

[0025] To this end, the inventors solved the problem through the following technical routes: (1) Clarify the regulatory effect of MMA on osteogenic differentiation and bone loss: Through in vitro (bone marrow mesenchymal stem cell osteogenic induction model) and in vivo (MMA accumulation animal model) experiments, detect the effect of MMA on osteogenic differentiation markers (such as Runx2, ALP, OCN), and combine Micro-CT to analyze bone microstructure changes to determine whether MMA directly inhibits bone formation and promotes bone loss. (2) Reveal the key role of CMA in MMA-mediated bone loss: By monitoring CMA activity indicators (LAMP-2A expression), evaluate whether MMA inhibits CMA function. Use CMA activators (such as CA77.1) to verify whether CMA restoration can reverse MMA-induced osteogenic differentiation disorders. (3) Develop intervention strategies targeting the MMA-CMA pathway: Explore the in vivo effect of CMA activators in resisting bone loss.

[0026] The present invention will be further described below with reference to the accompanying drawings and specific examples. The information of the drugs, reagents and consumables used in the following examples is as follows:

[0027] Methylmalonic acid (MMA, Sigma), osteogenic differentiation solution (Ciye Biosciences), DMSO (Sigma), CA77.1 (Selleck), α-MEM medium (Hyclone), fetal bovine serum (Thermo Fisher Scientific), penicillin / streptomycin double antibody solution (Gibico), standard PCR kit (Roche), real-time PCR primers (Sangon Biosciences), Tween-80 (Sigma), PEG300 (Selleck), ARS staining solution (Ciye Biosciences), and ALP staining kit (Beyotime Biosciences). MMA was dissolved in water, and CA77.1 was dissolved in DMSO.

[0028] Example 1: Effects of MMA on bone loss and osteogenic differentiation of BMMSCs

[0029] To investigate the effects of MMA on bone loss, young (2-month-old) and old (16-month-old) C57BL / 6 male mice were administered MMA via drinking water for 2 months. At the end of the experiment, the mice were euthanized and samples were collected for subsequent experiments. Femora were collected and fixed, and micro-CT scans were performed for 3D reconstruction and data analysis. Bone loss in the distal femoral cancellous bone was observed using 3D imaging, as well as bone morphological parameters such as bone mineral density (BMD) and bone volume fraction (BV / TV).

[0030] The results are as follows Figure 1 As shown in the data, after MMA administration in young and old mice, the three-dimensional structure of cancellous bone was significantly sparse, and the BMD and BV / TV levels were significantly reduced, showing severe bone loss, indicating that MMA can cause bone loss in mice.

[0031] ② Bone marrow mesenchymal stem cells (BMMSCs) were isolated from the bone marrow of 4-week-old SD rats and successfully identified by surface markers and trichodermal differentiation. BMSCs were induced to differentiate into osteoblasts in vitro. MMA (0, 0.1, 0.5, 1, and 5 mM) was added to the differentiation system. After 10 days of induction, ARS staining was performed to quantify calcium nodules and assess the inhibitory effect of MMA on late mineralization. ALP staining was performed after 5 days of induction to quantify ALP activity and assess the inhibitory effect of MMA on early osteogenesis. RNA was collected after 5 days of induction and analyzed by Q-PCR for the expression of osteogenic differentiation-related genes Sp7, Alp, Runx2, and Bglap.

[0032] The results are as follows Figure 2 As shown, rat MSCs were induced to differentiate into osteoblasts in vitro and stimulated with MMA. After MMA stimulation, calcium nodules and ALP activity decreased ( Figure 2 A and B), decreased expression of osteoblast markers ( Figure 2 C), indicating that MMA can inhibit osteogenic differentiation.

[0033] Example 2: Efficacy of CMA activators on MMA-induced bone loss

[0034] ①Bone marrow mesenchymal stem cells were isolated from SD rats and induced to differentiate into osteoblasts in vitro. MMA (1 mM) was added to the differentiation system. After 5 days of induction, the expression of the key CMA protein LAMP2a was detected by Western blot.

[0035] The results are as follows Figure 3 As shown in A, rat MSCs were induced to differentiate into osteoblasts in vitro and stimulated with MMA. After MMA stimulation, LAMP2A, a key marker of CMA, decreased, indicating that MMA inhibited the function of CMA.

[0036] ② A solution consisting of 10% DMSO, 5% Tween-80, 40% PEG-300, and 45% saline (% by volume) was prepared as a solvent, and the CMA activator CA77.1 was dissolved to the desired working concentration. Bone marrow mesenchymal stem cells (BMMSCs) isolated from SD rats were induced to differentiate into osteoblasts in vitro. MMA (1 mM) and the CMA activator CA77.1 were added to the differentiation system (the control group received MMA + DMSO treatment). After 10 days of induction, ARS staining and calcium nodule quantification were performed to analyze the role of CMA in MMA-regulated BMMSC osteogenic differentiation.

[0037] The results are as follows Figure 3 As shown in B, after CMA was given to stimulate CA77.1 on the basis of MMA treatment, the osteogenic differentiation inhibited by MMA was significantly alleviated.

[0038] To investigate the effects of CMA activation on MMA-induced bone loss and aging-related bone loss, aged (16-month-old) C57BL / 6 male mice were administered intraperitoneally with DMSO and CA77.1 for 3 months. After one month of CA77.1 administration, control (vehicle) and MMA were administered via drinking water until the end of the experiment. At the end of the experiment, mice were euthanized, and femurs were removed for microCT analysis, which revealed three-dimensional cancellous bone imaging and quantitative analysis of bone morphological parameters, including bone mineral density (BMD) and bone volume percentage (BV / TV).

[0039] The results are as follows Figure 3 As shown in C, old mice were administered CA77.1 simultaneously with MMA, showing that the BMD and BV / TV decreased by MMA were alleviated, indicating that CA77.1 can alleviate age-related and MMA-aggravated bone loss in mice.

[0040] The results of the above examples show that methylmalonic acid can induce bone loss by inhibiting the osteogenic differentiation of mesenchymal stem cells; methylmalonic acid limits the osteogenic differentiation of mesenchymal stem cells by inhibiting CMA; activation of CMA can alleviate methylmalonic acid-related bone loss, and targeted intervention of methylmalonic acid to regulate CMA is a potential new strategy for the treatment of bone loss-related diseases.

[0041] The specific embodiments described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above are only specific embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. Use of the MMA-CMA pathway as a target in the preparation of a drug for preventing or treating bone loss, characterized in that: By activating CMA function, it promotes bone formation while reversing the inhibitory effect of MMA on osteogenic differentiation, thereby alleviating bone loss symptoms.

2. Application of CMA activators in the preparation of drugs for preventing or treating bone loss.

3. The use according to claim 2, characterized in that The CMA activator is CA77.

1.

4. The use according to claim 3, characterized in that The CA77.1 acts on the MMA-CMA pathway, activates the CMA function to promote bone formation, and at the same time reverses the inhibitory effect of MMA on osteogenic differentiation, thereby alleviating MMA-induced bone loss.

5. Application of MMA in the preparation of preparations that inhibit the function of CMA in bone loss.

6. A pharmaceutical composition for treating, preventing or treating bone loss, characterized in that: The invention comprises an active ingredient and a pharmaceutically acceptable carrier, wherein the active ingredient is a CMA activator.

7. The pharmaceutical composition according to claim 6, characterized in that The CMA activator is CA77.1.

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