Application of lactoferrin as antioxidant in preparation of medicine for treating bone homeostasis imbalance disease
By using lactoferrin as an antioxidant to regulate osteoblast and osteoclast differentiation under oxidative stress, the problem of existing drugs being unable to simultaneously intervene in multiple pathogenic factors is solved, achieving effective regulation of bone homeostasis and cost reduction.
Patent Information
- Application Number
- CN202511242752.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-02
- Publication Date
- 2025-11-07
AI Technical Summary
Existing medications for treating bone homeostasis disorders cannot simultaneously address multiple pathogenic factors, have side effects, and are costly, making it difficult to meet comprehensive treatment needs.
Lactoferrin is used as an antioxidant to protect the differentiation of osteoblast precursors and inhibit osteoclast differentiation under oxidative stress, thereby regulating the balance between osteogenic and osteoclast processes.
Lactoferrin can enhance the expression of osteogenic genes and proteins under oxidative stress, inhibit osteoclast differentiation, reduce reactive oxygen species levels, maintain bone homeostasis, and reduce side effects and costs.
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Figure CN120899888A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The application belongs to the field of medicine, and particularly relates to application of lactoferrin as an antioxidant in preparation of a drug for treating a bone homeostasis imbalance disease. BACKGROUND
[0002] During the growth and development of the skeletal system, bone remodeling occurs continuously, which is crucial for maintaining bone morphology and repairing damaged bone. The bone homeostasis mediated by physiological bone remodeling is mainly maintained by the dynamic balance between bone formation mediated by osteoblasts and bone resorption mediated by osteoclasts. Osteoblasts mainly differentiate and mature from mesenchymal progenitor cells in the endosteum and periosteum and in the matrix of bone marrow, and can play an important osteogenic role by regulating the synthesis, secretion and mineralization of bone matrix. Osteoclasts are the only bone resorption cells in the body, and belong to tissue-specific multinucleated macrophages, which mainly differentiate from monocyte or macrophage precursors on the surface of bone or nearby. Dysfunction of any cell type involved in this process can lead to abnormal bone remodeling, and subsequently cause the occurrence of bone homeostasis imbalance related diseases, such as osteoporosis, bone fracture, bone necrosis, etc.
[0003] At present, the treatment of bone homeostasis imbalance related diseases in the clinic mainly uses anti-bone resorption drugs represented by bisphosphonates and bone formation promoting drugs represented by parathyroid hormone. Although these drugs have achieved certain effects in the clinic, they cannot reverse the disease progression. Moreover, the occurrence and development of bone homeostasis imbalance related diseases are affected by multiple pathogenic factors, and the existing drugs are difficult to intervene in multiple pathological links to meet the more comprehensive treatment needs. In addition, long-term use of such drugs can easily produce inevitable side effects such as gastrointestinal adverse reactions, visceral toxicity or drug resistance, and the synthesis cost of these drugs is high, the preparation process is complex, and the transportation and storage conditions are strict. Therefore, it is of great clinical significance to actively develop new multifunctional bone homeostasis regulating drugs. SUMMARY
[0004] The purpose of the present application is to provide application of lactoferrin as an antioxidant in preparation of a drug for treating a bone homeostasis imbalance disease.
[0005] Technical solution: The application of lactoferrin as an antioxidant in preparation of a drug for treating a bone homeostasis imbalance disease
[0006] Preferably, the drug is a bone homeostasis imbalance disease treatment drug for protecting osteoblast precursor osteogenic differentiation under oxidative stress environment.
[0007] Preferably, the drug is a bone homeostasis imbalance disease treatment drug for inhibiting osteoclast differentiation under oxidative stress environment.
[0008] Preferably, lactoferrin has an antioxidant effect to maintain bone homeostasis.
[0009] Preferably, the expression of osteogenic genes and proteins under oxidative stress is increased.
[0010] Preferably, early alkaline phosphatase activity is increased.
[0011] Preferably, the ability of late osteogenic mineralization is increased.
[0012] Preferably, the level of reactive oxygen species in osteoblast precursor cells under oxidative stress is reduced and the stability of mitochondrial membrane potential is maintained.
[0013] Preferably, the expression of osteoclast differentiation marker TRAP and the formation of multinucleated osteoclasts under oxidative stress are inhibited.
[0014] The drug for treating bone homeostasis imbalance diseases described in the present application comprises lactoferrin.
[0015] Principle of the invention: Oxidative stress is an important factor affecting bone homeostasis in the process of bone remodeling. When aging, hormone level changes or in pathological states such as abnormal glucose metabolism, inflammation, the body's antioxidant capacity is limited, ROS overaccumulates, which inhibits bone formation mediated by osteogenic mineralization of osteoblasts and promotes bone resorption mediated by osteoclast differentiation, thereby causing bone loss and aggravating bone homeostasis imbalance. Lactoferrin belongs to the class of transferrins, is easy to extract, has good biological activity and safety, and has multiple biological properties. The present application first found that lactoferrin LTF as an antioxidant has significant effects in treating bone homeostasis imbalance diseases, protecting osteoblast precursor osteogenic differentiation and inhibiting osteoclast differentiation under oxidative stress: lactoferrin can resist oxidation and coordinate the two biological processes of osteogenesis and osteoclastogenesis, on the one hand, it can increase the expression of osteogenic genes and proteins, early alkaline phosphatase activity and late osteogenic mineralization ability under oxidative stress, on the other hand, it can significantly inhibit the expression of osteoclast genes and proteins and excessive differentiation of osteoclasts under oxidative stress. This discovery can be applied to but not limited to osteoporosis, and has application potential in many other bone metabolism instability diseases characterized by oxidative stress.
[0016] Beneficial effects: Compared with the prior art, the present application has the following significant advantages: lactoferrin LTF in the present application has the effect of resisting oxidation to maintain bone homeostasis, on the one hand, it can protect the osteogenic differentiation ability of osteoblast precursor cells under oxidative stress, on the other hand, it can significantly inhibit the excessive differentiation of osteoclasts under oxidative stress. LTF used in the present application has been applied in many disease models such as Parkinson's disease, liver injury and kidney injury, and has high biological safety. At the same time, the synthesis method of LTF is mature, intensive production has been realized, the storage and transportation conditions are not high, which greatly reduces the cost of LTF as a drug. BRIEF DESCRIPTION OF DRAWINGS
[0017] Figure 1 Figure 1. The effect of LTF on the expression of osteogenic differentiation genes in osteoblast precursor cells under oxidative stress.
[0018] Figure 2 Figure 2. The effect of LTF on the expression of osteogenic differentiation proteins in osteoblast precursor cells under oxidative stress.
[0019] Figure 3 Figure 3. The effect of LTF on the oxidative stress level, early-stage alkaline phosphatase (ALP) activity, and late-stage osteogenic mineralization capacity of osteoblast precursor cells under oxidative stress.
[0020] Figure 4 Figure 4. The effect of LTF on the expression of osteoclast differentiation genes in bone marrow-derived macrophages (BMDMs) under oxidative stress.
[0021] Figure 5 Figure 5. The effect of LTF on the expression of osteoclast differentiation proteins in BMDMs under oxidative stress.
[0022] Figure 6 Figure 6. The effect of LTF on the oxidative stress level and osteoclast differentiation capacity of BMDMs under oxidative stress. DETAILED DESCRIPTION
[0023] The technical solutions of the present application will be further described below in combination with the accompanying drawings and examples.
[0024] Example 1
[0025] (1) Detection of the effect of LTF on the expression of osteogenic differentiation genes in osteoblast precursor cells under oxidative stress
[0026] Mouse primary osteoblast precursor cells were extracted and subcultured to the P1 generation and placed in a six-well plate. When the cell density reached 70%, 0 and 50 ng / ml of LTF were added to the culture medium, respectively, and an oxidative stress environment was induced using 200 μM TBHP. After 6 h of TBHP stimulation, the cells were removed and replaced with new cell culture solution, and after 24 h of continuous culture, the RNA of each group of cells was extracted, and the RNA was reverse transcribed into cDNA using a kit. The results of fluorescence quantitative PCR detection showed that the expression levels of osteogenic genes (Col1a1, Alp, Runx2) that had previously decreased under TBHP stimulation were significantly up-regulated after LTF treatment, as shown in Figure 1 , indicating that LTF can effectively increase the expression levels of osteogenic genes under oxidative stress.
[0027] (2) LTF on osteogenic protein expression of osteoblast precursor under oxidative stress environment
[0028] The mouse primary osteoblast precursor was extracted and subcultured to P1 generation and placed in a six-well plate. When the cell density reached 70%, 0, 50 ng / ml of LTF was added to the culture medium, and 200 μM TBHP was used to induce oxidative stress environment. After 6 hours of TBHP stimulation, the cell culture solution was removed and replaced with new one. After 48 hours of continuous culture, the protein of each group of cells was extracted by RIPA lysis buffer. Western Blot detection showed that the expression of osteogenic proteins (Col1, Alp, Runx2) that decreased under TBHP stimulation increased significantly after LTF treatment, as shown in Figure 2 , which showed that it could significantly increase the expression level of osteogenic proteins under oxidative stress environment.
[0029] (3) LTF on oxidative stress level, early ALP activity and late osteogenic mineralization ability of osteoblast precursor under oxidative stress environment
[0030] The mouse primary osteoblast precursor was placed in a six-well plate and a twelve-well plate, respectively. When the cell density reached 70%, 0, 50 ng / ml of LTF was added to the culture medium, and 200 μM TBHP was used to induce oxidative stress environment. After 6 hours of TBHP stimulation, the cell culture solution was removed and replaced with new one. ROS and JC-1 detection was performed immediately afterwards; ALP staining and ALP activity determination were performed after 7 days; after the culture of calcium nodules, alizarin red staining was performed, and after elution with 10% chlorohexylpyridine, the liquid was transferred to a 96-well plate, and the OD value was determined at 562 nm. The results showed that LTF could significantly reduce the ROS level under TBHP stimulation, maintain the stability of mitochondrial membrane potential to reduce the oxidative stress level, and improve the early ALP activity and late osteogenic mineralization ability, as shown in Figure 3 , which indicated that it could significantly reduce the oxidative stress level and had good osteogenic protection effect.
[0031] Example 2
[0032] (1) LTF on osteogenic protein expression of osteoblast precursor under oxidative stress environment
[0033] Primary mouse BMDM cells were extracted and placed in six-well plates. Osteoclast differentiation was induced by adding 50 ng / ml M-CSF and 50 ng / ml RANKL. Then, 0 ng / ml and 50 ng / ml LTF were added to the culture medium, and oxidative stress was induced using 20 μM TBHP. After TBHP stimulation for 24 h, the cell culture medium was removed, and RNA was extracted from each group of cells. The RNA was reverse transcribed into cDNA using a kit. Quantitative real-time PCR results showed that the expression levels of osteoclast genes (Nfatc1, Mmp9, Ctsk), which were previously elevated under TBHP stimulation, were significantly downregulated after LTF treatment. Figure 4 This indicates that LTF can significantly inhibit the elevated osteoclast gene expression level under oxidative stress.
[0034] (2) Detection of the effect of LTF on the expression of BMDM osteoclast differentiation protein under oxidative stress.
[0035] Primary mouse BMDM cells were extracted and placed in six-well plates. Osteoclast differentiation was induced by adding 50 ng / ml M-CSF and 50 ng / ml RANKL. Then, 0 ng / ml and 50 ng / ml LTF were added to the culture medium, and oxidative stress was induced using 20 μM TBHP. After TBHP stimulation for 48 h, the cell culture medium was removed, and cell proteins were extracted from each group using RIPA lysis buffer. Western blot analysis showed that the expression levels of osteoclast proteins (Nfatc1, Mmp9, Ctsk), which were previously elevated under TBHP stimulation, were significantly reduced after LTF treatment. Figure 5 This indicates that LTF can significantly inhibit the elevated osteoclast expression level under oxidative stress.
[0036] (3) Detection of the effect of LTF on the oxidative stress level of BMDM and its ability to differentiate into osteoclasts under oxidative stress.
[0037] Primary mouse BMDM cells were placed in 12-well plates, and osteoclast differentiation was induced by adding 50 ng / ml M-CSF and 50 ng / ml RANKL. Then, 0 ng / ml and 50 ng / ml LTF were added to the culture medium, and oxidative stress was induced using 20 μM TBHP. ROS and JC-1 levels were measured 12 h after TBHP stimulation; trap and phalloidin staining were performed 48 h later. The results showed that LTF significantly reduced ROS levels under TBHP stimulation, maintained mitochondrial membrane potential stability to decrease oxidative stress levels, and inhibited osteoclast differentiation. Figure 6 This indicates that LTF can reduce oxidative stress levels and inhibit excessive osteoclast differentiation.
Claims
1. Use of lactoferrin as an antioxidant in the preparation of a medicament for treating a bone homeostasis imbalance disease.
2. The use of lactoferrin as an antioxidant in the preparation of a medicament for the treatment of a bone homeostasis imbalance disease according to claim 1, characterized in that, The medicament is a bone homeostasis imbalance disease treatment medicament for protecting osteogenic differentiation of osteoblast precursors under oxidative stress environment.
3. The use of lactoferrin as an antioxidant in the preparation of a drug for the treatment of bone homeostasis imbalance diseases according to claim 1, characterized in that, The medicament is a bone homeostasis imbalance disease treatment medicament for inhibiting osteoclast differentiation under oxidative stress environment.
4. The use of lactoferrin as an antioxidant in the preparation of a drug for the treatment of bone homeostasis imbalance diseases according to claim 1, characterized in that, Lactoferrin has an antioxidant effect to maintain bone homeostasis.
5. The use of lactoferrin as an antioxidant in the preparation of a medicament for the treatment of bone homeostasis imbalance diseases according to claim 1, characterized in that, Improving osteogenic gene and protein expression under oxidative stress environment.
6. The use of lactoferrin as an antioxidant in the preparation of a medicament for the treatment of a bone homeostasis imbalance disease according to claim 1, characterized in that, Improving early alkaline phosphatase activity.
7. The use of lactoferrin as an antioxidant in the preparation of a medicament for the treatment of bone homeostasis imbalance diseases according to claim 1, characterized in that, Improving late osteogenic mineralization capacity.
8. The use of lactoferrin as an antioxidant in the preparation of a medicament for the treatment of a bone homeostasis imbalance disease according to claim 1, characterized in that, Reducing intracellular reactive oxygen species levels of osteoblast precursor cells under oxidative stress environment and maintaining mitochondrial membrane potential stability.
9. The use of lactoferrin as an antioxidant in the preparation of a medicament for the treatment of bone homeostasis imbalance diseases according to claim 1, characterized in that, Inhibiting the expression of osteoclast differentiation marker TRAP and the formation of multinucleated osteoclasts under oxidative stress environment.
10. A medicament for treating a bone homeostasis imbalance disease, characterized by, The medicament comprises lactoferrin.