Application of disulfiram in preparation of medicine for treating diabetic femoral bone loss

Abstract

The invention relates to the technical field of biological pharmacy, in particular to application of disulfiram in preparation of medicine for treating diabetic femoral bone loss, and the femoral bone loss is caused by diabetes. It is found through experiments that disulfiram can promote the multiplication capacity of bone marrow mesenchymal stem cells damaged by diabetes and the osteogenic differentiation capacity of the bone marrow mesenchymal stem cells damaged by diabetes, inhibit the adipogenic differentiation capacity of the bone marrow mesenchymal stem cells damaged by diabetes and can promote bone mass recovery of lost femur bone caused by diabetes; the medicine can be used for treating diabetes mellitus bone mass reduction.

Description

Technical Field

[0001] The invention relates to the technical field of biopharmaceuticals, and in particular to application of disulfiram in preparing a medicine for treating diabetic femoral bone loss. Background Art

[0002] Femoral bone loss is a bone metabolism disorder. The activities of osteoblasts and osteoclasts maintain the balance of human bone metabolism. Osteoclasts promote bone tissue absorption, and osteoblasts synthesize bone matrix to promote bone formation. Their interaction maintains the homeostasis of bone tissue. The only source of osteoblasts is bone marrow mesenchymal stem cells, which are a type of mesenchymal cells with the potential for proliferation, differentiation and self-renewal. Under the high-sugar and high-active oxygen environment of tissues caused by diabetes, the osteogenic differentiation ability of mesenchymal stem cells is impaired, thereby causing femoral bone loss in diabetic patients. Therefore, how to promote the proliferation and osteogenesis of bone marrow mesenchymal stem cells in diabetic patients is a key research topic in the treatment of such diseases.

[0003] Currently, drugs are commonly used to treat femoral bone loss in diabetes. The most commonly used drugs are bisphosphonates, which are a class of drugs that inhibit bone resorption. Their mechanism of action is to inhibit osteoclast formation and induce osteoclast apoptosis. However, long-term use of bisphosphonates will inhibit bone multicellular units and may cause bone loss and atypical fractures.

[0004] Disulfiram is a small molecule organic compound with a chemical formula of C 10 H 20 N 2 S 4 , with a relative molecular mass of 296.539. It is a sobering and alcohol abstinence drug, mainly used to prepare sobering drugs. At present, no research has shown the application and efficacy of disulfiram in the treatment of femoral bone loss caused by diabetes. Summary of the invention

[0005] The purpose of the present invention is to provide the use of disulfiram in preparing a medicine for treating diabetic femoral bone loss.

[0006] In order to solve the above technical problems, this application provides the following technical solutions: The present invention provides the use of disulfiram in preparing a medicine for treating diabetic femoral bone loss or a medicine for promoting the proliferation of bone marrow mesenchymal stem cells damaged by diabetes.

[0007] Wherein, the femoral bone loss is bone loss caused by diabetes.

[0008] Among them, disulfiram can promote the proliferation ability of bone marrow mesenchymal stem cells that are damaged by diabetes.

[0009] Among them, disulfiram can promote the osteogenic differentiation ability of bone marrow mesenchymal stem cells that is impaired by diabetes.

[0010] Among them, disulfiram can inhibit the adipogenic differentiation ability of diabetic bone marrow mesenchymal stem cells.

[0011] Among them, disulfiram can promote bone mass recovery after femoral bone loss caused by diabetes.

[0012] Compared with the prior art, the use of disulfiram in the present invention in the preparation of a drug for treating diabetic femoral bone loss has at least the following beneficial effects: The present invention has experimentally found that disulfiram can promote the osteogenic differentiation potential of mesenchymal stem cells damaged by diabetes, and inhibit the adipogenic differentiation ability of diabetic bone marrow mesenchymal stem cells; it can effectively promote the proliferation ability of diabetic bone marrow mesenchymal stem cells damaged by diabetes, can effectively treat bone loss caused by diabetes, restore bone mass, and can be used as a drug for treating diabetic femoral bone loss.

[0013] The application of disulfiram in the preparation of a medicament for treating diabetic femoral bone loss is further described below in conjunction with the accompanying drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The results of Ki67 immunofluorescence staining show that disulfiram promotes the proliferation of bone marrow mesenchymal stem cells damaged by diabetes. Ki67 represents the positive expression of Ki67 protein.

[0015] Figure 2 The results of ARS staining show that disulfiram promotes osteogenic differentiation of bone marrow mesenchymal stem cells impaired by diabetes. ARS stands for Alizarin Red Staining.

[0016] Figure 3 This is the result of Oil Red staining, showing that disulfiram inhibits the adipogenic differentiation of bone marrow mesenchymal stem cells impaired by diabetes.

[0017] Figure 4 The real-time fluorescence quantitative reverse transcription polymerase chain reaction was used to detect the expression of osteogenic and adipogenic genes in mesenchymal stem cells. The results showed that disulfiram could promote the expression of osteogenic-related genes ALP and OCN in bone marrow mesenchymal stem cells damaged by diabetes, and could inhibit the expression of adipogenic-related genes PPAR-γ and LPL.

[0018] Figure 5-Figure 6 The imaging test results of disulfiram-treated diabetic femoral bone loss show that disulfiram can effectively promote the efficient recovery of diabetic femoral bone loss. Among them, the healthy group represents healthy untreated mice, the diabetic group represents diabetic untreated mice, and the diabetic + disulfiram group represents diabetic mice treated with disulfiram. Among them, the diabetic group is type 2 diabetic mice induced by streptozotocin.

[0019] Figure 7 These are the histological HE staining results of disulfiram in the treatment of diabetic femoral bone loss, showing that disulfiram can effectively promote the efficient recovery of bone mass in diabetic femoral bone loss. DETAILED DESCRIPTION

[0020] The experimental materials and instruments involved in the present invention are as follows: α-MEM (Gibco), fetal bovine serum (Gibco), trypsin (Sigma), crystal violet stain (Solerbo), Ki67 antibody (Affinity), clean bench, Leica inverted microscope, carbon dioxide cell incubator (Thermo), micro-computed tomography (Bruker), real-time fluorescence quantitative reverse transcription polymerase chain reaction instrument (Thermo Fisher), reverse transcriptase (Thermo Fisher), laser confocal microscope (Leica), HE staining kit (Solerbo), ARS staining kit (Beyotime), Oil Red staining kit (Beyotime), disulfiram (Selleck), osteogenic induction medium (Thermo Fisher), adipogenic induction medium (Thermo Fisher), 6-well plate (Corning), C57BL / 6J mice (Viton River). Example 1 Disulfiram promotes the proliferation of diabetic bone marrow mesenchymal stem cells

[0021] (1) Preparation of disulfiram Disulfiram was purchased from Selleck, USA, with a purity of 99.91%. 1 mg was weighed and dissolved in 3.3722 mL of dimethyl sulfoxide (DMSO) to prepare a 1 mM stock solution.

[0022] (2) Extraction and in vitro culture of diabetic damaged bone marrow mesenchymal stem cells Femurs of healthy mice and diabetic mice were taken, and the muscle tissue attached to the bones was removed in a clean bench. The two ends were cut and the bone marrow cavity was flushed with culture medium. The suspension was filtered with a 100 μm cell sieve and inoculated into a 10 cm cell culture dish. When the cell clones were fused, the cells were passaged and the P2 generation cells were used for the experiment.

[0023] (3) Take P2 stem cells, place cell slides in a 12-well plate, and inoculate 4×10 5cells per well. When the cells adhered and fused to 50%, disulfiram was added to the experimental group at a final concentration of 50 nM. After 1 day of culture, the culture medium was removed, washed with phosphate buffered saline, fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 1% TritonX-100 for 10 minutes, blocked with 5% fetal bovine serum for 1 hour, and added with ki67 antibody dilution solution at a ratio of 1:200, and incubated in a refrigerator at 4°C overnight. The next day, the ki67 antibody was washed away with phosphate buffer, and the fluorescent antibody dilution solution was added and incubated at room temperature for 1 hour. After washing away the fluorescent antibody, the slides were sealed with a sealing agent, and the positive expression of ki67 was observed by immunofluorescence microscopy.

[0024] like Figure 1 As shown, 50 nM disulfiram can increase the expression of ki67 protein and the number of cells in diabetic damaged bone marrow mesenchymal stem cells, proving that disulfiram can effectively promote the proliferation of diabetic damaged bone marrow mesenchymal stem cells. Example 2 In vitro experiments demonstrate that disulfiram promotes osteogenic differentiation of diabetic bone marrow mesenchymal stem cells

[0025] (1) Osteogenesis induction As described in Example 1, femoral mesenchymal stem cells were extracted from healthy mice and diabetic mice, and the cells were divided into a healthy group, a diabetic group, and a diabetic + disulfiram group. The cells were seeded in a 12-well plate. When the cells were 70% confluent, osteogenic induction medium and disulfiram were added. RNA was extracted after 5 days of induction, and ARS staining was performed after 7 days of induction.

[0026] (2) ARS staining Take out the 12-well plate, remove the culture medium, add 4% paraformaldehyde to fix the cells. Add ARS staining solution, incubate for half an hour, and wash with distilled water to terminate the reaction. Use a scanner to scan the well plate and use a microscope to record the ARS staining.

[0027] like Figure 2 As shown, the ARS staining results showed that compared with the healthy group, the area of ​​ARS-positive staining in the diabetic group was significantly reduced. After disulfiram treatment, the area of ​​ARS-positive staining increased significantly and returned to the baseline level.

[0028] (3) Real-time fluorescence quantitative reverse transcription polymerase chain reaction After RNA extraction, real-time fluorescence quantitative reverse transcription polymerase chain reaction was performed to detect the expression of bone-related genes in the healthy group, diabetic group, and diabetic + disulfiram group. Figure 4 As shown in the figure, compared with the healthy group, the expression of osteogenic related genes ALP and OCN in the diabetic group was downregulated. After disulfiram treatment, the expression of osteogenic related genes increased. Example 3 In vitro experiments demonstrate that disulfiram inhibits the adipogenic differentiation of diabetic bone marrow mesenchymal stem cells

[0029] (1) Adipogenesis induction As described in Example 1, femoral mesenchymal stem cells were extracted from healthy mice and diabetic mice, and the cells were divided into a healthy group, a diabetic group, and a diabetic + disulfiram group. The cells were seeded in a 12-well plate. When the cells were 70% confluent, adipogenic induction medium and disulfiram were added. RNA was extracted after 5 days of induction, and oil red staining was performed after 14 days of induction.

[0030] (2) Oil red staining Take out the 12-well plate, remove the culture medium, add 4% paraformaldehyde to fix the cells. Add oil red staining solution and incubate for half an hour in the dark. Use a microscope to record the oil red staining.

[0031] like Figure 3 As shown, the results of oil red staining showed that compared with the healthy group, the number of lipid droplet-positive cells in the diabetic group increased significantly. After disulfiram treatment, the number of oil red-positive cells decreased significantly and returned to the baseline level.

[0032] (3) Real-time fluorescence quantitative reverse transcription polymerase chain reaction After RNA extraction, real-time fluorescence quantitative reverse transcription polymerase chain reaction was performed to detect lipid-related gene expression in the healthy group, diabetic group, and diabetic + disulfiram group. Figure 4 As shown in the figure, compared with the control group, the expression of adipogenesis-related genes PPAR-γ and LPL was upregulated in the diabetic group. After disulfiram treatment, the expression of adipogenesis-related genes was reduced. Example 4 In vitro experiments directly prove that disulfiram promotes efficient recovery of femoral bone loss in diabetic patients

[0033] (1) Preparation of disulfiram Disulfiram was purchased from Selleck, USA, with a purity of 99.91%. The drug was dissolved in dimethyl sulfoxide (DMSO). Taking 1 mL of working solution as an example, 50 μL of 100 mg / ml clear DMSO stock solution was added to 950 μL of corn oil and mixed evenly.

[0034] (2) Preparation of diabetic mice Adult C57BL / 6J male mice were fed a high-fat diet for 4 weeks. The mice were fasted overnight and intraperitoneally injected with streptozotocin at a dosage of 70 mg / kg. Fasting blood sugar was tested once every 3 days. When the fasting blood sugar was greater than 11.1 mmol / L, the type 2 diabetes model was successfully established. If the blood sugar level did not meet the standard, streptozotocin was re-injected at a dosage of 40 mg / kg until the blood sugar level reached the standard.

[0035] (3) Treatment of femoral bone loss in diabetic mice The disulfiram drug treatment was carried out on diabetic mice. The scheme was to use disulfiram dissolved in corn oil and gavage the mice every other day at a concentration of 100 mg / kg. The healthy group mice and the diabetic group were gavaged with corn oil alone. After 4 weeks of continuous treatment, the femurs of the mice were obtained and fixed overnight with 4% paraformaldehyde fixative, and scanned with Micro-CT for imaging analysis.

[0036] like Figure 5-Figure 6 As shown, two-dimensional and three-dimensional cross-sections of mouse femurs are shown. The cancellous bone mass was observed, and the results showed that the femoral bone mass of diabetic mice was severely damaged. After treatment with disulfiram, the bone mass was significantly restored and was similar to that of healthy mice. The femoral bone mass of mice was analyzed using software, and the results showed that the bone density, bone volume fraction, and number of trabeculae in diabetic mice were all reduced. After treatment with disulfiram, the above values ​​were restored, indicating an increase in bone mass after treatment.

[0037] like Figure 7 As shown, the mouse femur was sectioned and HE stained. Compared with the healthy group, the cancellous bone mass in the diabetic group was reduced, while the cancellous bone mass in the disulfiram treatment group was restored, proving that disulfiram has a high efficacy in treating diabetic femoral bone loss.

[0038] The embodiments described above are merely descriptions of preferred implementation modes of the present invention and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. The use of disulfiram in the preparation of drugs for treating diabetic femoral bone loss or promoting the proliferation of bone marrow mesenchymal stem cells.

2. The use according to claim 1, characterized in that: The femoral bone loss is bone loss caused by diabetes.

3. The use according to claim 2, characterized in that: The drug is a drug for promoting the proliferation of bone marrow mesenchymal stem cells damaged by diabetes.

4. The use according to claim 3, characterized in that: The disulfiram promotes the proliferation ability of bone marrow mesenchymal stem cells impaired by diabetes.

5. The use according to claim 3, characterized in that: The disulfiram promotes the osteogenic differentiation ability of bone marrow mesenchymal stem cells impaired by diabetes.

6. The use according to claim 3, characterized in that: The disulfiram can inhibit the adipogenic differentiation ability of diabetic bone marrow mesenchymal stem cells.

7. The use according to claim 3, characterized in that: The disulfiram promotes bone mass recovery after femoral bone loss induced by diabetes.

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