Application of palmatine or salt and derivative thereof in preparation of medicine for treating and / or relieving osteoporosis

Abstract

The invention belongs to the technical field of biotechnology and medicine, and particularly relates to application of palmatine or salt and derivative thereof in preparation of medicine for treating and / or relieving osteoporosis. It is found that palmatine and palmatine hydrochloride can promote osteogenic differentiation of bone marrow mesenchymal stem cells and inhibit adipogenic differentiation of the bone marrow mesenchymal stem cells, concentration dependence is achieved, and then the effect of treating and / or relieving osteoporosis is achieved. Therefore, the palmatine or the salt and the derivative thereof provided by the invention have the effect of regulating and controlling bone metabolism by influencing osteogenesis / adipogenic differentiation of the bone marrow mesenchymal stem cells and new application of the palmatine or the salt and the derivative thereof in treating the osteoporosis, and a new strategy can be provided for treating the osteoporosis.

Description

Technical Field

[0001] The present invention relates to the fields of biotechnology and medical technology, and in particular to application of bamipine or its salts and derivatives in preparing medicines for treating and / or alleviating osteoporosis. Background Art

[0002] Osteoporosis (OP) is a systemic bone disease characterized by decreased bone mass, damaged bone microstructure, increased bone brittleness, and susceptibility to fractures. The occurrence of osteoporosis is related to a variety of factors, including estrogen deficiency, calcium-phosphorus imbalance, lack of physical activity, and diseases and drugs that affect bone metabolism. Osteoporosis can occur at any age, but is more common in postmenopausal women and elderly men. The epidemiological survey report released by the National Health Commission in 2018 showed that the prevalence of OP in people aged 40 to 49 in my country was 3.2%, 19.2% in people over 50, and as high as 32.0% in people over 65. The basic treatment of osteoporosis includes a balanced diet, reasonable exercise, and supplementation of calcium and vitamin D. Drug treatment includes drugs that inhibit bone resorption, promote bone formation, and have both the former and the latter.

[0003] Anti-resorptive drugs can correct the imbalance of bone metabolism where bone absorption is greater than bone formation by inhibiting the process of bone absorption. Specifically, they include bisphosphonates, calcitonin, estrogen, and selective estrogen receptor modulators. The drug that promotes bone formation is mainly parathyroid hormone, which can induce osteoblast precursor cells to become osteoblasts, increase the number and activity of osteoblasts, and promote calcification, thereby promoting bone formation and increasing bone mass. Dual-action drugs include strontium salt drugs and vitamin K2, which have the dual effects of anti-bone absorption and promoting bone formation.

[0004] However, the above-mentioned therapeutic drugs still have many shortcomings, mainly manifested in the following aspects: (1) Long-term use of anti-bone resorption drugs is prone to adverse gastrointestinal reactions, electrolyte imbalance, mandibular osteonecrosis, urticaria, rash, itching, endometrial hyperplasia, high incidence of heart disease and breast cancer, etc.; (2) Drugs that promote bone formation can cause hypercalcemia, which in turn causes digestive system symptoms such as nausea and vomiting, and may also cause cardiovascular diseases such as arrhythmia and cardiac arrest. (3) Dual-acting drugs may cause side effects such as nausea, diarrhea, and ischemic heart disease.

[0005] In summary, given that the current common drug treatments have certain effects but most of them have side effects, there is an urgent need to develop drugs with good therapeutic effects and few side effects to treat or alleviate osteoporosis. Summary of the invention

[0006] In view of the shortcomings of the prior art, the purpose of the present invention is to provide the use of bamipine or its salts and derivatives in the preparation of drugs for treating and / or alleviating osteoporosis. Bamipine and bamipine hydrochloride can regulate bone metabolism by affecting the osteogenic / adipogenic differentiation of bone marrow mesenchymal stem cells, and thus be used to treat osteoporosis.

[0007] Another object of the present invention is to provide a drug for treating and / or alleviating osteoporosis.

[0008] In order to solve the above technical problems, the present invention provides the following technical solutions:

[0009] The present invention finds that bamipine and bamipine hydrochloride can promote the osteogenic differentiation of bone marrow mesenchymal stem cells and inhibit their adipogenic differentiation, and is concentration-dependent (within the range of 2.5 μM-10 μM, as the concentration increases, the stronger the ability of bamipine and bamipine hydrochloride to promote osteogenic differentiation, the stronger the ability to inhibit adipogenic differentiation), thereby achieving the effect of treating and / or alleviating osteoporosis. Through the establishment and treatment experiments of an osteoporosis model induced by bilateral ovariectomy (OVX) in mice, the results show that bamipine hydrochloride mainly achieves the effect of anti-osteoporosis by maintaining or increasing the bone mass of cancellous bones. Therefore, the present invention provides the effect of bamipine or its salts and derivatives on regulating bone metabolism by affecting the osteogenic / adipogenic differentiation of bone marrow mesenchymal stem cells and its new use for treating osteoporosis.

[0010] Specifically, the present invention seeks to protect the use of bamipine or its salts and derivatives in the preparation of drugs for treating and / or alleviating osteoporosis.

[0011] Furthermore, the drug treats and / or alleviates osteoporosis by inhibiting the adipogenic differentiation of bone marrow mesenchymal stem cells and promoting the osteogenic differentiation of bone marrow mesenchymal stem cells.

[0012] In one embodiment, the concentration of palmatine in the medicament is 2.5 μM to 20 μM.

[0013] Preferably, the concentration of bamipine in the drug is 2.5 μM-10 μM.

[0014] More preferably, the concentration of bamipine in the drug is 10 μM.

[0015] In another embodiment, the concentration of bamipine hydrochloride in the medicament is 2.5 μM-20 μM.

[0016] Preferably, the concentration of bamipine hydrochloride in the drug is 2.5 μM-10 μM.

[0017] More preferably, the concentration of bamipine hydrochloride in the drug is 10 μM.

[0018] The present invention also claims a drug for treating and / or alleviating osteoporosis, wherein the drug contains palmatine, and the concentration of the palmatine is 2.5 μM-20 μM.

[0019] Preferably, the concentration of bamipine is 2.5 μM-10 μM.

[0020] More preferably, the concentration of bamipine is 10 μM.

[0021] The present invention also claims a drug for treating and / or alleviating osteoporosis, wherein the drug contains bamipine hydrochloride, and the concentration of bamipine hydrochloride is 2.5 μM-20 μM.

[0022] Preferably, the concentration of bamipine hydrochloride is 2.5 μM-10 μM.

[0023] More preferably, the concentration of bamipine hydrochloride is 10 μM.

[0024] Compared with the prior art, the present invention has the following beneficial effects:

[0025] Bamitain and Bamitain hydrochloride can promote the osteogenic differentiation of bone marrow mesenchymal stem cells and inhibit their adipogenic differentiation, and are concentration-dependent (with the concentration increasing within the range of 2.5 μM-10 μM, the stronger the ability of Bamitain and Bamitain hydrochloride to promote osteogenic differentiation, the stronger the ability to inhibit adipogenic differentiation), thereby achieving the effect of treating and / or alleviating osteoporosis. Through the establishment and treatment experiments of the osteoporosis model induced by bilateral ovariectomy (OVX) in mice, the results show that Bamitain hydrochloride mainly achieves the effect of anti-osteoporosis by maintaining or increasing the bone mass of cancellous bones. Therefore, the present invention provides the effect of Bamitain or its salts and derivatives on regulating bone metabolism by affecting the osteogenic / adipogenic differentiation of bone marrow mesenchymal stem cells and its new use for treating osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is the structural formula of bamipine hydrochloride.

[0027] Figure 2Bamatin hydrochloride promotes the differentiation of BMSCs into osteoblasts and inhibits the differentiation of BMSCs into adipocytes. Among them, a is a cytotoxicity experiment, which shows that PaH at a concentration not exceeding 20μM does not affect the proliferation of BMSCs. b is that the cells treated with PaH have a deeper alizarin red staining, indicating that PaH promotes the differentiation of mouse BMSCs into osteoblasts. c is that the cells treated with PaH have a deeper oil red O staining, indicating that PaH inhibits the differentiation of mouse BMSCs into adipocytes. d is the change of alkaline phosphatase staining after treatment with different concentrations of PaH, indicating that PaH promotes the differentiation of mouse BMSCs into osteoblasts in a concentration-dependent manner. e is the change of alizarin red staining after treatment with different concentrations of PaH, indicating that PaH promotes the differentiation of mouse BMSCs into osteoblasts in a concentration-dependent manner. f is the oil red O staining and BODIPY staining after treatment with different concentrations of PaH, indicating that PaH inhibits the differentiation of mouse BMSCs into adipocytes in a concentration-dependent manner.

[0028] Figure 3 Bamatin hydrochloride rescues bone loss in ovariectomized osteoporosis model mice. a is CT three-dimensional reconstruction of distal femurs of mice in sham operation group, OVX osteoporosis model group and PaH treatment group, indicating that PaH treatment can significantly reduce bone loss in osteoporotic mice, restore the number of trabeculae in mice and improve bone microstructure. bg is the morphometric statistics of mouse bone tissue, indicating that PaH can increase bone density, bone volume fraction, trabecular number and trabecular thickness in OVX mice. hj is histopathological detection and statistics, indicating that PaH treatment can significantly increase bone mass in osteoporotic mice and reduce the accumulation of medullary adipose tissue.

[0029] Figure 4 Bamatin promotes the differentiation of BMSCs into osteoblasts and inhibits the differentiation of BMSCs into adipocytes. Among them, a is a cytotoxicity experiment, which shows that Pa with a concentration not exceeding 20μM does not affect the proliferation of BMSCs. b is the change of Alizarin Red staining after treatment with different concentrations of Pa, indicating that Pa promotes the differentiation of mouse BMSCs into osteoblasts in a concentration-dependent manner. c is the Oil Red O staining after treatment with different concentrations of Pa, indicating that PaH inhibits the differentiation of mouse BMSCs into adipocytes in a concentration-dependent manner. DETAILED DESCRIPTION

[0030] The present invention will be further described in detail below in conjunction with specific examples. The following examples are not intended to limit the present invention, but are only intended to illustrate the present invention. The experimental methods used in the following examples are generally conventional, unless otherwise specified, and the materials, reagents, etc. used in the following examples are commercially available, unless otherwise specified.

[0031] Terminology and explanation:

[0032] 1. Bone marrow mesenchymal stem cells (BMSCs): They are a type of multipotent stem cells that develop from the mesoderm and have the potential for self-renewal and multidirectional differentiation. Under certain conditions, they can differentiate into osteoblasts. Although BMSCs can replenish damaged or dead cells through self-proliferation and differentiation, their proliferation and differentiation abilities will change with the increase of individual age and changes in the state of the body. In patients with osteoporosis, BMSCs show an increase in the number of apoptotic cells, a decrease in their self-renewal ability, a slowdown in their proliferation rate, and a decrease in the number of osteoblasts differentiated from them. Whether it is postmenopausal osteoporosis patients or senile osteoporosis patients, their BMSCs show the common characteristics of weakened osteogenic differentiation and enhanced adipogenic differentiation. Related studies have shown that by regulating the osteogenic / adipogenic differentiation direction of BMSCs, enhancing the osteogenic ability of BMSCs and inhibiting their differentiation into fat can prevent bone loss in osteoporosis model animals, reduce fat accumulation in the bone marrow cavity and increase bone mass. Therefore, restoring the osteogenic / adipogenic balance of BMSCs differentiation is an effective strategy for the treatment of osteoporosis.

[0033] 2. Palmatine (Pa): Also known as Fibraureapierre, it is an alkaloid extracted from the dried stems of Fibraureapierre, a plant of the Menispermaceae family. It has a broad-spectrum antibacterial and antiviral effect, a multi-pharmacological effect of significantly increasing the phagocytosis of bacteria by leukocytes, and a good anti-inflammatory and immunity-enhancing effect. It has the function of clearing away heat and detoxifying, and is used for gynecological inflammation, bacillary dysentery, enteritis, respiratory and urinary tract infections, surgical infections, and conjunctivitis.

[0034] 3. Palmatine hydrochloride (PaH): Also known as palmatine hydrochloride, palmitoyl hydrochloride, and african tetrandrine hydrochloride, it is extracted and refined from the dried stems of Fibraureapierre, a plant of the Menispermaceae family, and has the effect of clearing away heat and detoxifying. Currently, palmatine hydrochloride is commonly used for gynecological inflammation, bacillary dysentery, enteritis, respiratory and urinary tract infections, surgical infections, and conjunctivitis.

[0035] Example 1 PaH drug cytotoxicity detection

[0036] PaH drug cytotoxicity detection, drug toxicity detection was performed using a CCK-8 detection kit.

[0037] 1. Add 100 μL of cell suspension containing 8,000 human BMSCs to each well of a 96-well plate. The complete culture medium for BMSCs is low-glucose DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) penicillin-streptomycin solution (100X, Beyotime, C0222). Pre-culture the culture plate in an incubator for 24 hours (37°C, 5% CO2).

[0038] 2. Use BMSCs culture medium to dilute the DMSO-dissolved PaH stock solution to 1.25μM, 2.5μM, 5μM, 10μM, 20μM, and each concentration of PaH solution contains 1‰ DMSO. BMSCs are treated with the above concentrations of PaH, and 3 replicates are performed for each concentration. The blank control group is treated with culture medium containing 1‰ DMSO at the same time.

[0039] 3. Incubate the plate in an incubator for 24 or 72 hours.

[0040] 4. Add 10 μL of CCK8 solution to each well.

[0041] 5. Continue to incubate the culture plate in the incubator for 1 hour.

[0042] 6. Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0043] The results are as follows Figure 2 As shown in a, the relative values ​​of absorbance after treatment with different PaH concentrations / control group absorbance were obtained. When the PaH concentration was 40μM and 80μM, the relative values ​​at 24 hours and 72 hours were significantly reduced, and there was no significant difference between other concentrations and the control group. This shows that PaH concentrations not exceeding 20μM do not affect the proliferation of BMSCs.

[0044] Example 2 Effect of PaH on osteogenic differentiation of mouse BMSCs

[0045] 1. Take mBMSCs from C57BL / 6 female mice at a rate of 1×10 5 The cells were seeded in 6-well plates at a density of 1 cell / well.

[0046] 2. When mBMSCs expanded to 80%, osteogenic induction medium (OIM) was prepared as follows: 50 μL dexamethasone (stock concentration 100 μM) + 500 μL ascorbic acid (stock concentration 5 mM) + 500 μL β-glycerophosphate (stock concentration 1 M) + 500 μL double antibody (100X, Beyotime, C0222) + 5 mL FBS, add low-glucose DMEM culture medium to 50 mL. 2 mL OIM was added to the control group, and 2 mL OIM containing 20 μM PaH was added to the treatment group for induction.

[0047] 3. Each group was changed every 3 days. After 14 days of induction, the induction was terminated, washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 10 minutes, washed three times with PBS, and then stained with 1 mL of Alizarin Red for 20 minutes per well. After discarding the staining solution, the cells were washed three times with PBS and photographed. Preparation of Alizarin Red (ARS) staining solution: 1.3692 g of Alizarin Red powder was dissolved in 100 mL of PBS, and the pH was adjusted to 4.1-4.3.

[0048] The results are as follows Figure 2 As shown in b, the ARS staining of cells treated with 20 μM PaH was darker, indicating that 20 μM PaH promoted the osteogenic differentiation of mBMSCs.

[0049] Example 3 Effect of PaH on adipogenic differentiation of mouse BMSCs

[0050] 1. Take mBMSCs from C57BL / 6 female mice at a rate of 1×10 5 The cells were seeded in 6-well plates at a density of 1 cell / well.

[0051] 2. When mBMSCs expanded to 95%, adipogenic differentiation was induced. The adipogenic induction medium (AIM) preparation scheme was as follows: 500 μL of dexamethasone (stock concentration 100 μM) + 125 μL of indomethacin (stock concentration 80 mM) + 2.5 mL of 1-methyl-3-isobutylxanthine (stock concentration 10 mM) + 100 μL of glargine insulin (stock concentration 10 mg / mL) + 500 μL of double antibody (100X, Biyuntian, C0222) + 5 mL of FBS, and low-glucose DMEM culture medium was added to 50 mL. The adipogenic induction maintenance medium was prepared as follows: 5 mL of FBS + 500 uL of double antibody + 100 μL of glargine insulin (stock concentration 10 mg / mL), and low-glucose DMEM culture medium was added to 50 mL. 2 mL of AIM was added to the control group, and 2 mL of AIM containing 20 μM PaH was added to the treatment group for induction.

[0052] 3. After every 2 days of induction, change to adipogenic maintenance medium for 1 day, repeat until the 10th day, stop the culture, wash twice with PBS, fix with 4% PFA at room temperature for 10 minutes, wash twice with PBS and dry as much as possible, let dry at room temperature, add oil red O dye solution at room temperature for 1 hour, rinse 3 times with 70% alcohol, observe and take pictures under the microscope. Preparation of 0.5% Oil Red O dye solution: add 0.25g Oil Red O powder to 50mL isopropanol, invert the centrifuge tube to mix and dissolve, filter with filter paper and set aside at room temperature, store away from light. When using, mix the newly filtered 0.5% (w / v) Oil Red O with deionized water in a ratio of 3:2, filter and dye at room temperature for 1 hour.

[0053] The results are as follows Figure 2As shown in c, there was almost no adipocyte formation in the cells treated with 20 μM PaH, while there were a large number of adipocytes stained with Oil Red in the control group, indicating that 20 μM PaH inhibited the adipogenic differentiation of mBMSCs.

[0054] Example 4 Effect of PaH on osteogenic differentiation of human BMSCs

[0055] 1. Human hBMSCs were obtained at a rate of 1×10 5 The cells were seeded in 6-well plates at a density of 1 cell / well.

[0056] 2. When hBMSCs expanded to 80%, osteogenic differentiation was performed. 2 mL OIM was added to the control group, and 2 mL OIM containing 2.5 μM, 5 μM, 10 μM, and 20 μM PaH was added to the treatment group for induction.

[0057] 3. Each group was changed every 3 days. The cells stained with alkaline phosphatase (ALP) were induced for 7 days and then stopped, washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 10 minutes, washed three times with PBS, and then 1 mL of alkaline phosphatase staining solution was added to each well. The cells were stained at 37°C for 20 minutes and then stopped. The cells were washed twice with PBS and photographed. The cells stained with alizarin red were induced for 21 days and then stopped, washed twice with PBS, fixed with 4% paraformaldehyde at room temperature for 10 minutes, washed three times with PBS, and then 1 mL of alizarin red was added to each well for 20 minutes. The staining solution was discarded, and then the cells were washed three times with PBS and photographed.

[0058] The results are as follows Figure 2 As shown in d and e, the ALP and ARS staining of cells treated with 2.5 μM PaH was darker than that of the control group. As the concentration increased, the staining became deeper, reaching the deepest at 10 μM, while 20 μM was shallower than 10 μM but still darker than the control group. This indicates that PaH promotes the osteogenic differentiation of hBMSCs in a concentration-dependent manner. In the range of 2.5 μM-10 μM, the ability to promote osteogenic differentiation increases with the increase in concentration.

[0059] Example 5 Effect of PaH on adipogenic differentiation of human BMSCs

[0060] 1. Human hBMSCs were obtained at a rate of 1×10 5 The cells were seeded in 6-well plates at a density of 1 cell / well.

[0061] 2. When hBMSCs expanded to 95%, adipogenic differentiation was induced. 2 mL AIM was added to the control group, and 2 mL AIM containing 2.5 μM, 5 μM, or 10 μM PaH was added to the treatment group for induction.

[0062] 3. After every 2 days of induction, change to adipogenesis maintenance medium for 1 day, and repeat this process until the 14th day when culture is terminated. Wash twice with PBS, fix with 4% PFA at room temperature for 10 min, wash three times with PBS and then dry as much as possible. After drying at room temperature, add oil red O staining solution at room temperature for 1 h, rinse three times with 70% alcohol, and observe and take pictures under a microscope.

[0063] 4. To observe the morphology of lipid droplets, a lipid droplet green fluorescence detection kit (BODIPY 493 / 503) was used for detection. The culture was terminated on the 14th day of induction, and the cells were washed twice with PBS, fixed with 4% PFA at room temperature for 10 min, washed three times with PBS, and then BODIPY 493 / 503 and Hoechst 33342 were added. The cells were stained at room temperature in the dark for 10 min. The cells were observed and photographed under a fluorescence microscope.

[0064] The results are as follows Figure 2 As shown in figure f, the number of adipocytes after treatment with 2.5μM-10μM PaH was significantly reduced, and the lipid droplets were sparse. In contrast, there were a large number of adipocytes stained with oil red in the control group, and the lipid droplets were round and plump, indicating that PaH inhibited the adipogenic differentiation of hBMSCs in a concentration-dependent manner. Within the concentration range of 2.5μM-10μM, the ability to inhibit adipogenic differentiation became stronger as the concentration increased.

[0065] Example 6 Establishment and treatment of osteoporosis model induced by bilateral ovariectomy (OVX) in mice

[0066] 1. Prepare 15 8-week-old C57BL / 6 female mice and anesthetize them by intraperitoneal injection of ketamine (100 mg / kg) and xylazine (10 mg / kg).

[0067] 2. Shave the hair on the back of the mice, disinfect the skin, make a longitudinal skin incision along the spine 0.5 cm above the sacroiliac joint (the incision is less than 1 cm), separate the subcutaneous fascia on both sides, and cut the muscles 0.5 cm-1 cm on both sides of the spine to reveal white fat masses. In the sham group, only the skin and muscles were incised and the muscles and skin were sutured directly, and the ovaries were not processed. In the 10 mice to be ovarian removed, the fat masses were carefully clipped out to reveal the slightly red cauliflower-shaped ovaries, and the ovarian arteries and fallopian tubes were ligated, and the ovaries and part of the fallopian tubes were cut along the ligation. After removing the ovary on the other side in the same way, the muscles and skin were sutured and the skin was disinfected.

[0068] 3. Pay attention to the condition of the mice within one week after surgery to ensure that they can eat freely and move well.

[0069] 4. Two weeks later, the 10 mice with ovariectomized were randomly divided into 2 groups: OVX group and OVX+PaH group, with 5 mice in each group. PaH was first fully dissolved with DMSO and then diluted in normal saline. DMSO of equal volume concentration was injected intraperitoneally every other day at 2 mg / kg body weight, and the Sham group and OVX group were injected with DMSO of equal volume concentration. The samples were collected after 4 weeks of continuous treatment. The mice were killed by cervical dislocation, the muscles on the surface of the femurs on both sides were stripped, and the mice were fixed in 4% PFA for 24 hours, and then replaced with 75% ethanol for transportation and storage.

[0070] 5. μCT detection and analysis: The left femur was selected for μCT scanning, and the scanning instrument SCANCOμCT 100 was used. The scanning conditions were: 70kV voltage, 200μA current, 300ms exposure time, and scanning layer thickness of 10μm. When analyzing cancellous bone, the 0.3mm-1.1mm area below the growth plate was selected as the region of interest, and the 2mm-3mm area below the growth plate was selected as the region of interest for cortical bone. The static parameters of cancellous bone analysis include bone mineral density (BMD), bone volume fraction (BV / TV), trabecular thickness (Tb.Th), trabecular separation (Tb.Sp), trabecular number (Tb.N), trabecular bone surface / bone volume (Tb.BS / BV) and cortical thickness (Ct.Th). The cancellous bone and cortical bone regions of interest were reconstructed in three dimensions.

[0071] The results are as follows Figure 3 As shown in ag, the bone density, bone volume fraction, number of trabeculae and thickness of trabeculae in the femur of OVX mice were significantly lower than those in the Sham group. The PaH intervention group rescued the cancellous bone loss caused by OVX, effectively restored the number of trabeculae in mice and improved bone microstructure. However, PaH intervention could not rescue the thinning of cortical bone thickness. This indicates that PaH mainly achieves the effect of anti-osteoporosis by maintaining or increasing the amount of cancellous bone.

[0072] 6. Histopathological examination: The right femur was selected for histopathological examination. The decalcified femur was dehydrated, transparent, paraffin-embedded, sliced, and stained with hematoxylin-eosin (HE) and ALP. The HE staining results are shown in Figure 3As shown in h, compared with the Sham group, the medullary cavity of the OVX group was full of residual vacuoles of fat cells, while the trabeculae were significantly sparse and smaller in diameter. ALP staining showed that the ALP staining near the growth plate and on the surface of trabecular bones in the OVX group of mice was significantly lighter, indicating that the number of osteoblasts attached to the bone surface was significantly reduced. In the PaH-treated group, the medullary fat vacuoles were significantly less than those in the OVX group, and the trabeculae were denser and thicker in diameter. ALP staining showed that the number of osteoblasts on the bone surface in the PaH-treated group was significantly more than that in the OVX group. The proportion of bone tissue area in the slice ( Figure 3 i) and lipid droplet area ratio ( Figure 3 j) showed that PaH treatment could inhibit the accumulation of medullary fat in OVX mice and rescue the bone loss in OVX mice. The above results are consistent with the results of μCT. Combined with the osteogenic / adipogenic differentiation experiment of BMSCs, it further shows that PaH may rescue the bone loss in OVX mice by inhibiting the adipogenic differentiation of BMSCs and promoting their osteogenic differentiation, thus exerting its anti-osteoporosis effect.

[0073] Example 7 Pa drug cytotoxicity detection

[0074] Pa drug cytotoxicity detection, drug toxicity detection was performed using a CCK-8 detection kit.

[0075] 1. Add 100 μL of cell suspension containing 8,000 human BMSCs to each well of a 96-well plate. The complete culture medium for BMSCs is low-glucose DMEM medium + 10% (v / v) fetal bovine serum + 1% (v / v) penicillin-streptomycin solution (100X, Beyotime, C0222). Pre-culture the culture plate in an incubator for 24 hours (37°C, 5% CO2).

[0076] 2. Use BMSCs culture medium to dilute the DMSO-dissolved Pa stock solution to 1.25μM, 2.5μM, 5μM, 10μM, and 20μM. Each concentration of Pa solution contains 1‰ DMSO. BMSCs are treated with the above concentrations of Pa, and 6 replicates are performed for each concentration. The blank control group is treated with culture medium containing 1‰ DMSO at the same time.

[0077] 3. Incubate the culture plate in an incubator for 72 hours.

[0078] 4. Add 10 μL of CCK8 solution to each well.

[0079] 5. Continue to incubate the culture plate in the incubator for 1 hour.

[0080] 6. Measure the absorbance at 450 nm using an enzyme-labeled instrument.

[0081] The results are as follows Figure 4As shown in a, the relative value of absorbance after treatment with different Pa concentrations / control group absorbance was obtained. When the Pa concentration was 40 μM, the relative value after 72 hours was significantly reduced. When the Pa concentration was 20 μM, the relative value was less than that of the control group, but there was no statistical difference. There was no significant difference between other concentrations and the control group. This shows that Pa concentrations not exceeding 20 μM do not affect the proliferation of BMSCs.

[0082] Example 8 Effect of Pa on Osteogenic Differentiation of Human BMSCs

[0083] 1. Human hBMSCs were obtained at a rate of 1×10 5 The cells were seeded in 6-well plates at a density of 1 cell / well.

[0084] 2. When hBMSCs expanded to 80%, osteogenic differentiation was performed. 2 mL OIM was added to the control group, and 2 mL OIM containing 2.5 μM, 5 μM or 10 μM Pa was added to the treatment group for induction.

[0085] 3. Each group was changed every 3 days. The cells were induced for 21 days before termination of induction. The cells were washed with PBS twice, fixed with 4% paraformaldehyde at room temperature for 10 min, washed with PBS three times, and stained with 1 mL of Alizarin Red for 20 min. The staining solution was discarded, and the cells were washed with PBS three times before taking pictures.

[0086] The results are as follows Figure 4 As shown in b, the ARS staining of cells treated with 2.5 μM Pa was darker than that of the control group, and the staining became deeper as the concentration increased, reaching the deepest at 10 μM. This indicates that Pa promotes the osteogenic differentiation of hBMSCs in a concentration-dependent manner, and the ability to promote osteogenic differentiation increases with the increase in concentration within the range of 2.5 μM-10 μM.

[0087] Example 9 Effect of Pa on Adipogenic Differentiation of Human BMSCs

[0088] 1. Human hBMSCs were obtained at a rate of 1×10 5 The cells were seeded in 6-well plates at a density of 1 cell / well.

[0089] 2. When hBMSCs expanded to 95%, adipogenic differentiation was induced. 2 mL AIM was added to the control group, and 2 mL AIM containing 2.5 μM, 5 μM, and 10 μM Pa was added to the treatment group for induction.

[0090] 3. After every 2 days of induction, change to adipogenesis maintenance medium for 1 day, and repeat this process until the 14th day when culture is terminated. Wash twice with PBS, fix with 4% PFA at room temperature for 10 min, wash three times with PBS and then dry as much as possible. After drying at room temperature, add oil red O staining solution at room temperature for 1 h, rinse three times with 70% alcohol, and observe and take pictures under a microscope.

[0091] The results are as follows Figure 4As shown in b, the number of adipocytes after treatment with 2.5μM-10μM Pa was significantly reduced, and the lipid droplets were sparse. In contrast, there were a large number of adipocytes stained with oil red in the control group, and the lipid droplets were round and plump, indicating that Pa inhibited the adipogenic differentiation of hBMSCs in a concentration-dependent manner. Within the concentration range of 2.5μM-10μM, the ability to inhibit adipogenic differentiation became stronger as the concentration increased.

[0092] In summary, the results of the embodiments of the present invention show that bamipine and bamipine hydrochloride can regulate bone metabolism by affecting the osteogenic / adipogenic differentiation of bone marrow mesenchymal stem cells, and thus can be used to treat osteoporosis. Therefore, bamipine or its salts and derivatives can be used as a new strategy for treating and / or alleviating osteoporosis.

[0093] Finally, it should be noted that the above content is only used to illustrate the technical solution of the present invention, rather than to limit the scope of protection of the present invention. Simple modifications or equivalent substitutions of the technical solution of the present invention by ordinary technicians in this field do not deviate from the essence and scope of the technical solution of the present invention.

Claims

1. Use of bamipine or its salts and derivatives in the preparation of drugs for treating and / or alleviating osteoporosis.

2. The use according to claim 1, characterized in that: The drug treats and / or alleviates osteoporosis by inhibiting the adipogenic differentiation of bone marrow mesenchymal stem cells and promoting the osteogenic differentiation of bone marrow mesenchymal stem cells.

3. The use according to claim 1 or 2, characterized in that: The concentration of palmatine in the drug is 2.5 μM-20 μM.

4. The use according to claim 3, characterized in that: The concentration of palmatine in the drug is 2.5 μM-10 μM.

5. The use according to claim 1 or 2, characterized in that: The concentration of bamipine hydrochloride in the drug is 2.5 μM-20 μM.

6. The use according to claim 5, characterized in that: The concentration of bamipine hydrochloride in the drug is 2.5 μM-10 μM.

7. A drug for treating and / or alleviating osteoporosis, characterized in that: The medicine contains bamipine, and the concentration of bamipine is 2.5 μM-20 μM.

8. The drug according to claim 7, characterized in that The concentration of palmatine is 2.5 μM-10 μM.

9. A drug for treating and / or alleviating osteoporosis, characterized in that: The medicine contains bamipine hydrochloride, and the concentration of bamipine hydrochloride is 2.5 μM-20 μM.

10. The drug according to claim 9, characterized in that The concentration of the palmatine hydrochloride is 2.5 μM-10 μM.

Images