Application of aryl turmerone in preparation of anti-osteoporosis medicine

Abstract

The invention relates to the technical field of medical health, in particular to application of aryl turmerone in preparation of an anti-osteoporosis medicine. The aryl turmerone plays an anti-osteoporosis effect through a dual action mechanism: in an in-vitro experiment, the aryl turmerone can inhibit the differentiation of RAW264.7 cells induced by RANKL to osteoclasts, promote the differentiation of bone marrow-derived mesenchymal stem cells to osteoblasts, and remarkably up-regulate the mRNA expression level of osteogenic differentiation related genes (such as COL-I, ALP, Runx2 and OCN). In an in-vivo experiment, the aryl turmerone effectively inhibits the bone loss phenomenon of an ovariectomy (OVX) induced osteoporosis mouse, and promotes osteoblast differentiation in the mouse. The results show that the aryl turmerone not only has a good anti-osteoporosis effect, but also shows excellent biological safety, and provides an important theoretical basis for further development of the aryl turmerone into medicines for clinically treating osteoporosis.

Description

Technical Field

[0001] The present invention relates to the field of medical health technology, and in particular to the application of turmeric flavonoids in the preparation of anti-osteoporosis drugs. Background Art

[0002] Osteoporosis is a systemic bone disease characterized by decreased bone density, destruction of bone microstructure, and increased bone brittleness. With the aging of the population, the incidence of osteoporosis has increased year by year, becoming an important health problem that seriously affects the quality of life and socioeconomic burden of the elderly. Therefore, it is particularly important to develop effective drugs for the treatment and prevention of osteoporosis.

[0003] The occurrence of osteoporosis is related to many factors, including genetics, endocrine, nutrition and lifestyle. Bone is an organ that is constantly growing and rebuilding, and its health depends on the dynamic balance between osteoblasts and osteoclasts. Osteoblasts are responsible for bone formation, while osteoclasts are responsible for bone absorption. When bone absorption exceeds bone formation, bone mass gradually decreases, eventually leading to the occurrence of osteoporosis.

[0004] At present, the treatment of osteoporosis mainly includes drug therapy and non-drug therapy. In terms of drug therapy, bisphosphonates, calcitonins and hormone replacement therapy are commonly used treatment methods. These drugs increase bone density and improve bone quality by inhibiting osteoclast activity, promoting bone formation or regulating bone metabolism. However, these drugs also have certain side effects, such as gastrointestinal reactions, renal impairment and tumor risks, which limit their long-term application.

[0005] In recent years, the pharmacological activity of natural products has attracted wide attention in the treatment of osteoporosis. Flavonoids, as a class of natural active ingredients widely found in plants, have a variety of pharmacological effects, including antioxidant, anti-inflammatory and anti-tumor effects, which suggest that flavonoids may have potential application value in the treatment of osteoporosis and provide a theoretical basis for the development of new low-toxic and highly effective anti-osteoporosis drugs. Summary of the invention

[0006] The purpose of the present invention is to solve the above technical problems and provide an application of ar-tumerone (ART) in the preparation of anti-osteoporosis drugs.

[0007] The technical solution of the present invention is as follows:

[0008] A use of turmeric flavonoids in the preparation of an anti-osteoporosis drug, wherein the structural formula of turmeric flavonoids is shown in Formula I:

[0009] The chemical formula of turmeric flavonoids is: 15 H20 O.

[0010] Preferably, the application is the application of aromatic turmeric flavonoids to inhibit TRAP activity and osteoclastogenesis in an osteoclast model in vitro;

[0011] The osteoclast model uses RANKL as an inducer and RAW264.7 cells as osteoclast precursor cells.

[0012] Preferably, the application is the application of turmeric flavonoids to promote the differentiation of mouse bone marrow-derived mesenchymal stem cells into osteoblasts in vitro.

[0013] Preferably, the application is the application of aromatic turmeric flavonoids to promote the expression of osteogenic differentiation-related genes COL-I, ALP, Runx 2 and OCN mRNA in mouse bone marrow-derived mesenchymal stem cells in vitro.

[0014] Preferably, the application is the application for preparing a drug for treating postmenopausal osteoporosis.

[0015] Furthermore, the drug is a drug that inhibits osteoclastogenesis.

[0016] The invention discloses an osteoporosis osteogenic drug, which contains a pharmaceutically effective dose of aromatic turmeric flavonoids.

[0017] Further, the effective dose of aromatic turmeric flavonoids is 20-60 μM.

[0018] Furthermore, the osteoporosis osteogenic drug uses turmeric as an effective ingredient and is used to promote the recovery of osteogenic parameters BMD, BV / TV, Ct.V, CT.Th, Tb.N and Tb.Th in postmenopausal osteoporosis patients.

[0019] Furthermore, the osteoporosis osteogenic drug uses turmeric as an effective ingredient and is used to inhibit the growth of Tb.Sp and SMI parameters in postmenopausal osteoporosis patients.

[0020] Furthermore, the osteoporosis osteogenic drug uses turmeric as an effective ingredient and is used to promote the increase of osteoblast number, bone formation rate and mineral apposition rate parameters in postmenopausal osteoporosis patients.

[0021] Preferably, the drug is in the form of tablets, powders, injections, capsules or aerosols; the auxiliary materials of the drug are any acceptable excipients in the drug.

[0022] The advantages of the present invention are:

[0023] The present invention provides an application of ART in the preparation of osteoporosis drugs. ART exerts anti-osteoporosis effects through a dual mechanism: on the one hand, ART can promote osteoblast differentiation, and on the other hand, it can effectively inhibit osteoclastogenesis. Specifically, in in vitro experiments, ART significantly inhibited the differentiation of RAW264.7 cells induced by RANKL into osteoclasts; at the same time, ART also promoted the differentiation of bone marrow-derived mesenchymal stem cells into osteoblasts and significantly upregulated the mRNA expression levels of osteogenic differentiation-related genes (such as COL-I, ALP, Runx 2 and OCN). In in vivo experiments, ART can inhibit the bone loss phenomenon of osteoporotic mice induced by ovariectomy (OVX), and promote osteoblast differentiation in mice. These results show that ART has good anti-osteoporosis effects and biosafety, providing theoretical support for its further development as a drug for the clinical treatment of osteoporosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a data chart showing the effect of ART on the proliferation of mBMSCs and RAW264.7.

[0025] Figure 2 The data of ART on the osteoclast differentiation of RAW264.7 cells treated with ALN are as follows: Figure 2 A is the tartrate-resistant acid phosphatase (TRAP) staining, scale bar: 50 μm; Figure 2 B is a quantitative analysis of TRAP activity.

[0026] Figure 3 The data of ART and ALN on the effect of mBMSCs differentiation into osteoblasts: Figure 3 A is the quantitative analysis diagram of ALP activity. Figure 3 B is the number of mineralized nodules stained with alizarin red. Figure 3 C is the free score of mineralized nodules stained with alizarin red; Figure 3 D is the result of Alizarin Red staining, scale bar: 200 μm.

[0027] Figure 4 The real-time fluorescence quantitative PCR was used to detect the osteogenic-related gene COL-I ( Figure 4 A) ALP Figure 4 B) Runx 2( Figure 4 C) OCN mRNA ( Figure 4 D) expression results.

[0028] Figure 5 This is an in vivo evaluation of the bone healing effects of ART and ALN on osteoporotic mice: Figure 5A is a representative micro-CT image of the distal femur; the trabecular parameters of the distal femur were analyzed by micro-CT. Figure 5 B is bone mineral density (BMD), Figure 5 C is bone volume / tissue volume (BV / TV), Figure 5 D is the volume of cortical bone (Ct.V), Figure 5 E is the thickness of cortical bone (CT.Th), Figure 5 F is the number of trabeculae (Tb.N), Figure 5 G is the trabecular thickness (Tb.Th), Figure 5 H is trabecular separation (Tb.Sp) and Figure 5 I is the structural model index (SMI).

[0029] Figure 6 The data graphs show that ART and ALN promote osteoblast differentiation in mice; Figure 6 A is the H&E staining and calcein staining of the tibia specimens of mice in each group. Figure 6 B is trabecular bone volume / tissue area, Figure 6 C is the number of osteoblasts per tissue area, Figure 6 D is the histomorphometric analysis of the mineral apposition rate (MAR), Figure 6 E is the bone formation rate.

[0030] Figure 7 This is a data chart of ART in vivo biosafety evaluation. DETAILED DESCRIPTION

[0031] The technical solutions described in the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the embodiments described in this specification are only part of the feasible technical solutions of the present invention. Other implementation methods obtained by ordinary technicians in this field based on the embodiments of the present invention without any creative work should be deemed to belong to the scope of protection of the present invention.

[0032] Example 1: MTT method to evaluate the effect of ART on the proliferation of mBMSCs cells and RAW264.7 cells

[0033] Primary mBMSCs were isolated from the femur and tibia of 6- to 8-week-old mice and cultured in minimal essential medium α (α-MEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin and streptomycin (P / S). RAW264.7 cells within passage 20 were cultured in DMEM complete medium (containing 10% FBS, 1% P / S) at 37°C and 5% CO. 2When the two cell types grew to 70% of the cell density, mBMSC and RAW264.7 cells were seeded into two 96-well plates, with 5000 cells in each well, at 37°C and 5% CO 2 , and cultured overnight under the conditions until the cells adhered to the wall. Using the gradient dilution method, ART was prepared into osteogenic induction medium (α-MEM containing 10% FBS, 1% P / S, 50μM L-ascorbic acid, 10mMβ-glycerophosphate and 0.1μM dexamethasone) of different concentrations, and 100μL of the solution was added to each well of the 96-well plate to make the final concentrations in the well plate 0, 20, 40, 60 and 80μM; after co-culture with cells for 2d / 4d, the medium was changed every two days. After the treatment, the original medium in the 96-well plate was discarded, and sterile MTT at a concentration of 5mg / mL was added under light-proof conditions, 20μL per well, and continued to be cultured in the incubator at 37C in the dark for 4 hours. After 4 hours, MTT was discarded, 150μL DMSO was added to fully dissolve the crystallized formazan in the well plate, and different absorbances were measured at 490nm using an enzyme marker to analyze cell viability.

[0034] Experimental results: Before the activity evaluation, the effect of ART on the cell proliferation of mBMSCs was observed. According to the results of MTT experiment Figure 2 As shown in (AB), at 20 and 40 μM concentrations of ART, mBMSCs and RAW264.7 cells proliferated compared with 0 μM, and the proliferation effect weakened at 60 and 80 μM. In general, ART treatment within the concentration range of 20-80 μM for 2 days and 4 days had no cytotoxicity to mBMSCs and RAW264.7 cells, and had a certain proliferation effect.

[0035] Example 2: Effects of ART on osteoclast differentiation of RAW264.7 cells and osteogenic differentiation of mBMSCs cells

[0036] 1. Effect of ART on osteoclast differentiation of RAW264.7 cells

[0037] RAW264.7 cells were seeded into 96-well plates at a density of 1000 / well and incubated at 37°C with 5% CO 2 The cells were cultured overnight in a cell culture incubator until the cells adhered to the wall. The corresponding drugs were added according to the grouping: Control group (PBS solution), ALN (20 μM), ART group (20, 40, 60 μM), and the culture medium used was osteoclast induction medium (α-MEM medium containing 10% FBS, 1% P / S and 50 ng / mL RANKL). After culturing for 5 days, the cell samples were collected and operated according to the instructions of the tartrate-resistant acid phosphatase (TRAP) detection kit and the staining kit.

[0038] Experimental results: TRAP is a marker enzyme for osteoclast differentiation and maturation. The status of osteoclast differentiation can be observed by staining and activity analysis of TRAP enzyme. In order to study the effect of ART on the differentiation of RAW264.7 cells into osteoclasts, and the difference in inhibiting osteoclast differentiation compared with the common clinical drug ALN, TRAP staining and activity detection were performed. Figure 2 As shown in A, compared with the Control group, ALN can reduce the number of osteoclasts, and the ART group showed a dose-dependent and significant inhibition of osteoclast formation, and the inhibitory effect of ART at 40 and 60 μM was better than that of the ALN group. The cell samples were further tested for TRAP activity, and the results were analyzed as follows Figure 2 As shown in B, compared with the Control group, ALN can inhibit the activity of TRAP in osteoclasts. The ART group also inhibited the production of TRAP in a dose-dependent manner, and the inhibitory effect of ART was better than that of ALN at concentrations of 40 and 60 μM. The above experimental results all indicate that ART can inhibit RANKL-induced differentiation of RAW264.7 cells into osteoclasts.

[0039] 2. Effect of ART on osteogenic differentiation of mBMSCs

[0040] (1) Alkaline phosphatase (ALP) activity assay: The mBMSCs cell density was adjusted to 2×10 4 cells / cm 2 The cells were seeded in 24-well plates, and after the cells adhered to the wall, the corresponding drugs were added according to the grouping: Control group (PBS solution), alendronate sodium (ALN) (20μM), ART group (20, 40, 60μM), and the culture medium used was osteogenic induction medium. After culturing for 14 days, the cell samples were collected and tested according to the instructions of the ALP detection kit.

[0041] (2) Alizarin red (ARS) staining: The cell density of mBMSCs was adjusted to 1×10 5 Cells / well were seeded in 6-well plates. After the cells adhered to the wall, the corresponding drugs were added according to the grouping: Control group (PBS solution), ALN (20μM), ART group (20, 40, 60μM), and the culture medium used was osteogenic induction medium. After culturing for 21 days, the cells in each group were fixed with 4% paraformaldehyde for 10 minutes, washed with PBS three times, stained with alizarin red for 30 minutes, washed with distilled water, observed and photographed, the number of mineralized nodules in different fields of view was randomly counted, and the area ratio of mineralized nodules was quantified using Image J software.

[0042] ALP is involved in the process of bone formation in bone metabolism and is a marker enzyme for osteoblast differentiation and maturation. In order to study the effect of ART on the osteogenic differentiation of mBMSCs cells and the difference in promoting osteoblast differentiation compared with the common clinical drug ALN. ALP activity detection and ARS staining were performed respectively. The results of the ALP detection experiment are shown in Figure 3 As shown in A, compared with the Control group, ALN and ART treatment of mBMSCs cells promoted their ALP activity, and ART promoted cell ALP activity best at a concentration of 40 μM.

[0043] Since mineralized nodules appear red after being stained with alizarin red, the present invention uses alizarin red staining to observe the formation of mineralized nodules in cells. Figure 3 (BD) shows that the number of mineralized nodules formed after ALN and ART treatment ( Figure 3 B) and mineralized area fraction ( Figure 3 C) were significantly higher than those in the Control group, which indicated that ART could not only enhance the mineralization ability of bone marrow mesenchymal stem cells, but also its promoting mineralization effect was stronger than that of ALN at a concentration of 40 μM. In addition, this conclusion was consistent with the results of Alizarin Red staining. Figure 3 The staining results of D were consistent: the red area in the ART group was significantly larger than that in the Control group and the ALN group, and the ability to promote the mineralization of mBMSCs was most significant at 40 μM.

[0044] Example 3: Effect of ART on mRNA expression of genes related to osteogenic differentiation of mBMSCs

[0045] Adjust the cell density of mBMSCs to 2×10 4 cells / cm 2 The cells were seeded in 6-well plates. After the cells adhered to the wall, the corresponding drugs were added according to the grouping: Control group (PBS solution), ALN (20μM), ART group (20, 40, 60μM), and the culture medium used was osteogenic induction medium. After culturing for 21 days, the cell pellets were collected and the total cell RNA was extracted for real-time fluorescence quantitative detection (RT-qPCR).

[0046] In the process of differentiation into osteoblasts, bone marrow-derived mesenchymal stem cells secrete type I collagen (COL-I) and osteocalcin (OCN) to assist osteoblasts in performing related functions. Runx2 is a key transcription factor that regulates osteoblast differentiation during bone formation and is a marker gene for osteoblast differentiation. In this regard, this application uses RT-qPCR to detect the mRNA expression of COL-I, OCN, Runx2 and ALP. The experimental results are as follows Figure 4(AD) It can be seen that after the cells were treated with ALN and ART, the related mRNAs of COL-Ⅰ, OCN, Runx2 and ALP showed an increasing expression trend compared with the Control group. Compared with the ALN group, the mRNAs of COL-Ⅰ, OCN, Runx2 and ALP in the ART group were higher, and ART promoted the highest expression of related genes at a concentration of 40μM, which showed that ART promoted the mRNA expression of genes related to osteogenic differentiation of mBMSCs cells and the effect was better than ALN, further indicating that ART has better prospects for medical treatment.

[0047] Example 4: In vivo evaluation of the effect of ART on bone healing in osteoporotic mice

[0048] Female C57BL / 6 mice aged 8 weeks were divided into 4 groups (n=15 per group), 3 groups underwent surgical ovariectomy (OVX) via a lateral retroperitoneal approach combined with ketamine / xylazine, and the remaining groups underwent sham surgery (Sham). Paracetamol was used to relieve postoperative pain within 24 hours. PBS solution was injected subcutaneously in the Sham and OVX groups, ART was injected subcutaneously in the OVX mice in the ART group at a dose of 10 μg / kg body weight, and ALN was injected subcutaneously in the OVX mice at a dose of 5 μg / kg body weight as a positive control. After 8 weeks, all mice were euthanized by cervical dislocation after anesthesia. The femurs and tibias of the mice were collected for CT scanning, H&E and calcein staining, and the hearts, livers, spleens, lungs and kidneys of the mice were collected for H&E staining.

[0049] The changes in bone mineral density (BMD) and bone volume parameters were observed by CT scanning of the femur and tibia of mice. Figure 5 As shown, BMD, BV / TV, Ct.V, CT.Th, Tb.N, Tb.Th ( Figure 5 AG) were significantly decreased; whereas Tb.Sp and SMI were significantly increased in OVX mice compared with the Sham group, however, these parameters were decreased by injection of ART and ALN ( Figure 5 These results suggest that ART, like ALN, can protect against bone loss induced by estrogen deficiency, and the data show that ART is more effective than ALN ​​in protecting mice from bone loss.

[0050] To examine the relationship between ART- and ALN-rescued OVX-induced bone loss and increased osteoblastic bone formation, parameters of osteoblastic bone formation were examined by histology, dual calcein labeling, and histomorphometric analysis. Figure 6 As shown in the present application, it was observed that the number of osteoblasts, mineral apposition rate (MAR), bone formation rate (BFR / BS), and trabecular bone volume were significantly reduced in OVX mice, while these parameters were restored by supplementation with ART and ALN ( Figure 6AE). These results suggest that ART and ALN can increase osteoblastic bone formation in OVX mice.

[0051] In order to observe the biological safety of ART in mice, the present invention collected the heart, liver, spleen, lung and kidney of mice for H&E staining and observation. Figure 7 As shown, compared with the OVX group injected with PBS solution, there was no significant difference in the organs of the ART group, indicating that ART did not produce obvious toxic side effects on mice, reflecting the good biosafety of ART.

[0052] In summary, the present invention provides an application of ART in the preparation of an anti-osteoporosis drug, which can inhibit osteoclastogenesis and promote osteoblast differentiation in vitro, inhibit bone loss in OVX-induced osteoporosis mice in vivo and promote osteoblast differentiation in mice, reflecting its good anti-osteoporosis effect and good biosafety, and is expected to be applied to the development and use of clinical osteoporosis drugs.

[0053] The above is only a preferred embodiment of the present invention. It should be pointed out that ordinary technicians in this technical field can make several improvements and supplements without departing from the principle of the present invention. These improvements and supplements should also be regarded as the scope of protection of the present invention.

Claims

1. An application of aromatic turmeric flavonoids in the preparation of anti-osteoporosis drugs.

2. The application according to claim 1, characterized in that: The aromatic turmeric flavonoids inhibit TRAP activity and osteoclastogenesis in an osteoclast model in vitro; the osteoclast model uses RANKL as an inducer and RAW264.7 cells as osteoclast precursor cells.

3. The application according to claim 1, characterized in that: The aromatic turmeric flavonoids promote the differentiation of mouse bone marrow-derived mesenchymal stem cells into osteoblasts in vitro.

4. The use according to claim 3, characterized in that: The aromatic turmeric flavonoids promote the expression of mRNA genes related to osteogenic differentiation of mouse bone marrow-derived mesenchymal stem cells in vitro, namely COL-I, ALP, Runx 2 and OCN.

5. The use according to claims 1-4, characterized in that: The medicine is a medicine for treating postmenopausal osteoporosis.

6. An osteoporosis osteogenic drug, characterized in that: The invention contains a pharmaceutically effective dose of aromatic turmeric flavonoids; the effective dose of the aromatic turmeric flavonoids is 20-60 μM.

7. The osteoporosis bone-promoting drug according to claim 6, characterized in that: The aromatic turmeric flavonoids are used as an effective ingredient to promote the recovery of bone formation parameters BMD, BV / TV, Ct.V, CT.Th, Tb.N and Tb.Th in postmenopausal osteoporosis patients.

8. The osteoporosis bone-promoting drug according to claim 6, characterized in that: The aromatic turmeric flavonoids are used as an effective ingredient to inhibit the growth of Tb.Sp and SMI parameters in postmenopausal osteoporosis patients.

9. The osteoporosis bone-promoting drug according to claim 6, characterized in that: The aromatic turmeric flavonoids are used as an effective ingredient to promote the increase of osteoblast number, bone formation rate and mineral apposition rate parameters in postmenopausal osteoporosis patients.

10. The drug according to claim 6 is in the form of tablets, powders, injections, capsules or aerosols; the auxiliary materials of the drug are any acceptable excipients in the drug.

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