Application of sodium taurocholate in preparation of medicine for coordinating bone fat metabolism balance

Abstract

According to the application of the sodium taurocholate in preparing the medicine for coordinating the metabolic balance of the bone fat, the sodium taurocholate is used for coordinating the metabolic balance of the bone fat by inhibiting the accumulation of bone marrow adipose tissues. According to the application, the bone marrow adipose tissue can be accurately targeted to inhibit abnormal accumulation of the bone marrow adipose tissue, and normal bone marrow adipose tissue is not influenced. And moreover, by inhibiting abnormal accumulation of bone marrow adipose tissues, the bone fat metabolism balance is synergistically regulated and controlled, and particularly, adipogenesis inhibition and continuous osteogenesis promotion are combined. Sodium taurocholate is an endogenous bile acid derivative, so that the safety is higher, and sodium taurocholate is a micromolecular drug, so that sodium taurocholate can be orally taken compared with the current macromolecular drug injection administration route. In the application of the sodium taurocholate in preparing the medicine for coordinating the metabolism balance of the bone fat, the volume reduction amount of bone marrow adipose tissue is not less than 40%, and the bone mass increase amount is not less than 30%.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of medicine, in particular to the use of sodium taurocholate in the preparation of drugs for coordinating bone lipid metabolism balance. BACKGROUND

[0002] In the bone marrow microenvironment, the content of bone marrow adipose tissue (also referred to as BMAT) is closely related to cell differentiation dynamics and bone structure homeostasis. When BMAT abnormally accumulates, it will squeeze out space, signal regulation, and microenvironment changes, breaking the balance between osteogenesis and osteoclasts. BMAT is not only an energy storage organ, but also a regulatory hub for bone metabolism balance. On the one hand, BMAT expansion directly squeezes out the osteogenic differentiation space of bone marrow mesenchymal stem cells, disrupting the bone marrow microenvironment; on the other hand, BMAT secretes inflammatory factors such as IL-6, CCL2, and TNF-α, which induce osteoblast apoptosis, promote osteoclast activation, and accelerate bone loss; furthermore, the excessive free fatty acids released by BMAT can induce lipotoxic microenvironments, which can directly interfere with normal metabolic signaling pathways in cells, and also produce excessive reactive oxygen species through mitochondria, reducing osteoblast differentiation and bone formation.

[0003] Currently, clinical drugs for treating osteoporosis are divided into synthetic metabolic drugs and anti-bone resorption drugs. Synthetic metabolic drugs include parathyroid hormone analogs and recombinant human bone morphogenetic protein-2 (rhBMP-2), and anti-bone resorption drugs include bisphosphonates (alendronate sodium), bone resorption inhibitors (denosumab), and calcitonin. However, these drugs have significant side effects and limitations when used long-term, and cannot reverse the abnormal accumulation of BMAT. The incidence of bisphosphonate jaw osteonecrosis in long-term drug users is 0.001%-0.1%, and long-term use of bisphosphonates is the strongest risk factor for atypical femoral fractures, with a median duration of 56.5 months. Long-term use of bone resorption inhibitor denosumab can cause hypocalcemia. Head-to-head comparison studies have shown that synthetic metabolic agents show stronger anti-fracture efficacy and significantly higher bone density compared to anti-bone resorption drugs. Teriparatide is a synthetic form of naturally occurring parathyroid hormone, and is currently the only synthetic metabolic or bone-building agent approved by the FDA in the United States. However, due to its potential risk of osteosarcoma, it is limited to continuous treatment time, and the cumulative use time in a lifetime should not exceed 24 months. Long-term use of calcitonin can increase the risk of cancer, and it is now rarely used for the prevention and treatment of osteoporosis. Concerns about side effects and long-term efficacy have also led to severe treatment deficiencies among osteoporosis patients. Existing drugs have a single mechanism of action, such as bisphosphonates, which only inhibit osteoclast activity, and teriparatide, which only temporarily activates osteogenic signals, and neither involves BMAT-osteoblast coupling regulation. Therefore, exploring the BMAT metabolic microenvironment and developing synthetic metabolic drugs that inhibit adipogenesis, promote osteogenesis, and increase bone mass are key directions for breaking through existing treatment bottlenecks.

[0004] Therefore, in order to solve the problems in the prior art, the application provides a use of sodium taurocholate in preparation of a medicine for coordinating bone lipid metabolism balance. SUMMARY

[0005] The application aims to avoid the problems in the prior art and provide a use of sodium taurocholate in preparation of a medicine for coordinating bone lipid metabolism balance. The use can accurately target bone marrow adipose tissue to inhibit abnormal accumulation of the bone marrow adipose tissue.

[0006] The above object of the application is achieved by the following technical solutions. The application provides a use of sodium taurocholate in preparation of a medicine for coordinating bone lipid metabolism balance.

[0007] Preferably, the sodium taurocholate can inhibit accumulation of bone marrow adipose tissue to coordinate bone lipid metabolism balance.

[0008] Preferably, the sodium taurocholate is administered orally.

[0009] Preferably, the sodium taurocholate is administered at a dose of 10 mg / kg to 100 mg / kg.

[0010] In the use of the sodium taurocholate in preparation of the medicine for coordinating bone lipid metabolism balance, the volume of the bone marrow adipose tissue is reduced by not less than 40%.

[0011] In the use of the sodium taurocholate in preparation of the medicine for coordinating bone lipid metabolism balance, the volume of the bone marrow adipose tissue is reduced by 50.67%.

[0012] In the use of the sodium taurocholate in preparation of the medicine for coordinating bone lipid metabolism balance, the bone mass is increased by not less than 30%.

[0013] The use of the sodium taurocholate in preparation of the medicine for coordinating bone lipid metabolism balance can accurately target bone marrow adipose tissue to inhibit abnormal accumulation of the bone marrow adipose tissue, and has no effect on normal bone marrow adipose tissue. Moreover, the application can inhibit lipid formation and continuously promote bone formation to coordinate and control bone lipid metabolism balance. The sodium taurocholate is an endogenous bile acid derivative and is safer. Moreover, the sodium taurocholate is a small molecule drug, and compared with a current macromolecular drug injection administration route, the sodium taurocholate can be administered orally. BRIEF DESCRIPTION OF DRAWINGS

[0014] The application is further described by using the accompanying drawings, but the content in the drawings does not constitute any limitation on the application.

[0015] Figure 1 It is a two-dimensional structure diagram of the sodium taurocholate.

[0016] Figure 2 is a three-dimensional structure diagram of sodium taurocholate, wherein Figure 2 (a) is a Sticks model diagram, Figure 2 (b) is a Ball and Stick model diagram, Figure 2 (c) is a Surface model diagram.

[0017] Figure 3 is a micro-CT trabecular bone three-dimensional reconstruction of the sham group, the OVX group, the NAT_10 group, the NAT_50 group and the NAT_100 group.

[0018] Figure 4 is a trabecular bone related parameter of the sham group, the OVX group, the NAT_10 group, the NAT_50 group and the NAT_100 group.

[0019] Figure 5 is a HE and Masson staining picture of the sham group, the OVX group and the NAT_100 group.

[0020] Figure 6 is a volume ratio diagram of BMAT in the proximal femur of the sham group, the OVX group and the NAT_100 group.

[0021] Figure 7 is a representation diagram of bone turnover biomarkers PINP and BALP of the sham group, the OVX group, the NAT_10 group, the NAT_50 group and the NAT_100 group.

[0022] Figure 8 is a relative expression amount of RNX2 of the bone marrow mesenchymal stem cells MSCs after 3 days and 7 days of intervention of sodium taurocholate.

[0023] Figure 9 is a relative expression amount of ALP of the bone marrow mesenchymal stem cells MSCs after 14 days and 21 days of intervention of sodium taurocholate.

[0024] Figure 10 is a relative expression amount of COL1 of the bone marrow mesenchymal stem cells MSCs after 14 days and 21 days of intervention of sodium taurocholate. DETAILED DESCRIPTION

[0025] The technical solutions of the present application are further described in combination with the following examples. The experimental methods in the following examples are all conventional methods unless otherwise specified. The raw materials, reagents and materials used in the following examples can be purchased from conventional biochemical reagent stores or pharmaceutical business enterprises unless otherwise specified.

[0026] Example 1 The application of sodium taurocholate in preparing a medicine for coordinating bone lipid metabolism balance. In the application, sodium taurocholate inhibits abnormal accumulation of bone marrow adipose tissue, thereby coordinating bone lipid metabolism balance.

[0027] The bone lipid metabolism balance of the application refers to inhibiting abnormal accumulation of bone marrow adipose tissue and simultaneously promoting osteogenic differentiation.

[0028] The administration route of sodium taurocholate of the application is oral administration. The original medical use of sodium taurocholate is a cholagogue, and the original medical use of sodium taurocholate is a cholagogue. The molecular formula of sodium taurocholate (abbreviated as NAT) is C 26 H 46 NNaO8S, the SMILES formula is C[C@H](CCC(=O)NCCS(=O)(=O)[O])[C@H]1CC[C@@H]2[C@@]1([C@H](C[C@H]3[C@H]2[C@@H](C[C@H]4[C@@]3(CC[C@H](C4)O)C)O)O)C.O.[Na+], a two-dimensional structure diagram and a three-dimensional structure diagram are shown in Figure 1 and Figure 2 The current sodium taurocholate is used for treating chronic cholestasis, and it is a conjugated bile acid sodium salt with a molecular weight of 537.68 Da. Bile acids are products of cholesterols oxidized and decomposed in the liver, synthesized in the liver, and stored in the gallbladder, and play an important role in maintaining the homeostasis of cholesterols in the body.

[0029] The application of the sodium taurocholate in preparing a medicine for coordinating bone lipid metabolism balance can accurately target bone marrow adipose tissue to inhibit abnormal accumulation of bone marrow adipose tissue without affecting normal bone marrow adipose tissue. Moreover, the application can coordinate and regulate bone lipid metabolism balance by inhibiting abnormal accumulation of bone marrow adipose tissue, specifically by inhibiting adipogenesis and continuously promoting osteogenesis. Since sodium taurocholate is an endogenous bile acid derivative, it is safer. Moreover, sodium taurocholate is a small molecule drug, which can be administered orally compared to the current large molecule drug injection administration route. In the application of sodium taurocholate in preparing a medicine for coordinating bone lipid metabolism balance, the volume of bone marrow adipose tissue is reduced by not less than 40%, and the bone mass is increased by not less than 30%.

[0030] Test example Effect of sodium taurocholate on promoting bone formation and bone marrow adipose tissue (BMAT).

[0031] The application analyzes the mechanism of sodium taurocholate (NAT) on bone marrow microenvironment, specifically 40 12-week-old mice are randomly divided into 5 groups (n=8 per group), as follows: The sham group: after incision to the muscle layer, suture, and reserve the ovary.

[0032] OVX group: bilateral ovariectomy, dH2O 0.2ml gavage (2 days / time).

[0033] NAT_10 group: bilateral ovariectomy, the concentration of NAT is 10mg / kg, and 0.2ml gavage (2 days / time).

[0034] NAT_50 group: bilateral ovariectomy, the concentration of NAT is 50mg / kg, and 0.2ml gavage (2 days / time).

[0035] NAT_100 group: bilateral ovariectomy, the concentration of NAT is 100mg / kg, and 0.2ml gavage (2 days / time).

[0036] 5 group starts gavage at 1 week after operation, and all mice are killed and sampled after 10 weeks of gavage for subsequent analysis.

[0037] 1, micro-CT quantitative analysis shows As shown in Figure 3 and Figure 4 , the three different concentration treatment groups of sodium taurocholate NAT, 10mg / kg, 50mg / kg, 100mg / kg, wherein the 100mg / kg group and the ovariectomy (OVX) group have significant differences in each index.

[0038] Compared with the OVX group, the proximal femur bone volume fraction (BV / TV, P<0.01), the trabecular bone number per unit volume (Tb.N, P<0.01) and the trabecular bone connection density (Conn.d, P<0.01) of the NAT treatment group (OVX-NAT, 100mg / kg) increased significantly, close to the sham operation group (Sham group); the trabecular bone separation degree (Tb.Sp) decreased to the Sham group level (P<0.01).

[0039] Since the present application is a fixed height reconstruction trabecular bone, there is no statistical difference in trabecular bone thickness (Tb.Th) between groups. Compared with the Sham group, there is no statistical difference in each bone parameter of the OVX-NAT group (100mg / kg), so it is suggested that sodium taurocholate can improve the bone loss caused by estrogen deficiency. Through verification, the bone mass of the sodium taurocholate NAT of the present application is greater than or equal to 30%. It can be seen that the proximal femur bone volume fraction of the NAT group increases, the bone mass increases, and the trabecular bone number per unit volume increases, and it is restored to the sham operation group level.

[0040] 2, histomorphological analysis shows As shown in Figure 5 and Figure 6As shown, the proportion of bone marrow adipose tissue in the OVX group reached 25.34%, significantly higher than that in the Sham group (10.53%, P<0.01) and the NAT group (12.5%, P<0.05). However, there was no statistically significant difference between the OVX-NAT group and the Sham group, suggesting that sodium taurocholate specifically inhibits abnormal accumulation of bone marrow adipose tissue under pathological conditions. Therefore, in pathological conditions, this invention reduces bone marrow adipose tissue volume by no less than 40%, specifically by 50.67%.

[0041] 3. Dynamic monitoring of bone metabolism markers like Figure 7 As shown, serum PINP and BALP levels were significantly increased in the OVX group (P<0.001), while they were significantly downregulated after NAT intervention (P<0.001), indicating that sodium taurocholate inhibits bone high turnover and promotes metabolic homeostasis reconstruction.

[0042] 4. In vitro experiments Primary mouse bone marrow mesenchymal stem cells were cultured in 6 / 12-well plates and divided into four groups for induction: normal culture medium group (CON), 1 μM NAT group (CON+N), osteogenic induction culture medium group (OB), and osteogenic induction culture medium + 1 μM NAT group (OB+N).

[0043] The specific methods for obtaining primary mouse bone marrow mesenchymal stem cells are as follows: (1) Select 4-6 week old C57 mice and kill them by cervical dislocation. Then soak these mice in 75% alcohol solution for 5-10 minutes. (2) The soaked mice were washed with sterile PBS to remove any alcohol residue. The skin of the mice was cut open, and then the tibia and femur were separated. The remaining muscle and connective tissue were then removed, and the tissues were placed in sterile PBS. (3) Gently cut off both ends of the long bone with surgical scissors to expose the medullary cavity; (4) Fill a 1ml syringe with PBS, insert the needle into the bone marrow cavity and rinse repeatedly. A red viscous substance can be seen flowing out, and the diaphysis of the long bone gradually turns white. (5) Transfer the PBS containing the bone marrow contents into a new EP tube, and prepare a 15ml centrifuge tube at the same time; (6) First, firmly fix the EP tube at the opening of the centrifuge tube, then tighten the cap to ensure the stability of the EP tube. Next, put the 15ml centrifuge tube into the centrifuge and centrifuge at 4℃ and 2000rpm for 10 minutes. After centrifugation, discard the supernatant of the EP tube, then add 3-5 times the volume of the precipitate of red blood cell lysis buffer, mix well, let stand at 4℃ for 5 minutes, and then centrifuge at 4℃ and 1500rpm for 5 minutes. (7) Discard the supernatant, add 2 ml of α-MEM complete medium containing 10% FBS, resuspend the cells by pipetting, and transfer them to a 10 cm culture dish, add 8 ml of α-MEM complete medium containing 10% FBS, and finally place them in a 37°C, 5% incubator; (8) After the cells are incubated for 4-5 days, the first liquid exchange can be performed, and the number and density of the cells are observed. Then, the α-MEM complete medium containing 10% FBS is replaced every three days.

[0044] The preparation method of the osteogenic induction medium of the application: according to the predetermined concentration, mix 10 mM of sodium β-glycerophosphate with 50 μg / ml of ascorbic acid.

[0045] The results of in vitro osteogenic differentiation show that, as Figures 8 to 10 shown in FIG. 6, the mRNA expression of the early osteogenic marker RUNX2 reaches a peak at 7 days of NAT intervention, and the mRNA expression of the middle and late osteogenic markers (COL1 and ALP) is enhanced at 21 days of NAT intervention.

[0046] In summary, sodium taurocholate can precisely target bone marrow adipose tissue to inhibit abnormal accumulation of bone marrow adipose tissue without affecting normal bone marrow adipose tissue, thereby synergistically regulating bone lipid metabolism balance, specifically by inhibiting adipogenesis and continuously promoting osteogenesis. In the use of sodium taurocholate in the preparation of a drug for regulating bone lipid metabolism balance, the volume of bone marrow adipose tissue is reduced by not less than 40%, and the bone mass is increased by not less than 30%.

[0047] Finally, it should be noted that the above examples are only used to illustrate the technical solutions of the application and not to limit the protection scope of the application. Although the application has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the application can be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the application.

Claims

1. Use of sodium taurocholate in the preparation of a drug for coordinating bone lipid metabolism balance.

2. Use of sodium taurocholate according to claim 1 for the preparation of a medicament for coordinating the balance of bone lipid metabolism, characterized by: The sodium taurocholate coordinates the bone lipid metabolism balance by inhibiting bone marrow adipose tissue accumulation.

3. Use of sodium taurocholate according to any one of claims 1 to 2 for the preparation of a medicament for coordinating the balance of bone lipid metabolism, characterized in that: The administration route of the sodium taurocholate is oral administration.

4. Use of sodium taurocholate according to any one of claims 1 to 2 for the preparation of a medicament for coordinating the balance of bone lipid metabolism, characterized in that: The administration amount of the sodium taurocholate is 10 mg / kg-100 mg / kg.

5. Use of sodium taurocholate according to claim 1 for the preparation of a medicament for coordinating the balance of bone lipid metabolism, characterized by: The bone marrow adipose tissue volume reduction amount is not less than 40%.

6. Use of sodium taurocholate according to claim 1 for the preparation of a medicament for coordinating the balance of bone lipid metabolism, characterized by: The bone marrow adipose tissue volume reduction amount is 50.67%.

7. Use of sodium taurocholate according to claim 1 for the preparation of a medicament for coordinating the balance of bone lipid metabolism, characterized by: The bone mass increase amount is greater than or equal to 30%.

Images