Application of tacalolactone A in preparation of medicine for preventing and treating osteoporosis

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of tacarrolactone A in preparation of medicines for preventing and treating osteoporosis. It is proved for the first time that the intestinal flora metabolite tacarrolactone A can remarkably promote osteogenic differentiation of bone marrow mesenchymal stem cells by regulating and controlling a Wnt / beta-catenin signal channel, the bone protection effect is achieved from the core link of promoting bone formation, the mechanism is different from an existing conventional treatment strategy which mainly inhibits bone resorption, and the treatment effect is remarkable. The invention provides a brand new bone metabolism regulation target and treatment path, opens up a new drug research and development direction for clinical prevention and treatment of osteoporosis, and has important scientific value and application prospect.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to application of tacarolide A in preparation of a medicine for preventing and treating osteoporosis. BACKGROUND

[0002] Osteoporosis, especially postmenopausal osteoporosis (PMOP), as a common chronic bone metabolism disease in clinic, is easy to cause brittle fracture, causes serious health hazards and heavy economic burden, and the population size of the disease is huge in China and the world, and the prevention and treatment demand is urgent. At present, the clinical treatment is mainly western medicine. Although such chemical drugs can play a certain role by promoting bone formation and inhibiting bone resorption, there are significant limitations: for patients with severe pain, it is difficult to achieve drug replacement due to long-term medication, and some patients have drug intolerance, resulting in poor treatment effect and limited clinical application.

[0003] PMOP is classified as "bone flaccidity" and "bone dryness" in traditional Chinese medicine, and the core prevention and treatment idea is to tonify the kidney. Although it can provide an auxiliary solution for patients with poor western medicine treatment effect, the current research on the mechanism of traditional Chinese medicine in preventing and treating PMOP is still not deep enough. It is known that intestinal flora, as an important material basis of the function of "spleen" in traditional Chinese medicine, is closely related to the theory of "kidney governing bone" and bone metabolism. The intestinal flora-bone axis can regulate bone metabolism through the immune system, endocrine system and nutrient absorption, and the Wnt / β-catenin signaling pathway is a key pathway for regulating bone homeostasis. Intestinal flora and its metabolites may affect bone metabolism through this pathway, and the specific correlation mechanism between the two has not been clarified.

[0004] Although existing research has confirmed that traditional Chinese medicine such as kidney-tonifying and blood-activating prescriptions has a significant preventive and therapeutic effect on PMOP, it is also speculated that intestinal flora may be an important link for traditional Chinese medicine to regulate bone metabolism. However, there is insufficient research on the specific role and mechanism of intestinal flora metabolites in the regulation of bone metabolism, especially the lack of research on intestinal flora metabolites regulating bone marrow mesenchymal stem cell osteogenic differentiation through the Wnt / β-catenin signaling pathway to prevent and treat osteoporosis. The specific active metabolites and their target points have not been clarified, which cannot provide new drug directions and mechanism support for the precise treatment of osteoporosis, and there is still a significant gap in existing technology. SUMMARY

[0005] The application aims to overcome the defects and deficiencies in the prior art and provide the use of tacaronolide A in the preparation of a drug for preventing and treating osteoporosis. The key intestinal flora metabolite tacaronolide A discovered through screening can be used as a beneficial supplement to the existing treatment method. The present study further clarifies the specific action mechanism of tacaronolide A: it plays a bone protection role by activating the Wnt / β-catenin signaling pathway, promoting the osteogenic differentiation of bone marrow mesenchymal stem cells, and enhancing bone formation. The discovery lays an experimental foundation for the development of new anti-osteoporosis drugs in the future and provides a new idea for the prevention and treatment of osteoporosis.

[0006] To achieve the above object, the technical scheme adopted by the present application comprises: In a first aspect, the present application provides the use of tacaronolide A in the preparation of a drug for preventing and treating osteoporosis.

[0007] Preferably, the tacaronolide A plays a role in preventing and treating osteoporosis by activating the Wnt / β-catenin signaling pathway.

[0008] Preferably, the tacaronolide A activates the Wnt / β-catenin signaling pathway by promoting the expression of Wnt5a, p-GSK3β / GSK3β and β-catenin.

[0009] Preferably, the osteoporosis includes postmenopausal osteoporosis.

[0010] Preferably, the tacaronolide A is an intestinal flora metabolite.

[0011] The present application clearly discloses the core action mechanism and key molecular target of tacaronolide A (TA) in preventing and treating osteoporosis, and provides a new path with scientificity and practicability for the treatment of osteoporosis (especially postmenopausal osteoporosis, PMOP).

[0012] From the action mechanism, the Wnt / β-catenin signaling pathway is the core pathway for regulating bone homeostasis, and its activation can directly promote the osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and inhibit the activity of osteoclasts. The present application proves by experiments that the intestinal flora metabolite TA can specifically up-regulate the expression of Wnt5a, p-GSK3β / GSK3β and β-catenin, precisely activate the Wnt / β-catenin signaling pathway, and then promote the osteogenic differentiation of bone marrow mesenchymal stem cells. The present study clearly clarifies the complete regulation chain of "intestinal flora metabolite-signaling pathway-bone formation", fills the gap in the prior art that the correlation mechanism of intestinal flora and its metabolite TA with bone metabolism is not clear, and opens up a new direction for the research and development of osteoporosis treatment drugs.

[0013] Preferably, the effective concentration of tacaronolide A is 0.2-0.8 μM.

[0014] It is found through experiments that tacaronolide A in the above-mentioned preferred concentration range can dose-dependently improve the cell viability of BMSCs, and the concentrations of 0.4 and 0.8 μM can significantly up-regulate the expressions of ALP, COL1A1 and RUNX2 in BMSCs, while the concentrations of 0.2-0.8 μM can effectively promote the expressions of Wnt5a, p-GSK3β / GSK3β and β-catenin, the above-mentioned effects from multiple aspects such as the enhancement of cell viability, the promotion of the expressions of key markers of osteogenic differentiation and the activation of the classical osteogenic signaling pathway, clearly verify the positive regulation of tacaronolide A on the osteogenic-related biological processes at the preferred concentration, and provide precise concentration basis and solid experimental support for the development of tacaronolide A as an efficient and targeted anti-osteoporosis drug.

[0015] More preferably, the effective concentration of tacaronolide A is 0.4 μM.

[0016] In the second aspect, the present application provides a drug for preventing and treating osteoporosis, and the effective component of the drug is tacaronolide A.

[0017] Preferably, the concentration of tacaronolide A in the drug is 0.2-0.8 μM.

[0018] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0019] Compared with the prior art, the present application has the following beneficial effects: The present application first confirms that the intestinal flora metabolite tacaronolide A can significantly promote the osteogenic differentiation of bone marrow mesenchymal stem cells by regulating the Wnt / β-catenin signaling pathway, and plays a bone protection role from the core link of promoting bone formation, which is different from the conventional treatment strategy of mainly inhibiting bone resorption, provides a brand-new bone metabolism regulation target and treatment path, opens up a new drug research and development direction for the clinical prevention and treatment of osteoporosis, and has important scientific value and application prospect. BRIEF DESCRIPTION OF DRAWINGS

[0020] Figure 1 Figure 1 is a result graph of the effect of tacaronolide A (TA) on the proliferation of BMSCs; *P<0.05, **P<0.01 vs. the control group (0 μM); Figure 2 Figure 2 is a result graph of ALP staining of the effect of different concentrations of tacaronolide A (TA) on the osteogenic differentiation of BMSCs; Figure 3 Figure 3 is a result graph of WB detection of the effect of different concentrations of tacaronolide A (TA) on the osteogenic differentiation of BMSCs; Figure 4 The image shows the Western blot results of the effects of different concentrations of tacalololide A (TA) on proteins related to the Wnt / β-catenin signaling pathway in BMSCs. Figure 5 The image shows the results of Western blot analysis of the effect of tacalololide A (TA) on proteins related to the Wnt / β-catenin signaling pathway after Wnt signaling blockade. Figure 6 The image shows the results of Western blot analysis of the effect of tacalololide A (TA) on the bone formation-related protein COL1A1 after Wnt signaling blockade. Detailed Implementation

[0021] To make the objectives, technical solutions, and effects of this invention clearer and more explicit, the invention will be further described in detail below with reference to the embodiments and accompanying drawings. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.

[0022] Unless otherwise specified, all reagents used in the examples are conventional reagents available in the art and can be purchased commercially. Experimental procedures not specifically described in the examples are conventional procedures in the art or can be understood or known by those skilled in the art based on their prior knowledge or common general knowledge.

[0023] The tacalololide A described in this invention was purchased from Shanghai Taoshu Biotechnology Co., Ltd., product number 108885-68-3.

[0024] Example 1 In this embodiment, the effect of tacalololide A (TA) on the proliferation of BMSCs was detected by CCK8 assay. Different TA concentrations (0, 0.2, 0.4, 0.8, 1.6, 3.2 μM) were set up to intervene BMSCs for 24 h, 48 h, and 72 h, respectively.

[0025] The results showed that, compared with the control group (0 μM), after TA intervention for 12, 24, and 48 hours, cell viability increased in a dose-dependent manner at TA concentrations of 0.2, 0.4, and 0.8 μM (P<0.05), significantly promoting cell viability; however, when the TA concentration increased to 1.6 and 3.2 μM, cell viability decreased compared with the 0.8 μM group (see results below). Figure 1 ).

[0026] Example 2 This embodiment investigates the intervention effect of tacalololide A (TA) on bone marrow mesenchymal stem cells (BMSCs). The expression of bone formation-related proteins and Wnt / β-catenin signaling pathway-related proteins was detected by Western blot, and the effect of TA on BMSCs was analyzed by ALP staining.

[0027] (1) Regulatory role of TA on osteogenic differentiation of BMSCs Bone marrow mesenchymal stem cells (BMSCs) play a crucial role in bone formation, possessing the ability to differentiate into multiple lineages, directly participating in bone formation by differentiating into bone cells (OBs). ALP staining results show ( Figure 2 Compared with the control group (0 μM), the blue-purple staining depth of cells treated with 0.2, 0.4, and 0.8 μM TA increased with increasing TA concentration under a microscope, suggesting that ALP activity increased with increasing TA concentration, and TA can promote the differentiation of BMSCs into osteogenic groups.

[0028] The expression of bone formation-related factors ALP, COL1A1, and RUNX2 was detected by Western blot. The results showed that ( Figure 3 Compared with the control group (0 μM), the effect of TA at a concentration of 0.2 μM on BMSCs was not significantly different, while the intervention groups of 0.4 and 0.8 μM TA upregulated the expression of ALP, COL1A1 and RUNX2 in BMSCs (P<0.01).

[0029] (2) Activation effect of TA on the Wnt / β-catenin signaling pathway in BMSCs The effects of different concentrations of TA on proteins related to the Wnt / β-catenin signaling pathway were detected by Western blot. The results showed that ( Figure 4 Compared with the control group, 0.2, 0.4, and 0.8 μM TA significantly promoted the protein expression of Wnt5a, p-GSK3β / GSK3β, and β-catenin (P<0.05), suggesting that TA can activate the Wnt / β-catenin signaling pathway in BMSCs.

[0030] Example 3 This embodiment investigates the effect of TA on BMSCs after blocking the Wnt / β-catenin signaling pathway.

[0031] (1) Effects of TA on proteins related to the Wnt / β-catenin signaling pathway after intervention with the pathway inhibitor CCT. Based on the aforementioned results, the inventors further explored the mechanism of TA's effect on osteogenic differentiation of BMSCs. The optimal TA concentration was selected as 0.4 μM for subsequent mechanistic studies. CCT, a potent inhibitor of Wnt signaling, was used to block the Wnt / β-catenin signaling pathway in BMSCs. A concentration of 5 μM CCT was used to intervene in BMSCs, and the effects of TA on Wnt signaling pathway-related proteins in BMSCs after pathway blockade were observed. The results are as follows... Figure 5As shown, compared with the control group, TA promoted the expression of Wnt5a and β-catenin, while CCT reduced β-catenin expression, but had no significant effect on Wnt5a. Compared with the TA group, the combined application of CCT and TA reduced β-catenin protein expression.

[0032] (2) Effects of TA on bone formation-related proteins through the Wnt / β-catenin signaling pathway Based on the aforementioned results, when the Wnt / β-catenin signaling pathway is inhibited, the regulatory effect of TA on the pathway is weakened. Next, the inventors used Western blot to examine the osteogenic differentiation effect of TA on BMSCs under pathway inhibition. The results are as follows: Figure 6 As shown, compared with the control group, TA can promote the expression of bone formation-related markers COL1A1, RUNX2 and ALP, while CCT intervention can inhibit the expression of RUNX2 and ALP proteins. Moreover, compared with the TA group, the combined use of CCT and TA can reduce the expression of COL1A1, ALP and RUNX2.

[0033] The above results indicate that the Wnt / β-catenin signaling pathway is a key pathway for TA to promote osteogenic differentiation of BMSCs, and blocking this pathway weakens the osteogenic effect of TA on BMSCs.

[0034] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Application of tacalololide A in the preparation of drugs for the prevention and treatment of osteoporosis.

2. The application as described in claim 1, characterized in that, Tacalololide A exerts its role in preventing and treating osteoporosis by activating the Wnt / β-catenin signaling pathway.

3. The application as described in claim 2, characterized in that, Tacalonolactone A activates the Wnt / β-catenin signaling pathway by promoting the expression of Wnt5a, p-GSK3β / GSK3β, and β-catenin.

4. The application as described in claim 1, characterized in that, The effective concentration of tacalololide A is 0.2-0.8 μM.

5. The application as described in claim 4, characterized in that, The effective concentration of tacalololide A is 0.4 μM.

6. The application as described in claim 1, characterized in that, Tacalololide A is a metabolite of intestinal flora.

7. The application as described in claim 1, characterized in that, The osteoporosis mentioned includes postmenopausal osteoporosis.

8. A drug for preventing and treating osteoporosis, characterized in that, The active ingredient of the drug is tacalololide A.

9. The medicament as described in claim 8, characterized in that, The concentration of tacalololide A in the drug is 0.2-0.8 μM.

10. The medicament as claimed in claim 8, characterized in that, The drug also includes pharmaceutically acceptable excipients.

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