Application of resibufogenin or pharmaceutically acceptable salt thereof in preparation of medicine for treating atherosclerosis

Abstract

The invention provides an application of resibufogenin or a pharmaceutically acceptable salt thereof in preparation of a medicine for treating atherosclerosis. The NLRP3 inflammasome is a multi-protein complex and participates in regulation and control of innate immune response, and abnormal activation of the NLRP3 inflammasome is related to occurrence and development of various diseases including atherosclerosis and the like. The resibufogenin targeted NLRP3 inflammasome is found for the first time and can be used for treating atherosclerosis. The NLRP3 inflammasome is used as a key regulatory factor of inflammatory response in atherosclerosis, and the abnormal activation of the NLRP3 inflammasome aggravates the disease progress. The invention provides a more effective anti-inflammatory treatment strategy by targeting the NLRP3 inflammasome.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to the use of resibufogenin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating atherosclerosis. Background Art

[0002] Currently, the treatment of atherosclerosis mainly focuses on aspects such as reducing blood lipids, anti - inflammation, and anticoagulation. Commonly used drugs include statins, anti - platelet drugs, or ACE inhibitors, etc. In addition, in recent years, with the deepening of the understanding of the role of inflammation in atherosclerosis, scientists have begun to pay attention to targeted therapies against inflammatory pathways. Among them, the NLRP3 inflammasome, as an important inflammatory regulatory factor, has become a research hotspot. Some studies have explored the potential of NLRP3 inhibitors in the treatment of atherosclerosis.

[0003] Although the existing treatment methods for atherosclerosis can slow down the disease process to a certain extent, there are still some deficiencies. Traditional therapies such as statins mainly relieve the symptoms of atherosclerosis by reducing blood lipids, but do not directly target the key pathological process of the inflammatory response. For the targeted therapy of the NLRP3 inflammasome, current research is still in the early stage, and many candidate drugs have deficiencies in terms of specificity, stability, and safety. In addition, some NLRP3 inhibitors may cause immunosuppression and increase the risk of infection. Therefore, developing an efficient and safe NLRP3 inflammasome inhibitor is of great significance for the treatment of atherosclerosis. Summary of the Invention

[0004] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide the use of resibufogenin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating atherosclerosis. The present invention provides a novel and effective method for treating atherosclerosis, opening up a new research direction for the treatment of cardiovascular diseases.

[0005] To achieve the purpose of this invention, the following technical solutions are adopted by the present invention:

[0006] In the first aspect, the present invention provides the use of resibufogenin or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating atherosclerosis.

[0007] Resibufogenin (RBG), resibufogenin, a natural compound extracted from toad secretions. RBG can be obtained by extraction and purification from toad secretions, or prepared by chemical synthesis methods. RBG is a compound with a polycyclic steroid structure, and its key feature is that it can form a covalent bond with the CYS - 279 site of the NLRP3 protein.

[0008] As a natural compound, RBG has important application value in inhibiting the activation and assembly of NLRP3 inflammasome. Especially in the treatment of atherosclerosis, it has unique application value and excellent effects in targeting inflammatory responses.

[0009] The present invention provides an innovative treatment strategy based on the natural compound RBG, providing a new theoretical basis and experimental support for the clinical intervention of atherosclerosis.

[0010] Preferably, the drug forms a covalent bond with the CYS-279 site of NLRP3 protein to inhibit the activation of NLRP3 inflammasome.

[0011] Experiments of the present invention have demonstrated that RBG shows excellent anti-atherosclerotic effects in the ApoE - / - mouse model, and verified its mechanism of inhibiting NLRP3 inflammasome in cell experiments. RBG forms a covalent bond with the CYS-279 site of NLRP3 protein to specifically inhibit the activation of inflammasome.

[0012] Preferably, the drug promotes the polarization transformation of macrophages from M1 to M2 type.

[0013] RBG can promote the polarization transformation of macrophages from M1 type to M2 type, reduce the release of inflammatory factors and promote tissue repair. This function provides multiple pharmacological effects for its application in atherosclerosis.

[0014] Preferably, the drug promotes tissue repair.

[0015] Existing treatment methods for atherosclerosis mostly focus on reducing blood lipid levels, but there is relatively little direct intervention in the core pathological process of inflammatory response. As a key regulatory factor of inflammatory response in atherosclerosis, the abnormal activation of NLRP3 inflammasome exacerbates the progression of the disease. The present invention provides a more effective anti-inflammatory treatment strategy by targeting NLRP3 inflammasome.

[0016] The present invention improves treatment specificity and safety: Existing NLRP3 inhibitors have deficiencies in terms of specificity and safety, which may lead to risks of immunosuppression and infection. By studying the covalent binding of resibufogenin (RBG) to specific sites of NLRP3, the present invention provides a highly specific and long-lasting NLRP3 inhibition mechanism, reducing the risk of adverse reactions.

[0017] Utilization of multiple pharmacological effects in the present invention: RBG not only has the effect of inhibiting the NLRP3 inflammasome, but also can regulate the polarization state of macrophages, thereby reducing the inflammatory response and promoting tissue repair. This multiple pharmacological effect provides new possibilities for the comprehensive treatment of atherosclerosis.

[0018] In the present invention, RBG can promote the stabilization of atherosclerotic lesions: by inhibiting the activation of the NLRP3 inflammasome; using RBG can reduce the inflammatory infiltration and foam cell formation in atherosclerotic lesions, thereby reducing the risk of plaque instability and rupture, which is of great significance for preventing the occurrence of cardiovascular events.

[0019] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0020] Preferably, the excipient includes any one or a combination of at least two of a shaping agent, a diluent, a carrier, a flavoring agent, a binder, a filler, a disintegrant or an acid-base regulator.

[0021] Preferably, the dosage form of the drug is any pharmaceutically acceptable dosage form, such as tablets, powders, suspensions, granules, capsules, solutions, enemas or emulsions, etc. The drugs of the above various dosage forms can be prepared according to the conventional methods in the pharmaceutical field.

[0022] Preferably, the drug can be introduced into the body such as muscle, intradermal, subcutaneous, intravenous or mucosal tissue by injection, spraying, nasal dropping, eye dropping, penetration, absorption, physical or chemical mediated methods; or be introduced into the body after being mixed or encapsulated by other substances.

[0023] In a second aspect, the present invention provides the use of cinobufagin or a pharmaceutically acceptable salt thereof in the preparation of a preparation for inhibiting the activation of the NLRP3 inflammasome.

[0024] According to the research results of the present invention, cinobufagin or a pharmaceutically acceptable salt thereof has a significant effect of inhibiting the activation of the LRP3 inflammasome. Therefore, this result indicates that cinobufagin or a pharmaceutically acceptable salt thereof can be used as a preparation for scientific research fields, such as for theoretical research on the activation behavior of the NLRP3 inflammasome, for screening more drugs for the treatment of atherosclerosis, and so on.

[0025] In a third aspect, the present invention provides the use of cinobufagin or a pharmaceutically acceptable salt thereof in the preparation of a preparation for promoting tissue repair.

[0026] According to the research results of the present invention, resibufogenin or its pharmaceutically acceptable salt has a significant effect of promoting tissue repair. Therefore, these results indicate that resibufogenin or its pharmaceutically acceptable salt can be used as a preparation for scientific research fields, such as for theoretical research on tissue repair behavior, for screening more drugs that promote tissue repair, and so on.

[0027] Fourthly, the present invention provides the use of resibufogenin or its pharmaceutically acceptable salt in the preparation of a preparation for promoting the polarization transformation of macrophages from M1 to M2 type.

[0028] According to the research results of the present invention, resibufogenin or its pharmaceutically acceptable salt has a significant effect of promoting the polarization transformation of macrophages from M1 to M2 type. Therefore, these results indicate that resibufogenin or its pharmaceutically acceptable salt can be used as a preparation for scientific research fields, such as for theoretical research on the polarization transformation behavior of macrophages from M1 to M2 type.

[0029] Fifthly, the present invention provides a method for reducing the inflammatory response in atherosclerotic lesions, the method comprising administering resibufogenin or its pharmaceutically acceptable salt into atherosclerotic lesions.

[0030] In the present invention, RBG is mainly used for the treatment of atherosclerosis. It inhibits the assembly and activation of NLRP3 inflammasome, reduces the release of inflammatory factors and the formation of foam cells, thereby slowing down the process of atherosclerosis.

[0031] Compared with the prior art, the present invention has the following beneficial effects:

[0032] (1) Targeting the inflammatory response: The present invention targets and inhibits the assembly and activation of NLRP3 inflammasome through Resibufogenin (RBG), significantly reducing the release of inflammatory factors in atherosclerosis. This mechanism effectively reduces the inflammatory response of the arterial wall, which is a powerful supplement to the traditional treatment method mainly focusing on lipid-lowering.

[0033] (2) Improving atherosclerotic symptoms: In the ApoE - / - mouse model, RBG treatment shows significant improvement in atherosclerotic symptoms, including weight loss, reduction of arterial plaque area, improvement of serum lipid levels, etc., indicating its effectiveness in slowing down the disease process.

[0034] (3) High specificity and persistence: RBG forms a covalent bond with the CYS-279 site of NLRP3 protein, providing a highly specific and persistent inhibitory effect, reducing the possible risks of immunosuppression and infection, and enhancing the safety of treatment.

[0035] (4) Multiple pharmacological effects: In addition to inhibiting the inflammasome, RBG also promotes the polarization transformation of macrophages from the M1 to the M2 type, which helps reduce the inflammatory response and promote tissue repair, further enhancing its comprehensive therapeutic potential as an anti-atherosclerotic drug.

[0036] (5) Discovery of new targets: This invention reveals the key role of the CYS-279 site in the NLRP3 protein as an inhibitory target, providing a new research direction for the development of other NLRP3 inhibitors. Description of the Drawings

[0037] Figure 1 For APOE - / - Changes in body weight and arterial pathology of APOE mice.

[0038] Figure 2 For APOE - / - Quantitative statistics of pathological injury area and changes in blood lipid levels in APOE mice.

[0039] Figure 3 RBG reduces Ox-LDL-induced BMDM foaming.

[0040] Figure 4 Effect of RBG on macrophage polarization induced by Ox-LDL.

[0041] Figure 5 Effect of RBG on macrophage polarization under the influence of LPS + IFN-γ.

[0042] Figure 6 Statistical chart of macrophage polarization.

[0043] Figure 7 Effect of RBG on inflammatory factors and TC, TG levels in the supernatant of BMDM cell culture medium.

[0044] Figure 8 Effect of RBG on NLRP3 pathway proteins in BMDM cells under Ox-LDL intervention.

[0045] Figure 9 Statistical chart of the expression levels of NLRP3 pathway-related proteins in BMDM cells.

[0046] Figure 10 Effect of RBG on the fluorescence intensity and co-localization level of NLRP3 inflammasome proteins in BMDM cells.

[0047] Figure 11 Effect of RBG on NF-κB nuclear translocation in BMDM cells.

[0048] Figure 12 Docking conformation diagram of RBG with human NLRP3 protein.

[0049] Figure 13 It is the docking conformation diagram of RBG and mutant human NLRP3 protein.

[0050] Figure 14 It is the surface plasmon experiment result of RBG and human NLRP3 protein.

[0051] Figure 15 It is the molecular dynamics simulation result of RBG and human NLRP3 protein. Detailed implementation manners

[0052] The technical solutions of the present invention will be further described below through specific implementation manners. Those skilled in the art should understand that the described embodiments are only for helping to understand the present invention and should not be regarded as specific limitations to the present invention.

[0053] For those not specifying specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in the art or according to the product specifications. For those reagents or instruments without indicating the manufacturer, they are all conventional products that can be obtained through regular commercial channels.

[0054] Example 1

[0055] 1. Preparation of animal model: Select ApoE - / - mice as the model of atherosclerosis, and feed them with a high-fat diet for 12 weeks to induce atherosclerosis.

[0056] 2. Compound treatment: Starting from the fifth week, different doses of RBG (high, medium, and low doses are 5 mg / kg, 3 mg / kg, and 1 mg / kg respectively) were given to the mice by gavage once a day for 8 consecutive weeks.

[0057] 3. Experimental analysis: After the treatment, the mice were dissected, and serum and aortic tissues were collected. The pathological plaques in the arteries were evaluated by HE staining, Masson staining, and Oil Red O staining. The expression levels of inflammasome-related proteins such as NLRP3, ASC, and caspase-1 were detected by methods such as Western Blot, ELISA, and immunofluorescence.

[0058] 4. Molecular docking, kinetic simulation, and surface plasmon resonance: Using techniques such as molecular docking, kinetic simulation, and surface plasmon resonance, analyze the binding mode of RBG and NLRP3, and confirm the covalent binding of RBG to the CYS-279 site.

[0059] 5. Data analysis and result verification: Combining the mouse model, cell experiments, and molecular simulation results, verify the mechanism by which RBG effectively slows down the process of atherosclerosis by inhibiting the NLRP3 inflammasome.

[0060] (1) Effects on mouse body weight, aortic plaques, lipid deposition in the aortic root, fibrotic pathological damage, and blood lipid levels

[0061] Figure 1 is APOE - / - Changes in mouse body weight and arterial pathological changes. Figure 2 is APOE - / - Quantitative statistics of the pathological damage area in mice and changes in blood lipid levels. Figure 1-2 Among them, (A) Establishment of the model and treatment protocol. (B) Mouse body weight-time curve (n = 10). (C and E) Representative Oil Red O staining of the aorta, quantification of the aortic lesion area (n = 3). (D, F, G, H) Representative images of HE, Masson, and Oil Red O staining, and quantification of the aortic sinus lesions, collagen, or lipid areas (n = 3). (I, J, K, L) Serum TG, TC, LDL-C, and HDL-C levels (n = 10) *P<0.05, **P<0.01, ***P<0.001,

[0062] ****P<0.01 and ns: not significant.

[0063] The results showed that RBG treatment effectively reduced the body weight of ApoE mice fed a high-fat diet - / - ( Figure 1 B in). Gross Oil Red O staining of the aorta showed that RBG treatment could protect ApoE - / - mice from atherosclerosis ( Figure 1 C in, Figure 2 E in). HE staining, Masson staining, and Oil Red O staining of the aortic root confirmed smaller plaques in RBG-treated mice ( Figure 1 D in, Figure 2 F, G, and H in). Serum analysis showed that RBG treatment partially improved the serum levels of triglyceride (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C) in ApoE - / - mice ( Figure 2 I, J, L in).

[0064] (2) RBG inhibits macrophage infiltration, reduces Ox-LDL-induced foam cell formation and NLRP3 inflammasome expression and assembly.

[0065] Figure 3 is that RBG reduces Ox-LDL-induced BMDM foaming. Figure 4 is the effect of RBG on macrophage polarization induced by Ox-LDL, Figure 5 is the effect of RBG on macrophage polarization under the influence of LPS+IFN-γ, Figure 6Statistical chart of macrophage polarization. Figure 7 Effects of RBG on inflammatory factors and TC and TG levels in the supernatant of BMDM cell culture medium. Figure 8 Effects of RBG on NLRP3 pathway proteins in BMDM cells under Ox-LDL intervention. Figure 9 Statistical chart of the expression levels of NLRP3 pathway-related proteins in BMDM cells. Figure 10 Effects of RBG on the fluorescence intensity and co-localization level of NLRP3 inflammasome proteins in BMDM cells. Figure 11 Effects of RBG on NF-κB nuclear translocation in BMDM cells.

[0066] In Figure 3-11 Among them, Figure 3 In A and B are representative images and statistical analysis of Oil Red O staining in BMDMs (n = 4). Figure 4 and Figure 5 , Figure 6 In D, E, F, G show that in the polarization of BMDM induced by ox-LDL or LPS + IFN-γ, RBG reduces the proportion of M1 macrophages and increases the proportion of M2 macrophages (n = 4). Figure 7 In H, I, J, K, L, M show the levels of il-18, il-1β, TNF-α, il-10, TG, and TC in the supernatant of BMDM culture (n = 8). Figure 8 , Figure 9 In O, P, Q, R, S, T, U, V are Western blot and statistical analysis of NLRP3, ASC total protein, ASC oligomerization, NEK7, Caspase-1, Cleaved Caspase-1, Pro IL18, and IL18 (N = 3). Figure 10 In W, a, b, c, d are representative images and statistical analysis of immunofluorescence staining of NLRP3, ASC, and Caspase-1 in BMDMs (n = 3). Figure 11 In X, Y are representative images of NF-κB immunofluorescence in BMDMs, the ratio of the fluorescence intensity of NF-κB in the nucleus to that in the cytoplasm (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.01, and ns: not significant.

[0067] This example determined three RBG dose gradients: 100 nM, 50 nM, and 25 nM. By intervening BMDM with Ox-LDL, a foam cell model was replicated. Oil Red O staining showed that RBG effectively reduced lipid accumulation and foam cell formation in BMDM ( Figure 3In (A) and (B). In addition, by intervening with Ox-LDL and LPS + IFN-γ, it was observed that RBG could reduce the infiltration of M1 macrophages and increase the infiltration of M2 macrophages in both cases ( Figure 4 and Figure 5 , Figure 6 in (D), (E), (F), and (G). ELISA results showed that RBG could increase the levels of IL18, IL1β, TNF-α, IL10, triglyceride (TG), and total cholesterol (TC) in the supernatant of Ox-LDL-induced BMDM cultures ( Figure 7 in (H), (I), (J), (K), (L), and (M). Western blot analysis showed that RBG could reduce the levels of NLRP3, ASC, ASC oligomerization, NEK7, Caspase-1, cleaved Caspase-1, pro-IL18, and IL18 in Ox-LDL-induced BMDM ( Figure 8 , Figure 9 in (N), (O), (P), (Q), (R), (S), (T), (U), and (V). Immunofluorescence results showed that RBG could reduce the expression and co-localization of NLRP3, ASC, and caspase-1 in Ox-LDL-induced BMDM ( Figure 10 in (W), (a), (b), (c), and (d). In addition, we found by immunofluorescence that RBG could reduce the nuclear translocation of NF-κB ( Figure 9 in (T) and (U). ( Figure 11 in (X) and (Y)) results showed that RBG reduced the nuclear translocation of NF-κB in Ox-LDL-induced BMDM cells.

[0068] (4) RBG forms a covalent chemical bond with the CYS-279 residue of NLRP3 protein through Michael addition reaction to reduce the nuclear translocation of NF-κB in Ox-LDL-induced BMDM cells.

[0069] Figure 12 is the docking conformation diagram of RBG with human NLRP3 protein. Figure 13 is the docking conformation diagram of RBG with mutant human NLRP3 protein, Figure 14 is the surface plasmon experiment result of RBG with human NLRP3 protein. Figure 15 is the molecular dynamics simulation result of RBG with human NLRP3 protein. Figure 12 In (A), the site map predicts the cavity of human NLRP3 protein and the covalent site map, where the spheres represent the CYS covalent reaction sites and the cyan blocks represent the pocket cavities. (B) The binding mode of RBG with wild-type human NLRP3 protein.

[0070] Figure 13 shows the binding mode of RBG with the R143A mutant of human NLRP3 protein.

[0071] Figure 14 Shows the SPR response of purified human NLRP3 protein to RBG.

[0072] Figure 15 In (D), the change of the root mean square deviation (RMSD) of the system with time during the molecular dynamics simulation. (E) The change of energy with time. (F) After 500 ns of extended simulation and R143A mutant simulation, the change of RMSD with time.

[0073] As Figure 12 As shown in A, we performed a visual analysis to identify five exposed cysteine residues on the NLRP3 NACHT domain, namely CYS-279, CYS-514, CYS-598, CYS-319, and CYS-673, which may undergo covalent reactions with the compound. Molecular docking studies were performed on these residues, and only CYS-279 and CYS-598 bound to RBG, with binding energies of -5.4 kcal / mol and -4.2 kcal / mol, respectively. Apparently, the binding at the CYS-279 site is the most favorable.

[0074] As Figure 12 As shown in B, the binding mode of RBG to NLRP3 indicates that RBG occupies a central position on NLRP3 and covalently binds to CYS-279. This covalent binding is a key factor for the stable interaction between RBG and the protein. In addition, we observed that RBG can form hydrogen bonds with residues such as ARG-147 on the protein, which helps to stabilize the molecular recognition pocket and further provides a stable platform for covalent binding. In summary, RBG interacts with the NLRP3 protein through covalent and hydrogen bond mechanisms. The root mean square deviation (RMSD) of the molecular dynamics simulation reflects the motion of the complex; a higher RMSD and larger fluctuations indicate more intense motion, while a lower RMSD indicates smoother motion.

[0075] As Figure 15 As shown in D, the RMSD system was monitored over time during the simulation, and three separate RMSD calculations were performed, each for 500 ns. The results showed that all three RMSD measurements fluctuated stably around indicating that RBG and the NLRP3-NACHT domain can bind stably. The system maintained low-fluctuation motion, indicating that RBG effectively inhibited the dynamic motion of the NLRP3-NACHT domain. During the simulation, the Coulomb potential energy and Lennard-Jones potential energy of the protein-ligand complex were also monitored.

[0076] As Figure 15As shown in Figure E, the Coulomb energy fluctuates between -30,000 and -35,000 kcal / mol, while the Lennard-Jones energy remains at around -3,000 kcal / mol. Notably, the stability of the system energy indicates that no decomposition has occurred, which confirms the correctness of the simulation and the data reference values.

[0077] To further illustrate the important role of ARG-147 in the binding of RBG to NLRP3, ARG-147 was mutated and a 100-ns molecular dynamics simulation was performed. Figure 15 The results shown in Figure F clearly indicate that in the initial simulation stage, the D value of the mutant is higher than that of the wild type (WT), indicating that the mutation disrupts the stable binding trend between the small molecule and the protein.

[0078] From Figure 15 Figure F, it can be observed that there is no interaction between the small molecule and ARG-147, which may lead to an increase in the mobility of RBG and cause instability. In addition, we extended the simulation to analyze the conformation after 500 nanoseconds. The extended simulation shows low and stable RMSD fluctuations, indicating that the interaction between RBG and NLRP3 can remain stable over time, further demonstrating that the small molecule can effectively inhibit the protein.

[0079] As Figure 13 shown, the binding mode of RBG to the R143A mutant indicates that RBC occupies a central position in the R143A mutation, far from the covalent binding site of CYS-279, making covalent binding less likely. Notably, molecular analysis shows that only R237 binds to the hydrogen bond in the pocket and no other interactions are observed. The weak binding interaction indicates that the combination of RBG and the ARG-147 mutant is unstable and may lead to off-target effects at any time.

[0080] In summary, in view of the deficiencies of the prior art in aspects such as inflammatory response control, treatment specificity, and safety, the present invention provides a novel and effective method for treating atherosclerosis by targeting the NLRP3 inflammasome, opening up a new research direction for the treatment of cardiovascular diseases.

[0081] The applicant declares that the above description is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily conceived within the technical scope disclosed by the present invention by those skilled in the art of this technology fall within the protection scope and the disclosure scope of the present invention.

Claims

1. Use of bisbufogenin or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating atherosclerosis.

2. The use according to claim 1, characterized in that: The drug forms a covalent bond with the CYS-279 site of the NLRP3 protein, inhibiting the activation of the NLRP3 inflammasome.

3. The use according to claim 1 or 2, characterized in that: The drug promotes the polarization transition of macrophages from M1 to M2 type.

4. The use according to any one of claims 1 to 3, characterized in that: The drug promotes tissue repair.

5. The use according to any one of claims 1 to 4, characterized in that: The drug also includes pharmaceutically acceptable excipients.

6. The use according to claim 5, characterized in that: The auxiliary materials include any one of excipients, diluents, carriers, flavoring agents, adhesives, fillers, disintegrants or acid-base regulators, or a combination of at least two of them.

7. Use of bisbufogenin or a pharmaceutically acceptable salt thereof in the preparation of a preparation for inhibiting the activation of NLRP3 inflammasome.

8. Use of bisbufogenin or a pharmaceutically acceptable salt thereof in the preparation of a preparation for promoting tissue repair.

9. Use of bisbufogenin or a pharmaceutically acceptable salt thereof in the preparation of a preparation for promoting the polarization transition of macrophages from M1 to M2.

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