Application of glycyrrhetinic acid or glycyrrhetinic acid liposome in preparation of abdominal aortic aneurysm protection medicine

Abstract

The invention belongs to the field of biological medicine, particularly discloses application of glycyrrhetinic acid or glycyrrhetinic acid lipidosome in preparation of an abdominal aortic aneurysm protection medicine, and reveals that glycyrrhetinic acid can effectively improve death of mice after aorta rupture caused by Ang II treatment, can obviously improve degradation of an aorta elastic fiber layer caused by Ang II treatment, and can improve the aortic aneurysm protection effect. The macrophages induced by lipopolysaccharide (LPS) can be promoted to be polarized to M2 type; the glycyrrhetinic acid is used for intervention before Ang II treatment, the occurrence of Ang II induced abdominal aortic aneurysm can be relieved, the survival rate of mice is increased, and it is indicated that the glycyrrhetinic acid can be used for preparing the medicine for relieving the abdominal aortic aneurysm. Furthermore, the glycyrrhetinic acid is entrapped into the liposome, so that the solubility and bioavailability of the glycyrrhetinic acid are improved, meanwhile, after glycyrrhetinic acid coating is carried out by utilizing a nanotechnology, the dosage of the glycyrrhetinic acid can be effectively controlled, and the curative effect of the glycyrrhetinic acid is further improved and enhanced.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, in particular to the use of glycyrrhetinic acid or liposomes of glycyrrhetinic acid in the preparation of drugs for protecting abdominal aortic aneurysm. Background Art

[0002] Current research shows that the occurrence mechanism of abdominal aortic aneurysm is related to macrophage-mediated inflammatory response. When stimulated by high-risk factors such as high sugar, high pressure and oxidative stress, irreversible dilation of the blood vessel wall will occur, and a large number of inflammatory cells such as mononuclear macrophages and chemokines will accumulate around the aneurysm blood vessels, mediating the inflammatory response, leading to vascular endothelial dysfunction, and then promoting the proliferation and migration of vascular smooth muscle cells, changing the physiological structure and function of the blood vessel wall, reducing vascular homeostasis and compliance, and ultimately promoting local dilation of the aortic blood vessel wall, resulting in the occurrence of abdominal aortic aneurysm. At present, the main treatment and intervention methods for abdominal aortic aneurysm are surgical treatment, replacing the diseased aortic segment through thoracotomy or laparotomy, or using minimally invasive vascular intervention technology to place a stent in the abdominal aortic aneurysm to reinforce the aneurysm wall and prevent further dilation and rupture. Although treatment with drugs such as antihypertensive drugs and β-blockers can reduce the risk of the aorta, it cannot directly eliminate abdominal aortic aneurysm. At present, there is a lack of effective drugs to intervene in the dilation of abdominal aortic aneurysm to eliminate abdominal aortic aneurysm.

[0003] Glycyrrhetinic acid is one of the important active ingredients of licorice, a pentacyclic triterpenoid saponin with an oleanane-type skeleton. It is a white needle crystal at room temperature, insoluble in water, and soluble in organic solvents such as methanol, ethanol, and chloroform. At present, a large number of studies at home and abroad have proved that glycyrrhetinic acid has a variety of biological activities, such as antiviral, anti-inflammatory, anti-ulcer, hypoglycemic, and lipid-regulating. Some studies have shown that glycyrrhetinic acid can reduce pulmonary edema and lung tissue pathological damage in hemorrhagic shock, and reduce the expression of inflammatory factors such as TNF-α and IL-1β in blood and bronchoalveolar lavage fluid. Glycyrrhetinic acid can also reduce the inflammation of microglia (BV2 cells) in mice treated with 1-methyl-4-phenylpyridine, showing an anti-inflammatory effect. Glycyrrhetinic acid can also reduce the plasma glucose and glycosylated hemoglobin levels in diabetic rats, regulate the activity of key enzymes in glucose metabolism in the liver, and maintain normal blood glucose levels. However, there is no report on the intervention and treatment effect of glycyrrhetinic acid on the course of abdominal aortic aneurysm. Although glycyrrhetinic acid has good biological activity, it also has adverse effects, mainly manifested as inhibition of cell life activities at too high concentrations; and due to its special molecular structure, it has high lipophilicity, low water solubility, low drug bioavailability, and difficult dose control. Therefore, it has a slow onset and insignificant curative effect in clinical treatment, which also limits its application in clinical practice. Summary of the Invention

[0004] An object of the present invention is to provide the use of glycyrrhetinic acid or glycyrrhetinic acid liposomes in the preparation of drugs for protecting abdominal aortic aneurysms. Through the intervention and treatment with glycyrrhetinic acid or glycyrrhetinic acid liposomes, the rupture of abdominal aortic aneurysms can be alleviated and the survival rate can be increased.

[0005] To achieve the above object, the present invention is implemented according to the following technical solutions: The first technical solution provided by the present invention is the use of glycyrrhetinic acid or glycyrrhetinic acid liposomes in the preparation of drugs for protecting abdominal aortic aneurysms.

[0006] Furthermore, the preparation method of the glycyrrhetinic acid liposomes comprises the following steps: S1. Take soybean lecithin and glycyrrhetinic acid according to a mass ratio of 25:1, fully dissolve them in an organic solvent, and completely evaporate the organic solvent by rotary evaporation to obtain a uniform lipid film. S2. Add 100 mg of the lipid film to 10 mL of 0.154 M NaCl solution and shake to form a suspension. Place the suspension in a 30-degree water bath and perform ultrasonic treatment at 200 W for 15 minutes for hydration to form small particle liposomes. S3. Dialyze and purify the obtained liposomes to obtain purified liposomes; subject the purified liposomes to membrane extrusion to obtain glycyrrhetinic acid liposomes with uniform particle sizes.

[0007] Preferably, the organic solvent is one or more of chloroform, methanol, ethanol, and ether.

[0008] Preferably, a dialysis membrane or dialysis bag with a cut-off molecular weight of 3500 Da is used for dialysis purification.

[0009] Preferably, the particle size of the glycyrrhetinic acid liposomes is 100 - 150 nm.

[0010] Compared with the prior art, the present invention has the following beneficial effects: The present invention reveals that glycyrrhetinic acid can effectively improve the death after the rupture of abdominal aorta in mice caused by Ang II treatment, can significantly improve the degradation of the elastic fiber layer of the abdominal aorta caused by Ang II treatment, and can promote the polarization of lipopolysaccharide (LPS)-induced macrophages into the M2 type; intervening with glycyrrhetinic acid before Ang II treatment can alleviate the occurrence of abdominal aortic aneurysms induced by Ang II and increase the survival rate of mice, indicating that glycyrrhetinic acid can be used to prepare drugs for alleviating abdominal aortic aneurysms.

[0011] Furthermore, in the present invention, glycyrrhetinic acid is encapsulated into liposomes, improving the solubility and bioavailability of glycyrrhetinic acid. Meanwhile, after coating glycyrrhetinic acid using nanotechnology, the dosage of the drug can be effectively controlled, further improving and enhancing the efficacy of glycyrrhetinic acid. Intervention with glycyrrhetinic acid liposomes before Ang II treatment can better alleviate the occurrence of AngII-induced abdominal aortic aneurysm and improve the survival rate of mice, indicating that glycyrrhetinic acid liposomes can be used to prepare drugs for alleviating abdominal aortic aneurysm, reducing the off-target effect while exerting the efficacy most rapidly. Description of the Drawings

[0012] Figure 1 Role of glycyrrhetinic acid in alleviating abdominal aortic aneurysm in mice: A, abdominal aortic B-ultrasound results; B, statistical results of the maximum diameter of the abdominal aorta; C, probability of abdominal aortic aneurysm occurrence; D, survival curve of mice after Ang II modeling.

[0013] Figure 2 Reduction of macrophage infiltration and aortic elastic fiber degradation in the abdominal aorta of mice by glycyrrhetinic acid: A, hematoxylin-eosin (HE) staining method shows the infiltration of immune cells in the aorta; B, elastic fiber and collagen fiber (EVG) staining method shows the degradation of elastic fibers in the aorta.

[0014] Figure 3 Expression of each marker in mouse alveolar cell line (MH-S cells) cultured with different drugs: A, CD80 antibody; B, CD163 antibody.

[0015] Figure 4 Detection results of glycyrrhetinic acid liposomes prepared by an embodiment of the present invention: A, transmission electron micrograph; B, particle size distribution, C, hydrated particle diameter; D, polydispersity; E, encapsulation efficiency; F, Zeta potential.

[0016] Figure 5 Alleviation of the occurrence of abdominal aortic aneurysm in mice by glycyrrhetinic acid liposomes: A, abdominal aortic B-ultrasound results; B, statistical results of the maximum diameter of the abdominal aorta; C, probability of abdominal aortic aneurysm occurrence. Detailed Embodiment

[0017] To make the objectives, technical solutions and advantages of the present invention clearer and more understandable, the following further details the present invention with reference to embodiments. The specific embodiments described herein are only used to explain the present invention and are not used to limit the invention.

[0018] In the following embodiments, glycyrrhetinic acid was purchased from Sigma Company; other related reagents used, unless otherwise specified, were commercially available drugs, and the methods involved, unless otherwise specified, were well-known methods.

[0019] Example 1. In-vivo experiment on animals: Abdominal aortic aneurysm modeling experiment Twenty 6-8-week-old specific pathogen-free (SPF) ApoE - / - C67BL / 6 male mice (purchased from Guangdong Vital River) were weighed and randomly divided into 4 groups of 5 mice each. After being caged separately, the mice were raised under the conditions of an artificial climate chamber (23-25 °C) and artificial lighting (14 hours of light and 10 hours of darkness), fed with standard pellet feed, and allowed free access to water and food. The mice were anesthetized with 0.67% pentobarbital solution, fixed in a prone position on the operating table after anesthesia, the skin and fur on the neck and back were disinfected, and an opening about 1 cm long was cut transversely to separate the subcutaneous tissue to form a space. A micro-osmotic pump loaded with Ang II at a dosing concentration of 1000 ng / kg / min was implanted subcutaneously in the back.

[0020] The skin incision was sutured with a suture and surface disinfected. After the mice woke up, they were returned to the cage and continued to be raised conventionally for 28 days for the development of abdominal aortic aneurysm. Three days after modeling, each group of mice was gavaged with medicine every other day: the first group was gavaged with DMSO and denoted as the Ang II group, the second group was gavaged with 50 mg / kg glycyrrhetinic acid (1% DMSO) and denoted as the Ang II + glycyrrhetinic acid group, and the third group was not modeled and the surgical operation was carried out normally. A micro-osmotic pump containing PBS was implanted subcutaneously in the back and denoted as the PBS group. On the 7th day after Ang II modeling, the mice were examined by B-ultrasound for the diameter of the abdominal aorta and the survival time was recorded.

[0021] As Figure 1 shown in A and B below: The B-ultrasound results showed that on the 7th day after Ang II modeling, the diameter of the abdominal aorta in the Ang II group of mice was significantly larger than that in the PBS group, while the diameter of the abdominal aorta in the Ang II + glycyrrhetinic acid group of mice was significantly reduced, indicating that glycyrrhetinic acid can effectively reduce the abdominal aortic dilation in mice caused by Ang II treatment.

[0022] On the 28th day after Ang II modeling, the mice were sacrificed and samples were taken. According to the incidence of abdominal aortic aneurysm in the mice after modeling as shown in Figure 1 C below, no abdominal aortic aneurysm was detected in the mice of the PBS group, the incidence of abdominal aortic aneurysm in the mice of the Ang II group was 100%, while the incidence of abdominal aortic aneurysm in the mice of the Ang II + glycyrrhetinic acid group was 40%, indicating that glycyrrhetinic acid treatment can significantly improve the incidence of abdominal aortic aneurysm induced by Ang II. The survival curve plotted according to the survival time of the mice after modeling is as shown in Figure 1As shown in D of [reference], mice in the Ang II group began to die after modeling, while mice in the Ang II + glycyrrhetinic acid group did not die, indicating that glycyrrhetinic acid can effectively improve the death caused by Ang II-induced rupture of the abdominal aorta in mice.

[0023] Example 2. HE and EVG staining of the abdominal aorta The mice were anesthetized with 0.67% pentobarbital solution and placed on a foam board to fix their limbs. After opening the chest cavity to expose the heart, the right hand held the needle and inserted it at the apex of the heart, and the left hand used an ophthalmic scissors to cut open the right auricle. About 30 - 40 mL of PBS was injected at the injection site. After the liver turned white and the outflowing liquid became clear, the vascular perfusion was completed. Open the abdominal cavity, remove other organs, then dissect out the entire aorta and soak it in formalin. Take it out for photography after 2 hours. Take the position of the abdominal aortic aneurysm for fixation, embedding, sectioning, and then perform HE staining to observe the changes of cells in the aorta. As Figure 1 As shown in A of [reference], compared with the abdominal aorta of the non-modeled control group, the abdominal aorta of mice in the Ang II group showed obvious lesions, including a decrease in vascular smooth muscle cells and a large accumulation of adventitial macrophages. However, the number of smooth muscle cells in the aorta of mice in the Ang II + glycyrrhetinic acid group was significantly improved, and the adventitial macrophages were not obvious. This shows that glycyrrhetinic acid can effectively improve the abdominal aortic lesions induced by Ang II in mice. Observe the degree of degradation of elastic fiber and collagen fiber layers by EVG staining. The degradation of the elastic fiber layer is divided into 4 levels from mild to severe (I - IV): Grade I, damage such as destruction of the elastic fiber layer and reduction of vascular smooth muscle cells is limited to 1 layer of the elastic fiber layer; Grade II, the damage involves two or all layers, but is limited to 1 / 4 of the blood vessel; Grade III, the damage involves all elastic fiber layers, but is limited to less than 1 / 2 of the blood vessel; Grade IV, the damage involves all elastic fiber layers and extends to more than 3 / 4 of the blood vessel. As Figure 2 As shown in B of [reference], the elastic fiber layer of the abdominal aorta of mice in the PBS group was intact. In mice in the Ang II group, the proportion of Grade I lesions was 20%, Grade II lesions was 20%, and Grade III lesions was 60%. In mice in the Ang II + glycyrrhetinic acid group, the proportion of Grade I lesions was 60% and Grade II lesions was 40%. It can be seen that treatment with glycyrrhetinic acid can significantly improve the degradation of the elastic fiber layer of the abdominal aorta induced by Ang II in mice.

[0024] Example 3. In vitro cell experiment: Detection of the polarization of LPS-induced MH-S cells by flow cytometry using glycyrrhetinic acid Take MH-S cells in the logarithmic growth phase and prepare them into a density of 3×10 5 / mL of cell suspension was inoculated into 12-well plates at 1 mL per well and placed at 37°C with 5% CO 2 Culture overnight in a cell culture incubator. Discard the original culture medium and add RPMI-1640 medium containing 0.1 μg / mL LPS to induce for 24 hours. Discard the culture medium and add RPMI-1640 medium containing 3 μg / mL glycyrrhetinic acid to culture at 37 ℃ and 5% CO 2 The cells were cultured in a cell culture incubator for 24 h, designated as the LPS+glycyrrhetinic acid group, using RPMI-1640 medium containing 0.1 μg / mL LPS at 37 °C and 5% CO 2 The cells cultured in the cell culture incubator for 24 hours were recorded as the LPS group, and the MH-S cells treated with PBS were recorded as the PBS group. After the incubation, the cell surface markers were stained with FITC-labeled anti-mouse CD80 antibody and APC-labeled anti-mouse CD163 antibody, and the expression of each marker was detected by flow cytometry. The results are shown in Figure 3 As shown, glycyrrhetinic acid can significantly reduce the expression of CD80 induced by LPS and upregulate the expression level of CD163, suggesting that glycyrrhetinic acid can promote the polarization of LPS-induced macrophages to M2 type.

[0025] Example 4. Preparation of glycyrrhetinic acid liposomes Soybean lecithin and glycyrrhetinic acid are taken in a mass ratio of 25:1, fully dissolved in anhydrous ethanol, and the anhydrous ethanol is completely evaporated by rotary evaporation to obtain a uniform lipid film; 100 mg of the lipid film is added to 10 mL of 0.154 M NaCl solution and oscillated to form a suspension, the suspension is placed in a 30-degree water bath, and hydrated by 200 W ultrasound for 15 minutes to form small-particle liposomes; the obtained liposomes are dialyzed and purified using a dialysis membrane or dialysis bag with a molecular weight cutoff of 3500 Da to obtain purified liposomes; the purified liposomes are extruded through a membrane to obtain glycyrrhetinic acid liposomes with a particle size of 100-150 nm.

[0026] The structure of glycyrrhetinic acid liposomes was detected by transmission electron microscopy, the encapsulation efficiency of glycyrrhetinic acid liposomes was detected by high performance liquid chromatography, and the particle size distribution of glycyrrhetinic acid liposomes was detected by dynamic light scattering experiment. Figure 4 As shown. Figure 4 As shown in A, glycyrrhetinic acid liposomes are round particles with a lipid bilayer structure, uniform size distribution, and a particle size of about 100 nm. Figure 4 From B and C in Figure 1, we can see that the particle size distribution of glycyrrhetinic acid liposomes is uniform and normal, and the hydrated particle diameter is 140.33±1.81 nm. Figure 4As can be seen from D in [reference], glycyrrhetinic acid liposomes have good polydispersity (PDI: 0.121 ± 0.011). From Figure 4 As can be seen from E in [reference], the encapsulation efficiency of glycyrrhetinic acid liposomes is 30.05 ± 0.41%, and from Figure 4 As can be seen from F in [reference], the Zeta potential of glycyrrhetinic acid liposomes is -38.42 ± 0.38 mV.

[0027] Example 5. Glycyrrhetinic acid liposomes alleviate the occurrence of abdominal aortic aneurysm We modeled the mice according to the same method in Example 1. Three days before and after modeling, each group of mice was intraperitoneally injected with drugs every other day: the first group was intraperitoneally injected with DMSO and recorded as the Ang II group, the second group was intraperitoneally injected with 5 mg / kg of glycyrrhetinic acid liposomes and recorded as the Ang II + glycyrrhetinic acid liposome group, and the third group was not modeled and the surgical operation was carried out normally. A micro-osmotic pump containing PBS was implanted subcutaneously in the back and recorded as the PBS group. On the 7th day after modeling, the diameter of the aorta of the mice was detected by B-ultrasound.

[0028] As Figure 5 shown in A and B in [reference]: The B-ultrasound results on the 7th day after Ang II modeling showed that the abdominal aortic diameter of the mice in the Ang II group was significantly increased compared with that of the mice in the PBS group, while the abdominal aortic diameter of the mice in the Ang II + glycyrrhetinic acid liposome group was significantly reduced, indicating that low-concentration glycyrrhetinic acid liposomes can effectively reduce the abdominal aortic dilation in mice caused by Ang II treatment. On the 28th day after Ang II modeling, the mice were sacrificed and samples were taken. According to the incidence results of abdominal aortic aneurysm in the mice after modeling as Figure 5 shown in C in [reference], no abdominal aortic aneurysm was detected in the non-modeled PBS group of mice, the incidence of abdominal aortic aneurysm in the Ang II group of mice was 80%, while the incidence of abdominal aortic aneurysm in the Ang II + glycyrrhetinic acid liposome group of mice was 20%, indicating that low-concentration glycyrrhetinic acid liposome treatment can significantly improve the incidence of abdominal aortic aneurysm induced by Ang II treatment.

[0029] In summary, intervention with glycyrrhetinic acid or glycyrrhetinic acid liposomes before Ang II treatment can alleviate the occurrence of abdominal aortic aneurysm induced by Ang II, improve the survival rate of mice, and reduce macrophage infiltration in the aorta, indicating that glycyrrhetinic acid or glycyrrhetinic acid liposomes can be used to prepare drugs for alleviating abdominal aortic aneurysm, and glycyrrhetinic acid can improve the bioavailability of glycyrrhetinic acid and the therapeutic effect of glycyrrhetinic acid on abdominal aortic aneurysm through liposome coating.

[0030] The technical solution of the present invention is not limited to the limitations of the above specific embodiments. Any technical deformation made according to the technical solution of the present invention falls within the protection scope of the present invention.

Claims

1. Application of glycyrrhetinic acid or glycyrrhetinic acid liposomes in the preparation of abdominal aortic aneurysm protective drugs.

2. The use according to claim 1, characterized in that: The preparation method of the glycyrrhetinic acid liposome comprises the following steps: S1. Soybean lecithin and glycyrrhetinic acid are taken in a mass ratio of 25:1, fully dissolved in an organic solvent, and the organic solvent is completely evaporated by rotary evaporation to obtain a uniform lipid film; S2, add 100 mg of lipid film to 10 mL of 0.154 M NaCl solution and shake to form a suspension, place the suspension in a 30 degree water bath, and sonicate at 200 W for 15 minutes to hydrate and form small particle liposomes; S3, purifying the obtained liposomes by dialyzing to obtain purified liposomes; and extruding the purified liposomes through a membrane to obtain glycyrrhetinic acid liposomes with uniform particle size.

3. The use according to claim 2, characterized in that: The organic solvent is one or more of chloroform, methanol, ethanol and ether.

4. The use according to claim 2, characterized in that: In step S3, dialysis purification is performed using a dialysis membrane or dialysis bag with a molecular weight cutoff of 3500 Da.

5. The use according to claim 2, characterized in that: The particle size of the glycyrrhetinic acid liposome is 100-150 nm.

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