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

Abstract

The present invention belongs to the field of biomedicine and specifically discloses the use of glycyrrhetinic acid or glycyrrhetinic acid liposomes in the preparation of a drug for protecting against abdominal aortic aneurysms. It reveals that glycyrrhetinic acid can effectively ameliorate the mortality of mice following aortic rupture caused by Ang II treatment, significantly improve the degradation of the aortic elastic fiber layer caused by Ang II treatment, and promote the polarization of lipopolysaccharide (LPS)-induced macrophages toward the M2 type. Intervention with glycyrrhetinic acid before Ang II treatment can alleviate the occurrence of Ang II-induced abdominal aortic aneurysms and improve the survival rate of mice, indicating that glycyrrhetinic acid can be used to prepare a drug for alleviating abdominal aortic aneurysms. Furthermore, the present invention improves the solubility and bioavailability of glycyrrhetinic acid by encapsulating it in liposomes. At the same time, after glycyrrhetinic acid is encapsulated using nanotechnology, its dosage can be effectively controlled, further improving and enhancing the therapeutic effect of glycyrrhetinic acid.

Description

Technical Field

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

[0002] Current research suggests that the pathogenesis of abdominal aortic aneurysms (ABAs) is linked to a macrophage-mediated inflammatory response. When stimulated by high-risk factors such as high glucose, high pressure, and oxidative stress, irreversible dilation of the vascular wall occurs. Inflammatory cells such as mononuclear macrophages and chemokines accumulate around the aneurysmal vessels, mediating an inflammatory response that leads to endothelial dysfunction. This in turn promotes the proliferation and migration of vascular smooth muscle cells, altering the physiological structure and function of the vascular wall, impairing vascular homeostasis and compliance, and ultimately promoting local dilation of the aortic wall, leading to the development of ABAs. Current treatment and intervention for ABAs primarily involves surgery, either through thoracotomy or laparotomy to replace the diseased aortic segment, or through minimally invasive vascular interventions to place stents within the ABA to reinforce the aneurysm wall and prevent further dilation and rupture. Although medications such as antihypertensive drugs and beta-blockers can reduce aortic risk, they cannot directly eliminate ABAs. Currently, there is a lack of effective pharmacological interventions to address ABA dilation and eliminate it.

[0003] Glycyrrhetinic acid, a key active ingredient in licorice, is a pentacyclic triterpenoid saponin with an oleanane-type skeleton. It appears as white, needle-shaped crystals at room temperature and is insoluble in water, but readily soluble in organic solvents such as methanol, ethanol, and chloroform. Numerous studies, both domestically and internationally, have demonstrated that glycyrrhetinic acid possesses diverse biological activities, including antiviral, anti-inflammatory, anti-ulcer, hypoglycemic, and lipid-regulating properties. Studies have shown that glycyrrhetinic acid can alleviate 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 also reduces inflammation in microglia (BV2) cells from mice treated with 1-methyl-4-phenylpyridine, demonstrating an anti-inflammatory effect. Glycyrrhetinic acid also reduces plasma glucose and glycosylated hemoglobin levels in diabetic rats, regulates the activity of key glucose metabolism enzymes in the liver, and maintains normal blood glucose levels. However, there are no reports on the interventional and therapeutic effects of glycyrrhetinic acid on the progression of abdominal aortic aneurysms. Although glycyrrhetinic acid has good biological activity, it also has adverse effects, the main manifestation of which is that excessive concentration will inhibit the vital activities of cells; and due to its special molecular structure, it has high lipophilicity and low water solubility, low drug bioavailability, and difficulty in controlling dosage. Therefore, it has a slow onset of effect and insignificant therapeutic effect in clinical treatment, which also limits its application in clinical practice. Summary of the Invention

[0004] One object of the present invention is to provide the use of glycyrrhetinic acid or glycyrrhetinic acid liposomes in the preparation of abdominal aortic aneurysm protective drugs. Through intervention treatment with glycyrrhetinic acid or glycyrrhetinic acid liposomes, abdominal aortic aneurysm rupture can be alleviated and the survival rate can be improved.

[0005] To achieve the above object, the present invention is implemented according to the following technical solutions:

[0006] The first technical solution provided by the present invention is the use of glycyrrhetinic acid or glycyrrhetinic acid liposomes in the preparation of abdominal aortic aneurysm protective drugs.

[0007] Furthermore, the preparation method of the glycyrrhetinic acid liposomes comprises the following steps:

[0008] S1. Soy 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;

[0009] 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°C water bath and sonicate at 200 W for 15 minutes to hydrate and form small-particle liposomes.

[0010] S3. The obtained liposomes are dialyzed and purified to obtain purified liposomes; the purified liposomes are extruded through a membrane to obtain glycyrrhetinic acid liposomes with uniform particle size.

[0011] Preferably, the organic solvent is one or more of chloroform, methanol, ethanol and diethyl ether.

[0012] Preferably, dialysis purification is performed using a dialysis membrane or dialysis bag with a molecular weight cut-off of 3500 Da.

[0013] Preferably, the particle size of the glycyrrhetinic acid liposome is 100-150 nm.

[0014] Compared with the prior art, the present invention has the following beneficial effects:

[0015] The present invention reveals that glycyrrhetinic acid can effectively improve the death of mice after abdominal aortic rupture 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 lipopolysaccharide (LPS)-induced macrophage polarization to M2 type; intervention with glycyrrhetinic acid before Ang II treatment can alleviate the occurrence of Ang II-induced abdominal aortic aneurysm and improve the survival rate of mice, indicating that glycyrrhetinic acid can be used to prepare drugs for alleviating abdominal aortic aneurysm.

[0016] Furthermore, the present invention improves the solubility and bioavailability of glycyrrhetinic acid by encapsulating glycyrrhetinic acid into liposomes. At the same time, after glycyrrhetinic acid is encapsulated using nanotechnology, its dosage can be effectively controlled, further improving and enhancing the therapeutic effect 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. This shows that glycyrrhetinic acid liposomes can be used to prepare drugs for alleviating abdominal aortic aneurysms, reducing off-target effects while being able to exert therapeutic effects as quickly as possible. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The effect of glycyrrhizic acid in alleviating abdominal aortic aneurysms in mice: A, abdominal aorta 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.

[0018] Figure 2 Glycyrrhetinic acid reduces macrophage infiltration and aortic elastic fiber degradation in the abdominal aorta of mice: A. Hematoxylin-eosin (HE) staining shows immune cell infiltration in the aorta; B. Elastic fiber and collagen fiber (EVG) staining shows elastic fiber degradation in the aorta.

[0019] Figure 3 Expression of various markers in mouse alveolar cell lines (MH-S cells) cultured with different drugs: A, CD80 antibody; B, CD163 antibody.

[0020] Figure 4 Test results of glycyrrhetinic acid liposomes prepared in one embodiment of the present invention: A, transmission electron microscopy; B, particle size distribution; C, hydrated particle diameter; D, multi-dispersibility; E, encapsulation efficiency; F, zeta potential.

[0021] Figure 5 Glycyrrhetinic acid liposomes alleviate the occurrence of abdominal aortic aneurysms in mice: A, abdominal aorta ultrasound results; B, statistical results of the maximum diameter of the abdominal aorta; C, probability of abdominal aortic aneurysm occurrence. DETAILED DESCRIPTION

[0022] In order to make the purpose, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0023] In the following examples, glycyrrhetinic acid was purchased from Sigma; other related reagents used, unless otherwise specified, are commercially available agents, and the methods involved, unless otherwise specified, are well-known methods.

[0024] Example 1. In vivo animal experiment: abdominal aortic aneurysm modeling experiment

[0025] Take 6-8 weeks old specific pathogen free (SPF) ApoE - / - Twenty C67BL / 6 male mice (purchased from Guangdong Weitong Lihua) were weighed and randomly divided into four groups of five mice each. Mice were housed in separate cages in a controlled room (23-25°C) under artificial lighting (14 hours light, 10 hours dark) and fed a standard pelleted diet with free access to food and water. Mice were anesthetized with 0.67% pentobarbital solution and secured in a prone position on a surgical table. The fur on the nape of the neck was disinfected, and a transverse incision approximately 1 cm long was made to separate the subcutaneous tissue. An osmotic micropump loaded with Ang II at a dosing concentration of 1000 ng / kg / min was implanted subcutaneously in the back.

[0026] The skin incision was closed with sutures and the surface was disinfected. After the mice regained consciousness, they were returned to their cages and housed as normal for 28 days to allow for the development of abdominal aortic aneurysms. Three days after modeling, each group of mice was gavaged every other day: the first group received DMSO (Ang II group), the second group received 50 mg / kg glycyrrhetinic acid (1% DMSO) (Ang II + Glycyrrhetinic Acid group), and the third group, which did not undergo modeling but underwent normal surgical procedures and received a subcutaneous microosmotic pump containing PBS, was designated the PBS group. Seven days after Ang II modeling, the diameter of the abdominal aorta was measured by ultrasound, and survival time was recorded.

[0027] like Figure 1 As shown in A and B: B-ultrasound results showed that on the 7th day after Ang II modeling, the diameter of the abdominal aorta of mice in the Ang II group was significantly increased compared with that in the PBS group, while the diameter of the abdominal aorta of mice in the Ang II + glycyrrhetinic acid group was significantly decreased, indicating that glycyrrhetinic acid can effectively alleviate the dilation of the abdominal aorta of mice caused by Ang II treatment.

[0028] On the 28th day after Ang II modeling, the mice were sacrificed and samples were collected. Figure 1 As shown in Figure C, no abdominal aortic aneurysms were detected in the mice in the PBS group, the incidence of abdominal aortic aneurysms in the mice in the Ang II group was 100%, and the incidence of abdominal aortic aneurysms in the mice in the Ang II + glycyrrhetinic acid group was 40%, indicating that glycyrrhetinic acid treatment can significantly improve the incidence of abdominal aortic aneurysms induced by Ang II. The survival curve drawn according to the survival time of the mice after modeling is shown in Figure 4. Figure 1As shown in D: The mice in the Ang II group began to die after modeling, while the 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 abdominal aorta rupture in mice.

[0029] Example 2: HE and EVG staining of abdominal aorta

[0030] The mice were anesthetized with 0.67% pentobarbital solution and placed on a foam board to fix their limbs. After opening the chest cavity, the heart was exposed. The needle was inserted into the apex of the heart with the right hand, and the right atrial appendage was cut open with ophthalmic scissors in the left hand. About 30-40 mL of PBS was injected at the needle insertion site, and vascular perfusion was completed after the liver turned white and the outflowing fluid became clear. After opening the abdominal cavity and removing other organs, the entire aorta was peeled out and immersed in formalin. It was taken out and photographed after 2 hours. The location of the abdominal aortic aneurysm was fixed, embedded, and sliced, and then HE staining was performed to observe the changes in cells in the aorta. Figure 1 As shown in Figure A, compared with the abdominal aorta of the non-modeled control group, the abdominal aorta of the mice in the Ang II group showed obvious lesions, including a decrease in vascular smooth muscle cells and a large accumulation of adventitial macrophages. In the aorta of mice in the Ang II + glycyrrhizic acid group, the number of smooth muscle cells was significantly improved, and the adventitial macrophages were not obvious. This shows that glycyrrhizic acid can effectively improve the abdominal aorta lesions induced by Ang II in mice. The degree of degradation of the elastic fiber and collagen fiber layers was observed by EVG staining. The degradation of the elastic fiber layer is divided into 4 levels (I-IV) from mild to severe: Grade I, damage such as destruction of the elastic fiber layer and reduction of vascular smooth muscle cells is limited to one layer of elastic fiber layer; Grade II, damage involves two layers or all, but is limited to 1 / 4 of the blood vessels; Grade III, damage involves all elastic fiber layers, but is limited to less than 1 / 2 of the blood vessels; Grade IV, damage involves all elastic fiber layers and extends to more than 3 / 4 of the blood vessels. As shown in Figure A Figure 2 As shown in Figure B, the elastic fiber layer of the abdominal aorta in mice treated with PBS was intact. In the Ang II group, grade I lesions were 20%, grade II lesions were 20%, and grade III lesions were 60%. In the Ang II + glycyrrhetinic acid group, grade I lesions were 60% and grade II lesions were 40%. This indicates that glycyrrhetinic acid treatment significantly ameliorated Ang II-induced degradation of the elastic fiber layer in the abdominal aorta of mice.

[0031] Example 3. In vitro cell experiment: Flow cytometry detection of glycyrrhetinic acid on the polarization of MH-S cells induced by lipopolysaccharide (LPS)

[0032] MH-S cells in the logarithmic growth phase were taken and cultured with RPMI-1640 complete medium (containing 0.05 mM β-mercaptoethanol) to a density of 3 × 10 5 / mL of cell suspension was inoculated into a 12-well plate at a rate of 1 mL per well, and cultured in a 37 ℃, 5% CO2 cell culture incubator overnight. The original culture medium was discarded, and RPMI-1640 culture medium containing 0.1 μg / mL of LPS was added for induction for 24 hours. The culture medium was discarded, and the cells were cultured in a 37 ℃, 5% CO2 cell culture incubator with RPMI-1640 culture medium containing 3 μg / mL of glycyrrhetinic acid for 24 hours, which was recorded as the LPS+glycyrrhetinic acid group; the cells were cultured in a 37 ℃, 5% CO2 cell culture incubator with RPMI-1640 culture medium containing 0.1 μg / mL of LPS for 24 hours, which was recorded as the LPS group; and the MH-S cells treated with PBS were recorded as the PBS group. After 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. Figure 3 As shown in the results, 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.

[0033] Example 4: Preparation of glycyrrhetinic acid liposomes

[0034] Soy lecithin and glycyrrhetinic acid were taken in a mass ratio of 25:1 and fully dissolved in anhydrous ethanol. The anhydrous ethanol was completely evaporated by rotary evaporation to obtain a uniform lipid film; 100 mg of the lipid film was added to 10 mL of 0.154 M NaCl solution and oscillated to form a suspension. The suspension was placed in a 30-degree water bath and sonicated at 200 W for 15 minutes to hydrate and form small-particle liposomes; the obtained liposomes were 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 were extruded through a membrane to obtain glycyrrhetinic acid liposomes with a particle size of 100-150 nm.

[0035] 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 lipid bilayer structure, uniform size distribution, and 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 4 From the D in the figure, we can see that glycyrrhetinic acid liposomes have good multi-dispersibility (PDI: 0.121±0.011). Figure 4 From the E in the figure, we can see that the encapsulation efficiency of glycyrrhetinic acid liposomes is 30.05±0.41%. Figure 4 From the F in the figure, we can see that the Zeta potential of glycyrrhetinic acid liposomes is -38.42±0.38 mV.

[0036] Example 5: Glycyrrhetinic acid liposomes alleviate the occurrence of abdominal aortic aneurysm

[0037] Modeling was performed using the same method as in Example 1. Three days before modeling and every other day thereafter, each group of mice received intraperitoneal injections of medication: the first group received intraperitoneal injections of DMSO (denoted as the Ang II group); the second group received intraperitoneal injections of 5 mg / kg glycyrrhetinic acid liposomes (denoted as the Ang II + glycyrrhetinic acid liposome group); and the third group, which did not undergo modeling but underwent normal surgical procedures and received a subcutaneous microosmotic pump containing PBS, was designated as the PBS group. Seven days after modeling, the aortic diameter of the mice was measured by ultrasound.

[0038] like Figure 5 As shown in A and B: On the 7th day after Ang II modeling, the B-ultrasound results showed that the diameter of the abdominal aorta of mice in the Ang II group was significantly larger than that of mice in the PBS group, while the diameter of the abdominal aorta of mice in the Ang II + glycyrrhetinic acid liposome group was significantly reduced, indicating that low-concentration glycyrrhetinic acid liposomes can effectively reduce the dilation of the abdominal aorta in mice caused by Ang II treatment. On the 28th day after Ang II modeling, the mice were sacrificed and samples were collected. The results of the incidence of abdominal aortic aneurysms in mice after modeling are shown in Figure 2. Figure 5 As shown in Figure C, no abdominal aortic aneurysm was detected in the mice in the unmodeled PBS group, the incidence of abdominal aortic aneurysm was 80% in the mice in the Ang II group, and the incidence of abdominal aortic aneurysm was 20% in the mice in the Ang II + glycyrrhetinic acid liposome group, indicating that low-concentration glycyrrhetinic acid liposome treatment can significantly improve the incidence of abdominal aortic aneurysm induced by Ang II treatment.

[0039] In summary, intervention with glycyrrhetinic acid or glycyrrhetinic acid liposomes before Ang II treatment can alleviate the occurrence of AngII-induced abdominal aortic aneurysm, improve the survival rate of mice, and reduce the infiltration of macrophages 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 through liposome coating, thereby improving the effect of glycyrrhetinic acid in treating abdominal aortic aneurysm.

[0040] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. Use of glycyrrhetinic acid or glycyrrhetinic acid liposomes in the preparation of a drug for protecting abdominal aortic aneurysm, characterized in that: The preparation method of the glycyrrhetinic acid liposome comprises the following steps: S1. Soy 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°C water bath and sonicate at 200 W for 15 minutes to hydrate and form small-particle liposomes. S3. The obtained liposomes were dialyzed and purified to obtain purified liposomes; the purified liposomes were extruded through a membrane to obtain glycyrrhetinic acid liposomes with uniform particle size and an encapsulation efficiency of 30.05±0.41%.

2. The use according to claim 1, characterized in that: The organic solvent is one or more of chloroform, methanol, ethanol and ether.

3. The use according to claim 1, characterized in that: In step S3, dialysis purification is performed using a dialysis membrane or dialysis bag with a molecular weight cut-off of 3500 Da.

4. The use according to claim 1, characterized in that: The particle size of the glycyrrhetinic acid liposome is 100-150 nm.

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