CN116407531A - Application of anhydroicaritin in preparation of drugs for inhibiting macrophage iron death to treat atherosclerosis - Google Patents

Abstract

The invention provides application of anhydroicaritin in preparation of medicines for inhibiting macrophage iron death to treat atherosclerosis, relates to the technical field of medicines, and aims to solve the problem of lack of medicines for clinically treating atherosclerosis at present. The invention uses the pharmacological and molecular biological experiments and experimental results of the anhydroicaritin to demonstrate the application of the anhydroicaritin in preparing the drugs for inhibiting macrophage iron death so as to treat atherosclerosis. Animal experiments prove that the ANH can play a role in treating atherosclerosis by inhibiting iron death of macrophages. According to in vitro experiments, from macrophages, the ANH has the effects of inhibiting macrophage apoptosis caused by RSL3 and inhibiting macrophage iron death caused by RSL3, and the ANH is proved to be a compound for inhibiting iron death, and Hmox1 iron metabolic genes are further screened out to serve as novel target genes for regulating and controlling macrophage iron death by the ANH, so that a novel drug target and a novel theory are provided for effective prevention and treatment of atherosclerosis.

Description

Application of anhydroicaritin in preparation of drugs for inhibiting macrophage iron death to treat atherosclerosis

Technical Field

The invention relates to the technical field of medicines, in particular to application of anhydroicaritin in preparation of a medicament for inhibiting macrophage iron death so as to treat atherosclerosis.

Background

Atherosclerosis (AS) is a type of chronic inflammatory disease characterized by lipid accumulation, and is a common pathological basis for a variety of cardiovascular and cerebrovascular diseases. Atherosclerotic cardiovascular disease is a global disease with high mortality and high disability rate, which seriously endangers human life and health. At present, no safe and effective means for preventing and treating atherosclerosis exists. Finding a suitable drug to treat or delay the onset and progression of atherosclerosis has therefore been a great challenge for clinical and basic medical workers. In the early stage of atherosclerosis, macrophages remove modified lipoproteins, cell fragments and dead cells in plaques through phagocytosis, so that plaque growth is limited, and the apoptosis of the macrophages in the plaques is increased along with the progress of the disease, and the removal function is reduced, and as apoptotic substances formed after apoptosis of macrophage-derived foam cells are difficult to remove, secondary necrosis and inflammatory reaction occur, the integrity of cells is destroyed, and lipid is mainly accumulated after the release of cytoplasmic contents to form a lipid pool, so that the formation and gradual enlargement of plaque centers are promoted, and therefore, the proliferation and the apoptosis of the macrophages have a close relationship with the development of atherosclerosis.

Iron death (ferrotosis) was first proposed by the university of columbia dr. Parent r. Stock well in 2012 to be iron dependent, as distinguished from novel apoptosis of apoptosis, cell necrosis, autophagy, and cell coke death. The essence is that glutathione is exhausted, the activity of glutathione peroxidase (GPX 4) is reduced, the oxidation resistance of cells is reduced, so that lipid peroxidation is increased, lipid Reactive Oxygen Species (ROS) are increased, and finally iron death is initiated. The prior art, "research method for the involvement of CN113186269a iron death in the process of atherosclerosis and foam cell formation in mice" discloses: during the formation of atherosclerosis, iron death occurs in macrophage-derived foam cells; meanwhile, studies have demonstrated that inhibiting iron death can alleviate lipid peroxidation and atherosclerosis of aortic endothelial cells of mice, but the occurrence and development of macrophage iron death in atherosclerotic plaques and the potential mechanism of action have not been reported, and drugs for treating atherosclerosis by inhibiting macrophage iron death are currently lacking clinically.

Anhydroicaritin (ANH) is an isoprene flavonoid compound, which is present in epimedium, a berberidaceae plant, and has the structural formula:

to date, there has been no report on the use of ANH for the preparation of a medicament for the treatment of atherosclerosis.

Disclosure of Invention

The invention aims to solve the technical problems that:

at present, the problem of lack of medicines for treating atherosclerosis clinically is solved, and the application of the anhydroicaritin in preparing medicines for inhibiting macrophage iron death so as to treat atherosclerosis is provided.

The invention adopts the technical scheme for solving the technical problems:

the invention provides an application of anhydroicaritin in preparing a medicament for inhibiting macrophage iron death so as to treat atherosclerosis. In order to better understand the essence of the present invention, the following uses of the pharmacological and molecular biological experiments and experimental results of ANH to illustrate its application in the preparation of drugs for inhibiting macrophage iron death to treat atherosclerosis.

Preparing an in-vivo atherosclerosis model of a mouse by giving the mouse a high-fat diet, and adopting ANH daily gastric lavage administration with the dosage of 30mg/kg to observe the arterial lipid deposition degree of the mouse, the size of the arterial plaque of the aortic root of the heart, the internal lipid deposition degree of the plaque and the expression condition of GPX 4; confirming from animal level the use of ANH to inhibit macrophage iron death to treat atherosclerosis;

in vitro experiments, iron death inducer RSL3 is adopted to induce iron death of macrophages, and the inhibition effect of ANH on iron death of the macrophages is confirmed by detecting the survival and apoptosis conditions of the macrophages under the action of ANH, and the results are respectively confirmed by MTT experiments and propidium iodide staining experiments; meanwhile, an iron death inducer RSL3 is adopted to induce iron death of macrophages, and the inhibition effect of the ANH on the iron death of the macrophages is further confirmed by detecting the protein level of GPX4, the content of lipid Reactive Oxygen Species (ROS) and the content of lipid peroxidation product Malondialdehyde (MDA) in the macrophages under the action of the ANH, and the result is respectively determined by an immunoblotting experiment, a fluorescent probe DCFH-DA experiment and an MDA content detection experiment;

iron death inducer RSL3 is adopted to induce iron death of macrophages, genes with expression differences and variation trends in RNA of the macrophages under the action of ANH are detected, a novel target gene Hmox1 gene of the atherosclerosis regulated by the ANH is determined, the inhibition effect of the ANH on the iron death of the macrophages by inhibiting the Hmox1 gene is confirmed, and the result is confirmed by a TRIzol method and a transcriptome sequencing technology (RNA-seq).

The principle and the advantages of the invention are as follows:

the animal experiments prove that the ANH can play a role in treating atherosclerosis by inhibiting the iron death of macrophages. According to in vitro experiments, starting from macrophages which are the key for regulating atherosclerosis, the ANH has the effects of inhibiting macrophage apoptosis caused by RSL3 and inhibiting macrophage iron death caused by RSL3, and further screening Hmox1 iron metabolic genes as novel target genes for regulating macrophage iron death by the ANH, so that a theoretical basis is laid for developing the ANH into medicines for clinically treating atherosclerosis.

Drawings

FIG. 1 is a schematic diagram of the results of inhibiting atherosclerosis by using a mouse experimental ANH according to the embodiment of the invention, wherein (a) is a comparison graph of the results of aortic artery disease of a mouse, (b) is a comparison graph of the results of atherosclerosis of aortic root of a mouse heart, and (c) is a comparison graph of the results of immunofluorescence detection of plaque of aortic root of a mouse heart;

FIG. 2 is a schematic diagram showing the results of up-regulating RSL3 by ANH in macrophages to cause GPX4 protein expression in the examples of the present invention;

FIG. 3 is a graph showing the results of reversing RSL 3-induced macrophage viability decline by ANH in macrophages in accordance with an embodiment of the present invention;

FIG. 4 is a graph showing the results of the ANH in macrophages reversing RSL 3-induced intracellular ROS aggregation in accordance with an embodiment of the present invention;

FIG. 5 is a graph showing the results of the ANH-reversed RSL3 in macrophages resulting in cell death in accordance with an embodiment of the present invention;

FIG. 6 is a graph showing the results of reversing MDA content in RSL 3-induced cells by ANH in macrophages according to an embodiment of the present invention;

FIG. 7 is a schematic illustration of the volcanic formation of genes involved in iron death of macrophages in which ANH can regulate a number of RSL3 in macrophages in accordance with an embodiment of the present invention.

Detailed Description

In order that the above objects, features and advantages of the invention will be readily understood, a more particular description of the invention will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings.

Example 1:

24 male ApoE ^-/- The mice were randomly divided into four groups of 6 mice, namely: normal Diet (ND), high Fat Diet (HFD), hfd+fer-1 (High fat diet+ferrostatin-1), and hfd+anh (High fat diet+anh). Wherein the ND group is fed by common feed, the HFD group is fed by high-fat feed containing 20% fat and 0.3% cholesterol, the HFD+Fer-1 group and the HFD+ANH group are respectively fed by high-fat diet for 4 weeks and then are simultaneously fed by Ferrostatin-1 and ANH for 8 weeks, and the ANH is fed by stomach irrigation every day with the dosage of 30mg/kg; ferrosistatin-1 is administered by intraperitoneal injection every day at a dose of 1mg/kg, and Ferrosistatin-1 is an inhibitor of iron death and is used as a positive control drug. After 12 weeks, each group of mice was sacrificed under ether anesthesia, hearts and aorta were taken and placed in 4% paraformaldehyde solution for soaking overnight, and after sugar deposition overnight, the aorta was stained with Oil Red O and the degree of lipid deposition in the aorta was observed for each group of mice; preparing frozen sections of heart aortic root by using H respectively&E staining for detecting size of aortic root plaque of heart, oil Red OStaining was used to detect lipid deposition in the aortic heart root plaque and immunofluorescence was used to detect GPX4 expression in the aortic heart root plaque. As shown in fig. 1, the results of combining graphs (a), (b) and (c) indicate that there is a large amount of lipid deposition at the aortic arch and abdominal aorta in the HFD group mice compared to the ND group; the aortic root has obvious atheromatous plaque formation, and a large amount of lipid droplets are deposited, which indicates that the atherosclerosis model of the mice is successfully constructed and the expression of GPX4 in macrophages of the aortic root is obviously reduced, thus indicating that the macrophages are subjected to iron death during AS of the mice; compared with the HFD group, the HFD+ANH group has the advantages of significantly reduced aortic lipid accumulation, significantly reduced aortic root arterial plaque and significantly increased GPX4 expression level, and the results of the example show that: ANH may exert an anti-AS effect by inhibiting macrophage iron death.

Since iron death is a regulated form of cell death, iron-dependent accumulation of lipid peroxidation reaches lethal levels. When the cell cystine transport protein is inhibited (such as Erastin), intracellular Glutathione (GSH) is exhausted, and finally, the inactivation of glutathione peroxidase (GPX 4) is caused, so that lipid peroxidation accumulation is caused, and cell death can be induced to a certain extent, and inhibition of GPX4 enzyme (such as RSL 3) can also directly cause the effect, so that GPX4 is used as a marker of iron death. This example demonstrates the use of ANH to inhibit macrophage iron death from animal levels to treat atherosclerosis.

The example used an H & E staining kit (bi yun, china), oil Red O detection kit (soribao, beijing).

Example 2:

macrophage RAW264.7 was selected as experimental cells, and the experimental group included control group (macrophage), RSL3 group (macrophage+rsl3 0.5 μm), rsl3+anh group (macrophage+rsl3+1, 3, 5, 10 μm different doses of ANH) and ANH group (macrophage+anh5 μm). The survival of macrophages in the experimental and control groups was compared using the MTT method.

The detection method comprises the following steps: macrophages of the experimental group and the control group are inoculated in a 96-well plate and are placed in a 37 ℃ incubator for culture so as to adhere cells. According to the experimental group, adding RSL3 and ANH with specified dosages into each cell, culturing in an incubator at 37 ℃ for 24 hours, adding 20 μl MTT solution into each cell (the final MTT concentration is 0.5 mg/ml), continuously culturing for 3 hours, carefully sucking out the culture solution in the cell, adding 150ul dimethyl sulfoxide into each cell, and oscillating at low speed on a micro oscillator for 10 minutes to fully dissolve the crystal. Finally, the absorbance of each well was measured with an ELISA, and the measurement wavelength was 490nm. As shown in FIG. 2, in contrast to the control group, RSL3 induced severe macrophage death, whereas in the RSL3+ ANH group, ANH significantly reversed RSL3 induced macrophage death, and the ANH group results were substantially consistent with the control group results, indicating that ANH had no inhibitory effect on normal macrophages.

The present example used the MTT kit (bi yun tian, china).

Example 3:

isolation and purification of peritoneal primary macrophages: c57BL/6 mice of about 8 weeks of age were selected, 3% thioglycolate broth was intraperitoneally injected, and after 4 days, the mice were sacrificed by cervical removal and immersed in 75% ethanol for 3-5 minutes. Taking out, placing in a sterile operation table, fixing the mouse in a supine position, opening the skin by surgical scissors, opening the abdominal cavity (to the sternum cavity and white xiphoid process) by ophthalmic scissors to avoid damaging diaphragm muscle, then exposing the intestinal tract, sucking pre-cooled phosphate buffer solution PBS by adopting a 1mL syringe, slowly instilling the solution into the abdominal cavity of the mouse, taking another syringe in the intestinal tract, carefully and fully stirring to avoid liquid overflowing the abdominal cavity, sucking the liquid after the liquid is turbid, and filtering the liquid into a 50mL centrifuge tube through a 70 mu m filter screen. Phosphate buffered saline PBS the abdominal cavity was washed 3-5 times and the washed solutions were combined into a 50ml centrifuge tube. The 50ml centrifuge tube was centrifuged at 1500rpm for 5min and the supernatant was discarded. The cells obtained were cultured in 10ml of DMEM medium containing 10% fetal bovine serum, resuspended and blown up uniformly, counted and plated. After attaching cells for 2 hours, discarding the culture solution, gently cleaning the cells once by using PBS, sucking and discarding the cells, and changing the cells into the culture solution containing differentiation promoting components to continue culturing for 24 hours to obtain the primary macrophages of the abdominal cavity.

The experimental process comprises the following steps: the primary macrophages of the abdominal cavity are used as experimental cells, and the experiments are divided into: control group (peritoneal primary macrophage), RSL3 group (peritoneal primary macrophage+rsl3 0.5μm), rsl3+anh group (peritoneal primary macrophage+rsl3 0.5μm+anh5 μm); the corresponding drugs are added to be cultured together with the cells for 24 hours in a cell culture box, and the apoptosis condition is detected by adopting Propidium Iodide (PI) staining experiment. The detection method comprises the following steps: firstly, absorbing cell culture solution, adding PBS for washing once, then adding 200 mu L of PBS, adding 10 mu L of propidium iodide staining solution for staining, gently mixing, incubating for 10-20 minutes at room temperature (20-25 ℃) in the dark, then placing in an ice bath, using aluminum foil for dark in the whole staining process, immediately observing under a fluorescent microscope, and photographing. As shown in fig. 3, the PI-stained dead cells were significantly increased in the RSL3 group compared to the control group, indicating that RSL3 caused massive death of peritoneal primary macrophages, while the ANH significantly reversed RSL 3-induced death of peritoneal primary macrophages in the rsl3+anh group.

The principle according to the embodiment is as follows: propidium Iodide (PI) can stain necrotic cells or cells with late apoptosis losing cell membrane integrity, causing them to appear red fluorescent.

In the embodiment, the primary abdominal macrophages are adopted, and because the primary abdominal macrophages are in a fusiform shape under the normal culture condition, cells are round when iron death occurs, and experimental results are easier to observe after dyeing.

Example 2 and example 3 iron death was induced in macrophages using iron death inducer RSL3 and inhibition of iron death by ANH was confirmed by examining the survival and apoptosis of macrophages under the action of ANH.

This example uses Propidium Iodide (PI) staining kit (bi yun tian, china).

Example 4:

macrophage RAW264.7 is selected as experimental cells and divided into an experimental group and a control group, wherein the experimental group is divided into five groups, each group of macrophages is given with RSL 3.5 mu M, and different doses of ANH 0, 1, 3, 5 and 10 mu M are given, the experimental group and the control group are respectively cultivated in six-hole plates containing 2ml of culture solution in each hole and having 60% of cell density, and after the experimental group and the control group are cultivated in a cell incubator at 37 ℃ for 24 hours, the total cell proteins of the experimental group and the control group are extracted, and immunoblotting experiments are carried out. As shown in fig. 4, the results demonstrate that RSL3 reduced the protein level of GPX4 compared to the control group; the expression level of GPX4 increased to varying degrees when different doses of ANH were added, and the effect was best with an ANH dose of 5. Mu.M by analysis of the results of multiple experiments. This example demonstrates that ANH can up-regulate the expression of the iron death marker GPX4 protein, demonstrating the iron death inhibitory effect of ANH.

The GPX4 antibody (Abcam, uk) used in this example.

Example 5:

macrophage RAW264.7 was used as experimental cell, and the experiments were grouped into: the control group (macrophage), RSL3 group (macrophage+RSL3 0.5. Mu.M), RSL3+ANH group (macrophage+RSL3 0.5. Mu.M+ANH 5. Mu.M) were cultured in a 37℃cell incubator for 24 hours, and the ROS activity of each group of cells was detected using a reactive oxygen species fluorescent probe DCFH-DA.

The detection method comprises the following steps: diluting DCFH-DA with serum-free culture solution according to the ratio of 1:1000 to make the final concentration of the DCFH-DA be 10 mu M/L; removing cell culture solution from cultured macrophage, adding 500 μl diluted DCFH-DA; incubating in a cell incubator at 37 ℃ for 20 minutes; the cells were washed three times with serum-free cell culture medium to sufficiently remove DCFH-DA that did not enter the cells, observed under a fluorescence microscope, and photographed. As shown in fig. 5, the green fluorescence in the RSL3 group was significantly enhanced compared to the control group, indicating that RSL3 significantly caused the aggregation of macrophage ROS, resulting in cell iron death, whereas in the rsl3+anh group, ANH significantly reversed the occurrence of ROS aggregation, indicating the iron death inhibition by ANH.

The fluorescent probe DCFH-DA detection kit (Biyun Tian, china) is adopted in the embodiment.

Example 6:

macrophage RAW264.7 is taken as an experimental cell, the experimental group comprises a control group (macrophage), an RSL3 group (macrophage+RSL3 0.5 mu M) and an RSL3+ANH group (macrophage+RSL3+ANH5 mu M), and MDA content of each group of macrophages is detected by adopting an MDA content detection method. The detection method comprises the following steps: macrophages were seeded in 96-well plates and incubated in an incubator at 37 ℃ to adhere the cells. According to the experimental grouping, adding the RSL3 and the ANH with the specified dosages into each cell, culturing for 24 hours in a 37 ℃ incubator, finally collecting cell lysate according to the step operation of an MDA kit, quantitatively detecting MDA in each cell lysate by an enzyme-labeled instrument, and measuring the wavelength to 532nm and 600nm. Since MDA is the final product of lipid peroxidation, MDA is an important marker for the occurrence of iron death and a key indicator for iron death detection. As shown in fig. 6, the MDA content in the RSL3 group was higher than that in the control group, and the ANH significantly reversed the MDA content in the RSL 3-induced macrophages, indicating that the ANH significantly inhibited macrophage iron death.

In this example, the MDA detection kit was (Soxhaust Bao, beijing).

Example 4, example 5 and example 6 iron death in macrophages was induced using iron death inducer RSL3, and inhibition of iron death in macrophages by ANH was further confirmed by measuring GPX4 protein levels, lipid Reactive Oxygen Species (ROS) levels, lipid peroxidation products Malondialdehyde (MDA) levels in macrophages under the action of ANH.

Example 7:

macrophage RAW264.7 was used as experimental cell, and the experiments were grouped into: control group (macrophage), RSL3 group (macrophage+RSL3.5. Mu.M), RSL3+ANH group (macrophage+RSL3.5. Mu.M+ANH5. Mu.M), experimental group and control group were cultured in cell incubator at 37℃for 24 hours, cell RNA was collected by TRIzol method, RNA-seq was performed, and genes with expression difference and a certain trend of variation in the group were selected to draw volcanic image. As shown in FIG. 7, the results demonstrate that RSL3 promotes the expression of twelve genes (Hmox 1, cat, plin2, prdx1, cyb5a, fth1, srxn1, slc48a1, blvrb, gstm1, sqstm1, ftl1ps 1) that are most significantly varied in macrophages, while ANH significantly reverses the expression of these genes. Since Hmox1 is a rate-limiting enzyme in the process of heme catabolism of iron porphyrin compounds, heme can be decomposed into carbon monoxide, biliverdin and ferrous ions, which are important components of iron death, ANH inhibits macrophage iron death by regulating Hmox1 gene.

The final study of the invention considers that: the ANH can inhibit iron death of macrophages by regulating HMOX1 gene, inhibiting iron accumulation in macrophages, improving mitochondrial function, inhibiting lipid ROS (reactive oxygen species) production, promoting GPX4 expression, and inhibiting content of Malondialdehyde (MDA) which is a lipid peroxidation reaction product in cells, so that iron death of macrophages is inhibited to achieve the effect of preventing and treating atherosclerosis.

The final research of the invention finds that: ANH can remarkably inhibit the generation of lipid reactive oxygen species ROS in macrophages by inhibiting Hmox1 genes, promote the expression of GPX4 and inhibit the content of Malondialdehyde (MDA) which is a lipid peroxidation reaction product in cells, so that iron death of the macrophages is inhibited and the AS-resistant effect is exerted.

Although the present disclosure is disclosed above, the scope of the present disclosure is not limited thereto. Various changes and modifications may be made by one skilled in the art without departing from the spirit and scope of the disclosure, and such changes and modifications would be within the scope of the disclosure.

Claims (2)

1. Application of anhydroicaritin in preparing medicine for inhibiting macrophage iron death to treat atherosclerosis is provided.

2. The use according to claim 1, characterized in that the medicament is prepared from anhydroicaritin and pharmaceutically acceptable excipients.

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