Application of lentinan in preparation of macrophage ferroptosis inhibitor

Abstract

The invention discloses application of lentinan in preparation of a macrophage ferroptosis inhibitor, and belongs to the technical field of biological medicines. Animal experiments prove that the LNT can play a role in treating atherosclerosis by inhibiting ferroptosis of macrophages. In-vitro experiments prove that LNT has the effect of inhibiting lipid deposition and ferroptosis of macrophages caused by ox-LDL from macrophages which are keys for regulating and controlling atherosclerosis, and prove that LNT is a natural product for inhibiting ferroptosis, and Slc7a11 is further screened as a target gene for regulating and controlling ferroptosis of macrophages by LNT. A drug target and a new theory are provided for effective prevention and treatment of atherosclerosis, and a theoretical foundation is laid for developing LNT into a drug for clinical treatment of atherosclerosis.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to the application of lentinan in the preparation of a macrophage ferroptosis inhibitor. Background Art

[0002] Atherosclerosis (AS) is a chronic inflammatory and vascular metabolic disease characterized by lipid accumulation and inflammatory infiltration. Macrophages play an important role in the pathogenesis of atherosclerosis. In the initial stage of atherosclerosis development, a large amount of low-density lipoprotein (LDL) leaks into the intima and is oxidized by free radicals, such as reactive oxygen species (ROS), to produce oxidized LDL (ox-LDL), which is then taken up by macrophages. When the intake of ox-LDL exceeds the clearance capacity of macrophage metabolism, it is transformed into foam cells. The death of macrophage-derived foam cells is an important feature of advanced plaques and a key pathological process for the formation of necrotic cores and plaque instability. Excessive accumulation of ROS can lead to ferroptosis of macrophages.

[0003] Ferroptosis is a new form of cell death triggered by lipid peroxidation in an iron-dependent manner. Its main mechanism is that under the action of divalent iron, unsaturated fatty acids are catalyzed to cause lipid peroxidation. In addition, it also manifests as a decrease in the regulatory capacity of the antioxidant system (glutathione system). Therefore, pharmacological targeting of macrophage ferroptosis is a potential therapeutic strategy that is beneficial for improving atherosclerotic vulnerable plaque rupture and secondary thrombosis, and reducing the mortality and disability rate of acute coronary syndrome.

[0004] There is a lack of safe and effective drugs for treating atherosclerosis by inhibiting macrophage ferroptosis in the clinic. Compared with chemically synthesized drugs, natural products have the advantages of simple structure, good stability, and low toxicity and side effects. At present, there have been many reports on the use of natural product extracts or monomers to improve macrophage dysfunction and inhibit their ferroptosis. Lentinan (LNT) is extracted from the fruiting bodies of Lentinan edodes and is reported to be an immunomodulator and antiviral drug. The basic substance that makes Lentinan biologically active is β-glucan, among which cereal β-glucan oat polysaccharide has been shown by a large number of studies to improve glucose and lipid metabolism, thereby protecting the cardiovascular system. So far, there has been no report on the use of Lentinan with similar compound structure to prepare drugs for inhibiting macrophage ferroptosis to treat atherosclerosis. Summary of the invention

[0005] The purpose of the present invention is to provide the use of Lentinan in the preparation of a macrophage ferroptosis inhibitor to solve the problems of the above-mentioned prior art. The present invention provides the use of Lentinan in the preparation of a drug for inhibiting macrophage ferroptosis to treat atherosclerosis.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] One of the technical solutions of the present invention is the use of Lentinan in the preparation of a macrophage ferroptosis inhibitor.

[0008] The second technical solution of the present invention is a macrophage ferroptosis inhibitor, comprising Lentinan.

[0009] Based on the above technical solution, the present invention has the following technical effects:

[0010] The present invention proves through animal experiments that LNT can play a role in treating atherosclerosis by inhibiting macrophage ferroptosis. Through in vitro experiments, starting from macrophages, which are the key to regulating atherosclerosis, it is proved that LNT has the effect of inhibiting lipid deposition and ferroptosis of macrophages caused by ox-LDL, and that LNT is a natural product that inhibits ferroptosis. Slc7a11 is further screened as a target gene for LNT to regulate macrophage ferroptosis, providing drug targets and new theories for the effective prevention and treatment of atherosclerosis, and laying a theoretical foundation for the development of LNT into a drug for the clinical treatment of atherosclerosis. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0012] Figure 1 is the blood lipid level of each group of mice in Example 1 of the present invention, wherein A is total cholesterol, B is high-density lipoprotein cholesterol, C is low-density lipoprotein cholesterol, and D is triglyceride.

[0013] Figure 2 The Oil Red O staining of the aorta of each group of mice in Example 2 of the present invention is shown in FIG. A is a staining diagram, and B is a statistical diagram.

[0014] Figure 3 The Oil Red O staining diagrams of aortic valves of each group in Example 2 of the present invention are shown in Figure 2. A is a staining diagram, and B is a statistical diagram.

[0015] Figure 4These are HE staining images of aortic valves in each group in Example 2 of the present invention, wherein A is the staining image and B is the statistical image.

[0016] Figure 5 The Masson staining diagrams of aortic valves of each group in Example 2 of the present invention are shown in Figure 2. A is the staining diagram and B is the statistical diagram.

[0017] Figure 6 This is a schematic diagram of the results of LNT improving cell death in atherosclerotic plaques in the animal experiment in Example 3 of the present invention. Among them, A is a staining diagram and B is a statistical diagram.

[0018] Figure 7 This is a schematic diagram of the results of LNT reducing iron deposition in atherosclerotic plaques in the animal experiment in Example 4 of the present invention. Among them, A is a staining diagram and B is a statistical diagram.

[0019] Figure 8 The figure is a schematic diagram of the results of LNT reversing the decrease in macrophage viability and cell death induced by ox-LDL in macrophages in Example 5 of the present invention. A is a cell viability test, B is a statistical graph of cell apoptosis detected by cell flow cytometry, and C is a scatter plot of cell apoptosis detected by cell flow cytometry.

[0020] Fig. 9 This is a schematic diagram of the results of LNT improving ox-LDL-induced iron deposition in macrophages in Example 6 of the present invention.

[0021] Fig.10 This is a schematic diagram of the results of LNT improving ox-LDL-induced lipid deposition in macrophages in Example 7 of the present invention.

[0022] Among them, A is the coloring map and B is the statistical map.

[0023] Fig.11 This is a schematic diagram of the results of LNT promoting cholesterol efflux to mature HDL in macrophages in Example 7 of the present invention.

[0024] Fig.12 This is a schematic diagram of the results of LNT promoting the translocation of SR-B1 to the cell membrane in macrophages in Example 8 of the present invention.

[0025] Among them, A is a schematic diagram of flow cytometry detection of the expression of macrophage SR-B1 total protein and membrane protein, B is a statistical graph of the expression level of macrophage SR-B1 membrane protein, and C is a statistical graph of the expression level of macrophage SR-B1 total protein.

[0026] Fig.13 This is a schematic diagram of the results of reducing the intracellular lipid ROS accumulation caused by ox-LDL in macrophages by LNT in Example 9 of the present invention. Among them, A is a schematic diagram of flow cytometry detection of intracellular lipid ROS accumulation in macrophages, and B is a statistical diagram.

[0027] Fig.14 This is a schematic diagram of the results of LNT reducing the intracellular MDA content caused by ox-LDL in macrophages in Example 10 of the present invention.

[0028] Fig.15 This is a schematic diagram of the results of LNT reversing the decrease of GSH / GSSG caused by ox-LDL in macrophages in Example 11 of the present invention.

[0029] Fig.16 The figure is a schematic diagram of the results of LNT reversing the mitochondrial morphological changes and membrane potential reduction caused by ox-LDL in macrophages in Example 12 of the present invention. A is a scatter plot of JC-1 detected by flow cytometry, B is a statistical graph of A, and C is a transmission electron microscopy detection of mitochondrial morphology.

[0030] Fig.17 It is the transcription level of inflammatory factors induced by ox-LDL in macrophages reversed by LNT in Example 13 of the present invention. Wherein, A is the relative mRNA level of IL-1β, and B is the relative mRNA level of TNFα.

[0031] Fig.18 The volcano diagram of differentially expressed genes and the GSEA diagram of the ferroptosis pathway in macrophages regulated by LNT in macrophages in Example 14 of the present invention are shown. A is the GSEA diagram of the ferroptosis pathway, and B is the volcano diagram of differentially expressed genes.

[0032] Fig.19 Schematic diagram of the results of up-regulating the expression of Slc7a11 protein in macrophages by LNT in Example 15 of the present invention. A is a schematic diagram of WB detection of the expression level of Slc7a11 protein in macrophages, and B is a statistical diagram.

[0033] Fig. 20 This is a schematic diagram of the results of LNT upregulating the expression of Slc7a11 protein in macrophages at atherosclerotic plaques in Example 15 of the present invention, wherein A is a staining diagram and B is a statistical diagram. DETAILED DESCRIPTION

[0034] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0035] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0036] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0037] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0038] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0039] The technical solutions described in the present invention, unless otherwise specified, are all conventional solutions in the art, and the reagents or raw materials used, unless otherwise specified, are purchased from commercial channels or have been disclosed.

[0040] The embodiment of the present invention provides the use of Lentinan in preparing a macrophage ferroptosis inhibitor.

[0041] In some specific embodiments, the macrophages comprise bone marrow-derived macrophages.

[0042] The embodiment of the present invention also provides a macrophage ferroptosis inhibitor, comprising Lentinan.

[0043] In some specific embodiments, a pharmaceutically acceptable excipient is also included.

[0044] In some specific embodiments, the dosage form is a liquid dosage form, a solid dosage form or a paste dosage form.

[0045] The present invention uses pharmacological and molecular biological experiments and experimental results of Lentinan to illustrate its application in the preparation of drugs for inhibiting macrophage ferroptosis to treat atherosclerosis. The present invention proves through animal experiments that Lentinan can play a role in treating atherosclerosis by inhibiting macrophage ferroptosis. Through in vitro experiments, starting from macrophages, it is proved that Lentinan has the effect of inhibiting macrophage ferroptosis caused by ox-LDL, and it is proved that Lentinan is a natural product that inhibits ferroptosis, and further screens out the key molecule Slc7a11 in the antioxidant system as the target gene for Lentinan to regulate macrophage ferroptosis, providing new drug targets and new theories for the effective prevention and treatment of atherosclerosis.

[0046] The present invention provides the use of lentinan in preparing a drug for inhibiting macrophage ferroptosis to treat atherosclerosis. In order to better understand the essence of the present invention, the pharmacological and molecular biological experiments and experimental results of LNT are used to illustrate its use in preparing a drug for inhibiting macrophage ferroptosis to treat atherosclerosis.

[0047] APOE- / - mice were given a high-fat diet to establish an in vivo atherosclerosis model, and LNT was administered freely in drinking water at a dose of 10 mg / kg. Atherosclerosis-related indicators (serum TC, TG, LDL-C, HDL-C, abdominal aorta Oil Red O staining, aortic valve Oil Red O, H&E, Masson staining, etc.) were comprehensively tested, and cell death and iron deposition in the aortic root plaques of mice were observed; the application of LNT in inhibiting macrophage ferroptosis to treat atherosclerosis was confirmed at the animal level.

[0048] In vitro experiments were conducted using ox-LDL to induce ferroptosis in macrophages, and Erastin, an ferroptosis inducer, was used as a positive control. The survival and apoptosis of macrophages under LNT intervention were detected to confirm the inhibitory effect of LNT on macrophage cell death. The results were confirmed by CCK8 assay and Annexin The results were determined by V / PI double staining experiment; the inhibitory effect of LNT on macrophage ferroptosis was confirmed by detecting iron deposition in macrophages under the intervention of LNT; at the same time, the lipid metabolism and lipid ROS content, lipid peroxidation product malondialdehyde (MDA) content, and reduced glutathione / oxidized glutathione (GSH / GSSG) content in macrophages under the intervention of LNT were detected to confirm the effect of LNT on lipid metabolism and peroxide clearance in macrophages, and these results were confirmed by Oil Red O staining, cholesterol efflux experiment, fluorescent probe C11-BODIPY experiment, MDA content detection experiment, and GSH / GSSG content detection experiment, respectively; in addition, the effect of LNT on endogenous ROS in macrophages was further confirmed by detecting the changes in mitochondrial morphology and membrane potential of macrophages under the intervention of LNT, and this result was confirmed by transmission electron microscopy and JC-1 staining experiments; the above in vitro experiments confirmed that LNT inhibited macrophage ferroptosis.

[0049] Macrophage ferroptosis was induced by ox-LDL. The target gene of LNT regulating macrophage ferroptosis was determined to be Slc7a11 through RNA sequencing (RNA-seq) data of genes with expression differences and change trends in macrophages under LNT intervention. In vitro and in vivo experiments confirmed that LNT inhibited macrophage ferroptosis by promoting the expression of Slc7a11.

[0050] Lentinan (LNT) used in the examples of the present invention was purchased from Yuanye Biotechnology, S23110.

[0051] Example 1

[0052] Animals and feeding in the experiment of the present invention: 48 male APOE - / - The mice were randomly divided into four groups, with 12 mice in each group, namely: normal chow diet (NCD), LNT administration group (LNT), high fat diet group (HFD) and high fat diet + LNT group (HFD + LNT).

[0053] The normal diet group and LNT administration group were fed with ordinary feed, and the high-fat diet group and high-fat diet+LNT group were fed with high-fat feed.

[0054] Among them, ordinary feed and high-fat feed were provided by Jiangsu Collaborative Pharmaceutical Bioengineering Co., Ltd.; LNT group and HFD+LNT group were given LNT freely in drinking water, with a dose of 10 mg / kg. After 12 weeks, the mice in each group were anesthetized, blood was collected from the eyeballs, and then the mouse hearts were perfused with normal saline, and then the hearts and entire aortas were removed in turn.

[0055] Blood lipid test: The blood samples were centrifuged at 3000 rpm / min for 15 min, and the upper serum was collected. The levels of triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and low-density lipoprotein cholesterol (LDL-C) in the serum of the above-mentioned groups of mice were measured by microplate spectrophotometer using the assay kits according to the instructions, and the data were statistically analyzed.

[0056] Results Figure 1 Compared with the NCD group, the serum TC, TC, and LDL-C levels of mice in the HFD group were significantly increased, and the HDL-C level was significantly decreased. After LNT intervention, the serum TC, TC, and LDL-C levels of mice were significantly decreased, and the HDL-C level was significantly increased. This indicates that LNT can effectively improve the blood lipid level of mice with atherosclerosis, reduce serum TC, TG, and LDL-C levels, and increase HDL-C levels.

[0057] This example uses a triglyceride assay kit (Nanjing Jiancheng Bioengineering Institute, A110-1-1), a total cholesterol assay kit (Nanjing Jiancheng Bioengineering Institute, A111-1-1), a high-density lipoprotein cholesterol assay kit (Nanjing Jiancheng Bioengineering Institute, A112-1-1), and a low-density lipoprotein cholesterol assay kit (Nanjing Jiancheng Bioengineering Institute, A113-1-1).

[0058] Example 2

[0059] The experimental groups included a normal chow diet group (NCD), a LNT administration group (LNT), a high fat diet group (HFD) and a high fat diet + LNT group (HFD + LNT). Feeding and administration were the same as in Example 1.

[0060] Histopathological examination: The aorta was removed from the aortic arch to the bifurcation of the common iliac artery, the adventitia and fat were separated, fixed with 4% paraformaldehyde overnight, and then incubated with saturated oil red O solution for 30 minutes for staining and observation of lipid deposition in the aorta of each group of mice; the heart tissue was collected and fixed in 4% paraformaldehyde for 24 hours. Then it was dehydrated with gradient concentrations of ethanol and xylene, then embedded in paraffin, and 4 μm thick sections were made. HE and Masson staining were performed according to the instructions to detect the aortic valve plaque area, fibrous cap, necrotic core and collagen fiber area respectively; the heart was removed, completely immersed in the embedding agent, and rapidly frozen at the liquid nitrogen-air interface until OCT was completely solidified, and the heart frozen tissue embedding block was prepared, and the liver was sliced ​​at -20℃. Then it was incubated with saturated oil red O solution for 30 minutes for staining, washed until there was no residual staining solution, and then placed under a microscope to observe lipid deposition and take pictures.

[0061] Results Figure 2 Compared with the NCD group, the HFD group had significantly more aortic plaques, and the plaques were mainly concentrated in the aortic arch. After LNT intervention, the aortic plaques of the mice were significantly reduced, indicating that LNT can significantly reduce the formation of aortic plaques.

[0062] Figure 3 Oil red O staining of the aortic valve of mice in each group showed that compared with the NCD group, the lipid deposition area at the aortic valve position in the HFD group was significantly increased. After LNT intervention, the lipid deposition at the aortic valve of the heart was significantly reduced.

[0063] like Figure 4 As shown, compared with the NCD group, the aortic valve of the mice in the HFD group had a large number of foam cell infiltrations, an increased necrotic core, and a decreased thickness of the fibrous cap. After LNT intervention, the foam cell infiltration of the aortic valve of the mice's hearts decreased, the necrotic core decreased, and the thickness of the fibrous cap increased, indicating that LNT has the effect of increasing plaque stability.

[0064] like Figure 5 As shown in the figure, compared with the NCD group, the collagen fiber content in the aortic valve position of the mice in the HFD group was significantly increased. After LNT intervention, the collagen fiber content in the aortic valve position of the mice's hearts was reduced.

[0065] This example uses a saturated Oil Red O solution (G1015, Wuhan Saiweier Biotechnology Co., Ltd.).

[0066] The results of Example 1 and Example 2 show that: APOE induced by high-fat diet - / - In mice, intervention with LNT can effectively improve hyperlipidemia, significantly reduce lipid accumulation in the mouse aorta, and significantly inhibit the occurrence and development of atherosclerotic plaques.

[0067] Example 3

[0068] The experimental groups included a normal diet group (NCD), a LNT administration group (LNT), a high fat diet group (HFD) and a high fat diet + LNT group (HFD + LNT). Feeding and administration were the same as in Example 1.

[0069] Tunel staining was used to detect cell death in cardiac aortic root plaques.

[0070] like Figure 6 As shown in the figure, compared with the NCD group, there was cell death in the aortic root plaques of the mice in the HFD group, and the number of cell death in the aortic root plaques of the mice in the HFD+LNT group was significantly reduced, indicating that cell death exists in the plaques during the progression of atherosclerosis in mice.

[0071] This example uses the TUNEL cell apoptosis detection kit (Keygen Biotechnology, KGA702-1).

[0072] Example 4

[0073] The experimental groups included a normal diet group (NCD), a LNT administration group (LNT), a high fat diet group (HFD) and a high fat diet + LNT group (HFD + LNT). Feeding and administration were the same as in Example 1.

[0074] DAB-enhanced Prussian blue staining was used to detect iron deposition in cardiac aortic root plaques.

[0075] like Figure 7 As shown, compared with the NCD group, the iron deposition in the aortic root plaques of the HFD group mice was increased, and the iron deposition in the aortic root plaques of the HFD+LNT group was reduced, indicating that ferroptosis exists in the plaques during the progression of atherosclerosis in mice.

[0076] This example uses DAB to enhance Prussian blue staining (Sevier Biotechnology).

[0077] The results of Example 3 and Example 4 show that LNT can exert its anti-atherosclerotic effect by inhibiting ferroptosis in plaques.

[0078] Example 5

[0079] Extraction and differentiation induction of primary bone marrow macrophages from mice: C57BL / 6 mice aged 6-8 weeks were selected, killed by cervical dislocation, and soaked in 75% ethanol for 3-5 minutes. Take out, place on a sterile operating table, fix the mice in supine position, separate the femur and tibia of the mice, collect bone marrow cells and filter through a 70μm filter, and then lyse red blood cells. The obtained cells were resuspended in DMEM complete medium containing 20ng / mL macrophage colony stimulating factor (M-CSF), blown evenly, counted, and plated. Fresh BMDM growth medium was replaced on the third day. After 7 days of cell culture, mature mouse primary bone marrow macrophages (Bone marrow-derived macrophages, BMDM) were used in subsequent experiments.

[0080] Experimental process: Bone marrow-derived macrophages (BMDM) were selected as experimental cells. The experimental groups included Control group (+PBS), oxidized low-density lipoprotein (ox-LDL) group (+ox-LDL 100μg / ml), ferroptosis inducer Erastin group (+Erastin 10μM), ox-LDL+LNT group (+ox-LDL 100μg / ml+LNT 100μg / ml) and ox-LDL+ferroptosis inhibitor Ferrostatin-1 group (+ox-LDL 100μg / ml+Ferrostatin-15μM).

[0081] The survival of macrophages in the experimental group and the control group was compared by CCK8 method and AnnexinV / PI double staining method. The detection method was as follows: macrophages were inoculated in a 96-well plate and cultured in a 37°C incubator to allow the cells to adhere to the wall. According to the experimental grouping, LNT was added to each well of cells for 2 hours before the specified dose of ox-LDL and Erastin was added. After 24 hours of culture in a 37°C incubator, CCK8 solution was added to each well and culture was continued. Finally, the absorbance of each well was measured by an enzyme-linked immunosorbent assay, and the wavelength was 450nm. After the same intervention, macrophages were stained with AnnexinV / PI double staining apoptosis detection kit, incubated at room temperature (20-25°C) in the dark for 10-20 minutes, and then placed in an ice bath. The whole staining process was kept away from light, and then measured by flow cytometry.

[0082] like Figure 8 As shown, compared with the Control group, ox-LDL and Erastin induced severe macrophage cell death, while in the ox-LDL+LNT and ox-LDL+Ferrostatin-1 groups, LNT significantly reversed macrophage cell death.

[0083] This example uses CCK8 kit (Yisun Biotechnology Company, 40203ES60); Annexin V / PI double staining cell apoptosis detection kit (Abbkine, KTA0002).

[0084] Example 6

[0085] BMDM was used as experimental cells, and the experimental groups included Control group (+PBS), ox-LDL group (+ox-LDL100μg / ml), Erastin group (+Erastin 10μM), ox-LDL+LNT group (+ox-LDL 100μg / ml+LNT 100μg / ml) and ox-LDL+Ferrostatin-1 group (+ox-LDL 100μg / ml+Ferrostatin-15μM).

[0086] The total cell iron colorimetric test kit was used, and the absorbance of each well was measured by an enzyme-linked immunosorbent assay (ELISA) at a wavelength of 593 nm.

[0087] like Fig. 9 As shown in the results, the total iron content in the ox-LDL and Erastin groups increased compared with the Control group, while LNT improved iron deposition in macrophages, further confirming the inhibitory effect of LNT on macrophage ferroptosis.

[0088] This example uses a total cellular iron colorimetric test kit (Elabscience, E-BC-K880-M).

[0089] Example 7

[0090] BMDM were used as experimental cells, and the experimental groups included Control group (+PBS), LNT group (+LNT 100 μg / ml), ox-LDL group (+ox-LDL 100 μg / ml), and ox-LDL+LNT group (+ox-LDL 100 μg / ml+LNT 100 μg / ml).

[0091] After 24 h of ox-LDL intervention, cell samples were collected, stained by incubation with saturated Oil Red O solution for 30 min, washed until there was no residual staining solution, and then placed under a microscope to observe lipid deposition and take pictures.

[0092] The results are as follows Fig.10 Compared with the Control group, there was obvious lipid droplet deposition in the macrophages of the ox-LDL group. After LNT intervention, the lipid droplet deposition in the macrophages decreased, indicating that LNT can significantly reduce lipid deposition in macrophages.

[0093] The cells were further divided into groups: ox-LDL group (+ox-LDL 100μg / ml) and ox-LDL+LNT (+ox-LDL 100μg / ml+LNT 100μg / ml). NBD-cholesterol probe was used to detect the intervention cells to load them with excess cholesterol, and mature high-density lipoprotein (HDL) was added as a cholesterol efflux receptor. The amount of cholesterol effluxed by each group of cells to mature HDL was detected by enzyme-linked immunosorbent assay.

[0094] The results are as follows Fig.11 As shown, LNT increased the efflux of excess intracellular cholesterol to mature HDL compared with the ox-LDL group.

[0095] This example uses NBD-cholesterol probe (Thermo Fisher Scientific, N1148); HDL (Guangzhou Yiyuan Biotechnology Co., Ltd., YB-003).

[0096] Example 8

[0097] Since scavenger receptor B1 (SR-B1) is the main transporter for cholesterol efflux to mature HDL, and SR-B1 is a cell membrane protein, its stable expression on the cell membrane is the key to its function. Therefore, BMDM was used as experimental cells, and the experimental groups included ox-LDL group (+ox-LDL 100μg / ml) and ox-LDL+LNT (+ox-LDL 100μg / ml+LNT 100μg / ml).

[0098] Flow cytometry antibody SR-B1 was used to label the total protein and membrane protein of macrophages, and the total protein and the expression level of SR-B1 on the cell membrane of each group of cells were detected by flow cytometer.

[0099] The results are as follows Fig.12 As shown, LNT promoted the cell membrane translocation of SR-B1 compared with the ox-LDL group.

[0100] This example uses SR-B1 antibody (Novus, NB400-101).

[0101] Examples 7 and 8 illustrate that LNT can promote the efflux of excess intracellular cholesterol to mature HDL by increasing the translocation of SR-B1 to the cell membrane, thereby significantly inhibiting the deposition of lipid peroxides in macrophages.

[0102] Example 9

[0103] BMDM was used as experimental cells, and the experimental groups included Control group (+PBS), ox-LDL group (+ox-LDL 100μg / ml), Erastin group (+Erastin 10μM), ox-LDL+LNT group (+ox-LDL 100μg / ml+LNT 100μg / ml) and ox-LDL+Ferrostatin-1 group (+ox-LDL 100μg / ml+Ferrostatin-15μM). The cells were cultured in a 37℃ cell culture incubator for 24 hours, and the lipid ROS activity of cells in each group was detected using C11 BODIPY 581 / 591 lipid peroxidation fluorescent probe. The detection method was as follows: C11BODIPY was diluted with serum-free culture medium at a ratio of 1:1000 to a final concentration of 10μM; the cell culture medium was removed from the BMDM after intervention, and C11 BODIPY working solution was added, and incubated at 37℃ for 1 hour. The cells were washed twice with PBS to remove excess dye, then trypsinized and resuspended in PBS containing 5% FBS. Fluorescence signals were measured using flow cytometry.

[0104] The results are as follows Fig.13 As shown in the figure, compared with the Control group, the fluorescence signals in the ox-LDL group and Erastin group were significantly enhanced, indicating that ox-LDL and Erastin obviously caused the aggregation of lipid ROS in macrophages, while in the ox-LDL+LNT and ox-LDL+Ferrostatin-1 groups, LNT significantly reversed the phenomenon of lipid ROS aggregation, indicating that LNT can significantly inhibit macrophage ferroptosis.

[0105] This example uses C11 BODIPY 581 / 591 lipid peroxidation fluorescent probe (Thermo Fisher Scientific, D3861).

[0106] Example 10

[0107] BMDM was used as experimental cells, and the experimental groups included Control group (+PBS), ox-LDL group (+ox-LDL 100μg / ml), Erastin group (+Erastin 10μM), ox-LDL+LNT group (+ox-LDL 100μg / ml+LNT 100μg / ml) and ox-LDL+Ferrostatin-1 group (+ox-LDL 100μg / ml+Ferrostatin-15μM).

[0108] The MDA content detection method was used to detect the MDA content of macrophages in each group. The detection method is: macrophages were inoculated in a 96-well plate and placed in a 37°C incubator to allow the cells to adhere to the wall. After intervening the cells according to the experimental grouping, the steps of the MDA kit were followed, and the cell lysate was finally collected. The microplate reader was used to quantitatively detect the MDA in the cell lysate of each group, and the measurement wavelength was 532nm and 600nm. Since MDA is the final product of lipid peroxidation, MDA is an important sign of ferroptosis and a key indicator for ferroptosis detection.

[0109] like Fig.14 As shown in the figure, compared with the Control group, the MDA content in the ox-LDL and Erastin groups was higher, and LNT could significantly reverse the MDA content in macrophages, indicating that LNT could significantly inhibit macrophage ferroptosis.

[0110] This example uses the MDA detection kit (Abbkine, KTB1050).

[0111] Embodiment 11

[0112] When the cell cystine / glutamate antiporter is inhibited (such as the inhibitor Erastin), intracellular glutathione (GSH) will be depleted, while oxidized glutathione (GSSG) will increase, and the GSH / GSSG ratio will decrease, leading to the accumulation of lipid peroxidation, which can induce cell death when reaching a certain level. Therefore, GSH / GSSG is used as a detection indicator of ferroptosis.

[0113] BMDM was used as experimental cells, and the experimental groups included Control group (+PBS), ox-LDL group (+ox-LDL 100μg / ml), Erastin group (+Erastin 10μM), ox-LDL+LNT group (+ox-LDL 100μg / ml+LNT 100μg / ml) and ox-LDL+Ferrostatin-1 group (+ox-LDL 100μg / ml+Ferrostatin-15μM).

[0114] GSH and GSSG detection kits were used to detect the GSH / GSSG content of macrophages in each group. The cells after intervention were washed with PBS, centrifuged to collect the cells, and the supernatant was aspirated. Protein removal reagent M solution was added in an amount 3 times the volume of the cell pellet and fully oscillated. The samples were quickly frozen and thawed twice using liquid nitrogen and a 37°C water bath. Place in an ice bath for 5 minutes. Centrifuge at 4°C, 10,000g for 10 minutes. The supernatant was used for the determination of total glutathione. Take part of the samples prepared above for the total glutathione content to be measured, add diluted GSH scavenging auxiliary solution at a ratio of 20μl of diluted GSH scavenging auxiliary solution per 100μl sample, and oscillate to mix immediately. Then add GSH scavenging working solution at a ratio of 4μl of GSH scavenging reagent working solution per 100μl sample, oscillate to mix immediately, and react at 25°C for 60 minutes before using for the determination of GSSG.

[0115] like Fig.15 As shown, compared with the Control group, the GSH / GSSG content in the ox-LDL and Erastin groups was decreased, and LNT could significantly increase the GSH / GSSG content in macrophages.

[0116] This example uses a GSH and GSSG detection kit (Biyuntian Biotechnology Company, S0053).

[0117] In Examples 9, 10 and 11, ox-LDL and the ferroptosis inducer Erastin were used to induce macrophage ferroptosis. The lipid ROS content, MDA content and GSH / GSSG content in macrophages under the intervention of LNT were detected to further confirm the inhibitory effect of LNT on macrophage ferroptosis.

[0118] Example 12

[0119] BMDM was used as experimental cells, and the experimental groups included Control group (+PBS), ox-LDL group (+ox-LDL 100μg / ml), Erastin group (+Erastin 10μM), ox-LDL+LNT group (+ox-LDL 100μg / ml+LNT 100μg / ml) and ox-LDL+Ferrostatin-1 group (+ox-LDL 100μg / ml+Ferrostatin-15μM).

[0120] The mitochondrial membrane potential of macrophages in each group was detected using the mitochondrial membrane potential detection kit (JC-1). The detection method is as follows: collect the macrophages after intervention in a flow tube, add JC-1 staining working solution, and mix thoroughly. Incubate at 37°C in a cell culture incubator for 20 minutes. Prepare an appropriate amount of JC-1 staining buffer and place it in an ice bath. After the incubation at 37°C, remove the supernatant and wash twice with JC-1 staining buffer. Add cell culture medium to resuspend, and use flow cytometry to detect the ratio of JC-1 aggregates to monomers.

[0121] like Fig.16 As shown, the results of Figure (A) show that LNT can increase the ratio of JC-1 aggregates to monomers in macrophages, indicating that LNT can reverse the decrease of mitochondrial membrane potential in macrophages. Transmission electron microscopy was used to detect the morphology of macrophage mitochondria. The culture medium of the macrophages treated was discarded, and the electron microscopy fixative was added. The cells were fixed at room temperature and away from light for 5 minutes. The cells were gently scraped in one direction with a cell scraper and the cell fluid was sucked into a centrifuge tube with a Pasteur pipette. The tube was centrifuged at low speed for 3 minutes in a centrifuge. After discarding the fixative, new electron microscopy fixative was added, and the cell clusters were blown off and resuspended. The cells were fixed at room temperature and away from light for 30 minutes, then transferred to 4°C for storage, and then embedded, sliced ​​and observed. The results of Figure (B) show that the mitochondrial electron density in the ox-LDL and Erastin groups was significantly increased, and the outer membrane structure was incomplete; after LNT intervention, this phenomenon was improved. This shows that LNT improved macrophage mitochondrial damage, reduced endogenous ROS production, and thus partially inhibited ferroptosis.

[0122] The JC-1 detection kit used in this example is (Biyuntian Biotechnology Company, C2006).

[0123] Embodiment 13

[0124] BMDM was used as experimental cells, and the experimental groups included Control group (+PBS), LNT group (+LNT 100μg / ml), ox-LDL group (+ox-LDL 100μg / ml), and ox-LDL+LNT group (+ox-LDL 100μg / ml+LNT 100μg / ml). Cell samples were collected 24 hours after ox-LDL intervention, extracted with chloroform, precipitated with isopropanol, washed with 75% ethanol, and dissolved in DEPC water. The concentration was determined using an ultra-micro nucleic acid quantifier. Total RNA was reverse transcribed into cDNA using PrimeScriptTMRTreagent Kit with gDNA Eraser (RR047A, Takara, Japan). Real-time fluorescence quantitative PCR (qRT-PCR) was performed according to the operating instructions of Takara's TB Premix Ex TaqTMII (TliRNaseH Plus). The PCR operating conditions were: 95℃, 5min, 40 cycles, each cycle was 95℃, 10s, 60℃, 30s. 2 -△△ct The relative expression levels of IL-1β and TNF-α were calculated by the method.

[0125] The primers used to detect the expression of inflammatory factor genes are as follows:

[0126] IL-1β:5'-TACCACTTCACAAGTCGGAGGC-3';

[0127] IL-1β:3'-CTGCAAGTGCATCATCGTTGTTC-5'.

[0128] TNF-α:5'-GGTGCCTATGTCTCAGCCTCTT-3';

[0129] TNF-α:3'-GCCATAGAACTGATGAGAGGGAG-5'.

[0130] Results Fig.17 Compared with the Control group, the levels of inflammatory factors IL-1β and TNF-α in the ox-LDL group were significantly increased. After LNT intervention, the transcription levels of inflammatory factors decreased, indicating that LNT can significantly inhibit the inflammatory level of macrophages.

[0131] Embodiment 14

[0132] BMDM was used as experimental cells, and the experimental groups included ox-LDL group (+ox-LDL 100μg / ml) and ox-LDL+LNT (+ox-LDL 100μg / ml+LNT 100μg / ml) groups. The experimental group and the control group were cultured in a cell culture incubator at 37°C for 24 hours, and the cell RNA was collected by TRIzol method for RNA-seq sequencing. Genes with differential expression and a certain change trend in the group were screened for drawing volcano maps.

[0133] like Fig.18 As shown, the GSEA analysis results in (A) show that the ferroptosis pathway is significantly expressed; since the cystine / glutamate antiporter SLC7A11 (also known as xCT) is a key molecule for importing cystine for glutathione biosynthesis and antioxidant defense, its increased expression can significantly inhibit ferroptosis. The results in (B) show that Slc7a11 is one of the genes with the most significant changes in LNT-promoted macrophages. Therefore, the target gene of LNT in inhibiting macrophage ferroptosis may be Slc7a11.

[0134] Embodiment 15

[0135] BMDM was used as experimental cells, and the experimental groups included Control group (+PBS), ox-LDL group (+ox-LDL 100μg / ml), Erastin group (+Erastin 10μM), ox-LDL+LNT (+ox-LDL 100μg / ml+LNT 100μg / ml) and ox-LDL+Ferrostatin-1 group (+ox-LDL 100μg / ml+Ferrostatin-15μM). The experimental and control groups were cultured in six-well plates with 2ml of culture medium in each well and a cell density of 60%. After culturing in a cell culture incubator at 37°C for 24 hours, the total protein of the experimental and control cells was extracted for immunoblotting experiments.

[0136] like Fig.19 As shown, the results showed that compared with the Control, ox-LDL and Erastin decreased the protein expression level of Slc7a11; when LNT was added, the protein expression level of Slc7a11 was upregulated to a certain extent.

[0137] In the in vivo experiment, the experimental groups included a high fat diet group (HFD) and a high fat diet + LNT group (HFD+LNT), and the feeding and administration were the same as in Example 1. Immunofluorescence was used to detect the expression of Slc7a11 in macrophages in the aortic root plaque.

[0138] like Fig. 20As shown, the results showed that compared with the HFD group, the expression of Slc7a11 co-stained with the macrophage marker f4 / 80 in the plaques of the HFD+LNT group was increased. This proved that LNT upregulated the expression of Slc7a11 in macrophages in vivo and in vitro, thereby inhibiting the ferroptosis of macrophages.

[0139] In summary, the present invention first determines that APOE induced by a high-fat diet - / - In mice, intervention with LNT can effectively improve hyperlipidemia, significantly reduce lipid accumulation in the mouse aorta, and significantly inhibit the occurrence and development of atherosclerosis. Secondly, it was found that LNT promoted SR-B1 cell membrane translocation, increased intracellular lipid peroxide efflux, and inhibited the level of inflammatory factors. At the same time, it upregulated the expression of Slc7a11, reduced iron deposition in macrophages, improved mitochondrial function, inhibited the production of lipid ROS, and inhibited the intracellular MDA content, thereby inhibiting macrophage ferroptosis to prevent and treat atherosclerosis.

[0140] The final study of the present invention found that LNT can significantly inhibit the production of lipid ROS in macrophages and the intracellular MDA content by upregulating the Slc7a11 gene, thereby inhibiting macrophage ferroptosis and exerting an anti-atherosclerotic effect, and can be used as a drug for clinical prevention, relief and / or treatment. In addition, LNT can bind to the dectin-1 receptor specifically expressed on activated myeloid cells, increasing the specificity of LNT targeting macrophages, thereby reducing the side effects of drug off-target. LNT is also a natural polymer material that not only exhibits biological activity, but also can self-assemble to form a series of supramolecular bioactive materials, such as nanoparticles, micelles, gels, etc. This makes LNT significantly surpass in function and biological activity, including enhancing solubility and stability, penetrating biological barriers, achieving sustained release and intelligent response release, synergy and reduced toxicity, etc. LNT has great potential in clinical application and transformation.

[0141] Obviously, the above embodiments of the present invention are merely examples for clearly illustrating the present invention, and are not intended to limit the implementation methods of the present invention. For ordinary technical users in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. Application of Lentinan in the preparation of macrophage ferroptosis inhibitors.

2. The use according to claim 1, characterized in that: The macrophages include bone marrow-derived macrophages.

3. A macrophage ferroptosis inhibitor, characterized in that Including Lentinan.

4. The macrophage ferroptosis inhibitor according to claim 3, characterized in that Pharmaceutically acceptable excipients are also included.

5. The macrophage ferroptosis inhibitor according to claim 3, characterized in that The dosage form is liquid dosage form, solid dosage form or paste dosage form.

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