Use of copg1 protein in preparation of a drug for promoting m2 polarization of macrophages

Abstract

The application discloses application of COPG1 protein in preparation of a drug for promoting M2 polarization of macrophages, and belongs to the technical field of biological medicines.The application firstly proposes that COPG1 can promote M2 polarization of macrophages, and that COPG1 in tumor-derived exosomes can inhibit the M2 polarization of macrophages.The strategy of regulating the COPG1 content in macrophages and exosomes and inhibiting the M2 polarization of macrophages to regulate the immune function of macrophages proposed by the application provides a theoretical and practical basis for developing an immune regulator by targeting COPG1.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of COPG1 protein in the preparation of drugs that promote M2 polarization of macrophages. Background Technology

[0002] Macrophages are immune cells that play a crucial role in regulating inflammatory responses, promoting tissue repair, and maintaining dynamic homeostasis. They recognize pathogen-associated molecules and identify damaged cells through pattern recognition receptors, thereby exerting immunomodulatory effects to clear pathogens, inhale allergens, and reduce infection. However, inappropriate activation or abnormal inflammatory responses of macrophages can disrupt their homeostatic function, leading to uncontrolled inflammatory responses, persistent tissue and organ damage, and are associated with the development of various chronic diseases, including malignancies, inflammatory diseases, metabolic diseases, and infectious diseases. In response to this abnormal immune microenvironment, several macrophage-related immunotherapies have been developed in recent years, such as those targeting macrophage polarization.

[0003] Macrophage polarization refers to the process by which mature macrophages develop different functional phenotypes under specific microenvironmental stimuli. These different phenotypes mainly include M1 and M2 types. M1 macrophages exhibit inflammatory and antibacterial properties, while M2 macrophages display anti-inflammatory and repair characteristics. Macrophage M2 polarization plays an important role in the occurrence and development of diseases. M2 macrophages have regulatory effects on various diseases. For example, M2 macrophages influence autoimmunity and inflammatory diseases by regulating anti-inflammatory factors, and they can also provide a favorable microenvironment for tumor cell growth and spread by secreting various growth factors and angiogenesis factors. In addition, M2 macrophages can also affect the occurrence and development of diseases through interactions with other immune cells.

[0004] The γ1 subunit encoding the coating protein complex (COPG1) is a key subunit of the COPI coating protein complex, involved in vesicle transport and playing a crucial role in cellular material transport and homeostasis. COPG1 can be secreted extracellularly via exosomes and delivered to immune cells in the microenvironment, such as macrophages, regulating their function and, in turn, influencing disease development. Currently, the association and specific mechanisms between COPG1 and macrophage polarization state remain unclear, and strategies for regulating macrophage function by modulating COPG1 to reshape macrophage polarization state have not been reported. Summary of the Invention

[0005] The purpose of this invention is to provide the application of COPG1 protein in the preparation of drugs that promote M2 polarization of macrophages, so as to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] One of the technical solutions of this invention is the application of COPG1 protein in the preparation of drugs that regulate M2 polarization of macrophages.

[0008] The second technical solution of the present invention is a drug for inhibiting macrophage M2 polarization, including inhibition of COPG1 protein.

[0009] The third technical solution of this invention is the application of substances that regulate COPG1 protein content in the preparation of drugs that regulate macrophage polarization.

[0010] The fourth technical solution of the present invention is a drug for regulating macrophage polarization, wherein the drug includes a substance that regulates the content of COPG1 protein in exosomes.

[0011] Based on the above technical solution, the present invention has the following technical effects:

[0012] This invention reveals that COPG1 can promote M2 polarization in THP-1-derived M0 macrophages. Extensive experimental results show that COPG1 protein promotes M2 polarization in THP-1-derived M0 macrophages, making them morphologically more M2-type macrophages. At the gene level, it increases the expression of M2 macrophage-related genes CD206, CD163, and TGM2, and at the protein level, it increases the expression of M2 macrophage-related proteins CD206 and TGM2. COPG1 is expressed in exosomes secreted by non-small cell lung cancer cells (NCI-H460 and NCI-H1299), promoting M2 polarization in macrophages. When COPG1 is knocked out in non-small cell lung cancer cells, the resulting exosomes inhibit M2 polarization, manifested as a reduction in morphologically M2-type macrophages and decreased expression of M2 macrophage-related genes CD206, CD163, and TGM2, as well as related proteins CD206 and TGM2. This invention is the first to propose that COPG1 can promote M2 polarization in macrophages, and that reducing COPG1 in tumor-derived exosomes can inhibit M2 polarization in macrophages. The strategy proposed in this invention, which regulates COPG1 levels in macrophages and exosomes and inhibits macrophage M2 polarization to modulate their immune function, provides a theoretical and practical basis for developing immunomodulators that target COPG1. Attached Figure Description

[0013] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0014] Figure 1This is a schematic diagram showing the effect of COPG1 protein on the level of polarization marker proteins in THP-1-derived M0 macrophages in Example 1.

[0015] Figure 2 This is a schematic diagram illustrating the effect of COPG1 protein on the gene levels of polarization markers in THP-1-derived M0 macrophages in Example 2. In the diagram, A represents the change in the M2 marker TGM2, B represents the change in the M2 marker CD206, and C represents the change in the M2 marker CD163.

[0016] Figure 3 This is a schematic diagram showing the effect of COPG1 protein on the polarization morphology of THP-1-derived M0 macrophages in Example 3.

[0017] Figure 4 This is a schematic diagram illustrating the effect of COPG1 knockout in lung cancer-derived exosomes on the protein levels of M2 polarization markers in THP-1-derived M0 macrophages, as described in Example 4. A shows the changes in COPG1 levels in NCI-H1299 non-small cell lung cancer cells with and without COPG1 knockout; B shows the changes in COPG1 levels in NCI-H460 non-small cell lung cancer cells with and without COPG1 knockout; C shows the expression of COPG1 in exosomes secreted by NCI-H1299 non-small cell lung cancer cells with and without COPG1 knockout; and D shows the exosomes secreted by NCI-H460 non-small cell lung cancer cells with and without COPG1 knockout. E shows the changes in M2 markers CD206 and TGM2 after exosomes secreted by NCI-H1299 non-small cell lung cancer cells with and without COPG1 knockout were administered to M0 macrophages, with M2 macrophages serving as a control. F shows the changes in M2 markers CD206 and TGM2 after exosomes secreted by NCI-H460 non-small cell lung cancer cells with and without COPG1 knockout were administered to M0 macrophages, with M2 macrophages serving as a control.

[0018] Figure 5 This is a schematic diagram illustrating the effect of COPG1 knockout on lung cancer-derived exosomes in Example 5 on the polarization morphology of THP-1-derived M0 macrophages.

[0019] Figure 6 This is a schematic diagram illustrating the effect of COPG1 knockout lung cancer-derived exosomes on the gene levels of M2 markers in THP-1-derived M0 macrophages, as shown in Example 6. The diagram shows the changes in mRNA levels of M2 markers TGM2 (A), CD206 (B), and CD163 (C) after exosomes secreted by non-small cell lung cancer cells NCI-H1299 and NCI-H460 (with and without COPG1 knockout) were administered to M0 macrophages. M2 macrophages served as a control.

[0020] Figure 7 This is a schematic diagram illustrating the reduction of COPG1 expression in lung cancer-derived exosomes by ginkgo biloba extract, the active ingredient in Example 7. In this diagram, A shows the changes in COPG1 expression in exosomes secreted by NCI-H460 non-small cell lung cancer cells after treatment with ginkgo biloba extract (GK) and isoginkgo biloba extract (ISO), and B shows the changes in COPG1 expression in exosomes secreted by NCI-H1299 non-small cell lung cancer cells after treatment with ginkgo biloba extract (GK) and isoginkgo biloba extract (ISO).

[0021] Figure 8 This is a schematic diagram illustrating how the active ingredients of Ginkgo biloba flavonoids reduce COPG1 expression in M2 macrophages, as shown in Example 8. Detailed Implementation

[0022] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0023] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0024] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0025] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be obvious to those skilled in the art. This application specification and embodiments are merely exemplary.

[0026] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0027] Unless otherwise specified, the technical solutions described in this invention are all conventional solutions in the field, and the reagents or raw materials used are all purchased from commercial channels or are publicly available unless otherwise specified.

[0028] This invention provides the application of COPG1 protein in the preparation of drugs that regulate M2 polarization of macrophages.

[0029] COPG1 protein encodes the γ1 subunit of the coating protein complex and promotes M2 polarization in THP-1-derived M0 macrophages. Exosomes extracted from non-small cell lung cancer cells (NCI-H460 and NCI-H1299) with COPG1 knockout inhibited M2 polarization in THP-1-derived M0 macrophages.

[0030] This invention also provides a drug for regulating macrophage M2 polarization, including COPG1 protein.

[0031] This invention also provides the application of substances that regulate COPG1 protein content in the preparation of drugs that regulate macrophage polarization.

[0032] In some specific implementations, the substances that regulate COPG1 protein content include ginkgo biflavonoids.

[0033] In some specific implementation schemes, increasing COPG1 protein content promotes macrophage M2 polarization; decreasing COPG1 protein content inhibits macrophage M2 polarization.

[0034] This invention also provides a drug for regulating macrophage polarization, wherein the drug includes a substance that regulates the content of COPG1 protein.

[0035] In some specific implementations, the substances that regulate COPG1 protein content include ginkgo biflavonoids.

[0036] Example 1

[0037] COPG1 protein promotes M2 polarization in THP-1-derived M0 macrophages at the protein level.

[0038] One million THP-1 cells were seeded in 6 cm culture dishes and cultured in 1640 complete medium. PMA 100 ng / ml was added, and after 24 hours, M0 macrophages were induced. M2 macrophages were induced by adding IL-425 ng / ml and IL-1625 ng / ml to the M0 macrophages for 72 hours. COPG1 protein was added to the experimental groups at concentrations of 100 ng / ml, 200 ng / ml, and 300 ng / ml, respectively. After 48 hours of culture, cells were collected and lysed. Protein quantification was performed using the BCA method, and the expression of polarization-related proteins CD206 and TGM2 in M2 macrophages was determined by Western blot.

[0039] See comparison results Figure 1 The results showed that COPG1 protein concentration-dependently increased the protein levels of M2 polarization markers in THP-1-derived M0 macrophages.

[0040] Example 2

[0041] COPG1 protein promotes M2 polarization in THP-1-derived M0 macrophages at the gene level.

[0042] 500,000 THP-1 cells were seeded into each well of a 6-well plate and cultured in 1640 complete medium. PMA 100 ng / ml was added, and after 24 hours, M0 macrophages were induced. M2 macrophages were induced by adding IL-425 ng / ml and IL-1625 ng / ml to the M0 macrophages for 72 hours. COPG1 protein was added to the experimental groups at concentrations of 100 ng / ml, 200 ng / ml, and 300 ng / ml, respectively, and cultured for 48 hours. Cells were collected, lysed, and intracellular RNA was extracted and reverse transcribed into cDNA. The expression of polarization-related genes CD206, TGM2, and CD163 in M2 macrophages was measured using qPCR.

[0043] See comparison results Figure 2 The results showed that COPG1 protein increased the mRNA levels of M2 polarization markers in THP-1-derived M0 macrophages in a gradient-dependent manner.

[0044] Example 3

[0045] COPG1 protein promotes the morphology of THP-1-derived M0 macrophages to tend towards the M2 polarized morphology.

[0046] 500,000 THP-1 cells were seeded into each well of a 6-well plate and cultured in 1640 complete medium. PMA 100 ng / ml was added, and after 24 hours, the cells were induced to become M0 macrophages. M0 macrophages were then induced to become M2 macrophages by adding IL-425 ng / ml and IL-1625 ng / ml, respectively, for 72 hours. The experimental groups were then treated with COPG1 protein at concentrations of 100 ng / ml, 200 ng / ml, and 300 ng / ml, respectively, and cultured for 48 hours. After washing once with 1×PBS, fresh medium was added, and cell morphology was observed under a microscope.

[0047] See comparison results Figure 3 The results showed that COPG1 protein promoted the M2 polarization morphology of THP-1-derived M0 macrophages.

[0048] Example 4

[0049] Exosomes secreted by non-small cell lung cancer cells that knock out COPG1 reduce the protein expression of M2 polarization markers in THP-1-derived M0 macrophages.

[0050] COPG1 knockout cell lines of NCI-H460 and NCI-H1299 were constructed using the CRISPR-Cas9 method. When the density of the knockout cell line or the non-knockout cell line reached 80%-90%, they were cultured in serum-free medium for 72 hours. After 72 hours, the cell culture medium was collected, and the cells were removed by centrifugation at 300g for 10 minutes at 4°C, followed by centrifugation at 2000g for 10 minutes at 4°C to remove cell debris, and centrifugation at 10000g for 30 minutes at 4°C to remove apoptotic bodies and membrane microparticles. The precipitate was discarded, and the supernatant was ultracentrifuged at 100000g for 1 hour at 4°C to extract exosomes. The exosomes were resuspended in 1×PBS and purified by ultracentrifugation at 100000g for 1 hour at 4°C. The purified exosomes were then resuspended in 100 μl of 1×PBS. THP-1-derived macrophages were induced into M0 macrophages by adding 100 ng / ml PMA for 24 hours. M2 macrophages were then induced by adding 25 ng / ml IL-4 and 25 ng / ml IL-1 for 72 hours. M0 macrophages were then treated with lung cancer-derived exosomes with or without COPG1 knockout for 48 hours, followed by cell collection and lysis. Protein quantification was performed using the BCA method, and the expression of polarization-related proteins CD206 and TGM2 in M2 macrophages was determined by Western blot.

[0051] See the appendix for comparison results. Figure 4The results showed that, compared with THP-1-derived M0 macrophages treated with lung cancer-derived exosomes without COPG1 knockout, intervention with lung cancer-derived M0 macrophages with COPG1 knockout suppressed the protein levels of M2 polarization markers.

[0052] Example 5

[0053] Knockout of COPG1 in lung cancer-derived exosomes inhibits the morphological tendency of THP-1-derived M0 macrophages to become M2 macrophages.

[0054] THP-1-derived macrophages were induced to M0 type macrophages by adding PMA 100 ng / ml for 24 hours. M2 type macrophages were induced by adding IL-4 25 ng / ml and IL-1 625 ng / ml to M0 type macrophages for 72 hours. M0 type macrophages were then treated with lung cancer-derived exosomes with or without COPG1 knockout for 48 hours. After washing once with 1×PBS, fresh culture medium was added, and cell morphology was observed under a microscope.

[0055] See the appendix for comparison results. Figure 5 The results showed that compared with THP-1-derived M0 macrophages treated with lung cancer-derived exosomes without COPG1 knockout, fewer cells with M2 macrophage morphology were observed after THP-1-derived M0 macrophages treated with lung cancer-derived exosomes with COPG1 knockout.

[0056] Example 6

[0057] Knocking out COPG1 in lung cancer-derived exosomes reduces the mRNA expression of M2 polarization markers in THP-1-derived M0 macrophages.

[0058] THP-1-derived macrophages were induced to M0 type macrophages by adding 100 ng / ml PMA for 24 hours. The negative control group consisted of M0 type macrophages. The positive control group consisted of M0 type macrophages induced to M2 type macrophages by adding IL-425 ng / ml and IL-1625 ng / ml for 72 hours. M0 type macrophages were treated with lung cancer-derived exosomes with or without COPG1 knockout for 48 hours. Cells were collected, lysed, and intracellular RNA was extracted and reverse transcribed into cDNA. The mRNA levels of polarization-related genes CD206, TGM2, and CD163 in M2 type macrophages were measured using qPCR.

[0059] See comparison results Figure 6The results showed that compared with THP-1-derived M0 macrophages treated with lung cancer-derived exosomes without COPG1 knockout, intervention with lung cancer-derived M0 macrophages with COPG1 knockout reduced the mRNA expression of M2 polarization markers.

[0060] Example 7

[0061] Ginkgo biloba flavonoids reduce COPG1 expression in lung cancer-derived exosomes.

[0062] Once the cell densities of NCI-H1299 and NCI-H460 cells reached 80%-90%, the culture medium was replaced with serum-free medium, and ginkgo biloba extract (GK) or isoginkgo biloba extract (ISO) was added. After culturing for 72 hours, lung cancer-derived exosomes were extracted by ultracentrifugation, with or without the drug-treated cells. The purified exosomes were then resuspended in 100 μl of 1×PBS. The exosomes were lysed, and after BCA protein quantification, the expression level of COPG1 in the exosomes was determined by Western blot. The results showed that GK could inhibit the COPG1 content in lung cancer-derived exosomes.

[0063] Example 8

[0064] Ginkgo biloba flavonoids reduce COPG1 expression in M2 macrophages.

[0065] THP-1-derived macrophages were induced to M0 type macrophages by adding PMA 100 ng / ml for 24 hours. M0 macrophages were then induced to M2 type macrophages by adding IL-4 25 ng / ml and IL-1 625 ng / ml for 72 hours. M2 macrophages were then treated with Ginkgo biloba extract. Western blot analysis was performed on COPG1 expression in each treatment group. The results showed that compared to M0 macrophages, M2 macrophages exhibited increased COPG1 levels, along with a significant increase in the M2 macrophage marker CD206 and a significant decrease in the M1 macrophage marker CD86, indicating a positive correlation between COPG1 and M2 polarization in macrophages. GK administration significantly reduced COPG1 expression in M2 macrophages, inhibited the expression of the M2 macrophage marker CD206, and increased the expression of CD86 in M1 macrophages, indicating that GK, while inhibiting COPG1, remodeled macrophages from M2 to M1 type.

[0066] 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 of the present invention. For those skilled in the art, other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively describe all embodiments here. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for inducing M0 macrophages into M2 macrophages in vitro, characterized in that, This includes the step of co-culturing M0 macrophages with COPG1 protein.

2. The method according to claim 1, characterized in that, The M0 macrophages are THP-1-derived M0 macrophages; the co-culture time is 48 hours, and the concentration of COPG1 protein is 100~400 ng / ml.

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