Application of COPG1 protein in preparation of medicine for promoting M2 polarization of macrophages

Abstract

The invention discloses application of COPG1 protein in preparation of drugs for promoting M2 polarization of macrophages, and belongs to the technical field of biological medicines. The invention proposes that COPG1 can promote M2 polarization of macrophages and reduce the M2 polarization effect of COPG1 in tumor-derived exosomes on inhibition of macrophages for the first time. The strategy for regulating the COPG1 content in the macrophage and the exosome, inhibiting the M2 polarization of the macrophage and regulating the immune function of the macrophage provides a theoretical and practical basis for developing an immunomodulator through targeted regulation of the COPG1.

Description

Technical Field

[0001] The present invention relates to the field of biomedical technology, and in particular to application of COPG1 protein in preparing a drug for promoting macrophage M2 polarization. Background Art

[0002] Macrophages are immune cells that play a key role in regulating inflammatory responses, promoting tissue damage repair, and maintaining dynamic homeostasis. They recognize pathogen-associated molecules and recognize damaged cells through pattern recognition receptors, thereby exerting immunomodulatory effects to clear pathogens, inhaled allergens, and reduce infections. However, improper activation of macrophages or abnormal inflammatory responses may disrupt their homeostatic functions, leading to uncontrollable inflammatory responses, triggering persistent tissue and organ damage, and are associated with the development of a variety of chronic diseases, including malignant tumors, inflammatory diseases, metabolic diseases, and infectious diseases. In response to this abnormal immune microenvironment, a variety of macrophage-related immunotherapies have been developed in recent years, such as regulating macrophage polarization.

[0003] Macrophage polarization refers to the process in which mature macrophages produce different functional phenotypes under the stimulation of a special microenvironment. These different phenotypes mainly include M1 and M2. M1 macrophages are inflammatory and antibacterial, while M2 exhibits anti-inflammatory and repair properties. Macrophage M2 polarization plays an important role in the occurrence and development of diseases. M2 macrophages have a regulatory effect on a variety of diseases. For example, M2 macrophages affect autoimmune and inflammatory diseases by regulating anti-inflammatory factors, and can provide tumor cells with a microenvironment that is conducive to their growth and spread by secreting various growth factors, angiogenic factors, etc. In addition, M2 macrophages can also affect the occurrence and development of diseases through interactions with other immune cells.

[0004] The protein encoding the gamma 1 subunit of the COPI complex (COPG1) is a key subunit of the COPI complex, which participates in the vesicle transport process and plays an important role in the transport of cellular substances and the maintenance of cell homeostasis. COPG1 can be secreted into the extracellular space through exosomes and delivered to immune cells in the microenvironment, such as macrophages, to regulate their functions, which in turn affects the occurrence and development of diseases. At present, the association and specific mechanism between COPG1 and the polarization state of macrophages have not been clarified, and there has been no report on the strategy of regulating the function of macrophages by regulating COPG1 to reshape the polarization state of macrophages. Summary of the invention

[0005] The purpose of the present invention is to provide the use of COPG1 protein in the preparation of a drug for promoting macrophage M2 polarization, so as to solve the problems existing in the above-mentioned prior art.

[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 COPG1 protein in the preparation of a drug for regulating macrophage M2 polarization.

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

[0009] The third technical solution of the present invention is the use of a substance that regulates the content of COPG1 protein in the preparation of a drug that regulates macrophage polarization.

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

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

[0012] The present invention found that COPG1 can promote the M2 polarization of THP-1-derived M0 macrophages. A large number of experimental results show that COPG1 protein promotes the M2 polarization of THP-1-derived M0 macrophages, which are morphologically more inclined to M2 macrophages, increase the expression of M2 macrophage-related genes CD206, CD163, and TGM2 at the gene level, and increase the expression of M2 macrophage-related proteins CD206 and TGM2 at the protein level. COPG1 expression exists in the exosomes secreted by non-small cell lung cancer cells (NCI-H460 and NCI-H1299), which can promote the M2 polarization of macrophages; when COPG1 is knocked out in non-small cell lung cancer cells, the exosomes produced can inhibit the polarization of M2, which is manifested as a decrease in morphological M2 macrophages, and a decrease in the expression of M2 macrophage-related genes CD206, CD163, TGM2 and related proteins CD206 and TGM2. The present invention proposes for the first time that COPG1 can promote the M2 polarization of macrophages, and reducing COPG1 in tumor-derived exosomes can inhibit the M2 polarization of macrophages. The strategy proposed in the present invention to regulate the content of COPG1 in macrophages and exosomes, inhibit the M2 polarization of macrophages and regulate their immune function provides a theoretical and practical basis for the development of immunomodulators by targeted regulation of COPG1. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] 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.

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

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

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

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

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

[0019] Figure 6 This is a schematic diagram of the effect of lung cancer exosomes with COPG1 knockout on the gene levels of M2 markers in THP-1-derived M0 macrophages in Example 6. 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, the changes in the mRNA of M2 markers TGM2 (A), CD206 (B), and CD163 (C) were observed, and M2 macrophages were used as controls.

[0020] Figure 7 This is a schematic diagram of the effective ingredients of Ginkgo biloba flavonoids reducing the expression of COPG1 in lung cancer-derived exosomes in Example 7. A shows the changes in COPG1 in exosomes secreted by Ginkgo biloba flavonoids (GK) and ISO-Ginkgo biloba flavonoids (ISO) after administration to non-small cell lung cancer cells NCI-H460, and B shows the changes in COPG1 in exosomes secreted by Ginkgo biloba flavonoids and ISO-Ginkgo biloba flavonoids after administration to non-small cell lung cancer cells NCI-H1299.

[0021] Figure 8 This is a schematic diagram of Example 8 in which the effective ingredients of ginkgo biloba flavonoids reduce the expression of COPG1 in M2 macrophages. DETAILED DESCRIPTION

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

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

[0027] 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.

[0028] The embodiment of the present invention provides the use of COPG1 protein in preparing a drug for regulating macrophage M2 polarization.

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

[0030] The embodiment of the present invention also provides a drug for regulating macrophage M2 polarization, including COPG1 protein.

[0031] The embodiments of the present invention also provide the use of a substance for regulating the content of COPG1 protein in the preparation of a drug for regulating macrophage polarization.

[0032] In some specific embodiments, the substance that regulates the COPG1 protein content includes ginkgo biflavonoids.

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

[0034] The embodiment of the present invention further provides a drug for regulating macrophage polarization, wherein the drug includes a substance for regulating the content of COPG1 protein.

[0035] In some specific embodiments, the substance that regulates the COPG1 protein content includes ginkgo biflavonoids.

[0036] Example 1

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

[0038] 1 million THP-1 cells were inoculated in 6 cm culture dishes and cultured with 1640 complete medium. PMA 100 ng / ml was added and induced into M0 macrophages after 24 hours. IL-4 25 ng / ml and IL-16 25 ng / ml were added to M0 macrophages to induce M2 macrophages for 72 hours. COPG1 protein of 100 ng / ml, 200 ng / ml and 300 ng / ml was added to the experimental groups respectively. After culturing for 48 hours, the cells were collected and lysed. After protein quantification by BCA method, the expression of M2 macrophage polarization-related proteins CD206 and TGM2 was determined by western blot.

[0039] The results of the comparison can be found in 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 the M2 polarization of THP-1-derived M0 macrophages at the genetic level.

[0042] 500,000 THP-1 cells were inoculated in each well of a 6-well plate and cultured with 1640 complete medium. PMA 100 ng / ml was added and induced into M0 macrophages after 24 hours. IL-4 25 ng / ml and IL-16 25 ng / ml were added to M0 macrophages to induce M2 macrophages for 72 hours. 100 ng / ml, 200 ng / ml, and 300 ng / ml of COPG1 protein were added to the experimental groups, respectively, and cultured for 48 hours. The cells were collected and lysed, and the intracellular RNA was extracted and reverse transcribed into cDNA. The expression of M2 macrophage polarization-related genes CD206, TGM2, and CD163 was determined by qPCR.

[0043] The results of the comparison can be found in Figure 2 : The results showed that COPG1 protein gradient-dependently increased the mRNA levels of M2 polarization markers in THP-1-derived M0 macrophages.

[0044] Example 3

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

[0046] 500,000 THP-1 cells were inoculated into 6-well plates in each well and cultured with 1640 complete medium. PMA 100 ng / ml was added and induced into M0 macrophages after 24 hours. IL-4 25 ng / ml and IL-16 25 ng / ml were added to M0 macrophages to induce M2 macrophages for 72 hours. COPG1 protein of 100 ng / ml, 200 ng / ml and 300 ng / ml were added to the experimental groups respectively and cultured for 48 hours. After washing with 1× PBS, fresh medium was added and cell morphology was observed under a microscope.

[0047] The results of the comparison can be found in Figure 3 The results showed that COPG1 protein promoted THP-1-derived M0 macrophages to present an M2 polarized morphology.

[0048] Example 4

[0049] Exosomes secreted by COPG1-deficient non-small cell lung cancer cells 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 by Crispr-Cas9 method. When the density of knockout cell lines or non-knockout cell lines reached 80%-90%, serum-free medium was replaced and cultured for 72 hours. After 72 hours, the cell culture medium was collected, centrifuged at 300g for 10 minutes at 4°C to remove cells, centrifuged at 2000g for 10 minutes at 4°C to remove cell debris, and centrifuged at 10000g for 30 minutes at 4°C to remove apoptotic bodies and membrane particles. 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 resuspended in 100μl 1×PBS. THP-1 derived macrophages were induced into M0 macrophages by adding PMA 100 ng / ml for 24 hours, and IL-4 25 ng / ml and IL-16 25 ng / ml were added to M0 macrophages for 72 hours to induce M2 macrophages. M0 macrophages were induced with or without COPG1 knockout lung cancer exosomes for 48 hours, and the cells were collected and lysed. After protein quantification by BCA method, the expression of M2 macrophage polarization-related proteins CD206 and TGM2 was determined by western blot.

[0051] The comparison results are shown in the attached Figure 4The results showed that compared with THP-1-derived M0 macrophages intervened with lung cancer-derived exosomes without COPG1 knockout, lung cancer-derived exosomes with COPG1 knockout intervened in THP-1-derived M0 macrophages and inhibited the protein levels of M2 polarization markers.

[0052] Example 5

[0053] Lung cancer exosomes derived from COPG1 knockout inhibit the morphology of THP-1-derived M0 macrophages toward M2 macrophages.

[0054] THP-1 derived macrophages were induced into M0 macrophages by adding PMA 100ng / ml for 24 hours. M0 macrophages were induced into M2 macrophages by adding IL-4 25ng / ml and IL-16 25ng / ml for 72 hours. M0 macrophages were induced with or without COPG1 knockout exosomes from lung cancer for 48 hours, washed with 1×PBS, added with fresh culture medium, and observed cell morphology under a microscope.

[0055] The comparison results are shown in the attached Figure 5 The results showed that compared with THP-1-derived M0 macrophages intervened by lung cancer-derived exosomes without COPG1 knockout, the number of cells with M2 macrophage morphology was morphologically observed to be reduced after THP-1-derived M0 macrophages were intervened by lung cancer-derived exosomes with COPG1 knockout.

[0056] Example 6

[0057] Lung cancer-derived exosomes with COPG1 knockout reduced the mRNA expression of M2 polarization markers in THP-1-derived M0 macrophages.

[0058] THP-1 derived macrophages were induced into M0 macrophages by adding PMA 100ng / ml for 24 hours, the negative control group was M0 macrophages, and the positive control group was induced into M2 macrophages by adding IL-4 25ng / ml and IL-16 25ng / ml on the basis of M0 macrophages for 72 hours. M0 macrophages were added with lung cancer exosomes with or without COPG1 knockout, induced for 48 hours, and the cells were collected and lysed, and the intracellular RNA was extracted and reverse transcribed into cDNA. The mRNA levels of M2 macrophage polarization-related genes CD206, TGM2, and CD163 were determined by qPCR.

[0059] The results of the comparison can be found in Figure 6The results showed that compared with THP-1-derived M0 macrophages intervened with lung cancer-derived exosomes without COPG1 knockout, lung cancer-derived exosomes with COPG1 knockout intervened in THP-1-derived M0 macrophages and reduced the mRNA expression of M2 polarization markers.

[0060] Example 7

[0061] Ginkgo biflavonoids active ingredients reduce COPG1 expression in lung cancer-derived exosomes

[0062] When the cell density of NCI-H1299 and NCI-H460 reached 80%-90%, serum-free medium was replaced, and Ginkgo biloba (GK) or ISO (ISO) was added for 72 hours. The lung cancer exosomes were extracted by ultra-high speed centrifugation, and the purified exosomes were resuspended in 100μl 1×PBS. The exosomes were lysed, and the protein expression of COPG1 in the exosomes was determined by western blot after protein quantification by BCA method. The results showed that GK could inhibit the content of COPG1 in lung cancer exosomes.

[0063] Example 8

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

[0065] THP-1 derived macrophages were induced into M0 macrophages by adding PMA 100ng / ml for 24 hours. M0 macrophages were induced into M2 macrophages by adding IL-4 25ng / ml and IL-16 25ng / ml for 72 hours. M2 macrophages were treated with Ginkgo biloba. Western blot was used to detect the expression of COPG1 in cells of each treatment group. The results showed that compared with M0 macrophages, the COPG1 content of M2 macrophages increased, while the macrophage M2 marker CD206 increased significantly, and the M1 marker CD86 decreased significantly, indicating that COPG1 in macrophages was positively correlated with M2 polarization. After GK was administered, GK could significantly reduce the expression of COPG1 in M2 macrophages, inhibit the expression of M2 macrophage marker CD206, and increase the expression of M1 macrophage CD86, indicating that GK reshapes the transformation of macrophages from M2 to M1 while inhibiting COPG1.

[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 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 COPG1 protein in the preparation of drugs for regulating macrophage M2 polarization.

2. A drug for regulating macrophage M2 polarization, characterized in that: Including COPG1 protein.

3. Application of substances that regulate COPG1 protein content in the preparation of drugs that regulate macrophage polarization.

4. The use according to claim 3, characterized in that: The substance that regulates the content of COPG1 protein includes ginkgo biflavonoids.

5. The use according to claim 3, characterized in that: Increasing the COPG1 protein content promotes macrophage M2 polarization; decreasing the COPG1 protein content inhibits macrophage M2 polarization.

6. A drug for regulating macrophage polarization, characterized in that: The medicine includes a substance that regulates the content of COPG1 protein.

7. The drug according to claim 6, characterized in that The substance that regulates the content of COPG1 protein includes ginkgo biflavonoids.

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