Application of secondary bile acid in preparation of inhibitor for killing activity of NK (Natural Killer) cells

Abstract

The invention relates to the technical field of biological medicine, and particularly discloses application of secondary bile acid in preparation of an inhibitor for NK cell killing activity. According to the application, mouse NK cells are isolated, cultured and activated in vitro, and the result of an in-vitro experiment shows that the secondary bile acid can inhibit the killing effect of the NK cells, so that the secondary bile acid has an immunosuppressive effect on the NK cells; in addition, spirolactone or progesterone can reverse the immunosuppression effect of iso-LCA on the NK cells, and the activity and the effector function of the NK cells can be regulated and controlled.

Description

Technical Field

[0001] The present application relates to the field of biomedicine technology, and in particular to the use of a secondary bile acid in the preparation of an inhibitor of NK cell killing activity. Background Art

[0002] There are different immune cell subsets in the tumor microenvironment, such as NK, NKT, CD8 + T, TAM and TAM, etc., these cells play an important role in tumor clearance or progression. NK cells (natural killer cells) are of great significance in the process of immune surveillance and tumor clearance. When tumor antigens are exposed, they can secrete cytotoxic cytokines such as interferon-γ, tumor necrosis factor-α, perforin and granulamycin to inhibit tumor cells. Compared with tissues in non-tumor areas, the proportion of NK cells in tumor-infiltrated areas drops sharply, and a lower proportion of NK cell infiltration is also correlated with the prognosis of patients with tumor-related diseases. In the tumor microenvironment, the cytotoxicity and cytokine secretion of NK cells are significantly inhibited. In addition, regulatory T cells, stromal cells and MDSC cells can also secrete immunosuppressive cytokines to inhibit the anti-tumor immune response of NK cells. NK cells are continuously exposed to immunosuppressive factors in the tumor microenvironment, such as immunosuppressive molecules, hypoxia, metabolic intermediates (kynuric acid / hydrogen peroxide), and the interaction between NK cell membrane surface receptors and ligands on the surface of tumor cells, which will lead to the exhaustion of NK cell function.

[0003] The factors that regulate NK tumor immunity in the tumor microenvironment are complex and varied. Screening compounds that inhibit NK killing function is of great significance for improving NK cell immunotherapy. Summary of the invention

[0004] The purpose of the present application is to overcome the shortcomings of the above-mentioned prior art and provide an application of secondary bile acid in the preparation of an inhibitor of NK cell killing activity. The secondary bile acid of the present application can inhibit the expression of NK cell killing cytokines, and the use of spironolactone or progesterone can reverse the immunosuppressive effect of secondary bile acid on NK cells, and can regulate the activity and effector function of NK cells.

[0005] To achieve the above purpose, the technical solution adopted by this application is:

[0006] The present application provides the use of secondary bile acid in the preparation of inhibitors of NK cell killing activity.

[0007] After extensive research and experiments, the inventors of the present application found that by isolating, culturing and activating mouse NK cells in vitro, it can be seen from the results of in vitro experiments that secondary bile acids can inhibit the killing effect of NK cells, indicating that secondary bile acids have an immunosuppressive effect on NK cells.

[0008] As a preferred embodiment of the application described in the present application, the mass concentration of the secondary bile acid is 100-200 μM.

[0009] The present application uses secondary bile acids within the above mass concentration range to better inhibit the expression of NK cell killer cytokines.

[0010] As a preferred embodiment of the application described in the present application, the mass concentration of the secondary bile acid is 100 μM.

[0011] When the secondary bile acid of the present application is preferably at the above mass concentration, it can more significantly inhibit the killing effect of NK cells and further inhibit immunity to NK cells.

[0012] As a preferred embodiment of the application described in the present application, the secondary bile acid includes at least one of ursocholic acid (apocholic acid), allocholic acid (allocholic acid), 3β-UDCA, isolithocholic acid (iso-LCA) and dehydrolithocholic acid (DehydroLCA).

[0013] The present application adopts the above-mentioned types of secondary bile acids, which have an inhibitory effect on the immunity of NK cells and inhibit the expression of NK cell killer cytokines.

[0014] As a preferred embodiment of the application described in the present application, the secondary bile acid is isolithocholic acid.

[0015] In vitro experiments have shown that isolithocholic acid (iso-LCA) can significantly inhibit the expression of NK cell cytotoxic cytokines and the immunosuppressive effect of iso-LCA on NK cells.

[0016] The present application also provides the use of secondary bile acid analogs in the preparation of reagents for reversing the immunosuppression of NK cells by secondary bile acids.

[0017] The present application discovered that secondary bile acid analogs can reverse the immunosuppressive effect of secondary bile acids on NK cells.

[0018] As a preferred embodiment of the use described in the present application, the secondary bile acid analogue includes spironolactone or progesterone.

[0019] Spironolactone is a clinical potassium-sparing diuretic and blood pressure-lowering drug; progesterone is a natural progestin that maintains pregnancy and breast development; the present application found that spironolactone or progesterone can reverse the immunosuppressive effect of iso-LCA on NK cells, regulate the activity and effector function of NK cells, and improve the immunotherapy effect of NK cells.

[0020] The results may suggest that spironolactone and progesterone can be used as potential drugs to enhance the activity and effector function of NK cells in clinical practice, which means that spironolactone and progesterone may have the potential to be used for multiple purposes in clinical practice.

[0021] As a preferred embodiment of the application described in the present application, the mass concentration of spironolactone is 100-200 μM; the mass concentration of progesterone is 100-200 μM.

[0022] As a preferred embodiment of the application described in the present application, the mass concentration of spironolactone is 100 μM; the mass concentration of progesterone is 100 μM.

[0023] The use of spironolactone or progesterone within the above mass concentration range can better reverse the immunosuppressive effect of iso-LCA on NK cells, thereby enhancing the activity and effector function of NK cells and improving the immune effect of NK cells.

[0024] As a preferred embodiment of the application described in the present application, the secondary bile acid is isolithocholic acid.

[0025] Compared with the prior art, this application has the following beneficial effects:

[0026] The present application provides an application of secondary bile acids in the preparation of inhibitors of NK cell killing activity. The present application isolates, cultures and activates mouse NK cells in vitro. From the results of in vitro experiments, it can be seen that secondary bile acids can inhibit the killing effect of NK cells, indicating that secondary bile acids have an immunosuppressive effect on NK cells; and spironolactone or progesterone can reverse the immunosuppressive effect of iso-LCA on NK cells, and can regulate the activity and effector function of NK cells to improve the immune effect of NK cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The figure shows the result of secondary bile acid inhibiting the expression of cytokine of NK cells;

[0028] Figure 2 This is a graph showing the results of secondary bile acid analogs reversing the immunosuppressive effect of isolithocholic acid (iso-LCA) on NK cells. DETAILED DESCRIPTION

[0029] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0030] In the present application, the technical features described in an open manner include closed technical solutions composed of the listed features, and also include open technical solutions containing the listed features.

[0031] In this application, when it comes to numerical ranges, unless otherwise specified, the above numerical ranges are deemed to be continuous and include the minimum and maximum values ​​of the range, as well as each value between such minimum and maximum values. Further, when a range refers to an integer, each integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be merged. In other words, unless otherwise specified, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0032] In the present application, there is no particular limitation on the specific dispersion and stirring treatment methods.

[0033] Unless otherwise specified, the components, raw materials or instruments used in the embodiments of the present invention are all commercially available raw materials or instruments, and the components and raw materials used in each parallel experiment are all of the same type.

[0034] The present application provides the use of secondary bile acid in the preparation of inhibitors of NK cell killing activity.

[0035] In some specific embodiments, the secondary bile acid comprises at least one of ursocholic acid, allocholic acid, 3β-UDCA, isolithocholic acid and dehydrolithocholic acid.

[0036] More preferably, the secondary bile acid is isolithocholic acid.

[0037] In some specific embodiments, the mass concentration of the secondary bile acid is 100-200 μM.

[0038] More preferably, the mass concentration of the secondary bile acid is 100 μM.

[0039] The present application also provides the use of secondary bile acid analogs in the preparation of reagents for reversing the immunosuppression of NK cells by secondary bile acids.

[0040] In some embodiments, the secondary bile acid analog comprises spironolactone or progesterone.

[0041] In some specific embodiments, the mass concentration of spironolactone is 100-200 μM; the mass concentration of progesterone is 100-200 μM.

[0042] More preferably, the mass concentration of spironolactone is 100 μM; the mass concentration of progesterone is 100 μM.

[0043] Example 1: Secondary bile acids inhibit the expression of killer cytokines in NK cells

[0044] 1. Isolation, culture and activation of mouse NK cells in vitro:

[0045] Methods: The spleen of mice was removed under sterile conditions and ground into a cell suspension. NK cells were labeled with an NK cell isolation kit and then separated by LS column. The NK cells were then supplemented with IL-2 (500 U ml -1 ) and IL-15 (20 ng ml -1 The isolated NK cells were cultured in 1640 medium containing cytokines from PeproTech. During this process, they were treated with 100 μM of different types of secondary bile acids (apocholic acid, allocholic acid, 3β-UDCA, isolithocholic acid (iso-LCA) and dehydrolithocholic acid (DehydroLCA)) for 48 hours, and ethyl alcohol was added as a control group. Before flow cytometry detection of cytokines, they were stimulated with immune cell activation reagents for 4 hours, and then the expression and secretion of cytokines were detected.

[0046] 2. Detection of NK cell cytokine expression by flow cytometry:

[0047] Flow cytometry was used to detect the expression of NK cell cytokines. The staining was performed according to the following color scheme (APC-Cy7-CD45.2, BV421-CD3, FITC-NK1.1, PE-Cy7-IFN-γ, Percp5.5-Tnf, PE-GranzymeB), and the results were analyzed using Flowjo.

[0048] like Figure 1 As shown in the data, compared with the control group, apocholic acid, allocholic acid, 3β-UDCA, iso-LCA and dehydro-LCA can inhibit the expression of cytotoxic cytokines of NK cells, among which iso-LCA most significantly inhibited the expression of cytotoxic cytokines of NK cells, indicating that iso-LCA has an inhibitory effect on the immunity of NK cells.

[0049] Example 2: Secondary bile acid analogs reverse the immunosuppressive effect of isolithocholic acid (iso-LCA) on NK cells

[0050] Mouse NK cells were isolated and cultured in the same manner as in Example 1. 100 μM isolithocholic acid (iso-LCA) was added to the culture medium, and 100 μM of different iso-LCA structural analogs (chenodeoxycholic acid, ulobetasol, cholic Acid, deoxycholic acid, brexanolone, spironolactone, progesterone, ursodeoxycholic acid) were also added. After culturing for 48 hours, the cells were treated with an immune cell activation reagent (Cellstimulation cocktail plus protein transport inhibitors Invitrogen Cat#00497593) for 4 hours, and the expression and secretion of cell-killing cytokines were detected by flow cytometry.

[0051] Results: Figure 2 As shown, compared with the control group (added DMSO), the addition of isolithocholic acid (iso-LCA) alone inhibited the expression of NK cell cytokines, while spironolactone and progesterone (100 μM) could more significantly reverse the immunosuppressive effect of iso-LCA, indicating that the use of specific types of iso-LCA structural analogs can better reverse the immunosuppressive effect of iso-LCA.

[0052] The present application isolates, cultures and activates mouse NK cells in vitro. From the results of in vitro experiments, it can be seen that secondary bile acids can inhibit the killing effect of NK cells, indicating that secondary bile acids have an immunosuppressive effect on NK cells; and spironolactone or progesterone can reverse the immunosuppressive effect of iso-LCA on NK cells, and can regulate the activity and effector function of NK cells.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. Application of secondary bile acids in the preparation of inhibitors of NK cell killing activity.

2. The use according to claim 1, characterized in that The mass concentration of the secondary bile acid is 100-200 μM.

3. The use according to claim 2, characterized in that The mass concentration of the secondary bile acid is 100 μM.

4. The use according to claim 1, characterized in that The secondary bile acid includes at least one of ursocholic acid, allocholic acid, 3β-UDCA, isolithocholic acid and dehydrolithocholic acid.

5. The use according to claim 4, characterized in that The secondary bile acid is isolithocholic acid.

6. Use of secondary bile acid analogs in the preparation of reagents for reversing the immunosuppression of NK cells by secondary bile acids.

7. The use according to claim 6, characterized in that The secondary bile acid analogs include spironolactone or progesterone.

8. The use according to claim 7, characterized in that The mass concentration of the spironolactone is 100-200 μM; the mass concentration of the progesterone is 100-200 μM.

9. The use according to claim 8, characterized in that The mass concentration of the spironolactone is 100 μM; the mass concentration of the progesterone is 100 μM.

10. The use according to claim 6, characterized in that The secondary bile acid is isolithocholic acid.

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