Use of asapholidin a1 in activating nk cells

Abstract

The application provides an application of dodartin A1 in activating NK cells. Through in-vitro experiments, it is proved that dodartin A1 can significantly promote the synthesis and release of IFN-gamma and cytotoxic substances (such as granzyme B and perforin) of NK92 cells, and increase the expression of the surface agonistic receptors NKp46 and NKG2D of the NK92 cells; through in-vivo experiments, it is proved that dodartin A1 can promote the expression of the NK cell activation related substances CD107a and NKp46 to play an anti-hepatoma effect; in conclusion, dodartin A1 has the effect of activating NK cells.

Description

Technical Field

[0001] This invention relates to the field of cell culture technology, specifically to the application of astragaloside A1 in activating NK cells. Background Technology

[0002] Natural killer cells (NK cells) are part of the innate immune system. They are a group of cells that can attack viruses and tumor cells without prior sensitization, possessing immune functions such as anti-infection, anti-tumor, and immunomodulation. Immunotherapy for tumors is a treatment method that eliminates tumor cells by enhancing or stimulating the body's immune response. In recent years, immunotherapy has been a hot research topic in the field of tumor treatment. From the launch of the first tumor immunotherapy drug TFN-α to the approval of new immunotherapies such as immune checkpoint inhibitors (PD-1 / PD-L1, CLTA-4, etc.), CAR-T cell therapy, and tumor vaccines, tumor immunotherapy has shown promising therapeutic effects. However, immune checkpoint inhibitors have shown low overall treatment response rates, CAR-T cell therapy has limited efficacy in solid tumors, and tumor vaccines carry the risk of causing gene mutations in normal cells, with significant individual variability in neoantigens. NK cell therapy, on the other hand, does not rely on the patient's own specific immune cells, has a wider range of cell sources, does not require MHC molecules to present antigens, and does not require antigen activation. It can target multiple pathogenic antigens and has stronger cytotoxicity, thus showing greater therapeutic potential.

[0003] Currently, NK cells can be activated through technologies such as cytokines (IL-2, IL-15, and IL-12, etc.) and gene engineering (CAR-NK therapy), enabling them to effectively recognize and attack tumor cells and enhance their ability to kill tumor cells. Developing new small molecule compounds that can activate NK cells is one of the ongoing hot topics in this field. Summary of the Invention

[0004] This invention provides the application of astragaloside A1 in activating NK cells.

[0005] In a first aspect, the present invention provides a method for activating NK cells in vitro, comprising contacting oxytocin A1 with NK cells in vitro to activate NK cells.

[0006] The method described above uses the CAS number 140147-77-9 of the herbal extract A1, and its chemical structure is shown in Formula 1:

[0007]

[0008] As described above, activating NK cells refers to stimulating isolated NK cells with higher cytotoxicity or cell-killing activity than before the treatment using oxytocin A1.

[0009] In the method described above, the NK cells are derived from mammals. The mammals can be selected from bovine, equine, feline, canine, lagomorphic, suidae, cameloid, rodent, and primate animals, including but not limited to cattle, horses, goats, sheep, cats, rabbits, pigs, camels, alpacas, rats, mice, guinea pigs, non-human primates (such as apes, monkeys, baboons, and orangutans), and humans, with cattle, horses, dogs, goats, sheep, pigs, camels, rats, mice, monkeys, and humans being preferred.

[0010] The NK cells described above include isolated autologous cells, isolated allogeneic cells, isolated allogeneic cells, or NK cell lines.

[0011] As described above, based on specific markers expressed on the surface of NK cells, NK cells can be divided into different subpopulations. Specifically, human NK cells can be divided into CD56^bright NK cells, which have high cytotoxicity but relatively low cell-killing activity, and CD56^dim NK cells, which have high cytotoxicity and cell-killing activity. Further, the NK cells are CD56^bright NK cells. Even further, the NK cells are NK92 cells.

[0012] The method described above, specifically contacting ascorbic acid A1 with NK cells in vitro, includes contacting ascorbic acid A1 with NK cells in an NK cell culture system. Further, the NK cell culture system can be a conventional NK cell culture medium in the art, such as human NK cell complete culture medium; the final concentration of ascorbic acid A1 in the culture system is not less than 2.5 μM, further not less than 5 μM, and further can be 10 μM or higher.

[0013] Secondly, the present invention provides a product for activating NK cells in vitro, comprising Astragalus membranaceus A1.

[0014] The product described above can specifically be an NK cell culture reagent or a cell culture kit.

[0015] It is understood that the application of products that activate NK cells in vitro in the in vitro activation of NK cells also falls within the scope of protection of this invention.

[0016] Thirdly, the present invention provides activated NK cells or NK cell subsets obtained according to the above method.

[0017] As mentioned above, NK cell subsets refer to NK cell subsets with specific phenotypes and functions isolated from activated NK cells. Examples include NK cell subsets with high levels of toxicity-related substances Granzyme B and Perforin on the NK cell surface, and increased expression of toxicity-related receptors NKG2D and NKp46, or NK cell subsets that can specifically kill other target cells.

[0018] Fourthly, the present invention provides cell preparations comprising the above-mentioned NK cells or NK cell subsets.

[0019] Fifthly, the present invention provides the use of Ashoka A1 in the preparation of a drug that inhibits the growth of liver cancer cells.

[0020] In the application described above, the liver cancer cells are Hepa 1-6 cells.

[0021] This invention provides the application of astragaloside A1 in activating NK cells in vitro. In vitro experiments have demonstrated that astragaloside A1 can significantly promote the synthesis and release of IFN-γ and cytotoxic substances (such as granzyme B and perforin) from NK92 cells, and increase the expression of NK92 cell surface agonist receptors NKp46 and NKG2D. In vivo experiments have demonstrated that astragaloside A1 can promote the expression of NK cell activation-related substances CD107a and NKp46 to exert an anti-hepatocellular carcinoma effect. In summary, astragaloside A1 has the effect of activating NK cells. Attached Figure Description

[0022] Figure 1 The effect of ascorbic acid A1 on IFN-γ production in NK92 cells is shown in the following figures: A represents the statistical results of IFN-γ secreted by NK92 cells after culturing NK92 cells with different concentrations of ascorbic acid A1; B represents the Western blotting results of IFN-γ after culturing NK92 cells with different concentrations of ascorbic acid A1; C represents the flow cytometry results of IFN-γ production by NK92 cells after culturing NK92 cells with 10 μM ascorbic acid A1; and D represents the relative content of IFN-γ released by NK92 cells after culturing NK92 cells with 10 μM ascorbic acid A1.

[0023] Figure 2The effect of asparagine A1 on the cytotoxicity of NK92 cells was investigated; where A represents the flow cytometry analysis results of granzyme B generated by NK92 cells after culturing with asparagine A1; and B represents the granzyme released by NK92 cells after culturing with asparagine A1. B represents the relative content of NK92 cells after culturing with Astragalus A1; C represents the flow cytometry analysis results of perforin produced by NK92 cells after culturing with Astragalus A1; D represents the relative content of perforin released by NK92 cells after culturing with Astragalus A1; E represents the flow cytometry analysis results of NKG2D produced by NK92 cells after culturing with Astragalus A1; F represents the relative content of NKG2D released by NK92 cells after culturing with Astragalus A1; G represents the flow cytometry analysis results of NKp46 produced by NK92 cells after culturing with Astragalus A1; H represents the relative content of NKp46 released by NK92 cells after culturing with Astragalus A1.

[0024] Figure 3 The effects of astragalus extract A1 on the expression levels of IFN-γ, granzyme B, perforin, NKG2D, and NKp46 genes in NK92 cells were investigated. Specifically, A represents the effect of astragalus extract A1 on the expression level of IFN-γ gene in NK92 cells; B represents the effect of astragalus extract A1 on the expression level of granzyme B gene in NK92 cells; C represents the effect of astragalus extract A1 on the expression level of perforin gene in NK92 cells; D represents the effect of astragalus extract A1 on the expression level of NKG2D gene in NK92 cells; and E represents the effect of astragalus extract A1 on the expression level of NKp46 gene in NK92 cells.

[0025] Figure 4 The effect of astragaloside A1 on the killing effect of NK92 on the hepatocellular carcinoma cell line K562;

[0026] Figure 5 The effects of ascorbic acid A1 on hepatocellular carcinoma mice are as follows: A represents the effect of ascorbic acid A1 on tumor growth in hepatocellular carcinoma mice; B represents the effect of ascorbic acid A1 on body weight in hepatocellular carcinoma mice; C represents the effect of ascorbic acid A1 on tumor volume in hepatocellular carcinoma mice; and D represents the effect of ascorbic acid A1 on tumor weight in hepatocellular carcinoma mice.

[0027] Figure 6The effects of astragaloside A1 on CD107a and NKp46 production in mice with hepatocellular carcinoma are as follows: A represents the flow cytometry results of CD107a on the surface of NK cells in the spleen tissue of mice with hepatocellular carcinoma after treatment with astragaloside A1; B represents the statistical results of the relative content of CD107a in the spleen tissue of mice with hepatocellular carcinoma after treatment with astragaloside A1; C represents the flow cytometry results of NKp46 on the surface of NK cells in the spleen tissue of mice with hepatocellular carcinoma after treatment with astragaloside A1; and D represents the statistical results of the relative content of NKp46 in the spleen tissue of mice with hepatocellular carcinoma after treatment with astragaloside A1.

[0028] Figures 1-6 In the diagram, * indicates the ratio of the treatment group to the control group, p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Detailed Implementation

[0029] To make the objectives, technical solutions, and advantages of this invention clearer, the technical solutions of this invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, embodiments of this invention, and should not be construed as limiting the invention. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention. In the description of this invention, it should be understood that the terminology used is for descriptive purposes only and should not be construed as indicating or implying relative importance.

[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0031] The cell lines NK-92 (natural killer cells from human patients with malignant non-Hodgkin's lymphoma) and Hepa 1-6 (mouse liver cancer cells) used in the following examples were both purchased from Wuhan Pronosai Life Science Technology Co., Ltd. NK-92 cells were cultured in human NK cell complete medium provided by Pronosai. Hepa 1-6 cells were cultured in 1640 complete medium containing 10% fetal bovine serum.

[0032] The C57BL / 6J mice (male, SPF grade, 18-20g) used in the following examples were purchased from Spiefol (Beijing) Biotechnology Co., Ltd. Male C57BL / 6 mice were housed in plastic cages filled with poplar shavings at a temperature of 25±1℃, with a 12h light / dark cycle, and provided with clean food and water.

[0033] The ascorbic acid A1 (EA1, CAS: 140147-77-9, purity 99.18%) used in the following examples was purchased from Shanghai Taoshu Biotechnology Co., Ltd. Ascorbic acid A1 was completely dissolved in DMSO to prepare a 100 mM DMSO solution. The concentration required for subsequent experiments can be directly diluted with an appropriate amount of DMSO (for cell-carrying concentrations below 0.1%) or culture medium. The prepared drug was stored at -20°C.

[0034] Example 1: Effect of Asparagine A1 on NK-92 cells

[0035] NK-92 cells were distributed at a ratio of 5 × 10⁶ cells per well. 4 NK-92 cells were seeded in 96-well plates, with a total volume of 100 μL per well, and cultured overnight in a cell culture incubator. The cells were divided into 8 groups, with 3 wells per group. Group 1 was the control group, with 0.1 μL of DMSO added and cells cultured for 18 hours. Groups 2-8 were astragalus A1 groups, with 0.1 μL of different concentrations of astragalus A1 in DMSO solution (final concentrations of astragalus A1: 100 nM, 200 nM, 500 nM, 1 μM, 2 μM, 5 μM, and 10 μM) added to each well and cells cultured for 18 hours. After 18 hours, 0.1 μL of the inhibitor Monensin Solution (purchased from Biolegend, catalog number: 420701) was added to each well for 6 hours of inhibition. After 6 hours, the cells were then cultured using... The expression level of IFN-γ in NK92 cells was detected by human IFN-γ ELISA kit, Western blotting, and flow cytometry.

[0036] The results are as follows Figure 1 As shown. According to Figure 1 As shown in Figure A, compared with the control group, Astragalus membranaceus A1 promoted the expression of IFNγ, a substance related to NK-92 cell activation, at concentrations of 1 μM, 2 μM, 5 μM, and 10 μM, and this promotion was dose-dependent. Figure 1 As shown in Figure B, the expression level of IFNγ protein in NK-92 cells treated with Astragalus membranaceus A1 (2, 5, 10 μM) was significantly increased compared with that in the control group.

[0037] NK-92 cells treated with 10 μM ascorbic acid A1 were analyzed by flow cytometry. The results are as follows: Figure 1 As shown in Figure C, aspheridine A1 promotes the increase of IFNγ content in NK-92 cells. These experiments preliminarily clarify that aspheridine A1 has the effect of activating NK-92 cells.

[0038] Example 2: Effect of Astragalus membranaceus A1 on NK-92 cell cytotoxicity

[0039] NK-92 cells were distributed at a density of 1.2 × 10⁶ cells per well. 6 Cells were seeded in 12-well plates with a total volume of 1 mL per well and incubated overnight in a cell culture incubator. NK-92 cells were divided into two groups of 3 wells each. Group 1 was the control group, in which 1 μL of DMSO was added to the cells and the cells were cultured for 18 hours. Group 2 was the ascorbic acid A1 group, in which 1 μL of DMSO solution of ascorbic acid A1 (final concentration of ascorbic acid A1 was 10 μM) was added to the cells and the cells were cultured for 18 hours. After 18 hours, 0.1 μL of the inhibitor Monensin Solution was added to block the cell growth for 6 hours. After 6 hours, the expression levels of NK-92 cytotoxicity-related substances Granzyme B, Perforin, and cytotoxicity-related receptors NKG2D and NKp46 were detected by flow cytometry.

[0040] Test results as follows Figure 2 As shown in the figure, compared with the control group, the levels of granzyme B and perforin in the astragalus A1 group were significantly increased; at the same time, 10 μM astragalus A1 significantly increased the expression of NK92 cytotoxicity-related receptors NKG2D and NKp46. These experiments indicate that astragalus A1 has the effect of increasing NK92 cell cytotoxicity.

[0041] Example 3: Effect of Asparagine A1 on the gene expression levels of activation-related substances in NK-92 cells

[0042] NK-92 cells were distributed at a density of 1.2 × 10⁶ cells per well. 6 1 mL of cells were seeded into 12-well plates and incubated overnight in a cell culture incubator. NK-92 cells were divided into 4 groups, with 3 wells in each group. Group 1 was the control group, in which 1 μL of DMSO was added to the cells and the cells were cultured for 18 hours. Groups 2-4 were ascorbic acid A1 groups, in which 1 μL of DMSO solution of ascorbic acid A1 (final concentrations of ascorbic acid A1 were 2.5 μM, 5 μM, and 10 μM, respectively) was added to the cells and the cells were cultured for 18 hours. After 18 hours, 0.1 μL of the inhibitor Monensin Solution was added to block the cells for 6 hours, and the cells were collected after 6 hours.

[0043] Add 500 μL of Trizol to each group of cells, lyse the cells thoroughly on ice, centrifuge at 12000g, 4℃ for 5 min, and collect the supernatant. Add 100 μL of chloroform, mix thoroughly, centrifuge at 12000g, 4℃ for 5 min, and collect the clear supernatant. Add an equal volume of isopropanol, mix thoroughly, centrifuge at 12000g, 4℃ for 5 min, and discard the supernatant. Add 500 μL of 75% ethanol, shake inverted, centrifuge at 7500 rpm, 4℃ for 5 min, discard the supernatant, evaporate the 75% ethanol at room temperature, and dissolve the precipitate in 30 μL of sterile, enzyme-free water to obtain RNA. Then, reverse transcribe the RNA into cDNA using the HiFiScript gDNA Removal cDNA Synthesis Kit. Prepare the DNA amplification system using the 2×SuperFast Universal SYBR Master Mix kit, and finally, use a PCR system to detect the relative mRNA expression levels of each gene. The calculation formula is 2. -ΔΔCt The internal reference gene is GAPDH.

[0044] Test results as follows Figure 3 As shown in the results of the study, compared with the control group, the relative mRNA levels of IFNγ, granzyme B, perforin, NKG2D, and NKp46 were increased in the astragalus A1 group. These experiments further clarified the role of astragalus A1 in activating NK-92 in vitro.

[0045] Example 4: Effect of Astragalus membranaceus A1 on the killing effect of NK92 cells

[0046] NK-92 cells were distributed at a ratio of 5 × 10⁶ cells per well. 4 0.1 mL of cells were seeded into 96-well plates and incubated overnight. NK-92 cells were divided into four groups of three wells each. Groups 1 and 2 were the control group, with 0.1 μL of DMSO added to the cells for 18 hours. Groups 3 and 4 were the astragaloside A1 group, with 0.1 μL of astragaloside A1 in DMSO solution (final concentration of astragaloside A1: 10 μM) added to the cells for 18 hours. After 18 hours, the cancer cell line K562 and NK cells were added to 96-well plates at different effector / target ratios (1:1 or 1:5) and co-cultured for 8 hours. 150 μg / mL of D-luciferin (PerkinElmer) was added to each well, and cell death was assessed using a luminometer.

[0047] Test results as follows Figure 4 As shown in the figure, compared with the untreated NK92 cell group, the killing effect of NK92 cells after intervention with Astragalus membranaceus A1 was significantly enhanced on the K562 liver cancer cell line.

[0048] Example 5, In vivo experiment

[0049] 1. Establishment of an in vivo mouse model of liver cancer and drug intervention

[0050] A hepatocellular carcinoma (HCC) model was established in C57BL / 6J mice after one week of adaptive feeding. The right back of the mice was prepared one day prior to the model. Logarithmic growth phase Hepatocellular carcinoma Hepatocellular Carcinoma (HCC) cells were harvested, digested, centrifuged, and diluted with PBS to a concentration of 1x10⁻⁶. 7 A single-cell suspension of mouse liver cancer Hepa 1-6 cells was drawn up with a 1 mL syringe and injected into the right back of each mouse at a rate of 200 μL to construct a mouse model of liver cancer.

[0051] Mice with hepatocellular carcinoma were randomly divided into a control group, a positive drug group (sorafenib), and a cyproheptadine A1 group, with 10 mice in each group. Intraperitoneal administration began 3 days after modeling. The dose of cyproheptadine A1 was set at 50 mg / kg, the dose of sorafenib at 10 mg / kg, and the control group received an equal volume of solvent. Administration continued for 20 days, once daily.

[0052] 2. Effects of Astragalus membranaceus A1 on body weight, tumor size, and tumor weight in mice with liver cancer

[0053] The body weight of mice in each group was recorded every 5 days. After the drug administration ended, on day 21, the mice were sacrificed and the tumors were removed. The tumors were weighed and the longest diameter (a) and shortest diameter (b) of the tumors were measured with calipers. The corresponding tumor volume (V = 0.52ab) was calculated. 2 ).

[0054] Test results are as follows Figure 5 As shown, there was no significant difference in body weight among the groups of mice. However, the tumor size in the asparagine A1 group was significantly smaller than that in the control group, and asparagine A1 also reduced the tumor weight in mice with liver cancer. These results preliminarily confirm that asparagine A1 has an anti-liver cancer effect.

[0055] 3. Effects of Asparagine A1 on the levels of CD107a and NKp46, NK activity-related substances, in spleen tissue of mice with hepatocellular carcinoma.

[0056] The spleen is the largest immune organ in the body, and detecting NK cell activation-related indicators in the spleen can, to some extent, elucidate drug effects. After euthanizing mice, the spleens were removed and placed in RPMI-1640 medium containing 1% serum. Cell suspension was collected by sieving through a sterile 40 μM sieve. The cells were centrifuged at 2000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS. The cells were then centrifuged again at 2000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in erythrocyte lysis buffer. Lysis was performed on ice for 5 minutes, followed by centrifugation at 2000 rpm for 5 minutes, the supernatant was discarded, and the cells were resuspended in PBS. The cells were then centrifuged again at 2000 rpm for 5 minutes, the supernatant was discarded, and the cells were washed with PBS and resuspended. An antibody mixture (AF700-CD3, PE / Cy7-NK1.1, BV711-CD107a, BV421-NKp46) was added, and the cells were stained at room temperature in the dark for 20 minutes, followed by centrifugation at 2000 rpm for 5 minutes. The supernatant was discarded, the cells were washed once with an appropriate amount of PBS, and the cells were resuspended in 200 μL of PBS for analysis. Flow cytometry results were analyzed using FlowJo V10.

[0057] Test results are as follows Figure 6 As shown, compared with the control group, the expression of CD107a, a NK cell activation-related substance, was significantly increased in the spleen tissue of mice in the astragalus root extract (A1) group. Simultaneously, the expression level of the NK cell activation receptor NKp46 was also higher in the astragalus root extract (A1) group than in the control group. These results indicate that astragalus root extract (A1) also has the effect of enhancing NK cell activity in vivo.

[0058] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for activating NK cells in vitro, characterized in that, This includes contacting oxytocin A1 with NK cells in vitro to activate NK cells.

2. The method according to claim 1, characterized in that, The NK cells are derived from mammals.

3. The method according to claim 1, characterized in that, The NK cells include isolated autologous cells, isolated allogeneic cells, isolated allogeneic cells, or NK cell lines.

4. The method according to any one of claims 1-3, characterized in that, The NK cells are at least one of CD56^bright NK cells and CD56^dim NK cells.

5. The method according to any one of claims 1-4, characterized in that, The NK cells mentioned are NK92 cells.

6. The method according to any one of claims 1-5, wherein asparagine A1 is contacted with NK cells in an NK cell culture system, wherein the final concentration of asparagine A1 in the culture system is not less than 2.5 μM.

7. A product for activating NK cells in vitro, characterized in that, Including Astragalus membranaceus A1.

8. Activated NK cells or NK cell subsets obtained by the method according to any one of claims 1-6.

9. Cell preparations comprising NK cells or NK cell subsets as described in claim 8.

10. Application of Chaohuodin A1 in the preparation of drugs that inhibit the growth of liver cancer cells.

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