New application of berberine

Abstract

The invention discloses a novel application of berberine as an STING agonist in anti-tumor immunotherapy. By activating an STING / IFN-beta signal channel, berberine induces calcium influx by up-regulating an STIM-1 / Orai1 calcium channel, and further activates a CRAC channel, so that release of T cell granzyme B is promoted; meanwhile, the expression of IFN-beta and IFN-gamma can be remarkably improved, and the expression of a T cell activation marker CD25 is enhanced.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of biological medicine, and particularly relates to a new use of berberine. BACKGROUND

[0002] The immune system is an important mechanism for the body to defend against foreign pathogens and abnormal cells, and can recognize and eliminate infected or tumor cells. In recent years, the mutual relationship between tumor and immunity has attracted widespread attention. Studies have shown that tumor cells can escape immune surveillance through various ways, leading to immune escape, thus promoting the growth and spread of tumors. Therefore, exploring how to enhance the recognition and attack ability of the immune system to tumors has become one of the important research directions of modern tumor treatment. Through immunotherapy, the body's anti-tumor immune response can be activated and enhanced, providing new treatment options for tumor patients and improving their survival rate and quality of life.

[0003] Tumor immunotherapy is a treatment method that uses the body's immune system to recognize and attack cancer cells. Its basic concept is to enhance or restore the function of the immune system so that it can effectively recognize tumor cells. In recent years, with the in-depth study of tumor immune microenvironment and the development of immune checkpoint inhibitors, tumor immunotherapy has become an important field of cancer treatment. The main types of tumor immunotherapy currently include: immune checkpoint inhibitors, which enhance T cell anti-tumor response by blocking inhibitory immune signals (such as PD-1, CTLA-4); tumor vaccines, which prevent or treat cancer by stimulating the body to produce immune response against specific tumor antigens; cell therapy, including CAR-T cell therapy and tumor infiltrating lymphocyte (TIL) therapy, which uses modified or expanded immune cells of patients to fight tumors; immune modulators, which enhance anti-tumor immune response by regulating the overall function of the immune system, such as the use of cytokines (IL-2, IFN-α).

[0004] Immunotherapy has become a research hotspot in recent years, especially immune cell therapy, which has shown high effectiveness and safety in the field of hematological tumors. However, the treatment of solid tumors still faces many challenges, such as difficulty in breaking through efficacy, risk of cytokine storm, and high treatment cost, which makes the current cancer treatment not widely used. Although tumor vaccines can effectively induce immune response, their killing rate is still low. At the same time, immune checkpoint inhibitors can achieve long-term control of cancer, but only a small number of patients can obtain positive response. In addition, clinical anti-tumor therapy also faces the problems of lack of immune cells in tumor microenvironment and immune cell exhaustion.

[0005] Therefore, there is an urgent need in the art to develop new drugs to effectively inhibit the growth and spread of tumor cells, thus providing new treatment options for cancer patients. SUMMARY

[0006] The present application aims to at least partly solve one of the technical problems existing in the prior art, and for this purpose, the present application provides a new use of berberine.

[0007] According to an aspect of the present application, there is provided a use of berberine in the preparation of a medicament for treating diseases related to the stimulator of interferon genes (STING) pathway.

[0008] According to an aspect of the present application, there is provided a use of berberine in the preparation of a medicament for treating diseases related to the STIM-1 / Orai1 signaling pathway.

[0009] According to an aspect of the present application, there is provided a method for inducing cells to synthesize type 1 interferon in vitro, wherein the cells are treated with berberine. Preferably, the cells are 293T cells transfected with the IFN-β gene.

[0010] According to an aspect of the present application, there is provided a method for inducing human immune cells to activate in vitro, wherein the cells are treated with berberine. Preferably, the cells are Thp-1 cells.

[0011] According to an aspect of the present application, there is provided a method for promoting Orai1 expression in cells in vitro, wherein the cells are treated with berberine. Preferably, the cells are Jurkat cells and T cells.

[0012] The present application discloses a new use of berberine as a STING agonist in antitumor immunotherapy. By activating the STING / IFN-β signaling pathway, berberine significantly promotes the expression of IFN-β and IFN-γ, and at the same time, berberine induces calcium influx by up-regulating the STIM-1 / Orai1 calcium channel, further activates the CRAC channel, and promotes the release of T cell granzyme B. In the in vitro co-culture experiment, the T cells treated with berberine show a significant killing effect on H1975 tumor cells. In the Lewis lung cancer mouse model, intraperitoneal injection of berberine (5-10 mg / kg) can dose-dependently inhibit tumor growth, activate dendritic cells (MHC-II / CD80↑) and CD8 + T cells (granzyme B / IFN-γ↑), enhance central memory and effector memory T cell responses. The present application provides a new STING targeted drug for tumor immunotherapy. BRIEF DESCRIPTION OF DRAWINGS

[0013] The accompanying drawings, which are included to provide a further understanding of the application and are incorporated in and constitute a part of this application, illustrate embodiments of the application and together with the description serve to explain the application. In the drawings:

[0014] Figure 1Figures showing in vitro data of berberine as a STING agonist according to embodiments of the present application;

[0015] Figure 2 Figures showing data of berberine inducing calcium ion influx through activating STIM-1 / Orai1 calcium channel to promote T cell activation according to embodiments of the present application;

[0016] Figure 3 Figures showing data of berberine significantly enhancing tumor cell killing effect in in vitro co-culture system after inducing T cell activation according to embodiments of the present application;

[0017] Figure 4 Figures showing in vivo anti-tumor data of berberine in Lewis lung cancer tumor-bearing mouse model according to embodiments of the present application;

[0018] Figure 5 Figures showing data of berberine inducing dendritic cell (DC) immune activation in Lewis lung cancer tumor-bearing mouse model according to embodiments of the present application;

[0019] Figure 6 Figures showing data of berberine inducing T cell immune response and exerting strong anti-tumor activity in Lewis lung cancer tumor-bearing mouse model according to embodiments of the present application. DETAILED DESCRIPTION

[0020] The following examples are set forth in order to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present application. It is understood that the examples are intended to be purely exemplary and are not intended to limit the scope of the application. The starting materials, reagents or devices mentioned in the following examples, if not otherwise specified, are commercially available or are prepared by known methods.

[0021] 1. Berberine is a potential STING agonist

[0022] STING / IFN-β signaling is an important immunomodulatory molecule. After the activation of STING, it promotes the production of IFN-β, and then induces the synthesis of type 1 interferon, thereby enhancing the anti-tumor immune activity. Therefore, the present application establishes a screening model of STING / IFN-β activator, aiming to obtain small molecule compounds that can effectively enhance the immune activity of the host. First, the present application uses IFN-β responsive luciferase reporter gene for high-throughput screening. 293T cells are used as host cells, and after transfection of the IFN-β reporter gene, the cells are treated with candidate drugs for a specific time, and the luciferase intensity is measured. HEK293T cells are transfected with IFN-β-PGL3-luc. 24 hours after transfection, different concentrations of berberine are administered for 24 hours, and then the activity of the expressed firefly luciferase in the cell lysate is evaluated according to the guidelines provided by the manufacturer (Tecan). To block the STING pathway in macrophages, STING inhibitor H151 is added 2 hours before berberine treatment. The IFN-γ level in macrophages is detected by flow cytometry, respectively.

[0023] Figure 1 (A) IFN-β-PGL3-luc reporter gene experiment: After different concentrations of berberine were administered to HEK293T cells transfected with IFN-β-PGL3-luc for 24 hours, the luciferase activity in the cell lysate expressing firefly luciferase was evaluated. (B) After different concentrations of berberine were administered to Thp-1 cells for 24 hours, the expression of IFN-β was detected by flow cytometry. (C) After different concentrations of berberine were administered to Thp-1 cells for 24 hours, the effect of berberine on STING and STING phosphorylation was detected by Western blotting. (D) After different concentrations of berberine were administered to Thp-1 cells for 24 hours, the effect of berberine on the induction of IFN-γ expression was detected by flow cytometry. In addition, the effect of berberine on IFN-γ expression was significantly weakened under the influence of STING blocker H151.

[0024] The present application observes that berberine effectively increases the expression of IFN-β induced luciferase reporter gene Figure 1 , A). At the same time, the present application also uses flow cytometry method to further verify the effect of berberine on IFN-β induction. After the present application gives different concentrations of berberine to Thp-1 cells, it is found that the expression of IFN-β in Thp-1 cells is significantly increased Figure 1 , B). In addition, Western blotting analysis shows that the expression of STING in cells treated with berberine is increased Figure 1 , C).

[0025] After STING activation, IFN-γ expression is induced, and the activity of immune cells in the body is improved. The results of the present application show that berberine can effectively promote the expression of IFN-γ in Thp-1 cells Figure 1 , D). Based on this, it can be determined that berberine enhances the activity of STING by promoting the production of IFN-β, thereby increasing the expression of IFN-γ. H151 is an inhibitor of the STING signaling pathway. When berberine and H151 act together, H151 can significantly inhibit the STING activity induced by berberine, thereby inhibiting the expression of IFN-γ Figure 1 , D). Further verified that berberine is a STING agonist.

[0026] 2. Berberine induces calcium influx through activation of STIM-1 / Orai1 calcium channels

[0027] In resting T cells, STING is located in the endoplasmic reticulum (ER) and interacts with the calcium sensor STIM-1. Before Ca 2+ release from the ER, STIM-1 is redistributed throughout the ER membrane. Among various Ca 2+ channels, the calcium release-activated calcium (CRAC) channel plays a unique role in T cells. STIM-1 is used to initiate store-operated calcium entry (SOCE) through activated CRAC channels in immune cells. Subsequently, the present application uses Western blotting to evaluate the expression of STIM-1.

[0028] Figure 2 (A) After different concentrations of berberine were given to Jurkat cells for 24 hours, PMA was used as a positive control, and Western blotting was used to detect the expression of Orai1 and STIM-1 in berberine. (B) After different concentrations of berberine were given to Jurkat cells for 24 hours, flow cytometry was used to detect the expression of Ca 2+ in berberine.(C-D) After different concentrations of berberine were given to Jurkat cells and T cells for 24 hours, flow cytometry was used to detect the expression of CD25 and granzyme B (GzmB) in berberine. In addition, under the influence of Ca 2+ blocker BTP2, flow cytometry was used to detect the expression of CD25 and granzyme B in berberine.

[0029] The results show that the expression of STIM-1 in Jurkat cells treated with berberine is significantly increased Figure 2, A). The binding of STIM-1 to Orai1 is a key step in SOCE. When STIM-1 detects a decrease in calcium concentration within the ER, it translocates to Orai1, causing Orai1 channels to open, which subsequently triggers calcium influx. This interaction between STIM-1 and Orai1 is crucial for the proper functioning of the immune system, particularly during T cell activation, which plays a vital role in generating an effective immune response. The present invention evaluated Orai1 protein levels using Western blotting. Cells were collected and lysed in RIPA lysis buffer containing protease inhibitors to extract total proteins. Proteins were separated by 10% SDS-PAGE, followed by transfer to nitrocellulose membranes and blocking with 5% skim milk powder for 1 hour. Primary antibodies were incubated overnight at 4°C. Then, secondary antibodies labeled with anti-rabbit IgG HRP were incubated for 1 hour at room temperature. After the addition of hypersensitive ECL chemiluminescent substrate (Beijing 4A Biotech Co., Ltd, China), protein expression was detected using an Amersham Imager 600 scanner (USA).

[0030] The results showed that berberine increased the expression level of Orai1 in Jurkat cells relative to the control group Figure 2 , A). To investigate the link between berberine-induced T cell activation and intracellular Ca 2+ level regulation, the present invention conducted experiments using the Jurkat cell line. Flow cytometry was used to assess the effect of berberine on intracellular Ca 2+ levels. The present invention found that berberine promoted an increase in Ca 2+ levels Figure 2 , B).

[0031] To investigate the effect of the interaction between Orai1 and T cell activation markers, the present invention used the Orai1 inhibitor YM-58483 (or BTP2) to inhibit Orai1 activity. Subsequent flow cytometry analysis showed that when Orai1 was inhibited, berberine-induced CD25 upregulation in Jurkat cells was significantly attenuated Figure 2 , C). In addition, the increase in granzyme B levels in T cells was also reduced Figure 2 , D). In summary, these results suggest that the expression of downstream cytokines after berberine activation of immune cells is mainly mediated through the activation of CRAC channels. Understanding this signaling pathway is crucial for elucidating how berberine improves T cell function.

[0032] 3. Berberine achieves tumor cell killing based on inducing T cell activation

[0033] To explore whether berberine can achieve the purpose of anti-tumor by enhancing the activity of immune cells, the present application carries out co-culture killing experiment. Luciferase-expressing H1975 tumor cells are co-cultured with T cells in the presence of berberine. The main purpose is to evaluate the effect of berberine on tumor cells. In the experiment of the present application, cells are co-cultured for 24 and 48 hours to evaluate the killing of berberine on tumor cells.

[0034] Cytotoxicity experiment uses 5000 luciferase-containing H1975 cells seeded in a 96-well plate. After overnight, the cells are then incubated with 10,000 T cells in different concentrations of berberine for 24 or 48 hours. After treatment, 150 μg / ml D-luciferin potassium salt working solution is added to the cells, and incubated at 37°C for 5 minutes. Then evaluate using microplate reader (Tecan, Morrisville, NC, USA) at all wavelengths.

[0035] Figure 3 (A-B) with human non-small cell lung cancer adenocarcinoma cells (H1975) expressing luciferase are co-cultured with or without T cells, and different concentrations of berberine are given, and the killing of berberine on H1975 cells is detected at 24 and 48 hours, respectively. The results of the study show that T cells treated with berberine exhibit significant cytotoxicity to H1975 tumor cells Figure 3 , A). Importantly, the cytotoxic response peaks at 48 hours Figure 3 , B). This indicates that berberine not only enhances the cytotoxic function of T cells, but also enhances its effectiveness over time.

[0036] 4. Berberine exhibits high anti-tumor activity by inducing DC and T cell immune response in Lewis lung cancer mouse model

[0037] To explore the effect of berberine in mice, the present application evaluates the tumor growth and progression of Lewis lung cancer in C57BL / 6J mice. To evaluate the anti-tumor effect of berberine, the present application measures tumor volume and weight as key indicators. Once the tumor grows to about 30-50 mm 3 in size, the mice are treated with various treatments, including PBS, berberine at a dose of 5 mg / kg injected intraperitoneally, and higher dose of berberine 10 mg / kg injected through the same route. The treatment regimen lasts for a total of 18 days, during which the present application carefully records the changes in tumor size and weight every three days. All animal experiments comply with animal ethics standards and use laboratory animals. In the experiment of different doses of berberine, about 5 x 10 5Lewis lung cancer (LLC) cells were subcutaneously injected into 6-8 week old male C57BL / 6J mice. 24 mice were randomly divided into the following intraperitoneal injection treatment groups (when the tumor volume reached 20 mm²). 3 The mice were injected once daily starting at day 18: control group (PBS), low-dose group (5 mg / kg), and high-dose group (10 mg / kg). Tumor growth was monitored every 3 days by measuring the long diameter (L) and short diameter (W), and tumor volume was calculated using the formula V = 1 / 2 × L × W². On day 18, tumor tissue, spleen, and blood were collected for pharmacodynamic analysis, with a focus on detecting the number of T cells and dendritic cells in the treatment group mice.

[0038] Figure 4 (A) Tumor size in lung cancer mice after administration of different concentrations of berberine, 5 mg / kg and 10 mg / kg. (B) Statistical graph of tumor size in lung cancer mice during treatment. (C) Tumor weight in lung cancer mice after 18 days of treatment. (D) Statistical graph of body weight in lung cancer mice during treatment. (E) Spleen weight in lung cancer mice after 18 days of treatment. The results of this study indicate that berberine treatment leads to a reduction in tumor weight and size, and the observed effect shows a clear dose-dependent relationship. Figure 4 (AD). Compared with the control group, the spleen volume of mice decreased after treatment ( Figure 4 (E). This shows that berberine can enhance immune system function and reduce spleen overactivity.

[0039] To investigate how berberine affects the immune system, lymphocytes were collected from mouse blood, spleen, and tumor tissue and then analyzed using flow cytometry.

[0040] Figure 5 In a mouse model of central anterior (AC) lung cancer treated with berberine for 18 days, immune cells were extracted from the tumor, blood, and spleen. Changes in dendritic cells (DCs) were detected by flow cytometry. Indicators of dendritic cell activation (CD80 and MHC-II) in mouse tumors, blood, and spleen were detected by flow cytometry. The results showed that berberine treatment increased the expression of dendritic cells. Figure 5 (AC). In mice with tumors, the activation of dendritic cells plays a crucial role in the anti-tumor immune response. The results of this invention indicate that the expression of MHC-II and CD80 in dendritic cells is significantly increased (AC). Figure 5 ,DF).

[0041] Figure 6 Mice with lung cancer (A) were euthanized after 18 days of treatment. Tumors, blood, and spleens were collected and analyzed by flow cytometry for the formation of mouse cytotoxic T lymphocytes (CD8+). + (T) Detection. (B) Detection of CD8 in mouse tumors by flow cytometry. +Indicators of T cell activation (granzyme B and IFN-γ). (B) CD8 + Indicators of T cell memory (CD44 and CD62L). The increase in MHC-II and CD80 enhances the strength of the anti-tumor immune response. Berberine administration at a dose of 10 mg / kg leads to an increase in CD8 + T cell proliferation ( Figure 6 , A), and promotes CD8 + T cells release granzyme B and IFN-γ, producing a strong anti-tumor response ( Figure 6 , B). This process leads to an increase in CD8 + T cells and their activation, enabling them to more effectively recognize and attack tumor cells. CD8 + T cells also express central memory and effector memory cells, which are able to rapidly produce an anti-tumor response upon re-exposure to the same antigen ( Figure 6 , C).

[0042] The above only the preferred embodiments of the present application, and is not intended to limit the present application, for those skilled in the art, the present application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the present application, should be included in the scope of protection of the present application.

Claims

1. Use of berberine in the preparation of a medicament for treating diseases related to the stimulator of interferon genes pathway after STING activation.

2. Use of berberine in the preparation of a medicament for treating diseases related to the STIM-1 / Orai1 signaling pathway.

3. A method of inducing cells to synthesize type 1 interferon in vitro, characterized in that, treating the cells with berberine.

4. The method of claim 3, wherein, The cells are 293T cells transfected with the IFN-β gene.

5. A method of inducing activation of human immune cells in vitro, characterized in that, treating the cells with berberine.

6. The method of claim 5, wherein, The cells are T cells.

7. A method of promoting Orai1 expression in a cell in vitro, comprising contacting the cell with a compound of any one of claims 1-6. 8 treating the cells with berberine.

8. The method of claim 7, wherein, The cells are Jurkat cells.

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