Application of cynarin as cGAS-STING signaling pathway inhibitor in preparation of anti-inflammatory drugs

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to application of cynarin as a cGAS-STING signal channel inhibitor in preparation of anti-inflammatory drugs. It is found for the first time that cynarin can inhibit phosphorylation of STING and translocation of STING by influencing the stability of STING spatial conformation, so that channel downstream protein activation is inhibited, nuclear translocation of IRF3 is hindered, expression of a series of interferon-induced related genes such as IFN-beta, CXCL10 and ISG15 is inhibited, finally secretion of IFN-beta can be hindered, cGAS-STING inflammatory response can be inhibited by targeting STING, and the application of cynarin in inhibiting cGAS-STING inflammatory response is achieved. A new thought and a new means are provided for developing an auxiliary treatment method aiming at STING related inflammations.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the use of cynarin as a cGAS-STING signaling pathway inhibitor in the preparation of anti-inflammatory drugs. Background Art

[0002] The cGAS-STING signaling pathway is an important upstream pathway for type I interferon response and NF-κB signaling pathway, and is a unique intracellular nucleic acid recognition and response mechanism. After specifically recognizing DNA from pathogens or cancer cells accumulated in the cytoplasm, cGAS can catalyze the synthesis of 2,3-cGAMP from ATP and GTP; the latter can bind to the STING protein on the endoplasmic reticulum membrane to promote its transport to the Golgi apparatus, where it activates the interferon regulatory factor IRF3 by phosphorylating TBK1 kinase, or activates the NF-κB signaling pathway by phosphorylating IKK, thereby initiating a series of immune responses that synthesize interferons and inflammatory cytokines, playing an important role in infection and tumor immunity.

[0003] However, excessive activation of cGAS or STING caused by abnormal DNA accumulation may also induce inflammatory cytokine storms, thereby damaging normal tissue cells. Inhibiting the excessive activation of the cGAS-STING signaling pathway and its mediated downstream innate immune response has been shown to be a potential target for the treatment of STING-related inflammation, but there is still a lack of drugs on the market that specifically inhibit this pathway.

[0004] Artichoke is a plant rich in phenolic compounds and has antioxidant capacity, which can be used as a healthy food. The phenolic compounds in artichoke include various types of caffeoylquinic acid, among which 1,3-dicaffeoylquinic acid (also known as cynarin) is considered to be the compound with the highest ability to inhibit low-density lipoprotein oxidation and cholesterol biosynthesis. Early studies have clearly reported the ROS scavenging ability of cynarin. In endothelial cells, artichoke extracts reduce low-density lipoprotein-induced oxidative stress and promote the expression of endothelial nitric oxide synthase. However, there are currently no reports on the correlation between cynarin and cGAS-STING inflammatory response. Summary of the invention

[0005] The purpose of the present invention is to overcome the shortcomings and deficiencies in the prior art and provide the use of cynarin as a cGAS-STING signaling pathway inhibitor in the preparation of anti-inflammatory drugs.

[0006] To achieve the above objectives, the technical solutions adopted by the present invention include:

[0007] In the first aspect, the use of cynarin or its derivatives in the preparation of inhibitors of the cGAS-STING signaling pathway.

[0008] In the second aspect, cynarin or its derivatives are used as inhibitors of the cGAS-STING signaling pathway in the preparation of anti-inflammatory drugs.

[0009] Preferably, the activation of the cGAS-STING signaling pathway is caused by cyclic GMP-AMP.

[0010] Preferably, the cynarin or its derivatives target STING to inhibit the cGAS-STING signaling pathway.

[0011] Preferably, the effective concentration of cynarin or its derivatives is 1-10 μM.

[0012] More preferably, the effective concentration of cynarin or its derivative is 5 μM.

[0013] Preferably, the drug is used in at least one of the following aspects:

[0014] a. Affect the stability of STING's spatial conformation to inhibit STING's phosphorylation and STING's translocation;

[0015] b. Inhibit activation of proteins in the cGAS-STING signaling pathway;

[0016] c. Block the nuclear translocation of IRF3;

[0017] d. Inhibit the expression of interferon-induced related genes;

[0018] e. Inhibit the expression and secretion of interferon.

[0019] Preferably, the interferon-induction-related gene includes at least one of CXCL10, IFI16, ISG54, ISG56 and ISG58; and the interferon includes IFN-β.

[0020] The present invention has found through experimental research that cynarin can specifically inhibit the STING pathway by inhibiting the excessive activation of the cGAS-STING signaling pathway and its mediated downstream innate immune response, thereby achieving a good therapeutic effect on STING-related inflammation, filling the gap in the current market for drugs that specifically inhibit this pathway.

[0021] In a third aspect, the present invention provides an anti-inflammatory drug, wherein the main active ingredient of the drug includes cynarin or its derivatives.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The present invention discovers for the first time that cynarin can inhibit the phosphorylation and translocation of STING by affecting the stability of STING's spatial conformation, thereby inhibiting the activation of downstream proteins in the pathway and hindering the nuclear translocation of IRF3, thereby inhibiting the expression of a series of interferon-induced related genes such as IFN-β, CXCL10, and ISG15, and ultimately hindering the secretion of IFN-β, thereby achieving targeted STING to inhibit the cGAS-STING inflammatory response, and providing new ideas and means for the development of auxiliary treatment methods for STING-related inflammation. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a result diagram of exploring the effective concentration of cynarin in inhibiting STING in Example 1;

[0025] Figure 2 This is a result diagram showing that cynarin inhibits the production of IFN-β, the end product of cGAS-STING inflammatory response, and the transcription of interferon-induced related genes in Example 2;

[0026] Figure 3 This is a result diagram of cynarin inhibiting the phosphorylation of related proteins in the cGAS-STING pathway and the translocation of STING in Example 3;

[0027] Figure 4 This is a result diagram of cynarin inhibiting the related protein interaction in the cGAS-STING pathway and the nuclear entry of IRF3 in Example 4;

[0028] Figure 5 This is a diagram showing the effect of cynarin on the conformational stability of STING protein. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and effect of the present invention clearer and more specific, the present invention is further described in detail with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0030] Unless otherwise specified, the reagents used in the examples are conventional reagents in the art and can be purchased through commercial channels. The experimental operations not specifically described in the examples are conventional operations in the art or can be understood or known by those skilled in the art based on the existing technology or common knowledge they master.

[0031] The cynarin described in the examples of the present invention was purchased from MedChemExpress, model HY-N0359.

[0032] Example 1. Determination of effective concentration of cynarin for inhibiting STING

[0033] The specific method is as follows:

[0034] In THP1 cells, cells were pretreated with cynarin at concentrations of 0.25, 0.5, 1, 5 and 10 μM for 24 hours, and cell proteins were extracted to detect the expression level of STING.

[0035] Depend on Figure 1 The results showed that cynarin could reduce the expression level of STING at a concentration between 1-10 μM.

[0036] Example 2. Study on the inhibition of cynarin on the production of IFN-β, the end product of cGAS-STING inflammatory response, and the transcription of interferon-induced related genes

[0037] The specific method is as follows:

[0038] The experiment was conducted using THP-1 cells, which were divided into three groups. The first group was the blank control group, which was treated with the same concentration of DMSO for the same period of time for observation; the second group was the positive control group, which was treated with the same concentration of DMSO and then treated with 2 mg / mL cGAMP for 18 hours for observation; the third group was the experimental group, which was pretreated with 5 μM cynarin for 12 hours and then treated with 2 mg / mL cGAMP for 18 hours to extract cell DNA. After reverse transcription, RT-PCR detection was performed.

[0039] Depend on Figure 2 (AF) It can be seen that cynarin significantly inhibited the transcription of interferon-γ induced protein 10 (CXCL10), interferon-γ induced protein 16 (IFI16), interferon-stimulated genes 54, 56, 58 (ISG54, ISG56, ISG58), but had no significant effect on interferon-stimulated gene 60 (ISG60), interleukin 6 (IL-6) and tumor necrosis factor α (TNFα), and when the downstream secreted IFN-β was detected by ELISA ( Figure 2 J), and it was also found that cynarin had an inhibitory effect on the secretion of IFN-β. Therefore, it is fully proved that cynarin can indeed effectively inhibit the expression and production of IFN-β.

[0040] Example 3. Study on the inhibition of phosphorylation of related proteins in the cGAS-STING pathway and STING translocation by cynarin

[0041] In THP-1 cells, cells were pretreated with cynarin at a concentration of 5 μM for 12 h, and then treated with cGAMP at a concentration of 2 mg / mL for 0 h, 2 h, and 4 h to detect the phosphorylation changes of related proteins in the pathway. The results are as follows: Figure 3As shown in A, after treatment with cynarin, the phosphorylation levels of STING, IRF3, and TBK1-related proteins caused by the activation of the STING signaling pathway were significantly inhibited.

[0042] In addition, after THP-1 cells were treated with different drugs, the inventors also explored the co-localization of endogenous STING protein and Golgi membrane protein GM130, such as Figure 3 As shown in B, after treatment with cGAMP, the STING protein is transported to the Golgi apparatus and gradually polymerizes and aggregates in the Golgi apparatus, thereby forming a functional signaling complex that leads to the phosphorylation of IRF3 and entry into the cell nucleus, promoting the expression and secretion of interferon and interferon-stimulated genes. After treatment with cynarin, the translocation process of the STING protein is significantly inhibited, and the aggregation of the STING protein in the Golgi apparatus is significantly reduced, thereby effectively inhibiting the subsequent expression of interferon and interferon-stimulated genes, and hindering the activation of the cGAS-STING pathway caused by cGAMP stimulation. In addition, by comparison, cynarin and the STING inhibitor C-170 have comparable inhibitory effects on STING translocation.

[0043] In addition, the inventors also transfected exogenous FLAG-STING plasmid into HEK293T cells and investigated the co-localization of exogenous FLAG-STING protein with Golgi membrane protein GM130 after treatment with different drugs. Figure 3 As shown in C, the translocation process of exogenous FLAG-STING protein was also significantly inhibited after treatment with cynarin.

[0044] Example 4. Study on the inhibition of cGAS-STING pathway related protein interactions and IRF3 nuclear entry by cynarin

[0045] The specific method is as follows:

[0046] THP1 or HEK293 cells were cultured in 10 cm cell culture dishes. HEK293 cells were transfected with plasmids and then treated with drugs 24 hours after plasmid transfection. The corresponding concentration of natural compounds was mixed into the complete culture medium, and then added to the cell culture dish containing the corresponding cells to pretreat the cells for 12 hours. Perm Buffer reagent was prepared in advance before the experiment (prepared and used immediately), Digitonin with a final concentration of 5 nM and 2 μg / mL cGAMP were added to Perm Buffer and mixed for use. After discarding the old culture medium, wash once with PBS, add the pre-prepared Perm Buffer, incubate at 37 ° C for 20 minutes, remove the incubated cells, discard the Buffer, wash once with PBS, and finally replace with complete culture medium containing the corresponding concentration of natural compounds. Continue incubation according to experimental requirements. Collect protein samples after 24 hours, and take 1 / 20 of the lysate as input. Take 1.2 mg of total protein from the remaining lysate, add STING antibody and mix well, place on a DNA rotating mixer in a 4°C refrigerator for antigen-antibody incubation overnight (12 h); take 30 μL of Protein A / G magnetic beads or agarose beads in a pre-cooled labeled 1.5 mL centrifuge tube, add 1 mL of IP Lysis Buffer to resuspend, and then centrifuge, repeat twice, and discard IP Lysis Buffer as much as possible for the last time; take out the sample incubated overnight and add it to a 1.5 mL centrifuge tube with activated Protein A / G magnetic beads or agarose beads. Place the centrifuge tube again in a 4°C refrigerator on a DNA rotating mixer and incubate at 15 rpm for 4 hours; centrifuge the Protein A / G mixture on a magnetic rack or centrifuge at 4°C, 1000g for 5 minutes, and repeat the washing of the Protein A / G beads 15 times (using IP Lysis Buffe); resuspend the resulting precipitate in 2× loading buffer and boil it in a metal bath at 100°C for 10 minutes, and store the sample at -30 / -80°C; take the reserved Input and IP samples for immunoblotting detection to detect the interaction between STING, TBK and TRF3, respectively.

[0047] Through immunoprecipitation experiments, it was found that whether it was endogenous STING in THP-1 cells or exogenous STING in HEK293T cells transfected with FLAG-tagged STING plasmid, the interaction between related proteins in the cGAS-STING pathway was reduced to a certain extent after treatment with cynarin ( Figure 4 AB), indicating that cynarin blocked the interaction between STING and TBK1 and IRF3, and the nuclear entry of IRF3 was significantly reduced in the cells treated with cynarin ( Figure 4C). To further verify, the inventors separated the cytoplasm and nucleus and performed Western Blot detection respectively. After cynarin treatment, there was no significant change in the phosphorylated IRF3 in the cytoplasm, but the phosphorylation of IRF3 in the nucleus was significantly reduced ( Figure 4 D). The above results all indicate that cynarin can inhibit the interaction between STING and key proteins in the pathway, thereby preventing IRF3 from phosphorylating and incorporating into the nucleus, and ultimately inhibiting the expression and secretion of interferon.

[0048] Example 5. Study on the effect of cynarin on the conformational stability of STING protein

[0049] (1) In THP-1 cells, cells were treated with cynarin at a concentration of 5 μM for different time periods (0, 1.5, 3, 6, 12, and 24 h), and the changes in STING protein in the cells were detected by Western Blot. The results are as follows: Figure 4 As shown in A, after treatment with cynarin, the concentration of STING protein gradually decreased with the extension of treatment time, indicating that cynarin has a certain effect on the stability of STING protein.

[0050] (2) THP-1 cells were used for the experiment and divided into three groups. The first group was the blank control group, in which THP-1 cells were treated with DMSO for 30 hours; the second group was the positive control group, in which THP-1 cells were treated with DMSO for 12 hours and then treated with 2 mg / mL cGAMP for 18 hours for observation; the third group was the experimental group, in which cells were pre-treated with cynarin at a concentration of 2 μM for 12 hours and then treated with 2 mg / mL cGAMP for 18 hours for observation. In the QPCR detection process, β-actin was used as the internal reference gene, + represents the use of the corresponding drug treatment, - represents the use of the corresponding drug treatment, the number of independent experiments was ≥3 times, *(P<0.05), **(P<0.01), ***(P<0.001), the results showed that the expression level of STING mRNA was significantly increased after cGAMP stimulation, while its mRNA expression level was significantly decreased after cynarin treatment ( Figure 5 B).

[0051] (3) Protein extracts from HEK293T, Hela, and THP-1 cells were mixed with cynarin and incubated at different temperatures (50, 55, 60, 65, 70, 75, and 80°C) for corresponding periods of time, and then the changes in STING protein levels were detected by Western Blot. Figure 5CE results showed that at different incubation temperatures, the protein level of TUBULIN protein in the cells did not change significantly after treatment with cynarin, while the level of STING protein in the cells gradually decreased with the increase of incubation temperature, indicating that cynarin has a certain effect on the stability of STING protein.

[0052] In summary, the present invention innovatively discovered for the first time that cynarin can inhibit the phosphorylation and translocation of STING by affecting the stability of STING's spatial conformation, thereby inhibiting the activation of downstream proteins in the pathway and hindering the nuclear translocation of IRF3, thereby inhibiting the expression of a series of interferon-induced related genes such as IFN-β, CXCL10, and ISG15, and ultimately hindering the secretion of IFN-β, thereby achieving targeted STING to inhibit the cGAS-STING inflammatory response, and providing new ideas and means for the development of auxiliary treatment methods for STING-related inflammation.

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

Claims

1. Application of cynarin or its derivatives in the preparation of inhibitors of cGAS-STING signaling pathway.

2. The use of cynarin or its derivatives as inhibitors of cGAS-STING signaling pathway in the preparation of anti-inflammatory drugs.

3. The use according to claim 1 or 2, characterized in that: Activation of the cGAS-STING signaling pathway is caused by cyclic GMP-AMP.

4. The use according to claim 1 or 2, characterized in that: The cynarin or its derivatives target STING and inhibit the cGAS-STING signaling pathway.

5. The use according to claim 1 or 2, characterized in that: The effective concentration of cynarin or its derivative is 1-10 μM.

6. The use according to claim 5, characterized in that The effective concentration of cynarin or its derivative is 5 μM.

7. The use according to claim 2, characterized in that The drug is used in at least one of the following aspects: a. Affect the stability of STING's spatial conformation to inhibit STING's phosphorylation and STING's translocation; b. Inhibit activation of proteins in the cGAS-STING signaling pathway; c. Block the nuclear translocation of IRF3; d. Inhibit the expression of interferon-induced related genes; e. Inhibit the expression and secretion of interferon.

8. The use according to claim 7, characterized in that The interferon-induction-related gene includes at least one of CXCL10, IFI16, ISG54, ISG56 and ISG58.

9. The use according to claim 7, characterized in that The interferons include IFN-β.

10. An anti-inflammatory drug, characterized in that: The main active ingredients of the medicine include cynarin or its derivatives.

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