Application of ursolic acid in preparation of medicine for preventing and treating cGAS-STING mediated diseases

Abstract

The invention belongs to the field of biomedicine and medicine, and particularly relates to application of ursolic acid to preparation of medicine for preventing and treating cGAS-STING mediated diseases. The invention discloses an application of ursolic acid (Ursolic acid) to inhibition of cGAS-STING through interaction with IRF3 (Interferon Regulating Factor 3) so as to improve diseases mediated by the cGAS-STING. Ursolic acid is wide in source, raw materials are easy to obtain, and the ursolic acid has various biological activities including anti-inflammatory, anti-oxidation and anti-tumor effects and the like. The invention provides a new medicine source for preventing, protecting, treating and researching cGAS-STING mediated diseases, is easy to popularize and apply, and can generate huge social benefits and economic benefits in a relatively short time.

Description

Technical Field

[0001] The invention belongs to the field of biomedicine and medicine, and relates to the application of ursolic acid (Ursolic Acid) in inhibiting cGAS-STING by interacting with IRF3 to improve cGAS-STING-mediated diseases, and particularly relates to the application of ursolic acid in preparing drugs for preventing and treating cGAS-STING-mediated diseases. Background Art

[0002] cGAS is a DNA recognition receptor present in the cell cytoplasm. It can recognize abnormal DNA in the cytoplasm, catalyze guanosine triphosphate (GTP) and adenosine triphosphate (ATP) to synthesize the second messenger cyclic GMP-AMP (cGAMP), further activate the simulator of interferon genes (STING), induce STING multimerization and produce a cascade reaction. On the one hand, STING oligomerization can recruit regulatory proteins such as TANK-binding kinase 1 (TBK1) and interferon regulatory factor 3 (IRF3), activate the TBK1-IRF3 pathway, and activate IRF3 to induce the expression and secretion of type I interferon and interferon-stimulated genes, and induce the production of inflammatory cytokines and IFN-β. On the other hand, cGAS-STING activation can also induce the activation of the NF-κB pathway, initiate the transcriptional maturation of inflammatory factors such as TNF-α and IL-6, and further release a large number of inflammatory factors. The cGAS-STING pathway can regulate the body's immunity, but excessive activation can lead to severe inflammatory responses. In lung sections of patients with fibrotic interstitial lung disease, STING overactivation and TBK1-IRF3 phosphorylation associated with cGAS-STING pathway activation were found. Further, in animal models, it was demonstrated that the proinflammatory type I IFN response induced by cGAS-STING pathway activation is an important mechanism for promoting lung inflammation and fibrosis; the abnormal type I IFN response induced by cGAS-STING is an important cause of cell death and tissue necrosis in the late stage of COVID-19, and is the main driving factor for the adverse consequences of COVID-19; the activation of the cGAS-STING pathway releases a large number of proinflammatory factors, leading to lysosomal dysfunction and triggering abnormal autophagy, which is the main reason for promoting the development of sepsis ALI disease.

[0003] Current studies have shown that a variety of small molecule compounds, such as quercetin, 4-octyl isocyanate, C-176, perillaldehyde, etc., have been shown to have potential inhibitory effects on cGAS-STING pathway activation. The above compounds have been tested in animal models of human diseases and have shown potential therapeutic effects. Among these compounds, C-176 is a specific inhibitor of STING, which can target and inhibit the activation of the cGAS-STING pathway, inhibit the body's inflammatory response, and play an anti-ALI effect; perillaldehyde can inhibit the cGAS-STING pathway-mediated IRF3-NF-κB signal transcription to treat sepsis ALI. In general, cGAS-STING pathway inhibitors show great potential in the treatment of cGAS-STING-mediated diseases.

[0004] Ursolic acid is a pentacyclic triterpenoid bioactive ingredient, which exists in a variety of natural plants such as fruits, vegetables and Chinese herbal medicines. Ursolic acid is distributed in about 7 families, 46 genera and 62 species of plants in free form or in the form of glycosides combined with glycosides. It can be extracted from rosemary leaves of Lamiaceae, Ligustrum lucidum of Oleaceae, pulp and peel of apple and papaya of Rosaceae, white snake grass of Rubiaceae and eucalyptus bark of Myrtaceae. It has multiple biological activities, including anti-inflammatory, antioxidant and anti-tumor effects. Ursolic acid has been shown to activate the Nrf-2-ARE pathway, reduce ROS, improve mitochondrial function, and reduce protein and lipid oxidation. It can regulate inflammatory cell function by inhibiting Toll-like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs), p38 mitogen-activated protein kinase (MAPK), phosphatidylinositol-3-hydroxylase (PI3K), nuclear factor-κB (NF-KB) and other pathways, and reduce the release of inflammatory factors such as tumor necrosis inducing factor-α (TNF-α), interleukin (IL)-1, IL-6, IL-8, IL-12, and interferon-γ (IFN-γ). It can also significantly inhibit the expression of local focal adhesion kinase genes and proteins, and prevent fibroblast activation. Although ursolic acid has shown obvious beneficial effects, its potential mechanisms and direct targets remain to be elucidated. Summary of the invention

[0005] The purpose of the present invention is to provide a new use of ursolic acid. The molecular formula of ursolic acid in the present invention is as follows:

[0006]

[0007] The present invention provides the use of ursolic acid in preparing a medicine for preventing and / or treating a cGAS-STING-mediated disease.

[0008] cGAS-STING-mediated diseases include acute lung injury, cGAS-STING-mediated autoimmune diseases, cGAS-STING-mediated neurodegenerative diseases, tumors, and cGAS-STING-mediated cardiovascular diseases.

[0009] The present invention also provides the use of ursolic acid in preparing a cGAS-STING inhibitor.

[0010] Specifically, the ursolic acid is used in at least one of the following:

[0011] 1) Application in the preparation of drugs for inhibiting HT-DNA-induced cGAS-STING signaling pathway activation;

[0012] 2) Application in the preparation of drugs for inhibiting poly(I:C)-induced activation of the cGAS-STING signaling pathway;

[0013] 3) Application in the preparation of drugs for inhibiting DMXAA-induced IRF3 nuclear translocation;

[0014] 4) Application in the preparation of drugs for inhibiting cGAMP-induced IRF3 nuclear translocation.

[0015] The present invention provides a pharmaceutical composition, comprising ursolic acid and a pharmaceutically acceptable carrier or excipient, and the pharmaceutical composition is used for preventing and / or treating cGAS-STING-mediated diseases.

[0016] As an improvement, the pharmaceutical composition is in the form of oral, injectable, inhalable or other pharmaceutically acceptable preparations.

[0017] The advantages of the present invention are:

[0018] The present invention discloses the application of ursolic acid (Ursolic Acid) which can inhibit cGAS-STING by interacting with IRF3 to improve cGAS-STING mediated diseases. Ursolic acid has a wide range of sources, and the raw materials are easily available. It has multiple biological activities, including anti-inflammatory, antioxidant and anti-tumor effects. The present invention provides a new source of drugs for preventing, protecting, treating and studying cGAS-STING mediated diseases, and is easy to promote and apply, and can generate huge social and economic benefits in a relatively short period of time. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1Figure 2 shows the effect of ursolic acid (UA) on the activation of cGAS-STING signaling pathway induced by activators HT-DNA and poly(I:C) in mouse primary bone marrow macrophages (BMDM); Figure A is the molecular structure diagram of ursolic acid, Figure B is the effect of ursolic acid on the activation of cGAS-STING signaling pathway induced by HT-DNA; Figure C is the effect of ursolic acid on the activation of cGAS-STING signaling pathway induced by poly(I:C); Figures DG are the effects of ursolic acid on the mRNA expression levels of IFN-β(D), TNF-α(E), ISC15(F), and IL-6(G) induced by HT-DNA; Figures HK are the effects of ursolic acid on the mRNA expression levels of IFN-β(D), TNF-α(E), ISC15(F), and IL-6(G) induced by poly(I:C).

[0020] Figure 2 Figure 3 shows the effect of ursolic acid (UA) on the activation of cGAS-STING signaling pathway induced by HT-DNA and poly(I:C) in human mononuclear macrophages (THP-1). Figure A shows the effect of ursolic acid on the activation of cGAS-STING signaling pathway induced by HT-DNA. Figure B shows the effect of ursolic acid on the activation of cGAS-STING signaling pathway induced by poly(I:C). Figures CF show the effects of HT-DNA-induced IFN-β (C), TNF-α (D), ISC15 (E), and IL-6 (F) mRNA expression levels. Figures GJ show the effects of ursolic acid on poly(I:C)-induced IFN-β (G), TNF-α (H), ISC15 (I), and IL-6 (J) mRNA expression levels. n=3. * p<0.05, ** p<0.01, *** p<0.0001.

[0021] Figure 3 This is a study on the mechanism of ursolic acid inhibiting the activation of cGAS-STING signaling pathway; Figure A is the immunofluorescence result; Figure B is the structure of immunoblotting; Figure C is the specific binding effect of ursolic acid on IRF3, Sting, and TBK-1; Figure DF is the specific binding effect of ursolic acid on Trex1 - / - Effects of ursolic acid on the mRNA expression levels of IFN-β (D), CXCL-10 (E), and ISC15 (F) in primary mouse bone marrow macrophages; n=3, *p<0.05, ***p<0.0001.

[0022] Figure 4Figure 1 is the effect of ursolic acid on the LPS-induced acute lung injury mouse model; Figure A is the result of H&E staining; Figures B and C are ELISA detection of the expression of inflammatory factors in bronchoalveolar lavage fluid; Figure D I is q-RCR detection of the effect of ursolic acid on inflammatory factors in the lung tissue of mice with acute lung injury, n = 3, * p<0.05, ** p<0.001, *** p<0.0001, **** p<0.0001. DETAILED DESCRIPTION

[0023] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.

[0024] Example 1. Extraction and preparation of ursolic acid

[0025] Ursolic acid is a pentacyclic triterpenoid bioactive ingredient, which exists in a variety of natural plants such as fruits, vegetables and Chinese herbal medicines. Ursolic acid is distributed in about 7 families, 46 genera and 62 species of plants in free form or in the form of glycosides combined with glycosides. It can be extracted from rosemary leaves of Lamiaceae, Ligustrum lucidum of Oleaceae, pulp and peel of apple and papaya of Rosaceae, white snake grass of Rubiaceae and eucalyptus bark of Myrtaceae.

[0026] The following is an introduction to the extraction and preparation method of ursolic acid in this method:

[0027] Take 500g of dry rosemary coarse powder, add 8 times the amount of anhydrous ethanol, reflux extraction for 2.5 hours, and filter to obtain the first extract; add 6 times the amount of anhydrous ethanol to the residue, reflux extraction for 1.5 hours; filter to obtain the second extract; combine the two extracts, concentrate under reduced pressure until there is basically no alcohol taste, cool and stand to precipitate, and filter to obtain the precipitate.

[0028] The precipitate was dissolved with chloroform, applied to silica gel column chromatography, and then eluted with chloroform, then with a chloroform-acetone (4:1) mixed solvent, and then eluted with a chloroform-acetone (3:1) mixed solvent and concentrated to obtain ursolic acid.

[0029] Example 2: Ursolic acid in vitro cell culture test

[0030] 1. Preparation of experimental materials

[0031] Mouse bone marrow primary macrophages (BMDM), human mononuclear macrophages (THP-1), ursolic acid (UA), activator HT-DNA and poly(I:C);

[0032] Cell culture media and related reagents;

[0033] ELISA kit (for detecting cytokines such as IFN-β);

[0034] Western blot reagents and antibodies (used to detect protein expression and phosphorylation levels of cGAS, STING, IRF3, NF-κB, etc.).

[0035] 2. Experimental Procedure

[0036] 2.1 Cell culture

[0037] BMDM were extracted from mouse bone marrow and cultured, and THP-1 cells were cultured in RPMI1640 medium containing 10% fetal bovine serum.

[0038] 2.2 Cell treatment

[0039] BMDM and THP-1 cells were divided into control group, HT-DNA treatment group, poly(I:C) treatment group, UA+HT-DNA group, and UA+poly(I:C) group;

[0040] Among them, the added amounts of ursolic acid (UA) in the UA+HT-DNA group and the UA+poly(I:C) group were 10μM, 20μM, and 40μM, respectively.

[0041] 2.3 Collecting samples

[0042] The cell supernatant was collected for ELISA detection, and the cell lysate was collected for Western blot analysis.

[0043] 3. Data Collection and Analysis

[0044] 3.1ELISA

[0045] ELISA kits were used to detect the secretion levels of cytokines such as IFN-β and IL-6 in the cell supernatant of each group.

[0046] 3.2 Western blot analysis

[0047] The expression and phosphorylation levels of STING, p-IRF3 and other proteins were analyzed to evaluate the activation of the cGAS-STING signaling pathway.

[0048] 4. Experimental Replication

[0049] To ensure the reliability of the experimental results, each experiment was repeated at least three times.

[0050] The test results are as follows:

[0051] In primary mouse bone marrow macrophages (BMDM), Figure 1 As shown, B: UA can dose-dependently reduce HT-DNA-induced IRF3 phosphorylation; C: UA can dose-dependently reduce HT-DNA-induced IRF3 phosphorylation; (DG) UA can dose-dependently reduce the mRNA expression levels of HT-DNA-induced IFN-β (D), TNF-α (E), ISC15 (F), and IL-6 (G); (HK) UA can dose-dependently reduce the mRNA expression levels of poly(I:C)-induced IFN-β (H), TNF-α (I), ISC15 (J), and IL-6 (K); n = 3, * p<0.05, ** p<0.01, *** p<0.0001.

[0052] In human mononuclear macrophages (THP-1), Figure 2 As shown, A: UA can dose-dependently reduce HT-DNA-induced IRF3 phosphorylation; B: UA can dose-dependently reduce HT-DNA-induced IRF3 phosphorylation; (CF) UA can dose-dependently reduce the mRNA expression levels of HT-DNA-induced IFN-β (C), TNF-α (D), ISC15 (E), and IL-6 (F); (GK) UA can dose-dependently reduce the mRNA expression levels of poly(I:C)-induced IFN-β (G), TNF-α (H), ISC15 (I), and IL-6 (J); n = 3, * p<0.05, ** p<0.01, *** p<0.0001.

[0053] Example 3: Study on the mechanism of UA inhibiting the activation of cGAS-STING signaling pathway

[0054] The test steps are as follows:

[0055] 1. Preparation of experimental materials

[0056] Mouse bone marrow primary macrophages (including Trex1- / - mouse bone marrow primary macrophages);

[0057] Ursolic acid (UA);

[0058] activators DMXAA and cGAMP;

[0059] Cell culture media and related reagents;

[0060] Immunofluorescence staining reagents and antibodies (against IRF3);

[0061] Western blot reagents and antibodies (against IRF3);

[0062] RT-qPCR reagents and primers (for IFN-β, CXCL-10, ISC15).

[0063] 2. Experimental Procedure

[0064] 2.1 Cell culture

[0065] Primary macrophages were extracted from mouse bone marrow and cultured in DMEM medium containing 10% fetal bovine serum.

[0066] 2.2 Preprocessing

[0067] The cells were divided into control group, UA treatment group, DMXAA treatment group, cGAMP treatment group, and UA+DMXAA, UA+cGAMP treatment groups.

[0068] The cells in the UA treatment group were treated with UA for a certain period of time (eg, 1 hour) in advance.

[0069] 2.3 Stimulating cells

[0070] The control group received no treatment.

[0071] The cells in the DMXAA-treated group and the cGAMP-treated group were stimulated with DMXAA and cGAMP, respectively.

[0072] The UA+DMXAA treatment group and the UA+cGAMP treatment group were pretreated with UA and then stimulated with DMXAA and cGAMP.

[0073] 2.4 Immunofluorescence

[0074] The cells were fixed and permeabilized, and immunofluorescence staining was performed using an antibody against IRF3 to observe the nuclear localization of IRF3.

[0075] 2.5 Western blot

[0076] Cell lysates were collected and subjected to Western blot analysis using an antibody against IRF3 to assess the nuclear localization of IRF3.

[0077] 2.6 Co-immunoprecipitation

[0078] Co-immunoprecipitation experiments were performed to verify the specific binding of UA to IRF3 protein.

[0079] 2.7 mRNA expression level detection

[0080] Primary bone marrow macrophages from Trex1- / - mice were collected, and RT-qPCR was used to detect the expression levels of IFN-β, CXCL-10, and ISC15 mRNA.

[0081] 3. Data Collection and Analysis

[0082] Collect immunofluorescence results using flow cytometry or fluorescence microscopy.

[0083] Western blot analysis software was used to analyze the nuclear localization of IRF3.

[0084] RT-qPCR analysis software was used to calculate the relative expression levels of IFN-β, CXCL-10, and ISC15 mRNA.

[0085] like Figure 3 As shown, A: Immunofluorescence results showed that UA could inhibit the nuclear translocation of IRF3 induced by the activator DMXAA in primary bone marrow macrophages of mice; B: Western blotting showed that UA could reduce the nuclear translocation of IRF3 induced by cGAMP; (C) UA could specifically bind to IRF3 protein; (DF) In primary bone marrow macrophages of Trex1- / - mice, UA could significantly reduce the mRNA expression levels of IFN-β (D), CXCL-10 (E), and ISC15 (F); n=3, *p<0.05, ***p<0.0001.

[0086] Example 4: Animal Test of Ursolic Acid

[0087] Purpose

[0088] To evaluate the protective effects of ursolic acid (UA) on LPS-induced acute lung injury mouse model, including the effects on lung tissue pathological changes, inflammatory factor expressions and related gene mRNA levels.

[0089] 1. Preparation of experimental materials

[0090] Clean grade mice, lipopolysaccharide (LPS), C-176, ursolic acid (UA), H&E staining reagents, ELISA kits (for detection of IL-6 and TNF-α, Western blot reagents and antibodies (for IRF3), RT-qPCR reagents and primers (for IL-6, CXCL-10, Ifit-1, IFN-β, ISG-15, TNF-α).

[0091] 2. Experimental Animal Grouping

[0092] The mice were randomly divided into a control group, an LPS-induced group, an LPS+C-176 group, an LPS+UA (20 mg / kg) group, and an LPS+UA (40 mg / kg) group (each group had at least 3 mice, n=3).

[0093] 3. Induction of Acute Lung Injury

[0094] The control group was given normal saline.

[0095] The LPS-induced group was given LPS to induce acute lung injury.

[0096] The UA-treated group was given different doses of UA at the same time as LPS.

[0097] The LPS+C-176 group was given 15 mg / kg of C-176 at the same time as LPS.

[0098] 4. Sample Collection

[0099] 24 h after administration, lung tissues and bronchoalveolar lavage fluid were collected from mice.

[0100] 5. Experimental Procedure

[0101] 5.1 Pathological evaluation

[0102] Lung tissue sections were stained with H&E staining to observe and evaluate the hemorrhage and inflammatory infiltration of lung tissue.

[0103] 5.2 Detection of inflammatory factors

[0104] ELISA kits were used to detect the expression levels of IL-6 and TNF-α in bronchoalveolar lavage fluid.

[0105] 5.3 Protein expression analysis

[0106] Western blot was used to analyze the effect of UA on cGAMP-induced IRF3 nuclear translocation.

[0107] 5.4 Gene expression analysis

[0108] RT-qPCR was used to detect the expression levels of IL-6, CXCL-10, Ifit-1, IFN-β, ISG-15, and TNF-α mRNA in lung tissues.

[0109] like Figure 4As shown, A: H&E staining results, UA can reduce the bleeding and inflammatory infiltration of lung tissue in mice with acute lung injury in a dose-dependent manner; (B, C) ELISA detected the expression of inflammatory factors in bronchoalveolar lavage fluid, and UA could dose-dependently reduce the production of serum IL-6 (B) and TNF-α (C) in mice with acute lung injury model; Western blotting showed that UA could reduce the nuclear entry of IRF3 induced by cGAMP; (D I) q-RCR detected the effect of UA on inflammatory factors in lung tissue of mice with acute lung injury, and UA could significantly reduce the mRNA expression levels of IL-6 (D), CXCL-10 (E), Ifit-1 (F), IFN-β (G), ISG-15 (H), and TNF-α (I) in lung tissue; n = 3, * p<0.05, ** p<0.001, *** p<0.0001, **** p<0.0001.

[0110] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. Use of ursolic acid in the preparation of drugs for preventing and / or treating cGAS-STING mediated diseases.

2. The application according to claim 1, characterized in that: The cGAS-STING mediated diseases include acute lung injury.

3. Application of ursolic acid in the preparation of cGAS-STING inhibitors.

4. The use according to claim 3, characterized in that: The application of ursolic acid in at least one of the following: 1) Application in the preparation of drugs for inhibiting HT-DNA-induced cGAS-STING signaling pathway activation; 2) Application in the preparation of drugs for inhibiting poly(I:C)-induced activation of the cGAS-STING signaling pathway; 3) Application in the preparation of drugs for inhibiting DMXAA-induced IRF3 nuclear translocation; 4) Application in the preparation of drugs for inhibiting cGAMP-induced IRF3 nuclear translocation.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises ursolic acid and a pharmaceutically acceptable carrier or excipient, and the pharmaceutical composition is used for preventing and / or treating cGAS-STING-mediated diseases.

6. A pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition is in the form of oral, injection, inhalation or other pharmaceutically acceptable preparations.

7. The use according to claim 1, characterized in that: The molecular formula of the ursolic acid is:

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