Use of dihydrotanshinone I in the preparation of a drug for treating and / or preventing a TREX1 gene defect related self-inflammatory disease

Abstract

Use of dihydrotanshinone I or a pharmaceutically acceptable salt thereof in the preparation of a drug for treating and / or preventing a TREX1 gene defect related self-inflammatory disease. It is found that dihydrotanshinone I can effectively inhibit the activation of the cGAS-STING pathway and inhibit the nuclear entry of IRF3 and P65 in diABZI induced BMDMs cells. Further, it is found that dihydrotanshinone I can significantly alleviate the inflammatory response caused by the deletion of the Trex1 gene in mice ‑ / ‑ significantly inhibit the expression of type I interferon related genes (IFN-beta, ifit1, ifit2, Isg15 and Rsad2) in mouse bone marrow derived BMDMs, and dihydrotanshinone I can significantly alleviate the inflammatory response caused by the deletion of the Trex1 gene in mice ‑ / ‑ inflammatory response caused by the deletion of the TREX1 gene in mice.

Description

Technical Field

[0001] This article relates to the field of biomedical technology, and in particular to the use of dihydrotanshinone I or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the treatment and / or prevention of autoinflammatory diseases associated with TREX1 gene defects. Background Technology

[0002] Autoinflammatory diseases (AUIDs) are inflammatory disorders caused by excessive activation of inflammation without antigen dependence. Based on their triggering factors, they can be broadly categorized into diseases mediated by abnormalities in inflammasomes and related IL-1 family cytokines, abnormal interferon production and signal transduction, and abnormal NF-κB or TNF-α activity. Abnormal interferon production and signal transduction is the most common type. Current research indicates that AUIDs are primarily related to genetic and environmental factors, with gene mutations playing a crucial role in their development. TREX1 is a major cytoplasmic nuclease that mediates the degradation of 3'-terminal mismatched oligonucleotides, playing a vital role in cytoplasmic DNA clearance and base mismatch recombination repair. TREX1 gene mutations lead to cytoplasmic DNA aggregation, binding of DNA to the cGAS receptor, and subsequent activation of the cGAS-STING (stimulator of interferon genes) pathway, thus inducing AUIDs. Multiple clinical studies have shown that human TREX1 gene mutations are associated with the development of various AUIDs. Mutations in the N-terminal DNase domain of TREX1 leading to loss of enzyme activity are a key mechanism inducing Aicardi-Goutieres (AGS) syndrome.

[0003] Currently, there is an urgent need to develop a highly effective and safe drug for treating autoinflammatory diseases caused by TREX1 gene mutations. Summary of the Invention

[0004] Based on the above background, this application screened natural small molecule compounds based on the cGAS-STING signaling pathway and discovered that dihydrotanshinone I, an active ingredient in the traditional Chinese medicine Danshen, can significantly inhibit the activation of the cGAS-STING signaling pathway. Further research confirmed that dihydrotanshinone I can significantly alleviate Trex1. - / - The multi-organ inflammatory response in mice suggests that dihydrotanshinone I could be a potential therapeutic agent for TREX1 gene deletion-related autoinflammatory diseases.

[0005] In one aspect, this application provides the use of dihydrotanshinone I or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the treatment and / or prevention of autoinflammatory diseases associated with TREX1 gene defects.

[0006] On the other hand, this application also provides the use of dihydrotanshinone I or a pharmaceutically acceptable salt thereof in the preparation of a medicament that inhibits the aberrant activation of the cGAS-STING signaling pathway.

[0007] Dihydrotanshinone I (DHT) has the following structural formula:

[0008]

[0009] In this application, the applicant constructed an HT-DNA-mediated cGAS-STING pathway activation model using human monocyte-macrophages (THP-1) and normal mouse bone marrow-derived macrophages (BMDMs). The expression and phosphorylation levels of STING and IRF3 proteins were detected by Western blotting, the IFN-β content in the cell supernatant was detected by ELISA, and the inhibitory effect of dihydrotanshinone I on the mRNA levels of related genes (IFN-β, IL-6, Cxcl10, and TNF-α) was assessed by RT-qPCR. It was found that dihydrotanshinone I inhibited cGAS-STING pathway activation. It was also verified that dihydrotanshinone I can inhibit the activation of the cGAS-STING pathway induced by multiple STING agonists (HT-DNA, DMXAA, diABZI, and cGAMP).

[0010] In addition, the applicant used Trex1 - / - Mouse bone marrow-derived BMDMs, when treated with dihydrotanshinone I, were found to significantly inhibit the expression of type I interferon-related genes (IFN-β, ifit1, ifit2, Isg15, and Rsad2).

[0011] Animal experiments used 3-week-old Trex1 cells - / - Mice were intraperitoneally injected with dihydrotanshinone I for 2 weeks. The effects of dihydrotanshinone I on target organ damage in mice, including the heart, liver, tongue, spleen, lungs, and kidneys, were analyzed. The effect of dihydrotanshinone I on the expression of genes related to the cGAS-STING pathway was analyzed using RT-PCR. The improvement of inflammation in each organ was evaluated using H&E staining. The overall effect of dihydrotanshinone I on systemic inflammatory response induced by TREX1 gene deficiency was comprehensively evaluated. It was found that dihydrotanshinone I significantly inhibited TREX1. - / - In mice, TREX1 gene deletion led to elevated mRNA levels of type I interferon-related genes (IFN-β, Isg15, and Rsad2); on the other hand, H&E staining results indicated that dihydrotanshinone I significantly alleviated TREX1... - / - Systemic inflammatory response induced by TREX1 gene deletion in mice. This suggests that dihydrotanshinone I may be a therapeutic agent for autoinflammatory diseases associated with TREX1 gene deficiency.

[0012] Other features and advantages of this application will be set forth in the following description, and will be apparent in part from the description, or may be learned by practicing the application. Other advantages of this application can be realized and obtained by means of the solutions described in the description and the accompanying drawings. Attached Figure Description

[0013] The accompanying drawings are used to provide an understanding of the technical solutions of this application and constitute a part of the specification. They are used together with the embodiments of this application to explain the technical solutions of this application and do not constitute a limitation on the technical solutions of this application.

[0014] Figure 1 This study demonstrates that dihydrotanshinone I significantly inhibits the activation of the cGAS-STING pathway in mouse bone marrow macrophages (BMDMs). HT-DNA is the activator of the cGAS-STING signaling pathway, p-IRF3 is the phosphorylated form of IRF3 protein, and p-STING is the phosphorylated form of STING protein. Increased p-IRF3 and p-STING indicate enhanced activation of the cGAS-STING signaling pathway. HSP90 is an internal control protein.

[0015] Figure 2 This study demonstrated that dihydrotanshinone I significantly inhibited the activation of the cGAS-STING pathway in human monocyte-macrophages (THP-1).

[0016] Figure 3 This study demonstrated that dihydrotanshinone I significantly inhibited the activation of the cGAS-STING pathway induced by multiple factors. Among them, HT-DNA, DMXAA, diABZI, and cGAMP were agonists of the cGAS-STING signaling pathway, while DMSO served as a blank control.

[0017] Figure 4 This demonstrates that dihydrotanshinone I inhibits Trex1. - / - Activation of the cGAS-STING pathway in mouse BMDMs cells.

[0018] Figure 5 This study demonstrated that dihydrotanshinone I significantly alleviated the systemic inflammatory response induced by TREX1 gene deletion. Specifically, WTvehicle represents wild-type mice injected with vehicle (5% DMSO, 20% PEG300, and 75% saline), WTDHT represents wild-type mice injected with dihydrotanshinone I, and KO vehicle represents gene knockout mice (i.e., TREX1). - / - Mice were injected with a vehicle (5% DMSO, 20% PEG300 and 75% saline), and KO DHT knockout mice were injected with dihydrotanshinone I. Detailed Implementation

[0019] Unless otherwise stated, the technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art to which this application pertains. When a quantity, concentration, or other value or parameter is expressed as a range, preferred range, or preferred upper and lower numerical limits, it should be understood that this is equivalent to specifically disclosing any range by combining any pair of upper or preferred values ​​with any lower or preferred value, regardless of whether the range is specifically disclosed. Unless otherwise stated, the numerical ranges listed herein are intended to include the endpoints of the range and all integers and fractions (decimals) within that range.

[0020] When used with a numerical variable, the terms "about" or "approximately" usually mean that the value of the variable and all values ​​of the variable are within the experimental error (e.g., within a 95% confidence interval for the mean) or within ±10% of the specified value, or a wider range.

[0021] The expression "comprising," or similar expressions such as "including," "containing," and "having," is open-ended and does not exclude additional unlisted elements, steps, or components. The expression "consisting of," excludes any unspecified elements, steps, or components. The expression "substantially consisting of," limits the scope to the specified elements, steps, or components, plus optional elements, steps, or components that do not materially affect the essential and novel features of the claimed subject matter. It should be understood that the expression "comprising" encompasses both the expressions "substantially consisting of" and "consisting of."

[0022] The expression "at least one" or "one or more" indicates 1, 2, 3, 4, 5, 6, 7, 8, 9 or more kinds.

[0023] Mutations at different sites in the TREX1 gene can lead to various types of diseases. Mutations in the N-terminal DNase domain of the TREX1 gene mainly occur in patients with AGS. Mutations in its catalytic core and C-terminal domains are involved in mediating the development of systemic lupus erythematosus (SLE). In addition, frameshift mutations in the C-terminal region of TREX1 are involved in mediating retinal vascular disease and retinal leukodystrophy (RVCL). Although C-terminal mutations do not affect the catalytic function of the nuclease, they can cause the protein to be unable to be properly localized in the endoplasmic reticulum in the internuclear space. It is evident that both the loss of TREX1 enzyme activity and altered protein localization can lead to the development of related autoinflammatory diseases.

[0024] Mutations in the TREX1 gene lead to loss of enzyme activity and site alterations, preventing TREX1 from functioning properly. This results in cytoplasmic DNA aggregation, activating cGAS. Activated cGAS catalyzes the synthesis of cGAMP, which then binds to STING, inducing its oligomerization and phosphorylation. Activated STING recruits proteins such as TANK-binding kinase 1 (TBK1), IκB kinase (IKK), and interferon regulatory factor 3 (IRF3), mediating the phosphorylation of p65 and IRF3 into the nucleus, thereby inducing the expression of inflammatory factors such as IFN-β and TNF-α. Studies show that activation of the cGAS-STING pathway not only mediates TREX1 gene-deficient inflammatory diseases but also participates in inducing liver inflammation and fibrosis, and can mediate non-alcoholic fatty liver disease by inducing insulin resistance and lipid deposition. Furthermore, cGAS / cGAMP-independent STING activation can also induce the sustained expression and secretion of downstream inflammatory factors, leading to tissue inflammatory damage and participating in the induction of diseases such as COPA syndrome caused by mutations in the colysate protein complex α subunit COPα, NPC caused by loss of function of Niemann-Pick disease type C1 (NPC1), and STING-associated vasculopathy with onset ininfancy (SAVI) caused by gain-of-function mutations in STING. Therefore, the sustained activation of the cGAS-STING pathway is involved in the occurrence of various inflammatory diseases. Targeted inhibition of cGAS-STING can not only be used for the treatment of TREX1 gene defect-related inflammatory diseases, but also has important value for the prevention and treatment of other diseases related to the cGAS-STING pathway.

[0025] Due to Trex1 - / - It can cause sustained activation of the cGAS-STING pathway and induce inflammatory pathological damage in multiple organs such as the liver, kidneys, and heart in mice, ultimately leading to death from cardiovascular inflammation in young mice. Multiple studies have shown that knocking out the cGAS / Sting / Irf gene can effectively improve the autoinflammatory disease caused by TREX1 gene deficiency in mice and successfully rescue Trex1. - / - The death of mice and the inhibition of disease progression such as myocardial inflammation indicate that the cGAS-STING pathway is continuously activated in Trex1. - / - TREX1 plays a crucial role in the development of autoinflammatory diseases in mice, and targeting and inhibiting the cGAS-STING pathway is an important strategy and target for treating TREX1 gene-related autoinflammatory diseases.

[0026] The continuous activation of the cGAS-STING pathway is involved in the occurrence of various diseases, with TREX1 gene-deficient autoinflammatory diseases being the most typical example. Therefore, this application first elucidates the effect and target mechanism of dihydrotanshinone I in inhibiting the cGAS-STING pathway.

[0027] TREX1 gene mutations and loss of activity lead to cytoplasmic DNA accumulation, which binds to the cGAS receptor, thereby activating the cGAS-STING pathway and inducing autoinflammatory diseases. Therefore, targeting and inhibiting the cGAS-STING pathway is an important target and strategy for treating TREX1 gene deficiency-related autoinflammatory diseases. This application focuses on dihydrotanshinone I to improve TREX1 gene deficiency autoinflammatory diseases and inhibit the cGAS-STING pathway, ultimately demonstrating the objectivity of dihydrotanshinone I in treating TREX1 gene deficiency autoinflammatory diseases, and providing a potential drug candidate for the development of therapeutic drugs for TREX1 gene deficiency autoinflammatory diseases.

[0028] In one aspect, this application provides the use of dihydrotanshinone I or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the treatment and / or prevention of autoinflammatory diseases associated with TREX1 gene defects. A "pharmaceutically acceptable salt" is a salt of a compound that can be formulated for use in a medicament, including, for example, metal salts (sodium, potassium, magnesium, calcium, etc.) and salts of ammonia or organic amines.

[0029] In some implementations, autoinflammatory diseases associated with TREX1 gene deficiency include Aicardi-Guoutieres syndrome (AGS), familial lupus erythematosus (FCL), and systemic lupus erythematosus (SLE). In some implementations, the autoinflammatory disease associated with TREX1 gene deficiency is Aicardi-Guoutieres syndrome.

[0030] In some implementations, the drug is administered orally, intravenously, intradermally, transdermally, intrathecally, intra-arterially, intraperitoneally, intranasally, intravaginally, intrarectally, intrabladderally, intratumorally, locally, intramuscularly, subcutaneously, via mucosally, by inhalation, injection, infusion, or any combination thereof.

[0031] In some embodiments, the pharmaceutical preparations prepared in this application may be administered intravenously, intradermally, percutaneously, intrathecally, intra-arterially, intraperitoneally, intranasally, intravaginally, intrarectally, intravesically (e.g., directly administered to the bladder via injection or intravesical infusion), intratumorally, locally, intramuscularly, subcutaneously, mucosally, orally, locally, inhaled (e.g., aerosol inhalation), injected, infused, continuously infused, via catheter, via irrigation with emulsion, via direct local perfusion with liquid composition (e.g., liposomes), or by other methods as known to those skilled in the art or any combination thereof.

[0032] In some implementations, the dosage form of the drug is selected from one or more of the following: capsules, tablets, pills, liquids, powders, granules, fine granules, film-coated agents, pills, lozenges, sublingual tablets, gels, sublingual preparations, pastes, syrups, suspensions, elixirs, emulsions, coatings, ointments, plasters, mud dressings, transdermal preparations, lotions, inhalers, aerosols, injections, or suppositories.

[0033] In some embodiments, the drug also comprises pharmaceutically acceptable excipients. "Pharmaceutically acceptable excipients" means excipients that are generally safe, non-toxic, and desirable for use in the preparation of pharmaceutical compositions, and includes excipients acceptable for both veterinary and human use. Such excipients can be solid, liquid, semi-solid, or, in the case of aerosol compositions, gaseous. Pharmaceutical-grade organic or inorganic carriers and / or diluents suitable for oral and topical application can be used to formulate compositions containing therapeutically active compounds. Diluents known in the art include aqueous media, vegetable and animal oils, and fats. Stabilizers, wetting agents and emulsifiers, salts for altering osmotic pressure or buffers for ensuring appropriate pH, and skin penetration enhancers can be used as adjuvants. Non-limiting examples of excipients include granulators, binders, lubricants, disintegrants, sweeteners, gliding agents, anti-sticking agents, antistatic agents, surfactants, antioxidants, gums, coating agents, colorants, flavoring agents, plasticizers, preservatives, suspending agents, emulsifiers, antimicrobial agents, plant cellulose materials, and pelleting agents, and any combination thereof.

[0034] In some implementations, the subjects are mammals; optionally, mammals include humans, rats, mice, cats, dogs, horses, sheep, cows, or monkeys. Illustrative examples of subjects in some implementations include primates, particularly humans; companion animals such as cats and dogs and similar animals; working animals such as horses, donkeys, and similar animals; livestock animals such as sheep, cattle, goats, pigs, and similar animals; laboratory test animals such as rabbits, mice, rats, guinea pigs, hamsters, and similar animals; and captive wild animals such as captive wild animals in zoos and wildlife parks, deer, dingoes, and similar animals.

[0035] In some implementations, the subjects are humans.

[0036] On the other hand, this application also provides the use of dihydrotanshinone I or a pharmaceutically acceptable salt thereof in the preparation of a medicament that inhibits the aberrant activation of the cGAS-STING signaling pathway.

[0037] In some embodiments, the drug prepared in this application is an inhibitor of abnormal activation of the cGAS-STING signaling pathway. Abnormal activation of the cGAS-STING signaling pathway includes Trex1 gene mutations, such as Trex1 gene defects, and induction by interferon inducers. Abnormal activation of the cGAS-STING signaling pathway can induce various diseases and complications, including but not limited to AGS syndrome. An inhibitor is a compound that inhibits, partially or completely blocks stimulation or activation, reduces, prevents, delays activation, inactivates, desensitizes, or downregulates physiological / cellular processes. An agonist is a compound that induces, activates, stimulates, increases, promotes, enhances activation, sensitizes, or upregulates at least one physiological / cellular process. The inhibitor prepared in this application can reduce the level of type I interferon by inhibiting abnormal activation of the cGAS-STING signaling pathway.

[0038] This application describes several embodiments, but these descriptions are exemplary and not restrictive, and it will be apparent to those skilled in the art that many more embodiments and implementations are possible within the scope of the embodiments described herein. Although many possible combinations of features are shown in the accompanying drawings and discussed in the detailed description, many other combinations of the disclosed features are also possible. Unless specifically limited, any feature of any embodiment may be used in combination with any other feature in any other embodiment, or may replace any other feature in any other embodiment.

[0039] This application includes and contemplates combinations of features known to those skilled in the art. The embodiments and features disclosed in this application can also be combined with any conventional features to form a unique inventive scheme as defined by the claims. Any feature of any embodiment can also be combined with features from other inventive schemes to form another unique inventive scheme as defined by the claims. Therefore, it should be understood that any feature shown and / or discussed in this application can be implemented individually or in any suitable combination. Therefore, the embodiments are not limited except by the limitations imposed by the appended claims and their equivalents. Furthermore, various modifications and changes can be made within the scope of the appended claims.

[0040] In addition, when describing representative embodiments, the specification may have presented the method and / or process as a particular sequence of steps. However, to the extent that the method or process does not depend on the particular order of the steps described herein, the method or process should not be limited to the particular order of steps described. As will be understood by those of ordinary skill in the art, other step sequences are possible. Therefore, the particular order of steps set forth in the specification should not be construed as a limitation on the claims. In addition, the claims directed to the method and / or process should not be limited to performing their steps in the order written, as those skilled in the art can readily understand that these orders can vary and still remain within the spirit and scope of the embodiments of the present application.

[0041] For the experimental methods without specific conditions indicated in the following embodiments, they are generally determined according to national standards. The experimental materials without sources indicated in the following embodiments are all commercially available raw materials. The equipment used in each step of the following embodiments is all conventional equipment. If there is no corresponding national standard, it is carried out according to general international standards, conventional conditions, or according to the conditions recommended by the manufacturer. Unless otherwise defined or stated, all professional and scientific terms used in this application have the same meaning as those familiar to persons skilled in the art. In addition, any methods and materials similar or equivalent to the recorded content can be applied to the methods of this application.

[0042] Embodiment

[0043] 1. Reagents and Cells

[0044] Dihydrotanshinone I (HY-N0360, MCE); DMEM (Dulbecco's Modified Eagle's Medium, CM10013) and RPMI-1640 (Roswell Park Memorial Institute-1640, CM10040) culture media were purchased from Zhongke Maichen (Beijing) Technology Co., Ltd.; mouse IFN-β ELISA kit ((luex-mIFN-β v2, InvivoGen); StarFect transfection reagent (C101-10) was purchased from Genstar; serum (FBS, C04001) was purchased from VivaCell; DMXAA, cGAMP and diABZI were purchased from Sigma-Aldrich; THP-1 cells were purchased from ATCC.

[0045] 2. Animals

[0046] 8-10 week-old C57BL / 6 mice, purchased from Spf Biotechnology Co., Ltd. (Beijing, China), with the license number SCXK (Beijing 2019-0010). Trex1 - / -Mice were purchased from Jackson Laboratory (Genetics Research, BarHarbor, Maine, USA).

[0047] 2. Experimental Methods

[0048] In this application, immunoblotting, ELISA, and RT-qPCR are used to detect the expression levels of various proteins or mRNAs. These methods are all conventional methods well known to those skilled in the art.

[0049] Example 1. Dihydrotanshinone I inhibited HT-DNA-induced activation of the cGAS-STING pathway and the level of type I interferon-related genes in BMDMs cells.

[0050] Humerus bones were harvested from 8-10 week old C57BL / 6 mice. The bone marrow was washed three times, and the bone marrow cells were cultured in DMEM medium for 5 days (supplemented with 50 ng / ml M-CSF). M-CSF was supplemented once during the culture period to obtain BMDMs suitable for experimental use. The cells were cultured at a rate of 1 x 102... 6 BMDMs were seeded into 24-well plates at the specified density and incubated overnight. The liquid in the plates was discarded, and Opti-MEM containing dihydrotanshinone I (1.25 μmol / L, 2.5 μmol / L, 5 μmol / L) was added. After 1 hour, 1 μg / mL of HT-DNA was added. After 2 hours, the supernatant was discarded, and SDS buffer was added for subsequent experiments.

[0051] The inhibitory effect of dihydrotanshinone I on the cGAS-STING signaling pathway was assessed by immunoblotting. Western blot assay: Cells were lysed, and the expression levels of phosphorylated IRF3 (p-IRF3), IRF3, phosphorylated STING (p-STING), STING, and HSP90 proteins in the cell lysate were detected.

[0052] Cell supernatant was collected, and the inhibitory effect of dihydrotanshinone I on IFN-β levels in BMDMs cell supernatant was assessed by ELISA. Cells were lysed, and the inhibitory effect of dihydrotanshinone I on the mRNA levels of related genes (IFN-β, IL-6, Cxcl10, and TNF-α) was assessed by RT-qPCR.

[0053] Experimental results:

[0054] Depend on Figure 1It was found that dihydrotanshinone I dose-dependently inhibited the expression of p-IRF3 and p-STING in BMDM cell lysates, but had no effect on the expression of IRF3 and STING. ELISA results showed that dihydrotanshinone I dose-dependently inhibited the expression of IFN-β in BMDM cell supernatant. RT-qPCR results showed that dihydrotanshinone I dose-dependently inhibited the expression of IFN-β and interferon-stimulated genes (IFN-β, IL-6, Cxcl10, and TNF-α) mRNA levels in BMDM cell lysates, indicating that dihydrotanshinone I dose-dependently inhibited the activation of the cGAS-STING pathway in BMDM cells.

[0055] Example 2. Dihydrotanshinone I inhibited HT-DNA-induced activation of the cGAS-STING pathway and the level of type I interferon-related genes in THP1 cells.

[0056] THP1 cells were grown at a concentration of 1.5 x 10⁻⁶. 6 The concentration of the HT-DNA will be seeded in 48-well plates overnight. The liquid in the plates will be discarded, and Opti-MEM containing dihydrotanshinone I (1.25 μmol / L, 2.5 μmol / L, 5 μmol / L) will be added. After 1 hour, 1 μg / mL of HT-DNA will be added. After 2 hours, the supernatant will be discarded, and SDS buffer will be added for subsequent experiments.

[0057] The inhibitory effect of dihydrotanshinone I on the cGAS-STING signaling pathway was assessed by immunoblotting. Western blot assay: Cells were lysed, and the expression levels of phosphorylated IRF3 (p-IRF3), IRF3, phosphorylated STING (p-STING), STING, and HSP90 proteins in the cell lysate were detected.

[0058] Cell supernatant was collected, and the inhibitory effect of dihydrotanshinone I on IFN-β levels in THP1 cell supernatant was assessed by ELISA. Cells were lysed, and the inhibitory effect of dihydrotanshinone I on mRNA levels of related genes (IFN-β, IL-6, Cxcl10, and TNF-α) was assessed by RT-qPCR.

[0059] Experimental results:

[0060] Depend on Figure 2It was found that dihydrotanshinone I dose-dependently inhibited the expression of p-IRF3 and p-STING in THP1 cell lysates, but had no effect on the expression of IRF3 and STING. ELISA results showed that dihydrotanshinone I dose-dependently inhibited the expression of IFN-β in THP1 cell supernatant. RT-qPCR results showed that dihydrotanshinone I dose-dependently inhibited the expression of IFN-β and interferon-stimulated genes (IFN-β, IL-6, Cxcl10 and TNF-α) mRNA levels in THP1 cell lysates, indicating that dihydrotanshinone I dose-dependently inhibited the activation of the cGAS-STING pathway in THP1 cells.

[0061] Example 3. Dihydrotanshinone I inhibits the activation of the cGAS-STING pathway in BMDMs cells induced by multiple factors.

[0062] Humerus bones were harvested from 8-10 week old C57BL / 6 mice. The bone marrow was washed three times, and the bone marrow cells were cultured in DMEM medium for 5 days (supplemented with 50 ng / ml M-CSF). M-CSF was supplemented once during the culture period to obtain BMDMs suitable for experimental use. The cells were cultured at a rate of 1 x 102... 6 BMDMs were seeded into 24-well or 12-well plates at the specified density and incubated overnight. The liquid in the plates was discarded, and Opti-MEM containing dihydrotanshinone I (5 μmol / L) was added. DMSO was added as a blank control. After 1 hour, HT-DNA (1 μg / mL), DMXAA (25 μg / mL), diABZI (25 μg / mL), and cGAMP (2 μg / mL) were added. After 2 or 4 hours, the supernatant was discarded, and SDS buffer or Trizol reagent was added for subsequent experiments.

[0063] The inhibitory effect of dihydrotanshinone I on the cGAS-STING signaling pathway was assessed by immunoblotting. Western blot assay: Cells were lysed, and the expression levels of phosphorylated IRF3 (p-IRF3), IRF3, phosphorylated STING (p-STING), STING, and HSP90 proteins in the cell lysate were detected.

[0064] Cell supernatant was collected, and the inhibitory effect of dihydrotanshinone I on IFN-β levels in BMDMs cell supernatant was assessed by ELISA. Cells were lysed, and the inhibitory effect of dihydrotanshinone I on the mRNA levels of related genes (IFN-β, IL-6, Cxcl10, and TNF-α) was assessed by RT-qPCR.

[0065] Experimental results:

[0066] Depend on Figure 3It was found that dihydrotanshinone I inhibited the expression of p-IRF3 and p-STING in BMDM cell lysates induced by various STING agonists (HT-DNA, DMXAA, diABZI, and cGAMP), but had no effect on the expression of IRF3 and STING. ELISA results showed that dihydrotanshinone I also inhibited the expression of IFN-β in BMDM cell supernatant. RT-qPCR results showed that dihydrotanshinone I also inhibited the expression of IFN-β and interferon-stimulated genes (IFN-β, IL-6, Cxcl10, and TNF-α) mRNA levels in BMDM cell lysates, indicating that dihydrotanshinone I can inhibit the activation of the cGAS-STING pathway induced by various agonists.

[0067] Example 4. Dihydrotanshinone I inhibits Trex1 - / - Activation of the cGAS-STING pathway in mouse BMDMs cells

[0068] Next, we will investigate the effect of dihydrotanshinone I on TREX1 gene knockout (Trex1). - / - The effect of type I interferon secretion in BMDMs of 3-week-old Trex1 mice. - / - Mouse humeral bone marrow was washed three times, and bone marrow cells were cultured in DMEM medium for 5 days (supplemented with 50 ng / ml M-CSF), with one additional M-CSF addition during the period, to obtain Trex1 cells suitable for experimental use. - / - BMDMs in mice. (According to 1x10) 6 The density of Trex1 - / - Mouse BMDMs were seeded in 12-well plates and incubated overnight. The liquid in the wells was discarded, and opti-MEM containing dihydrotanshinone I (5 μmol / L) was added. DMSO was added as a blank control. After 8 hours, the supernatant was discarded, and SDS buffer was added for subsequent experiments.

[0069] Cells were lysed, and wild-type mouse BMDMs were used as negative controls. The inhibition of mRNA levels of type I interferon-related genes (IFN-β, ifit1, ifit2, Isg15, and Rsad2) by dihydrotanshinone I treatment was assessed by RT-qPCR.

[0070] Experimental results:

[0071] Depend on Figure 4It was found that after TREX1 gene knockout, the expression of type I interferon-related genes (IFN-β, ifit1, ifit2, Isg15 and Rsad2) in BMDMs cells was significantly increased, but dihydrotanshinone I treatment could significantly inhibit the expression of type I interferon-related genes (IFN-β, ifit1, ifit2, Isg15 and Rsad2).

[0072] Example 5. Dihydrotanshinone I significantly alleviated systemic inflammatory response caused by TREX1 gene deletion.

[0073] In Trex1 - / - In an autoimmune inflammatory response experiment in mice, 3-week-old Trex1 mice were used. - / - Mice were intraperitoneally injected with dihydrotanshinone I at a dose of 40 mg / kg daily for 14 consecutive days. On day 14, mice were sacrificed one hour after the dihydrotanshinone I injection, and tissues (tongue, spleen, lung, heart, liver, and kidney) were collected. One portion of the tissues was stained with Hematoxylin and eosin (H&E) to assess the degree of pathological damage, while RNA was extracted from the other portion of the tissues. The mRNA levels of interferon-related genes (Isg15, Ifit1, Ifit2, Cxcl10, and Rsad2) were detected by qPCR. Wild-type mice were used as negative controls.

[0074] Experimental results:

[0075] Depend on Figure 5 It was found that dihydrotanshinone I had no effect on the mRNA levels of wild-type type I interferon-related genes (IFN-β, Isg15, and Rsad2), but it significantly inhibited the increase in the mRNA levels of these genes caused by TREX1 gene deletion. Furthermore, H&E staining results showed that mice with TREX1 gene deletion exhibited partial hemorrhage, increased inflammatory cell infiltration, and increased inflammatory factors, while wild-type mice showed no abnormalities in their liver tissue. These further results indicate that dihydrotanshinone I significantly alleviated the inflammatory response induced by TREX1 gene deletion, demonstrating that it can significantly alleviate the autoinflammatory response caused by TREX1 gene deletion. Therefore, dihydrotanshinone I may be considered a therapeutic drug for autoinflammatory diseases caused by TREX1 gene deletion.

Claims

1. Use of dihydrotanshinone I or a pharmaceutically acceptable salt thereof in the preparation of medicaments for the treatment and / or prevention of Ecardi-Gutierrez syndrome.

2. The use according to claim 1, wherein, The drug is administered orally, intravenously, intradermally, transdermally, intrathecally, intra-arterially, intraperitoneally, intranasally, intravaginally, intrarectally, intrabladderally, intratumorally, intramuscularly, subcutaneously, via inhalation, or any combination thereof.

3. The use according to claim 1, wherein, The dosage form of the drug is selected from one or more of the following: capsules, tablets, pills, liquids, powders, granules, lozenges, sublingual tablets, oral preparations, pastes, emulsions, coatings, ointments, plasters, mud pastes, transdermal preparations, inhalants, injections, or suppositories.

4. The use according to claim 1, wherein, The drug further comprises a pharmaceutically acceptable excipient selected from one or more of granulating agents, binders, lubricants, disintegrants, sweeteners, gliding agents, anti-adhesives, antistatic agents, surfactants, antioxidants, gums, coating agents, colorants, flavoring agents, plasticizers, preservatives, suspending agents, emulsifiers, antimicrobial agents, plant cellulose materials, or pelleting agents, and any combination thereof.

5. The use according to claim 1, wherein, The drug is administered to a subject, who is a mammal.

6. The use according to claim 5, wherein, The mammals mentioned are humans, rats, mice, cats, dogs, horses, sheep, cows, or monkeys.

7. The use according to claim 6, wherein, The subjects were humans.

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