Medical application of chalcone derivative CTG25 in preparation of NLRP3 inflammasome inhibitor

Abstract

The invention provides medical application of a chalcone derivative CTG25 in preparation of an NLRP3 inflammasome inhibitor, and belongs to the technical field of biological medicine. The invention provides an application of a chalcone derivative CTG25 in preparation of an NLRP3 inflammasome inhibitor and a medicine for preventing or treating diseases related to abnormal activation of NLRP3 inflammasome. The chalcone derivative CTG25 has a remarkable inhibition effect on NLRP3 inflammasome activation, is a potential and more effective NLRP3 inflammasome inhibitor, shows a remarkable treatment effect in a mouse model of NLRP3 mediated diseases, and is expected to be possibly developed into a candidate medicine for treating NLRP3 inflammasome related inflammatory diseases.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of biological medicine, and particularly relates to the medical application of chalcone derivative CTG25 in preparation of NLRP3 inflammasome inhibitor. BACKGROUND

[0002] Inflammation is a key protective immune response initiated by the body when facing external stimuli, which is closely related to the activation of the immune system, especially the activation of the innate immune system. Innate immunity, as the first line of defense against external threats, plays an extremely important role in maintaining body homeostasis. The innate immune system mainly recognizes the conserved structures of pathogens, i.e. pathogen-associated molecular patterns (PAMPs) and danger-associated molecular patterns (DAMPs), through a series of pattern recognition receptors (PRRs). In this process, part of the pattern recognition receptors can recruit downstream adaptor proteins, and then form a complex called inflammasome, promoting the maturation and secretion of inflammatory mediators such as IL-1β and IL-18, thereby triggering the inflammatory response. Moderate inflammatory response is crucial for maintaining the health of the body. If the inflammatory response is excessive, it may cause damage to the body, and even induce a variety of major diseases, such as hepatitis, enteritis, arthritis, diabetes, tumors and neurodegenerative diseases, etc. Therefore, in-depth exploration of the occurrence, development and regulation mechanism of the inflammatory response has extremely important significance for elucidating the pathogenesis and providing intervention means for treatment.

[0003] Inflammatory responses can be induced by a variety of inflammasomes, among which NLRP1, NLRP3, NLRC4, AIM2 and Pyrin inflammasomes are the most studied types. As a key regulatory protein, NLRP3 inflammasome, as a key regulatory protein, is an important part of innate immunity. NLRP3 inflammasome is mainly composed of intracellular NOD-like receptor family protein 3 (NLRP3), apoptosis-associated speck-like protein (ASC) and cysteine aspartate-specific protease 1 (caspase-1) and other core proteins. The activation of NLRP3 inflammasome needs to be mediated by two key signals: the cell recognizes "signal 1" (such as lipopolysaccharide LPS) through pattern recognition receptor TLR, and then activates the NF-κB signaling pathway, prompting the expression of NLRP3, pro-IL-1β and other inflammasome component proteins to be up-regulated. Subsequently, under the induction of "signal 2" (such as nigericin), NLRP3 recruits ASC and pro-caspase-1 to assemble into an active inflammasome, allowing pro-caspase-1 to self-cleave and activate. Activated caspase-1 mediates cell apoptosis by cleaving Gasdermin D on one hand; on the other hand, it cleaves and activates pro-IL-1β and pro-IL-18, promotes their secretion, and then recruits other inflammatory cells to play a role in innate immunity. Unlike other inflammasomes, NLRP3 inflammasome not only recognizes viruses or bacteria and other pathogenic microorganisms, but also recognizes a variety of dangerous signals, which indicates that it plays an important role in the body's resistance to pathogen invasion and the occurrence and development of a variety of inflammation-related diseases, such as gout, type 2 diabetes, atherosclerosis and neurodegenerative diseases. Therefore, intervention in the abnormal activation of NLRP3 inflammasome in diseases is considered a promising disease treatment strategy.

[0004] In recent years, the research on NLRP3 inflammasome inhibitors has made rapid progress, and a large number of NLRP3 inflammasome inhibitors have been developed. However, due to various problems, these inhibitors have not been applied to the clinic. For example, MCC950 is the most effective and specific NLRP3 inflammasome inhibitor, but it was terminated in the clinical phase II experiment due to its hepatotoxicity. Although NLRP3 inflammasome is closely related to a variety of major diseases in the human body, there is still a lack of clinical drugs that directly target NLRP3 inflammasome. Therefore, it is particularly urgent to find safe and effective NLRP3 inflammasome inhibitors. SUMMARY

[0005] The purpose of the present application is to provide the medical application of chalcone derivative CTG25 in the preparation of NLRP3 inflammasome inhibitors, and to provide technical support for the development of NLRP3 inflammasome inhibitors.

[0006] In order to achieve the above-mentioned object of the present application, the present application provides the following technical solutions. The present application provides a medical application of chalcone derivative CTG25, and the application is any one of the following: (a) preparing an NLRP3 inflammasome inhibitor; (b) preparing a drug for preventing or treating a disease related to abnormal activation of NLRP3 inflammasome; The chemical structural formula of the chalcone derivative CTG25 is as follows: .

[0007] Preferably, the disease related to abnormal activation of NLRP3 inflammasome includes gout, neurodegenerative disease, type 2 diabetes, atherosclerosis, and infectious inflammatory disease.

[0008] Preferably, the infectious inflammatory disease includes sepsis or acute systemic inflammation.

[0009] Preferably, the sepsis and the acute systemic inflammation are induced by LPS.

[0010] Preferably, the drug further comprises a pharmaceutically acceptable excipient.

[0011] Preferably, the dosage form of the drug includes a solid dosage form and a liquid dosage form.

[0012] Compared with the prior art, the present application has the following beneficial effects: The present application provides the use of chalcone derivative CTG25 as an effective NLRP3 inflammasome inhibitor, which can significantly inhibit the activity of caspase-1 and the release of LDH in BMDM cells, and has a broad-spectrum and specific inhibitory effect, and can effectively inhibit the activation of NLRP3 inflammasome. Chalcone derivative CTG25 exerts anti-inflammatory activity under NLRP3-related inflammatory conditions, such as LPS-induced fatal sepsis and acute systemic inflammation. These findings prove that chalcone derivative CTG25 can effectively inhibit the activation of NLRP3 inflammasome in vivo, and can be used for preparing a drug for preventing and treating NLRP3-mediated related inflammatory diseases such as gout, neurodegenerative disease, and infectious inflammatory disease.

[0013] The present application provides chalcone derivative CTG25 has inhibitory effect on activation of NLRP3 inflammasome, and further studies the application of chalcone derivative CTG25 in preparation of drugs for preventing and treating NLRP3-mediated diseases. The chalcone derivative CTG25 has significant inhibitory effect on activation of NLRP3 inflammasome, is a potential and more effective NLRP3 inflammasome inhibitor, shows significant therapeutic effect in a mouse model of NLRP3-mediated disease, and is expected to be developed as a candidate drug for treating NLRP3 inflammasome-related inflammatory diseases. BRIEF DESCRIPTION OF DRAWINGS

[0014] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the drawings needed to be used in the embodiments or prior art description will be briefly introduced below. Obviously, the drawings in the following description are only embodiments of the present application, and other drawings can also be obtained according to the provided drawings without creative labor for those skilled in the art.

[0015] Figure 1Chalcone derivative CTG25 inhibits NLRP3 inflammasome activation in BMDMs and THP-1; Wherein, A is the structural formula of CTG25; B~C is the activity of BMDM cells (B) and THP-1 cells (C) treated with different doses of CTG25 for 24 hours evaluated using cell viability assay; D~F is the Western blot analysis of pro-caspase-1 (p45), pro-IL-1β, NLRP3 and ASC in whole cell lysates (WCL) and secretion of activated caspase-1 p20 and IL-1β p17 in culture supernatants (SN) of BMDMs primed with LPS for 4 hours, treated with CTG25 for 30 minutes, and then stimulated with Nigericin for 25 minutes (D), the activity of caspase-1 in SN (E) and the release of LDH (F) were determined; G~I is the Western blot analysis of pro-caspase-1 (p45), pro-IL-1β, NLRP3 and ASC in whole cell lysates (WCL) and secretion of activated caspase-1 (p20) and IL-1β (p17) in culture supernatants (SN) of THP-1 treated with PMA overnight, treated with CTG25 for 1 hour, and then stimulated with Nigericin for 45 minutes (G), the activity of caspase-1 in SN (H) and the release of LDH (I) were measured. Coomassie blue staining was used as supernatant loading control, and Lamin B was used as lysate loading control. Data are presented as mean ± SEM of biological replicates (n=3), data were analyzed using one-way ANOVA with Dunnett's post-hoc test. P<0.1 compared with the concentration of 0 mM; P<0.01 compared with the concentration of 0 mM; P<0.001 compared with the concentration of 0 mM; P<0.0001 compared with the concentration of 0 mM; ns: not significant.

[0016] Figure 2CTG25 is a specific inhibitor of NLRP3 inflammasome; wherein, A~B BMDMs were primed with LPS, then treated with CTG25 (5 mM) for 30 min, and then stimulated with Nigericin, ATP, SiO2or poly(l:C). Western blot analysis of pro-caspase-1 (p45), pro-IL-1 b, NLRP3 and ASC in whole cell lysates (WCL); secretion of activated caspase-1 p20 in the medium supernatant (SN) of BMDMs (A), caspase-1 activity was measured in the SN (B); C~D BMDMs primed with Pam3CSK4, then treated with CTG25 (5 mM), and then transfected with LPS, Western blot analysis of pro-caspase-1 (p45), pro-IL-1 b, NLRP3 and ASC in whole cell lysates (WCL); secretion of activated caspase-1 p20 in the medium supernatant (SN) of BMDMs (C), caspase-1 activity was measured in the SN (D); E~F LPS-primed BMDMs were treated with CTG25 (5 mM) for 30 min, then stimulated with Nigericin for 30 min, or poly(dA:dT) / bacterial flagellin for 5 h. Western blot analysis of pro-caspase-1 (p45), pro-IL-1 b, NLRP3 and ASC in whole cell lysates (WCL); secretion of activated caspase-1 p20 in the medium supernatant (SN) of BMDMs (E), caspase-1 activity was measured in the SN (F). Data are presented as mean ± SEM of biological replicates (n = 3), data were analyzed using one-way ANOVA with Dunnett’s post-test. P < 0.01 compared to the concentration of 0 mM; P < 0.001 compared to the concentration of 0 mM; P < 0.0001 compared to the concentration of 0 mM; ns: not significant.

[0017] Figure 3Representing CTG25 inhibits ASC oligomerization during NLRP3 inflammasome activation; wherein, A is a western blot analysis of proteins in cell lysates of BMDMs stimulated with LPS for 3 hours followed by treatment with CTG25 for 1 hour or first treated with CTG25 for 1 hour followed by stimulation with LPS for 3 hours; B is a western blot analysis of cross-linked ASC in Triton X insoluble particles of LPS primed BMDMs pretreated with CTG25 (5 mM) for 30 minutes followed by stimulation with Nigericin for 30 minutes; C is a western blot analysis of cross-linked ASC in Triton X insoluble particles in LPS primed BMDMs pretreated with CTG25 (5 mM) followed by stimulation with Nigericin, ATP, poly(I:C), and SiO2; D is a western blot analysis of cross-linked ASC in Triton X insoluble particles in Pam3CSK4 primed BMDMs treated with CTG25 (5 mM) followed by LPS transfection primed BMDMs; E is a western blot analysis of cross-linked ASC in Triton X insoluble particles in LPS primed BMDMs pretreated with CTG25 (5 mM) followed by stimulation with Nigericin, poly(dA:dT), or bacterial flagellin.

[0018] Figure 4 Representing CTG25 inhibits NLRP3 inflammasome activation and improves LPS-induced sepsis in vivo; wherein, A-J are the levels of IL-1β (A, B), IL-18 (C, D), IL-6 (E, F), TNF-α (G, H), CXCL1 / KC (I, J) in serum and peritoneal lavage fluid measured using ELISA in mice pretreated with CTG25 (15 mg / kg), CTG25 (30 mg / kg), MCC950 (40 mg / kg), or CTG25 (30 mg / kg) + MCC950 (40 mg / kg) for 1 hour followed by injection with LPS (20 mg / kg) for 5 hours. Data are presented as mean ± SD. Significance of differences was analyzed using the log-rank test (A), one-way ANOVA with Sidak’s post-test (B-G), or unpaired t-test. DETAILED DESCRIPTION

[0019] The technical solutions provided by the present application are described in detail below in conjunction with the embodiments, but they should not be understood as limiting the scope of protection of the present application.

[0020] Experimental design: This study mainly evaluates the therapeutic effect of chalcone derivative CTG25 on NLRP3 inflammasome-mediated diseases. The effect of CTG25 on the activation of NLRP3 inflammasome in mouse BMDMs and human THP-1 cells. Evaluated by immunoblotting, activity measurement, etc. And evaluate the therapeutic effect in mouse models of NLRP3 inflammasome-mediated diseases.

[0021] Reagents, materials used: Chalcone derivative CTG25, molecular formula is C 20 H 20O4, molecular weight 324.37, light yellow powder, soluble in DMSO, purchased from MCE company (CAS number: 2577388-38-4). MCC950 (HY-12815A), murine macrophage colony-stimulating factor (MCSF) (HY-P7085) were purchased from MedChemExpress (New Jersey, USA). Nigericin, ATP, silica, poly(dA:dT), poly(I:C), PMA and DMSO were purchased from Sigma-Aldrich. Pam3CSK4, ultra-pure LPS, Flagellin were purchased from InvivoGen. Caspase-1 activity assay kit (G9951) and LDH assay kit (G1780) were purchased from Promega. Anti-human IL-1β antibody (p17, 12242S), caspase-1 antibody (4199S), NLRP3 antibody (15101S) and anti-ASC antibody (sc-22, 514-R) were purchased from Cell Signaling Technology. Active caspase-1 antibody (AG-20B-0042) was purchased from Adipogen, and anti-mouse IL-1β antibody (AF-401-NA) was purchased from R&D Systems. Anti-Lamin B antibody (66,095-1-Ig) was purchased from Proteintech. Disuccinimidyl suberate (DSS) (21,655) was purchased from Thermo Scientific. Certified fetal bovine serum (FBS) was purchased from VivaCell (Shanghai, China). Protease inhibitor (C0001) was purchased from TargetMol. StarFect transfection reagent (C101-01) was purchased from Genstar. Mouse TNF-α Elisa kit (1217202), mouse IL-1β Elisa kit (1210122), mouse IL-6 Elisa kit (1210602) were purchased from Dakewe. Mouse IL-18 Elisa kit (ml002294) was purchased from Enzyme-Linked Biotechnology. Mouse CXCL1 / KC kit (EK296 / 2-96) was purchased from Union Biomed. C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0022] Statistical methods: All experiments were performed randomly and blindly. Data analysis was performed using GraphPad Prism (Graphpad Prism 10 software). All experimental results were expressed as mean ± SEM. Statistical significance between groups was analyzed using one-way ANOVA for multiple groups and t-test for two groups. Differences were considered statistically significant when P < 0.05.

[0023] Example 1 Chalcone derivative CTG25 inhibits NLRP3 inflammasome activation in murine BMDM and human THP-1 cells in a dose-dependent manner

[0024] 1. Experimental operation method

[0025] 1.1 Culture of BMDM, THP-1 cells

[0026] Culture and differentiation of mouse bone marrow-derived macrophages (BMDMs): 10-12-week-old C57BL / 6 mice were taken, and after dislocation, the bilateral femurs of the mice were separated in a clean bench and the bone marrow cells were flushed out with DMEM medium using a fine needle. After repeatedly blowing the cells, they were transferred to a 50 mL centrifuge tube, and after centrifugation at room temperature, the supernatant was removed, and the cells were resuspended in DMEM medium containing 10% fetal bovine serum (fetal bovine serum, FBS) and 1% double-antibiotic, and macrophage colony stimulating factor (macrophage colony stimulating factor, M CSF) was added to a final concentration of 25 ng / mL. After 5 days of cell culture, BMDMs were obtained.

[0027] THP1 cells were purchased from ATCC company and cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-streptomycin (CM10040, Macgene) at 5% CO2 and 37°C in a humidified atmosphere.

[0028] 1.2 BMDMs, THP-1 cell viability assay

[0029] BMDMs were collected by trypsin and EDTA (2:1) digestion and seeded in 96-well plates at a density of 1.2 x 10 6 cells / mL in 100 μL / well overnight. The next day, the culture medium was discarded, and chalcone derivative CTG25 was treated at different concentration gradients, and incubated at 37°C in a 5% CO2 environment for 6 h. Then, half of the old culture medium was discarded, and an equal volume of ATP detection reagent working solution was added, and incubated at 37°C in a 5% CO2 environment for 15 min. Then, an equal amount of liquid was aspirated from each well into a 96-well white plate, and the fluorescence value was detected using an enzyme-labeled instrument.

[0030] Cells were stimulated with PMA (100 nM) and 1.2 × 10⁻⁶ NM. 6 Seeds were generated at a density of 100 μL / well in 96-well plates and incubated overnight. The following day, the procedure was the same as for BMDMs cell viability assay.

[0031] 1.3 Sample processing, Western blotting analysis, caspase-1 and LDH activity assay

[0032] BMDMs and THP-1 cells were administered at 1.2 × 10⁻⁶ cells per cell line. 6 Cells were seeded at a density of 100 cells / mL in 12-well plates overnight. The next day, cells were stimulated with LPS (50 ng / mL) for 4 hours (this step is not required for THP-1 cells). BMDMs were treated with chalcone derivative CTG25 for 30 minutes and THP-1 cells for 1 hour, followed by stimulation with nigericin (10 μM) for 30 minutes and THP-1 cells for 50 minutes. After stimulation, the culture supernatant and cell lysis buffer of BMDMs and THP-1 cells were collected, centrifuged at 5000 rpm for 5 minutes, and 1 / 4 volume of trichloroacetic acid (TCA) was added to the supernatant. The cells were then stored in a refrigerator. After incubating overnight at 20°C, centrifuge at 13000 rpm for 15 min at 4°C, discard the supernatant, wash once with ice-cold acetone, evaporate the acetone in a 105°C metal bath, add 40 μL of 1× loading buffer after evaporation, vortex to mix, boil in a water bath, and cool to obtain the supernatant sample. For adherent cells, wash the plate twice with PBS, place on ice, add 200 μL of 1× loading buffer to each well, scrape off the cells after 10 min, collect the cell lysis buffer, boil in a water bath for 20 min, and cool to obtain the cell lysis sample.

[0033] Protein extracts were lysed with 1× loading buffer, and protein samples were separated onto 12% or 10% SDS-PAGE gels in electrophoresis buffer. These gels were then transferred to polyvinylidene fluoride (PVDF) membranes using a wet transfer system. The membranes were blocked for 1 hour with 5% skim milk prepared in Tris-buffered saline-Tween (TBST) solution, and then incubated overnight at 4°C with the primary antibody against the target protein. After washing, the membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody, developed using a chemiluminescent reagent, and visualized by X-ray.

[0034] The Caspase-Glo 1 Inflammasome Assay and LDH reagent were prepared according to the reagent instructions. Supernatants from LPS-stimulated nigerin-induced BMDMs and THP-1 cells were collected, and caspase-1 activity was measured using the caspase-1 activity assay kit and the LDH assay kit, respectively. 1 p20 enzyme activity and LDH release, detection was performed according to the kit instruction.

[0035] 2. Experimental procedure and results

[0036] We first tested CTG25 (chemical formula as shown in Figure 1 A) for cytotoxicity in mouse bone marrow-derived macrophages (BMDM) and human THP-1 cells. Cell viability assay showed that CTG25 did not exhibit any cytotoxicity in BMDM and THP-1 at doses lower than 20 μΜ ( Figure 1 B, C). Next, we further tested caspase-1 activation, IL-1β secretion and LDH release in BMDM cells upon nigericin stimulation by WB and activity assay. WB results showed that chalcone derivative CTG25 could effectively inhibit the protein content of caspase-1 p20 and IL-1β p17 in BMDM cell supernatant, and the inhibitory effect increased with the increase of concentration, and it completely inhibited the protein content of caspase-1 p20 and IL-1β p17 at 5 μΜ, correspondingly, the protein levels of NLRP3 and pro-IL-1β in whole cell lysate were not affected ( Figure 1 D). We also tested caspase-1 activation and LDH release in BMDM cell supernatant by activity assay. The results showed that chalcone derivative CTG25 inhibited caspase-1 activation ( Figure 1 E) and LDH release ( Figure 1 F) in a dose-dependent manner. Chalcone derivative CTG25 significantly inhibited NLRP3 inflammasome activation at a concentration of 5 μΜ.

[0037] Because some compounds cannot be applied to clinical practice due to interspecies differences, we further evaluated the effect of chalcone derivative CTG25 on NLRP3 inflammasome activation in human THP-1 cells. WB results showed that chalcone derivative CTG25 could effectively inhibit the protein content of caspase-1 p20 and IL-1β p17 in THP-1 cell supernatant, and the inhibitory effect increased with the increase of concentration, and it completely inhibited the protein content of caspase-1 p20 and IL-1β p17 at a concentration of 20 μΜ, correspondingly, the protein levels of NLRP3 and pro-IL-1β in whole cell lysate were not affected ( Figure 1G). We also detected the activation of caspase-1 and the release of LDH in THP-1 cell supernatants by the method of activity. The results showed that chalcone derivative CTG25 inhibited the activation of caspase-1 in a dose-dependent manner (Fig. 4A) Figure 1 and the release of LDH (Fig. 4B) in THP-1 cell supernatants. It had a good inhibitory effect at a concentration of 10 μM, and the inhibitory effect was enhanced with increasing concentration. Figure 1

[0038] In summary, chalcone derivative CTG25 significantly inhibited the activation of caspase-1, the secretion of IL-1β and the release of LDH induced by nigericin in human THP-1 cells. Thus, it can be concluded that chalcone derivative CTG25 can significantly inhibit the activation of NLRP3 inflammasome induced by nigericin in both mouse and human cells, and there is no species difference.

[0039] Example 2 Chalcone derivative CTG25 inhibits NLRP3 activation induced by classical and non-classical agonists

[0040] 1. Experimental operation method

[0041] 1.1 The operation of cell culture and inoculation, Western blot analysis and caspase-1 activity determination method is the same as that in Example 1.

[0042] 1.2 Sample treatment

[0043] BMDMs were inoculated in 12-well plates at a density of 1.2 x 10 6 cells / mL overnight. The next day, cells were stimulated with LPS (50 ng / mL) or Pam3CSK4 (400 ng / mL) for 4 hours. After treating BMDM cells with chalcone derivative CTG25 for 30 minutes, they were stimulated with nigericin (nigericin 10 μM) and ATP (5 mM) for 30 minutes, SiO2 (250 μg / mL), poly (I:C) (2 μg / mL), ultra-LPS (1 μg / mL), poly (dA:dT) (2 μg / mL), Flagellin (10 μg / mL) for 4 hours. After the stimulation time was over, the samples were collected and the operation was the same as in Example 1.

[0044] 2. Experimental process and results

[0045] ​NLRP3 inflammasome is a multi-protein complex, the activation of NLRP3 can be induced by a variety of stimuli, including the activation of NLRP3 inflammasome induced by classic and non-classic agonists. Therefore, we further studied whether chalcone derivative CTG25 inhibited the activation of NLRP3 inflammasome induced by other agonists in addition to nigericin. As can be seen from the WB results, 5 μM chalcone derivative CTG25 effectively inhibited the protein content of caspase-1 p20 in the supernatant of BMDM cells induced by classic NLRP3 stimuli, such as nigericin, ATP, SiO2, poly (I:C) (Fig. 2A); we also detected the activation of caspase-1 in the supernatant of BMDM cells by activity assay, and found that chalcone derivative CTG25 effectively inhibited the activation of caspase-1 induced by classic stimuli (Fig. 2B); at the same time, the protein content of caspase-1 p20 in the supernatant of BMDM cells induced by non-classical NLRP3 inflammasome stimuli Pam3CSK4 pretreatment and LPS was also significantly inhibited (Fig. 2C), and the activation of caspase-1 in the supernatant of BMDM cells was detected by activity assay, and the results showed that chalcone derivative CTG25 effectively inhibited the activation of caspase-1 induced by non-classical stimuli (Fig. 2D). Figure 2 Figure 2 Figure 2 Figure 2

[0046] In summary, we can conclude that chalcone derivative CTG25 can effectively inhibit the activation of classic and non-classical NLRP3 inflammasome, and is a broad-spectrum inhibitor of NLRP3 inflammasome activation.

[0047] Example 3 Chalcone derivative CTG25 is a specific inhibitor of NLRP3 inflammasome

[0048] 1. Experimental operation method: same as example 2.

[0049] 2. Experimental process and results

[0050] Considering that multiple NLR and ALR families can regulate inflammasomes, we next studied the effect of chalcone derivative CTG25 on the activation of other inflammasomes, such as AIM2 and NLRC4 inflammasomes. As can be seen from the WB results, 5 μM concentration of chalcone derivative CTG25 effectively inhibited the protein content of caspase-1 p20 in the supernatant of BMDM cells induced by nigericin; but had almost no effect on the protein content of caspase-1 p20 in the supernatant of BMDM cells induced by poly (dA:dT) transfection and bacterial flagellin (Fig. 3A). Figure 2 ​​​​Nigericin-induced caspase-1 activation in BMDM cell supernatant. We found that 5 μΜ concentration of chalcone derivative CTG25 effectively inhibited Nigericin-induced caspase-1 activation in BMDM cell supernatant, but had little effect on poly(dA:dT)-transfected and Flagellin-induced caspase-1 activation in BMDM cell supernatant. Figure 2 Nigericin-induced caspase-1 activation in BMDM cell supernatant. We found that 5 μΜ concentration of chalcone derivative CTG25 effectively inhibited Nigericin-induced caspase-1 activation in BMDM cell supernatant, but had little effect on poly(dA:dT)-transfected and Flagellin-induced caspase-1 activation in BMDM cell supernatant.

[0051] In summary, we can conclude that chalcone derivative CTG25 can effectively inhibit Nigericin-induced NLRP3 inflammasome activation in BMDM cells, but has no effect on poly(dA:dT)-transfected AIM2 inflammasome and Flagellin-induced NLRC4 inflammasome activation. This indicates that chalcone derivative CTG25 has no inhibitory effect on AIM2 and NLRC4 inflammasomes, but has a significant inhibitory effect on NLRP3 inflammasome. Chalcone derivative CTG25 specifically inhibits NLRP3 inflammasome.

[0052] Example 4 Chalcone derivative CTG25 inhibits the NF-κB pathway in the Prime phase of NLRP3 inflammasome

[0053] 1. Experimental operation method

[0054] BMDMs were seeded in 24-well plates at a density of 1.2 x 10 6 Cells were stimulated with LPS (50 ng / mL) for 3 hours, and then treated with chalcone derivative CTG25 for 1 hour. The operation of cell lysis was the same as in Example 1.

[0055] 2. Experimental process and results

[0056] Many upstream mechanisms can affect the oligomerization of ASC as triggers, so we further explored the possible mechanism of chalcone derivative CTG25 inhibiting the activation of NLRP3 inflammasome. In the early stage of NLRP3 inflammasome activation, there is a Prime stage as the first signal of NLRP3 inflammasome activation. In the Prime stage, cells induce NF-κΒ activation by recognizing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), triggering the transcriptional expression of NLRP3 and pro-IL-Ιβ. Therefore, we detected the NLRP3-related proteins in the BMDM cell lysate by WB to explore whether chalcone derivative CTG25 affects the expression of NLRP3 and pro-IL-Ιβ dependent on NF-κΒ. The results showed that after giving LPS stimulation for 3 h, under the condition of treating with different concentrations of chalcone derivative CTG25 for 1 h, we found that chalcone derivative CTG25 did not affect the expression of NLRP3 and pro-IL-Ιβ proteins that had been produced; while under the condition of first treating with different concentrations of chalcone derivative CTG25 for 1 h and then giving LPS co-stimulation for 3 h, we found that chalcone derivative CTG25 could inhibit the expression of NLRP3 and pro-IL-Ιβ proteins that had been formed, and the inhibition effect became stronger with the increase of concentration Figure 3 Fig. 5A). It can be seen that treating BMDM cells with chalcone derivative CTG25 before LPS stimulation can inhibit the expression of NLRP3 and pro-IL-Ιβ proteins. It can be shown that chalcone derivative CTG25 can inhibit the NF-κΒ signaling pathway, and the inhibitory effect of chalcone derivative CTG25 on NLRP3 inflammasome is not through down-regulating the expression of NLRP3 and pro-IL-Ιβ proteins.

[0057] Example 5 Chalcone derivative CTG25 blocks NLRP3-dependent ASC oligomerization

[0058] 1. Experimental operation method

[0059] 1.1 The steps of NLRP3 inflammasome activation are the same as in Example 1.

[0060] 1.2 Triton buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 0.5% Triton X-100 and protease inhibitor cocktail without EDTA) for 15 min, and the cells were scraped from the well plates and centrifuged at 6000 g for 15 min at 4°C. The supernatant and the pellet fraction were referred to as Triton X-soluble and Triton X-insoluble, respectively. The Triton X-insoluble fraction was washed and resuspended in 200 μL PBS and cross-linked with 2 mM DSS for 30 min at 37°C. The pellet was centrifuged at 6000 g for 15 min at 4°C, and then collected and solubilized in 1x Triton loading buffer for Western blot analysis.

[0061] 2. Experimental procedures and results

[0062] Oligomerization of ASC, the inflammasome adaptor protein, is a key event in the assembly and activation of NLRP3 inflammasome. To investigate the effect of chalcone derivative CTG25 on ASC oligomerization, we detected the expression of ASC oligomerization during the activation of NLRP3 inflammasome induced by various classical and non-classical stimuli. The results showed that chalcone derivative CTG25 could dose-dependently inhibit ASC oligomerization during the activation of NLRP3 inflammasome induced by nigericin in BMDM cells (Fig. 2A). Figure 3 Chalcone derivative CTG25 could inhibit ASC oligomerization during the activation of NLRP3 inflammasome induced by nigericin, ATP, SiO2, poly(I:C) and Pam3CSK4+LPS in BMDM cells (Fig. 2B, C, D); but had no effect on ASC oligomerization during the activation of AIM2 and NLRC4 inflammasome induced by poly(dA:dT) transfection and flagellin, respectively (Fig. 2E). Figure 3 Figure 3

[0063] In summary, it can be concluded that chalcone derivative CTG25 can inhibit ASC oligomerization during the activation of NLRP3 inflammasome in a broad spectrum, and has no effect on ASC oligomerization during the activation of AIM2 and NLRC4 inflammasome. This indicates that chalcone derivative CTG25 can specifically inhibit NLRP3 inflammasome by inhibiting ASC oligomerization or upstream events of ASC oligomerization.

[0064] Example 6 CTG25 can improve LPS-induced fatal sepsis and acute systemic inflammation ​​

[0065] 1. Experimental operation method

[0066] C57BL / 6 mice, 7~8 weeks, female, 42, were randomly divided into 7 groups, respectively, blank control group, only CTG25 group, LPS model group, CTG25 treatment group 1 (15mg / kg) and CTG25 treatment group 2 (30mg / kg), positive drug MCC950 treatment group (40mg / kg), CTG25 (30mg / kg) and MCC950 (40mg / kg) synergistic treatment group; the blank control group, only CTG25 group, model group were injected with equal amount of normal saline, and the rest of the groups were injected with CTG25 and positive drug MCC950 dissolved in 5% DMSO; 1 hour later, except for the blank control group and the only CTG25 group injected with equal amount of PBS solution, the rest of the groups were injected with LPS normal saline solution with a mass concentration of 20mg / kg, 6 hours later, the eyeball was taken out to take blood and abdominal lavage fluid, 4℃ centrifugation to take serum, ELISA detection of IL 1β, IL-18, IL-6, TNF α, CXCL1 / KC content.

[0067] 2. Experimental process and results

[0068] Intraperitoneal injection of LPS triggers the activation of NLRP3 inflammasome in mice, and the levels of various indicators in serum and abdominal cavity increase. Next, we studied the therapeutic potential of CTG25 for LPS-induced fatal sepsis. The results show that in control mice, NLRP3 inflammasome drives the production of IL-1β after 5h of LPS treatment, but CTG25 pretreatment reduces the production of IL-1β, IL-18 in serum and abdominal cavity, and is dose-dependent, and its effect is similar to that of positive drug MCC950 (Fig. Figure 4 A~D). In contrast, the production of TNF-α, IL-6 and CXCL1 / KC, which are independent of NLRP3 inflammasome activation, is not affected by CTG25 administration (Fig. Figure 4 E~J). These results show that CTG25 can inhibit the activation of NLRP3 inflammasome in vivo and reduce the inflammatory response induced by LPS in mice.

[0069] In summary, we found that chalcone derivative CTG25 could dose-dependently and specifically inhibit the activation of NLRP3 inflammasome, and its inhibitory effect had a broad spectrum. At the same time, in vivo, CTG25 played an anti-inflammatory activity under the condition of NLRP3-related inflammation, such as LPS-induced fatal sepsis, acute systemic inflammation. These findings proved that chalcone derivative CTG25 was a specific NLRP3 inflammasome inhibitor, which had great potential to develop as a clinical drug for treating NLRP3 inflammasome-mediated diseases.

[0070] The above merely describes the preferred embodiments of the present application, and it should be pointed out that for those skilled in the art, without departing from the principles of the present application, a number of improvements and refinements can be made, and these improvements and refinements should also be considered as the protection scope of the present application.

Claims

1. The medical use of chalcone derivative CTG25, characterized in that, The application is any one of the following: (a) preparing an NLRP3 inflammasome inhibitor; (b) preparing a drug for preventing or treating a disease associated with abnormal activation of NLRP3 inflammasome; The chemical structural formula of the chalcone derivative CTG25 is: 。 2. Use according to claim 1, wherein The disease associated with abnormal activation of NLRP3 inflammasome includes gout, neurodegenerative disease, type 2 diabetes, atherosclerosis, infectious inflammatory disease.

3. Use according to claim 2, wherein the compound is ###0002### The infectious inflammatory disease includes sepsis or acute systemic inflammation.

4. The use according to claim 3, wherein the compound is ###0002### The sepsis and the acute systemic inflammation are induced by LPS.

5. The use according to claim 1, wherein The drug also includes a pharmaceutically acceptable excipient.

6. The use according to claim 1, wherein The dosage form of the drug includes a solid dosage form and a liquid dosage form.

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