Application of 1, 5-anhydro-D-sorbitol in inhibition of NLRP3 inflammasome activation

Abstract

The invention discloses application of 1, 5-anhydro-D-sorbitol (1, 5-anhydroglaucitol, called 1, 5-AG for short) in inhibition of activation of NLRP3 inflammasomes, and particularly discloses application of 1, 5-anhydro-D-sorbitol (1, 5-AG for short) in inhibition of activation of NLRP3 inflammasomes. The invention relates to an application of 1, 5-anhydro-D-sorbitol in preparation of a medicine for inhibiting activation of NLRP3 inflammasomes, in particular to an application of 1, 5-anhydro-D-sorbitol. The NLRP3 inflammasome activation related diseases are selected from septicaemia induced by LPS, gouty arthritis induced by MSU, non-alcoholic fatty liver disease, systemic inflammatory response syndrome or inflammatory bowel disease. The 1, 5-AG is combined with an NATCH structural domain of the NLRP3 protein, so that the interaction between the 1, 5-AG and NEK7 and ASC is inhibited. The invention relates to an application of 1, 5-anhydro-D-sorbitol in preparation of functional food for inhibiting activation of NLRP3 inflammasomes. The 1, 5-AG is natural in source, high in oral bioavailability and high in safety, and has the advantage of being further developed into inflammation regulation drugs or dietary supplements.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for inhibiting NLRP3 inflammasome activation based on 1,5-AG and its application in the prevention or treatment of inflammatory diseases, especially applicable to diseases such as infectious sepsis and gouty arthritis caused by abnormal activation of NLRP3 inflammasome. Background Technology

[0002] The NLRP3 inflammasome is an important signaling platform in the innate immune system, capable of sensing various pathogen-associated and damage-associated molecular patterns, activating downstream Caspase 1, thereby promoting the maturation and release of pro-inflammatory factors such as IL-1β and IL-18. Aberrant activation of the NLRP3 inflammasome is considered closely related to various inflammatory diseases, including sepsis, autoimmune diseases, gout, atherosclerosis, and neurodegenerative diseases. Currently, there is a lack of widely applicable, highly targeted drugs with minimal side effects for regulating the NLRP3 inflammasome. Although some small molecule compounds (such as MCC950) have been reported to selectively inhibit NLRP3, these drugs are still in the early stages of research and suffer from instability issues. Therefore, the search for new, natural, highly effective, and safe NLRP3 inflammasome regulatory molecules has significant value for basic research and translational applications.

[0003] 1,5-AG is an endogenous cellular metabolite widely present in humans and animals, and its application in metabolic monitoring of diabetes is already established. However, its role in immune regulation, particularly in the regulation of the NLRP3 inflammasome, has not been clearly reported. Summary of the Invention

[0004] The purpose of this invention is to provide a novel use of 1,5-AG in inhibiting NLRP3 inflammasome activation, to reveal its mechanism of action as a natural immune regulatory molecule, and to further expand its application potential in the treatment of inflammatory diseases, especially NLRP3-related diseases (such as sepsis, gouty arthritis, etc.).

[0005] The objective of this invention is achieved through the following technical solution: This invention is the first to discover that 1,5-AG is significantly consumed during NLRP3 inflammasome activation, and that exogenous supplementation of 1,5-AG can inhibit inflammasome assembly and activation by specifically binding to the NATCH domain of the NLRP3 protein, thereby exerting an anti-inflammatory effect and demonstrating its potential as a novel immunomodulatory molecule. This invention provides a method for inhibiting NLRP3 inflammasome activation using 1,5-AG, which involves applying 1,5-AG to macrophages or animal models in a pre-inflammasome activation state to observe its effect on IL-1β expression.

[0006] Experiments showed that 1,5-AG could significantly inhibit the activation of the NLRP3 inflammasome.

[0007] Co-IP, CETSA, and molecular dynamics simulations revealed that 1,5-AG can inhibit the inflammasome assembly process by directly binding to the NLRP3 protein and blocking its oligomerization with NEK7 and ASC.

[0008] SPR experiments further verified the specific binding relationship between 1,5-AG and NLRP3 protein.

[0009] The present invention also provides the use of 1,5-AG in the preparation of medicaments for treating or alleviating NLRP3 inflammasome-related diseases, including but not limited to: LPS-induced infectious sepsis, MSU-induced gouty arthritis, and other diseases caused by abnormal activation of NLRP3 (such as atherosclerosis, non-alcoholic fatty liver disease, autoimmune diseases, etc.).

[0010] Animal experiments have shown that 1,5-AG can significantly improve the inflammatory response in the above-mentioned disease models, and this effect is significantly weakened in NLRP3-deficient mice, suggesting that its action depends on the NLRP3 pathway.

[0011] Use of 1,5-dehydrated-D-sorbitol in the preparation of functional foods for inhibiting NLRP3 inflammasome activation.

[0012] The beneficial effects of this invention are as follows: 1. For the first time, 1,5-AG was discovered to have biological activity as an endogenous metabolite in the activation of the NLRP3 inflammasome; 2. The mechanism of direct binding between 1,5-AG and NLRP3 protein was revealed (NATCH domain-specific binding, KD=3.407×10⁻). 4 M); 3. This study confirmed the broad intervention potential of 1,5-AG for inflammasome-related diseases and demonstrated its promising application prospects. 4.1,5-AG is naturally derived, has high oral bioavailability and high safety, and has the advantage of being further developed into an inflammation-regulating drug or dietary supplement. Attached Figure Description

[0013] Figure 1A PCA plots are shown in the non-targeted metabolomics detection macrophage control group and macrophage NLRP3 inflammasome activation (LPS+ATP) group. Figure 1B The image shows a volcano plot of differentially expressed metabolites in the non-targeted metabolomics analysis control group and the NLRP3 inflammasome activation group. Figure 1CThe differences in the effects of five carbohydrates before and after NLRP3 inflammasome activation were shown; Figure 1D RNA-seq data of macrophages in the CON and LPS groups were analyzed, and heatmaps focused on the 1,5-AG transporter gene families Slc2a and Slc5a.

[0014] Figure 2A The level of IL-1β secretion in cell supernatant after 1,5-AG treatment was detected by ELISA. Figure 2B The effects of ATP-activated NLRP3 inflammasome activation were shown by Western blot analysis of Caspase1 activation, GSDMD cleavage, and NLRP3 protein expression levels after 1,5-AG treatment of cells. Figure 2C The study showed that IL-1β secretion and GSDMD cleavage levels were observed in cells treated with 1,5-AG after activation of the NLRP3 inflammasome by multiple agonists using Western blot analysis.

[0015] Figure 3A The study showed the IL-1β secretion level in the cell supernatant of cells treated with 1,5-AG after TRG5 interference, as detected by ELISA. Figure 3B The intracellular chloride ion level was measured using a chloride ion assay kit after cells were treated with 1,5-AG. Figure 3C The intracellular potassium level was measured using a potassium ion assay kit after 1,5-AG treatment of cells. Figure 3D The image shows mitochondrial morphology after 1,5-AG treatment of cells with TOMM20 labeling and immunofluorescence detection. Figure 3E This shows the formation of ASC spots after 1,5-AG cell treatment using immunofluorescence.

[0016] Figure 4A This study demonstrates the effect of 1,5-AG treatment on the thermostability of NLRP3 protein in cells, analyzed using a CETSA assay. Figure 4B -C shows the docking of 1,5-AG with NLRP3 protein molecules using AutoDock and PyMol, and the results show the docking site and pocket of 1,5-AG with the NATCH domain; Figure 4D The interaction between 1,5-AG and the PYD domain of NLRP3 protein in vitro was analyzed by surface plasmon resonance (SRP) experiments. Figure 4E The interaction between 1,5-AG and the NATCH domain of the NLRP3 protein in vitro was shown by SRP assay.

[0017] Figure 5A The effect of 1,5-AG pretreatment on serum IL-1β levels in LPS-induced septicemia mice was shown by ELISA. Figure 5B The effects of 1,5-AG pretreatment on intestinal and liver pathological damage in LPS-induced septicemia mice were analyzed using HE staining experiments. Figure 5C The results show the effects of Western blot analysis on intestinal Caspase 1 activation and pyroptosis protein GSDMD cleavage in LPS-induced septicemia mice from 1,5-AG pretreated mice. Figure 5D This study shows the activation of liver Caspase 1 and cleavage of pyroptosis protein GSDMD in LPS-induced septicemia mice by Western blot analysis of 1,5-AG pretreated mice.

[0018] Figure 6A This shows the effect of 1,5-AG pretreatment on ankle swelling in mice with MSU-induced arthritis. Figure 6B This demonstrates the effect of 1,5-AG pretreatment on Caspase1 activity in ankle tissue of mice with MSU-induced arthritis, as detected by a Caspase1 activity assay kit. Figure 6C The effects of 1,5-AG pretreatment on the pathological damage of ankle joint tissue in mice with MSU-induced arthritis were analyzed using HE staining experiments. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of the present invention clearer, the present invention will be further described in detail below with reference to specific embodiments and accompanying drawings.

[0020] Unless otherwise specified, the experimental methods used in the following implementation cases are standard experimental methods. Unless otherwise specified, the experimental materials used in the following implementation cases were all purchased from regular biochemical reagent stores.

[0021] In this invention, the term "NLRP3 inflammasome activation" refers to the assembly of NLRP3, ASC and Pro-Caspase1 into a multi-protein functional complex under the action of an activator (such as LPS+ATP). Pro-Caspase1 self-splices to the active form Caspase1 p20 / p10. The activated Caspase1 splices cytokine precursors such as Pro-IL-1β and Pro-IL-18 into active factors and is released into the extracellular space.

[0022] The following examples use GraphPad 8.0.2 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. The data are analyzed using the Column Test, and pairwise comparisons are performed using the unpaired T-test. 0.01 ≤ P ≤ 0.05 (*) indicates that there is a difference between the groups, P < 0.01 (**) indicates that there is a significant difference between the groups, and P < 0.001 (***) indicates that there is a highly significant difference between the groups.

[0023] Unless otherwise specified, the experimental methods used in the following implementation cases are all conventional experimental methods. Unless otherwise specified, the experimental materials used in the following implementation cases were all purchased from regular biochemical reagent stores, as shown in the table below.

[0024]

[0025] C57BL / 6J wild-type mice and NLRP3- / - mice were purchased from Jiangsu Jicui Pharmaceutical Co., Ltd.

[0026] Example 1: Screening of differential endogenous metabolites under macrophage NLRP3 inflammasome activation 1. Metabolomics screening of key endogenous metabolites beneath the NLRP3 inflammasome in macrophages (1) One day in advance, J774A.1 cells were seeded in 6-well plates. The next day, the cells were pretreated with Opti-MEM medium (containing 2 μm / mL 1,5-AG) for 1 hour. Then, the cells were treated with LPS at a final concentration of 500 ng / mL for 4 hours, and then stimulated with 2.5 mM ATP for 30 minutes.

[0027] (2) Collect cell lysate and investigate the changes in endogenous metabolites before and after NLRP3 inflammasome activation by non-targeted metabolomics detection.

[0028] like Figure 1A As shown, the overall metabolic patterns of macrophages before and after activation of the NLRP3 inflammasome are significantly different; for example... Figure 1B As shown, among the 262 detectable metabolomes, 50 showed significant downregulation and 41 showed significant upregulation; Figure 1BAs shown in -C, among the 50 downregulated metabolites, 5 were carbohydrates, including 1,5-AG.

[0029] II: Transcriptomic investigation of the regulatory mechanism of 1,5-AG under macrophage NLRP3 inflammasome activation. (1) One day in advance, J774A.1 cells were seeded in 6-well plates. The next day, the cells were pretreated with Opti-MEM medium (containing 2 μm / mL 1,5-AG) for 1 hour. Then, the cells were treated with LPS at a final concentration of 500 ng / mL for 4 hours, and then stimulated with 2.5 mM ATP for 30 minutes.

[0030] (2) Collect cell lysate, extract RNA, and investigate the changes in endogenous metabolite 1,5-AG metabolism-related genes before and after NLRP3 inflammasome activation through transcriptomics.

[0031] like Figure 1D As shown, under inflammatory stimulation, Slc2a3, Slc2a4, and Slc5a3, all genes in the Slc2a and Slc5a families related to 1,5-AG transport, were significantly downregulated. This indicates that LPS stimulation suppresses intracellular 1,5-AG levels by inhibiting the Slc2a3, Slc2a4, and Slc5a3 genes involved in 1,5-AG transport.

[0032] Example 2: In vitro study of the inhibitory effect of 1,5-AG on NLRP3 inflammasome. 1. 1,5-AG inhibits ATP-induced IL-1β secretion and pyroptosis. (1) One day in advance, J774A.1 cells were seeded in 6-well plates. The next day, the cells were pretreated with Opti-MEM medium (containing 2 μm / mL 1,5-AG) for 1 hour. Then, the cells were treated with LPS at a final concentration of 500 ng / mL for 4 hours, and then stimulated with 2.5 mM ATP for 30 minutes.

[0033] (2) Collect cell supernatant and cell lysate, and use ELISA and Western Blot to detect the IL-1β secretion level and GSDMD protein cleavage in the supernatant.

[0034] like Figure 2A ELISA results showed that 1,5-AG significantly inhibited ATP-induced IL-1β secretion, such as Figure 2B As shown in Table 1, the Western blot results indicate that 1,5-AG significantly inhibited ATP-induced N-GSDMD cleavage. These results demonstrate that 1,5-AG can significantly inhibit ATP-induced IL-1β secretion and pyroptosis.

[0035] 2. 1,5-AG inhibits NLRP3 inflammasome activation induced by multiple agonists. (1) One day in advance, J774A.1 cells were seeded in 6-well plates. On the second day, the cells were pretreated with Opti-MEM medium (containing 2 μm / mL 1,5-AG) for 1 hour. Then, the cells were treated with LPS to a final concentration of 500 ng / mL for 4 hours, and then stimulated with Alum (300 μg / mL, 6 hours), Nigericin (20 μM, 30 minutes) and MSU (200 μg / mL, 6 hours).

[0036] (2) Collect cell supernatant and cell lysate, and use ELISA and Western Blot to detect the IL-1β secretion level and Caspase1 and GSDMD protein cleavage in the supernatant.

[0037] like Figure 2C As shown in Table 1, the Western blot results indicate that 1,5-AG significantly inhibited Alum, Nigericin, and MSU-induced IL-1β secretion and N-GSDMD cleavage. These results demonstrate that 1,5-AG can significantly inhibit NLRP3 inflammasome activation induced by multiple agonists.

[0038] Example 3: Mechanism of 1,5-AG inhibiting NLRP3 inflammasome activation 1. Effects of the TGR5 pathway on the inhibition of IL-1β secretion by 1,5-AG (1) One day in advance, J774A.1 cell lines were seeded in 12-well plates. 50 μL of Opti-MEM medium was added to two RNA-free 1.5 mL EP tubes, followed by 2.5 μL of siRNA (20 μM) to one tube and 1 μL of Lipo3000 to the other. The tubes were incubated for 5 minutes, then mixed and incubated for another 20 minutes. Simultaneously, the culture medium in the cell plate was replaced with 400 μL of Opti-MEM, followed by the addition of the siRNA mixture. The cells were incubated for 6 hours. Then, the medium was replaced with DMEM complete medium. After 40 hours, the cells were pretreated with Opti-MEM medium (containing 2 μM / mL 1,5-AG) for 1 hour. Then, the cells were treated with LPS to a final concentration of 500 ng / mL for 4 hours, followed by stimulation with ATP to a final concentration of 2.5 mM / mL for 30 minutes. The target sequence of the siRNA used was: Tgr5: CCTACCTCTACCTGGAAGTTT.

[0039] (2) Collect cell supernatant and use ELISA to detect the IL-1β secretion level in the supernatant.

[0040] like Figure 3AAs shown, TGR5 inhibition can significantly increase IL-1β secretion, but 1,5-AG treatment can still inhibit IL-1β secretion, and the absence of TGR5 does not affect the level of 1,5-AG inhibition of IL-1β secretion. This indicates that 1,5-AG does not inhibit NLRP3 inflammasome activation in a TGR5-dependent manner.

[0041] II: Effects of 1,5-AG on ion flow and mitochondrial function (1) One day in advance, J774A.1 cells were seeded in 6-well plates. The next day, the cells were pretreated with Opti-MEM medium (containing 2 μm / mL 1,5-AG) for 1 hour. Then, the cells were treated with LPS at a final concentration of 500 ng / mL for 4 hours, and then stimulated with ATP at a final concentration of 2.5 mM / mL for 30 minutes.

[0042] (2) Discard the supernatant, add 1 mL ddH2O to each well, and lyse at 37°C for 10 minutes. Then, transfer to a 1.5 mL EP tube and freeze-thaw twice in liquid nitrogen and a 37°C water bath. Centrifuge at 4°C and 8,000 rpm for 10 minutes to remove the lower layer of cell debris, and then transfer to a new EP tube. Mix 50 μL of lysis buffer with 50 μL of MQAE (20 μM) and add to a white 96-well plate. Detect the fluorescein intensity using a microplate reader; this result characterizes the chloride ion concentration.

[0043] (3) Collect cell supernatant. Follow the instructions of the kit: add protein precipitation reagent, centrifuge at 3500 rpm for 5 minutes, take the supernatant, add 50 μL of supernatant to a 96-well plate, add 200 μL of potassium ion working solution, and then detect the OD value by microplate reader. This result represents the potassium ion concentration.

[0044] (4) After treatment, the cells were fixed with 4% formaldehyde for 10 minutes, followed by washing with PBS. They were then infiltrated with 0.1% Triton X-100 for 8 minutes at room temperature, followed by blocking with 3% BSA for 30 minutes. The cells were co-incubated with TOMM20 antibody overnight at 4°C, and then incubated with Alexa Fluor 594 (abcam, ab150080) goat anti-rabbit secondary antibody for 30-45 minutes at room temperature. The cell nuclei were stained with DAPI (1:4000 dilution) for 5-10 minutes. Coverslips were placed on the slides, and the cells were observed and photographed using a Zeiss LSM710 confocal microscope. This result represents the functional morphology of mitochondria.

[0045] like Figure 3B As shown, the chloride ion assay kit indicated that 1,5-AG did not affect the chloride ion concentration during NLRP3 inflammasome activation; Figure 3CAs shown, potassium ion assay results indicate that 1,5-AG does not affect potassium ion concentration during NLRP3 inflammasome activation; Figure 3D As shown, immunofluorescence of TOMM20 mitochondrial markers indicates that 1,5-AG does not affect mitochondrial function during NLRP3 inflammasome activation. These results collectively demonstrate that the mechanism by which 1,5-AG inhibits NLRP3γ inflammasome activation is independent of alterations in macrophage ion flow and mitochondrial function.

[0046] 3: 1,5-AG inhibits ASC oligomerization (1) One day in advance, J774A.1 cells were seeded in 6-well plates. On the second day, the cells were pretreated with Opti-MEM medium (containing 2 μm / mL 1,5-AG) for 1 hour. Then, the cells were treated with LPS at a final concentration of 500 ng / mL for 4 hours, and then stimulated with ATP at a final concentration of 2.5 mM / mL for 30 minutes.

[0047] (2) After treatment, the cells were fixed with 4% formaldehyde for 10 minutes, followed by washing with PBS. They were then infiltrated with 0.1% Triton X-100 for 8 minutes at room temperature, followed by blocking with 3% BSA for 30 minutes. The cells were incubated overnight at 4°C with ASC antibody, and then incubated with Alexa Fluor 594 goat anti-rabbit secondary antibody for 30-45 minutes at room temperature. The cell nuclei were stained with DAPI (1:4000 dilution) for 5-10 minutes. Coverslips were placed on the slides, and the cells were observed and photographed using a Zeiss LSM710 confocal microscope.

[0048] like Figure 3E As shown, the immunofluorescence results of ASCs indicate that 1,5-AG can significantly inhibit the formation of ASC spots during the activation of the NLRP3 inflammasome, suggesting that 1,5-AG may inhibit the activation of the NLRP3 inflammasome by inhibiting ASC oligomerization.

[0049] Example 4: Study on the binding mode and binding region of 1,5-AG and NLRP3 1. 1,5-AG can be directly combined with NLRP3 (1) One day in advance, J774A.1 cells were seeded in 6-well plates. On the second day, the cells were pretreated with Opti-MEM medium (containing 2 μm / mL 1,5-AG) for 1 hour. Then, the cells were treated with LPS at a final concentration of 500 ng / mL for 4 hours, and then stimulated with ATP at a final concentration of 2.5 mM / mL for 30 minutes.

[0050] (2) Protein samples were directly lysed and collected, and evenly distributed into 6 PCR tubes. The samples were then subjected to three freeze-thaw cycles in liquid nitrogen and a 37°C water bath, followed by centrifugation at 4°C and 10,000 rpm for 20 minutes to remove lower-layer cell debris. The samples were then transferred to new PCR tubes. Each sample was heated in a PCR instrument at a temperature gradient (40, 42, 44, 46, 48, 50°C) for 3 minutes, then cooled to 20°C. The samples were then transferred to EP tubes and centrifuged at 4°C and 10,000 rpm for 20 minutes. The supernatant was collected, and an appropriate amount of 5*loading buffer was added. The tubes were then incubated in a 100°C metal bath for 10 minutes. Western blotting (WB) was then performed.

[0051] like Figure 4A As shown, Western blot results indicate that 1,5-AG can significantly alter the thermostability of the NLRP3 protein, suggesting that 1,5-AG may directly interact with the NLRP3 protein.

[0052] 2. Interaction between the 1,5-AG and NLRP3 NATCH domains (1) Download the 3D structures of the NLRP3 structural protein (Q8R4B8·NLRP3_MOUSE) and 1,5-AG from the Uniprot and PubChemx databases, respectively. Remove water and heteroatoms from the NLRP3 structural protein using PyMol. Define the active site, perform docking using AutoDock, and refine and display the docking results using PyMol.

[0053] (2) The binding affinity of 1,5-AG to the PYD or NATCH structures was determined using the SRP assay. The binding affinity of the small molecule 1,5-AG to the NLRP3-PYD or NLRP3-NATCH domains was evaluated using the Biacore T200 system. His-SUMO-tagged mouse NLRP3-PYD and NLRP3-NATCH proteins were immobilized on the CM5 chip surface via EDC / NHS chemical coupling, with a final binding amount of 7306.8 RU. Binding analysis was performed manually, with 5 concentrations of 1,5-AG (2-fold serial dilution) set at a flow rate of 30 μL / min, and binding and dissociation times of 120 s each. Data were acquired using a multi-cycle method and fitted using a 1:1 Langmuir binding model to calculate the equilibrium dissociation constant (KD).

[0054] like Figure 4B As shown in -C, molecular docking results indicate that 1,5-AG is most likely to interact with the ARG41 and GLU45 sites of PYD, or the GLU638 and ASP639 sites of NATCH; Figure 4DAs shown in Figure -E, 1,5-AG does not bind to the PYD domain but does bind to the NATCH domain, with a binding force of KD = 3.407E-4. These results indicate that 1,5-AG inhibits the interaction between NLRP3 and NEK7 and ASC by interacting with the NATCH domain of NLRP3, thereby suppressing the activation of the NLRP3 inflammasome.

[0055] Example 5: In vivo study of 1,5-AG inhibiting the NLRP3 inflammasome 1. 1,5-AG inhibits LPS-induced sepsis in mice (1) Six- to eight-week-old C57BL6 wild-type mice and NLRP3- / - mice of the same size (used to demonstrate that 1,5-AG inhibits LPS-induced sepsis in a manner dependent on NLRP3) were selected. The C57BL6 wild-type mice and NLRP3- / - mice were divided into three groups of six mice each. The specific grouping and treatment were as follows: Group 1 was injected intraperitoneally with 200 μL PBS; Group 2 was injected intraperitoneally with 200 μL LPS (25 mg / kg, dissolved in 50 μL PBS); Group 3 was first injected intraperitoneally with 1,5-AG, and then injected intraperitoneally with 200 μL LPS (25 mg / kg, dissolved in 50 μL PBS) 2 hours later.

[0056] (2) Six hours after LPS injection, mice were sacrificed, orbital blood was collected, and liver and intestinal tissues were collected. HE staining was used to analyze the pathological damage of the mouse tissues and organs.

[0057] like Figure 5A As shown, LPS injection significantly increased serum IL-1β levels in wild-type mice, and the addition of 1,5-AG effectively alleviated this increase, with levels similar to those in NLRP3- / - mice. Figure 5B As shown, LPS injection significantly increased the level of inflammatory cell infiltration in the intestinal and liver tissues of wild-type mice, and the addition of 1,5-AG effectively alleviated this infiltration, with levels similar to those in the intestinal and liver tissues of NLRP3- / - mice; Figure 5C As shown in Figure D, LPS injection significantly enhanced Caspase 1 activation and GSDMD cleavage in the intestinal and liver tissues of wild-type mice. The addition of 1,5-AG effectively alleviated these Caspase 1 activation and GSDMD cleavage. These results indicate that 1,5-AG effectively inhibits LPS-induced sepsis.

[0058] 2: 1,5-AG inhibits MSU-induced arthritis in mice (1) Six- to eight-week-old C57BL6 wild-type mice and NLRP3- / - mice of the same size (used to demonstrate that 1,5-AG inhibits MSU-induced arthritis in a manner dependent on NLRP3) were selected. The C57BL6 wild-type mice and NLRP3- / - mice were divided into three groups of eight mice each. The specific grouping and treatment were as follows: Group 1 was injected with 20 μL PBS into the ankle joint; Group 2 was injected with 1 mg MSU (dissolved in 50 μL PBS) into the ankle joint; Group 3 was first injected with 1,5-AG into the ankle joint, and then 1 mg MSU (dissolved in 50 μL PBS) into the ankle joint 30 minutes later.

[0059] (2) The width of the mouse ankle joint was measured with calipers at 2, 4, 6 and 8 hours after MSU treatment. After 12 hours, the mice were sacrificed, the ankle joint tissue was collected, and the activation of Caspase1 in the ankle joint tissue was detected by a Caspase1 activity assay kit. HE staining was used to examine the pathological changes in the ankle joint tissue.

[0060] like Figure 6A As shown, after injection of MSU, wild-type mice exhibited significant ankle swelling, which was effectively alleviated by the addition of 1,5-AG, and the degree of swelling was consistent with that in NLRP3- / - mice; Figure 6B As shown, after injection of MSU, the activation level of Caspase1 in the ankle joint tissue of wild-type mice was significantly increased. The addition of 1,5-AG effectively inhibited Caspase1 activation, and this activation level was similar to that of NLRP3- / - mice. Figure 6C As shown, injection of MSU significantly increased the level of inflammatory cell infiltration in the ankle joint tissue of wild-type mice. The addition of 1,5-AG effectively inhibited inflammatory cell infiltration in the ankle joint tissue, and the level was similar to that in NLRP3- / - mice. These data indicate that 1,5-AG effectively inhibits MSU-induced arthritis.

[0061] The embodiments described above can be further combined or replaced, and these embodiments are merely descriptions of preferred embodiments of the present invention, not limitations on the concept and scope of the present invention. Various changes and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the inventive concept are all within the protection scope of the present invention. The protection scope of the present invention is given by the appended claims and any equivalents.

Claims

1. Use of 1,5-anhydroglucitol (1,5-AG) in the preparation of drugs for inhibiting NLRP3 inflammasome activation.

2. The use according to claim 1, characterized in that, The NLRP3 inflammasome activation-related diseases are selected from LPS-induced sepsis, MSU-induced gouty arthritis, non-alcoholic fatty liver disease, systemic inflammatory response syndrome, or inflammatory bowel disease.

3. The use according to claim 1 or 2, characterized in that, The 1,5-AG inhibits the interaction of the NLRP3 protein with NEK7 and ASC by binding to the NATCH domain of the NLRP3 protein.

4. The use according to any one of claims 1 to 3, characterized in that, The drug is an oral formulation, injection, sustained-release microparticle, liposome, transdermal formulation, or other pharmaceutically acceptable dosage form.

5. A drug for inhibiting NLRP3 inflammasome activation, the active ingredient of which is 1,5-dehydro-D-sorbitol.

6. The drug according to claim 5, characterized in that, This drug is used to treat or alleviate diseases caused by abnormal activation of the NLRP3 inflammasome, including LPS-induced sepsis, MSU-induced gouty arthritis, non-alcoholic fatty liver disease, systemic inflammatory response syndrome, and inflammatory bowel disease.

7. The drug according to claim 5 or 6, characterized in that, The drug may further contain pharmaceutically acceptable excipients or carriers.

8. The medicament according to any one of claims 5 to 6, characterized in that, The drug can be formulated as an injection, oral solution, sustained-release granules, nanoemulsion, or micelle formulation.

9. Use of 1,5-anhydroglucitol (1,5-AG) in the preparation of functional foods for inhibiting NLRP3 inflammasome activation.

10. A method for inhibiting NLRP3 inflammasome activation using 1,5-AG, characterized in that, When 1,5-AG is applied to macrophages or animal models in a pre-inflammation body activation state, 1,5-AG inhibits the inflammation body assembly process by directly binding to the NLRP3 protein and blocking the oligomerization of NLRP3 with NEK7 and ASC.

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