Application of xanthiatin in medicine for preventing or treating NLRP3 inflammasome-mediated inflammatory diseases

Abstract

The invention provides application of xanthiatin in a medicine for preventing or treating NLRP3 inflammasome mediated inflammatory diseases, and belongs to the technical field of biological medicine. The natural product xanthiatin shows good activity of inhibiting the NLRP3 inflammasome, in in-vivo experiments, the xanthiatin can also effectively inhibit activation of the NLRP3 inflammasome, and the xanthiatin can be used for preparing drugs for preventing and treating NLRP3-mediated related diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and more particularly to the use of Xanthium sibiricum in the prevention or treatment of NLRP3 inflammasome-mediated inflammatory diseases. Background Technology

[0002] Inflammation is a protective immune response to exogenous stimuli, and its mechanism is closely related to the activation of the immune system, especially the innate immune system. As the body's first line of defense, the innate immune system plays a central role in maintaining homeostasis by recognizing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) through pattern recognition receptors (PRRs). Some PRRs can recruit downstream adaptor proteins to form multi-protein complexes—inflammasomes—which in turn promote the maturation and secretion of pro-inflammatory factors such as interleukin-1β (IL-1β) and interleukin-18 (IL-18). A moderate inflammatory response is crucial for clearing pathogens and danger signals, while an imbalanced response (too weak or too strong) can lead to persistent infection or tissue damage, and even induce various serious diseases, including hepatitis, enteritis, arthritis, diabetes, tumors, and neurodegenerative diseases. Therefore, a deep understanding of the regulatory mechanisms of inflammatory responses is of great significance for elucidating the pathological mechanisms of related diseases and developing novel therapeutic strategies.

[0003] Among the various inflammasomes identified, the NLRP3 inflammasome has become a research hotspot due to its extensive activation mechanisms and important pathophysiological functions. This complex consists of nucleotide-binding oligomerization domain-like receptor protein 3 (NLRP3), apoptosis-associated speckle-like protein (ASC), and caspase-1. Its activation requires a dual signal: the first signal (such as LPS) activates the NF-κB pathway via a Toll-like receptor, upregulating the expression of NLRP3 and pro-IL-1β; the second signal (such as nigrain) induces NLRP3 to assemble with ASC and pro-caspase-1 to form a functional inflammasome. Activated caspase-1 induces pyroptosis by cleaving Gasdermin D, while simultaneously promoting the maturation and secretion of IL-1β and IL-18. It is worth noting that the NLRP3 inflammasome can not only recognize pathogens, but also respond to a variety of endogenous danger signals, which makes it a dual-purpose target for infectious diseases and a variety of chronic inflammatory diseases such as gout, type 2 diabetes, atherosclerosis and neurodegenerative diseases.

[0004] Despite the promising results shown by NLRP3 inflammasome inhibitors (such as MCC950) in preclinical studies, their clinical translation remains a significant challenge. The termination of MCC950's Phase II clinical trial due to hepatotoxicity highlights the importance of developing highly selective, low-toxicity inhibitors. Currently, the pharmaceutical industry is significantly increasing its investment in NLRP3-targeted drug development, but no specific inhibitor has yet been approved for clinical use. Therefore, discovering novel, safe, and effective NLRP3 inflammasome inhibitors not only has important theoretical value but also provides new intervention strategies for the clinical treatment of related diseases.

[0005] Nonalcoholic steatohepatitis (NASH), one of the most common chronic liver diseases worldwide, affects 3-6% of the adult population. It is a serious, progressive liver disease that can progress to cirrhosis, liver failure, and liver cancer. It is also an emerging risk factor for type 2 diabetes, cardiovascular disease, and end-stage renal disease. Currently, there are no effective treatments for NASH patients, and no drugs have been approved. Clinically, basic treatments such as weight control and lifestyle modifications are generally recommended. Therefore, the development and design of drugs targeting NASH is a current hot topic and challenge in the field of liver disease research. Modern medical research shows that persistent or excessive activation of the NLRP3 inflammasome is an important mechanism of NASH, and targeted regulation of the NLRP3 inflammasome can effectively prevent and reverse NASH-related inflammation, damage, and pathological processes. However, there are currently no clinical drugs that specifically target the NLRP3 inflammasome. Based on the significant anti-inflammatory activities of various traditional Chinese medicines and the crucial role of the NLRP3 inflammasome in the development and progression of NASH, our research group previously used a novel high-throughput NLRP3 inflammasome activity screening system to discover that the small molecule Xanthium sibiricum derived from traditional Chinese medicine can significantly inhibit NLRP3 inflammasome activation. Xanthium sibiricum is a naturally occurring bicyclic sesquiterpene lactone, mainly isolated from the plant Xanthium sibiricum L. (family Asteraceae). In recent years, scholars at home and abroad have conducted in-depth research on the bioactivity of Xanthium sibiricum, finding that it has significant effects in antibacterial, anti-inflammatory, anti-insect, anti-tumor, and antiviral activity. The inventors systematically elucidated the target and molecular mechanism of Xanthium sibiricum's inhibition of the inflammasome. Based on this, and combined with a mouse model of NASH, the researchers systematically analyzed the potential of Xanthium sibiricum in preventing and treating NASH, providing potential candidate drugs for the prevention and treatment of NASH and other NLRP3 inflammasome-related diseases. This also provides a theoretical basis for the anti-inflammatory efficacy and resource development of traditional Chinese medicine and its related preparations. Summary of the Invention

[0006] The purpose of this invention is to provide the application of Xanthium sibiricum in the preparation of a drug for the prevention or treatment of NLRP3 inflammasome-mediated inflammatory diseases, which has a good inhibitory effect on the activity of NLRP3 inflammasome and has excellent therapeutic effect on the diseases mediated by it.

[0007] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the use of Xanthium sibiricum in the preparation of a medicament for the prevention or treatment of NLRP3 inflammasome-mediated inflammatory diseases.

[0008] Preferably, the NLRP3 inflammasome-mediated inflammatory diseases include, but are not limited to: gout, neurodegenerative diseases, infectious inflammatory diseases, type 2 diabetes, atherosclerosis, or non-alcoholic steatohepatitis.

[0009] Preferably, the use of Xanthium sibiricum in the preparation of medicaments for the prevention or treatment of non-alcoholic steatohepatitis.

[0010] Preferably, the molecular formula of the oxalis extract is C 15 H 18 O3, with a molecular weight of 246.3, has the following chemical structural formula: .

[0011] Preferably, the drug comprises Xanthium sibiricum and pharmaceutically acceptable excipients.

[0012] Beneficial effects

[0013] The Xanthium sibiricum provided by this invention can specifically target the NLRP3 inflammasome: it only inhibits the activation of the NLRP3 inflammasome, without inhibiting other inflammasomes such as AIM2 and NLRC4, thus avoiding the side effects caused by non-target inhibition and having better safety; it broadly covers the NLRP3 activation pathway: it can inhibit the classical NLRP3 activation induced by nigericin, ATP, MSU, and poly(I:C), and it can also block the non-classical NLRP3 activation induced by Pam3CSK4 pretreatment combined with LPS transfection, making it applicable to a wider range of scenarios; it works by blocking NLRP3-dependent ASC oligomerization (a key step in inflammasome activation), and in in vitro experiments, a concentration of 10 μM can almost completely inhibit NLRP3 activation and the release of inflammatory factors such as IL-1β and caspase-1, with high inhibition efficiency, providing a potential candidate drug for the treatment of NLRP3 inflammasome-mediated diseases. Attached Figure Description

[0014] Figure 1 This is a diagram showing the results of Xanthium sibiricum inhibiting the activation of NLRP3 inflammasomes in BMDM in Example 1; Figure 2The graph shows the results of Xanthium sibiricum inhibiting classical and non-classical NLRP3 activation in Example 2 (red: DMSO control group; black: Xan-treated group). Figure 3 The graph shows the results of the specific inhibition of NLRP3 inflammasome by Xanthium sibiricum in Example 3 (red: DMSO control group; black: Xan administration group). Figure 4 This is a graph showing the results of Xanthium sibiricum inhibiting ASC oligomerization during NLRP3 inflammasome activation in Example 4; Figure 5 This is a graph showing the results of Xanthium sibiricum inhibiting NLRP3 inflammasome activation and improving MCD-induced non-alcoholic hepatitis in vivo in Example 5. Detailed Implementation

[0015] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0016] Xanthatin, with the molecular formula C 15 H 18 O3, with a molecular weight of 246.3, is a white crystalline solid, soluble in DMSO. Its chemical structure is shown below: .

[0017] Experimental Design

[0018] This study primarily evaluated the therapeutic effect of xanthatin on NLRP3 inflammasome-mediated diseases. The effect of xanthatin on NLRP3 inflammasome activation in mouse BMDM was investigated. Evaluation was conducted using methods such as Western blotting, activity assays, and enzyme-linked immunosorbent assay (ELISA), and the therapeutic effect was assessed in a mouse model of NLRP3 inflammasome-mediated diseases.

[0019] animal

[0020] Eight-week-old C57BL / 6 mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). Researchers were unaware of the animal experiments conducted and randomly selected and grouped the mice, placing them in a specific sterile facility (12-hour / 12-hour light / dark cycle; 20±2℃). All animal experiments were approved by the Laboratory Animal Welfare and Ethics Committee of Capital Medical University.

[0021] reagents

[0022] Xanthatin (T3S0153), MCC950 (HY-12815A), and murine macrophage colony-stimulating factor (MCSF) (HY-P7085) were purchased from MedChemExpress (New Jersey, USA). Nigerian styraxin, ATP, MSU, poly(dA:dT), poly(I:C), and DMSO were purchased from Sigma-Aldrich. Pam3CSK4, ultrapure LPS, and Lfn-Flic were purchased from InvivoGen. Caspase-1 activity assay kit (G9951) and LDH assay kit (G1780) were purchased from Promega. NLRP3 antibody (15101S) and ASC antibody (sc-22, 514-R) were purchased from Cell Signaling. Proteintech. Active caspase-1 antibody (AG-20B-0042) and anti-mouse IL-1β antibody (AF-401-NA) were purchased from Adipogen and R&D Systems, respectively. Anti-LaminB 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. Mouse TNF-α ELISA kit (1217202) was purchased from Dakewe. Mouse IL-1β ELISA kit (MLB00C) was purchased from R&D Systems. StarFect transfection reagent (C101-01) was purchased from Genstar. Mouse alanine aminotransferase (ALT) was also purchased. The aminotransferase (ALT) kit (C009-2-1) and the mouse aspartate transaminase (AST) kit (C010-2-1) were purchased from Nanjing Jiancheng Biotechnology Institute.

[0023] Statistical methods

[0024] All experiments were conducted in a randomized, blinded manner. Data analysis was performed using GraphPad Prism (GraphPad Prism 10 software). All experimental results are expressed as mean ± SEM. Statistical significance between groups was analyzed using multiple one-way ANOVA and two-way t-tests. A p-value < 0.05 was considered statistically significant.

[0025] Example 1: Xanthium sibiricum inhibited the activation of the NLRP3 inflammasome in BMDM in a dose-dependent manner in mice.

[0026] 1.1 Cell Culture

[0027] Primary bone marrow cells were isolated from the femoral bone marrow of 8-week-old male C57BL / 6 mice and cultured for 6 days in DMEM complete medium (10% fetal bovine serum FBS, 1% penicillin) and mouse macrophage colony-stimulating factor (M-CSF, 50 ng / ml) to differentiate into mouse primary bone marrow-derived macrophages (BMDMs). Cells were cultured at 37°C and 5% CO2. BMDM cells were collected by digestion with trypsin and EDTA (2:1) and cultured at 1.2 × 10⁻⁶ cells / mL. 6 The cells were seeded at a density of 10 cells / ml in 12-well plates and incubated overnight.

[0028] 1.2 Cell viability assay

[0029] BMDMs were seeded into 96-well cell culture plates and incubated overnight. The next day, the culture medium was discarded, and the plates were treated with different concentration gradients of Xanthium sibiricum and incubated at 37°C and 5% CO2 for 6 hours. Half of the old culture medium was then discarded, and an equal volume of ATP assay working solution was added. The plates were then incubated at 37°C and 5% CO2 for 15 minutes. Finally, an equal volume of the solution was aspirated from each well into a white 96-well plate, and the fluorescence value was detected using a microplate reader.

[0030] 1.3 Detection of NLRP3 inflammasome activation and related inflammatory factors

[0031] Replace the culture medium with a concentration of 50 ng / mL. 1 Lipopolysaccharide (LPS) was pretreated in DMEM medium for 4 hours. After LPS stimulation was removed, cells were treated with different concentrations of Xanthium sibiricum for 30 minutes, followed by stimulation with nigericin (an NLRP3 inflammasome activator) for 45 minutes. Cell supernatants were collected after stimulation and analyzed using an IL-1β and TNF-α ELSIA assay kit. Caspase-Glo 1 Inflammasome Assay and LDH reagent were prepared according to the reagent instructions.

[0032] 1.4 Western Blot Analysis

[0033] Sample processing and Western blot determination of NLRP3 inflammasome-related protein expression: Repeat the above steps, collect cell culture supernatant and cell lysate, and incubate at 5000 r·min⁻¹ 1After centrifugation, add 1 / 4 volume of trichloroacetic acid (TCA) to the supernatant, incubate overnight at -20°C, then centrifuge at 12000 rpm. 1 Centrifuge at 4℃ for 15 min, discard the supernatant, wash once with ice-cold acetone, evaporate the acetone in a metal bath at 105℃, add 70 μL of 1× loading buffer after acetone evaporation, vortex to mix, boil in a water bath, and cool to obtain the supernatant sample. Add 200 μL of 1× loading buffer to each well of adherent cells, 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. Take 30 μL of protein sample for SDS-polyacrylamide gel electrophoresis, transfer the separated proteins in the gel to a PVDF membrane, block with 10% skim milk at room temperature for 0.5 h, add primary antibody solutions of pro-IL-1β, caspase-1p20, IL-1β, ASC, NLRP3, and Lamin B respectively, and incubate overnight at 4℃. Add horseradish peroxidase-labeled secondary antibody solutions and incubate at room temperature for 1 h, then develop with chemiluminescent solution and X-ray.

[0034] First, in order to study the burdock ( Figure 1 A) Effects on NLRP3 inflammasome activation: We first tested the cytotoxicity of Xanthium sibiricum in mouse bone marrow-derived macrophages (BMDM). Cell viability assays showed that Xanthium sibiricum at doses below 20 μM in BMDM did not exhibit any cytotoxicity (e.g., Figure 1 As shown in B).

[0035] Next, we evaluated the effect of Xanthium sibiricum on NLRP3 inflammasomes (results are shown below). Figure 1 As shown in Figure C), Western blot and bioassay results revealed that Xanthium sibiricum inhibited caspase-1 activation under nigericin stimulation. At a concentration of 10 μM, it almost completely inhibited the secretion of mature IL-1β (IL-1β, p17) and active caspase-1 (Casp-1 p20) from BMDM cells induced by nigericin. Bioassay and ELISA results indicated that Xanthium sibiricum inhibited caspase-1 activation in a dose-dependent manner. Figure 1 D), IL-1β secretion ( Figure 1 In addition, we also examined another indicator following inflammasome activation—pyroptosis. By detecting LDH released during cell death, we observed that nigericin significantly induced LDH release, while xanthine blocked LDH release from cells activated by inflammasomes. Figure 1 F). At the same time, we can also observe inflammatory factors that are not regulated by inflammasomes, and the secretion of TNF-α is not affected by Xanthium sibiricum (F). Figure 1 G).

[0036] Figure 1 A represents the structural formula of Xanthium sibiricum; B represents the cell viability assay used to evaluate the activity of BMDM cells treated with different doses of Xanthium sibiricum for 12 hours. CG represents the Western blot analysis of pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC in whole-cell lysate (WCL) after BMDM cells were pretreated with LPS for 4 hours, treated with Xanthium sibiricum for 30 minutes, and then stimulated with nigericin for 25 minutes, as well as the secretion of activated caspase-1 (p20) and IL-1β (p17) in the culture supernatant (SN) of BMDM cells (C). Caspase-1 activity (D), IL-1β secretion (E), LDH release (F), and TNF-α content (F) in SN were measured. Coomassie brilliant blue staining was used as a supernatant loading control, while lamin B was used as a lysate loading control. Data are expressed as mean ± SEM of biological replicates (n=3). Data are expressed as mean ± SD. Compared with 0 μM, P < 0.05. Compared with 0 μM, P < 0.01; the significance of the difference was determined using one-way ANOVA.

[0037] It is evident that Xanthium sibiricum has a significant inhibitory effect on inflammasomes in cells, and this inhibitory effect is specific to a certain extent.

[0038] Example 2: Xanthium sibiricum inhibits classical and non-classical NLRP3 activation

[0039] 2.1 Cell Culture and NLRP3 Inflammasome Activation

[0040] Primary bone marrow cells were isolated from the femoral bone marrow of 8-week-old male C57BL / 6 mice and cultured for 6 days in DMEM complete medium (10% fetal bovine serum FBS, 1% penicillin) and mouse macrophage colony-stimulating factor (M-CSF, 50 ng / ml) to differentiate into mouse primary bone marrow-derived macrophages (BMDMs). Cells were cultured at 37°C and 5% CO2. BMDM cells were collected by digestion with trypsin and EDTA (2:1) and cultured at 1.2 × 10⁻⁶ cells / mL. 6 Cells were seeded at a density of [number] cells / ml in 12-well plates overnight. The next day, cells were stimulated with LPS (50 ng / ml) for 4 hours. After treatment with the compound for 30 minutes, cells were stimulated with nigericin (10 μM) and ATP (5 mM) for 30 minutes, followed by stimulation with MSU (250 μg / ml) and poly(I:C) (2 μg / ml) for 4 hours. Cell supernatant was collected after stimulation and analyzed using an IL-1β and TNF-α ELSIA assay kit.

[0041] 2.2 Western Blot analysis of proteins and detection of related inflammatory factors

[0042] Sample processing and Western blot determination of NLRP3 inflammasome-related protein expression: Same as 1.3 and 1.4 in Example 1.

[0043] The NLRP3 inflammasome is a multiprotein complex, and its activation can be induced by various stimuli, including both classical and non-classical stimuli. Therefore, we further investigated whether xanthamine inhibited NLRP3 inflammasome activation induced by various stimuli other than nigrain. Western blotting results showed that 40 μM xanthamine effectively inhibited the decrease in caspase-1 p20 protein levels in the supernatant of BMDM cells induced by classical NLRP3 stimuli, such as nigrain, ATP, MSU, and poly(I:C). Figure 2 A); We also detected IL-1β and TNF-α in BMDM cell supernatant using ELISA. The results showed that compared with DMSO alone (black), (DMSO control group) Xanthium sibiricum (red) effectively inhibited the classical stimulation-induced secretion of IL-1β. Figure 2 B), but it has no effect on TNF-α, a cytokine not regulated by the activation of inflammasomes. Figure 2 C). The above results demonstrate that oxaliplatin can act as a broad-spectrum inhibitor of the NLRP3 inflammasome in mouse cells.

[0044] Figure 2 AC consisted of BMDMs pretreated with LPS, then treated with xanthine (10 μM) for 30 min, followed by stimulation with nigrain, ATP, poly(I:C), or MSU. Western blot analysis was performed on pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC in whole-cell lysates (WCL); secretion of activated caspase-1 (p20) in the culture supernatant (SN) of BMDMs was measured (A). IL-1β (B) and TNF-α secretion in SN were measured (C). Data are presented as mean ± SEM of biological replicates (n=3). Compared with 0 μM, P < 0.05. Compared with 0 μM, P < 0.01; the significance of the difference was determined using one-way ANOVA and Dunnett's post-hoc test.

[0045] Example 3: Xanthium sibiricum is a specific inhibitor of the NLRP3 inflammasome.

[0046] 3.1 Cell culture and activation of various inflammasomes

[0047] Primary bone marrow cells were isolated from the femoral bone marrow of 8-week-old male C57BL / 6 mice and cultured for 6 days in DMEM complete medium (10% fetal bovine serum FBS, 1% penicillin / streptomycin) and mouse macrophage colony-stimulating factor (M-CSF, 50 ng / ml) to differentiate into mouse primary bone marrow-derived macrophages (BMDMs). Cells were cultured at 37°C and 5% CO2. BMDM cells were collected by digestion with trypsin and EDTA (2:1) and cultured at 1.2 × 10⁻⁶ cells / mL. 6 Cells were seeded overnight in 12-well plates at a density of 10 cells / ml. The next day, cells were stimulated for 4 hours with LPS (50 ng / ml) or Pam3CSK4 (400 ng / ml). After treating the cells with the compound for 30 minutes, they were stimulated for 30 minutes with Nigericin (10 μM), and then for 4 hours with ultra-LPS (1 μg / ml), poly(dA:dT) (2 μg / ml), and bacterial flagellin flagelin.

[0048] 3.2 Western blotting analysis and detection of related inflammatory factors

[0049] Sample processing and Western blot determination of NLRP3 inflammasome-related protein expression: Same as 1.3 and 1.4 in Example 1.

[0050] Considering that multiple NLR and ALR families can regulate inflammasomes, we then investigated the effect of xanthine on the activation of other inflammasomes, such as the AIM2 and NLRC4 inflammasomes. Western blotting results showed that 10 μM xanthine effectively inhibited the protein content of caspase-1 p20 in the supernatant of BM1DM cells stimulated by nigericin; however, it 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 flagelin. Figure 3 A). Simultaneously, we constructed a non-classical NLRP3 inflammasome activation model induced by Pam3CSK4 pretreatment combined with LPS transfection. Compared to treatment with DMSO (black), the DMSO group, treated with 10 μM xanthate (red), almost completely inhibited the secretion of mature IL-1β (IL-1β p17) and active Caspase-1 (Casp-1 p20) generated by Pam3CSK4 combined with LPS transfection-induced non-classical NLRP3 inflammasome activation. Figure 3A). We also detected IL-Iβ activation in BMDM cell supernatant using ELISA. The results showed that 10 μM xanthine effectively inhibited IL-Iβ activation in BMDM cell supernatant induced by *Bacillus alginolyticus* stimulation and LPS co-transfection with Pam3CSK4; however, it had almost no effect on IL-Iβ activation in BMDM cell supernatant induced by poly(dA:dT) transfection and bacterial flagellin flagellin. Figure 3 B) has no effect on the cytokine TNF-α. Figure 3 C). In summary, we can conclude that Xanthium sibiricum effectively inhibits the activation of the NLRP3 inflammasome induced by Nigeriacin stimulation and the non-classical NLRP3 inflammasome activation induced by Pam3CSK4 combined with LPS transfection in BMDM cells, but has no effect on the activation of the AIM2 inflammasome induced by poly(dA:dT) transfection and the NLRC4 inflammasome induced by bacterial flagellin. This indicates that Xanthium sibiricum has no inhibitory effect on the AIM2 and NLRC4 inflammasomes, but has a significant inhibitory effect on the NLRP3 inflammasome, suggesting that Xanthium sibiricum specifically inhibits the NLRP3 inflammasome.

[0051] Figure 3 In the AC group, BMDM cells were pretreated with LPS, then treated with xanthine (10 μM) for 30 min, followed by stimulation with nigrain for 30 min, poly(dA:dT), bacterial flagellin flagellin, or Pam3CSK4 pretreatment, and then transfected with LPS for 5 h. Western blot analysis was performed on pro-caspase-1 (p45), pro-IL-1β, NLRP3, and ASC in whole-cell lysate (WCL); secretion of activated caspase-1 (p20) in the culture supernatant (SN) of BMDM cells was measured (A). IL-1β (B) and TNF-α secretion in SN were measured (C). Data are presented as mean ± SEM of biological replicates (n=3). Compared with 0 μM, P<0.05, Compared with 0 μM, P < 0.01; the significance of the difference was determined using one-way ANOVA and Dunnett's post-hoc test.

[0052] Example 4: Xanthium sibiricum blocks NLRP3-dependent ASC oligomerization and inhibits the NF-κB pathway in the Prime stage of the NLRP3 inflammasome.

[0053] 4.1 Cell culture and activation of various inflammasomes

[0054] The steps are the same as 1.1 in Example 1 and 3.1 in Example 3.

[0055] 4.2 ASC Oligomerization Test

[0056] The NLRP3 inflammasome activation procedure was the same as the experimental procedures for cell culture and NLRP3 inflammasome activation. Cells were lysed for 15 min with Triton buffer (150 mM NaCl, 50 mM Tris-HCl [pH 7.5], 0.5% Triton X-100, and a mixture of EDTA-free protease inhibitors). Cells were scraped from the wells and centrifuged at 6000 g for 15 min at 4 °C. The supernatant and precipitate were designated Triton X-soluble and Triton X-insoluble, respectively. The Triton X-insoluble fraction was washed and resuspended in 200 μl PBS and crosslinked with 2 mM DSS at 37 °C for 30 min. The precipitate was centrifuged at 6000 g for 15 min at 4 °C, then collected and dissolved in 1×Triton loading buffer for Western blot analysis.

[0057] 4.3 Western Blot Analysis

[0058] Same as 1.4 in Example 1.

[0059] ASC oligomerization is a key intermediate step in the activation of the inflammasome. Therefore, we evaluated the effect of Xanthium sibiricum on ASC oligomerization during the activation of the NLRP3 inflammasome and found that it could inhibit ASC oligomerization during NLRP3 activation in a concentration-dependent manner. Figure 4 A), but it has no effect on ASC oligomerization during the activation process of NLRC4 and AIM2 inflammasomes (A). Figure 4 B). This suggests that Xanthium sibiricum may specifically inhibit the NLRP3 inflammasome by suppressing ASC oligomerization or by inhibiting upstream events that trigger ASC oligomerization. Therefore, further investigation into its upstream mechanisms is needed.

[0060] Many upstream mechanisms can act as triggers to influence ASC oligomerization, so we further investigated the possible mechanism by which Xanthium sibiricum inhibits NLRP3 inflammasome activation. In the early stages of NLRP3 inflammasome activation, there is also the Prime phase, which serves as the first signal for NLRP3 inflammasome activation. In the Prime phase, cells induce NF-κB activation by recognizing pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs), triggering the transcriptional expression of NLRP3 and pro-IL-1β. Therefore, we used Western blotting to detect NLRP3-related proteins in BMDM cell lysates to investigate whether Xanthium sibiricum affects NF-κB-dependent NLRP3 and pro-IL-1β expression. The results showed that, under the condition of LPS stimulation for 3 h followed by treatment with different concentrations of oxaliplatin for 1 h, oxaliplatin did not affect the expression of already formed NLRP3 and pro-IL-1β proteins; however, under the condition of treatment with different concentrations of oxaliplatin for 1 h followed by LPS co-stimulation for 3 h, we found that oxaliplatin could inhibit the expression of already formed NLRP3 and pro-IL-1β proteins, and the inhibitory effect became stronger with increasing concentration. Figure 4 C). It can be seen that treating BMDM cells with Xanthium sibiricum before LPS stimulation can inhibit the expression of NLRP3 and pro-IL-1β proteins, which indicates that Xanthium sibiricum can inhibit the NF-κB signaling pathway. However, under the phenotype we tested, the expression of NLRP3 and pro-IL-1β proteins was not observed. Therefore, it can be concluded that the inhibitory effect of Xanthium sibiricum on the NLRP3 inflammasome does not work by downregulating the expression of NLRP3 and pro-IL-1β proteins.

[0061] Figure 4 In Figure A, LPS-induced BMDM cells were pretreated with xanthine (10 μM) for 30 minutes and then stimulated with nigrain for 30 minutes. Western blot analysis of cross-linked ASCs in Triton X insoluble particles was performed. In Figure B, LPS-induced BMDM cells were pretreated with xanthine (10 μM), then with nigrain, poly(dA:dT), or bacterial flagellin flagellin and Pam3CSK4, followed by Western blot analysis of cross-linked ASCs in Triton X insoluble particles. In Figure C, LPS-induced BMDM cell lysates were obtained by either LPS stimulation for 3 hours followed by xanthine treatment for 1 hour or by first treating with xanthine for 1 hour followed by LPS stimulation for 3 hours.

[0062] Example 5: Xanthium sibiricum can improve MCD-induced non-alcoholic steatohepatitis (NASH).

[0063] 5.1 Construction of the NASH animal model and determination of liver function and inflammatory factors

[0064] Thirty-six 8-week-old male C57BL / 6 mice were purchased from Beijing Sibefore Biotechnology Co., Ltd. After a week of acclimatization in the laboratory, they were randomly divided into six groups (n=6): a methionine- and choline-deficient diet group (fed MCD diet) and a diet with adequate methionine and choline (fed MCS (methionine-choline-sufficient diet)). The MCS diet group consisted of a control group and a group receiving Xanthium sibiricum (30 mg / kg) treatment; the MCD diet group consisted of a control group, a group receiving Xanthium sibiricum (30 mg / kg) treatment, and a group receiving MCC950 (40 mg / kg) treatment. Mice were fed the diet for one week, and the above dosages were administered once daily. After one week, the diet was continued daily, with treatment every other day. Five weeks later, blood samples were collected from the mice to detect serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) liver function indicators. Real-time quantitative PCR was used to detect the expression levels of Il-1β and Tnf-α mRNA in liver tissue.

[0065] Sustained or excessive activation of the NLRP3 inflammasome is a crucial mechanism of NASH, and targeted regulation of the NLRP3 inflammasome can effectively prevent and reverse NASH-related inflammation, damage, and pathological processes. Therefore, we fed mice daily a diet lacking choline and methionine (which can be converted into choline). Because fat in the mouse liver could not be normally transported into the bloodstream, it accumulated in the liver cells and intercellular spaces, forming lipid droplets, which in turn led to NASH. The choline and methionine-deficient diet (MCD) induced non-alcoholic fatty liver disease (NAFLD) mouse model is a classic NLRP3 inflammasome-related disease model. We used this model to evaluate the preventive and therapeutic effects of Xanthium sibiricum on NAFLD. The results showed that 30 mg / kg of Xanthium sibiricum and the known NLRP3 inhibitor MCC950 significantly reduced liver function loss caused by NAFLD in mice. Figure 5 AB) and expression of inflammatory molecules ( Figure 5 (CD) abnormalities. Furthermore, we found no significant difference between the group receiving Xanthium sibiricum and MCC950 in combination and the groups receiving Xanthium sibiricum or MCC950 alone. The experimental results indicate that Xanthium sibiricum can effectively inhibit the NLRP3 inflammasome and improve MCD-induced non-alcoholic fatty liver disease in vivo, and its effect is mainly achieved through the inhibition of the NLRP3 inflammasome.

[0066] Figure 5In the AD group, mice were fed a 4-week MCD diet and then treated with intraperitoneal injections of either Xanthium sibiricum (30 mg / kg), MCC950 (40 mg / kg), or a combination of Xanthium sibiricum (30 mg / kg) and MCC950 (40 mg / kg) for 1 hour. The levels of ALT (A), AST (B), IL-1β (C), and TNF-α (D) in the liver were measured using ELISA. Data are expressed as mean ± SD. Compared with 0 μM, P < 0.05. Compared with 0 μM, P < 0.01; the significance of the difference was determined using one-way ANOVA and Dunnett's post-hoc test.

[0067] In summary, we found that Xanthium sibiricum can specifically inhibit the activation of the NLRP3 inflammasome in a dose-dependent manner, and its inhibitory effect is broad-spectrum, but it does not inhibit the activation of other inflammasomes, such as AIM2 and NLRC4. Furthermore, in animals, Xanthium sibiricum exerts anti-inflammatory activity under NLRP3-related inflammatory conditions, such as MCD-induced non-alcoholic steatohepatitis. These findings demonstrate that Xanthium sibiricum is a specific NLRP3 inflammasome inhibitor with great potential for development as a clinical drug for treating NLRP3 inflammasome-mediated diseases.

[0068] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of Xanthium sibiricum in the preparation of a medicament for the prevention or treatment of NLRP3 inflammasome-mediated inflammatory diseases.

2. The application as described in claim 1, characterized in that, The NLRP3 inflammasome-mediated inflammatory diseases include, but are not limited to: gout, neurodegenerative diseases, infectious inflammatory diseases, type 2 diabetes, atherosclerosis, or non-alcoholic steatohepatitis.

3. The application as described in claim 2, characterized in that, The use of Xanthium sibiricum in the preparation of drugs for the prevention or treatment of non-alcoholic steatohepatitis.

4. The application as described in any one of claims 1 to 3, characterized in that, The molecular formula of the cocklebur is C 15 H 18 O3, with a molecular weight of 246.3, has the following chemical structural formula: 。 5. The application as described in claim 1, characterized in that, The drug includes Xanthium sibiricum and pharmaceutically acceptable excipients.

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