Natural small molecule inhibitors targeting nlrp3 inflammasome and uses thereof

Abstract

The embodiment of the application discloses a natural small molecule inhibitor targeting NLRP3 inflammasome and application thereof, and belongs to the technical field of biological medicine. Chuanxiongdiolide R11 is a phthalide dimer compound with clear stereochemistry found in traditional Chinese medicine Chuanxiong, which can target NLRP3 inflammasome, prevent the activation and assembly of the inflammasome, thereby inhibiting caspase-1 activation, GSDMD cleavage, pyroptosis and the maturation and release of IL-1beta / IL-18. The compound can be used for preparing a drug for treating NLRP3 related inflammatory diseases such as systemic inflammation and peritonitis, and has a good clinical application prospect, and provides a new idea for developing and applying NLRP3 inhibitors in clinic.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, specifically to a natural small molecule inhibitor targeting the NLRP3 inflammasome and its application. More specifically, the natural small molecule inhibitor is a phthalide dimer small molecule compound Chuanxiongdiolide R11 derived from Ligusticum chuanxiong, and its use in the preparation of drugs for the prevention or treatment of NLRP3 inflammasome-mediated inflammatory diseases. Background Technology

[0002] The NLRP3 (NOD-like receptor family pyrin domain-containing protein 3) inflammasome, as a core intracellular sensor of the innate immune system, is a key driver of the development and progression of inflammatory diseases. Its abnormal activation is closely related to the pathological processes of various chronic inflammations, degenerative diseases, and autoimmune / inflammatory syndromes. Activation of the NLRP3 inflammasome is mediated primarily through two well-defined mechanisms: the classical pathway and the non-classical pathway. In the classical pathway, after cells come into contact with pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) (such as adenosine triphosphate (ATP), nigericin, monosodium urate (MSU) crystals, abnormal ion flow, reactive oxygen species (ROS) generation, and lysosomal damage), NEK7-dependent NLRP3 oligomerization is initiated via proximal signal transduction, which in turn recruits the adaptor protein ASC (apoptosis-associated speckle-like protein) and promotes its polymerization, ultimately mediating the activation of pro-caspase-1. In the non-classical pathway, lipopolysaccharide (LPS) in the cytoplasm can specifically activate caspase-4 / 5 / 11. These caspases further cleave pyroptosis D (GSDMD), indirectly triggering NLRP3 inflammasome activation through downstream signaling. The core common link between the two pathways is that NLRP3 specifically binds to NEK7 and forms oligomers. The oligomerized NLRP3 interacts isomorphically with the PYD domain of ASC through the PYD domain. ASC then recruits pro-caspase-1 through the CARD domain and assembles into a mature NLRP3 inflammasome. Subsequently, pro-caspase-1 undergoes self-cleavage activation. The activated caspase-1 mediates the cleavage of GSDMD to produce an N-terminal active fragment, inducing pyroptosis. On the other hand, it cleaves pro-IL-1β and pro-IL-18 into mature pro-inflammatory cytokines and releases them into the extracellular space, forming a persistent inflammatory cascade that ultimately exacerbates tissue damage and disease progression.

[0003] Currently, the development of inhibitors targeting the NLRP3 inflammasome has become a hot topic in the biopharmaceutical field, with many candidate drugs entering clinical research stages, the vast majority of which are structural analogs of MCC950. The mechanism of action of these inhibitors mainly involves targeting the NACHT ATPase domain of NLRP3, inhibiting its ATPase activity to prevent inflammasome assembly. Although MCC950 has shown significant anti-inflammatory efficacy in preclinical disease models such as rheumatoid arthritis, multiple sclerosis, and CAPS, its development was forced to be terminated in subsequent clinical trials due to severe hepatotoxicity, limiting its clinical translation and application. Furthermore, the chemical structures of existing NLRP3 inhibitors are mostly concentrated in the sulfonylurea backbone centered on MCC950, resulting in narrow chemical space and insufficient structural diversity. This not only easily leads to drug resistance but also increases the risk of off-target effects due to the homology of the NACHT domain in NLR family proteins, potentially causing side effects such as immune dysfunction.

[0004] Chuanxiongdiolide R11 is a phthalide dimer-like natural compound isolated and purified from the non-medicinal part of the rootlets of the traditional Chinese medicine Ligusticum chuanxiong Hort. Our prior patent (Patent No.: ZL202210856953.2, Authorization Announcement No.: CN115160335 B) has reported that it can inhibit LPS-induced NO production in Raw264.7 macrophages, exhibiting certain anti-inflammatory activity, and also possesses pharmacological activities such as vasodilatory effects. However, existing research only focuses on preliminary anti-inflammatory effects at the cellular level, without elucidating its anti-inflammatory molecular mechanism, and there are no reports on targeted regulation of the NLRP3 inflammasome pathway. Summary of the Invention

[0005] Therefore, embodiments of the present invention provide the use of Chuanxiongdiolide R11, a phthalide dimer small molecule compound derived from Ligusticum chuanxiong. This invention is the first to discover that Chuanxiongdiolide R11 can act as a naturally derived, specific NLRP3 inhibitor, blocking inflammasome assembly and downstream signal transduction to exert a highly effective anti-inflammatory effect. This discovery not only clarifies the mechanism of action of Chuanxiongdiolide R11 but also provides a novel candidate molecule for NLRP3 inflammasome-mediated inflammatory diseases, offering a new technical solution and strategy to address the shortcomings of existing inhibitors.

[0006] To achieve the above objectives, the embodiments of the present invention provide the following technical solutions:

[0007] Use of Chuanxiongdiolide R11 in the preparation of medicaments for the prevention or treatment of NLRP3 inflammasome-mediated inflammatory diseases, wherein Chuanxiongdiolide R11 has the structure shown in formula (I):

[0008] (I).

[0009] Furthermore, the Chuanxiongdiolide R11 inhibits caspase-1 activation, GSDMD cleavage, HMGB1 release, pyroptosis, and the maturation and release of IL-1β and IL-18 by blocking the assembly of the NEK7-NLRP3-ASC complex.

[0010] Furthermore, the Chuanxiongdiolide R11 can selectively inhibit both the classical and non-classical activation pathways of the NLRP3 inflammasome.

[0011] Furthermore, the NLRP3 inflammasome-mediated inflammatory diseases include LPS-induced systemic inflammation or MSU-induced peritonitis.

[0012] Furthermore, the drug includes a pharmaceutically acceptable carrier.

[0013] Furthermore, the dosage forms of the drug include tablets, granules, capsules, pills, solutions, emulsions, suspensions, injections, aerosols, powder inhalers, ointments, lotions, eye drops, patches, nasal drops, mouthwashes, sublingual suppositories, and suppositories.

[0014] The embodiments of the present invention have the following advantages:

[0015] The compound Chuanxiongdiolide R11 provided by this invention is a well-defined phthalide dimer from the traditional Chinese medicine Ligusticum chuanxiong. Using the phthalide dimer as the core framework expands the chemical space for NLRP3 inhibitors and avoids the inherent defects of sulfonylurea chemical types. This compound can specifically target the NLRP3 inflammasome, achieving multiple precise regulation of the NLRP3 inflammasome pathway by blocking the assembly of the NEK7-NLRP3-ASC complex, inhibiting caspase-1 activation, GSDMD cleavage, pyroptosis, and the maturation and release of IL-1β and IL-18. In vitro and in vivo experiments have confirmed its significant NLRP3-dependent anti-inflammatory activity. As a naturally derived small molecule, Chuanxiongdiolide R11 has advantages such as low toxicity and high biocompatibility, improving the poor drug-likeness of synthetic small molecule inhibitors and facilitating faster drug development and clinical translation. This compound can be used to prepare drugs for treating systemic inflammation, peritonitis, and other NLRP3-related inflammatory diseases, providing a new direction for the clinical development of novel NLRP3 inhibitors and showing promising application prospects. Attached Figure Description

[0016] To more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are merely exemplary, and those skilled in the art can derive other embodiments based on the provided drawings without creative effort.

[0017] Figure 1 This is a diagram showing the results of Chuanxiongdiolide R11 inhibiting the activation of the NLRP3 inflammasome in THP-1 cells, as provided in Example 1 of this invention.

[0018] Figure 2 This is a graph showing the results of Chuanxiongdiolide R11 inhibiting classical and non-classical NLRP3 activation, provided in Embodiment 2 of the present invention.

[0019] Figure 3 The diagram shows the results of Chuanxiongdiolide R11 inhibiting caspase-1 activation to suppress NLRP3 inflammasome-mediated pyroptosis, as provided in Example 3 of this invention.

[0020] Figure 4 This is a graph showing the results of Chuanxiongdiolide R11 inhibiting ASC oligomerization during NLRP3 inflammasome activation, as provided in Example 4 of this invention.

[0021] Figure 5This is a graph showing the in vivo inhibition of systemic inflammation in NLRP3-dependent mice by Chuanxiongdiolide R11, as provided in Example 5 of this invention.

[0022] Figure 6 The image shows the in vivo inhibition of NLRP3-dependent mouse peritonitis by Chuanxiongdiolide R11, as provided in Example 5 of this invention. Detailed Implementation

[0023] The following specific embodiments illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0024] The materials used in the following embodiments are shown below:

[0025] 1. Medicine

[0026] Chuanxiongdiolide R11 (abbreviated as CDR11) is directly isolated from the rootlets of Ligusticum chuanxiong, with the molecular formula C. 24 H 28 O4, molecular weight 380.20, is a yellow oily substance, soluble in methanol and DMSO. Its structural formula is as follows:

[0027] ,

[0028] This compound is a novel compound isolated from the rootlets of Ligusticum chuanxiong in the applicant's previous patent (Patent No.: ZL 202210856953.2, Authorization Announcement No.: CN115160335 B, Invention Title: A Phenylphthalide Dimer and Its Preparation Method and Application). The extraction and separation method has been disclosed in detail in that patent, and will not be repeated here in this invention.

[0029] 2. Reagents and materials

[0030] Lipopolysaccharide (LPS), adenosine triphosphate (ATP), nigericin, monosodium urate (MSU), MCC950, phorbol 12-myristate 13-acetate (PMA), and Cell Counting Kit-8 (CCK-8) were all purchased from Selleck Chemicals (Shanghai, China). The enzyme-linked immunosorbent assay (ELISA) kits for interleukin-1β (IL-1β), interleukin-18 (IL-18), tumor necrosis factor-α (TNF-α), and lactate dehydrogenase (LDH) were purchased from AmyJet Scientific Inc. (Wuhan, Hubei, China). The Caspase-1 FLICA probe (FAM-YVAD-FMK) was purchased from Immunochemistry Technologies (Davis, California, USA). Antibodies against NLRP3, ASC, NEK7, caspase-1 p20, GSDMD, high mobility group box 1 (HMGB1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-actin, and α-tubulin were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA).

[0031] Example 1: Chuanxiongdiolide R11 dose-dependently inhibited the activation of the NLRP3 inflammasome in THP-1 cells.

[0032] 1.1 Cell Culture

[0033] Human mononuclear cell line THP-1 cells were purchased from the Cell Bank of the Chinese Academy of Sciences and cultured routinely in a cell culture incubator at 37°C, 5% CO2, and saturated humidity in RPMI-1640 medium (purchased from Gibco) containing 10% fetal bovine serum (FBS, purchased from Gibco), 100 U / mL penicillin, and 100 μg / mL streptomycin.

[0034] 1.2 Cell viability assay

[0035] THP-1 cells were used at a rate of 1×10 5 Cells were seeded at a density of 100 μL / well in 96-well cell culture plates, and PMA was added to each well to a final concentration of 100 nM. The plates were then incubated overnight at 37°C with 5% CO2. The next day, the original culture medium was discarded, and fresh complete culture medium containing different concentrations of Chuanxiongdiolide R11 (0, 1.25 μM, 2.5 μM, 5 μM, 10 μM, 20 μM, 40 μM) was added to each well, with three replicates per concentration and a final volume of 100 μL per well. After incubation at 37°C with 5% CO2 for 24 h, 10 μL of CCK-8 reagent was added to each well, and incubation continued for another 2 h. The absorbance (OD value) of each well was measured using a microplate reader (Bio-Rad). Cells without the drug were used as a control group. Cell viability was calculated as follows: Cell viability (%) = (OD value of drug-treated group / OD value of control group) × 100%.

[0036] 1.3 NLRP3 inflammasome activation

[0037] To induce cell differentiation into macrophage-like cells, THP-1 cells in logarithmic growth phase were harvested and treated with 2 × 10⁻⁶ cells. 6 Cells were seeded at a density of [number] cells / mL in 6-well culture plates, and PMA was added to a final concentration of 100 nM. The next day, the PMA-containing medium was discarded, and the cells were treated with Opti-MEM medium containing 100 ng / mL LPS for 3 hours to initiate the NLRP3 inflammasome initiation phase. Then, different concentrations of Chuanxiongdiolide R11 (2 μM, 4 μM, 8 μM) were added for 1 hour each. Finally, ATP, the classical activator of the NLRP3 inflammasome pathway, was added to a final concentration of 5 mM, and stimulation was continued for 30 minutes to induce NLRP3 inflammasome activation. The supernatant and cells were collected for subsequent ELISA and Western blotting.

[0038] 1.4 Detection of relevant inflammatory factors

[0039] Collect the cell supernatant from each well in section 1.3, centrifuge at 4℃ and 12000 rpm for 10 min, and use the supernatant for later use. Follow the instructions of the IL-1β, IL-18, and TNF-α ELISA kits to detect the concentration of each cytokine in the supernatant. Each sample was tested in triplicate. The OD value at 450 nm was read using a microplate reader, and the actual concentration was calculated based on the standard curve.

[0040] 1.5 Western blot analysis of proteins

[0041] After treating cells according to the method in Section 1.3, collect the cell supernatant and cell pellet for separation by 10%-15% SDS-PAGE gel electrophoresis. After electrophoresis, transfer the proteins to a PVDF membrane under constant current of 200 mA and ice bath conditions for 2 h. After transfer, place the PVDF membrane in TBST buffer containing 5% skim milk powder and block at room temperature for 1 h. Discard the blocking solution, wash the membrane three times (5 min each time) with TBST buffer, and add diluted primary antibodies: NLRP3, ASC, NEK7 antibody, caspase-1 p20, IL-1β antibody, GSDMD antibody, HMGB1 antibody, GAPDH antibody, or β-actin antibody, and incubate overnight at 4°C. The next day, wash the membrane three times (5 min each time) with TBST buffer, add HRP-labeled secondary antibody of the corresponding species, and incubate at room temperature for 1 h. The membrane was washed three times with TBST buffer (5 min each time), and ECL chemiluminescence reagent was added. Protein bands were photographed using a chemiluminescence imaging system (Bio-Rad). Gray-scale quantitative analysis was performed using ImageJ software. The relative expression level of the target protein was calculated using GAPDH, β-actin, or α-tubulin as internal controls.

[0042] First, the inhibitory effect of Chuanxiongdiolide R11 on ATP-induced NLRP3 inflammasome activation in LPS-pretreated THP-1 macrophages was evaluated. The results showed that Chuanxiongdiolide R11 potently and dose-dependently inhibited IL-1β release, with an IC50 concentration of [missing value]. 50 = 3.16 ± 0.03 μM ( Figure 1 AB). To rule out cytotoxicity as a potential confounding factor, the viability of THP-1 cells treated with Chuanxiongdiolide R11 was assessed using a CCK-8 assay. No significant cytotoxicity was observed at concentrations up to 20 μM, indicating that the inhibitory effect of Chuanxiongdiolide R11 on IL-1β secretion was not due to impaired cell viability. Figure 1 C). Meanwhile, Western blot analysis ( Figure 1 D) showed that the levels of cleaved caspase-1 (p20) and mature IL-1β in the supernatant decreased in a concentration-dependent manner. These data indicate that Chuanxiongdiolide R11 inhibits the caspase-1-mediated IL-1β cleavage and maturation process, suggesting that it directly inhibits the activation of the NLRP3 inflammasome. Furthermore, Chuanxiongdiolide R11 also significantly inhibited the secretion of IL-18 (p20). Figure 1 E), a caspase-1-mediated cytokine, supports its role as a negative regulator of classical inflammasome activation. In contrast, Chuanxiongdiolide R11 had no significant effect on TNF-α expression. Figure 1 (F), indicating that Chuanxiongdiolide R11 does not interfere with upstream NF-κB-mediated initiation signaling. In summary, these findings suggest that Chuanxiongdiolide R11 selectively inhibits the secretion of IL-1β and IL-18.

[0043] Example 2: Chuanxiongdiolide R11 selectively inhibits NLRP3 inflammasome activation via both classical and non-classical pathways.

[0044] 2.1 Cell culture and activation of various inflammasomes

[0045] Take THP-1 cells in logarithmic growth phase, at 2×10⁻⁶ 6Cells were seeded at a density of cells / mL in 6-well culture plates, and PMA was added to a final concentration of 100 nM. After incubation overnight, the PMA-containing medium was discarded. The next day, the PMA-containing medium was discarded, and the cells were treated with Opti-MEM medium containing 100 ng / mL LPS for 3 hours. The canonical NLRP3 inflammasome was activated by incubation with 5 mM ATP for 30 min, 10 μM Nigericin for 1 h, or 150 μg / mL MSU for 6 h. After prestimulation with Pam3CSK4, cells were transfected with 1 μg / mL LPS using Lipofectamine 3000 transfection reagent and incubated for 16 h to activate the non-canonical NLRP3 inflammasome. For AIM2 inflammasome activation, 400 ng / mL poly(dA:dT) was transfected into THP-1 cells using Lipofectamine 3000 transfection reagent and incubated for 3 h. For NLRP1 inflammasome activation, THP-1 cells pretreated with LPS were treated with 10 μM Val-boroPro for 24 h. Chuanxiongdiolide R11 or MCC950 was added 1 h before stimulation.

[0046] 2.2 Immunoblot analysis of relevant inflammatory factors and proteins

[0047] The levels of IL-1β were measured by ELISA and the expression of NLRP3 inflammasome-related protein was measured by Western blot, as in 1.4 and 1.5 of Example 1.

[0048] To clarify whether Chuanxiongdiolide R11 has a broad-spectrum inhibitory effect on NLRP3 inflammasome activation induced by different stimuli, this study used LPS-pretreated THP-1 cells as a model and stimulated them with three classic NLRP3 activators: ATP, Nigericin, or MSU. Enzyme-linked immunosorbent assay (ELISA) results showed that treatment with 4 μM Chuanxiongdiolide R11 significantly reduced the IL-1β secretion level in the cell supernatant of the above three activation models. Figure 2 A). Immunoblot analysis simultaneously confirmed that the release of caspase-1 activation fragment p20 and mature IL-1β was significantly reduced in all models. Figure 2(B) indicates that Chuanxiongdiolide R11 has broad-spectrum inhibitory activity against conventional stimulation-induced NLRP3 inflammasome activation. Further investigation into the effect of Chuanxiongdiolide R11 on the non-canonical NLRP3 inflammasome pathway, in which intracellular LPS can activate human caspase-4 / 5, thereby triggering secondary NLRP3 activation, was conducted. In this study, a non-canonical activation model was constructed by transfecting LPS into THP-1 cells using Lipofectamine 3000. The results showed that Chuanxiongdiolide R11 dose-dependently inhibited p20 fragment release and IL-1β secretion (B). Figure 2 CD) confirmed that it can block the activation of the NLRP3 inflammasome downstream of intracellular LPS sensing, further expanding its inhibitory spectrum. To evaluate the specificity of Chuanxiongdiolide R11 on the NLRP3 inflammasome, this study also examined its effects on two other types of inflammasome pathways, NLRP1 and AIM2. THP-1 cells were stimulated with the DPP8 / 9 inhibitor Val-boroPro (an NLRP1 inflammasome-specific activator). The results showed that Chuanxiongdiolide R11 treatment had no significant effect on caspase-1 cleavage activation or IL-1β release. Figure 2 Similarly, when the AIM2 inflammasome was activated by poly(dA:dT) transfection, Chuanxiongdiolide R11 did not have a significant regulatory effect on the activation of the inflammasome. Figure 2 In summary, Chuanxiongdiolide R11 can broadly inhibit NLRP3 inflammasome activation induced by classical stimuli such as ATP, Nigericin, and MSU, as well as non-classical stimuli mediated by intracellular LPS. It can also specifically target the NLRP3 pathway without interfering with the function of other inflammasomes such as NLRP1 and AIM2, demonstrating outstanding targeting selectivity.

[0049] Example 3: Chuanxiongdiolide R11 inhibits NLRP3 inflammasome-mediated pyroptosis by inhibiting caspase-1 activation.

[0050] 3.1 Caspase-1 activity assay

[0051] Activated Caspase-1 was detected using the FAM-YVAD-FMK FLICA probe. LPS-stimulated THP-1 cells were co-incubated with different doses of Chuanxiongdiolide R11 for 1 h, followed by ATP activation for 30 min. The cell culture supernatant was discarded, and the cells were washed three times with PBS. The FAM-YVAD-FMK FLICA probe was added, and the cells were incubated at 37°C for 1 h. The probe was then discarded, and the cells were washed again. The cell nuclei were counterstained with Hoechst 33342 staining solution at 37°C for 10 min. The cells were washed three times with PBS, and images were acquired and analyzed under a fluorescence microscope.

[0052] 3.2 Mitochondrial ROS detection and mitochondrial integrity assessment

[0053] THP-1 cells were seeded on glass coverslips and, after inflammatory stimulation, were treated with either Chuanxiongdiolide R11 or MCC950 according to experimental groups. Subsequently, 5 μM MitoSOX staining solution was added, and the cells were incubated at 37°C for 30 min to label mitochondrial reactive oxygen species (ROS). Simultaneously, 20 nM MitoTracker staining solution was used for incubation at 37°C for 30 min to assess mitochondrial integrity. After staining, the cells were washed three times with PBS, fixed with 4% paraformaldehyde at room temperature, counterstained with DAPI, mounted with anti-fluorescence quenching mounting medium, and images were acquired and analyzed under a fluorescence microscope.

[0054] 3.3 Cell viability staining detection

[0055] THP-1 cells were seeded into 8-well glass culture plates. Stimulation and grouping with Chuanxiongdiolide R11 and MCC950 were performed according to the experimental protocol. The culture supernatant from each well was discarded, and the cells were gently washed three times with pre-cooled PBS. A mixed staining working solution containing 1× Hoechst 33342, 1× PI, and 1× Calcein AM was prepared, and sufficient staining solution was added to each well to completely cover the cells. The culture plates were placed in a 37°C, 5% CO2 cell culture incubator and incubated for 10 min in the dark. After incubation, the cells were washed three times with PBS, leaving an appropriate amount of PBS in each well to prevent cell drying. Imaging was performed under a confocal fluorescence microscope to observe the morphology of live cells, dead cells, and nuclei.

[0056] 3.4 Western blot analysis of proteins

[0057] Same as 1.5 in Example 1.

[0058] Activation of the NLRP3 inflammasome induces pro-caspase-1 protein hydrolysis and activation, which in turn cleaves GSDMD to release its N-terminal fragment (GSDMD-N). GSDMD-N accumulates in the cell membrane, forming pore structures and ultimately inducing pyroptosis. To clarify the regulatory role of Chuanxiongdiolide R11 in this pathway, its activity was detected using a caspase-1-specific fluorescent inhibitor probe (FLICA), which specifically binds to the activated form of caspase-1. The results showed that LPS / ATP co-stimulation significantly enhanced the intracellular green fluorescence signal, indicating that caspase-1 was significantly activated. Figure 3 (AB), and Chuanxiongdiolide R11 pretreatment dose-dependently reduced fluorescence intensity, suggesting that it can directly inhibit caspase-1 activity. This result is consistent with immunoblotting analysis, where the level of the caspase-1 activation fragment p20 in the cell culture supernatant was significantly downregulated after Chuanxiongdiolide R11 treatment. To further explore the downstream execution process of pyroptosis, this study examined the cleavage of GSDMD. Immunoblotting results showed that LPS / ATP stimulation could induce specific cleavage of GSDMD, producing a 35 kDa GSDMD-N active fragment, confirming that pyroptosis was effectively activated, while Chuanxiongdiolide R11 treatment significantly inhibited this cleavage event and the accumulation of the GSDMD-N fragment (AB). Figure 3 C), indicating that it can block the transmission of the caspase-1-GSDMD signaling axis. HMGB1 is a key damage-associated molecular pattern (DAMP) molecule released during pyroptosis. Experimental results showed that after LPS / ATP stimulation, the HMGB1 content in the supernatant of THP-1 cells was significantly increased; while Chuanxiongdiolide R11 treatment almost completely inhibited the release of HMGB1 ( Figure 3 C), further corroborating its inhibitory effect on pyroptosis signaling. Finally, the pyroptosis rate was quantitatively assessed using the Calcein-AM / propidium iodide (PI) / Hoechst 33342 three-color fluorescence staining method. The results showed that after LPS / ATP stimulation, the pyroptosis index of THP-1 cells increased significantly, with approximately 55% of cells showing PI positivity (increased membrane permeability); while Chuanxiongdiolide R11 treatment reduced this proportion to approximately 10% ( Figure 3 DE. Simultaneous LDH release assays confirmed that Chuanxiongdiolide R11 significantly reduced LPS / ATP-induced LDH release levels ( Figure 3The results (F) indicate that it can effectively alleviate NLRP3 inflammasome-mediated pyroptosis. In summary, Chuanxiongdiolide R11 effectively inhibits the pyroptosis cascade triggered by NLRP3 inflammasome activation by inhibiting caspase-1 activation, blocking GSDMD cleavage and HMGB1 release.

[0059] Example 4: Chuanxiongdiolide R11 blocks the assembly of the NEK7-NLRP3-ASC complex.

[0060] 4.1 Cell culture and activation of various inflammasomes

[0061] Same as 2.1 in Example 2.

[0062] 4.2 ASC Oligomerization Detection

[0063] The oligomerization level of ASCs was assessed using a detergent-insoluble crosslinking method: THP-1 cells treated with Chuanxiongdiolide R11 or MCC950 were lysed in NP-40 lysis buffer containing a protease inhibitor. The detergent-insoluble precipitate was collected by centrifugation, washed, and then 2 mM disuccinimide octanoate (DSS) was added. The crosslinking reaction was completed at room temperature. The crosslinked protein was resuspended in loading buffer, heated for denaturation, and ASC protein was detected by Western blot. Simultaneously, the soluble fraction after lysis was collected as a standardized control for protein loading.

[0064] 4.3 ASC Spot Formation Detection

[0065] THP-1 cells were seeded on glass coverslips, treated with inflammatory stimulation and Chuanxiongdiolide R11 or MCC950, and then fixed with 4% paraformaldehyde at room temperature. After permeabilization and blocking, the cells were incubated overnight at 4°C with anti-ASC primary antibody, and then incubated at room temperature the next day with fluorescently labeled secondary antibody. The cell nuclei were counterstained with DAPI staining solution. After staining, the coverslips were inverted and mounted in anti-fluorescence quenching mounting solution. Images were acquired using a confocal fluorescence microscope to assess the number of ASC spots and the positive rate.

[0066] 4.4 Immunoprecipitation and NLRP3 oligomerization

[0067] The target cell lysate was prepared using the aforementioned method. The lysate was mixed with anti-NLRP3 specific antibody and incubated overnight at 4°C. The following day, protein A+G agarose beads were added to the incubation system, and immunoprecipitation was continued at 4°C for 4 hours. The immunoprecipitate product was collected, and the binding of NEK7 and ASC proteins was detected by Western blotting. Simultaneously, an equal volume of total cell lysate was used as an input control, and Western blotting was performed concurrently to calibrate the experimental results.

[0068] 4.5 NLRP3 Oligomerization Detection

[0069] THP-1 cells after induction treatment were lysed thoroughly with Triton X-100 lysis buffer containing a mixture of phenylmethylsulfonyl fluoride (PMSF) and protease inhibitors. The lysate was centrifuged at 13000×g for 5 min at 4°C, and the precipitate was discarded, retaining the supernatant to remove cell debris. The supernatant was mixed with 5× loading buffer (containing 2.5x TBE buffer, 50% glycerol, 10% SDS, and 0.0025% bromophenol blue) in a specific ratio. The mixture was then loaded onto a 1.5% vertical agarose gel and electrophoresed for 1 h at a constant temperature of 4°C and a constant voltage of 80 V using 1×TBE containing 0.1% SDS as the running buffer. After electrophoresis, the proteins on the gel were transferred to a PVDF membrane. After standard immunoblotting procedures including primary antibody incubation and HRP-labeled secondary antibody incubation, the signal was read using chemiluminescence to detect NLRP3 oligomerization levels.

[0070] 4.6 Western blot analysis of proteins

[0071] Same as 1.5 in Example 1.

[0072] Mitochondrial dysfunction and excessive production of mitochondrial reactive oxygen species (mtROS) are classic upstream signals for NLRP3 inflammasome activation. This study investigated the target of Chuanxiongdiolide R11 in an LPS-prestimulated, ATP-triggered THP-1 macrophage model using MitoSOX (mtROS-specific probe) and MitoTracker (mitochondrial integrity probe) staining experiments. Results showed that combined LPS / ATP stimulation significantly induced mitochondrial fragmentation damage and increased mtROS production levels. Figure 4A); however, Chuanxiongdiolide R11 treatment failed to reverse mitochondrial morphological abnormalities or reduce mtROS content, indicating that its inhibitory effect on NLRP3 inflammasome activation is independent of mitochondrial function repair or ROS clearance mechanisms. Given that ASC oligomerization is a core step in NLRP3 inflammasome assembly, this study further investigated the effect of Chuanxiongdiolide R11 on this step. Western blot analysis showed that LPS / ATP stimulation significantly induced caspase-1 cleavage activation, IL-1β maturation and release, and ASC oligomerization (…). Figure 4 B). Immunofluorescence staining results simultaneously confirmed that ATP activation significantly increased the formation of intracellular ASC spots, while treatment with Chuanxiongdiolide R11 effectively inhibited the formation of these spots. Figure 4 C), suggesting that it can block ASC polymerization and the assembly of the inflammasome core structure. Furthermore, chemical cross-linking experiments showed that ATP activation significantly enhances NLRP3 dimer formation (a key prerequisite for inflammasome initiation), while Chuanxiongdiolide R11 effectively blocks this process. Figure 4 D). Considering the indispensable bridging role of NEK7 between NLRP3 dimer and ASC, the effect of Chuanxiongdiolide R11 on protein-protein interactions was evaluated using an immunoprecipitation assay. The results showed that ATP treatment significantly promoted the binding of NLRP3 to NEK7 and ASC. Figure 4 Treatment with Chuanxiongdiolide R11 or the positive control drug MCC950 significantly disrupted the aforementioned interactions. In summary, the anti-inflammatory body effect of Chuanxiongdiolide R11 is not mediated by mitochondrial reactive oxygen species, but rather by inhibiting NLRP3 subunit aggregation, blocking ASC recruitment, and disrupting the NEK7-NLRP3-ASC interaction network, thereby disrupting the assembly process of the NLRP3 inflammasome.

[0073] Example 5: In vivo inhibition of NLRP3-dependent acute inflammation by Chuanxiongdiolide R11

[0074] 5.1 Animals

[0075] 8-10 week old C57BL / 6 wild-type (WT) mice and Nlrp3 gene knockout (Nlrp3) - / -C57BL / 6 mice (background), weighing 18–22 g, were purchased from Shanghai Model Organisms Research Center Co., Ltd. All mice were housed in an SPF-grade animal facility under a 12-hour light / 12-hour dark circadian rhythm at a temperature of 22±2℃, with free access to food and water. All animal experimental protocols in this study were approved by the Experimental Animal Ethics Committee of Chengdu University of Traditional Chinese Medicine and met animal ethics and welfare requirements (Ethics No.: 2026017).

[0076] 5.2 Mouse model of systemic inflammation

[0077] Take 8-week-old C57BL / 6 WT and Nlrp3 - / - Mice were randomly divided into four groups of six per group: solvent control group, LPS model group, LPS + MCC950 treatment group, and LPS + CDR11 treatment group. Mice were first intraperitoneally injected with either MCC950 (30 mg / kg) or CDR11 (20 mg / kg), followed by intraperitoneal injection of LPS (30 mg / kg) to induce the LPS model. The solvent control group received an equal volume of solvent, while the model group received only LPS. Mice were sacrificed 4 h after LPS stimulation, and serum and peritoneal exudate were collected. Flow cytometry was used to analyze Ly6G... + CD11b + The markers were used to quantify neutrophil counts; serum and peritoneal exudate were clarified by centrifugation and used to detect IL-1β levels.

[0078] 5.3 Mouse model of peritonitis

[0079] Take 8-week-old C57BL / 6 WT and Nlrp3 - / - Mice were randomly divided into four groups of six per group: solvent control group, MSU model group, MSU + MCC950 treatment group, and MSU + CDR11 treatment group. Mice were first intraperitoneally injected with either MCC950 (30 mg / kg) or CDR11 (20 mg / kg), followed by intraperitoneal injection of MSU (10 mg / kg) to induce the MSU model. The solvent control group received an equal volume of solvent, while the model group received only MSU. Six hours after MSU stimulation, mice were sacrificed, and serum and peritoneal exudate were collected. Flow cytometry was used to analyze the samples using Ly6G... + CD11b + The markers were used to quantify neutrophil counts; serum and peritoneal exudate were clarified by centrifugation and used to detect IL-1β levels.

[0080] To evaluate the regulatory efficacy of Chuanxiongdiolide R11 on NLRP3 inflammasome activation in vivo, experiments were conducted using two classic NLRP3 inflammasome activation models: LPS-induced systemic inflammation and MSU-induced peritonitis.

[0081] In an LPS-induced systemic inflammation model, intraperitoneal injection of LPS in mice significantly increased IL-1β levels in serum and peritoneal exudate; intervention with 20 mg / kg Chuanxiongdiolide R11 significantly inhibited IL-1β secretion in the aforementioned sites, with inhibitory effects comparable to those of MCC950. Figure 5 AB) confirmed its good systemic anti-inflammatory activity. Flow cytometry results showed that LPS stimulation could induce intraperitoneal CD11b + Ly6G + Numerous neutrophil infiltration was observed, and treatment with Chuanxiongdiolide R11 and MCC950 significantly reduced the degree of neutrophil infiltration. Figure 5 CD), suggesting that it can inhibit NLRP3-mediated neutrophil inflammatory response. To clarify the NLRP3-dependent nature of this effect, this study used Nlrp3 gene knockout (Nlrp3) - / - Repeated experiments in mice showed that, in the absence of NLRP3, neither Chuanxiongdiolide R11 nor MCC950 could reduce IL-1β levels or inhibit neutrophil recruitment, indicating that their anti-inflammatory effects in the LPS model are strictly dependent on the presence of the NLRP3 inflammasome. Figure 5 AD).

[0082] To further verify the inhibitory effect of Chuanxiongdiolide R11 on the NLRP3 inflammasome, an MSU-induced aseptic peritonitis model (MSU is a classic activator of the NLRP3 inflammasome in gout) was used for verification, with MCC950 as a positive control. Results showed that MSU stimulation significantly increased IL-1β levels in mouse serum and peritoneal lavage fluid; after intervention with Chuanxiongdiolide R11, IL-1β secretion was significantly inhibited, with no significant difference in effect compared to MCC950. Figure 6 AB). Meanwhile, the typical characteristic of MSU-induced peritonitis—peritoneal neutrophil aggregation—was also effectively suppressed after treatment with Chuanxiongdiolide R11 and MCC950. Figure 6 CD). And it is consistent with the results of the LPS model, Nlrp3 - / - Mice showed no significant response to treatment with Chuanxiongdiolide R11 and MCC950. Figure 6The results suggest that its anti-inflammatory effect is entirely dependent on the functional NLRP3 inflammasome pathway. In summary, the above in vivo experimental results confirm that Chuanxiongdiolide R11 improves the acute inflammatory response in mice in an NLRP3-dependent manner, and its mechanism of action is highly consistent with that of the classic NLRP3 inhibitor MCC950.

[0083] In summary, this study found that Chuanxiongdiolide R11 can inhibit the activation of the NLRP3 inflammasome in a dose-dependent manner, and this inhibition is broad-spectrum, without interfering with upstream NF-κB-mediated initiation signaling or affecting other inflammasomes such as NLRP3 and AIM2. Chuanxiongdiolide R11 disrupts the assembly of the NEK7-NLRP3-ASC complex, thereby inhibiting caspase-1 activation, GSDMD cleavage, HMGB1 release, pyroptosis, and the maturation and release of IL-1β / IL-18. Simultaneously, in mice, Chuanxiongdiolide R11 improved LPS-induced systemic inflammation and MSU-induced peritonitis in an NLRP3-dependent manner. These findings indicate that Chuanxiongdiolide R11 is a specific inhibitor of the NLRP3 inflammasome and can be used to prepare drugs for the prevention or treatment of NLRP3 inflammasome-mediated inflammatory diseases, showing broad prospects for clinical translation.

[0084] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The use of Chuanxiongdiolide R11 in the preparation of medicaments for the prevention or treatment of NLRP3 inflammasome-mediated inflammatory diseases, characterized in that, The Chuanxiongdiolide R11 has the structure shown in equation (I): （I）。 2. The use according to claim 1, characterized in that, The Chuanxiongdiolide R11 inhibits caspase-1 activation, GSDMD cleavage, HMGB1 release, pyroptosis, and the maturation and release of IL-1β and IL-18 by blocking the assembly of the NEK7-NLRP3-ASC complex.

3. The use according to claim 1, characterized in that, The Chuanxiongdiolide R11 can selectively inhibit both the classical and non-classical activation pathways of the NLRP3 inflammasome.

4. The use according to claim 1, characterized in that, The NLRP3 inflammasome-mediated inflammatory diseases include LPS-induced systemic inflammation or MSU-induced peritonitis.

5. The use according to claim 1, characterized in that, The drug includes a pharmaceutically acceptable carrier.

6. The use according to claim 5, characterized in that, The dosage forms of the drugs include tablets, granules, capsules, pills, solutions, emulsions, suspensions, injections, aerosols, powder inhalers, ointments, lotions, eye drops, patches, nasal drops, mouthwashes, sublingual tablets, and suppositories.

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