Rubimainllin targets NLRP3 to suppress inflammasome activation and attenuate NLRP3-driven inflammatory diseases

Rubimainllin targets NLRP3 to suppress inflammasome activation and attenuate NLRP3-driven inflammatory diseases | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 15 October 2025 V1 Latest version Share on Rubimainllin targets NLRP3 to suppress inflammasome activation and attenuate NLRP3-driven inflammatory diseases Authors : Jing Xiao , Ran Zhang , Maoyuan Jiang , Xinhua Chen , Bin Jia , Huaiping Tang , Xinyu Bao , Yun Xu , Linjie Yu , Sen-Lin Ji , and Xiao-lei Zhu 0000-0003-4696-9696 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.176053563.32309600/v1 341 views 84 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Background: The NLR family pyrin domain-containing 3 (NLRP3) inflammasome hyper-activation is a shared pathogenic driver of multiple inflammatory, metabolic and neurodegenerative disorders, yet clinically approved inhibitors remain lacking. Purpose: To investigate whether Rubimainllin (Rub), a naphthoquinone isolated from Rubia cordifolia L., selectively inhibits NLRP3 inflammasome activation and exert therapeutic effects in relevant disease models. Methods: Macrophages and microglia were primed with Lipopolysaccharide (LPS) for 3 h, then stimulated with Nigericin (Nig) (2 h) or ATP (40 min) to induce NLRP3 inflammasome assembly and pyroptosis; caspase-1 activation, ASC speck formation, IL-1β release, and LDH secretion were quantified. The therapeutic potential of Rubimainllin was assessed in murine models of NLRP3-driven inflammation. Animals received intraperitoneal Rub (5 or 10 mg mg/kg) or vehicle in models of (i) LPS-induced peritonitis, (ii) monosodium urate crystal (MSU)-induced gouty arthritis (RA), and (iii) hippocampal Aβ 1-42 -induced Alzheimer’s disease (AD). Diseases severity was evaluated by histopathology, cytokine profiling, and behavioral tests. Results: Rub dose-dependently suppressed NLRP3-mediated pyroptosis and IL-1β secretion in macrophages and microglia without affecting AIM2 or NLRC4 pathways. Mechanistically, Rub directly bound NLRP3, blocked ASC oligomerization and NLRP3 oligomer formation, thereby preventing caspase-1 activation and [gasdermin D](https://www.sciencedirect.com/topics/immunology-and-microbiology/gasdermin-d) (GSDMD) cleavage. In vivo, Rub prolonged survival and attenuated lung injury in LPS-sepsis, reduced paw swelling and bone erosion in MSU-gout, and ameliorated cognitive deficits and neuroinflammation in Aβ-induced Alzheimer’s disease (AD) mice. Conclusion: Rub selectively targets NLRP3 and constitutes a promising natural lead compound for the treatment of NLRP3-driven inflammatory diseases including sepsis, gout and Alzheimer’s disease. 1.Introduction NLRP3 inflammatory vesicles are intracellular multiprotein complexes mainly composed of NLRP3 sensors, apoptosis-associated speck-like proteins (ASCs), and cysteine asparagin-1 (Caspase-1) (Swanson et al., 2019). Its core function is to recognize pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) and activate Caspase-1, which in turn shears the precursors of interleukin-1β (IL-1β) and IL-18, prompting their maturation and release into the extracellular compartment, triggering inflammatory responses (Huang et al., 2021). Pyroptosis is a mode of programmed cell death characterized by cell expansion, cell membrane rupture, and the release of inflammatory factors. NLRP3 inflammasome is closely related to pyroptosis. Activation of NLRP3 induces the activation of caspase-1, which cleaves Gasdermin D (GSDMD), leading to an increase in membrane permeability and cellular pyroptosis (Broz and Dixit, 2016). This process is accompanied by the maturation and release of inflammatory factors IL-1β and IL-18, further amplifying the inflammatory response. The abnormal activation of the NLRP3 inflammasome serves as a central pathological mechanism in numerous human diseases. In rheumatoid arthritis, NLRP3 expression and activity are markedly elevated in synovial tissue (Lee et al., 2024, Liu et al., 2023). Activated caspase-1 processes pro-inflammatory cytokines such as IL-1β and IL-18, thereby promoting persistent inflammation, cartilage degradation, and bone erosion. In Alzheimer’s disease, amyloid-beta (Aβ) functions as a damage-associated molecular pattern (DAMP), which activates the NLRP3 inflammasome (McManus and Latz, 2024, Abbate et al., 2020), leading to neuroinflammation and accelerated neuronal injury. Moreover, NLRP3 hyperactivation is closely associated with the progression of sepsis, gout, inflammatory bowel disease, multiple sclerosis, and type 2 debate (Li et al., 2023a, Zeng et al., 2022). Therefore, targeted suppression of NLRP3 has emerged as a key therapeutic strategy. Although MCC950 analogs, such as GDC-2394 and NT-0796, have advanced into clinical trials, safety concerns including hepatotoxicity and nephrotoxicity remain significant barriers to their widespread use (Coll et al., 2015). These challenges have prompted ongoing efforts to refine lead compounds to improve selectivity and long-term tolerability. Rubimainllin (Rub) is a major active ingredient in Rubia cordifolia (Rubia cordifolia L.) and belongs to the naphthoquinone class of compounds (Idhayadhulla et al., 2014). Rubia cordifolia has a long history of medicinal use, and its roots and rhizomes are widely used in traditional Chinese medicine for cooling the blood, stopping bleeding, activating blood circulation, removing blood stasis, and promoting menstruation (Li et al., 2020). As one of the main active ingredients of Rubia cordifolia, Rub plays an important role in blood coagulation, antitumor activity and anti-inflammatory effects (Hong et al., 2018, Li et al., 2023b). However, the mechanisms are not fully defined. In the present study, we found that Rub was able to inhibit the composition of the NLRP3 inflammasome by binding to NLRP3, and attenuated NLRP3 associated diseases progression. 2.Materials and methods 2.1 Rub treatment Rub (#S9162, Selleck, Houston, TX, USA) was first dissolved in dimethyl sulfoxide (DMSO; ST038, Beyotime, Shanghai, China) to prepare a 10 mg/ml stock solution. For in vivo studies, the stock solution was diluted with vehicle to concentrations of 0.5 mg/ml or 1 mg/ml and administered via intraperitoneal injection to experimental mice at doses of 5 mg/kg or 10 mg/kg, respectively. 2.2 Cell culture Immortalized bone marrow-derived macrophages (iBMDMs) and THP-1 cells (ATCC® TIB-202™, Manassas, VA, USA) were obtained from ATCC. iBMDMs were maintained in Dulbecco’s modified Eagle’s medium (DMEM; high glucose; Gibco, 31985-070, Grand Island, NY, USA), whereas THP-1 cells were cultured in Roswell Park Memorial Institute-1640 medium (RPMI-1640; Gibco, 11875119, Grand Island, NY, USA). Bone marrow-derived macrophages (BMDMs) were grown in DMEM (high glucose) supplemented with 30% L929-conditioned medium prepared by pooling supernatants collected from days 1 to 3. All media were supplemented with 10% fetal bovine serum (FBS; Gibco, 10091141, Grand Island, NY, USA) and 1% penicillin–streptomycin (P/S; Gibco, 15640055, Grand Island, NY, USA). Cells were incubated at 37 °C in a humidified atmosphere containing 5% CO₂. 2.3 Animals C57BL/6 mice were obtained from the Model Animal Research Center of Nanjing University and housed in a specific pathogen-free (SPF) facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International. All animal procedures were performed in compliance with the Animal Research: Reporting of In Vivo Experiments (ARRIVE) guidelines and were approved by the Animal Care Committee of Nanjing Drum Tower Hospital. 2.4 BMDM differentiation Primary BMDMs were isolated using the established protocols. Briefly, 8–10-week-old C57BL/6 mice were euthanized and immersed in 75% ethanol for 5 min. Bone marrow cells were harvested from the femurs and tibiae and cultured in DMEM induction medium. On day 3, half of the medium was replaced with a fresh medium. After 7 days of differentiation, BMDMs were gently scraped, centrifuged at 1,500 × g, transferred to 6-well plates, and allowed to adhere for overnight. The cells were then treated with either Rub or the vehicle. The differentiation status was confirmed the following day. 2.5 Induction of inflammasome NLRP3 inflammasome stimulation: Cells were pre-treated with 200 ng/mL LPS (Sigma-Aldrich, L2630, St. Louis, MO, USA) for 3 h, followed by incubation with Rub at various concentrations for 1 h. NLRP3 inflammasome activation was induced by one of the following: (1) 4 mmol/L ATP (Sigma-Aldrich, A6419, St. Louis, MO, USA) for 40 min; or (2) 10 mmol/L nigericin (Sigma-Aldrich, 481990, St. Louis, MO, USA) for 2 h. AIM2 inflammasome stimulation: After LPS priming and Rub treatment, the cells were transfected with 0.1 mg/L poly(dA:dT) (Cell Signaling Technology, 47945S, Danvers, MA, USA) using Lipofectamine™ 3000 (Invitrogen, L3000075, Carlsbad, CA, USA) and incubated for 4 h. NLRC4 inflammasome stimulation: Following LPS priming and Rub treatment, the cells were transfected with 0.1 mg/L flagellin (Beyotime, P7388, Shanghai, China) using Lipofectamine™ 3000 and stimulated for 8 h. 2.6 Cell Counting Kit-8 (CCK-8) assay The cytotoxicity of Rub was evaluated using a CCK-8 cell viability assay kit (Beyotime, C0039, Shanghai, China) following the manufacturer’s instructions. In brief, the cells were treated with different concentrations of Rub for 24 h. Then, 10 µL of CCK-8 reagent was added to each well, and the plates were incubated at 37 °C for 1–4 h in the dark. Absorbance was measured at 450 nm using a CytoTox 96 Non-Radioactive plate reader (≥4 replicates per group). 2.7 Lactate dehydrogenase (LDH) release assay Supernatants from the stimulated cells were collected, and LDH release was quantified using an LDH Cytotoxicity Assay Kit (Beyotime, C0017, Shanghai, China) according to the manufacturer’s protocol. Briefly, 60 µL of supernatant was mixed with 30 µL of assay reagent per well and incubated at 37 °C in the dark for 30 min. Absorbance was measured at 450 nm with a reference wavelength of 570 nm using a CytoTox 96 Non-Radioactive Plate Reader. 2.8 Enzyme-linked immunosorbent assay (ELISA) After model establishment or behavioral testing, supernatants from cultured cells, hippocampal tissue, or mouse serum were collected. The levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) were quantified using commercial ELISA kits according to the manufacturer’s instructions. All kits for human IL-1β (CEK1731), mouse IL-1β (CEK1788), mouse IL-6 (CEK1785), and mouse TNF-α (CEK1783) were purchased from Bioworld (Nanjing). 2.9 Propidium iodide (PI) staining Cells were seeded in 6-well plates and allowed to adhere for overnight. The following day, the cells were treated according to the NLRP3 inflammasome stimulation protocol. After treatment, the cells were washed thrice with PBS. The staining solution was prepared by adding 5 µL of PI (Dojindo, C542, Kumamoto, Japan) to 5 mL of DMEM. This mixture was added to the wells and incubated at room temperature in the dark for 10–20 min. Cells were rinsed three times with PBS, and three to five random fields per well were imaged using a laser confocal microscope. Data were analyzed using GraphPad Prism. Cell viability was determined by comparing PI-positive (dead) and PI-negative (live) cells across groups. 2.10 Immunofluorescence staining After fixation with 4% paraformaldehyde for 30 min, cell-seeded coverslips or brain sections were permeabilized with 0.1% Triton X-100 for 10 min at room temperature. Non-specific binding was blocked by incubating the cells in PBS containing 5% normal goat serum for 1 h at room temperature. Primary antibodies were applied and incubated overnight at 4 °C. After thorough washing, species-appropriate Alexa Fluor 488 or 594 secondary antibodies were applied for 1 h at room temperature. Nuclei were counterstained with DAPI (Bioworld, BD5010, Nanjing, Jiangsu, China) for 10 min, and images were acquired using a confocal laser-scanning microscope. 2.11 Western blotting After treatment, adherent cells were collected and lysed immediately in ice-cold NP-40 buffer (Beyotime, P0013F, Shanghai, China) supplemented with PMSF (Beyotime, ST506, Shanghai, China) for 30 min. Lysates were centrifuged at 13,000 × g for 20 min at 4 °C, and the supernatants were retained. Protein concentrations were determined using a BCA assay and were normalized. An equal volume of 5× Laemmli buffer was added, and the samples were denatured at 95 °C for 5 min. For secreted proteins, the supernatants were precipitated with chloroform–methanol: one-quarter volume of chloroform and an equal volume of methanol were added, followed by vertexing and centrifugation at 13,000 × g for 5 min at 4 °C. The upper methanol phase was discarded, fresh methanol was added to the interphase, and the proteins were pelleted by recentrifugation. Pellets were resuspended in 1× loading buffer (Beyotime, P0015L, Shanghai, China) and heated at 95 °C for 5 min. Proteins were separated by SDS-PAGE and transferred to PVDF membranes (Thermo Fisher, 88518, Shanghai, China). Membranes were blocked with 5% non-fat milk in TBST for 2 hours, incubated with primary antibodies overnight at 4 °C, washed three times with TBST, and probed with HRP-conjugated secondary antibodies for 1 hour at room temperature. After additional washes, immunoreactive bands were visualized using the SuperSignal West Pico ECL substrate. 2.12 Cross-linking of ASC oligomers iBMDMs were plated in 6-well dishes and incubated overnight at 37 °C under 5% CO₂. After treatment, the cells were lysed in NP-40 buffer and centrifuged at 13,000 × g for 20 min at 4 °C. Pellets were washed twice with ice-cold PBS, resuspended in 500 µL of 2 mM DSS (Selleck, S0657, Houston, TX, USA), and crosslinked on an orbital shaker for 60 min at room temperature. Pellets were then solubilized in NP-40 lysis buffer containing SDS-PAGE loading buffer, heated at 100 °C for 5–10 min, and analyzed by western blotting. 2.13 Assay of NLRP3 oligomerization After treatment, iBMDMs were collected and lysed in ice-cold NP-40 buffer with PMSF for 30 min. Lysates were centrifuged at 13,000 × g for 20 minutes at 4 °C, and supernatants were retained. Protein concentrations were normalized using the BCA assay. After adding 5× non-reducing loading buffer (Beyotime, P0016, Shanghai, China), samples were separated on BeyoGel™ Plus precast PAGE gels. 2.14 Cellular thermal shift assay (CETSA) After treatment, the residual medium was removed, and the cells were gently washed three times with ice-cold PBS. Cells were detached using 0.25% trypsin–EDTA, collected, and pelleted by centrifugation at 200 × g for 3 min. The pellets were resuspended in an equal volume of PBS and aliquoted into six microtubes. Each aliquot was heated for 3 min at one of the following six temperatures: 37, 42, 47, 52, 57, or 62 °C. Samples were snap-frozen in liquid nitrogen, thawed at 37 °C, and subjected to five freeze–thaw cycles. Lysates were centrifuged at 3,000 × g for 20 min at 4 °C, and the supernatants were analyzed using western blotting. 2.15 Drug affinity responsive target stability (DARTS) After treatment, the cells were lysed in ice-cold NP-40 buffer containing PMSF for 30 min. Lysates were centrifuged at 13,000 × g for 20 min at 4 °C, and the supernatants were collected. Equal aliquots were incubated with Pronase E (Sigma-Aldrich, 1.07433, St. Louis, MO, USA) at the specified concentrations for 10 min at 37 °C. Proteolysis was halted by adding 5× Laemmli buffer and denaturing at 95 °C for 5 min. Samples were analyzed by SDS-PAGE and Western blotting. 2.16 Molecular docking Rubimaillin was docked to the human NLRP3 receptor (PDB ID: 6NPY) using AutoDock Vina (v.1.2.0). The crystal structure was obtained from the Protein Data Bank, and the binding poses were visualized using PyMOL (v.2.5). 2.17 H&E (Hematoxylin and eosin) staining After modeling, the mice were transcardially perfused with ice-cold PBS, followed by 4% paraformaldehyde. Livers and lungs were harvested, post-fixed overnight in the same fixative at 4 °C, dehydrated through a graded ethanol series (70%, 80%, 95%, 100%), cleared in xylene, and embedded in paraffin. Sections (5 µm) were cut, deparaffinized, rehydrated, and stained with hematoxylin and eosin (H&E) for histopathological evaluation of inflammatory infiltration. 2.18 LPS-induced sepsis model Eight-week-old male C57BL/6 mice received an intraperitoneal (i.p.) injection of Rub 24 h prior to the study and a second injection 8–12 h later. Sixty minutes after the second injection, the mice were challenged with 0.3 mg LPS in saline (i.p.). Systemic inflammation was allowed to develop for 12 hours post-LPS, after which serum was collected for ELISA quantification of IL-1β, IL-6, and TNF-α. Mice were perfused with PBS followed by 4% paraformaldehyde, and the lungs and livers were harvested, processed, and stained with H&E for histopathological analysis. 2.19 MSU-induced gouty arthritis (RA) model Eight-week-old male C57BL/6 mice received a priming dose of Rub (i.p.) 24 h before experimentation, followed by a second injection 8–12 h later. One hour after the second injection, 0.6 mg monosodium urate (MSU) crystals (Invitrogen, Tlrl-MSU, Carlsbad, CA, USA) in sterile saline were injected subcutaneously into the left metatarsal joint, and the contralateral hind paw served as an untreated control. Joint swelling was measured hourly for 6 h using a digital caliper, with arthritis severity defined as the increase relative to the untreated paw. After the observation period, the mice were euthanized under deep anesthesia, and the foot joints were excised for IL-1β ELISA and H&E staining. 2.20 Aβ 1-42 -induced Alzheimer’s Disease (AD) model Eight-week-old male C57BL/6 mice were anesthetized and received bilateral stereotactic hippocampal injections of oligomeric Aβ 1-42 (2 µg per hemisphere; #AG968-1MG, Merck). After 24 h, the mice were administered Rub or PBS (i.p.) daily for 15 consecutive days. Behavioral assessments, including the open-field test, novel object recognition task, and Morris water maze, were performed sequentially. 2.21 Statistical analysis Data were analyzed using GraphPad Prism 8.0 and are presented as mean ± SEM. Comparisons between two groups were made using two-tailed unpaired Student’s t-tests; multiple groups were compared using one- or two-way ANOVA, followed by Tukey’s post hoc test. Statistical significance was defined as *p < 0.05, **p < 0.01, ***p < 0.001; NS indicated not significant. All experiments were performed in at least three independent replicates per group. 3. Results 3.1 Rub reduces pyroptosis and IL-1β release in mouse bone marrow primary macrophages (BMDMs) and human macrophage cell line THP-1 cells To explore the neuroprotective effects of Rub, we first accessed the cytotoxicity of Rub (Fig. 1A) on mouse BMDMs using the CCK8 assay (Fig. 1B), and the results showed that Rub didn’t affect the viability of BMDMs at concentrations below 100 μM. To investigate the effect of Rub on NLRP3 inflammatory vesicle activation, we detected the LDH release and IL-1β secretion in BMDMs pretreated with LPS and Rub and then induced by ATP or Nig to activate NLRP3 mediated pyroptosis. The results showed that Rub significantly decreased LDH release in BMDMs (Fig. 1C, F) and inhibited IL-1β secretion in the cell culture supernatants (Fig. 1D, G). In addition, immunoblot analysis showed that Rub inhibited the generation of cleaved Caspase-1 p20 and IL-1β p17 fragments, but did not affect the expression levels of NLRP3, Pro-Caspase-1, Pro-IL-1β, and apoptosis-associated speck-like protein ASC (Fig. 1E, H). Considering that Rub significantly inhibited IL-1β secretion, PI staining was performed on BMDMs after NLRP3 vesicle activation. Fluorescent images and analysis confirmed that Rub treatment reduced the number of dead BMDM cells after LPS + Nig stimulation (Fig.1 I, J). Taken together, these findings suggest that Rub effectively reduces pyroptosis and IL-1β release in BMDMs. Similar results were observed in the mouse macrophage cell line, iBMDMs (Fig. s1A- I) and human macrophage cell line THP-1 cells (Fig. s2A-E), indicating that Rub dose-dependently reduced NLRP3 inflammatory vesicle activation. 3.2 Rub inhibits primary microglia pyroptosis and NLRP3 inflammatory vesicle activation Since Rub can penetrate through the blood-brain barrier (Jia et al., 2025), we tested whether Rub inhibited LPS+ ATP-induced pyroptosis in microglia. The results showed that the Rub decreased LDH release in primary microglia (Fig. 2A) and effectively inhibited IL-1β secretion in the supernatants (Fig. 2B). Western blot analysis showed that the level of cleaved GSDMD was elevated in contrast to the active N-terminal form (N-GSDMD), and Rub treatment significantly suppressed this active form. Rub also inhibited the amount of cleaved Caspase-1 p20 and IL-1β p17 fragment production without affecting the expression levels of NLRP3, precursor Caspase-1, precursor IL-1β, and apoptosis-associated speck-like protein ASC (Fig. 2C). In addition, the DSS cross-linking assay showed that Rub inhibited the formation of ASC oligomers (Fig. 2D), and the immunofluorescence assay indicated that Rub attenuated the fluorescence signal of ASC speckles, suggesting that Rub inhibited the formation of ASC speckles in primary microglia induced by LPS + Nig (Fig. 2E). Therefore, our results showed that Rub inhibited NLRP3 inflammatory vesicle activation by suppressing the assembly of NLRP3 inflammatory vesicles in primary microglia. 3.3 Rub inhibits the activation of NLRP3 inflammatory vesicles by blocking the assembly To investigate the specificity of Rub for inhibiting NLRP3, we investigated the effect of Rub on other inflammasome vesicles such as AIM2 and NLRC4 by inducing the activation of AIM2 and NLRC4 inflammasome vesicles with Poly (dA:dT) and Flagellin, respectively (Oh et al., 2023). The results showed that Rub had no effect on the release of LDH induced by AIM2 inflammasome (Fig. 3A) and the expression of activated caspase-1 and IL-1β fragments in the supernatant (Fig. 3B, C). In addition, Rub didn’t affect the release of LDH induced by NLRC4 (Fig. 3D) and the expression of activated caspase-1 and IL-1β fragments in the supernatant (Fig. 3E, F). These data suggested that Rub did not affect AIM2 or NLRC4 inflammatory vesicle activation. During NLRP3 activation, ASC oligomerization is a critical step in the activation of caspase-1(Fu and Wu, 2023). To investigate the effect of Rub on the interaction between NLRP3 and ASC, we examined the effect of Rub on ASC spots in BMDMs after LPS + Nig stimulation by immunofluorescence, and the results showed that Rub attenuated the fluorescence signal of ASC spots (Fig. 3H, I), indicating that Rub inhibited Nig mycobacteria-induced BMDMs cellular of ASC oligomerization. Because Rub did not alter ASC expression in cell lysates, we next examined whether it impaired NLRP3-dependent ASC oligomerization. We detected ASC protein multimerization using a DSS cross-linking assay on stimulated BMDMs, and the experimental results showed that Rub suppressed ASC oligomerization without altering the expression of ASC in the cell lysates (Fig. 3G). These results suggested that Rub inhibited the activation of NLRP3 inflammatory vesicles by compromising the assembly of inflammatory vesicle components. 3.4 Rub directly binds to NLRP3 and inhibits NLRP3 inflammatory vesicle activation Since similar ASC data were obtained in THP-1 cells treated with LPS + Nig (Fig. 4A, B), we next examined the effect of Rub on the oligomerization of NLRP3 inflammatory vesicles using the native-PAGE assay technique (Broz and Dixit, 2016). As shown in Fig. 4C, Rub inhibited NLRP3 oligomerization in LPS + Nig-treated macrophages. Subsequently, we investigated whether Rub directly inhibited NLRP3 activation by interacting with NLRP3. Binding affinity was measured using CETSA and DARTS, two well-established experiments used to assess intracellular drug-target protein interactions. In DARTS experiments (Pai et al., 2015), preincubation of Rub with BMDMs reduced pronase-induced NLRP3 protein hydrolysis, but did not affect GSDMD, NEK7, ASC, or caspase-1 levels (Fig. 4D), which suggested that Rub specifically interacted with NLRP3. Moreover, in the CETSA experiment (Feng et al., 2023), BMDMs pretreated with Rub exhibited higher thermal stability than controls under the same temperature conditions (Fig. 4E). The results of CETSA and DARTS experiments indicated a direct interaction between Rub and NLRP3. The molecular docking tests showed that Rub had a good binding affinity with NLRP3, targeting the NACHT domain of NLRP3, with a binding energy of -7.1 kcal/mol (Fig. 4F). To further validate these results, a surface plasmon resonance (SPR) assay was used to investigate the affinity between Rub and purified NLRP3 protein, and the results showed a high-affinity interaction between Rub and NLRP3 protein (Fig. 4G). Taken together, these results suggested that Rub directly bound to NLRP3 and inhibited its oligomerization to attenuate the activation of NLRP3 inflammatory vesicles. 3.5 Rub ameliorates LPS-induced sepsis and IL-1β production in mice To explore whether Rub had an effect on LPS-induced sepsis model (Li et al., 2021), we pretreated with Rub for 2 h and found that Rub significantly prolonged the survival time of septic mice (Fig. 5A). In addition, the result of H&E staining showed that inflammatory infiltration of the lungs was ameliorated after Rub treatment (Fig. 5B). Furthermore, the Elisa analysis showed that the levels of inflammatory factor cells IL-1β and TNF-α were significantly reduced in the serum of Rub-treated mice, and there was no significant change in IL-6 level (Fig. 5C-E). These findings suggested that Rub ameliorated LPS-induced sepsis in mice and reduced the production of pro-inflammatory cytokines. 3.6 Rub inhibited MSU-induced gouty arthritis (RA) in mice RA is a chronic autoimmune disease characterized pathologically by synovial inflammation, hyperplasia, and destruction of articular cartilage and bone tissue. The NLRP3 inflammasome plays a key role in gouty arthritis (RA), and multiple factors in the synovial membrane of RA patients can activate the NLRP3 inflammasome (Liu et al., 2023). To assess the therapeutic effect of Rub on MSU-induced gouty arthritis, we measured footpad thickness hourly for 6 h after MSU treatment (Fig. 6A), and the results showed that MSU injections caused significant footpad swelling in the mice, which was dose-dependently reduced by Rub treatment (Fig. 6B). Compared with the control group, H&E staining showed that Rub treatment significantly attenuated the destruction of bone tissue by inflammatory cells (Fig. 6C). Furthermore, Rub treatment significantly reduced IL-1β levels in mice (Fig. 6D). Overall, our data showed that Rub ameliorates the gouty arthritis in mice. 3.7 Rub ameliorates cognitive deficits in Aβ 1-42 induced AD mice Numerous clinical and animal model studies have shown that NLRP3 inflammatory vesicles and cellular pyroptosis play important roles in neurodegenerative diseases (Han et al., 2023). In a clinical study of patients with AD, cerebrospinal fluid levels of IL-1β and IL-18 were positively correlated with cognitive decline. To investigate whether Rub attenuated the pathological features of AD mice, we stereotactically injected 8-week-old B6 mice with Aβ 1-42 , followed by intraperitoneal injection of Rub for 2 weeks (Fig. 7A). The absent field experiment showed that there was no significant difference in the time in the center (Fig. s3A) and corner (Fig. s3B). In addition, the NOR tests showed that Rub-treated mice explored the new objects for a longer time (Fig. 7B) compared with mice that were not treated with Rub. To determine the effect of Rub on spatial learning and memory in AD mice, the Morris water maze test was performed (Fig. 7C). On the training trial, Rub treatment significantly reduced escape latency (Fig. 7D). On the probe trial, the average swimming speed (Fig. 7E), and total distance (Fig. s3C) were not significantly changed in all groups, indicating that locomotor ability was not altered. Rub treatment significantly decreased the latency to arrive at the target area (Fig. 7F) and crossed the platform more times (Fig. 7G). Moreover, Rub-treated AD mice spent more time in the target quadrant (Fig. 7H), and the time to reach the target quadrant for the first time was shorter (Fig. 7I). These results suggested that Rub improved spatial memory and cognitive function in AD mice. 3.8 Rub exerts neuroprotective effects in Aβ 1-42 AD mice by inhibiting NLRP3 inflammatory vesicles Since neuroinflammatory response is a key pathological feature of AD, we examined the effects of Rub on the activation of microglia and astrocytes in the hippocampus of AD mice using immunofluorescence, and the results showed that Rub treatment reduced microglial and astrocyte activation in Aβ 1-42 AD mice (Fig. 8A-D). Givern that Rub directly bound to NLRP3 in microglia, we determine whether the neuroprotective effect of Rub was associated with inhibition of NLRP3 inflammatory vesicles. Rub administration resulted in a gradual reduction in ASC spots compared with untreated Aβ 1-42 AD mice (Fig. 8E, F), suggesting that Rub has an inhibitory effect on NLRP3 inflammatory vesicles in the hippocampal region of AD mice. Meanwhile, the ELISA assay showed that the level of the inflammatory factor IL-1β was significantly reduced in the serum of Rub-treated mice (Fig. 8G). These results suggested that Rub exerted neuroprotective effects by inhibiting the activation of NLRP3 inflammatory vesicles in AD mice. 4. Discussion The NLRP3 inflammasome is a cytosolic multi-protein complex that activates caspase-1, thereby releasing IL-1β/IL-18 and executing GSDMD-driven pyroptosis (Li et al., 2022). Excessive NLRP3 activation underlies autoinflammatory, metabolic and neurodegenerative disorders (Mangan et al., 2018), yet no clinically approved inhibitor exists. Current candidates such as MCC950, OLT1177 and RRX-001 are hampered by hepatotoxicity, low oral exposure or unverified anti-inflammatory efficacy (Zahid et al., 2019). But Rub, as a monomer of traditional Chinese medicine, has a novel structure and avoids the hepatotoxicity related to the MCC950 skeleton. In this study we demonstrate that Rub is a selective NLRP3 antagonist. In vitro, Rub binds NLRP3 with high affinity, blocks ASC oligomerization and suppresses inflammasome activation without affecting AIM2 or NLRC4. These findings position Rub as a natural, brain-penetrant NLRP3 inhibitor with therapeutic potential. Physiologic NLRP3 activation is required for host defense (Biasizzo and Kopitar-Jerala, 2020), yet excessive assembly underlies sepsis, gout, multiple sclerosis and neurodegeneration (Chen et al., 2024). We therefore evaluated Rub in vivo across relevant disease models. Sepsis is an extremely serious disease, characterized by an uncontrolled immune response triggered by severe infection, which in turn leads to dysfunction of organs (Shi et al., 2021). Worldwide, sepsis has become one of the main causes of death among patients in hospitals. The prognostic impact of antibiotics, fluid resuscitation and organ support therapy on patients with sepsis is limited. Patients with sepsis may die due to immunosuppression or excessive inflammation caused by primary infection. The activation of NLRP3 plays an important mediating role in the process of organ failure caused by sepsis (Tang et al., 2025). In the LPS-induced sepsis model, Rub markedly attenuated pulmonary inflammation and lowered serum IL-1β and TNF-α, indicating potent suppression of NLRP3-driven cytokine release. Gout is a chronic disease caused by the deposition of MSU crystals, characterized by arthritis attacks and disability. In RA, the activation of NLRP3 inflammasome leads to the activation of Caspase-1, which in turn triggers pyroptosis and releases inflammatory cytokines such as IL-1β and IL-18 (Liu et al., 2023). These inflammatory factors promote the proliferation of synovial cells and the formation of pannus by activating other immune cells, ultimately leading to the destruction of articular cartilage and bone tissue. Clinical research data show that the levels of inflammation-related molecules such as NLRP3 and IL-1β in the serum and joint fluid of RA patients are significantly elevated and are positively correlated with disease activity and the degree of joint injury (Chen et al., 2025). In the MSU-induced gouty arthritis model, Rub significantly reduced paw oedema, bone erosion and synovial IL-1β, confirming its anti-inflammatory efficacy against NLRP3-mediated pyroptosis. Collectively, these data demonstrate that Rub effectively mitigates organ injury and inflammatory damage in models where NLRP3 hyper-activation is a central pathogenic driver. Neurodegenerative disorders are chronic, progressive conditions characterized by irreversible neuronal loss that impairs cognition and shortens lifespan. In AD, extracellular Aβ plaques and intracellular hyper-phosphorylated tau tangles constitute the principal pathological substrates of neurotoxicity. Accumulating evidence demonstrates that soluble Aβ oligomers trigger assembly of the NLRP3 inflammasome in both microglia and neurons, thereby initiating caspase-1–dependent pyroptosis and perpetuating neuroinflammation (McManus and Latz, 2024). Mechanistically, Aβ activates NLRP3 through at least two convergent pathways: (i) it evokes intracellular reactive oxygen species (ROS) accumulation and mitochondrial dysfunction, which converge on the NLRP3 NACHT domain to promote oligomerization; and (ii) it engages cell-surface pattern-recognition or scavenger receptors, thereby activating downstream signaling cascades that facilitate NLRP3 inflammasome assembly. Post-mortem analyses of AD cerebral cortex reveal elevated NLRP3, cleaved caspase-1, and mature IL-1β levels that correlate strongly with disease severity, underscoring the pathogenic significance of this inflammatory axis. In the Aβ 1-42 -injected AD model, Rub treatment significantly improved spatial memory and cognition in behavioral tests. Subsequent immunofluorescence and ELISA analyses revealed that Rub exerted neuroprotection by suppressing NLRP3 inflammasome activation. Collectively, these findings position Rub as a promising therapeutic candidate for AD. Rubimainllin (Rubia cordifolia L.), as the main active ingredient of rubia cordifolia, has the advantages of natural source, clear mechanism, low toxicity and high efficiency (Idhayadhulla et al., 2014, Li et al., 2020). It has the dual effects of ”anti-inflammation and neuroprotection” and can cross the cell membrane and blood-brain barrier (Li et al., 2023b, Jia et al., 2025). It is expected to become a candidate lead compound for NLRP3-driven diseases, providing a basis for multi-indication development for gout, sepsis and AD, etc. However, this study also has some limitations, (i) the mechanism needs to be further explored, such as the exploration of whether Rub affects the interaction between NLRP3-NEK7 (He et al., 2016). In addition, further investigations are needed to explore the impact of Rub on several key upstream mechanisms of NLRP3 inflammasome activation, such as potassium efflux, mitochondrial reactive oxygen species generation, and lysosomal destruction. (ii) Comprehensive pharmacokinetic and safety data for Rub remain incomplete. Definitive gaps must be addressed by executing GLP-compliant acute and 28- to 90-day repeat-dose toxicology studies in both rodent and canine species, coupled with systematic evaluations of CYP-enzyme and transporter-mediated drug–drug interactions and brain PET-imaging to fully characterize central nervous system exposure. (iii) This study has yet to assess Rub in experimental autoimmune encephalomyelitis (EAE) or in other chronic inflammatory disorders such as high-fat-diet-induced diabetes mellitus and non-alcoholic fatty liver disease. Future studies will evaluate its anti-inflammatory efficacy and mechanisms in these disorders to broaden its clinical scope. In conclusion, this study demonstrated the inhibitory effect of Rub on NLRP3 inflammatory vesicle activation and its potential therapeutic effects in various inflammatory diseases models. These findings provide an important theoretical basis and experimental support for the use of Rub as a novel drug for the treatment of NLRP3-related inflammatory diseases. Future studies should further explore the mechanisms of action of Rub and evaluate its safety and efficacy for clinical applications. 5. CRediT authorship contribution statement Jing Xiao: Conceptualization, Investigation, Data curation, Formal analysis, Writing – original draft. Ran Zhang: Conceptualization, Validation, Writing – review & editing. Maoyuan Jiang: Methodology, Project administration. Xinhua Chen:Writing – original draft. Bin Jia: Validation. Huaiping Tang: Formal analysis. Xinyu Bao: Resources. Yun Xu: Resources. Xiaolei Zhu: Methodology, Funding acquisition. Sen-Lin Ji: Resources, Visualization. Linjie Yu: Methodology, Software. All authors read and approved the final manuscript. 6. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. 7. Acknowledgements This work was supported by grants from the National Natural Science Foundation of China (82471460 to X.Z. and 82101417 to S-L.J.), the STI2030-Major Projects (2022ZD0211800 to Y.X.), the Natural Science Foundation of Jiangsu Province of China (BK20231120 to X.Z.) and Nanjing Medical Science and Technology Development Foundation (ZKX22025 X.Z.). 8. References ABBATE, A., TOLDO, S., MARCHETTI, C., KRON, J., VAN TASSELL, B. W. & DINARELLO, C. A. 2020. Interleukin-1 and the Inflammasome as Therapeutic Targets in Cardiovascular Disease. Circ Res, 126 , 1260-1280.BIASIZZO, M. & KOPITAR-JERALA, N. 2020. Interplay Between NLRP3 Inflammasome and Autophagy. Front Immunol, 11 , 591803.BROZ, P. & DIXIT, V. M. 2016. Inflammasomes: mechanism of assembly, regulation and signalling. Nat Rev Immunol, 16 , 407-20.CHEN, P., WANG, Y., TANG, H., LIU, Z., WANG, J., WANG, T., XU, Y. & JI, S. L. 2024. Gastrodenol suppresses NLRP3/GSDMD mediated pyroptosis and ameliorates inflammatory diseases. Cell Immunol, 405-406 , 104888.CHEN, P., WANG, Y., TANG, H., ZHOU, C., LIU, Z., GAO, S., WANG, T., XU, Y. & JI, S. L. 2025. New applications of clioquinol in the treatment of inflammation disease by directly targeting arginine 335 of NLRP3. J Pharm Anal, 15 , 101069.COLL, R. C., ROBERTSON, A. A., CHAE, J. J., HIGGINS, S. C., MUñOZ-PLANILLO, R., INSERRA, M. C., VETTER, I., DUNGAN, L. S., MONKS, B. G., STUTZ, A., CROKER, D. E., BUTLER, M. S., HANEKLAUS, M., SUTTON, C. E., NúñEZ, G., LATZ, E., KASTNER, D. L., MILLS, K. H., MASTERS, S. L., SCHRODER, K., COOPER, M. A. & O’NEILL, L. A. 2015. A small-molecule inhibitor of the NLRP3 inflammasome for the treatment of inflammatory diseases. Nat Med, 21 , 248-55.FENG, F., ZHANG, W., CHAI, Y., GUO, D. & CHEN, X. 2023. Label-free target protein characterization for small molecule drugs: recent advances in methods and applications. J Pharm Biomed Anal, 223 , 115107.FU, J. & WU, H. 2023. Structural Mechanisms of NLRP3 Inflammasome Assembly and Activation. Annu Rev Immunol, 41 , 301-316.HAN, Y. H., LIU, X. D., JIN, M. H., SUN, H. N. & KWON, T. 2023. Role of NLRP3 inflammasome-mediated neuronal pyroptosis and neuroinflammation in neurodegenerative diseases. Inflamm Res, 72 , 1839-1859.HE, Y., ZENG, M. Y., YANG, D., MOTRO, B. & NúñEZ, G. 2016. NEK7 is an essential mediator of NLRP3 activation downstream of potassium efflux. Nature, 530 , 354-7.HONG, K. B., KIM, D., KIM, B. K., WOO, S. Y., LEE, J. H., HAN, S. H., BAE, G. U. & KANG, S. 2018. CF₃-Substituted Mollugin 2-(4-Morpholinyl)-ethyl ester as a Potential Anti-inflammatory Agent with Improved Aqueous Solubility and Metabolic Stability. Molecules, 23.HUANG, Y., XU, W. & ZHOU, R. 2021. NLRP3 inflammasome activation and cell death. Cell Mol Immunol, 18 , 2114-2127.IDHAYADHULLA, A., XIA, L., LEE, Y. R., KIM, S. H., WEE, Y. J. & LEE, C. S. 2014. Synthesis of novel and diverse mollugin analogues and their antibacterial and antioxidant activities. Bioorg Chem, 52 , 77-82.JIA, X., NAN, J., ZHANG, K. & ZHANG, L. 2025. Mollugin attenuates oxygen-glucose deprivation/reperfusion-induced brain microvascular endothelial cell death and permeability through activation of BDNF/TrkB-modulated Akt pathway. J Bioenerg Biomembr .LEE, J., SASAKI, F., KOIKE, E., CHO, M., LEE, Y., DHO, S. H., LEE, J., LEE, E., TOYOHARA, E., SUNAKAWA, M., ISHIBASHI, M., HUNG, H. H., NISHIOKA, S., KOMINE, R., OKURA, C., SHIMIZU, M., IKAWA, M., YOSHIMURA, A., MORITA, R. & KIM, L. K. 2024. Gelsolin alleviates rheumatoid arthritis by negatively regulating NLRP3 inflammasome activation. Cell Death Differ, 31 , 1679-1694.LI, J., ZHANG, J. L., GONG, X. P., XIAO, M., SONG, Y. Y., PI, H. F. & DU, G. 2020. Anti-inflammatory Activity of Mollugin on DSS-induced Colitis in Mice. Curr Med Sci, 40 , 910-916.LI, Q., TAN, Y., CHEN, S., XIAO, X., ZHANG, M., WU, Q. & DONG, M. 2021. Irisin alleviates LPS-induced liver injury and inflammation through inhibition of NLRP3 inflammasome and NF-κB signaling. J Recept Signal Transduct Res, 41 , 294-303.LI, Q., ZHAO, Y., GUO, H., LI, Q., YAN, C., LI, Y., HE, S., WANG, N. & WANG, Q. 2023a. Impaired lipophagy induced-microglial lipid droplets accumulation contributes to the buildup of TREM1 in diabetes-associated cognitive impairment. Autophagy, 19 , 2639-2656.LI, X., HOU, R., DING, H., GAO, X., WEI, Z., QI, T. & FANG, L. 2023b. Mollugin ameliorates murine allergic airway inflammation by inhibiting Th2 response and M2 macrophage activation. Eur J Pharmacol, 946 , 175630.LI, Z., JI, S., JIANG, M. L., XU, Y. & ZHANG, C. J. 2022. The Regulation and Modification of GSDMD Signaling in Diseases. Front Immunol, 13 , 893912.LIU, Y. R., WANG, J. Q. & LI, J. 2023. Role of NLRP3 in the pathogenesis and treatment of gout arthritis. Front Immunol, 14 , 1137822.MANGAN, M. S. J., OLHAVA, E. J., ROUSH, W. R., SEIDEL, H. M., GLICK, G. D. & LATZ, E. 2018. Targeting the NLRP3 inflammasome in inflammatory diseases. Nat Rev Drug Discov, 17 , 688.MCMANUS, R. M. & LATZ, E. 2024. NLRP3 inflammasome signalling in Alzheimer’s disease. Neuropharmacology, 252 , 109941.OH, S., LEE, J., OH, J., YU, G., RYU, H., KIM, D. & LEE, S. 2023. Integrated NLRP3, AIM2, NLRC4, Pyrin inflammasome activation and assembly drive PANoptosis. Cell Mol Immunol, 20 , 1513-1526.PAI, M. Y., LOMENICK, B., HWANG, H., SCHIESTL, R., MCBRIDE, W., LOO, J. A. & HUANG, J. 2015. Drug affinity responsive target stability (DARTS) for small-molecule target identification. Methods Mol Biol, 1263 , 287-98.SHI, X., TAN, S. & TAN, S. 2021. NLRP3 inflammasome in sepsis (Review). Mol Med Rep, 24.SWANSON, K. V., DENG, M. & TING, J. P. 2019. The NLRP3 inflammasome: molecular activation and regulation to therapeutics. Nat Rev Immunol, 19 , 477-489.TANG, H., ZOU, X., CHEN, P., WANG, Y., GAO, S., WANG, T., XU, Y. & JI, S. L. 2025. Broxyquinoline targets NLRP3 to inhibit inflammasome activation and alleviate NLRP3-associated inflammatory diseases. Int Immunopharmacol, 156 , 114687.ZAHID, A., LI, B., KOMBE, A. J. K., JIN, T. & TAO, J. 2019. Pharmacological Inhibitors of the NLRP3 Inflammasome. Front Immunol, 10 , 2538.ZENG, B., HUANG, Y., CHEN, S., XU, R., XU, L., QIU, J., SHI, F., LIU, S., ZHA, Q., OUYANG, D. & HE, X. 2022. Dextran sodium sulfate potentiates NLRP3 inflammasome activation by modulating the KCa3.1 potassium channel in a mouse model of colitis. Cell Mol Immunol, 19 , 925-943. Fig. 1. Rub can dose-dependently reduce pyroptosis and IL-1β release in BMDMs. (A) Rub structure. (B) BMDM cells were treated with different concentrations of Rub for 24 hours and cell viability was assessed by CCK-8 assay (n=5). (C, F) The BMDM cells stimulated by LPS for 3 hours were cultured with Rub for 1 hour, then stimulated with ATP and Nig for 40min or 2 hours, respectively. The supernatant was collected and treated with according to the instructions of the LDH test kit. The absorbance was measured at 450nm (n=4). (D, G) The concentration of IL-1β in supernatant was detected by ELISA (n=3). (E, H) The levels of pro-IL-1β, pro-caspase-1 and NLRP3 in BMDM cell lysate and IL-1β and caspase-1 in supernatant were detected by western blot analysis. (I) BMDM cells stimulated with LPS for 3 hours were cultured with Rub for 1 hour and then stimulated with Nig for 2 hours. Dead and alive cells were detected by immunofluorescence staining. (J) The proportion of PI positive cells in total cells (relative to LPS + Nig) (n=4). Data are presented mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, NS not significant, as assessed by one-way ANOVA (relative to LPS+ATP/Nig). Fig. 2. Rub inhibits LPS/ ATP-induced pyroptosis of microglia and activation of NLRP3 inflammasomes. (A) The Microglia cells stimulated by LPS for 3 h were cultured with Rub for 1 h, then stimulated with ATP for 40 min. The supernatant was collected and treated with according to the instructions of the LDH test kit. The absorbance was measured at 450nm (n=4). (B) The concentration of IL-1β in supernatant was detected by ELISA (n=3). (C) The levels of pro-IL-1β, pro-caspase-1 and NLRP3 in Microglia cell lysate and IL-1β and caspase-1 in supernatant were detected by western blot analysis. (D) Western blot analysis of ASC oligomerization levels after cross-linking np-40 insoluble microspheres of Microglia with DSS. (E) LPS-induced Microglia cells were incubated with different doses of Rub for 1 h and then stimulated with Nig for 2 h. ASC and NLRP3 spot immunofluorescence detection. Immunofluorescence staining was used to observe the formation of ASC spots (green), NLRP3 (red), and DAPI (blue). The formation of ASC spots and NLRP3 was observed by immunofluorescence method. (F) The percentage of ASC spots measured by image J (relative to LPS + Nig) (n=5). Data are presented mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, NS not significant, as assessed by one-way ANOVA (relative to LPS+ATP/Nig). Fig. 3. Rub affects the activation of NLRP3 inflammasomes by inhibiting their assembly. (A, D) The BMDM cells stimulated by LPS for 3 h were cultured with Rub for 1 h and then stimulated with Poly (dA: dT)/Flagellin for 6-8 h or 2-4 h, respectively. Supernatant was collected and treated with according to the instructions of LDH test kit (n=4). Absorbance was determined at 450nm. (B, E) The concentration of IL-1β in supernatant was detected by ELISA (n=3). (C, F) The levels of pro-IL-1β, pro-caspase-1 and NLRP3 in iBMDM cell lysate and IL-1β and caspase-1 in supernatant were detected by western blot analysis. (G) Western blot analysis of ASC oligomerization levels after cross-linking np-40 insoluble microspheres of BMDMs with DSS. (H) LPS-induced BMDM cells were incubated with different doses of Rub for 1 h and then stimulated with Nig for 2 h. ASC spot immunofluorescence detection. BMDMs is stained with ASC antibodies (green) and DAPI (blue). The formation of ASC spots was observed by immunofluorescence method. (I) The percentage of ASC spots measured by image J (relative to LPS + Nig) (n=5). Data are presented mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, NS not significant, as assessed by one-way ANOVA (relative to LPS + Poly (dA: dT)/Flagellin). Fig. 4. Rub specifically affects the activation of NLRP3 inflammasomes by directly binding to NLRP3. (A) LPS-induced THP-1 cells were incubated with different doses of Rub for 1 h and then stimulated with Nig for 2 h. ASC spot immunofluorescence detection. THP-1 is stained with ASC antibodies (green) and DAPI (blue). The formation of ASC spots was observed by immunofluorescence method. (B) The percentage of ASC spots measured by image J (relative to LPS + Nig) (n=5). (C) Oligomerization of NLRP3 was measured by the native-PAGE analysis. (D) DARTS tests with Pronase E (0-5 ug/ml) in the presence or absence of varying doses of Rub. Western blotting analysis of NLRP3, caspase-1, NEK7 and ASC protein levels. (E) The thermal stability of NLRP3, GSDMD, pro-caspase-1, NEK7 and ASC proteins in BMDM treated with or without Rub was determined by CETSA method. (F) Molecular docking analysis of Rub bound to NLRP3. 2D pose view of the interaction between Rub and NLRP3 in the molecular docking model. (G) Real-time binding kinetics of Rub with immobilized recombinant human NLRP3 were quantified by single-cycle surface plasmon resonance (SPR). Data are presented mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, NS not significant, as assessed by one-way ANOVA. Fig. 5. Rub can improve LPS-induced sepsis in mice and the production of IL-1β. (A) LPS was administered to the peritoneum of mice and continuously monitored for 72 h. (B) H&E staining of mouse lung tissue. (C-E) The concentrations of serum IL-1β, IL-6 and TNF-α in mice were detected by ELISA kit 6h after LPS injection (n=4). Data are presented mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, NS not significant, as assessed by one-way ANOVA (Relative to the group without Rub). Fig. 6. Rub substantially ameliorates MSU-induced gouty arthritis in mice. (A) The thickness of the foot pad measured with a vernier caliper six hours after MSU treatment. (B) Statistical chart (n=5). (C) Interleukin-1 beta (IL-1β) levels in the joint culture supernatant (SN) (n=4). (D) Representative hematoxylin and eosin (H&E) staining of mice footpads. Data are presented mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, NS not significant, as assessed by one-way ANOVA (Relative to the group without Rub). Fig. 7. Rub has a significant improvement on cognitive deficits in AD mice. (A) The timeline diagram of the entire AD model. (B) In NOR tests, the percentage of time to explore new objects was recorded. (C-I) The Morris water maze in each group (n=9). (C) Representative moving patterns in the probe trials. (D) In the acquisition trial, the escape latency was analyzed in the MWM tests. (E) Average swimming speed. (F)latency to the platform. (G) In probe tests, the number of target platform crossings. (H)The total time elapsed through the platform. (I) Latency to target quadrant. Data are presented mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, NS not significant, as assessed by one-way ANOVA (Relative to the group without Rub). Fig. 8. Rub exerts neuroprotective effects by inhibiting the NLRP3 inflammasomes in AD mice. (A) Immunofluorescence staining of Iba-1 (red, microglia marker) and DAPI (blue, nucleus) in the hippocampal regions of WT, AD model, and Rub-treated AD mice. (B) The fluorescence intensity of Iba-1 measured by image J (relative to Aβ) (n=4). (C) Immunofluorescence staining of GFAP (red, astrocyte marker) and DAPI (blue, nucleus) in the hippocampal regions. (D) The fluorescence intensity of GFAP measured by image J (relative to Aβ) (n=4). (E) Immunofluorescence staining of ASC (red) and DAPI (blue, nucleus) in the hippocampal regions. (F) The fluorescence intensity of ASC measured by image J (relative to Aβ) (n=4). (G) ELISA of IL-1β secretion in the hippocampal regions of WT, AD model, and Rub-treated AD mice (n=5). Data are presented mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, NS not significant, as assessed by one-way ANOVA (Relative to the group without Rub). Information & Authors Information Version history V1 Version 1 15 October 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Jing Xiao Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital View all articles by this author Ran Zhang Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital View all articles by this author Maoyuan Jiang Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital View all articles by this author Xinhua Chen Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital View all articles by this author Bin Jia Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital View all articles by this author Huaiping Tang Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital View all articles by this author Xinyu Bao Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital View all articles by this author Yun Xu Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital View all articles by this author Linjie Yu Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital View all articles by this author Sen-Lin Ji Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital View all articles by this author Xiao-lei Zhu 0000-0003-4696-9696 [email protected] Nanjing University Medical School Affiliated Nanjing Drum Tower Hospital View all articles by this author Metrics & Citations Metrics Article Usage 341 views 84 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Jing Xiao, Ran Zhang, Maoyuan Jiang, et al. Rubimainllin targets NLRP3 to suppress inflammasome activation and attenuate NLRP3-driven inflammatory diseases. Authorea . 15 October 2025. DOI: https://doi.org/10.22541/au.176053563.32309600/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . Format Please select one from the list RIS (ProCite, Reference Manager) EndNote BibTex Medlars RefWorks Direct import Tips for downloading citations document.getElementById('citMgrHelpLink').addEventListener('click', function() { popupHelp(this.href); return false; }); $(".js__slcInclude").on("change", function(e){ if ($(this).val() == 'refworks') $('#direct').prop("checked", false); $('#direct').prop("disabled", ($(this).val() == 'refworks')); }); View Options View options PDF View PDF Figures Tables Media Share Share Share article link Copy Link Copied! Copying failed. Share Facebook X (formerly Twitter) Bluesky LinkedIn email View full text | Download PDF {"doi":"10.22541/au.176053563.32309600/v1","type":"Article"} Now Reading: Share Figures Tables Close figure viewer Back to article Figure title goes here Change zoom level Go to figure location within the article Download figure Toggle share panel Toggle share panel Share Toggle information panel Toggle information panel Go to previous graphic Go to next graphic Go to previous table Go to next table All figures All tables View all material View all material xrefBack.goTo xrefBack.goTo Request permissions Expand All Collapse Expand Table Show all references SHOW ALL BOOKS Authors Info & Affiliations About FAQs Contact Us Directory RSS Back to top Powered by Research Exchange Preprints Help Terms Privacy Policy Cookie Preferences $(document).ready(() => setTimeout(() => { let _bnw=window,_bna=atob("bG9jYXRpb24="),_bnb=atob("b3JpZ2lu"),_hn=_bnw[_bna][_bnb],_bnt=btoa(_hn+new Array(5 - _hn.length % 4).join(" ")); $.get("/resource/lodash?t="+_bnt); },4000)); (function(){function c(){var b=a.contentDocument||a.contentWindow.document;if(b){var d=b.createElement('script');d.innerHTML="window.__CF$cv$params={r:'9ffda0b03ad61640',t:'MTc3OTQ3MTg1NA=='};var a=document.createElement('script');a.src='/cdn-cgi/challenge-platform/scripts/jsd/main.js';document.getElementsByTagName('head')[0].appendChild(a);";b.getElementsByTagName('head')[0].appendChild(d)}}if(document.body){var a=document.createElement('iframe');a.height=1;a.width=1;a.style.position='absolute';a.style.top=0;a.style.left=0;a.style.border='none';a.style.visibility='hidden';document.body.appendChild(a);if('loading'!==document.readyState)c();else if(window.addEventListener)document.addEventListener('DOMContentLoaded',c);else{var e=document.onreadystatechange||function(){};document.onreadystatechange=function(b){e(b);'loading'!==document.readyState&&(document.onreadystatechange=e,c())}}}})();