Abstract

   Dysregulated activation of the NLR family pyrin domain-containing 3
   (NLRP3) inflammasome contributes to the pathogenesis of numerous
   inflammatory and infectious diseases; however, effective targeted
   therapies remain elusive. In this study, we identify emodin—a bioactive
   anthraquinone derived from Rheum palmatum (radix Rhei) and Polygonum
   cuspidatum (Polygonaceae)—as a potent and selective inhibitor of NLRP3
   inflammasome activation. Notably, emodin disrupts the assembly of the
   NLRP3 complex without impairing inflammasome priming. Transcriptomic
   profiling via RNA sequencing reveals that emodin reprograms
   mitochondrial quality control pathways, markedly enhancing mitophagy
   flux. Mechanistically, emodin suppresses casein kinase II
   (CK2)-mediated phosphorylation of FUNDC1, a pivotal mitophagy receptor,
   thereby promoting mitochondrial clearance and preventing mitochondrial
   reactive oxygen species-induced NLRP3 inflammasome assembly. Both
   genetic silencing of FUNDC1 and pharmacological inhibition of mitophagy
   with 3-methyladenine abrogated abrogate the inhibitory effects of
   emodin, establishing a direct mechanistic link between FUNDC1-dependent
   mitophagy and NLRP3 regulation. In vivo, emodin confers significant
   protection in sepsis models, with these protective effects being lost
   in NLRP3-deficient mice or upon macrophage-specific deletion of FUNDC1.
   Collectively, our findings uncover a novel CK2-FUNDC1-mitophagy axis
   through which emodin inhibits NLRP3 inflammasome activation,
   highlighting its promise as a clinically translatable candidate for the
   treatment of NLRP3-driven inflammatory diseases.

   Keywords: Emodin, NLRP3 inflammasome, Mitochondrial homeostasis,
   Mitophagy, FUNDC1, Sepsis.

Introduction

   The NLR family pyrin domain (PYD)-containing 3 (NLRP3) inflammasome
   functions as a pivotal cytoplasmic surveillance complex, consisting of
   three essential components: the sensor molecule NLRP3, the adaptor
   protein apoptosis-associated speck-like protein containing a CARD
   (ASC), and the effector enzyme caspase-1 [43]^1. Acting as a sentinel
   of intracellular stress, the NLRP3 inflammasome detects a wide array of
   danger-associated molecular patterns (DAMPs) and pathogen-associated
   molecular patterns (PAMPs) generated during microbial invasion or
   sterile tissue injury [44]^2. Its activation follows a well-defined
   two-step signaling model. The initial priming phase, initiated by PAMPs
   (e.g., lipopolysaccharide) or pro-inflammatory cytokines (e.g., tumor
   necrosis factor alpha [TNF-α]), induces nuclear factor kappa B
   (NF-κB)-mediated transcriptional upregulation of pro-interleukin-1 beta
   [IL-1β], pro-IL-18, and NLRP3 itself, thereby licensing the
   inflammasome for activation [45]^3^-[46]^5. A subsequent activation
   signal, such as extracellular adenosine triphosphate (ATP), nigericin,
   or monosodium urate crystals, then promotes NLRP3 oligomerization and
   ASC speck formation via PYD interactions. This process enables the
   recruitment of pro-caspase-1 to ASC's CARD domain, facilitating its
   auto-proteolytic cleavage into active caspase-1 [47]^6^, [48]^7. The
   ensuing cascade culminates in the maturation and secretion of the
   pro-inflammatory cytokines IL-1β and IL-18, which orchestrate host
   defense mechanisms essential for pathogen clearance and tissue repair
   [49]^8^-[50]^11. However, dysregulated, or persistent activation of the
   NLRP3 inflammasome drives pathological inflammation, contributing to
   tissue injury and organ dysfunction in a range of diseases including
   sepsis, gout, acute peritonitis, and neurodegenerative disorders
   [51]^12^, [52]^13. Therefore, precise regulation of NLRP3 inflammasome
   activity is indispensable for maintaining immune homeostasis, striking
   a critical balance between effective host defense and the prevention of
   excessive tissue damage [53]^14.

   Beyond their canonical role in cellular bioenergetics, mitochondria
   have emerged as dynamic signaling organelles that integrate metabolic
   stress cues to modulate immune responses [54]^15. Accumulating evidence
   highlights mitochondrial dysfunction as a central driver of NLRP3
   inflammasome activation. Pathological stimuli— including microbial
   infection, metabolic perturbations, and oxidative stress—induce
   mitochondrial damage and trigger the release of DAMPs. Notably,
   mitochondrial reactive oxygen species (mtROS) have been identified as
   key mediators that facilitate the assembly of the NLRP3 inflammasome
   complex [55]^14^, [56]^16^, [57]^17. This mechanistic link is
   reinforced by pharmacological studies demonstrating that scavenging
   mtROS with antioxidants markedly attenuates NLRP3 activation
   [58]^18^-[59]^20, thus establishing mitochondrial oxidative stress as a
   proximal trigger of inflammasome signaling. Complementing this pathway,
   mitophagy— a selective form of autophagy targeting damaged
   mitochondria— acts as an intrinsic brake on NLRP3 activation. By
   eliminating dysfunctional mitochondria and limiting mtROS accumulation,
   mitophagy curbs aberrant inflammasome activity. Disruption of
   mitophagy, either genetically or pharmacologically, leads to mtROS
   accumulation, exacerbating NLRP3 activation and promoting inflammatory
   disease pathogenesis [60]^21^-[61]^24. Conversely, pharmacological
   enhancement of mitophagy suppresses mtROS production, restrains
   inflammasome activation, and alleviates inflammation in preclinical
   models. Collectively, these findings position mitophagy as a promising
   therapeutic axis for fine-tuning NLRP3-driven inflammation across
   diverse pathological contexts, including sepsis and neurodegenerative
   diseases [62]^20^, [63]^25^, [64]^26.

   Mitophagy, the autophagic clearance of damaged mitochondria, plays an
   essential role in maintaining cellular redox balance and preventing
   excessive ROS buildup. This process is orchestrated by intricate
   molecular networks that govern mitochondrial recognition and
   autophagosome formation [65]^27^-[66]^30. Central to this machinery is
   FUNDC1, an outer mitochondrial membrane receptor containing an
   N-terminal LC3-interacting region motif, which directly recruits
   autophagosome LC3 to initiate mitophagosome biogenesis. Emerging
   evidence suggests a functional interplay between FUNDC1-mediated
   mitophagy and NLRP3 inflammasome regulation [67]^31^-[68]^33.
   Specifically, FUNDC1-driven mitochondrial quality control has been
   shown to reduce IL-1β secretion in macrophages, inhibit hepatocellular
   carcinoma progression, and confer cardio-protection following ischemic
   injury— effects attributed to suppression of NLRP3 activation
   [69]^33^-[70]^35. Notably, FUNDC1 activity is tightly regulated via
   post-translational modifications. Casein kinase II (CK2), a
   constitutively active serine/threonine kinase, phosphorylates FUNDC1 at
   serine 13 (Ser13), sterically hindering its interaction with LC3 and
   thus inhibiting mitophagy [71]^36. Conversely, dephosphorylation of
   Ser13 enhances FUNDC1-LC3 binding, promoting mitochondrial clearance
   and reducing ROS levels. This phosphorylation-dependent molecular
   switch provides a rapid and adaptable means to regulate mitophagy flux
   in response to cellular stress. Consequently, pharmacological
   modulation of FUNDC1 phosphorylation emerges as a promising strategy to
   control NLRP3 inflammasome activity and mitigate its pathological
   consequences [72]^31.

   Emodin (1,3,8-trihydroxy-6-methylanthraquinone; Fig. [73]1A), a major
   active compound extracted from Rheum palmatum (radix Rhei) and
   Polygonum cuspidatum (Polygonaceae), embodies the pharmacological
   legacy of traditional Chinese medicine (TCM) [74]^37. These botanicals
   have been extensively utilized in TCM formulations such as the
   Da-Cheng-Qi decoction, historically prescribed for the treatment of
   gastrointestinal disorders, infectious diseases, and inflammatory
   conditions [75]^38^, [76]^39. Modern pharmacological research has
   elucidated the multifaceted bioactivity of emodin, including potent
   anti-inflammatory, antimicrobial, antioxidant, and anticancer effects.
   Mechanistically, emodin exerts its immunomodulatory functions by
   inhibiting the NF-κB signaling pathway, thereby suppressing the
   production of pro-inflammatory cytokines such as TNF-α and IL-6
   [77]^40^, [78]^41. Beyond its established role in NF-κB modulation,
   recent studies have implicated emodin in the inhibition of NLRP3
   inflammasome activation, leading to decreased IL-1β secretion and
   attenuation of the inflammatory cascade [79]^42. However, the precise
   molecular targets and cytoprotective mechanisms underlying emodin's
   inflammasome-suppressive effects remain incompletely defined.
   Intriguingly, emodin has been identified as a selective inhibitor of
   CK2, binding competitively to the ATP-binding pocket of the kinase
   [80]^43. Given the emerging link between CK2 activity and NLRP3
   inflammasome regulation, a critical knowledge gap persists regarding
   whether emodin exerts its anti-inflammatory effects through modulation
   of FUNDC1- mediated mitophagy, a downstream effector pathway of CK2
   signaling.

Figure 1.

   [81]Figure 1
   [82]Open in a new tab

   Emodin inhibits NLRP3 inflammasome activation in both BMDMs and human
   PBMCs. (A) Chemical structure of emodin (Emo). (B) BMDMs maintained
   viability after treatment with various doses of emodin (n = 5 per
   group). (C-F) Western blot analysis reveals alterations in NLRP3,
   pro-caspase-1, ASC, and pro-IL-1β expression in lysates (Lys) and IL-1β
   (p17), caspase-1 (p20) in supernatants (Sup) of BMDMs treated with
   emodin. The levels of IL-1β, LDH, and TNF-α in BMDM were quantified (n
   = 6 per group). (G-H) Western blot analysis reveals alterations in
   NLRP3, pro-caspase-1, ASC, and pro-IL-1β expression in Lys and IL-1β
   (p17), caspase-1 (p20) in Sup of BMDMs treated with emodin (G).
   Pam3CSK4 and transfected with LPS after emodin and IL-1β levels in Sup
   (n = 6 per group) (H). (I-K) Western blot analysis reveals alterations
   in pro-caspase-1 and pro-IL-1β expression in Lys and IL-1β (p17),
   caspase-1 (p20) in Sup of PBMCs treated with emodin(I). Quantification
   revealed reduced IL-1β (J) and LDH (K) levels in Sup (n = 6 per group).
   Data are presented as mean ± SEM from three independent experiments
   with statistical significance at ***P<0.001.

   In this study, we demonstrate that emodin potently suppresses NLRP3
   inflammasome activation and delineate a novel mechanistic pathway
   whereby emodin enhances mitochondrial quality control via CK2-dependent
   dephosphorylation of FUNDC1, thereby promoting mitophagy. In vivo,
   emodin confers significant protection in sepsis models, with these
   protective effects being lost in NLRP3-deficient mice or upon
   macrophage-specific deletion of FUNDC1. Collectively, our findings
   provide compelling evidence that emodin effectively attenuates
   NLRP3-driven inflammation, establishing it as a promising candidate for
   the prevention of inflammatory and infectious diseases associated with
   aberrant NLRP3 inflammasome activation.

Materials and Methods

Animals

   C57BL/6 wild-type (WT) mice and NLRP3 knockout (NLRP3^-/-) mice,
   weighing between 20 and 25 grams and aged 6 to 8 weeks, were procured
   from Beijing SPF Biotechnology Co., Ltd. (Beijing, China) and Shanghai
   Model Organisms Center, Inc. (Shanghai, China), respectively. The mice
   were maintained in a SPF facility before the commencement of
   experimental procedures. All animal experimentation was conducted in
   strict adherence to the guidelines and protocols sanctioned by the
   Animal Care and Use Committee of the Scientific Research Board at
   Tianjin Medical University (IRB2022-DWFL-472).

Reagents and antibodies

   The reagents utilized in this study were sourced from various suppliers
   as follows: Sigma-Aldrich (Munich, Germany) provided lipopolysaccharide
   (LPS, Escherichia coli strain 0111:B4, L2630), emodin (E7881),
   adenosine triphosphate (ATP, A1852), and dimethyl sulfoxide (DMSO,
   D2650). InvivoGen (Toulouse, France) supplied nigericin (tlrl-nig),
   monosodium urate (MSU, tlrl-msu), Pam3CSK4 (tlrl-pms), poly(dA:dT)
   (tlrl-patn), and flagellin (tlrl-stfla). MitoTracker (M7514,
   [83]M22426) and MitoSOX ([84]M36008) were obtained from Invitrogen
   (Carlsbad CA, USA). Adipogen (San Diego, CA, USA) furnished anti-mouse
   caspase-1 (1:1000 dilution, AG-20B-0042) and anti-NLRP3 (1:1000,
   AG-20B-0014). Anti-mouse IL-1β (1:1000, AF-401-NA) was acquired from
   R&D Systems (Minneapolis, MN, USA), while Santa Cruz Biotechnology Inc.
   (Dallas, TX, USA) provided anti-ASC (1:1000, sc-22514-R). Anti-human
   caspase-1 (1:1000, 2225S), anti-human IL-1β (1:1000, 12703), anti-human
   CK2α (1:1000,2656), and anti-LC3A/B (1:1000, 12741T) were sourced from
   Cell Signaling Technology (Boston, MA, USA). ABclonal (Wuhan, China)
   supplied antibodies against FUNDC1 (1:1000, a22001), LC3B (1:1000,
   a196665), and Flag (1:1000, AE005), as well as secondary antibodies:
   ABflo 488-conjugated goat anti-mouse IgG (1:1000, AS037), ABflo
   555-conjugated goat anti-mouse IgG (1:1000, AS057), and ABflo
   Cy3-conjugated goat anti-rabbit IgG (1:1000, AS007). Anti-GAPDH
   (1:1000, C1312) was purchased from Pulilai Gene Technology (Beijing,
   China), and a customized anti-p-FUNDC1 antibody (1:1000) was produced
   by Abiocenrer (Beijing, China).

Cell culture

   Bone marrow-derived macrophages (BMDMs) were extracted from the femur
   bone marrow of 6-week-old male C57BL/6 mice and subsequently cultured
   in DMEM containing 50 ng/mL of M-CSF for 1 week [85]^44. Peripheral
   blood mononuclear cells (PBMCs) were isolated from freshly drawn
   peripheral venous blood utilizing Human Lymphocyte Separation Medium,
   adhering to the manufacturer's protocol. Prior to any stimulation,
   these PBMCs were allowed to adapt in RPMI 1640 medium overnight. Human
   THP-1 cells, sourced from Suzhou Cyagen Biosciences Inc., were
   maintained in RPMI 1640, and differentiated using 100 nM PMA overnight.
   All cell cultures were maintained in a 37°C, 5% CO[2] humidified
   incubator (HEARcell 150i by Thermo Fisher Scientific).

Inflammasome activation

   BMDMs, THP-1 cells, and PBMCs were plated in 6-well plates at
   concentrations of 1 × 10^5 cells/mL, 5 × 10^5 cells per well, and 2.5 ×
   10^5 cells per well, respectively, and left to attach overnight. On the
   subsequent day, the medium was changed, and the cells were stimulated
   with 50 ng/mL of LPS for a duration of 4 h. Afterward, the medium was
   replaced with Opti-MEM containing emodin for 1 h. To activate the
   canonical NLRP3 inflammasome, the cells were exposed to nigericin at 10
   μM for 30 min, ATP at 5 mM for 45 min, or MSU at 200 μg/mL for 6 h. For
   NLRC4 inflammasome activation, the cells were treated with Lfn-Flic at
   200 ng/mL for 6 h. To induce AIM2 inflammasome activation, poly(dA:dT)
   at 2 μg/mL was transfected into the cells using Lipofectamine 2000 for
   6 h. For noncanonical NLRP3 inflammasome activation, BMDMs were first
   primed with Pam3CSK4 at 1 μg/mL for 4 h, followed by replacement of the
   medium with Opti-MEM containing emodin for 1 hour, and subsequently
   transfected with 1 μg/mL of LPS using Lipofectamine 2000 for 6 h.

RNA sequencing and bioinformatics analyses

   Cellular RNA was extracted from BMDMs using the TRIzol reagent
   (Invitrogen, USA). Samples that passed the basic quality metrics were
   used for RNA-Seq analysis. The sequencing data quality was evaluated
   using FastQC (version 0.11.5), followed by filtering out poor-quality
   sequences using NGSQC (version 2.3.3). StringTie (version 1.3.3b) was
   utilized for gene expression analysis, while DESeq2 (version 1.40.2)
   was employed to identify differentially expressed genes (DEGs) across
   the samples. The criteria for identifying DEGs included a ≥ 2-fold
   change in transcript abundance (|log2FC| ≥ 1) and an adjusted P-value <
   0.01. The volcano plot and heatmap were generated using R4.3.1
   software. Gene ontology (GO) functional enrichment analysis and gene
   set enrichment analysis (GSEA) were performed using the R package
   'cluster Profiler'. The M2 and M5 gene sets were downloaded from the
   MSigDB database. Subsequently, the gene sets were identified as
   significantly enriched after normalization, with a P < 0.05 and a false
   discovery rate (FDR) < 0.25.

In vivo LPS challenge

   C57BL/6 and NLRP3^-/- mice were administered 20 mg/kg emodin or vehicle
   control via intraperitoneal injection 1 h prior to 10 mg/kg LPS or PBS.
   The levels of IL-1β and TNF-α in peritoneal lavage fluid (PLF) and
   serum were detected using ELISA 8 h after LPS treatment. Lung and liver
   tissue sections underwent hematoxylin and eosin staining 24 h following
   LPS injection. Additional lung sections were harvested for
   quantification of NLRP3 and mitochondrial autophagy-related protein
   expression and western blot.

Adeno-associated virus gene construction and infection of C57BL/6 mice

   An adeno-associated virus (AAV) vector carrying a macrophage-specific
   FUNDC1 deletion plasmid was generated by GeneChem Co., Ltd. (Shanghai,
   China). The AAV9 system incorporates a macrophage-specific synthetic
   promoter, F4/80p, driving the expression of short hairpin RNA (shRNA)
   specifically targeting FUNDC1 based on miR155 sequences, along with an
   enhanced green fluorescent protein (EGFP) reporter. The nucleotide
   shRNA of FUNDC1 were cloned using the following sequence:
   5'-GCAGCACCTGAAATCAACAAT-3', 5'-CCACTGGTGGAATCGAG TATT- 3', and
   5'-AGGTG GTGGTTTCCTTCTTCT-3'. The recombinant vector was verified by
   DNA sequencing to confirm the accuracy of the cloned sequences.
   Four-week-old male mice were injected via the tail vein with
   AAV-shFUNDC1 or AAV-Ctrl shRNA virus at a dosage of 1 × 10^12 vector
   genomes per mouse diluted in 50 μl of PBS. On day 21 post-injection,
   GFP expression in infected macrophages was analyzed using flow
   cytometry, and FUNDC1 expression was evaluated with western blot.

Statistical analysis

   Data analysis was carried out using GraphPad Prism version 8.0.2. The
   experimental outcomes are presented as mean ± standard error of the
   mean. An unpaired two-tailed t-test was applied for comparisons between
   two groups. For experiments with multiple groups and a single
   independent variable, one-way analysis of variance was used, followed
   by the Bonferroni post-hoc test to determine statistical significance.
   The log-rank (Mantel-Cox) test was applied for survival analysis.
   Statistical significance was defined as a P-value < 0.05. * P < 0.05,
   ** P < 0.01, *** P < 0.001.

   Additional details for all methods are provided in the
   [86]Supplementary Methods.

Results

Emodin suppresses both canonical and noncanonical activation of the NLRP3
inflammasome

   The cytotoxicity of emodin in BMDMs was assessed using the CCK-8 assay.
   Concentrations up to 50 μM did not significantly affect cell viability
   compared to untreated controls (Fig. [87]1B), supporting their
   selection for subsequent experiments. To investigate the inhibitory
   effects of emodin on NLRP3 activation, BMDMs were primed with LPS,
   pretreated with emodin (12.5-50 μM), and subsequently stimulated with
   nigericin. Immunoblot analysis revealed a dose-dependent decrease in
   secreted mature IL-1β and cleaved caspase-1 (Fig. [88]1C), indicating
   that emodin suppresses NLRP3-mediated caspase-1 activation. Notably,
   emodin did not alter intracellular levels of NLRP3, ASC, pro-caspase-1,
   or pro-IL-1β (Fig. [89]1C), suggesting its action is confined to the
   activation stage. Consistently, emodin significantly reduced the
   release of IL-1β and lactate dehydrogenase (LDH) into the supernatant
   (Fig. [90]1D and E). To confirm inflammasome-specific effects, we
   measured TNF-α secretion, a cytokine dependent on priming but
   independent of inflammasome activation. Emodin had no effect on TNF-α
   levels (Fig. [91]1F), validating its selective inhibition of
   NLRP3-mediated IL-1β processing. To evaluate the broad-spectrum
   inhibitory potential of emodin against NLRP3 activation, BMDMs were
   stimulated with alternative NLRP3 activators, including ATP and MSU.
   Emodin suppressed IL-1β release and caspase-1 cleavage in a
   dose-dependent manner ([92]Fig. S1A and B, D and E), without affecting
   TNF-α production ([93]Fig. S1C, F), demonstrating its efficacy across
   diverse NLRP3 stimuli. We further explored emodin's impact on
   noncanonical NLRP3 activation, wherein intracellular LPS activates
   caspase-11, leading to IL-1β maturation [94]^45. Emodin pretreatment
   dose-dependently inhibited caspase-1 cleavage and IL-1β secretion
   following LPS transfection (Fig. [95]1G and H), indicating that emodin
   suppresses both canonical and noncanonical pathways of NLRP3
   activation. To assess specificity, we examined the effects of emodin on
   the AIM2 and NLRC4 inflammasomes. BMDMs were primed with LPS and
   subsequently stimulated with poly(dA:dT) (an AIM2 activator) or
   transfected with Lfn-Flic (an NLRC4 activator). Emodin did not affect
   IL-1β or LDH release ([96]Fig. S2B and C, E and F), nor did it inhibit
   caspase-1 cleavage ([97]Fig. S2A, D), confirming its specificity for
   the NLRP3 inflammasome.

   Finally, to validate translational relevance, we tested emodin in human
   PBMCs from healthy donors. Consistent with murine BMDMs, emodin
   dose-dependently suppressed caspase-1 cleavage, IL-1β secretion, and
   LDH release (Fig. [98]1I-K). Collectively, these results establish
   emodin as a selective NLRP3 inflammasome inhibitor with potential
   translational application in NLRP3-driven human diseases.

Emodin does not inhibit NLRP3 priming but disrupts ASC oligomerization during
NLRP3 activation

   Activation of the NLRP3 inflammasome involves a two-step process: a
   priming phase, in which pro-IL-1β and NLRP3 is upregulated, followed by
   an activation phase that triggers pro-caspase-1 cleavage and IL-1β
   maturation [99]^8. To determine whether emodin influences the priming
   step, BMDMs were treated with 12.5, 25, or 50 μM emodin either before
   or after LPS priming. Western blot analysis revealed no notable changes
   in the protein levels of NLRP3, ASC, pro-caspase-1, or pro-IL-1β under
   any condition, confirming that emodin does not impair LPS-induced
   priming at concentrations sufficient to inhibit inflammasome activation
   (Fig. [100]2A). During the activation phase, NLRP3 promotes ASC speck
   formation through PYD-PYD interactions, a critical step in inflammasome
   assembly [101]^1. Immunofluorescence microscopy revealed robust
   perinuclear ASC speck formation in activated cells, which was markedly
   attenuated by emodin pretreatment (Fig. [102]2B). Consistently, western
   blot analysis further confirmed that emodin significantly suppresses
   nigericin-induced ASC oligomerization (Fig. [103]2C). Collectively,
   these findings indicate that emodin selectively disrupts ASC
   polymerization and inflammasome assembly, while leaving the priming
   phase intact.

Figure 2.

   [104]Figure 2
   [105]Open in a new tab

   Emodin does not suppress NLRP3 priming but impedes NLRP3-mediated ASC
   oligomerization. (A) Western blot analysis was conducted on the
   specified proteins in Lys from BMDMs that were either exposed to LPS
   for 4 h prior to treatment with varying concentrations of emodin for 1
   h (emodin post-LPS) or pretreated with different doses of emodin for 1
   h followed by LPS stimulation for 4 h (emodin pre-LPS). (B)
   Immunofluorescence images depict the subcellular localization of ASC
   (green) in LPS-primed BMDMs stimulated with nigericin, with or without
   emodin. Nuclei are counterstained with DAPI (blue); scale bar: 20 μm.
   The proportion of cells exhibiting ASC specks (green) in BMDMs was
   quantified. (C) Western blot analysis was carried out to assess
   cross-linked ASC in BMDMs subjected to the same treatment as in (B).
   Data are expressed as the mean ± SEM from three independent
   experiments, with biological duplicates for (B), or are representative
   of three independent experiments for (A, C). **P<0.01 vs the control.

Emodin preserves mitochondrial homeostasis during NLRP3 inflammasome
activation

   To elucidate the mechanism by which emodin modulates NLRP3 inflammasome
   activation, we performed transcriptomic profiling of BMDMs primed with
   LPS and stimulated with nigericin, with or without emodin pretreatment.
   Volcano plot analysis identified 2,259 differentially expressed genes
   (DEGs, P < 0.01) in emodin-treated cells relative to NLRP3-activated
   controls, including 998 upregulated and 1,261 downregulated genes (Fig.
   [106]3A). Notably, emodin-upregulated genes were enriched in pathways
   related to mitochondrial function maintenance (Sod2, Cycs, Anxa5),
   oxidative stress response (Hifa, Foxo3, Abl1), mitophagy regulation
   (Atg9a, Atg4a, Atg5), and ROS metabolism (Jun, Ache, Ccl2) (Fig.
   [107]3B). GSEA further demonstrated significant enrichment of
   emodin-upregulated genes in pathways associated with mitochondrial
   respiratory chain complex assembly, autophagosome maturation, selective
   autophagy, and mitochondrial translation, with normalized enrichment
   scores (NES) of 1.74, 1.51, 1.75, and 1.51, respectively (Fig.
   [108]3C). GO enrichment analysis underscored the involvement of DEGs in
   immune regulation and oxidative stress management (Fig. [109]3D).
   Collectively, these data suggest that emodin reinforces mitochondrial
   homeostasis during NLRP3 inflammasome activation.

Figure 3.

   [110]Figure 3
   [111]Open in a new tab

   Emodin modulates mitochondrial homeostasis and mitophagy upon NLRP3
   inflammasome activation. (A) Volcano plots illustrate the differential
   gene expression between the LPS + nigericin and LPS + nigericin +
   emodin treatment groups. Genes up-regulated and down-regulated are
   highlighted in red and blue, respectively. (B) A heatmap presents the
   relative mRNA expression of genes associated with mitophagy,
   mitochondrial function, oxidative stress, and reactive oxygen species
   in the two treatment groups: LPS + nigericin and LPS + nigericin +
   emodin. (C) Gene Set Enrichment Analysis (GSEA) was conducted to
   identify significantly up-regulated genes in the LPS + nigericin +
   emodin group versus the LPS + nigericin group. (D) GO Pathway
   Enrichment Analysis (focusing on Biological Process, BP) was performed
   based on the differentially expressed genes (DEGs).

   Beyond their role in energy metabolism, mitochondria are increasingly
   recognized as central regulators of NLRP3 activation. Mitochondrial
   dysfunction and subsequent mtROS accumulation have been identified as
   key triggers of NLRP3 inflammasome activation [112]^46. Transcriptome
   analysis implied that emodin promotes mitochondrial integrity. To
   experimentally validate this, we quantified mtROS production using
   MitoSOX Red™ staining. As expected, LPS/nigericin stimulation elevated
   mtROS levels, whereas emodin pretreatment markedly reduced mtROS
   accumulation ([113]Fig. S3A and B). Furthermore, dual MitoTracker
   staining (Deep Red for active mitochondria, Green for total
   mitochondrial mass) revealed that NLRP3 activation increased the
   proportion of damaged mitochondria, which was significantly diminished
   by emodin treatment ([114]Fig. S3C and D). These findings substantiate
   the transcriptomic predictions and demonstrate that emodin preserves
   mitochondrial homeostasis during NLRP3 inflammasome activation.

Emodin promotes mitophagy, thereby suppressing NLRP3 inflammasome activation

   Mitophagy, an evolutionarily conserved protective mechanism,
   selectively eliminates damaged or dysfunctional mitochondria to reduce
   mtROS production and preserve mitochondrial homeostasis [115]^47^,
   [116]^48. Our bioinformatics analysis indicated that emodin upregulates
   the expression of genes associated with selective autophagy and
   autophagosome maturation (Fig. [117]3A-D). Based on these findings, we
   investigated whether emodin modulates NLRP3 inflammasome activation via
   mitophagy enhancement. During autophagosome formation, cytosolic LC3-I
   undergoes lipidation to generate LC3-II, a hallmark of autophagy
   induction [118]^49. In BMDMs stimulated with LPS and nigericin, emodin
   treatment significantly elevated LC3-II expression (Fig. [119]4A).
   Correspondingly, emodin markedly promoted the co-localization of
   mitochondria with LC3 in LPS/nigericin-stimulated BMDMs (Fig. [120]4B).
   To further substantiate the role of mitophagy in emodin-mediated
   suppression of NLRP3 activation, BMDMs were treated with
   3-methyladenine (3-MA), a pharmacological inhibitor of mitophagy. As
   expected, 3-MA treatment significantly reduced LC3-II levels and
   abolished the emodin-induced enhancement of mitochondrial-LC3
   co-localization, indicating impaired mitophagy (Fig. [121]4A and B).

Figure 4.

   [122]Figure 4
   [123]Open in a new tab

   Emodin enhances mitophagy to preserve mitochondrial homeostasis,
   subsequently inhibiting NLRP3 inflammasome activation. BMDMs primed
   with LPS were pretreated with 3-MA (5mM for 1 h) prior to emodin
   treatment (25μM for 1 h), followed by nigericin stimulation (10 μM for
   30 min). (A) Western blot analysis was conducted on the specified
   proteins in Lys. (B) Confocal microscopy was employed to observe the
   colocalization of LC3 (red) with mitochondria (green). Scale bar: 10μm.
   (C-D) BMDMs were labeled with Mitotracker Green (for total
   mitochondria) and Mitotracker Deep Red (for respiring mitochondria),
   and subsequently analyzed using flow cytometry. (E-F) BMDMs were
   stained with MitoSOX and examined under a fluorescence microscope.
   Scale bar: 40μm. (G-I) Western blot analysis was performed to detect
   IL-1β (p17), caspase-1 (p20) in Sup, and pro-IL-1β (p30), caspase-1
   (p45), NLRP3, and ASC in Lys (G). The levels of IL-1β (H) and LDH (I)
   in the Sup were quantified. Data are presented as mean ± SEM from three
   independent experiments with biological duplicates for (D, F, H, I), or
   are representative of three independent experiments for (A, G).
   **P<0.01 compared to control. ***P<0.001 compared to control. ns, not
   significant.

   Moreover, emodin treatment decreased the accumulation of damaged
   mitochondria in inflammasome-activated BMDMs, an effect that was
   effectively reversed by 3-MA co-treatment (Fig. [124]4C and D).
   Consistently, 3-MA administration led to a pronounced increase in
   MitoSOX-positive cells and elevated mtROS levels (Fig. [125]4E and F).
   Notably, the inhibitory effects of emodin on caspase-1 activation,
   IL-1β secretion, and LDH release were also markedly reversed by 3-MA
   treatment (Fig. [126]4G-I). Collectively, these findings suggest that
   emodin-induced mitophagy is a pivotal mechanism that eliminates damaged
   mitochondria, reduces mtROS production, restores mitochondrial
   homeostasis, and ultimately suppresses NLRP3 inflammasome activation.

Emodin suppresses NLRP3 inflammasome activation by enhancing FUNDC1-dependent
mitophagy

   Mitophagy, a specialized form of autophagy targeting dysfunctional
   mitochondria, operates via two primary mechanisms: Parkin-dependent and
   receptor-mediated pathways. Among the latter, FUNDC1 serves as a
   crucial stress-responsive receptor that recruits LC3-II to
   mitochondria, facilitating mitophagy [127]^50. Post-translational
   regulation of FUNDC1 by CK2-mediated phosphorylation at Ser13
   critically modulates its activity— phosphorylation impairs LC3-II
   binding and inhibits mitophagy, whereas dephosphorylation promotes this
   process [128]^36. Given the established CK2-inhibitory properties of
   emodin, we hypothesized that emodin may potentiate FUNDC1-dependent
   mitophagy by disrupting CK2-mediated phosphorylation.

   To test this, we first assessed CK2 kinase activity and observed that
   emodin inhibited CK2 in a dose-dependent manner (Fig. [129]5A).
   Subsequently, we performed affinity purification using
   emodin-conjugated CNBr-activated Sepharose beads to capture
   emodin-binding proteins from BMDM lysates. Immunoblotting confirmed
   specific enrichment of CK2 in the precipitates, while FUNDC1, LC3-I,
   and LC3-II were absent (Fig. [130]5B), indicating a direct interaction
   between emodin and CK2, excluding non-specific binding to core
   mitophagy effectors. Further mechanistic investigations revealed that
   NLRP3 inflammasome activation promoted FUNDC1 phosphorylation at Ser13
   in THP-1 cells, an effect significantly attenuated by emodin treatment
   (Fig. [131]5C). Concordant with enhanced mitophagy, emodin stabilized
   FUNDC1-LC3 complexes (Fig. [132]5D and E), elevated LC3-II expression,
   reduced mtROS accumulation, and improved mitochondrial quality control.
   To determine the essential role of FUNDC1 in emodin-induced mitophagy,
   we performed siRNA-mediated knockdown of FUNDC1 (Fig. [133]5F). FUNDC1
   silencing abrogated the effects of emodin, as evidenced by impaired
   mitochondrial-LC3 co-localization (Fig. [134]5G), accumulation of
   damaged mitochondria and mtROS ([135]Fig. S4A-D), and reversal of
   emodin's inhibitory effects on caspase-1 cleavage and IL-1β maturation
   (Fig. [136]5H). Together, these findings delineate a
   CK2-FUNDC1-mitophagy axis underlying NLRP3 inflammasome regulation. By
   directly inhibiting CK2, emodin promotes FUNDC1-mediated mitophagy,
   facilitates mitochondrial clearance, and effectively suppresses NLRP3
   inflammasome activation.

Figure 5.

   [137]Figure 5
   [138]Open in a new tab

   Emodin suppresses NLRP3 inflammasome activation via FUNDC1-mediated
   mitophagy. (A) Cell lysates of LPS-primed THP-1 with or without
   nigericin incubated with emodin-Sepharose. The pull-down samples and
   input were analyzed by western blot. (B) Effect of emodin on the ATPase
   activity of CK2. After incubation CK2 plus indicated different
   concentrations of free emodin (12.5 μM, 25 μM, and 50μM), ATP was
   measured by CellTiter-Glo Assay Kit and normalized to the control. (C)
   THP-1 cells, primed with LPS, were exposed to emodin or a vehicle
   control, followed by nigericin stimulation. Western blot analysis was
   conducted to assess levels of LC3II, FUNDC1, and p-FUNDC1. (D) THP-1
   cells were transfected with Flag-tagged FUNDC1 and subjected to the
   same treatment as in (C). Confocal microscopy was used to examine the
   colocalization of LC3 (red) and Flag-FUNDC1 (green). Scale bars: 10 μm.
   (E) Cell Lys underwent immunoprecipitation with an anti-LC3 antibody,
   and subsequent western blot was performed to evaluate the interaction
   between LC3 and FUNDC1 in THP-1 cells treated as in (C). (D-F) THP-1
   cells were transfected with FUNDC1 siRNA or a negative control siRNA
   for 24 h, followed by the treatment regimen outlined in the Figure.
   Western blot analysis was employed to measure FUNDC1 expression in Lys
   (D). Confocal microscopy was used to visualize the colocalization of
   LC3 (red) and Mitotracker (green). Scale bars: 10 μm (E). Western blot
   analysis was conducted to detect IL-1β (p17), caspase-1 (p20) in the
   Sup and pro-IL-1β (p30), caspase-1 (p45), LC3II in Lys (F). The data
   presented are representative of three independent experiments. **P<0.01
   compared to control. ***P<0.001 compared to control. ns, not
   significant.

Emodin protects against LPS-induced sepsis via suppressing NLRP3 inflammasome
activation

   To validate the physiological relevance of our in vitro observations,
   we employed a well-established murine model of LPS-induced sepsis,
   which is driven by NLRP3 inflammasome-mediated inflammatory responses.
   Mice were pretreated with emodin via intraperitoneally 1 h prior to LPS
   challenge (Fig. [139]6A). Survival analysis revealed that emodin
   pretreatment significantly improved 72-hour survival rates compared to
   vehicle controls (Fig. [140]6B), aligning with prior findings.
   Quantification of inflammatory markers demonstrated substantial
   increases in IL-1β and TNF-α levels in both PLF and serum following LPS
   exposure, which were markedly attenuated by emodin treatment (Fig.
   [141]6C and D). Histopathological changes further confirmed these
   effects, showing severe lung and liver tissue damage in LPS-challenged
   mice, which was notably alleviated by emodin administration (Fig.
   [142]6E and F). Western blot analysis of lung tissues revealed that LPS
   stimulation induced robust upregulation of cleaved caspase-1, but
   significantly downregulated LC3II expression. These LPS-induced changes
   were reversed upon emodin treatment (Fig. [143]6G).

Figure 6.

   [144]Figure 6
   [145]Open in a new tab

   Emodin demonstrates protective efficacy against sepsis induced by LPS.
   (A) Schematic overview of the experimental design. (B) The survival
   rates of male C57BL/6 mice were monitored for 72 hours after
   intraperitoneal injection of LPS (10 mg/kg), with or without
   pretreatment with emodin (20 mg/kg). Statistical analysis was performed
   using the log-rank test. (C-D) Concentrations of IL-1β and TNF-α in
   serum (C) and PLF (D) were quantified using ELISA. (E-F) Representative
   H&E-stained histological images of lung (E) and liver (F) tissues are
   shown. Scale bar: 100 μm. (G) Western blot analysis was conducted to
   evaluate the protein expression levels of NLRP3, pro-caspase-1 (p45),
   caspase-1 (p20), and LC3II in lung tissue. Data are expressed as mean ±
   SEM or are representative of at least two independent experiments, with
   5-7 animals per group. *P<0.05 compared to the control. ***P<0.001
   compared to the control.

   To determine whether emodin's protective effects were dependent on
   NLRP3, we utilized NLRP3 knockout (NLRP3^-/-) mice. Genetic ablation of
   NLRP3 significantly suppressed IL-1β and TNF-α production in both PLF
   and serum, mitigated organ injury, and improved survival outcomes
   compared to WT controls after LPS challenge ([146]Fig. S5A-F). Notably,
   emodin treatment provided minimal additional benefit in NLRP3^-/- mice
   across all assessed parameters ([147]Fig. S5A-F), indicating that
   emodin's protective effects are predominantly mediated through NLRP3
   inflammasome inhibition. Together, these in vivo findings establish
   that emodin confers protection against LPS-induced sepsis primarily by
   suppressing NLRP3 inflammasome activation, thereby dampening downstream
   inflammatory responses.

FUNDC1-dependent mitophagy contributes to emodin-mediated inhibition of NLRP3
inflammasome activation in LPS-induced sepsis

   To elucidate the mechanisms underlying emodin's protective effects in
   sepsis, we investigated its role in regulating FUNDC1-mediated
   mitophagy in the lungs of septic mice. Western blot analysis revealed
   increased LC3II levels in the lungs of emodin-treated septic mice,
   indicative of enhanced mitophagy (Fig. [148]6G). To directly assess the
   contribution of FUNDC1, we employed an AAV system with a macrophage
   specific F4/80 promoter to deliver shRNA targeting FUNDC1 (Fig. [149]7A
   and B). Efficient delivery and specific knockdown of FUNDC1 in
   pulmonary macrophages were confirmed (Fig. [150]7C and D).
   Macrophage-specific FUNDC1 knockdown significantly impaired emodin's
   protective efficacy, as demonstrated by worsened organ injury and
   decreased survival in septic mice (Fig. [151]7E, H and I).

Figure 7.

   [152]Figure 7

   [153]Figure 7
   [154]Open in a new tab

   Emodin's effects on LPS-induced sepsis rely on FUNDC1-mediated
   mitophagy. (A) Schematic representation of the
   AAV-F4/80p-EGFP-MIR155(RNAi)-SV40-PolyA construct (AAV-shFUNDC1). (B)
   Experimental design overview: Mice were administered tail vein
   injections of either AAV-shFUNDC1 or AAV-Ctrl-shRNA. After 21 days,
   mice were pretreated with emodin (20 mg/kg), followed by an
   intraperitoneal injection of LPS (10 mg/kg) one hour later. (C) Flow
   cytometry was used to evaluate GFP expression in lung macrophages from
   mice infected with AAV-Ctrl-shRNA or AAV-shFUNDC1 on day 21. (D)
   Western blot analysis of FUNDC1 expression in lung tissue from mice
   infected with AAV-shFUNDC1 or AAV-Ctrl-shRNA. (E) Survival rates of
   male C57BL/6 mice were tracked over 72 hours. Statistical significance
   was assessed using the log-rank test. (F-G) IL-1β and TNF-α levels in
   serum (F) and PLF (G) were quantified by ELISA. (H-I) Representative
   H&E-stained images of lung (H) and liver (I) tissues. Scale bar: 100
   μm. (J) Western blot analysis was conducted to measure the expression
   levels of NLRP3, pro-caspase-1 (p45), caspase-1 (p20), FUNDC1, and
   LC3II proteins in lung tissue. Data are expressed as mean ± SEM.
   Results are representative of at least two independent experiments,
   with 5-7 animals per group. *P<0.05 compared to control. **P<0.01
   compared to control. ***P<0.001 compared to control. ns, not
   significant.

   Furthermore, emodin-mediated reductions in IL-1β and TNF-α levels in
   both PLF and serum, as well as decreased cleaved caspase-1 expression
   in lung tissues, were reversed by FUNDC1 knockdown (Fig. [155]7F and G,
   J). Notably, FUNDC1 knockdown also abrogated emodin-induced
   upregulation of LC3II expression, indicating that emodin enhances
   mitophagy via a FUNDC1-dependent mechanism (Fig. [156]7J). In line with
   these findings, the inhibitory effects of emodin on NLRP3 inflammasome
   activation were nullified by FUNDC1 knockdown, as evidenced by
   increased caspase-1 cleavage and elevated IL-1β levels (Fig. [157]7F
   and G, J). Taken together, these results demonstrate that emodin
   protects against LPS-induced sepsis by promoting FUNDC1-dependent
   mitophagy, thereby suppressing NLRP3 inflammasome activation and
   mitigating the ensuing inflammatory response in mice.

Discussion

   The NLRP3 inflammasome has emerged as a pivotal molecular platform that
   not only functions as a frontline sentinel in host defense against
   pathogenic insults but also serves as a critical driver in the
   pathogenesis of a broad array of inflammatory diseases. This dual role
   encompasses a wide spectrum of clinical disorders, including systemic
   conditions such as sepsis and gout, acute inflammatory syndromes, and
   chronic neurodegenerative diseases [158]^1^, [159]^51^, [160]^52. Such
   dichotomous functionality underscores the immense therapeutic potential
   of targeting NLRP3 as a novel strategy for controlling pathological
   inflammation [161]^53. In recent years, pharmacological advances have
   led to the identification of several promising small-molecule NLRP3
   inhibitors, including MCC950, OLT1177, NT-0796, oridonin, cardamonin,
   and scoparone, all of which have demonstrated preclinical efficacy
   [162]^26^, [163]^54^-[164]^57. However, despite these encouraging
   developments, no NLRP3-targeted therapy has yet progressed to clinical
   approval. MCC950, the most advanced candidate to date, exhibited potent
   anti-inflammatory activity in NLRP3-driven disease models but was
   discontinued in Phase II trials due to hepatotoxicity. Meanwhile,
   clinical development of OLT1177 and NT-0796 remains at early stages
   [165]^53. These limitations highlight a critical unmet need for novel
   NLRP3 inhibitors that can deliver both robust efficacy and acceptable
   safety profiles.

   Emodin, a naturally occurring anthraquinone derivative primarily
   extracted from traditional medicinal plants such as Rheum palmatum
   (Radix Rhei) and Polygonum cuspidatum (Polygonaceae), has been widely
   recognized in East Asian ethnomedicine. In recent years, it has
   garnered significant scientific interest due to its potent
   anti-inflammatory properties [166]^4^, [167]^37^, [168]^58^, [169]^59.
   Preliminary studies have suggested that emodin may suppress NLRP3
   inflammasome activation and reduce IL-1β production [170]^60^-[171]^62.
   Building upon these early findings, our current study provides
   comprehensive mechanistic insights into emodin's broad-spectrum
   inhibitory effects on NLRP3 inflammasome activation. Specifically, our
   data demonstrate that emodin robustly inhibits caspase-1 activation and
   IL-1β maturation induced by both canonical and noncanonical NLRP3
   stimuli in BMDMs. Importantly, this inhibitory effect exhibits high
   specificity for the NLRP3 inflammasome, as emodin did not impair
   caspase-1 activation or IL-1β secretion mediated by the AIM2 or NLRC4
   inflammasomes. Moreover, we found that the concentrations of emodin
   required to suppress NLRP3 activation did not affect NF-κB-dependent
   priming signals, such as the induction of pro-IL-1β, NLRP3, or TNF-α
   production in BMDMs. These observations rule out non-specific
   suppression of inflammasome priming as a mechanism underlying emodin's
   anti-inflammatory effects [172]^61^-[173]^63. Mechanistically, our data
   reveal that emodin targets a critical upstream step in inflammasome
   assembly. ASC oligomerization is an essential event in the formation
   and activation of the NLRP3 inflammasome complex. Our findings
   demonstrate that emodin markedly reduces ASC speck formation and
   oligomerization in response to NLRP3 activation, suggesting that emodin
   exerts its anti-inflammatory effects, at least in part, by disrupting
   inflammasome assembly at this key molecular checkpoint. Collectively,
   these results establish emodin as a promising candidate for the
   development of therapeutics aimed at NLRP3-driven inflammatory
   diseases. Its dual features of broad-spectrum NLRP3 inhibition and
   inflammasome pathway specificity, combined with its natural origin,
   highlight emodin's potential as an attractive lead compound. Further
   investigation into its pharmacodynamic properties, bioavailability, and
   safety profile is warranted to facilitate its translation into clinical
   applications.

   Beyond their canonical functions in bioenergetics, mitochondria have
   recently emerged as pivotal regulators of NLRP3 inflammasome
   activation. They serve not only as structural scaffolds for
   inflammasome assembly but also as critical sensors of cellular stress.
   Mitochondrial damage and the subsequent generation of mtROS represent
   potent triggers for NLRP3 inflammasome assembly, playing indispensable
   roles in initiating inflammatory signaling cascades [174]^1^,
   [175]^17^, [176]^23^, [177]^64. This paradigm underscores the
   therapeutic promise of targeting mitochondrial quality control
   mechanisms to modulate NLRP3-driven pathologies [178]^65. Central to
   this quality control network is mitophagy, a specialized autophagy
   pathway responsible for the selective elimination of damaged
   mitochondria and excessive mtROS production, thereby acting as a
   critical brake on NLRP3 inflammasome activation [179]^1. Our RNA-seq
   analysis revealed a robust correlation between emodin-mediated NLRP3
   suppression and the modulation of mitochondrial homeostasis and
   mitophagy pathways. Functional validation demonstrated that emodin
   significantly attenuated mitochondrial damage and mtROS accumulation in
   BMDMs stimulated with LPS and nigericin, thereby preserving
   mitochondrial integrity. Furthermore, emodin enhanced mitophagy flux,
   as evidenced by elevated LC3-II/I ratios and increased co-localization
   of damaged mitochondria with autophagosomes. Importantly,
   pharmacological inhibition of mitophagy using 3-MA abolished emodin's
   protective effects on mitochondrial integrity and reversed its
   inhibitory action on NLRP3 activation in stimulated BMDMs.
   Collectively, these findings establish a causal link between
   emodin-induced mitophagy and NLRP3 inflammasome inactivation, proposing
   a mechanistic axis wherein emodin disrupts the pathological cycle of
   mitochondrial damage, mtROS production, and inflammasome activation via
   enhanced mitophagy.

   Mitophagy, a meticulously regulated cellular process governed by
   intricate molecular crosstalk, serves as a linchpin for maintaining
   mitochondrial homeostasis under physiological and pathophysiological
   conditions [180]^47^, [181]^66^, [182]^67. Among the diverse mitophagy
   pathways, the FUNDC1-dependent axis emerges as a highly dynamic
   stress-responsive mechanism, critically contributing to mitochondrial
   quality surveillance and suppression of NLRP3 inflammasome activation
   [183]^68. This mitochondrial outer membrane protein functions as a
   mitophagy receptor through direct interaction with
   microtubule-associated protein LC3 [184]^69. Central to this process is
   the phosphorylation status of FUNDC1: CK2-mediated phosphorylation at
   Ser13 disrupts FUNDC1-LC3 binding, thereby suppressing mitophagy flux.
   Conversely, dephosphorylation of FUNDC1 restores mitophagy,
   underscoring this post-translational modification (PTM) as a critical
   molecular switch for mitophagy regulation [185]^31^, [186]^36^,
   [187]^70. Notably, recent pharmacological investigations have
   identified emodin as a selective CK2 inhibitor that competitively
   occupies the ATP-binding pocket of the kinase [188]^71. Building on
   these observations, we demonstrated that emodin dose-dependently
   attenuated CK2 enzymatic activity in BMDMs and formed stable complexes
   with CK2, while exhibiting negligible off-target interactions with
   FUNDC1 or LC3. These findings collectively implicate emodin as a
   potential modulator of FUNDC1-dependent mitophagy through disruption of
   CK2-mediated phosphorylation events. Furthermore, functional validation
   revealed that emodin treatment elicited rapid FUNDC1 dephosphorylation,
   augmented FUNDC1-LC3 co-immunoprecipitation, and enhanced
   mitochondrial-LC3 colocalization. Silencing FUNDC1 significantly
   abrogated emodin-mediated preservation of mitochondrial integrity and
   reinstated NLRP3 inflammasome activation, thereby substantiating the
   functional primacy of FUNDC1-dependent mitophagy in these processes.
   Integrating these observations, our study uncovers a novel regulatory
   paradigm wherein emodin suppresses CK2 activity, promoting FUNDC1
   dephosphorylation and mitophagy flux. This cascade restores
   mitochondrial homeostasis while concomitantly attenuating NLRP3
   inflammasome activation, highlighting a previously unrecognized role
   for CK2-FUNDC1 axis modulation in inflammation resolution. Remarkably,
   this PTM-driven regulatory mechanism operates with exceptional temporal
   efficiency, mirroring the rapid activation kinetics of the NLRP3
   inflammasome.

   Our in vitro studies have elucidated a novel mechanism by which emodin
   suppresses NLRP3 inflammasome activation, specifically through the
   modulation of FUNDC1-mediated mitophagy. To assess the translational
   relevance of these findings, we employed a murine sepsis model, induced
   by LPS-triggered NLRP3 inflammasome activation. Emodin administration
   demonstrated robust anti-inflammatory efficacy, as evidenced by
   significantly reduced pro-inflammatory cytokine levels and attenuated
   histopathological injuries in both pulmonary and hepatic tissues.
   Importantly, emodin conferred a marked survival advantage in mice
   challenged with lethal doses of LPS, emphasizing its protective
   capacity in sepsis pathogenesis. Crucially, emodin exhibited minimal
   impact on inflammatory cytokine profiles, tissue damage, or survival
   outcomes in NLRP3-deficient mice. This genetic specificity highlights
   that emodin's beneficial effects in LPS-induced sepsis are
   fundamentally reliant on NLRP3 inflammasome activation. To further
   investigate the mechanistic basis of emodin's action, we utilized
   macrophage-targeted AAV-mediated knockdown of FUNDC1. This genetic
   intervention abolished emodin's ability to induce mitophagy, suppress
   NLRP3 inflammasome activation, and improve survival in septic mice.
   These findings not only validate emodin's therapeutic potential in
   NLRP3-driven sepsis but also establish FUNDC1-mediated mitophagy as a
   crucial effector pathway. The convergence of in vitro mechanistic
   insights and in vivo validation strongly supports the development of
   emodin-based strategies for targeting NLRP3-driven inflammatory
   diseases.

   Despite the promising findings presented in this study, several
   important limitations should be acknowledged. First, although we
   demonstrated that emodin suppresses NLRP3 inflammasome activation and
   promotes FUNDC1-mediated mitophagy, these results were primarily
   derived from animal models and ex vivo analyses using human PBMCs. The
   clinical relevance of these observations remains to be validated in
   human disease contexts. Second, the pharmacokinetic properties of
   emodin, particularly its poor oral bioavailability and metabolic
   instability, pose significant challenges to its therapeutic
   development. Moreover, the toxicological mechanisms of emodin are not
   yet fully understood and require further investigation. Future studies
   should focus on structural optimization of emodin or development of
   targeted delivery systems to improve its bioavailability and safety
   profile. Finally, well-designed clinical studies are essential to
   determine the feasibility and efficacy of emodin-based interventions in
   inflammatory diseases. Addressing these challenges will be critical for
   translating our findings into potential clinical applications.

Conclusion

   In summary, our findings elucidate a novel mechanism through which
   emodin exerts potent suppression of NLRP3 inflammasome activation.
   Specifically, emodin enhances mitochondrial quality control by
   promoting mitophagy via CK2-mediated dephosphorylation of FUNDC1.
   Functionally, emodin demonstrated robust protective effects in
   LPS-induced septic shock, whereas these effects were abolished in
   NLRP3-deficient mice or upon macrophage-specific deletion of FUNDC1.
   Collectively, these results not only establish the molecular
   underpinnings of emodin's anti-inflammatory activity, but also
   highlight its therapeutic potential in treating NLRP3-driven
   inflammatory and infectious diseases. Further investigation of emodin's
   pharmacological properties is warranted to explore its translational
   potential in clinical settings.

Supplementary Material

   Supplementary figures and tables.
   [189]ijbsv21p3631s1.pdf^ (2.8MB, pdf)

Acknowledgments