Rosthornin B alleviates inflammatory diseases via directly targeting NLRP3

Abstract

Aberrant activation of the NLRP3 inflammasome contributes to the evolution of diverse inflammatory diseases. Inhibition of the NLRP3 inflammasome has been proven to be an effective treatment strategy for NLRP3‐driven diseases. This study revealed that multiple natural diterpenes from Isodon plants can inhibit the NLRP3 inflammasome, among which Rosthornin B (Ros B) exhibited the best inhibitory effect, with an IC₅₀ of 0.39 μM. Further study revealed that Ros B directly interacts with NLRP3, thereby restraining NEK7‐NLRP3 interaction and inhibiting NLRP3 inflammasome assembly and activation. Remarkably, Ros B had a significant therapeutic benefit in mouse models of NLRP3‐driven septic shock, peritonitis, and colitis. Our study has identified a series of natural diterpenes that target the NLRP3 inflammasome. These natural diterpenes, especially those with low IC₅₀ values, may lead to the development of new drugs and potential clinical therapies for diseases driven by NLRP3 inflammasome activation.

This study shows that Rosthornin B (Ros B), one natural diterpene from Isodon plants, can inhibit the activation of NLRP3 inflammasome. Ros B directly interacts with NLRP3, thereby blocking NEK7‐NLRP3 interaction and inhibiting NLRP3 inflammasome assembly and activation. Remarkably, Ros B exhibits significant therapeutic benefit in mouse models of NLRP3‐driven septic shock, peritonitis, and colitis.

Abbreviations

ASC

apoptosis‐associated speck‐like protein containing a CARD

BMDM

bone marrow‐derived macrophage

CCK‐8

Cell Counting Kit‐8

DAI

disease activity index

DAMPs

damage‐associated molecular patterns

DSS

dextran sulfate sodium

ELISA

enzyme‐linked immunosorbent assay

GSDMD

Gasdermin D

HE

hematoxylin and eosin

HMGB1

high mobility group box 1

IC₅₀

half maximal inhibitory concentration

IL‐18

interleukin‐18

IL‐1β

interleukin‐1β

LDH

lactate dehydrogenase

LPS

lipopolysaccharide

MLNs

mesenteric lymph nodes

MSU

monosodium urate

NEK7

NIMA‐related kinase 7

NLRP3

NOD‐like receptor family pyrin domain containing 3

PAMPs

pathogen‐associated molecular patterns

ROS

reactive oxygen species

Ros B

Rosthornin B

SPR

surface plasmon resonance

1. INTRODUCTION

The NLRP3 inflammasome, a polymeric protein complex composed of NLRP3, pro‐caspase‐1, and ASC, has become a pivotal mediator of inflammation in various pathologies. ¹ Various DAMPs and PAMPs result in activation of the NLRP3 inflammasome. This induces the autocleavage of pro‐caspase‐1 into caspase‐1, which further cleaves GSDMD, pro‐IL‐18, and pro‐IL‐1β. ² Cleaved GSDMD forms transmembrane pores that enable the secretion of IL‐18 /1β and drive lytic cell death, termed “pyroptosis.” Aberrant NLRP3 activation can drive the development of diverse inflammatory diseases, and targeting NLRP3 or its downstream signaling pathways has the potential for therapeutic benefits. ³ , ⁴

Currently, the clinical drugs available for treating NLRP3‐driven diseases mainly target IL‐1β. ³ However, the activation of the NLRP3 inflammasome not only induces IL‐1β secretion but also initiates pyroptosis, IL‐18 release, and other DAMPs, such as HMGB1. ⁵ In addition, IL‐1β is an important cytokine that can also be induced by other inflammasomes in response to pathogen infection. ⁶ Therefore, targeting NLRP3 would lead to fewer immunosuppressive effects than current anti‐IL‐1β therapies and hence could be more beneficial for the therapy of NLRP3‐driven diseases. ⁷

The identification of NLRP3‐specific inhibitors presents a promising avenue for the clinical application and treatment of NLRP3‐driven inflammatory diseases. Previous studies have identified several small molecule compounds, such as CY‐09, MCC950, oridonin, and its analogues, that bind to NLRP3 and alleviate NLRP3‐driven inflammatory diseases in a mouse model. ⁸ , ⁹ , ¹⁰ , ¹¹ However, their clinical pharmacological effects and safety need to be evaluated further, and the exploitation of novel NLRP3 inhibitors with superior safety and specificity for clinical applications is still important. Bioactive compounds derived from natural products represent valuable resources with therapeutic potential, offering prospects for developing novel drugs. ¹²

Diterpenes are a type of natural product with broad bioactivities. ¹³ , ¹⁴ , ¹⁵ Previous studies have identified several diterpenes and their derivatives that exert anti‐inflammatory effects via inhibiting NLRP3‐related signaling pathways through different mechanisms. For example, DIH regulates the priming stage of NLRP3 inflammasome activation, ¹⁶ whereas oridonin restrains NLRP3 inflammasome activation by impeding the NEK7‐NLRP3 interaction. ¹⁰ However, most diterpenes only possess NLRP3 inhibitory properties at high concentrations, raising concerns about off‐target effects.

On the basis of previous studies, we hypothesized that a series of natural diterpenes that share a similar backbone structure may bind to NLRP3 and inhibit the NLRP3 inflammasome directly. To explore this possibility, the inhibitory effects of multiple classes of diterpenes on NLRP3 activation were assessed. Our results revealed that multiple diterpenes from different classes can inhibit the NLRP3 agonist‐induced IL‐1β secretion and that several tetracyclic diterpenes from Isodon plants showed better inhibitory potential. Among them, Rosthornin B (Ros B) has an optimal inhibitory effect, with an IC₅₀ value of 0.39 μM. Further experiments revealed that Ros B is a highly potent and specific inhibitor of NLRP3, that restrains inflammasome activation by directly binding to NLRP3 and blocking the NEK7‐NLRP3 interaction. In vivo experiments also demonstrated that Ros B has remarkable pharmacological effects on multiple mouse models of NLRP3‐related diseases. Our study revealed that a series of natural diterpenes from Isodon inhibit the NLRP3 inflammasome by impeding the NEK7‐NLRP3 interaction. This series of natural diterpenes, especially those with low IC₅₀ values, could be regarded as lead compounds for discovering drugs aimed at targeting NLRP3 inflammasome‐driven diseases.

2. MATERIALS AND METHODS

2.1. Mice

Male C57BL/6J mice were obtained from Gempharmatech in Jiangsu, China, and housed in a facility that was free of specific pathogens. The lighting regimen was 12 h of illumination (8 a.m. to 8 p.m.) and 12 h of darkness. For our study, we selected mice aged between 8 and 10 weeks. The Bengbu Medical College's Animal Research Ethics Committee granted approval for this use (number 057 [2020]).

2.2. Reagents

Ros B was obtained from Desite, Sichuan, China. All other diterpenes were purchased from TargetMol, USA. M‐CSF was supplied by Novoprotein (Cat. No: CB34). LPS (Cat. No: tlrl‐eklps), poly (A:T) (Cat. No: tlrl‐patn), and Pam3CSK4 (Cat. No: tlrl‐pms) were supplied by InvivoGen. Protein G agarose was obtained from Millipore. A range of compounds, such as nigericin (Cat. No: N7143), MSU (Cat. No: U0881), LPS (Cat. No: L2630), ATP (Cat. No: A2383), and PMA (Cat. No: 79346), were acquired from Sigma. The antibodies used included anti‐mouse NLRP3 (Cat. No: AG‐20B‐0014) and anti‐mouse caspase‐1 (Cat. No: AG‐20B‐0042) from Adipogen. Anti‐β‐actin (Cat. No: P30002) was obtained from Abmart. Anti‐NEK7 (Cat. No: ab133514) was obtained from Abcam. Additionally, Cell Signaling Technology provided antibodies such as anti‐mouse IL‐1β (Cat. No: 12507S), anti‐mouse ASC (Cat. No: 67824S), and anti‐human caspase‐1 (Cat. No: 2225).

2.3. Cell culture and activation procedures

THP‐1 cells were purchased from Procell, Wuhan, and no Mycoplasma contamination was detected after identification. THP‐1 cells were cultivated in RPMI 1640 medium enhanced with 10% fetal bovine serum (FBS) and 1% penicillin–streptomycin. THP‐1 differentiation and maturation were induced by overnight treatment with PMA at a concentration of 400 ng/mL. BMDMs of male mice aged 6–8 weeks were cultured in DMEM supplemented with M‐CSF at 50 ng/mL for a period of 5–7 days. Subsequently, these bone marrow‐derived macrophages were counted and seeded in 12‐well plates at a rate of 5 × 10⁵ cells/mL. Unless noted otherwise, BMDMs were always primed with LPS (50 ng/mL) for 3 h and subsequently treated with Ros B or other diterpenes for an additional 30 min. Additional stimuli included MSU (300 μg/mL) for 3 h, ATP (5 mM) or nigericin (10 μM) for 30 min, or poly (A:T) transfection (0.5 μg/mL) for 4 h to initiate the activation of NLRP3 or AIM2 inflammasome. For the activation of noncanonical NLRP3 inflammasome, BMDMs were primed with Pam3CSK4 (400 ng/mL) and then transfected with LPS (500 ng/mL) for 16 h. Immunoblotting was used to examine the cell culture supernatants and lysates.

2.4. Immunoblotting

Following cell stimulation, we collected the supernatant and extracted its proteins via a chloroform–methanol mixture. The cell lysates were then heated to 100°C for 10 min. We separated proteins by their molecular weights via SDS–PAGE with the appropriate gel concentration. The electrophoresis was initially performed at 80 V for approximately half an hour and then increased to 120 V for an hour. Afterward, the proteins were moved to a PVDF membrane at 90 V for 1 h. The membrane was soaked in 5% BSA solution (prepared in PBST) for 1 h, then shaken at 4°C with primary antibodies in the same solution overnight. Following three 10‐min washes in PBST, the membrane was exposed to secondary antibodies in 5% BSA at ambient temperature for an hour and then washed three times for 10 min in PBST. Finally, protein visualization and image documentation were conducted via the ChemiDoc Imaging System from Bio‐Rad.

2.5. Immunoprecipitation

Following the activation of the NLRP3 inflammasome, 300 μL of NP‐40 buffer was used for cell lysis on a shaker at 4°C, lasting between 30 and 60 min. The cell lysate was moved to a centrifuge tube and centrifuged at 6080 × g for 10 min. Next, 230 μL of the supernatant of the lysate was combined with Protein G agarose and the selected antibody at a 1:100 ratio, and this combination was rotated at 4°C for the whole night. The samples that underwent immunoprecipitation were further analyzed using immunoblotting techniques.

2.6. Confocal microscopy

Coverslips (Biosharp) in a 12‐well plate were seeded with 2 × 10⁵ cells/mL BMDMs and cultured overnight. The following day, the media were replaced with 1% FBS in DMEM containing 100 ng/mL LPS for 3 hrs. The cells were then treated with Ros B (2 μM) for 30 min and further incubated with MitoTracker (200 nM) or MitoSOX (1 μM). After an additional incubation of 40 min with 10 μM nigericin, the cells were rinsed three times with PBS, subsequently fixed with 4% PFA at room temperature for 15 min, and rinsed with PBST three times. Then the cells were imaged under the Olympus FV‐1200MPE SHARE confocal laser microscope.

2.7. Intracellular potassium determination

BMDM cells were plated overnight into 6‐well plates at a density of 1 × 10⁶/mL. The next day, the NLRP3 inflammasome was activated via established procedures. Subsequently, the cells were washed with potassium‐free PBS and completely lysed with nitric acid. The lysate was then heated using a magnetic stirring heater until a light yellow powder remained in the beaker. The light yellow powder was dissolved in deionized water, and the concentration of potassium in the solution was analyzed via plasma atomic spectrometry.

2.8. Histological analysis

Mouse colons were fixed by immersion in 4% PFA for 72 h, followed by dehydration, transparency, wax embedding, sectioning, staining with hematoxylin and eosin (HE), observation under an optical microscope, and photography.

2.9. ELISA

Supernatants from BMDMs, mouse peritoneal fluid, and serum were collected and analyzed. The expression levels of IL‐6 (R&D, DY406), IL‐1β (R&D; DY401), and TNF‐α (Invitrogen, 88–7324‐22) were determined via enzyme‐linked immunosorbent assay (ELISA). Additionally, the supernatants of THP‐1 cells were detected for IL‐1β and TNF‐α (R&D, DY201) concentrations with similar ELISA procedures.

2.10. LDH activity assay

The LDH (Lactate Dehydrogenase) amount that was released in the cell culture supernatants was assessed by an LDH assay kit (Promega, G1780) according to the user guide.

2.11. Cell viability assay

BMDMs were seeded into a 96‐well plate, the cell density was approximately 1 × 10⁵ cells per well. After culturing for 24 h, added various diterpenes at the concentration of 5 μM and incubated for 1 h, then added CCK‐8 reagent (10 μL per well), and the mixture was incubated at 37°C in an incubator for 1 h, away from light. Use a microplate spectrometer to detect the absorbance values at 450 nm.

2.12. LPS‐induced systemic inflammation

The mice received an intraperitoneal (i.p.) injection of Ros B (10 mg/kg) 50 min before being administered LPS (20 mg/kg). Serum samples were collected 4 h post‐LPS injection and assayed for IL‐1β and TNF‐α production via ELISA.

2.13. DSS‐induced colitis

Male mice were randomly allocated into three groups, with eight mice in each group. Acute colitis was established by adding DSS at 3% (w/v) to the drinking water for seven consecutive days. Group I was given autoclaved plain water, Group II received 3% DSS in drinking water, and Group III received a daily intraperitoneal injection of Ros B (10 mg/kg) along with the DSS treatment. Body weights were monitored daily. Disease activity index (DAI) evaluations were conducted using an established scoring system. On the eighth day, DSS water was discontinued for Groups II and III. All mice were euthanized on day 10, and the colons were removed for measurement. The quantification of caspase‐1 and IL‐1β in the colonic samples was carried out. The rest of the tissue was subjected to histological analysis. The proportion of Th17 cells was detected via flow cytometry in the mesenteric lymph nodes (MLNs) of the mice.

2.14. MSU‐induced peritonitis

To construct a peritonitis mouse model, the mice were given an intraperitoneal (i.p.) injection of Ros B (10 mg/kg) 50 min before receiving an MSU injection (100 mg/kg). Six hours later, serum and peritoneal lavage fluid were obtained from the mice and analyzed for IL‐1β and TNF‐α via ELISA.

2.15. Flow cytometry

Approximately 1 × 10⁶ cells were initially treated with purified anti‐CD16/32 (BioLegend) for 15 min at 4°C. This was followed by staining with a series of fluorochrome‐conjugated antibodies for 20 min at the same temperature. The antibody mixture used included markers such as Pacific blue‐CD3, PerCP/Cy5.5‐CD11c, FITC‐Ly6G, APC/Cy7‐CD19, PE‐F4/80, and PE/Cy7‐CD11b. For intracellular cytokine detection, 1.5 × 10⁶ cells were resuspended in RPMI‐1640 containing 10% FBS and treated with a Cell Stimulation Cocktail (eBioscience) at 37°C for four 4 h. After the initial treatments, the cells were stained with FITC‐CD8β, PerCP/Cy5.5‐CD4, APC/Cy7‐CD19, and PE/Cy7‐CD3 and then fixed and permeabilized with specific buffers from BioLegend. The cells were then harvested and intracellularly stained with APC‐IL‐17A and its isotype control antibody from BioLegend.

2.16. Surface plasmon resonance (SPR)

SPR with a BIAcore 3000 instrument (GE Healthcare, Sweden) was employed to test the binding affinity between some compounds and the recombinant human NLRP3 protein. The NLRP3 protein was coupled to a CM5 chip with an amine as a ligand, and the binding affinities (K_D) between the aptamers were studied. Different concentrations of either oridonin or Ros B were used as analytes under a typical flow of samples on the chip surface, and the instrument continuously monitored mass changes, revealing signs of binding. Once bound, a switching buffer was applied to the chip to facilitate detachment of the analytes from the ligand, and this dissociation was monitored in real time by measuring the response values.

2.17. Statistical analysis

All the data are presented as the means ± standard errors of the means (SEMs). Statistical analyses were conducted via the unpaired Student's t‐test for comparisons between the two groups and one‐way ANOVA was used for comparisons among multiple groups. GraphPad Prism version 6.0 (GraphPad Software, USA) was performed for statistical analysis. A p value <.05 was deemed significant.

3. RESULTS

3.1. Tetracyclic diterpenes from Isodon plants suppress nigericin‐induced NLRP3 inflammasome activation

As several diterpenes and derivatives are known to display anti‐inflammatory activities via inhibiting NLRP3 inflammasome activation, we were curious about whether other categories of diterpenes could have similar anti‐inflammatory properties. To evaluate the inhibitory effects of multiple classes of diterpenes on NLRP3 inflammasome activation, we investigated the impacts of several types of diterpenes on the release of IL‐1β driven by nigericin (Figure S1A–H). We found that multiple diterpenes from different classes were able to inhibit nigericin‐induced IL‐1β secretion at different doses without exhibiting potential toxicity (Figure 1A and Figure S1I). Some diterpenes, such as Andrographolide (And), inhibited NLRP3 inflammasome activation at high concentrations, which is consistent with other studies. ¹⁷ Several tetracyclic diterpenes, including oridonin and Rosthornin B (Ros B), showed better inhibitory potential. More importantly, these tetracyclic diterpenes that can inhibit the NLRP3 inflammasome are all from Isodon plants, and we found that most diterpenes from Isodon plants could also inhibit the NLRP3 inflammasome (Figure 1B–D). Notably, Ros B displayed the most effective inhibition, with an IC₅₀ value of 0.39 μM (Figure S2A).

FIGURE 1.

Multiple terpenoids of Isodon suppress nigericin‐induced NLRP3 inflammasome activation. (A) BMDMs were exposed to varying concentrations of different diterpenes for 30 min prior to nigericin induction. The levels of IL‐1β in the supernatants were quantified via ELISA. The terpenoids tested included Phytol (Phy), Geranylgeraniol (Ger), Crocetin (Cro), Retinol (Ret), Retinoic acid (RA), Sclareol (Scl), Andrographolide (And), Forskolin (For), Carnosic acid (CarA), Pleuromutilin (Ple), Triptonide (Tri), Gibberellin A3 (GibA3), Stevioside (Ste), Rosthornin B (Ros B), Oridonin (Ori), Incensole (Inc), Docetaxel (Doc), and Rhodojaponin II (Rho‐II). (B) Depiction of the structural chemistry of representative tetracyclic diterpenes. (C) BMDMs were pretreated with different doses of tetracyclic diterpenes of the Isodon species for 30 min before nigericin challenge. The supernatants were harvested for IL‐1β detection via ELISA. (D) BMDMs were pretreated with a uniform dose (10 μM) of each tetracyclic diterpene from Isodon prior to nigericin stimulation. The supernatants were tested for cleaved caspase‐1 (p20), and pro‐caspase‐1 in the cell lysates was assessed via immunoblotting. All the results presented are from three separate experiments and were statistically analyzed via an unpaired t‐test. *p < .05, **p < .01, ***p < .001.

3.2. Ros B specifically and effectively blocks NLRP3 inflammasome activation

Through the screening above, we revealed that Ros B is the most efficient diterpene‐derived blocker of NLRP3 inflammasome activation. To further confirm the result, we evaluated the effects of Ros B on maturation of caspase‐1 and secretion of IL‐1β in BMDMs. Treatment with Ros B dose‐dependently inhibited caspase‐1 cleavage, IL‐1β secretion (Figure 2A,B) and pyroptosis (Figure S2B,C) mediated by nigericin in LPS‐primed BMDMs. However, treatment with Ros B did not affect the generation of inflammasome‐independent cytokines TNF‐α and IL‐6 (Figure 2C,D). We also observed that Ros B inhibited nigericin‐mediated NLRP3 inflammasome activation in human monocyte THP‐1 cell lines (Figure 2E). In addition to nigericin, Ros B also inhibited caspase‐1 cleavage and IL‐1β secretion in response to other NLRP3 inflammasome stimuli, such as ATP and MSU (Figure 2F,G). Moreover, Ros B inhibited intracellular LPS (cLPS)‐induced noncanonical NLRP3 inflammasome activation (Figure 2H,I). In contrast, Ros B did not exhibit any inhibitory effect on the AIM2 inflammasome induced by poly (A:T) (Figure S3A,B).

FIGURE 2.

Ros B suppresses the activation of NLRP3 inflammasome. (A–D) BMDMs were pretreated with the indicated amounts of Ros B for 30 min prior to nigericin stimulation. (A) Supernatants were immunoblotted for the presence of p20 and lysates of BMDMs were immunoblotted for pro‐caspase‐1. (B–D) Supernatants of BMDMs were assayed via ELISA for the levels of IL‐1β (B), TNF‐α (C), and IL‐6 (D). (E) THP‐1 cells differentiated with PMA were stimulated with varying concentrations of Ros B prior to nigericin treatment. P20 in the supernatants and pro‐caspase‐1 in the lysates were analyzed via immunoblotting. (F and G) BMDMs were pretreated with Ros B (2 μM), followed by stimulation with MSU, nigericin, and ATP. (F) Immunoblotting for p20 in the supernatants (SN) and pro‐caspase‐1 in the lysates (Input) of BMDMs. (G) ELISA quantified for IL‐1β secretion in the supernatants of BMDMs. (H and I) Pam3‐primed BMDMs were treated with titrated doses of Ros B before stimulation with transfected cytosolic LPS (cLPS). (H) Immunoblotting for p20 in the supernatants (SN) and pro‐caspase‐1 in the lysates (Input) of BMDMs. (I) ELISA was used to quantify IL‐1β in the supernatants. All the results presented are from three separate experiments and were statistically analyzed via an unpaired t‐test. *p < .05, **p < .01, ***p < .001.

In subsequent experiments, we investigated whether Ros B affects the initiation of LPS‐induced inflammasome priming. The addition of Ros B prior to LPS treatment significantly diminished LPS‐induced pro‐IL‐1β and IL‐6 expression but had no effect on NLRP3 or TNF‐α expression (Figure S3C–E). The addition of Ros B after the administration of LPS did not impact the production of ASC, NLRP3, pro‐IL‐1β, or pro‐caspase‐1, or the release of IL‐6 and TNF‐α (Figure S3C–E). These results showed that Ros B does not modulate the LPS‐driven priming signal in our experimental system. Collectively, our results indicate that Ros B specifically suppresses NLRP3 inflammasome activation in a dose‐dependent fashion.

3.3. Ros B attenuates NLRP3 inflammasome assembly via blocking NEK7‐NLRP3 interaction

Based on the above experiments, we selected Ros B as the most promising candidate among the tetracyclic diterpenes sourced from Isodon and investigated its role and mechanism in NLRP3 inflammasome activation. Our findings showed that Ros B can effectively inhibit NLRP3 inflammasome activation in both BMDMs and THP‐1 cells. To elucidate Ros B's mechanism of action, we investigated the role of mitochondrial damage and potassium (K⁺) efflux, which are crucial upstream signals triggering the activation of NLRP3 inflammasome. ¹⁸ However, Ros B treatment had no significant effect on restoring the intracellular potassium level (Figure 3A). Moreover, Ros B did not reduce mitochondrial damage or reactive oxygen species (ROS) production induced by nigericin (Figure 3B). These results indicate that Ros B does not affect the events upstream of NLRP3 activation. Next, we evaluated the effect of Ros B on NLRP3 inflammasome assembly. The NEK7‐NLRP3 interaction is a crucial step in NLRP3 inflammasome assembly. ¹⁹ We found that Ros B inhibited endogenous NEK7‐NLRP3 and subsequent NLRP3‐ASC interactions in response to NLRP3 inflammasome activation in BMDMs (Figure 3C,D). Moreover, we confirmed that Ros B was capable of impeding the exogenous NEK7‐NLRP3 interaction, but not the exogenous NLRP3‐NLRP3 interaction in HEK‐293 T cells (Figure 3E,F). Our experiments revealed that the mechanism underlying the inhibition of NLRP3 inflammasome formation and activation by Ros B involves blockade of the NEK7‐NLRP3 interaction rather than affecting signals upstream of the NLRP3 inflammasome. Thus, on the basis of our findings, Ros B prevents NLRP3 inflammasome formation and activation by disrupting the NEK7‐NLRP3 interaction.

FIGURE 3.

Ros B disrupts NLRP3 inflammasome assembly. (A) Measurement of intracellular potassium levels via ICP‐OES in BMDMs following treatment with varying doses of Ros B and subsequent stimulation with nigericin. (B) Confocal microscopy was used to assess mitochondrial damage (MitoTracker) and ROS production (MitoSOX) in BMDMs treated with Ros B (2 μM), nigericin, or Ros B plus nigericin. (C) The endogenous NEK7‐NLRP3 interaction in BMDMs was examined through immunoblotting and immunoprecipitation after treatment with different concentrations of Ros B and nigericin stimulation. (D) The endogenous NLRP3‐ASC interaction in BMDMs was examined through immunoblotting and immunoprecipitation after treatment with different concentrations of Ros B and nigericin stimulation. (E) The NEK7‐NLRP3 interaction in HEK‐293 T cells was investigated through Western blotting and immunoprecipitation following treatment with varying doses of Ros B. (F) Western blotting and immunoprecipitation were used to study the NLRP3‐NLRP3 interaction in HEK‐293 T cells treated with different concentrations of Ros B. All the results presented are from three separate experiments and were statistically analyzed via an unpaired t‐test. ***p < .001; ns, no significant difference.

Another important issue is whether other tetracyclic diterpenes from Isodon also inhibit the activation of NLRP3 inflammasome through blockade of the NEK7‐NLRP3 interaction. Our experiments revealed that multiple tetracyclic diterpenes from Isodon also had no influence on the upstream signaling events of NLRP3 activation, including K⁺ efflux, mitochondrial damage, and ROS generation (Figure S4A,B). Moreover, most diterpenes from Isodon blocked the endogenous binding of NEK7 and NLRP3 in BMDMs but had no influence on the NLRP3‐NLRP3 interaction in HEK‐293 T cells (Figure S4C,D). Hence, our results suggest that not only Ros B but also other diterpenes from Isodon can efficiently disrupt NLRP3 inflammasome assembly and thus NLRP3 inflammasome activation by abrogating the NEK7‐NLRP3 interaction.

3.4. Ros B directly binds to NLRP3

To confirm whether the impact of Ros B on NLRP3 inflammasome complex activation is reversible, LPS‐sensitized BMDMs were pretreated with Ros B for 15 min and subsequently we washed away the unbound Ros B before nigericin induction. We found that removing Ros B also abrogated IL‐1β release (Figure 4A), suggesting that the inhibition of Ros B on NLRP3 inflammasome complex activation is irreversible. To further confirm the target protein of Ros B, we hypothesized that Ros B might bind to NEK7 or NLRP3, given that Ros B directly inhibits the interaction between exogenous NEK7 and NLRP3. To validate our hypothesis, we utilized SPR assays, which can detect specific binding between a target protein and its ligand. As a positive control, we used oridonin, a diterpene derived from Isodon that has been previously demonstrated to inhibit NLRP3 inflammasome complex activation by binding to NLRP3 and blocking the NEK7‐NLRP3 interaction. ¹⁰ The results revealed that both oridonin and Ros B bind directly to recombinant human NLRP3 (Figure 4B). Ros B presented a K_D value of 5.491 μM, similar to that of oridonin. To further confirm that Ros B binds to NLRP3, by using AutoDock4, we investigated the molecular interaction between Ros B and the human NLRP3 template structure (AF: Q96P20‐F1). Our results revealed that Ros B was docked into NLRP3 with a binding energy of −6.08 kcal/mol (Figure 4C). Furthermore, Ros B was predicted to form hydrogen bonds with NLRP3 at leucine 451 (L451) and arginine 454 (R454) (Figure 4D). Thus, these observations suggest that Ros B can directly bind to NLRP3 and that its inhibitory effect is irreversible.

FIGURE 4.

Ros B directly binds to NLRP3. (A) BMDMs were exposed to various concentrations of Ros B for 15 min, washed thoroughly three times, and then stimulated with nigericin. The IL‐1β levels in the supernatants were subsequently quantified via ELISA. (B) SPR was utilized to determine the binding affinities between the specified compounds and the recombinant human NLRP3 protein. (C) A depiction of the docking complex between NLRP3 and Ros B, where Ros B is highlighted in yellow and displayed in stick form. (D) Illustration of the two‐dimensional binding mode between NLRP3 and Ros B. All the results presented are from three separate experiments and were statistically analyzed via an unpaired t‐test. ***p < .001.

3.5. Ros B effectively attenuates LPS‐driven septic shock and MSU‐driven peritonitis in mice

As shown above, Ros B inhibits NLRP3 inflammasome activation in vitro. Next, we explored whether Ros B can alleviate NLRP3‐associated inflammatory diseases in vivo. Numerous researches have demonstrated that NLRP3 inflammasome activation plays a critical role in LPS‐induced septic shock. ³ The secretion levels of the inflammatory cytokines IL‐1β and TNF‐α in the serum of the mice were measured. The results revealed that Ros B (10 mg/kg) significantly inhibited the production of IL‐1β (Figure 5A), but it had no inhibitory impact on the inflammasome‐independent cytokine TNF‐α (Figure 5B). In addition, we also observed the survival of the mice after LPS injection at specific time points. Compared with the mice in the LPS‐only group, the mice in the Ros B plus group had longer survival times (Figure 5C). These results confirm that, in vivo, Ros B inhibits inflammation and septic shock by interrupting NLRP3 inflammasome activation. MSU induces NLRP3‐dependent IL‐1β production and neutrophil recruitment intraperitoneally and is commonly used to induce peritonitis in mice. ²⁰ Flow cytometry analysis demonstrated that Ros B treatment significantly reduced neutrophil recruitment, and our ELISA analysis revealed a significant reduction in intraperitoneal IL‐1β levels in mice with peritonitis (Figure 5D,E). In summary, these results demonstrated that Ros B effectively alleviates NLRP3‐mediated septic shock and peritonitis in vivo.

FIGURE 5.

Ros B alleviates LPS‐driven septic shock and MSU‐driven peritonitis in mice. (A and B) ELISA was used to measure IL‐1β (A) and TNF‐α (B) levels in the serum of 10‐week‐old male C57BL/6J mice after injection with LPS (20 mg/kg), with or without preadministration of Ros B (10 mg/kg), with nine mice per group. (C) The survival rate of the mice after being injected with LPS (20 mg/kg) with or without Ros B (10 mg/kg) was recorded, with six mice per group. (D) Flow cytometry analysis of neutrophil numbers in the peritoneal fluid of 9‐week‐old male C57BL/6J mice injected with MSU (100 mg/kg) with or without Ros B (10 mg/kg), with six mice in each group. (E) The levels of IL‐1β in the peritoneal fluid of mice injected with MSU (100 mg/kg) with or without Ros B (10 mg/kg) were quantified via ELISA, with six mice per group. All the results shown are from three separate experiments and were statistically analyzed via an unpaired t‐test. *p < .05, **p < .01, ns, no significant difference.

3.6. Ros B alleviates DSS‐driven colitis in mice

The NLRP3 inflammasome is an important factor in dextran sulfate sodium (DSS)‐induced colitis. ²¹ The mice were given 3% DSS and an intraperitoneal injection of Ros B (10 mg/kg), and their body weights and disease activity index (DAI) scores were recorded on a daily basis. The results revealed that Ros B mitigated weight loss and symptoms of colitis in mice (Figure 6A,B). In addition, Ros B significantly improved the appearance and length of the colon in the mice (Figure 6C). Ros B was highly effective at blocking the secretion of caspase‐1 and IL‐1β in the colon (Figure 6D,E). Histological analysis revealed that Ros B treatment reduced intestinal epithelial destruction (Figure 6F). The overactivation of Th17 cells plays a crucial role in the etiopathogenesis of colitis. ²² Flow cytometry data revealed that Ros B significantly reduced the percentage of Th17 (IL‐17A⁺) cells in MLNs (Figure 6G). Based on these results, we conclude that Ros B is efficacious in mitigating DSS‐induced colitis and other inflammatory diseases associated with the NLRP3 inflammasome in mice.

FIGURE 6.

Ros B alleviates DSS‐driven colitis in mice. (A–G) C57BL/6J mice were subjected to 3% DSS in water for 7 days, followed by euthanasia on day 10. (A and B) Observations of body weight fluctuations and the DAI for each group postcolitis induction using DSS. (C) Examination of colon morphology and measurement of colon length in each group. (D) Western blotting was conducted to detect p20 in the colonic tissue of the mice. (E) IL‐1β levels in the colonic tissues were quantified via ELISA. (F) Colonic sections were prepared and stained with hematoxylin and eosin for histological analysis. (G) Proportions of IL‐17A⁺ CD4⁺T cells in MLNs were determined via flow cytometry. All the results shown are from three separate experiments and were statistically analyzed via an unpaired t‐test. The data are shown as the mean ± SEM values (n = 8). *p < .05, **p < .01, ***p < .001.

4. DISCUSSION

Diterpenes belong to an extensive and diverse class of natural products that have diverse bioactivities, including antineoplastic, anti‐inflammatory, and antimicrobial activities. ²³ , ²⁴ Following a screening process to assess the inhibitory effects of multiple classes of diterpenes on NLRP3 inflammasome activation, we discovered that the majority of diterpenes derived from Isodon plants could inhibit the NLRP3 inflammasome. Among these, Ros B had the best inhibitory effect, with an IC₅₀ value of 0.39 μM, and it decreased NLRP3 inflammasome activation via directly binding to NLRP3 and blocking the NEK7‐NLRP3 interaction.

Furthermore, the majority of diterpenes derived from Isodon, including Ros B, disrupted the NEK7‐NLRP3 interaction without affecting the upstream signaling events of NLRP3 activation or the NLRP3‐NLRP3 interaction, suggesting a common inhibitory mechanism. Nevertheless, more research may be necessary to determine whether all these diterpenes from Isodon hinder the NEK7‐NLRP3 interaction through direct binding to NLRP3.

Multiple lines of evidence suggest that Ros B can directly bind to NLRP3. By using AutoDock4, it is predicted that Ros B forms hydrogen bonds with NLRP3 at the L451 and R454 sites (Figure 4D). Further experimental evidence is needed to validate this prediction. However, whether mutant NLRP3 at the L451 or R454 site can initiate the assembly and activation of the NLRP3 inflammasome as wild‐type NLRP3 does remains to be determined. Moreover, whether the binding and inhibitory activity of Ros B on NLRP3 depend on the L451 or R454 site also needs to be further tested. In addition, a previous study showed that oridonin, another diterpene derived from Isodon, could inhibit the activation of the NLRP3 inflammasome by covalently binding to cysteine 277 on NLRP3. ¹⁰ The mechanisms resulting from these different binding sites also need to be explored in detail. Once it is determined that the L451 or R454 of NLRP3 is crucial for the binding and inhibitory activity of Ros B, exploring whether other natural or endogenous molecules can also inhibit NLRP3 through a similar mechanism will greatly advance the development of NLRP3‐related inhibitors. High‐throughput docking site prediction and molecular screening will be helpful for identifying these new NLRP3‐related inhibitors.

Our in vivo experiments demonstrated that Ros B has remarkable pharmacological effects on mouse models of NLRP3‐driven diseases, including LPS‐induced septic shock, MSU‐induced peritonitis, and DSS‐induced colitis, suggesting that Ros B is a promising candidate for treating these diseases. However, the clinical pharmacological effects and safety of Ros B need to be evaluated further before its use in clinical trials and applications. Further research is needed to determine whether Ros B also has potent pharmacological effects on other inflammasome‐related diseases, including atherosclerosis, type 2 diabetes, and other conditions.

Although our results revealed that the addition of Ros B after LPS stimulation did not impact the LPS‐driven priming signal in our experimental system, we also found that the addition of Ros B prior to LPS treatment significantly diminished LPS‐induced pro‐IL‐1β and IL‐6 expression. These findings indicate that, in addition to targeting NLRP3, Ros B can also inhibit other proteins involved in the LPS‐TLR4 pathway. Many NLRP3‐related inhibitors, such as oridonin and tranilast, have also been shown to inhibit LPS‐TLR4 signaling. ²⁵ , ²⁶ Future research is essential to identify other potential targets and inhibitory mechanisms for these inhibitors, which will guide the optimization of these inhibitors to increase their specificity and diminish off‐target effects.

In the past, several natural products have been adopted as valuable lead compounds for new drug discovery. For example, multiple important drugs, such as morphine, vinblastine, and paclitaxel, originate from natural sources. ²⁷ , ²⁸ Our study revealed that a series of natural diterpenes from Isodon inhibit the NLRP3 inflammasome by blocking the NEK7‐NLRP3 interaction. This series of natural diterpenes, especially those with low IC₅₀ values, may also act as lead compounds for new drug development for the treatment of NLRP3 inflammasome‐driven diseases. Our study revealed a range of natural diterpenes from Isodon that effectively inhibit the NLRP3 inflammasome via blocking the NEK7‐NLRP3 interaction. These natural diterpenes, particularly those with low IC₅₀ values, could serve as promising lead compounds for the development of novel drugs targeting NLRP3 inflammasome‐driven diseases. Structural optimization of these lead compounds that inhibit NLRP3 activation involves modifying their chemical structure to increase their potency, selectivity, pharmacokinetics, and safety profiles, making them more suitable for clinical use.

The main limitation of our study concerns the clinical pharmacological effects and safety of the natural diterpenes derived from Isodon plants, particularly Ros B. Although we demonstrated that natural diterpenes from Isodon plants, represented by Ros B, show good anti‐inflammatory effects via the inhibition of NLRP3 in human cells and mouse models, their potential clinical application in inflammatory diseases needs to be explored further. Moreover, although these natural diterpenes share a similar structure, the backbone structure that is responsible for inhibiting the NLRP3 inflammasome has yet to be defined. In addition, exploring the potential of modified compounds derived from the backbone to improve the pharmacological effects and safety is also worth investigating.

AUTHOR CONTRIBUTIONS

Y. Yang and D. Liu performed experiments and were responsible for the accuracy of the data analysis. H. Cao, L. Lu, W. Zhang, C. Liu, Y. Zeng, and F. Shang contributed to some experiment progress and data analysis. Y. Tao and B. Zhao contributed to the revision. Y. Yang and M. Deng designed the research, wrote, and edited the manuscript. F. Wang and T. Tang supervised the project. All authors approved the final manuscript.

DISCLOSURES

The authors declare no competing financial interests.

Supporting information

Figure S1.

Figure S2.

Figure S3.

Figure S4.

Text S1.

Yang Y, Liu D, Cao H, et al. Rosthornin B alleviates inflammatory diseases via directly targeting NLRP3 . The FASEB Journal. 2024;38:e70248. doi: 10.1096/fj.202401198R

DATA AVAILABILITY STATEMENT

The data supporting the findings of our study can be obtained from the corresponding author upon request.