Homotherapy for heteropathy: therapeutic effect of Butein in NLRP3-driven diseases

Abstract

Background: Aberrant inflammatory responses drive the initiation and progression of various diseases, and hyperactivation of NLRP3 inflammasome is a key pathogenetic mechanism. Pharmacological inhibitors of NLRP3 represent a potential therapy for treating these diseases but are not yet clinically available. The natural product butein has excellent anti-inflammatory activity, but its potential mechanisms remain to be investigated. In this study, we aimed to evaluate the ability of butein to block NLRP3 inflammasome activation and the ameliorative effects of butein on NLRP3-driven diseases.

Methods: Lipopolysaccharide (LPS)-primed bone-marrow-derived macrophages were pretreated with butein and various inflammasome stimuli. Intracellular potassium levels, ASC oligomerization and reactive oxygen species production were also detected to evaluate the regulatory mechanisms of butein. Moreover, mouse models of LPS-induced peritonitis, dextran sodium sulfate-induced colitis, and high-fat diet-induced non-alcoholic steatohepatitis were used to test whether butein has protective effects on these NLRP3-driven diseases.

Results: Butein blocks NLRP3 inflammasome activation in mouse macrophages by inhibiting ASC oligomerization, suppressing reactive oxygen species production, and upregulating the expression of the antioxidant pathway nuclear factor erythroid 2-related factor 2 (Nrf2). Importantly, in vivo experiments demonstrated that butein administration has a significant protective effect on the mouse models of LPS-induced peritonitis, dextran sodium sulfate-induced colitis, and high-fat diet-induced non-alcoholic steatohepatitis.

Conclusion: Our study illustrates the connotation of homotherapy for heteropathy, i.e., the application of butein to broaden therapeutic approaches and treat multiple inflammatory diseases driven by NLRP3.

Keywords: Butein; Colitis; NLRP3 inflammasome; Non-alcoholic steatohepatitis; Nrf2; Peritonitis.

Fig. 1

Butein inhibits caspase-1 activation and IL-1β secretion. a The structure of butein. b Cell viability of butein. c BMDMs were primed with LPS for 4 h and then treated with butein for 1 h before stimulation with nigericin for 1 h. Western blot analyses of pro-IL-1β, caspase-1 p45, NLRP3, and ASC in lysates (Input) and IL-1β p17, caspase-1 p20 in culture supernatants (SN). d-f Caspase-1 activity (d), IL-1β (e) and TNF-α (f) secretion were measured in the SN. ^###P < 0.001 vs. the group of LPS. ^***P < 0.001 vs. the group of LPS + nigericin

Fig. 2

Butein prevents caspase-1-dependent GSDMD cleavage and pyroptosis. a, b LPS-primed BMDMs were treated with butein for 1 h prior to stimulation with nigericin for 1 h. BMDMs were then stained with Hoechst 33,342 followed by PI. Cells were observed by fluorescence microscope (a), and the PI-positive cells were counted (b). c Effect of butein on LDH release. d Western blot analysis of cleaved GSDMD in the input of BMDMs. e Relative intensity of cleaved GSDMD described in (d). ^###P < 0.001 vs. the group of LPS. ^***P < 0.001 vs. the group of LPS + nigericin

Fig. 3

Butein blocks multiple agonist-induced inflammasomes activation. a LPS-primed BMDMs were treated with butein prior to stimulation with nigericin, ATP, MSU, poly(I: C), or Pam3CSK4-primed BMDMs were treated with butein and followed by transfection with ultrapure LPS. Western blot analyses of pro-IL-1β, caspase-1 p45, NLRP3, and ASC in input and IL-1β p17, caspase-1 p20 in SN. b, c Relative intensity of IL-1β p17 (b) and caspase-1 p20 (c) described in (a). d-f Caspase-1 activity (d), IL-1β (e) and TNF-α (f) secretion were measured in the SN. g LPS-primed BMDMs were treated with butein before stimulation with nigericin, poly (dA: dT), and flagellin. Western blot analyses of pro-IL-1β, caspase-1 p45, NLRP3, and ASC in input and IL-1β p17, caspase-1 p20 in SN of BMDMs. h, i Relative intensity of IL-1β p17 (h) and caspase-1 p20 (i) described in (g). j-l Caspase-1 activity (j), IL-1β (k) and TNF-α (l) secretion were measured in the SN. ^***P < 0.001

Fig. 4

Butein inhibits NLRP3 inflammasome assembly. a BMDMs were treated with LPS for 4 h and then stimulated with butein for 1 h (butein after LPS) or treated with butein for 1 h and then stimulated with LPS for 4 h (butein before LPS). Western blot analysis of pro-IL-1β, caspase-1 p45, NLRP3, ASC in input. b, c TNF-α (b) and IL-6 (c) secretion in SN was measured by ELISA. d LPS-primed BMDMs were treated with butein for 1 h and then stimulated with nigericin for 1 h. Western blot analysis of cross-linked ASC in the Triton X-insoluble pellet. e LPS-primed BMDMs were treated with butein and then stimulated with nigericin, followed by the addition of HNO₃ to lyse the BMDMs. Intracellular K⁺ was detected by ICP-MS. f, g Detection of ROS by flow cytometry (f), and the intracellular ROS were counted (g). h, i Western blot analyses of Nrf2 and HO-1 levels in BMDMs (h), and the relative intensity of Nrf2 and HO-1 (i). ^###P < 0.001 vs. the group of LPS. ^*P < 0.05, and ^***P < 0.001 vs. the group of LPS + nigericin

Fig. 5

Butein alleviates DSS-induced colitis model. a-d DSS was dissolved in daily drinking water to induce colitis in C57BL/6 male mice for 9 days. Mice received butein (10, or 20 mg/kg) by intragastric gavage during the modeling period. Body weight change (a), DAI (b), representative colon image (c) and the colon lengths (d) of the mice were evaluated (n = 10 for each group). e Western blot analysis of caspase-1 p20, IL-1β p17, NLRP3, ASC, Nrf2, and HO-1 in colon tissues after homogenization of protein content. f, g Relative intensity of caspase-1 p20, IL-1β p17, NLRP3, ASC (f), and Nrf2 and HO-1(g). h, i HE staining (scale bar: 500/100 µm) (h) and histological score in colon sections (i). j Immunohistochemical staining (scale bar: 50 μm) of IL-1β and caspase-1 in colon sections. k ELISA analysis of serum IL-1β production. ^#P < 0.05, ^##P < 0.05, and ^###P < 0.001 vs. the group of Control. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 vs. the group of DSS

Fig. 6

Butein is effective in HFD-induced NASH model. a LPS-primed BMDMs were treated with butein and then stimulated with PO (300 µM) for 6 h. Western blot analyses of pro-IL-1β, caspase-1 p45, NLRP3, and ASC in input and IL-1β p17, caspase-1 p20 in SN. b Caspase-1 activity in the SN. c-h HFD was used to induce a NASH model in C57BL/6 male mice for 12 weeks. At 9–12 weeks, mice received butein (10, or 20 mg/kg) by intragastric gavage. Changes in body weight (c), liver weight (d), ALT (e), AST (f), TC (g), and TG (h) were evaluated (n = 10 for RD and HFD groups, n = 5 for high and low dose of butein intervention groups). i HE staining, oil red O staining, and Masson’s staining (scale bar: 20 μm) of liver tissues. j ELISA analysis of serum IL-1β production. k Western blot analysis of caspase-1 p20, IL-1β p17, NLRP3, ASC, Nrf2, and HO-1 in colon tissues after homogenization of protein content. l, m Relative intensity of caspase-1 p20, IL-1β p17, NLRP3, ASC (L), and Nrf2 and HO-1 (m) as described in (k). ^###P < 0.001 vs. the group of RD. ^*P < 0.05, ^**P < 0.01, and ^***P < 0.001 vs. the group of HFD

Fig. 7

Butein ameliorates LPS-induced peritonitis. a-d ELISA analysis of IL-1β production in serum (a) and peritoneal lavage fluids (b), and TNF-α production in serum (c) and peritoneal lavage fluids (d). e, f Flow cytometric analysis of the proportion of neutrophils in peritoneal lavage fluid (Ly6G⁺CD11b⁺). ^###P < 0.001 vs. the group of control. ^*P < 0.05 and ^***P < 0.001 vs. the group of LPS