Crocin Attenuates NLRP3 Inflammasome Activation by Inhibiting Mitochondrial Reactive Oxygen Species and Ameliorates Monosodium Urate-Induced Mouse Peritonitis

Abstract

Crocin is a hydrophilic carotenoid pigment found in the stigma of Crocus sativus or the fruit of Gardenia jasminoides. In this study, we investigated the effects of Crocin on the activation of the nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain containing 3 (NLRP3) inflammasome in J774A.1 murine macrophage cells and monosodium urate (MSU)-induced peritonitis. Crocin significantly inhibited Nigericin-, adenosine triphosphate (ATP)-, MSU-induced interleukin (IL)-1β secretion, and caspase-1 cleavage without affecting pro-IL-1β and pro-caspase-1. Crocin also suppressed gasdermin-D cleavage and lactate dehydrogenase release and enhanced cell viability, indicating that Crocin reduces pyroptosis. Similar effects were observed in primary mouse macrophages. However, Crocin did not affect poly(dA:dT)-induced absent in melanoma 2 (AIM2) and muramyl dipeptide-induced NLRP1 inflammasomes. Crocin decreased Nigericin-induced oligimerization and the speck formation of apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC). Crocin also dramatically alleviated the ATP-induced production of mitochondrial reactive oxygen species (mtROS). Finally, Crocin ameliorated the MSU-induced production of IL-1β and IL-18 and the recruitment of neutrophils during peritoneal inflammation. These results suggest that Crocin suppresses NLRP3 inflammasome activation by blocking mtROS production and ameliorates MSU-induced mouse peritonitis. Thus, Crocin may have therapeutic potential in various NLRP3 inflammasome-related inflammatory diseases.

Keywords: Crocin; NLRP3 inflammasome; mitochondrial membrane potential; mitochondrial reactive oxygen species; peritonitis; pyroptosis.

Figure 1

Effects of Crocin on NLRP3 inflammasome. LPS-primed J774A.1 cells were pretreated with various concentrations of Crocin (Cro, μM) for 3 h or 0.1 μM of MCC950 (M) for 1 h, and then incubated with Nigericin (Nig, 4 μM) for 1 h, ATP (5 mM) for 1 h, or MSU (300 μg/mL) for 6 h. (A) After incubation, the cell culture supernatant was collected and the amount of IL-1β was measured by ELISA. Data are expressed as the mean ± SEM and represent four experiments. * p < 0.05 vs. the group treated with Nigericin, ATP or MSU. (B) Simultaneously, the cell culture supernatant was precipitated and the level of cleaved caspase-1 p20 and IL-1β p17 was detected by Western blot. The levels of pro-caspase-1 and pro-IL-1β in cytosolic extract were also detected. The relative intensity of caspase-1 p20 and IL-1β p17 was quantified using image analysis software, ImageJ (http://rsb.info.nih.gov/ij, accessed on 28 February 2023).

Figure 2

Inhibitory effects of Crocin on GSDMD cleavage, LDH release, and cell viability. LPS-primed J774A.1 cells were pretreated with various concentrations of Crocin (Cro, μM) for 3 h or 0.1 μM of MCC950 (M) for 1 h, and then incubated with Nigericin (Nig, 4 μM) for 1 h, ATP (5 mM) for 1 h, or MSU (300 μg/mL) for 6 h. (A) Cleaved GSDMD and pro-GSDMD were detected with cell lysates by Western blotting. Relative intensity of cleaved GSDMD was quantified using image analysis software, ImageJ (http://rsb.info.nih.gov/ij, accessed on 28 February 2023). (B) LDH release into the cell culture supernatant was measured. (C) Cell viability was assessed by the MTT assay. The data are expressed as the mean ± SEM and represent four experiments. * p < 0.05 vs. the group treated with Nigericin, ATP or MSU.

Figure 3

Inhibitory effects of Crocin on NLRP3 inflammasome in mouse peritoneal macrophages. LPS-primed mouse peritoneal macrophages were pretreated with various concentrations of Crocin (Cro, μM) for 3 h, and then incubated with Nigericin (4 μM) for 1 h. (A) IL-1β in the cell culture supernatant was measured by ELISA. (B) Cleaved caspase-1 p20 and IL-1β p17 in the cell culture supernatant and GSDMD, pro-caspase-1 and pro-IL-1β in cytosolic extract were detected by Western blot. Relative intensity of caspase-1 p20, IL-1β p17, and cleaved GSDMD was quantified using image analysis software, ImageJ (http://rsb.info.nih.gov/ij, accessed on 28 February 2023). LDH release (C) and Cell viability (D) were assayed as described in Figure 2. The data are expressed as the mean ± SEM and represent three experiments. * p < 0.05 vs. the group treated with Nigericin.

Figure 4

Effects of Crocin on AIM2 and NLRP1 inflammasomes. LPS-primed J774A.1 cells were pretreated with Crocin (Cro, μM) for 3 h and transfected with MDP or poly(dA:dT). (A) After 6 h, cleaved caspase-1 p20 and IL-1β p17 were detected in the cell culture supernatant by Western blotting. (B) IL-1β in cell culture supernatant was measured by ELISA. (C) LDH release was assayed as described in Figure 2. The data are expressed as the mean ± SEM and represent three experiments.

Figure 5

Effects of Crocin on ASC oligomerization and ASC speck formation. LPS-primed J774A.1 cells were pretreated with various concentrations of Crocin for 3 h, and then incubated with Nigericin (4 μM) for 1 h. (A) DSS-crosslinked ASC in the NP-40-insoluble pellet was analyzed by Western blot. (B) Evidence of ASC specks by fluorescence microscopy. The last column shows a negative control represented by secondary antibody treatment alone. Bars indicate 20 μm. (C) Quantification of ASC specks. The data are expressed as the mean ± SEM and represent three experiments. * p < 0.05 vs. group treated with Nigericin.

Figure 6

Effects of Crocin on mtROS production and mitochondrial membrane potential. (A) LPS-primed J774A.1 cells were pretreated with Crocin (125 μM or 250 μM) for 3 h, and then incubated with ATP (5 mM) for 1 h. Cells were then incubated with MitoSOX for 10 min, and mitochondrial ROS were determined by flow cytometry. (B) Representative flow cytometry histograms. The data are expressed as the mean ± SEM and represent three experiments. * p < 0.05 vs. group treated with ATP.

Figure 7

Suppressive effects of Crocin on MSU-induced peritonitis. C57BL/6 mice were intraperitoneally injected with Crocin (125 mg/kg or 250 mg/kg). After 2 h, peritonitis was induced by intraperitoneal injection with MSU (2 mg) for 6 h. (A) IL-1β, (B) IL-18, (C) Total cell number, and (D) Neutrophil number from peritoneum exudates. The data are expressed as the mean ± SEM. Each group had five mice. (E) Representative flow cytometry histograms. * p < 0.05 vs. the group injected with MSU.