Zerumbone Suppresses the LPS-Induced Inflammatory Response and Represses Activation of the NLRP3 Inflammasome in Macrophages

Chia-Cheng Su,I-Chen Chen,Po-Len Liu,Shih-Hua Fang,Ching-Chih Liu,Ming-Yii Huang,Fang-Yen Chiu,Chia-Yang Li,Shu-Pin Huang,Po-Yen Lee,Shu-Chi Wang,Hsin-Chih Yeh,Chi-Han Huang,Wei-Chung Cheng,Kuan-Hua Huang

doi:10.3389/fphar.2021.652860

Abstract

Zerumbone is a natural product isolated from the pinecone or shampoo ginger, Zingiber zerumbet (L.) Smith, which has a wide range of pharmacological activities, including anti-inflammatory effects. However, the effects of zerumbone on activation of the NLRP3 inflammasome in macrophages have not been examined. This study aimed to examine the effects of zerumbone on LPS-induced inflammatory responses and NLRP3 inflammasome activation using murine J774A.1 cells, murine peritoneal macrophages, and murine bone marrow-derived macrophages. Cells were treated with zerumbone following LPS or LPS/ATP treatment. Production of nitric oxide (NO) was measured by Griess reagent assay. The levels of IL-6, TNF-α, and IL-1β secretion were analyzed by ELISA. Western blotting analysis was performed to determine the expression of inducible NO synthase (iNOS), COX-2, MAPKs, and NLRP3 inflammasome-associated proteins. The activity of NF-κB was determined by a promoter reporter assay. The assembly of NLRP3 was examined by immunofluorescence staining and observed by confocal laser microscopy. Our experimental results indicated that zerumbone inhibited the production of NO, PGE2 and IL-6, suppressed the expression of iNOS and COX-2, repressed the phosphorylation of ERK, and decreased the activity of NF-κB in LPS-activated J774A.1 cells. In addition, zerumbone suppressed the production of IL-1β and inhibited the activity of NLRP3 inflammasome in LPS/ATP- and LPS/nigericin-activated J774A.1 cells. On the other hand, we also found that zerumbone repressed the production of NO and proinflammatory cytokines in LPS-activated murine peritoneal macrophages and bone marrow-derived macrophages. In conclusion, our experimental results demonstrate that zerumbone effectively attenuates the LPS-induced inflammatory response in macrophages both in vitro and ex vivo by suppressing the activation of the ERK-MAPK and NF-κB signaling pathways as well as blocking the activation of the NLRP3 inflammasome. These results imply that zerumbone may be beneficial for treating sepsis and inflammasome-related diseases.

Highlights

Inflammasomes are multi-protein complexes mainly located in macrophages, dendritic cells, and some other immune cells, which mediate activation of the proteolytic enzyme caspase-1 and subsequently regulate the secretion of the proinflammatory cytokines, interleukin (IL)-1β and IL-18, as well as actuating pyroptosis, a form of cell death induced by bacterial pathogens (Franchi et al, 2009)
Our experimental results showed that LPS treatment induced the expression of iNOS and COX-2 compared with untreated cells (Figures 1C–E), and zerumbone significantly suppressed the expression of iNOS (20 and 40 μM) and COX-2 (40 μM) in LPSactivated J774A.1 cells (Figures 1C–E)
Macrophages are extremely important immune cells involved in the host defense mechanism during the inflammatory response, which secrete various inflammatory mediators such as Nitric oxide (NO) and proinflammatory cytokines, TNF-α, IL-6, and IL-1β during activation (Hung et al, 2019)

Summary

Introduction

Inflammasomes are multi-protein complexes mainly located in macrophages, dendritic cells, and some other immune cells, which mediate activation of the proteolytic enzyme caspase-1 and subsequently regulate the secretion of the proinflammatory cytokines, interleukin (IL)-1β and IL-18, as well as actuating pyroptosis, a form of cell death induced by bacterial pathogens (Franchi et al, 2009). Lipopolysaccharide, LPS) that induce mitogen-activated protein kinase (MAPK) signaling [e.g., the extracellular signal-regulated kinases (ERK), the c-Jun N-terminal kinases (JNK), and the p38 MAPKs (p38)] and the nuclear factor-κB (NF-κB)-mediated NLRP3 and pro-IL-1β expression (Yang et al, 2019). The subsequent activation step leads to NLRP3 inflammasome oligomerization and triggers the assembly of a multi-protein complex comprised of NLRP3, ASC, and procaspase-1. Three main activating mechanisms have been proposed to induce the activation of subsequent activation steps: K+ efflux, mitochondrial dysfunction and generation of mitochondria-derived reactive oxygen species (ROS), and phagolysosomal destabilization in response to particulates (Ratsimandresy et al, 2013)

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