Elsevier

Free Radical Biology and Medicine

Volume 91, February 2016, Pages 10-24
Free Radical Biology and Medicine

Original Contribution
A low toxicity synthetic cinnamaldehyde derivative ameliorates renal inflammation in mice by inhibiting NLRP3 inflammasome and its related signaling pathways

https://doi.org/10.1016/j.freeradbiomed.2015.12.003Get rights and content

Abstract

Uncontrolled inflammation is a leading cause of various chronic diseases. Cinnamaldehyde (CA) is a major bioactive compound isolated from the essential oil of the leaves of Cinnamomum osmophloeum kaneh that exhibits anti-inflammatory activity; however, the use of CA is limited by its cytotoxicity. Here, we synthesized three CA derivatives and identified 4-hydroxycinnamaldehyde-galactosamine (HCAG) as a low toxicity anti-inflammatory compound in vitro (HCAG IC50≫1600 µM; CA IC50=40 µM) and in vivo. HCAG reduced pro-inflammatory mediator expression in LPS-activated macrophages by inhibiting MAPK and PKC-α/δ phosphorylation, decreasing ROS generation and reducing NF-κB activation. HCAG also reduced NLRP3 inflammasome-derived IL-1β secretion by inhibiting the ATP-mediated phosphorylation of AKT and PKC-α/δ. In a mouse model of LPS-induced renal inflammation, we observed reduced albuminuria and a mild degree of glomerular proliferation, glomerular sclerosis and periglomerular inflammation in the HCAG-treated mice compared with the vehicle-treated mice. The underlying mechanisms for these renoprotective effects involved: (1) inhibited NLRP3 inflammasome activation; (2) decreased superoxide anion levels and apoptosis; and (3) suppressed activation of NF-κB and related downstream inflammatory mediators.

Introduction

Inflammation is one of the most important host defense mechanisms in mammals and is also required for wound healing; however, uncontrolled inflammation is harmful to health. Many studies have shown that inflammation is a risk factor for metabolic diseases [1]. Recently, a caspase-1-containing multi-protein complex called the NLRP3 inflammasome was identified that controls the release of IL-1β and IL-18 during the inflammatory processes [2]. A strong link between the NLRP3 inflammasome and the development of metabolic diseases is becoming increasingly evident [3], [4]. For example, the NLRP3 inflammasome promotes renal inflammation and contributes to chronic kidney disease [5], [6], [7], [8], [9], [10]. A recent study reported that peripheral blood mononuclear cells from chronic kidney disease patients showed higher expression of NLRP3 inflammasome components (NLRP3, caspase-1, and ASC) and products (IL-1β and IL-18) compared with healthy subjects [11]. These results suggest that the NLRP3/IL-1β pathway may be a potential therapeutic target for renal disease in humans.

Corticosteroids, some cytotoxic agents, and cyclosporine A are commonly used in the clinic to treat chronic kidney disease [12]; however, the prevention of the progression of renal lesions is accompanied by unsatisfactory side effects in the patients, which remains a major concern [13]. Thus, the development of new agents with sufficient therapeutic effects and negligible side effects is clinically important. Some small molecules or bioactive ingredients isolated from natural products ameliorated renal damage by inhibiting the NLRP3 inflammasome [14], [15], [16]. Recently, we isolated several small molecules from natural products and demonstrated their renal protective functions in various mouse chronic kidney disease models, including IgA nephropathy, focal segmental glomerulosclerosis, and lupus nephritis [17], [18], [19], [20], [21], [22].

Cinnamaldehyde (CA) is a major bioactive compound isolated from the essential oil of leaves of Cinnamomum osmophloeum kaneh [23]. CA exhibits immune modulation properties in bacteria-infected zebrafish by enhancing the host’s defenses against pathogen infection [24], in rat cerebral microvascular endothelial cells by decreasing IL-1β-induced COX-2 activity and PGE2 production [25], and in TNF-α-treated endothelial cells by inhibiting the adhesion of monocytes to the endothelial cells [26]. Our previous study showed that CA inhibited cytokine secretion from lipopolysaccharide (LPS)-activated macrophages but was cytotoxic at concentrations ≥40 μM [23]. Additionally, CA induced caspase-3-dependent apoptosis of human hepatoma cells by enhancing ROS generation and disrupting mitochondrial function [27]. Notably, one study showed that food consumption and the body weights of rats and mice fed CA were reduced, suggesting the possibility of a side effect associated with CA [28]. Another study indicated that CA induced eryptosis of erythrocytes and hemolysis [29]. These results indicate that safety should be a concern when CA is used as an anti-inflammatory agent. Here, we report the development of a nontoxic synthetic CA derivative (4-hydroxycinnamaldehyde-galactosamine, HCAG) that inhibits the LPS-induced inflammation in macrophages and reduces the LPS-induced renal inflammation in mice.

Section snippets

Ethics statement

All animal experiments were performed after approval by the Institutional Animal Care and Use Committee of The National Ilan University, Taiwan (Permit number: NIU 102-7) and were consistent with the NIH Guide for the Care and Use of Laboratory Animals.

Chemistries used in compound preparation

The goal of this study is to develop the nontoxic CA derivatives that can be used in ameliorating renal inflammation by inhibiting inflammation and NLRP3 inflammasome. To synthesize the CA derivatives, all reactions were conducted in dried

Synthesis of (E)-3-phenyl-2-propenoyl-β-d-galactosamine (EPPG)

EPPG can be readily made in three steps from cinnamic anhydride; the synthetic strategy is demonstrated in Scheme 1. Cinnamic anhydride was prepared by treating the mixture of cinnamic acid and thionyl chloride under basic conditions according to known methods [36]. Subsequently, N-linked glycosylation was conducted using galactosamine and cinnamic anhydride at room temperature to produce a moderate yield of EPPG.

Synthesis of N-cinnamyl-β-d-galactosamine (NCAG)

The direct substitution of galactosamine to the allyl position of cinnamyl bromide

Discussion

LPS-induced TLR4 activation in macrophages triggers ROS release, which leads to the activation of NF-κB and induces a rapid cytokine storm, including spikes in TNFα, IL-1β, and IL-6 [51], [52], [53]. These cytokines, in turn, activate a large number of immune cells that produce more ROS and inflammatory factors and damage the surrounding tissue [54], [55]. LPS not only activates macrophages but also promotes the activation of intrinsic renal cells, such as mesangial and tubular epithelial

Acknowledgments

This study was supported by grants 102-2628-B-197-001-MY3, 103-2923-B-197-001-MY3, MOST 103-2321-B-016-002 and NSC 102-2320-B-016-006-MY3 from the Ministry of Science and Technology, and MAB104-016 from National Defense Medical Center, Taipei, Taiwan, ROC.

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