This investigation demonstrated that the 50% ethanolic extract of Curcuma longa (CLE) appeared to have anti-neuroinflammatory effect against LPS-induced inflammation in BV2 microglial cells. The anti-neuroinflammatory effect of CLE was associated with the inactivation of NF-κB and MAPK (p38, ERK, and JNK) signaling pathways, resulting in the inhibition of pro-inflammatory mediators including NO, PGE2, iNOS, COX-2, and pro-inflammatory cytokines such as including IL-1β, IL-6 and TNF-α. Moreover, CLE induced HO-1 proteins by activating Nrf2 signaling, and inhibiting HO-1 expression using SnPP reversed the inhibitory effects of CLE, indicating the anti-neuroinflammatory effect of CLE was mediated by HO-1/Nrf2 signaling pathway.
Neuroinflammation is generally characterized by the excessive production of pro-inflammatory mediators including NO, PGE2, iNOS, COX-2, and pro-inflammatory cytokines . NO is catalyzed by the enzymatic activity of iNOS which converts L-arginine to NO and L-citrulline via the intermediate N-hydroxy-L-arginine , and PGE2 is synthesized from arachidonic acid (AA) by the enzymatic effect of COX and PGE synthases (PGES) . Pro-inflammatory cytokines are small secreted proteins from various immune cells, and they play multiple roles in CNS function including the regulation of sleep, neuronal development, and inflammatory responses against bacterial and viral infections of either the brain or the periphery . Therefore, the inactivation of these pro-inflammatory mediators could be one of the targets for treatment and prevention of neuroinflammatory diseases. This study sought to elucidate the anti-neuroinflammatory effects of CLE in LPS-induced BV2 microglial cells. The overproduction of NO/PGE2, and the protein expression of iNOS/COX-2 were inhibited by pre-treatment with CLE (Fig. 2). In addition, CLE decreased the overproduction of pro-inflammatory molecules and their mRNA expression including of IL-1β, IL-6, and TNF-α in LPS-induced BV2 microglial cells (Fig. 3).
NF-κB represents a family of inducible transcription factors, which regulates a large array of genes involved in different processes of the immune and inflammatory responses . It is composed of five different members including p65 (RelA), RelB, c-Rel, NF-κB1 (p50/p105), and NF-κB2 (p52/p100), which mediates transcription of target genes by binding to a specific DNA element, κB enhancer, as various hetero- or homo-dimers . NF-κB is activated via two major signaling pathways including the classical/canonical NF-κB pathway, and the alternative/non-canonical NF-κB pathway, and they are both important for regulating inflammatory responses [19, 20]. The canonical NF-κB pathway which are most extensively studied is regulated by the activation of a variety of cell surface receptors including IL-1 receptor, Toll-like receptors (TLRs), TNF receptor, as well as T-cell receptor and B-cell receptor , whereas the non-canonical NF-κB pathway can be triggered by a specific group of stimuli including ligands of subset of TNF-receptor superfamily members such as B cell activating factor (BAF), CD40, lymphotoxin β (LTβ) receptor, and receptor activator of NF-κB (RANK) . NF-κB can be activated by a wide variety of stimuli including viruses, bacterial toxins, UV light, oxidative stresses, inflammatory stimuli, cytokines, carcinogens, tumor promoters, and various mitogens [21, 22], and it regulates the expression of inflammatory-related genes including iNOS, COX-2, lipoxygenase (LOX), cytokines, adhesion molecules, cell cycle regulatory molecules, and angiogenic factors . Therefore, the inactivation of NF-κB is one of the important targets for the treatment of neuroinflammation. In this study, pre-treatment with CLE attenuated LPS-induced activation of NF-κB signaling pathway by inhibiting the phosphorylation and degradation of IκB-α, and the translocation into the nucleus of p65 subunit in BV2 microglial cells (Fig. 4).
MAPKs comprise a family of serine/threonine protein kinases that have been implicated in the regulation of key cellular processes including gene induction, cell survival/apoptosis, proliferation and differentiation as well as cellular stress and inflammatory responses . MAPK signaling pathways are triggered by the activation of TLRs, toll-interleukin receptor (TIR), or the TNF receptor families by primary inflammatory stimuli and cytokines . In mammals, MAPKs pathways consist of three major subunits including p38, ERK, and JNK . ERK1 and ERK2 which are homologous isoform and expressed in nearly all tissues are activated by MAPK kinase (MKK) and MKK2 and have important role in proliferation, cell death, cytoskeletal remodeling, and regulating cell shape and motility [25, 26]. JNKs consist of at least ten isoforms derived from alternatively spliced mRNAs of three genes including JNK1, JNK2, and JNK3 . They are also called as stress-activated kinases (SAPKs) because of their activation in response to cell stress . The activation of JNK is regulated by MKK4 and MKK7, and is important for survival of cells and replication of viruses . Four p38 isoforms (p38α, p38β, p38γ, and p38δ) are found in mammals. p38α and p38β isoforms are expressed in most of tissues, whereas p38γ, and p38δ isoforms are expressed in kidney, skin and muscle cells . They are activated by MKK3, MKK4, and MKK6 , and the activation of p38 isoforms leads to the phosphorylation of transcription factors which regulate pro-inflammatory mediators . The inhibition of MAPK signaling pathway emerge as attractive anti-inflammatory agents, because they can reduce reducing the synthesis of inflammation mediators at multiple levels and are effective in blocking inflammatory cytokine signaling . Our results showed that pre-treatment with CLE inhibited LPS-induced activation of p38, ERK, and JNK MAPKs by inhibiting the phosphorylation of them in BV2 microglial cells (Fig. 5).
HO has three distinct isoforms of HO including the only inducible form HO-1, which is the only inducible form and is known as heat-shock protein 32 (Hsp-32), and constitutively expressed HO-2, and HO-3 [31, 32]. In particular, HO-1 is a detoxifying phase II anti-oxidant enzyme, which is up-regulated in various pathological conditions including cellular stresses and stimuli including ischemia, hypoxia, oxidative stress, and inflammatory cytokines [33, 34]. Under oxidative injury and inflammatory conditions, HO-1 acts the rate-limiting enzyme in the catabolism of heme conversing into carbon monoxide (CO), ferrous ion (Fe2+), and biliverdin, which act as anti-oxidant and anti-inflammatory mediators and reported alleviating extent of oxidative stress and related disorders [33, 35]. Moreover, it has also been demonstrated that HO-1 expression is up-regulated by anti-inflammatory cytokines , indicating that HO-1 may be a therapeutic target in neurodegenerative diseases and brain infection . HO-1 expression is controlled by the Nrf2 signaling pathway. Nrf2 is a member of the cap-n-collar (CNC) transcription factor family of basic leucine zipper proteins . It is a crucial factor in regulation of cellular redox homeostasis, oxidative stress and immune inflammation [39, 40]. In resting state, Nrf2 is bound to the endogenous inhibitor Kelch-like ECH-associated protein 1 (Keap 1) in the cytoplasm, which induces ubiquitination and proteasomal degradation of Nrf2 [40, 41]. Under oxidative stress or inflammatory conditions, Nrf2 dissociates from Keap1, translocates into the nucleus, forms a heterodimer with the small Maf proteins that recognize and binds to antioxidant response elements (ARE) in the promoter site of phase II detoxifying enzymes and cytoprotective genes including including HO-1, NAD(P)H quinone oxidoreductase 1 (NQO1), peroxiredoxin (PRX), thioredoxin (Trx), glutathione S-transferase (GST), and glutathione peroxidase (GPx) [40, 42]. In addition, Nrf2-ARE binding also regulates the expression of genes related to pro- and anti-inflammatory enzymes including iNOS and COX-2 . Various kinds of natural products have been reported to up-regulate HO-1 expression by activating Nrf2 to bind with the ARE such as berberine from Coptidis chinensis, 7,8-dihydroxyflavone, or tryptanthrin in astrocytes, myoblast, and microglial cells [35, 44, 45]. In the present study, CLE induced HO-1 expression, as well as the accumulation of Nrf2 in the nucleus. In addition, pre-treatment with SnPP, a HO-1 inhibitor, abolished the CLE-induced inhibition of secretion of NO and PGE2 as well as the expression of iNOS and COX-2 proteins. Taken together, these results indicate that CLE-induced activation of HO-1/Nrf2 signaling pathway plays a crucial role in downregulating neuroinflammatory responses.
As it is known, C. longa is composed of various compounds including diarylheptanoids (including curcuminoids), diarylpentanoids, monoterpenes, sesquiterpenes, diterpenes, triterpenoids, alkaloid, and sterols, of which curcuminoids are the most abundant . Therefore, in order to analyze the content of curcuminoids that are the most abundant and exhibit various physiological activities among the components contained in C. longa, we established a quantitative analysis method using the ethanolic extract of C. longa grown in Korea and verified the content of curcuminoids in previous study . The ethanolic extract of C. longa is an optimal condition for analyzing the content of curcuminoids, and its physiological activities may be due to curcuminoids. However, since C. longa also contains other compounds such as monoterpenes or sesquiterpenes, it is necessary to further verification which compound is responsible for anti-neuroinflammatory effects of CLE.
In this investigation, setting the maximum concentration of CLE to 150 µg/mL could be considered an appropriate measure. First of all, our study evaluated the cytotoxicity of CLE in BV2 cells as shown in Fig. 1, and the results confirmed that it was toxic at 200 µg/mL but not at 150 µg/mL. Second, in the previous report confirming the anti-oxidant effect in BV2 microglial cells using the hexane extract of C. longa, the experiment was conducted by setting the maximum concentration of the hexane extract of C. longa to 500 µg/mL . Finally, other studies that examined the anti-neuroinflammatory effects in BV2 microglial cells using the extract of other natural products, the concentration much higher than 150 µg/mL used in this study was set as the highest concentration [47, 48]. Therefore, the 150 µg/mL of CLE used in this investigation is considered to be sufficient to examine the anti-neuroinflammatory activity.