6.1.2. E ffects on Inflammatory Signaling

When pattern recognition receptors (PRRs) interact with pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs), intracellular signal transduction pathways are induced to activate and translocate transcription factors such as nuclear factor (NF)-κB, activator protein (AP)-1, signal transducer and activator of transcription 3 (STAT3), and interferon regulatory factor 3 (IRF3) into the nucleus to stimulate the expression of pro-inflammatory genes, thereby producing an inflammatory response [56]. NF-κB is one of the transcription factors that expresses pro-inflammatory genes and is involved in both innate and adaptive immune responses. NF-κB can be activated through canonical and non-canonical signaling pathways. The canonical NF-κB pathway is mostly involved in immune response, while the non-canonical NF-κB pathway is only involved in parts of the adaptive immune system [57].

Park et al. [15] used an immunoblotting technique to show that the ethanol extract of *T. impetiginosa* suppressed the activation of Src and spleen tyrosine kinase (Syk). Furthermore, to determine the direct molecular targets, they conducted kinase assays and found that both Syk and Src were suppressed by *T. impetiginosa* [46]. However, Byeon et al. [8] discovered that the water extract of *T. impetiginosa* did not function in the NF-κB pathway due to non-inhibition of phospho-Iκ<sup>B</sup> and the upstream molecules that activate phosphorylation of IκB and AKT. Results from Park et al. [15] showed inhibition of phospho-IκB, even though they did not assess AKT expression. However, phospho-Syk and phospho-Src upstream of AKT were inhibited by ethanol extracts of *T. impetiginosa*. These results could vary depending on the proportion of active components contained in extracts using different solvents [58].

Park et al. [59] investigated the effect of anthraquinone, a main component of *T. impetiginosa*. They specifically focused on anthraquinone-2-carboxlic acid (9,10-dihydro-9,10-dioxo-2-anthracenecarboxylic acid) (AQCA: **1**) and discovered through immunoblotting that an inhibitor of IκB (IKK) and IκBα decreased when treated with 100 μM of AQCA in LPS-induced RAW264.7 cells. They repeated the kinase assay and found that Syk and Src were inhibited by treatment with AQCA [59].

Another pathway, the mitogen-activated protein kinase (MAPK) pathway, activates the activator protein (AP)-1 transcription factor that can lead to expression of pro-inflammatory genes. The MAPK pathway consists of three families: extracellular-signal-regulated kinases (ERKs), c-Jun *N*-terminal kinases (JNKs)/stress-activated protein kinases (SAPKs), and p38s. ERKs can be divided into two subgroups: classic ERKs that include ERK1 and ERK2 and larger ERKs such as ERK5. Classic ERKs are mainly responsible for cell growth, survival, differentiation, and development. JNK family members, which include JNK1, JNK2, and JNK3, are stress-activated [60].

Anthraquinone-2-carboxlic acid (AQCA) was identified as one of the major anthraquinones in *T. impetiginosa*. Administration of AQCA to mice treated with HCl/EtOH and aspirin resulted in reduced expression of phospo-p38 and interleukin 1 receptor associated kinase 1 (IRAK1) [61]. Treatment with AQCA reduced the expression of phospo-p38, c-JNK, mitogen-activated protein kinase 3/6 (MKK3/6), and transforming growth factor β-activated kinase (TAK1) in RAW264.7 cells. However, the expression of ERK was not inhibited. The upstream level of TAK1 was inhibited, as evidenced by degradation of IRAK1. These findings were confirmed using a conventional kinase assay with purified enzyme, and results showed potent suppression of IRAK1 by AQCA. Transfection was performed using HEK293 cells with the IRAK1 gene to validate the results, and treatment with AQCA suppressed phosphorylation of p38 protein without altering FLAG and IRAK1 protein levels. Taken together, these findings sugges<sup>t</sup> that downregulation of IRAK1 by AQCA contributes to an anti-inflammatory effect [59]. Results are summarized in a pathway chart (Figure 3).

**Figure 3.** Inhibitory targets of *Tabebuia impetiginosa* in the NF-κB and AP-1 pathways.
