## 4 Discussion  
Using a well-established cellular model of inflammation, the aim of our study was to evaluate the potential of EACEE to reduce the inflammatory effects of LPS, such as the activation of NF-\(\kappa\)B and MAPK signaling pathways, in RAW 264.7 macrophages. Macrophages play a key role in innate and adaptive immune responses [18]. For this reason, macrophage cell lines are most commonly used to study inflammation and oxidative stress [19; 20]. LPS is a potent stimulator of monocytes/macrophages, resulting in the activation of a series of inflammatory mediators [21]. Inductive stress produces ROS, NO, and signaling molecules that mediate cellular responses [22]. A previous study demonstrated the mechanism responsible for oxidative stress-induced iNOS gene transcription and promoter activity [23]. The overexpression of iNOS is likely to be an important factor in the development of inflammation-associated chronic diseases [24]. Our data indicated that LPS-induced NO production and iNOS expression are inhibited by EACEE treatment in macrophages (Figure 1). It can, therefore, be assumed that EACEE might inhibit the oxidative stress-induced development of inflammatory disease. NF-\(\kappa\)B is a key transcription factor that mediates infection, inflammation, and cell proliferation. LPS exposure leads to the activation and translocation of NF-\(\kappa\)B into the nucleus of macrophages [25]. In our study, treatment with EACEE suppressed the activation of the NF-\(\kappa\)B cascade and translocation of NF-\(\kappa\)B p65 in LPS-stimulated macrophages (Figure 2). An imbalance between the NF-\(\kappa\)B and Nrf pathways contributes to several  
Figure 5: Effects of EACEE on MAPK pathway in LPS-stimulated RAW 264.7 macrophages. Cells were pretreated with EACEE for 1 h and stimulated with LPS for 1 h. Levels of (**A**) p-p38 and p38, (**B**) p-ERK and ERK, and (**C**) p-JNK and JNK were detected by Western blotting. Densitometric analysis was performed using ImageJ software. Data are shown as mean \(\pm\) S.D. of three independent experiments. \({}^{***}\)_p_ < 0.001 vs. vehicle-treated LPS-stimulated macrophages; \({}^{***}\)_p_ < 0.001 vs. LPS-stimulated macrophages. conditions, including septic shock, inflammatory diseases, and cancer. Numerous studies have noted that the Nrf and NF-kB pathways must cooperate through molecular reciprocal action [26; 27]. Moreover, a previous study identified the crosstalk between NF-kB p65 and Nrf2 in the redox system, in which p65 regulates the ubiquitination of Nrf2 [28]. Additionally, Nrf2 knockout macrophages exhibit an enhanced inflammatory response, whereas its upregulation inhibits proinflammatory responses via the regulation of the NF-kB pathway [29]. The results of our study also demonstrated a consistent association between NF-kB and Nrf2 pathways, showing the upregulation of Nrf2 protein expression by EACEE treatment in LPS-stimulated macrophages (Figure 4A). Interestingly, treatment with EACEE also upregulated HO-1 protein expression in LPS-stimulated macrophages (Figure 4B). HO-1, an Nrf2 target gene, is central to Nrf2-mediated NF-kB inhibition [26]. It can, therefore, be assumed that EACEE has anti-inflammatory and antioxidant activities via the simultaneous regulation of NF-kB and Nrf2 pathways. A previous study has shown that LPS triggers oxidative burst in the Nrf2-dependent oxidative stress response [30]. Several studies have also documented that LPS stimulation drives the production of ROS and causes gene alterations in macrophages [31; 32; 33]. In our study, treatment with EACEE suppressed LPS-induced excess ROS production (Figure 3). Therefore, in agreement with the aforementioned studies, we predict that EACEE may exert ROS-scavenging activity via the regulation of the NF-kB and Nrf2 pathways in LPS-stimulated macrophages. LPS-stimulated immune cells also lead to the activation of Nov4, which drives the development of innate immunity disorders [34].