## 4 Discussion
TNF-\(\alpha\) is a major cytokine involved in the initiation of the inflammatory response. Its functions include the induction of the expression of other cytokines, such as IL-1 and IL-6, upregulation of the expression of adhesion molecules, and regulation of arachidonic acid metabolism. CHA suppresses both the mRNA expression and release of TNF-\(\alpha\) and IL-1\(\beta\) in LPS-stimulated primary microglia and macrophages [8; 33]. A significant inhibitory effect on the mRNA expression of both _Tuf-\(\alpha\)_ and _ll-1b_ was observed only when high doses of CHA were delivered in a sustained manner. A notable difference between the present study and previous studies is the timing of the CHA treatment. In previous studies, pretreatment with CHA one or two hours before LPS exposure was predominantly adopted [8; 27; 33]. However, in the present study, to illustrate the effect of CHA administered using different delivery models, CHA was delivered to the culture medium immediately after LPS exposure. This may have influenced the regulatory effects of CHA on the inflammatory response. In terms of the release of TNF-\(\alpha\), an inhibitory effect was observed when a combination of a high dose of CHA and a sustained delivery model was employed. However, IL-1\(\beta\) secretion showed a different pattern. Low doses of CHA administered via the sustained delivery model and high doses of CHA administered using both models inhibited the release of IL-1\(\beta\) into the conditioned medium, and no significant differences were observed among different treatments. This suggests that CHA modulates the secretion of IL-1\(\beta\) via both transcriptional and post-transcriptional regulation. HMOX1 catalyzes the degradation of heme groups in several proteins, such as hemoglobin, myoglobin, and cytochrome p450. Its expression is induced by oxidative stress, cytokines, and other mediators produced during the inflammatory processes. [34]. The induction of the expression of HMOX1 by both LPS and CHA has been reported previously [35; 36; 37; 38]. A clear variance of CHA treatments on the mRNA expression of _Hmox1_ but not other antioxidant genes measured in this study may be attributed to the mediation of _Hmox1_ by anti-inflammatory pathways [39; 40]. Other experiments, such as the intracellular oxidative status measurement, are needed to better understand the effect of different delivery motions on antioxidant efficacy. The regulatory effect of CHA on MAPK and NF-kB pathways in LPS-treated macrophages has been reported in several studies [10; 11; 12; 27]. In the present study, the inhibition of LPS-induced phosphorylation of p38 by CHA was enhanced more by sustained delivery than by higher doses of CHA. However, neither a high dose of CHA nor sustained delivery altered the nuclear translocation of NF-\(\kappa\)B. These findings suggested that the phosphorylation of p38 was more sensitive to CHA treatment than to the nuclear translation of NF-\(\kappa\)B. The does-dependent anti-inflammatory effect of CHA has been reported [7; 41; 42]. This may be attributed to a higher uptake of CHA by macrophages at higher does. Although sustained delivery and instantaneous delivery of low dose CHA showed similar CHA concentrations two hours after starting delivery, slow delivery motion may enhance the overall bioavailability of CHA. This is corroborated the discovery that a slow release of encapsulated apigen and curcumin showed a stronger anti-inflammatory effect than instantly released free apigen and curcumin [43]. The mechanism of how sustainable release enhances anti-inflammatory effect of bioactive compounds needs further investigation. Notably, sustained delivery of a high dose of CHA led to a higher CHA concentration in the conditioned medium two hours after starting delivery compared to those observed with the instantaneous delivery model, which may contribute to its stronger anti-inflammatory activity.