## 4 Discussion  
In the present study, a novel palm-sized windup syringe pump was utilized to actualize the sustained delivery of CHA to LPS-stimulated macrophages over two hours. The sustained delivery model is a simplified simulation of the in vivo pharmacokinetic profile of CHA after oral administration. Compared to traditional instantaneous delivery through a pipette, sustained delivery model showed a stronger inhibition on several LPS-induced inflammatory markers, including iNOS, TNF-\(\alpha\), and IL-1\(\beta\). Additionally, p38 MAPK signaling pathway may be involved in this enhanced anti-inflammatory efficacy of sustained delivery. CHA significantly inhibited LPS-induced elevation of iNOS mRNA and protein expression. As a driver of inflammation, iNOS catalyzes the overproduction of nitric oxide, which plays a pivotal role in the pathogenesis of inflammation [23]. The expression of iNOS is a direct consequence of an inflammatory process, and the activation of iNOS leads to organ destruction in some inflammatory and autoimmune diseases. Therefore, iNOS has been targeted to improve the efficacy of immunotherapies [24; 25]. A low dose of CHA in the sustained delivery model showed stronger inhibition of iNOS mRNA and protein expression than that observed with the regular instantaneous model. This difference was not observed when a high dose of CHA was used. This finding suggests that the inhibitory effect of CHA on iNOS would be saturated at a certain dose. This is supported by similar iNOS mRNA expression levels observed between samples not subjected to the LPS challenge and those treated with 400 uM CHA in an instantaneous manner. COX mediates inflammation by catalyzing arachidonic acid metabolism and prostaglandin synthesis. Among the three isoforms of COX, the expression of COX-2 is selectively induced by proinflammatory stimuli, such as LPS [26]. COX-2 inhibitors are potential therapeutics for inflammatory diseases. Although both the inhibitory and non-inhibitory effects of CHA on LPS-induced COX-2 protein expression have been reported previously [7; 8; 27], our results showed that CHA exhibited no effect on LPS-induced COX-2 protein expression at concentrations up to 400 uM. Noticeably, Cox-2 mRNA levels were elevated by certain CHA treatments. This may be related to the ability of CHA to activate protein kinase A (PKA) and the cAMP-responsive element-binding protein (CREB) [28]. Cox-2 mRNA expression is regulated at various stages in a complex manner. Apart from p38 MAPK and NF-\(\kappa\)B, activation of the protein kinase A (PKA) pathway targets CREB, another major transcription factor for the _Cox-2_ gene [29]. Activation of PKA and CREB by CHA has been reported under in vitro and in vivo conditions [28; 30; 31]. Figure 7: Effect of different delivery models on CHA concentration in the conditioned medium. (**a**) Upper panel: Representative HPLC chromatogram of standard CHA. Middle panel: Representative HPLC chromatogram of the conditioned medium with CHA observed 2 h after starting delivery. Lower panel: Representative HPLC chromatogram of the conditioned medium without CHA. (**b**) The concentration of CHA in the conditioned medium observed 2 h after starting delivery. Statistical analysis was performed using Student’s \(t\)-test (L vs. LS; H vs. HS). (**c**) A qualitative model of the pharmacokinetics of CHA in the conditioned medium with two different delivery models. CHA: chlorogenic acid; LPS: lipopolysaccharide-NF: nuclear factor; L: low dose administered via pipette; LS: low dose administered via syringe pump; H: high dose administered via pipette; HS: high dose administered via syringe pump. Although CHA inhibits the activation of p38 and NF-\(\kappa\)B, it may increase the transcription of Cox-2 via the PKA/CREB signaling pathway. Cytokines are small proteins produced by almost all cells that regulate and influence the immune response. Pro-inflammatory cytokines, such as TNF-\(\alpha\) and IL-1\(\beta\), are used as biomarkers to indicate inflammation progress [32].