## 4 Discussion
(2021) demonstrated the potential of Diphorethohydroxcarmalol isolated from Ishige okamurae to inhibit PM-Induced skin damage by regulating NF-\(\kappa\)B, AP-1, and MAPKs signaling pathways in vitro in human dermal fibroblasts. Taken together, in the present study, we have attempted to evaluate the protective effects of HEBI against UPM-activated inflammatory responses in MH-S macrophages. Elevated NO secretion is a typical inflammatory response of macrophages during inflammatory events [35]. Several studies reported that exposure of macrophages to UPM resulted in up-regulated production of PGE\({}_{2}\) and pro-inflammatory cytokines [26]. The production of NO and PGE\({}_{2}\) mainly depend on the expression levels of iNOS and COX2 proteins, respectively. The production of NO in the cellular environment depends on the conversion of l-arginine to l- citruline by the nitric oxide synthase (NOS). To date, three isoforms of NOS were identified and called iNOS, neuronal NO synthase (nNOS), and endothelial NO synthase (eNOS). In general, the activation of nNOS and eNOS are responsible for producing nano-molar concentrations of NO for short periods (seconds or minutes); however, the activation of iNOS caused the secretion of large amounts of NO (\(\upmu\)M range) for relatively long periods (from hours to days) [36]. Besides the iNOS; COX2 is the product of an \"immediate-early\" gene that is rapidly inducible and tightly regulated in cellular environments [37]. Moreover, COX2 is the inducible form of cyclo-oxygenase and plays an important role in inflammation by stimulating the conversion of arachidonic acid to prostaglandamlations. In normal situations, COX2 cannot secrete from the cells, and expression is avoided. During the inflammatory responses, macrophages secrete a large amount of COX2 to up-regulate inflammatory responses [37; 38]; therefore, we compared the transcription levels of _iNOS_ and _COX2_ as well as the protein expression levels of iNOS and COX2 with or without UPM stimulation. As expected, UPM up-regulates the gene transcriptions and protein expression levels of COX2 and iNOS; however, HEBI reduced elevated levels of COX2 and iNOS observed in UPM-exposed cells. After identifying basic anti-inflammatory properties, we attempted to evaluate possible anti-inflammatory signal pathways of HEBI against UPM-exposed macrophages. The principal role of toll-like receptors (TLRs) is reorganizing specific molecular patterns identical to microbial components. The activation of TLRs controls the distinct patterns of gene expressions linked with the protective mechanisms. TLRs are involved in stimulating innate immunity and the development of antigen-specific acquired immunity [39]. According to the previous observations, up-regulated expression of TNF-\(\upalpha\) triggers NF-\(\upkappa\)B and MAPK protein transcription via activating TLR dependent inflammatory pathways [40]. Moreover, TNF-\(\upalpha\) strongly stimulates the activation of NF-\(\upkappa\)B, and MAPKs stimulate the TNF-\(\upalpha\) secretion from cells. This positive feedback is key to the pathogenesis of inflammatory complications, including asthma, rheumatoid arthritis, cancer, and inflammatory bowel disease [40]. Interestingly, many studies demonstrated the potential of UPM to induce transcription of pro-inflammatory cytokines such as TNF-\(\upalpha\), IL-1\(\upbeta\), and IL-6 from macrophages [41]. Additionally, [PERSON] and [PERSON] (2004) reported that the activation of TLR 2 and 4 was responsible for the production of inflammatory cytokines involved in MyD88-dependent NF-kB pathway activation. Upon the activation of TLRs, TLR/MyD88 complex dislocate, resulting in downstream activation of the transcription factors related to NF-kB and MAPKs, which are the primary contributors to pro-inflammatory gene expression [42]; therefore, the up-regulated inflammatory responses observed in MH-S lung macrophages after UPM exposure should be related to the TLRs mediated NF-kB and MAPK protein expressions.