## 3 Results
### The Seaweed Extracts Induce Inflammation-Related Cytokine Expression in RAW 264.7 Cells  
It was reported previously that macrophages are major cells of the innate immune system. When they are exposed to external stimuli such as foreign particles or pathogens, they secrete cytokines such as TNF-\(\upalpha\), IL-1\(\upbeta\), IL-6, and IL-12 in addition to releasing leukotrienes, chemokines, prostaglandandins, and complement proteins. Together, these molecules increase vascular permeability and recruit inflammatory cells to fight infections [5]. To determine whether these seaweed extracts can induce these cytokines, RAW 264.7 cells were treated with 0, 50, 200, and 400 \(\upmu\)g/mL of three algal extracts (SH, UP, and SF) separately for 24 h. The transcription levels of the above-mentioned cytokines were observed. We found that all three extracts exhibit immunomodulatory properties, as all of them showed increased mRNA expression levels of TNF-\(\upalpha\), IL-1\(\upbeta\), IL-6, and IL-12 when compared with the control. SH significantly induced IL-1\(\upbeta\) expression in a concentration-dependent manner. UP showed higher expression of IL-1\(\upbeta\) at 100 \(\upmu\)g/mL as compared to other concentration groups of the same extract, but SF induced IL-1\(\upbeta\) only at 200 and 400 \(\upmu\)g/mL. In addition, SH and UP showed significant concentration-dependent effects in inducing TNF-\(\upalpha\), but SF induced TNF-\(\upalpha\) at a concentration of 200 and 400 \(\upmu\)g/mL. SH significantly increased IL-6 mRNA expression levels at 200 and 400 \(\upmu\)g/mL. UP and SF showed significant induction in IL-6 mRNA expression levels at 200 and 400 \(\upmu\)g/mL,  
Figure 1: Seaweed extracts induce cell proliferation in RAW 264.7 cells. RAW 264.7 cells were exposed to 50, 100, 200, and 400 \(\upmu\)g/mL of (**a**) SH, (**b**) UP, and (**c**) SF for 24 h. Cytotoxicity was observed using the CCK-8 assay. * \(p\) < 0.05, ** \(p\) < 0.01, *** \(p\) < 0.001.  
respectively. IL-12 was also found to be significantly upregulated in the presence of different concentrations of SH (100-400 \(\upmu\)g/mL) and SF (50, 200 and 400 \(\upmu\)g/mL), but not in the presence of UP (Figure 2a-d). The inflammatory response could be beneficial for the host only when the cytokines are generated in optimum amounts. The deregulation of these cytokines can also cause serious toxicity. IL-10 is another cytokine produced by macrophages that regulates inflammation [48]. Its main function is to suppress excessive macrophage activation by suppressing various other cytokines, namely IL-1\(\upmu\)g, IL-12, TNF-\(\upalpha\), IL-8, and IL-6 [6]. In our study, we found that SH significantly induced the expression levels of IL-10 at different concentrations (100-400 \(\upmu\)g/mL), but similar effects were not observed with UP and SF (Figure 3a-c). Figure 2: Seaweed extracts upregulate transcriptional levels of pro-inflammatory markers in RAW 264.7 cells. RAW 264.7 cells were exposed to 50, 100, 200, and 400 \(\upmu\)g/mL of SH, UP, and SF for 24 h. The transcription expression levels of **(a)** IL-1\(\upmu\)g, **(b)** TNF-\(\upalpha\), **(c)** IL-6, and **(d)** IL-12 were measured by RT-PCR. * \(p<0.05\), **\(p<0.01\), *** \(p<0.001\). ELISA was performed to observe the protein expression of the two major pro-inflammatory cytokines, TNF-\(\alpha\) and IL-6. It was found that, compared to the control, SH was able to induce TNF-\(\alpha\) cytokine production significantly at 100, 200, and 400 \(\upmu\)g/mL. However, UP showed similar significant effects even at lower concentrations of 50 \(\upmu\)g/mL with respect to the control. SF significantly upregulated TNF-\(\alpha\) cytokine production at 200 and 400 \(\upmu\)g/mL compared to the control. It was found that all three algal extracts induced significant IL-6 production at 200 and 400 \(\upmu\)g/mL (Figure 4a,b). Figure 4: Seaweed extracts upregulate protein expression levels of pro-inflammatory markers in RAW 264.7 cells.