## 3 Results and Discussion
### Correlation Between Gene Down-Regulation and Secretion of Pro-Inflammatory Cytokines  
The above-mentioned results suggest that the extracts down-regulate the expression of the genes involved in pro-inflammatory cytokine production, thereby inhibiting the secretion of the relevant cytokines. Many other studies have reported similar results. However, there are no published data on the quantitative correlation between gene regulation and protein secretion, since it is considered to be natural that the amounts of the cytokines secreted from BV-2 cells measured by ELISA analysis only support the results of gene expression from PCR analysis. Therefore, Figure 8 shows the comparison of the down-regulation of the expression of TNF-\(\alpha\) (Figure 8a), IL-6 (Figure 8b), and IL-1\(\beta\) (Figure 8c) genes, which are the cytokines most closely involved in the inflammation of brain nerve cells and resultant secretion of cytokines according to two different extraction processes, such as ultrasonic and conventional extraction processes. As shown in Figure 8a, for TNF-\(\alpha\), gene expression and protein production decrease proportionally according to the concentrations of extracts administered with high correlations. In particular, the degree of downregulation of gene expression and TNF-\(\alpha\) production is constant at all concentrations of the extracts without any large difference among concentrations. This means that the extracts act on gene regulation the most directly leads to the control of TNF-\(\alpha\) production. Therefore, it can be inferred that _Spirulina_ extracts inhibit TNF-\(\alpha\) production directly from transcription to induce anti-inflammatory effects. In contrast, for IL-6 (Figure 8b), although gene expression and IL-6 production inhibition occurred, the decreasing rates were not significantly proportional to each other. In particular, the analysis indicated that the degree of IL-6 production inhibition was higher than the degree of gene expression inhibition indicating the possibility that inhibition of IL-6 production from BV-2 cells occurs through pathways than gene expression or compositely unlike the TNF-\(\alpha\). Unlike the previous examples, IL-1\(\beta\) shows higher correlations between gene expression and inhibition of IL-6 production, but it has lower correlations than TNF-\(\alpha\). Therefore, it can be assumed that the extracts first control the amplification of TNF-\(\alpha\) and IL-1\(\beta\) genes thereby directly inhibiting the production of cytokines. Additionally, the production of IL-6 is more effectively reduced because of the effects on the IL-6-producing gene and the reduction of TNF-\(\alpha\) and IL-1\(\beta\) production, which have already been inhibited. The basic data on these mutual relationships have been presented for the first time in this study, and more detailed studies on the mutual relationships between _Spirulina_ extracts and the three pro-inflammatory cytokines are needed to confirm the findings. However, the data shows for the first time that although _Spirulina_ extracts are involved in inhibiting the secretion of three cytokines, rather than acting simultaneously, there are differences in the degree of inhibition mechanisms, and order of action. Therefore, these results suggest that rather than uniformly influencing the secretion of all pro-inflammatory cytokines, the natural extracts selectively affect the production. Future studies on the anti-inflammatory effects of the extracts are necessary to investigate more subdivided inhibition of target cytokines. In particular, stimulation by LPS is most directly involved in inflammatory diseases, such as septic shock, rheumatoid arthritis, insulin resistance, and cachexia, by maximizing TNF-\(\alpha\) production from macrophages [55; 56]. Therefore, since the ultrasonic extract inhibits TNF-\(\alpha\) secretion most effectively, the anti-inflammatory effects of these extracts are very high.