*3.4. 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-α (Figure 8a), IL-6 (Figure 8b), and Il-1β (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-α, 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-α 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-α production. Therefore, it can be inferred that *Spirulina* extracts inhibit TNF-α 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-α. Unlike the previous examples, IL-1β shows higher correlations between gene expression and inhibition of IL-6 production, but it has lower correlations than TNF-α. Therefore, it can be assumed that the extracts first control the amplification of TNF-α and IL-1β 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-α and IL-1β 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-α production from macrophages [55,56]. Therefore, since the ultrasonic extract inhibits TNF-α secretion most effectively, the anti-inflammatory effects of these extracts are very high. In particular, the effects of

the *Spirulina* extracts, containing chlorophyll on the inflammatory mediator and inflammatory cytokine production inhibition, were shown. This strongly implies that in addition to the antioxidant effects of chlorophyll and *Spirulina* extracts that are already known, the extracts also have an ability to protect against nerve cell inflammation. Therefore, the neuroprotective effects of *Spirulina* extracts were caused by inhibiting the inflammation of mouse nerve cells via antioxidant properties, potentially improving cognitive activities. The results would suggest another possible mechanism of the *Spirulina* extracts on its neuroprotection process.

**Figure 8.** Quantitative comparison of the secretion and mRNA expression of TNF-a (**a**), IL-6 (**b**) and IL-1beta (**c**). EE, 70% ethanol extraction at 80 ◦C for 12 h, UE, ultrasonic pretreatment with 70% ethanol at 40 kHz and room temperature for 8 h, and further extraction at 65 ◦C for 4 h. Values are presented as means ±SD; \* *p* < 0.05 and \*\* *p* < 0.01 compared with the non-treatment group.

## **4. Conclusions**

This work first showed that the *Spirulina* extract from the UE enhanced anti-inflammation activities in mouse microglial cells associated with its antioxidant effects, compared with that from the conventional 70% ethanol extract at high temperature. The non-thermal ultrasonic extract contained more than two-fold higher amounts of extremely heat-sensitive chlorophylls than the conventional extract and exhibited a two-fold higher extraction yield. Specifically, the extract effectively inhibited the production of inflammation mediators, such as NO and PGE2, while the conventional extract also showed a relatively high ability to inhibit their production. Strong anti-inflammatory effects of the extract from the UE were also demonstrated by the significant down-regulation of mRNA expression of the pro-inflammatory cytokines, TNF-α, IL-6 and IL-1β. Moreover, we quantitatively demonstrated that inhibition of the gene expression was closely related to the suppression of pro-inflammatory cytokine secretion even though ELISA analysis supports PCR results in general; in particular, TNF-α had a very strong relationship while IL-6 demonstrated the least correlation between the down-regulation of gene expression and the suppression of cytokine secretion. Notably, the effect on TNF-α expression was greater than that on IL-1β and IL-6, which changed the least. This result suggests that the extract first controls TNF-α secretion and later inhibits IL-6, but this hypothesis should be further validated with a more detailed mechanism of the anti-inflammation cascade within nerve cells. However, we clearly showed that the extract from the ultrasonic process was more effective at suppressing the secretion of both inflammatory mediators and pro-inflammatory cytokines, as reflected by its neuroprotective activities. Conclusively, the high anti-inflammatory effects of the extracts were closely correlated with the high amounts of chlorophylls, which have strong antioxidant activities, obtained only through a non-thermal ultrasonic process. The high anti-inflammatory effects of the *Spirulina* extract with mouse microglia cell line could be employed to develop a possible mechanism for in vivo anti-inflammation effects along with the results from primary nerve cells. However, the expression of anti-inflammatory cytokines, such as IL-10 and TGF-β, should also be studied to further elucidate more exact anti-inflammation effects of this extract. This study also provides useful information for developing functional foods from heat-labile natural resources.

**Author Contributions:** W.Y.C., J.-H.S., J.-Y.L. and D.H.K. carried out all of the experiments and H.Y.L. designed the whole experiments and drafted the manuscript. All authors read and approved the final manuscript.

**Funding:** This work has been supported by a research grant from the Marine Biotechnology Program funded by the Ministry of Oceans and Fisheries (PM61400), and all of the authors deeply appreciate their financial support.

**Conflicts of Interest:** The authors declare no conflict of interest.
