2.2.4. Cytokine Analysis

In Figure 4A, the concentration of TNF-α of cells treated with LPS after 24 h showed a statistically significant increase compared to that of the control group or the cell culture medium (DMEM). Placebo PC-liposomes were close to that of cells treated with LPS. However, the TNF-α concentration in the group of cells pretreated with PS-EGCG-liposomes or PS-EGCG-VE-liposomes followed by the LPS activation showed a statistically significant decrease compared to that of cells treated with LPS. This decreasing concentration of TNF-α indicates that EGCG-loaded liposomes can reduce the activation of microglial cells induced by LPS. The inhibitory effect of PS-EGCG-VE-liposomes was better than that of PS-EGCG-liposomes.

The phospholipids on the cell membrane can be hydrolyzed by cytosolic phospholipase A2 (cPLA2) to produce arachidonic acid. The cyclo-oxygenase (COX) is a key enzyme that converts arachidonic acid to prostaglandin. COX-2 and cPLA2 are often generated from inflammation or malignant disease [31–34]. In Figure 4B, the inflammation was induced by LPS of 5–50 ng/mL for 24 h, and the expression of cPLA2 increased when the

LPS concentration increased. Figure 4C showed the increased activities of COX-2, induced by LPS (5–50 ng/mL) for 24 h. The expression of COX-2 increased from 5–25 ng/mL LPS, while it decreased at 50 ng/mL. In Figure 4D, the cPLA2 expression was reduced when BV-2 cells were pretreated with EGCG and induced by LPS. The expression of COX-2 when LPS activated BV-2 cells was increased compared to the control group (Figure 4E). There was a significant decrease of COX-2 in the group of cells pretreated with EGCG, placebo PS-liposomes, PS-EGCG-liposomes, and PS-EGCG-VE-liposomes, followed by LPS inflammatory induction. Especially, expression of COX-2 of cells pretreated with placebo PS-liposomes had a statistically significant difference from that of cells induced by LPS.

**Figure 4.** In LPS-induced BV-2 cells, expression of (**A**) TNF-α (compared with the liposome (−) and LPS (+) group, \* *p* < 0.05; \*\* *p* < 0.01, *n* = 3), (**B**) cytosolic phospholipase A2 (cPLA2), and (**C**) cyclo-oxygenase-2 (COX-2). In BV-2 cells pretreated by EGCG or liposomes and then induced by LPS, expression of (**D**) cPLA2 and (**E**) COX-2 (compared with LPS (+) group \*\* *p* < 0.01, \*\*\* *p* < 0.001, *n* = 3).
