## 3 Results
### SGP Increased Immune-Enhancing Activity through NO and iNOS Level in RAW264.7 Macrophages  
Polysaccharides isolated from ginseng were reported to exert immune-enhancing effects [31; 32]. iNOS is an important factor to produce NO and activate macrophages. In this study, we investigated NO and iNOS levels in SGP-treated RAW264.7 macrophages. RAW264.7 macrophages were stimulated with 500 or 1000 \(\upmu\)g/mL SGP for 22 h (for NO) or 18 h (for iNOS). The results showed that treatment with 500 and 1000 \(\upmu\)g/mL SGP slightly increased NO (Figure 4A) and iNOS levels. As shown in the quantification graph of iNOS intensity, SGP increased iNOS level in RAW264.7 macrophages (Figure 4B). SGP Increased mRNA Expression and Cytokine Secretion of IL-6 and TNF-\(\alpha\) in RAW264.7 Macrophages  
To evaluate the effect of SGP on the secretion of IL-6 and TNF-\(\alpha\) in RAW264.7 macrophages, ELISA and RT-qPCR analysis were performed. The results indicated that secretion of IL-6 and TNF-\(\alpha\) levels increased in RAW264.7 cells after treatment with SGP at 500 and 1000 \(\upmu\)g/mL (Figure 5A,B). In addition, we investigated IL-6 and TNF-\(\alpha\) mRNA levels in SGP in RAW264.7 cells, both of which increased at SGP concentrations of 500 and 1000 \(\upmu\)g/mL in RAW264.7 macrophages (Figure 5C,D). Figure 3: Effect of SGP on the proliferation of RAW264.7 macrophages. RAW264.7 cells were stimulated with indicated concentration of SGP for 22 h, and cell proliferation was evaluated an EZ-Cytox reagent. SGP Increased Immune-Enhancing Activity through Phosphorylation of MAPKs and NF-\(\kappa\)B Signaling Pathways in RAW264.7 Macrophages  
To investigate whether SGP has an effect on the MAPK phosphorylation in RAW264.7 macrophages, the phosphorylation levels of ERK, JNK, and p38 were evaluated by immunoblotting. JNK, ERK, and p38 are members of the MAPK, and when MAPKs are activated, they regulate cell functions including cell growth, gene expression, and differentiation [33]. In addition, they can activate macrophages by phosphorylating transcription factors. Our results confirmed JNK, ERK, and p38 MAPK signaling pathway phosphorylation in macrophages activated by SGP. We also found that SGP activated the NF-\(\kappa\)B signaling pathway. NF-\(\kappa\)B is an essential expression factor which regulates genes involved in innate and adaptive immune responses [34]. RAW264.7 cells were inserted into SGP at each concentration (500 and 1000 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) for 30 min. Immunohistochemistry bands were quantified and expressed as the ratios of p-ERK/ERK, p-JNK/JNK, p-p38/p38, and p-p65/p65 intensity. Phosphorylation of p38, ERK, JNK, and p65 was significantly increased by SGP treatment (500 and 1000 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) in RAW264.7 macrophages (Figure 6A). The quantification graphs of p-ERK, p-JNK, p-p38, and p-p65/p65 intensity show an SGP for each concentration (500 and 1000 \(\mathrm{\SIUnitSymbolMicro g}\)/mL) in RAW264.7 macrophages (Figure 6B-E). Figure 4: Effects of SGP on NO and iNOS levels in RAW264.7 macrophages. RAW264.7 macrophages were inserted into 500 and 1000 \(\mathrm{\SIUnitSymbolMicro g}\)/mL SGP for 22 h (for NO) or 18 h (for iNOS). After 22 h, cell supernatants were used for NO production analysis. Supernatants were used according to the Griess method (**A**). After 18 h, cells were collected to evaluate iNOS protein levels; expression levels were determined through immunoblotting and quantification of iNOS/\(\beta\)-actin intensity (**B**). Shown are the means \(\pm\) SD of triplicate experiments. * \(p\) < 0.05 and *** \(p\) < 0.0001, compared to the controls. Figure 5: Effect of SGP on cytokine production and mRNA levels of IL-6 and TNF-\(\alpha\) in RAW264.7 macrophages. RAW264.7 cells were stimulated with SGP at 500 and 1000 \(\mathrm{\SIUnitSymbolMicro g}\)/mL for 24 h. Production of IL-6 and TNF-\(\alpha\) was determined using an ELISA (**A,B**).