## 3 Results and Discussion
### Effect of the Sericin and MSM Blends Treatment on LPS-Stimulated Macrophages
#### 3.2.3 COX-2/iNOS Expression in Raw264.7 Cells Stimulated with LPS  
COX-2/iNOS expression at the protein level, in Raw264.7 cells stimulated with LPS, was analyzed. COX-2 expression was higher in all four blends than in the control. However, higher MSM concentrations (5.0 and 10.0 mg/mL) resulted in lower COX-2 expression at the protein level (Figure 4A, \(p<0.05\)). NOS expression was significantly higher in LPS treatment than in control. However, 0.1S + 0.1M, 0.1S + 0.5M and 0.1S + 1.0M treatments showed significantly lower than LPS-only treatment. (Figure 4B, \(p<0.05\)).  
Inflammation is a biological defense mechanism that occurs during wound infection, and it neutralizes wound tissue through an inflammatory response and regenerates damaged tissue [24; 25]. It is promoted by oxidative stress generated in the body and the upregulation of certain genes in cells. There are several pathways of inflammation, of which NF&B/COX-2/iNOS pathway is a well-recognized mechanism. In particular, nuclear factor kappa B (NF&B) regulates the inflammatory stage through the regulation of cytokines (IL-1, TNF-\(\alpha\)) and several other factors, including inducible nitric oxide synthase (iNOS) [11; 26; 27].  
LPS is an inflammatory mediator that activates NF&B, an intracellular transcription factor in macrophages, and the induced NF&B moves to the nucleus, resulting in the induced expression of inflammatory cytokines, iNOS, and COX-2, which further induce gene expression [28]. NO is an indicator of the inflammatory response and is synthesized by iNOS, while COX-2 generates various mediators of the inflammatory response when inflammation is induced by harmful stimuli, infection, or trauma [28; 29]. MSM inhibits nitric oxide prostaglandin E2 production by inhibiting iNOS and COX-2 expression, and is involved in the immune response by inhibiting IL-6 and TNF-\(\alpha\) production through transcription factor NF&B [19]. [PERSON] et al. [8] reported that COX-2/iNOS expression decreased as the concentration of silk sericin increased, while COX-2/iNOS expression decreased at sericin concentrations of 2.5-10 mg/mL [19].  
Figure 3: NO assay based on the ratio of the sericin and MSM blends using Raw 264.7 macrophage. Cell treated with 1.0 (0.1S + 0.1M), 5.0 (0.1S + 0.5M), and 10.0 (0.1S + 1.0M) mg/mL MSM at 1.0 mg/mL sericin, stimulated with 1 mg/mL LPS. All values are presented as the mean \(\pm\) standard deviation (n = 3). * \(p<0.05\). S \(p<0.05\) vs. 0.1S + 1.0M.  
In this study, the effect of the sericin and MSM blends on the expression of COX-2/iNOS was investigated by stimulating Raw264.7 macrophages with LPS and then treating the cells with the sericin and MSM blends. Although differences in expression were observed based on the concentration of MSM, NO production and COX-2/iNOS expression were significantly decreased in the group treated with MSM at 5.0-10.0 mg/mL (Figures 3 and 4). Sericin was confirmed to prevent the decrease in cell viability caused by an increase in the concentration of MSM (Figure 1), and it also suppressed the expression of COX-2/iNOS, which likely affected the anti-inflammatory activity of macrophages. These findings indicate that the synergistic effect increased because of the inclusion of sericin in the blends.