## 3 Results  
### SHE Inhibits NO Production and iNOS and COX-2 Expression in RAW 264.7 Cells by LPS Treatment  
Because inflammation is associated with an abnormal release of NO from hyperactivated macrophages [19], we investigated the efficacy of SHE on NO produced by LPS treatment in RAW 264.7 cells. First, SHE had no cytotoxic affect at the given concentration in RAW 264.7 cells (Figure 1A). SHE dose-dependently decreased NO production by LPS significantly (Figure 1B). Because NO production is regulated by iNOS [20], we investigated the effect of SHE on increasing iNOS expression in RAW 264.7 cells due to LPS treatment. It can be seen from Figure 1C that the increased iNOS due to LPS treatment decreased as the concentration of SHE increased. Figure 1: Effects of _Sorseris hirsuta_ extract (SHE) on lipopolysaccharide-inducible nitrite production, inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2) expressions, and cell viability in RAW 264.7 cells. (**A**) SHE does not incur toxicity to RAW 264.7 cells at the given concentration for 24 h. (**B**) SHE effectively inhibited the increased nitrite from LPS treatment in a concentration-dependent manner to RAW 264.7 cells. (**C**) LPS-induced iNOS expression was suppressed by SHE treatment, but COX-2 did not. Cells were treated with SHE 1 h before exposure to LPS for 24 h. \(\#\)\(p<0.05\) between the LPS-only group and the control group; \(\star\)\(p<0.05\) and \(\star\)\(p<0.001\) between the SHE and LPS co-treatment groups and the LPS-only group. Data are presented as the mean \(\pm\) SD from \(n=3\). SHE Suppressed the Phosphorylation of NF-kB Signaling Cascade and MAPK in LPS-Stimulated RAW 264.7 Cells  
NF-kB is a signaling molecule, regulating the transcription of inflammatory genes, including _COX-2_ and _iNOS_[21]. We detected the efficacy of SHE on LPS-induced NF-kB signaling pathways. As demonstrated in Figure 2A, SHE inhibited LPS-induced phosphorylation of IKK\(\alpha\)/\(\beta\) and p65 in RAW 264.7 cells. The hyper-activation of MAPK signaling pathways by LPS is also critical for inflammation [5; 22]. SHE defended phosphorylation of LPS-induced JNK1/2 and p38; thus, the p-ERK1/2 was not decreased by SHE (Figure 2B). In RAW 264.7 Cells, Cytosol to Nuclear Translocation of p65 Due to LPS Treatment Is Inhibited by SHE  
In order to regulate the expression of inflammatory genes, such as _iNOS_ and _COX-2_, the p65 subunit of NF-kB translocates from the cytosol to the nuclear and binds to specific promoter sequences [23; 24]. Here, we assessed the efficacy of SHE cytosol to nuclear translocation of p65 due to LPS treatment in RAW 264.7 cells. After separating the cytosolic and nuclear fractions, we performed Western blot and immunofluorescence assays and found that LPS-induced nucleus translocation of p65 was significantly suppressed by SHE (Figure 3A,B). Figure 2: Effects of _Soroseris hirsuta_ extract (SHE) on lipopolysaccharideaccharide (LPS)-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) and mitogen-activated protein kinase (MAPK) signaling pathways in RAW 264.7 cells. (**A**) SHE inhibited the LPS-induced phosphorylation of the inhibitor of nuclear factor xB kinase (IKK) and p65. SHE protected dose-dependent degradation of IxB by LPS treatment. Cells were treated with SHE 1 h before exposure to LPS (1 \(\mu\)g/mL) for 30 min. (**B**) SHE inhibited the LPS-induced phosphorylation of p38 and c-JUN N-terminal kinase (JNK) 1/2, but did not affect the phosphorylation of extracellular signal-regulated kinase 1/2 in RAW 264.7 cells. Cells were treated with SHE 1 h before exposure to LPS for 30 min. The phosphorylation and expression of each protein were detected by Western blotting using specific antibodies. SHE Has an Antioxidant Activity Characteristic by Regulating ROS Production, Radicals, and NRF2/HO-1 Signaling Pathway in RAW 264.7 Cells  
ROS, which are chemically unstable, cause damage and inflammation in mammalian cells [25; 26].