# Anti-Inflammatory Effects of Novel _Glycyrrhiza_ Variety Wongam In Vivo and In Vitro
## 3 Results
### WG Inhibits the Expression Levels of ProInflammatory Cytokines from HMC-1 and RBL-2H3 Cells  
To determine the inhibitory effects of WG on proinflammatory cytokine production, we investigated its effects on TNF-\(\alpha\) and IL-6 production and mRNA levels. PMACI significantly induced higher levels of cytokine production (Figure 4A,B) and mRNA expression (Figure 4C,D). However, these levels were decreased by pretreatment with WG in PMACI-stimulated HMC-1 cells. We obtained similar results from RBL-2H3 cells, in which DNP-HAS increased the mRNA levels of TNF-\(\alpha\), IL-6, and IL-1\(\beta\), whereas WG downregulated the levels of cytokines; however, the change in IL-1\(\beta\) expression was not significant (Figure 4E). These results indicated that WG regulated the stimulator-induced expression of proinflammatory cytokines via transcriptional inhibition. WG Downregulates the Expression Levels of Chemokines, Cell Surface Antigens, and Th2 Cytokines in PMACI-Stimulated HMC-1 Cells  
Chemokines are a group of chemotactic cytokines that play a critical role in directing inflammatory cell recruitment during allergic reactions [21]. Moreover, the allergic inflammatory response is characterized by Th2 effector cell proliferation and recruitment, while Th2-related cytokines and chemokines can be found in the serum of patients suffering allergic disorders [22]; thus, we investigated the effects of WG on chemokines and Th2 cytokines. The expression levels of CC chemokines such asotaxin, MIP-2, and MCP-1, as well as Th2 cytokines IL-4, IL-5, and IL-13, were increased by PMACI stimulation; however, these were significantly decreased by WG treatment in HMC-1 cells (Figure 5A,B). In addition to proinflammatory cytokines, several cell surface antigens, such as CD63 and CD203c, are highly relevant to an IgE-mediated allergic reaction correlating with histamine [23],  
Figure 3: Effects of WG on the allergic mediators in PMACI-stimulated HMC-1 and RBL-2H3 cells. (**A**) HMC-1 and RBL-2H3 cells were treated with different concentrations of WG and the cell viability levels were determined using MTS/MTT assay. Note: * \(p\) < 0.05 and ** \(p\) < 0.01 vs. the non-treated group. (**B**) The mRNA level of \(\beta\)-tryptase was determined by quantitative qRT-PCR. (**C,D**) Histamine release was measured using ELISA kit. The data shown represent means \(\pm\) S.D. of three independent experiments. Note: *** \(p\) < 0.001 vs. the control group; ** \(p\) < 0.01 and *** \(p\) < 0.001 vs. stimulator-treated group. Our results showed that pretreatment with WG downregulated PMACI-induced CD63 and CD203c expression in HMC-1 cells (Figure 5C). WG Inhibits the Activation of MAPKs and NF-\(\kappa\)B Signaling Pathway in PMACI-Stimulated HMC-1 Cells  
To investigate whether WG prevents the activation of the MAPK pathway, we measured the phosphorylation levels of ERK and JNK. We found that cells pretreated with WG had significantly suppressed phosphorylation of ERK and JNK compared with those treated with PMACI alone (Figure 6A). As nuclear factor kappa B (NF-\(\kappa\)B) is involved in cytokines, chemokines, enzymes, and key transcription factors in inflammatory pathways, we examined the effects of WG on PMACI-stimulated degradation of IxB-\(\alpha\) and phosphorylation of IKK. As shown in Figure 6B, PMACI induced the phosphorylation of IKK and degradation of IxB\(\alpha\); however, WG pretreatment suppressed the PMACI-induced activation of IKK and IxB\(\alpha\) in HMC-1 cells. These results indicated that the inhibition of MAPK and NF-\(\kappa\)B signaling pathways is involved in WG's mechanisms in PMACI-induced allergic inflammatory responses in HMC-1 cells. Figure 4: Effects of WG on the proinflammatory cytokines in PMACI-stimulated HMC-1 and RBL-2H3 cells. (**A**,**B**) TNF-\(\alpha\) and IL-6 cytokines production was measured using ELISA kit. The mRNA levels of (**C**) TNF-\(\alpha\) and (**D**) IL-6 were determined by quantitative qRT-PCR from HMC-1 cells and (**E**) RBL-2H3 cells. The data shown represent means \(\pm\) S.D.