## 3 Results
### Effects of HR2302-30E and C3G on the FccRI Signaling Pathway  
When allergens bind to IgE bound to FccRI, FccRI aggregates, activating the mast cells. Aggregation of FccRI\(\beta\) and FccRI\(\gamma\) induces the activation of Src family tyrosine kinases, Lyn and Fyn, which phosphorylate the tyrosine residues in ITAM, leading to Syk activation. This further causes PLC\(\gamma\) and PKC8 activation and intracellular calcium release, contributing to the activation of the MAPK pathway. PLC\(\gamma\) and PKC8 stimulate the secretion of \(\beta\)-hexosaminidase and histamine, and MAPK leads to the synthesis of inflammatory cytokines [21,62,63]. The MAPK family, including ERK, p38, and JNK, is activated by various stimuli that induce inflammatory responses. These MAPKs are closely associated with degranulation and the secretion of inflammatory cytokines in mast cells [64]. Notably, ERK is activated by IgE receptor stimulation and plays a critical role in mast cell differentiation and proliferation. p38 stimulates mast cell migration and the secretion of IL-4, while JNK plays a role in the release of inflammatory cytokines, including IL-6 and IFN-\(\gamma\)[65]. Therefore, we assessed the effects of HR2302-30E and C3G on the activation of the FccRI pathway using Western blotting. HR2302-30E and C3G significantly reduced the expression levels of the FccRI subunits FccRI\(\beta\) and FccRI\(\gamma\) (Figure 5a,b) and inhibited the phosphorylation of Syk, Fyn, and Src in a dose-dependent  
Figure 4: Effects of HR2302-30E and C3G on (**a**) \(\beta\)-hexosaminidase and (**b**) histamine release in RBL-2H3 mast cells. IgE-primed RBL-2H3 cells were incubated with 250 ng/mL of DNP-HSA and HR2302-30E (500, 1000, and 2000 \(\upmu\)g/mL) and C3G (5 and 10 \(\upmu\)g/mL). Negative control: non-IgE-primed RBL-2H3 cells without DNP-HSA; positive control: IgE-primed RBL-2H3 cells with DNP-HSA and PP2. Data are represented as the mean \(\pm\) SEM of the three independent experiments. The statistical analyses were performed using the Dunnett \(t\)-test. PP2 is a general Src family kinase inhibitor. \({}^{*}\)\(p<0.05\), \({}^{**}\)\(p<0.01\) and \({}^{***}\)\(p<0.001\) vs. anti-DNP IgE plus DNP-HSA (IgE/Ag); \({}^{***}\)\(p<0.001\) vs. control. –, No treatment, +, Treatment. manner (Figure 5c,d). Furthermore, HR2302-30E and C3G decreased the phosphorylation of key components of the MAPK pathway, including p38, ERK, and JNK (Figure 6a,b), and significantly reduced the phosphorylation of PLCy and PKC6, which are involved in mast cell degranulation (Figure 6c,d). These results suggest that HR2302-30E and C3G contribute to the inhibition of mast cell degranulation by suppressing Fc\(c\)RI receptor activation and blocking the initial signaling pathways involving Syk, Fyn, and Src. Additionally, HR2302-30E and C3G may exhibit anti-inflammatory effects by inhibiting the secretion of cytokines that trigger inflammatory responses and the release of \(\beta\)-hexosaminidase and histamine during the mast cell degranulation process. The anti-allergic properties of HR2302-30E and C3G were confirmed through their inhibition of the Fc\(c\)RI signaling pathway and the mechanisms underlying the production of inflammatory cytokines and histamine (Figure 7). Figure 5: _Cont._  
Figure 5: Effects of HR2302-30E and C3G on the high-affinity IgE receptor (Fc\(\varepsilon\)RI) signaling cascade in RBL-2H3 cells. IgE-primed RBL-2H3 cells were incubated with 250 ng/mL of DNP-HSA and HR2302-30E (1000 and 2000 ug/mL) and C3G (5 and 10 ug/mL). The protein expression of (**a,b**) Fc\(\varepsilon\)RI \(\beta\) and Fc\(\varepsilon\)RI \(\gamma\), (**c,d**) Syk, Fyn, and Src were determined using immunoblotting. Data are represented as the mean \(\pm\) SEM of the three independent experiments. The statistical analyses were performed using the Dunnett \(t\)-test. PP2 is a general Src family kinase inhibitor. * \(p\) < 0.05, ** \(p\) < 0.01, and *** \(p\) < 0.001 vs. anti-DNP IgE plus DNP-HSA (IgE/Ag); *** \(p\) < 0.001 vs. control. \(-\), No treatment, +, Treatment.