## 3 Results
### Elisa and mRNA Analysis of Inflammatory Cytokines  
Besides the tissue damage in the histopathological image, the colitis is generally accompanied by the abnormal secretion of proinflammatory cytokines as well as immune chemokines [24] As showed in Figure 5, the stimulation of DSS significantly increased the levels of TNF-\(\alpha\), IL-6, IL-1\(\beta\), MCP-1 and LPS than NC group (\(p\)\(<\) 0.05), whereas the supplementation of GP notably inhibited the DSS-induced production of TNF-\(\alpha\), IL-6, IL-1\(\beta\), MCP-1, and LPS as compared with PC. For instance, the TNF-\(\alpha\) content in serum increased from 411 pg/mL (NC) to 1021 pg/mL (PC), whereas the pretreatment with 300 mg/kg/day (GP-2) significantly inhibited the TNF-\(\alpha\) content to be 403 pg/mL. Simultaneously, the content of IL-6 and MCP-1 in serum was also suppressed in GP-2 group, and the dose of 100 mg/kg/day in GP-1 showed no significant differences compared with PC (\(p\)\(>\) 0.05). According to the inflammatory cytokines in serum, the gavage of GP had a dose manner with the effective dosage of 300 mg/kg/day. Compared with NC, the stimulation of DSS also increased the mRNA expression of inflammatory cytokines, such as TNF-\(\alpha\), COX-2, and MCP-1, as shown in Figure 6. In GP groups, the aberrant secretion of biomarkers was significantly alleviated as compared with PC (\(p\)\(<\) 0.05). For instance, the expression of TNF-\(\alpha\) in GP-2 group was decreased than PC, meanwhile, the COX-2 and Toll-like receptor 4 (TLR-4) was also decreased respectively (\(p\)\(<\) 0.05). According to the protein expressions in Figure 7, the COX-2 was also demonstrated to be increased in PC and the supplement of GP showed to have a decreasing effect without significant differences. Indeed, the TLR-4 showed a same tendency as revealed in RT-PCR, where the PC groups expressed higher content than NC, and the GP treatment exhibited a suppressing effect on TLR-4. During the formation of IBD, TNF-\(\alpha\)plays a remarkable role in the pathogenesis, which could recruit more inflammatory factors to infiltrate the intestinal tissue and result in intestinal tissue lesions and inflammatory responses [25]. Thus, the anti-TNF-\(\alpha\) component has also been reported as a possible therapy to reduce the pathology in IBD patients. Currently, the GP-2 was measured to suppress the TNF-\(\alpha\) secretion with the intake of 300 mg/kg, implying a significant role in the IBD. Figure 5: GP reduced inflammatory cytokines secretion in serum of DSS-colitis mice. The levels of TNF-\(\alpha\) (**A**), IL-6 (**B**), IL-1\(\beta\) (**C**), MCP-1 (**D**), LPS (**E**) in serum were determined by ELISA. All values represent the means \(\pm\) SD (\(n\) = 8). According to the one-way ANOVA (Tukey’s Multiple Comparison Test), the different letters mean significant differences of \(p\)\(<\) 0.05. Simultaneously, the DSS stimulation also inhibited the mRNA expression of tight junction proteins occludin and ZO-1 (\(p<0.05\)), which is consistent in the histological image along with the DSS-induced intestinal mucosal damage. In the GP group, the ZO-1 and occludin mRNA expression was recovered than PC, implying the protective effect of GP on the mucosal tissue. As revealed in Figure 7C, the expression of occludin was also demonstrated to be improved compared with PC group (\(p<0.05\)). Tight junction proteins played a major component for the intestinal tract barrier with the function of controlling the paracellular spaces as well as mucosal tightness. The intercellular bridge is established by multiple proteins such as occludin, junctional adhesion molecules, claudins, ZO-1, and others. In special, the ZO-1 is the main linkage between occludin and other apical prejunctional proteins [26]. The peptides from mucosal protein were investigated to induce the extent of the mucosal barrier as well as pathological features in DSS-induced colitis mice, where the ZO-1 and occludin proteins were enhanced compared with that of DSS group [27].