*2.1. Ech A Treatment Exerted a Protective E*ff*ect against DSS-Induced Colitis In Vivo*

To evaluate the therapeutic effects of Ech A on IBD, we gave a single intravenous (i.v) injection of Ech A or vehicle to DSS-induced colitis mice at day 1 and monitored the survival rate, body weight and disease activity index for 12 days. We found that a high dose (10 mg/kg; E10) of Ech A could significantly reduce body weight loss and increase the survival rate of colitis affected mice when compared with vehicle (+) and a low dose of Ech A (1 mg/kg; E1) treated groups (Figure 1A). According to the disease activity index score, clinical symptoms were also improved by Ech A treatment in a dose-dependent manner (Figure 1B). In the gross examination of the large intestine, pathologic shortening of the colon length due to colitis was reversed by the administration of Ech A (Figure 1C). H&E staining-based histopathological analysis revealed that typical pathologic signs of the colitis-affected damaged colon such as loss of epithelial structure and irregular morphology of crypts were ameliorated upon the administration of 10 mg/kg Ech A (Figure 1D). In addition, we found that excessive accumulation of immune cells within the epithelial and mesenchymal layer of the damaged colon was prevented upon Ech A treatment, suggesting that Ech A could exhibit anti-inflammatory roles in the in vivo mouse model (Figure 1D). Therefore, we concluded that Ech A could alleviate disease symptoms and play protective roles in DSS-induced colitis mice.

**Figure 1.** Echinochrome A (Ech A) administration provided therapeutic effects on DSS-induced colitis mice in a dose-dependent manner. (**A**) Body weight and survival rate monitoring results are shown. The body weight at day 0 considered was as 100%. Numbers in parentheses represent the percentage of dead mice. (**B**) Disease activity index score for colitis severity at day 12 was significantly reduced in the E10 group. (**C**) Colon length measurement results showing the protective role of Ech A against DSS-induced colonic damage. (**D**) The histopathological score of the colitis-affected and normal colon was evaluated with H&E stained tissue section. In total, six animals per group were used. Results are shown as mean ± SD. In (**B**) and (**C**), p-value significance was calculated by comparing other groups against the (+) group (marked as +). \* *P* < 0.05, \*\* *P* < 0.01, \*\*\* *P* < 0.001.

#### *2.2. Ech A Suppressed the Proliferation of Human MNCs and T Lymphocytes In Vitro*

Based on the fact that excessive activation of the intestinal mucosal immune system triggered by increased epithelial permeability leads to aggravation of IBD progression [15,16], we next investigated whether Ech A could regulate the proliferation, differentiation and activation of various immune cells in vitro to explore the underlying therapeutic mechanisms of Ech A on the colitis model. First, we performed a mixed lymphocyte reaction (MLR) to evaluate the effect of Ech A on the expansion of human mononuclear cells (MNCs) and T lymphocytes. To stimulate in vitro proliferation, MNCs were treated with a non-specific mitogen concanavalin A (Con A) and cell proliferation capacity was evaluated using flow cytometry at day 5. Interestingly, the proliferation of Con A-treated MNCs was inhibited upon Ech A administration in a dose-dependent manner (Figure 2A,B). The percentage of proliferating cells in the control group was 60.4%, while it decreased to 49.3% and 37% upon 5 μM and 10 μM of Ech A treatment, respectively. Similarly, Ech A reduced the proliferation rate of CD3/CD28-stimulated T cells when compared with vehicle-treated samples (VC group: 72.2%; 5 μM Ech A group: 63.6%; 10 μM Ech A group: 54.9%) (Figure 2C,D), implying that Ech A could regulate immune cell proliferation in vitro.
