*3.5. Animals*

Male C57BL/6 SPF mice (6 weeks, 18 ± 2 g) were purchased from Jinan Pengyue Experimental Animal Breeding Co., Ltd. (Shandong, China, License ID: SCXK2014-0007). The mice were adapted to a specific pathogen-free condition (12 h light/dark cycle, 22 ± 2 ◦C) for 7 days before the experiments, with free access to drinking water and a commercial diet. All procedures for this experiment were approved by the Animal Ethics Committee of Ocean University of China (certificate no. SYXK20120014).

#### *3.6. The DSS-Induced Colitis Model*

The colitis was induced by orally administering 2% ( *w*/*v*) DSS drinking water for 5 days. The mice were randomly divided into five groups (*n* = 10)—the normal group (N group, drinking water), model group (M group, 2% DSS water), positive control group (PC group, 2% DSS water + 50 mg/kg 5-ASA), low dose LMW-ulvan group (LP group, 2% DSS water + 50 mg/kg LMW-ulvan), and high dose LMW-ulvan group (HP group, 2% DSS water + 100 mg/kg LMW-ulvan). The mice's body weight, stool condition, and fecal bleeding were recorded daily. On the 13th day, the mice were sacrificed after fasting for eight hours.

#### *3.7. Assessment of Severity of Colitis*

The length of the colon was measured from the ileocecal junction to the anal verge [27]. The disease activity index (DAI) was calculated by average scores for changes in body weight loss, stool condition, and fecal bleeding, according to the DAI scoring system [44]. Briefly, loss in body weight was scored as follows: (i) weight loss: 0, no weight loss; 1, 1–5% loss; 2, 5–10% loss; 3, 10–15% loss; 4, more than 15% loss, (ii) stool consistency: 0, normal; 2, loose stools; 4, diarrhea, and (iii) fecal bleeding: 0, no blood; 2, positive hemoccult; 4, severe bleeding. The spleen and thymus were immediately weighted to calculate the spleen and thymus indices. Thymus or spleen index = thymus or spleen weight mg/body weight g.

#### *3.8. Cytokines and Activities of Antioxidant Enzyme Assay*

Blood was taken from the ocular orbit and centrifuged at 4000 rpm for 40 min at 4 ◦C to obtain serum. To the colon was added PBS to make 10% tissue homogenate. Then, the colonic homogenate was centrifuged at 4000 rpm for 15 min and the supernatant was taken for biochemical determination. The expression levels of the inflammatory cytokines, including IL-1β, IL-4, and IFN-γ, both in the serum and colon tissue, were determined by ELISA kits, according to the manufacture's protocol. The level of MDA and activity of CAT and GPx in the colon were measured according to the manufacturer's instructions.

#### *3.9. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) Analysis*

Colon tissue was ground with liquid nitrogen and 10 μL β-mercaptoethanol and 500 μL Bu ffer GTC was added to it. Total RNA was then extracted according to the manufacturer's instructions. The extracted RNA was reverse-transcribed into cDNA (5X All-InOneMasterMix; abm, Vancouver, Canada) and qRT-PCR amplification was performed using the SYBR Green (TOYOBO, Osaka, Japan) reagen<sup>t</sup> to examine the mRNA relative expressions of ZO-1, occludin, and claudin [40]. The mRNA expression from each sample was calculated by normalizing with β-actin as an endogenous control. The primer sequences are listed in Table 2.


**Table 2.** Primer sequences for qRT-PCR.

#### *3.10. Western Blot (WB) Assay*

According to the method described by Tian et al. (2020) [45], the WB procedure was as follows. Colon tissue was ground in the presence of liquid nitrogen and then lysed in RIPA lysis bu ffer for 30 min on ice. The lysates were centrifuged under the condition of 12,000 rpm at 4 ◦C for 10 min. The protein concentrations of the supernatants were detected by using a BCA protein assay kit. Then, the same amount of protein was separated by 12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes (Hybond, Sunnyvale, CA, USA) using a semidry transfer system (Bio-Rad, Hercules, CA, USA).The membranes were incubated with specific antibodies against β-actin, ZO-1,occluding, and claudin-1 overnight at 4 ◦C, and then HRP-conjugated secondary antibodies were incubated for 1 h at room temperature. All of the antibodies were diluted in tris bu ffered saline (TBS). The protein signals were analyzed using an ECL detection system (Tanon, Nanjing, China).
