Ulcerative colitis (UC), a significant form of inflammatory bowel disease (IBD), is a chronic inflammatory disorder of the colonic mucosa. It usually starts in the rectum and spreads to the colon proximally, in a continuous manner [1
]. The global prevalence of IBD is increasing every year—more than 0.3% of the population suffers from the disease [2
]. The pathogenesis of IBD is unclear; however, many researches claim that it is a result of genetic defects, unhealthy lifestyles, imbalance of intestinal microbiota, or immune dysbiosis [3
]. Typical features of UC include structural and functional impairment of the intestinal mucosa, accompanied by diarrhea, rectal bleeding, and other symptoms [6
]. Currently, surgery and drugs (including corticosteroids, aminosalicylic acid, and immunosuppressive agents) are the main strategies from a clinical aspect [7
]. However, in addition to the high costs and incomplete treatment issues, these drug interventions have certain side-effects and have been seen to lose efficacy in long-term use patients [8
]. Consequently, more efforts should be made to enrich treatment and disease prevention methods.
Numerous reports have testified to inflammatory reactions being a crucial factor causing UC [9
]. The cytokines secreted by intestinal epithelial cells are important markers of the intestinal immune system and regulate their inflammatory responses [10
]. Pro-inflammatory cytokines such as tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and interleukin (IL)-1β can promote an inflammatory response, eventually causing colon tissue injury, while anti-inflammatory cytokines such as IL-4 and IL-10 can reduce inflammation [11
]. Excessive pro-inflammatory cytokines increase the production of reactive oxygen species (ROS), which ultimately cause intestinal epithelial cell injury and aggravate pathogenesis [12
]. Hence, materials with the ability to recover abnormal levels of cytokines and oxidative stress may be promising therapeutics for IBD.
Ulvan is a kind of sulphated polysaccharide located in the cell walls of Ulva
; it represents an abundant marine resource distributed worldwide. Ulvan extracted from different species of Ulva
consists primarily of Rha (16.8–45.0%), GlcA (6.5–19.0%), Xyl (2.1–12.0%), iduronic acid (IdoA) (0.7–9.1%), Glc (0.5–6.8%), and sulphate (14.3–23.2%) and its backbone is mostly composed of α-1,4- and α-1,2,4- linked L-rhamnose, β-1,4- and terminally linked D-glucuronic acid and β-1,4-linked D-xylose [13
]. Like other sulfated polysaccharides, ulvan exerts various therapeutic activities, such as antibacterial, immunostimulatory, antitumor, antioxidant, antihyperlipidemic, and anticoagulant properties [14
]. Qi et al. (2005) reported that high sulfate content ulvan showed a stronger scavenging activity of superoxide and hydroxyl radicals, reducing power, and metal chelating ability [16
]. In a research work by Li et al. (2018), the ulvan extracted from Ulva pertusa
showed significant protection against liver damage by oxidative stress induced by a cholesterol-rich diet [17
]. De Araújo et al. (2016) found that the enzymatic digestion of ulvan extracted from Ulva lactuca
had a vascular anti-inflammatory effect by decreasing TNF-α and IL-1 levels [18
]. Additionally, Berri et al. (2017) reported that ulvan extracted from Ulva armoricana
was able to initiate and amplify the protective immune responses of the host, and regulate mucosal immunity against intestinal pathogens [19
Reports on the antiinflammatory and antioxidant effects of ulvan are readily available, but few reports have been published on its protective effects in rats with DSS-induced colitis. In this study, LMW-ulvan produced by the enzymatic method was purified and characterized, and its potential to alleviate IBD symptoms in DSS-induced mice and protect the intestinal epithelial barrier were investigated.
3. Materials and Methods
3.1. Materials and Reagents
Ulva pertusa was collected from the coast near Weihai, China, in 2019. Ulvan lyase was provided by the Applied Microbiology Laboratory (Ocean University of China). DSS (36–50 kDa) and 5-aminosalicylic acid (5-ASA) were purchased from MP Biomedicals (Solon, CA, USA) and Sigma-Aldrich (St. Louis, MO, USA), respectively. ELISA detection kits for IL-1β, IL-4, IFN-γ, malonic dialdehyde (MDA), catalase (CAT), and glutathione peroxidase (GPx) were obtained from Dakewe Biotech Corporation (Beijing, China). Primary antibodies specific for β-actin, ZO-1, occluding, and claudin-1 were purchased from Protein Tech Group (Wuhan, China). The BCA Protein Assay Kit was purchased from Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). All other reagents were of analytical grade.
3.2. Enzymatic Preparation and Purification of LMW-Ulvan
The ulvan was extracted according to a method described by Qiao et al. (2020) [37
]. It was then degraded though an enzymatic method described by Chi et al. (2020) [20
] and crude enzymatic fractions with average molecular weight of 1–5 kDa were obtained by nanofiltration membrane system. The obtained fraction was purified using ÄKTAprime plus (GE Healthcare, Woburn, MA, USA) equipped with DEAE-Sepharose CL-6B column (1.6 × 10 cm). The fraction was pre-equilibrated with ultrapure water and then eluted with a linear gradient of 0–1 M NaCl at 1.0 mL/min. The purified fraction was then dialyzed, lyophilized, and nominated as LMW-ulvan.
3.3. Characterization of LMW-Ulvan
The total sugar content of LMW-ulvan was determined by phenol-sulfuric acid method [38
]. Sulfate content was determined after hydrolysis of trifluoroacetic acid [39
]. The molecular weights of LMW-ulvan were measured according to a method described by Ye et al. (2019) [40
]. The Fourier transform infrared (FI-IR) spectra of LMW-ulvan were recorded using the Magna-IR560 spectrometer (Nicolet Instrument Corp., Madison, WI, USA) according to research by Cui et al. (2019) [41
]. Its monosaccharide composition was measured by reversed-phase HPLC (Agilent Technologies, Santa Clara, CA, USA) after pre-column derivatization, according to a method described by Yu et al. (2017) [22
3.4. Methylation Analysis of LMW-Ulvan
Desulfation of the LMW-ulvan was achieved according to Falshaw and Furneaux (1998) [42
]. Briefly, the LMW-ulvan was converted to the pyridinium salt form by dialysis against pyridinium hydrochloride (0.1 M, adjusted to pH 6.8), then against distilled water, and finally lyophilised. The resulting materials was dissolved in 89:10:1 v
SO-MeOH-pyridine, and heated for 4 h at 100 °C. After cooling, the desulfated product was recovered by dialysis, freeze-dried, and designated as dsLMW-ulvan.
Methylation analysis was performed as the reported method with some modification (Li et al., 2019) [23
]. Briefly, 1 mg sample (LMW-ulvan or dsLMW-ulvan) dried by P2
was dissolved in 2.0 mL DMSO, and then 100 mg anhydrous NaH was added under nitrogen atmosphere. The mixture was stirred at 25 °C for 2 h under nitrogen atmosphere, and then 1 mL CH3
I was added dropwise in an ice-cold water bath. The mixture was incubated in the dark at 25 °C, stirring for 3 h. Finally, 1 mL distilled water was added to terminate the reaction, and then extracted with CHCl3
. The extract was washed with distilled water and evaporated to dryness. The methylated polysaccharide was converted into partially methylated alditol acetates, which were analysed by GC-MS (Thermo Fisher Scientific, MA, USA). The GC-MS analysis designed for methylation analysis was performed according to a report by Lin et al. (2016) [43
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 Buffer 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) reagent 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
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 buffer 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 buffered saline (TBS). The protein signals were analyzed using an ECL detection system (Tanon, Nanjing, China).
3.11. Statistics Analysis
All data were analyzed by using SPSS software 22 and expressed as the mean ± standard deviation of at least three separate experiments. Statistical significance was identified by one-way analysis of variance (ANOVA) and the Waller-Duncan test. The value of p < 0.05 was accepted as statistically different.
Our results revealed that LMW-ulvan enzymatic production of ulvan extracted from green algae U. pertusa consists of 57.23% rhamnose, 28.76% xylose, 7.42% glucuronic acid, and 1.77% glucose. Its backbone contained (1→3,4)-linked Rha, (1→4)-linked Xyl, and (1→4)-linked GlcA with small amounts of (1→4)-linked Rha residues; sulfate substitution was at C-3 of rhamnose. In addition, LMW-ulvan was able to relieve intestinal inflammation and oxidative damage caused by DSS. Meanwhile, LMW-ulvan was able to significantly increase mRNA levels of claudin, occluding, and ZO-1, which improve intestinal mucosal permeability. Consequently, the results of this study support the potential application of LMW-ulvan as a functional food ingredient for IBD.