Smilax china L. Polysaccharide Alleviates Dextran Sulphate Sodium-Induced Colitis and Modulates the Gut Microbiota in Mice

This work aimed to investigate the preventive effect of Smilax china L. polysaccharide (SCP) on dextran sulfate sodium (DSS)-induced ulcerative colitis (UC) in mice. Smilax china L. polysaccharide was isolated by hot water extraction, ethanol precipitation, deproteinization, and purification using DEAE-cellulose column chromatography to yield three polysaccharides: SCP_C, SCP_A, and SCP_N. Acute colitis was induced by administering 3% (w/v) DSS in drinking water for 7 days. Sulfasalazine, SCP_C, SCP_A, and SCP_N were administered by gavage for 9 days. SCP_C, SCP_A, and SCP_N could significantly improve symptoms, as evidenced by the declining disease activity index (DAI), decreased spleen weight, increased length of the colon, and improved colonic histology. Moreover, SCP_C, SCP_A, and SCP_N increased serum glutathione and decreased the levels of pro-inflammatory cytokines, malondialdehyde, nitric oxide, and myeloperoxidase in colon tissues. Additionally, SCP_C, SCP_A, and SCP_N modulated gut microbiota via ascending the growth of Lachnospiraceae, Muribaculaceae, Blautia, and Mucispirillum and descending the abundance of Akkermansiaceae, Deferribacteraceae, and Oscillibacter in mice with UC. The results suggested that Smilax china L. polysaccharide ameliorates oxidative stress, balances inflammatory cytokines, and modulates gut microbiota, providing an effective therapeutic strategy for UC in mice.


Introduction
Inflammatory bowel diseases (IBDs), which include Crohn's disease (CD) and ulcerative colitis (UC), are relapsing and chronic intestinal inflammations that lead to mucous ulceration and tissue damage and increase the risk of colon carcinogenesis [1]. As a chronic and non-specific IBD, UC is accompanied by weight loss, bloody diarrhea, and abdominal distress [2]. Although the pathophysiology of UC remains unclear, the pathogenesis is multifaceted and includes oxidative stress, genetic predisposition, intestinal barrier dysfunction, and colonic mucosal damage [2,3]. Currently, the treatment of UC generally relies on conventional drugs and biological preparations. For example, sulfasalazine (SSZ) is used to treat IBD and has a broad spectrum of bacteriostatic properties [4]. SSZ can lessen immunological pathogenic damage and inflammation of the intestinal mucosa [5]. Sun et al. [6] used SSZ as a positive control and reported that it alleviated the IBD symptoms. Long-term use of the medications, however, has toxic side effects such as abdominal pain, kidney damage, and hepatotoxicity [7]. Therefore, it is necessary to discover new and safer natural substances to prevent UC. Natural polysaccharides and polyphenols, which are bioactive compounds, are emerging as promising candidates for preventing UC [8].
Gut microbiota, which are referred to as "super-organisms" or the "second genome" of humans, are associated with the onset and progression of UC [9]. Patients with UC have increased intestinal permeability, which causes gut bacteria translocation across the

Isolation and Purification of Polysaccharides
Smilax china L. powder was soaked for 24 h in a clean plastic bucket with 2000 mL of ethanol (80%) to remove alcohol-soluble substances, such as small-molecule carbohydrates, lipids, polyphenols, and pigments. After that, the ethanol-soaked residue was air-dried and extracted twice for 2 h with a ratio of 1:20 (w/v) in 80 • C distilled water. After mixing the extracts, they were then centrifuged at 8400× g for 10 min to separate the desired supernatant from the residue. The concentrated liquid was then evaporated in a rotary evaporator until the volume reached 1/3 of the original solution (55 • C). The precipitated polysaccharides were dissolved in hot water, centrifuged, and deproteinated by the Sevag method [19], and then starch was removed with high-temperature α-amylase. Next, combine ethanol at a concentration of 80% (v/v), and keep the mixture at 25 • C overnight. Centrifugation at 8400× g for 10 min was used to collect the precipitate, which was then redissolved in ultrapure water and suspended to remove alcohol at 55 • C. The solution was then dialyzed for 48 h in distilled water with a dialysis bag, followed by 12 h in ultrapure water. Finally, the concentrate was lyophilized in a vacuum freeze dryer to obtain crude polysaccharide (named SCP_C).
SCP_C was further purified using a DEAE-cellulose column (2.6 × 35 cm). Experimental conditions were as follows: SCP_C (6 g, 500 mL) was prepared and poured into a DEAE-resin column for adsorption over 5 h. Sequentially, fractions were eluted with distilled water, 0.05 mol/L NaCl, and 0.1 mol/L NaCl solutions at a flow rate of 1.5 mL/min. The elute was collected and dialyzed in distilled water for 24 h and freeze-dried. Two polysaccharide fractions, named SCP_A (0.05M NaCl eluted) and SCP_N (aqueous eluted), were separated. The fractions, SCP_A and SCP_N, were dialyzed and lyophilized, respectively. The yield of 0.1 mol/L NaCl solution was low, so SCP_A and SCP_N were used in the subsequent studies.

Molecular Weight and Monosaccharide Composition Determination
The molecular weight of SCP_A and SCP_N was determined by high-performance gel permeation chromatography (HPGPC), which was performed in an Agilent 1260 HPLC system equipped with an KS-802 column (8 mm × 300 mm) detected by a refractive index detector. The monosaccharide composition was determined by HPAEC pulsed amperometric detection (PAD), a CarboPac TM PA10 column (2.0 mm × 250 mm), and a Dionex ICS-2500 system. Standard monosaccharides (Fuc, Rha, Ara, Gal, Glc, Xyl, Man, Fru, Gala, and Glca) were used as references.

UV and FT-IR Spectra Analysis
The UV-visible spectra of polysaccharide solution (1 mg/mL) were recorded using an ultraviolet-visible spectrophotometer (model UV-5200PC) in the range of 200-400 cm −1 . An amount of 1 mg of sample was mixed with 100 mg KBr powder, ground, and then pressed into flakes to record the FT-IR spectra (Nicolet iS5 FT-IR, Thermo Electron Co., Madison, WI, USA) absorbance from 4000-400 cm −1 in 64 scans with a resolution of 4 cm −1 [20].

Experimental Design
Male BALb/c mice, weighing 20 ± 2 g, were purchased from Hunan Silaike Laboratory Animal Co., Ltd. (Changsha, China SCXK <Xiang> 2016-0002). Mice were free to receive water and food, and they were kept under a standard environment (temperature 24 ± 2 • C, humidity 50 ± 10%, and 12 h light/dark cycle). All experimental involving animals were approved by the Animal Care and Use Committee of Jiangxi Agricultural University (Reg. No. 2018-001). After 5 days of adaptive feeding, all mice were randomly divided into six groups (n = 10): normal chow diet (NCD, received AIN-93M diet), DSS, SSZ (50 mg/kg bw/day), 200 mg/kg bw/day of SCP_C, SCP_A, and SCP_N (the dose was selected based on the results of the pre-experiment). The NCD group was given the same amount of distilled water, and the other groups were given 3% DSS dissolved in distilled water by gavage for 7 days to induce acute colitis. An experimental flow diagram is shown in Figure 2A. Throughout the experimental period, the mice were monitored daily for mental status, water consumption, body weight, fecal consistency, and fecal properties. After that, blood was collected from the orbit under isoflurane anesthesia at the end of the experiment, and the mice were then sacrificed by cervical dislocation. Subsequently, the spleen and colon tissue were quickly excised and weighed from each mouse. The colon length was measured. The caecum contents were collected and then partitioned for future research before storage at −80 • C. The compositions of diet are shown in Table S1.

Disease Activity Index (DAI) and Colonic Damage
The body weight, stool consistency, and gross bleeding at the anus or in the stool were monitored daily, based on the DAI scoring system in Table 1 [21] and calculating via the formula as follows: DAI = (weight loss score + stool consistency score + gross bleeding score)/3

Histopathologic Analysis of Colon Tissue
For histological analysis, colon tissues were fixed in 4% paraformaldehyde for 24 h. Afterwards, all fixed samples were paraffin embedded, sliced, and stained with hematoxylin eosin (H & E). The colonic structure was observed at 100× magnification by optical microscopy. Histological evaluation was performed according to the reported method [22].

Analysis of the Oxidative Stress Index
The serum superoxide dismutase (SOD) and glutathione (GSH) levels and the contents of malondialdehyde (MDA), nitric oxide (NO), and myeloperoxidase (MPO) in colon tissues were measured using a corresponding assay kit according to the manufacturer's instructions.

Measurements of Inflammation Cytokines Levels
The colon tissue was weighed and homogenized in a homogenizer in a saline solution. The homogenates were centrifuged 3000× g for 15 min at 4 • C. The supernatant was separated, and inflammatory cytokines (including IL-6, IL-10, IL-1β, and TNF-α) were detected using ELISA kits (Boster Biological Technology, Co., Ltd., Wuhan, China).

16S rDNA Gene Sequencing
Caecum contents were sent to Majorbio Bio-Pharm Technology Co., Ltd., (Shanghai, China) for DNA extraction, PCR amplification, and Illumina MiSeq sequencing. Briefly, microbial DNA was extracted using the DNA extraction kit (D4015, Omega, Inc., Norcross, GA, USA). After amplification and sequencing of the V3-V4 regions of the bacterial 16S rDNA were amplified using primers 341F (5 -CCTACGGGNGGCWGCAG-3 ) and 805R (5 -GACTACHVGGGGTATCTAATCC-3 ) by a thermocycler PCR system. The program for PCR was 1 cycle of 98 • C for 30 s, followed by 32 cycles of denaturing at 98 • C for 10 s, annealing at 54 • C for 30 s, extension at 72 • C for 45 s, and a final extension at 72 • C for 10 min. Prior to 16S rDNA data analysis, the raw reads were demultiplexed, quality-filtered by fqtrim (V0.94), and merged by FLASH version 1.2.7.

Statistics
The data were presented as mean ± standard error of the mean (SEM). Comparisons among different groups were evaluated with one-way analysis of variance (ANOVA) followed by the Dunn's multiple comparison test and post hoc LSD (SPSS 22, IBM Corp., Armonk, NY, USA). Different letters (a-d) mean statistical significance (p < 0.05).

Purification and Identification
Crude polysaccharide was isolated through hot water extraction and ethanol precipitation, deproteinization, dialysis, and drying. The SCP_C extraction yield was 7.1 ± 0.1%. SCP_C was further purified using distilled water, 0.05 M NaCl solution, and 0.1 M NaCl solution, respectively, on a DEAE cellulose column. After concentration and freeze-drying, SCP_N and SCP_A were obtained ( Figure S1). Table 2 shows the contents of total sugar, protein, and uronic acid in SCP_A and SCP_N. SCP_A, which contained 88.5% of total sugar and 24.8% of uronic acid, was composed of Fuc, Rha, Glu, Gal, Xyl, Gala, and Glca with a molecular ratio of 1.01, 3.45, 1.45, 7.41, 0.12, 1.47, and 1.45, respectively. SCP_N, which contained 86.7% of total sugar and no uronic acid, was mainly composed of Fuc, Rha, Glu, Gal, and Xyl with a molecular ratio of 0.18, 0.37, 42.89, 1.11, and 0.79, respectively. The molecular weights of SCP_A and SCP_N were 14.8 kDa and 31.2 kDa, respectively ( Table 2).

UV Visible Spectra and FT-IR Spectra
In Figure 1A,B, no UV/vis absorption peak was observed at 260 nm, indicating the absence of nucleic acid in SCP_A and SCP_N. In the UV spectrum, SCP_A and SCP_N showed weak UV absorption at 280 nm, suggesting that they contained a small amount of protein.

Polysaccharide Alleviates the Clinical Symptoms of DSS-Induced Colitis
In the DSS-induced UC model, 1 or 2 days after the start of DSS administration, mice were accompanied by symptoms of weakness, dim hair luster, and severe bloody stools in the anus ( Figure S2). The weights of mice in the DSS group were significantly lower than those in the SCP_C, SCP_A, and SCP_N groups on the 2nd day. After the 7 th day, the body weight of DSS mice consistently decreased and was lower than that of SCP_C-and SCP_A-treated mice ( Figure 1B). Figure 1C showed that from day 1, the DAI scores of the DSS-induced groups increased in comparison with those of the NCD group. The DAI score of the NCD group was stable at 0 during the whole experiment, and the growth status performed well. With the development of colitis, the DAI values showed a slow upward trend, manifesting in body weight loss, diarrhea, and bloody stools. A significant

Polysaccharide Alleviates the Clinical Symptoms of DSS-Induced Colitis
In the DSS-induced UC model, 1 or 2 days after the start of DSS administration, mice were accompanied by symptoms of weakness, dim hair luster, and severe bloody stools in the anus ( Figure S2). The weights of mice in the DSS group were significantly lower than those in the SCP_C, SCP_A, and SCP_N groups on the 2nd day. After the 7th day, the body weight of DSS mice consistently decreased and was lower than that of SCP_Cand SCP_A-treated mice ( Figure 2B). Figure 2C showed that from day 1, the DAI scores of the DSS-induced groups increased in comparison with those of the NCD group. The DAI score of the NCD group was stable at 0 during the whole experiment, and the growth status performed well. With the development of colitis, the DAI values showed a slow upward trend, manifesting in body weight loss, diarrhea, and bloody stools. A significant difference between the DSS and treatment groups was seen after four days of treatment. Supplementation with SCP_C, SCP_A, and SCP_N reduced the DAI scores. The shortening of the colon and increase in spleen weight are one of the clinical signs of colitis. As displayed in Figure 2D,E, compared with the DSS group, SCP_C, SCP_A, and SCP_N increased the length of colon and decreased spleen weight. The results indicated that polysaccharide could significantly alleviate the clinical symptoms of DSS-induced UC in mice.
After H & E staining, the sections of the colon were subjected to a pathological investigation about the effect of SCP C, SCP A, and SCP N on the severity of inflammation and colon morphology in UC induced by DSS ( Figure 2G). DSS treatment caused crypt loss, blurred goblet cells, and inflammatory cell infiltration, resulting in histopathological scores that increased significantly compared to the NCD group. After the intervention with SCP_C, SCP_A, and SCP_N, the DSS-induced pathological alterations are lessened. In particular, SCP_N reduced inflammatory cells' infiltration and alleviated goblet cell disappearance. Although SCP_A alleviated symptoms to some extent, there was still local inflammatory cell infiltration and goblet cell loss. Histological score analysis also revealed that SCP_C and SCP_N decreased the histopathologic damage ( Figure 2F).

Impact of Polysaccharide on Oxidative Stress in DSS-Induced Mice
Inhibition of oxidative stress and improvement of cellular anti-oxidant capacity are effective ways to treat UC [23]. SCP_C, SCP_A, and SCP_N increased the GSH content compared with the DSS group ( Figure 3A). There were no significant (p > 0.05) differences in the level of SOD between the DSS group and the treatment group ( Figure 3B). MDA and MPO activities were used as oxidative damage markers. Excessive MDA induces the release of NO, which leads to an increase in oxygen free radicals and tissue injury, and ultimately triggers inflammatory responses [24]. MPO is an important biomarker of acute inflammation and neutrophil infiltration, and its level rises when colitis occurs [25]. As depicted in Figure 3C-E, SCP_C, SCP_A, and SCP_N decreased oxidative stress as revealed by a reduction in MDA, MPO, and NO levels compared with that of the DSS group. The data indicated that SCP alleviates colon damage caused by oxidative stress.

mice.
After H & E staining, the sections of the colon were subjected to a pathological investigation about the effect of SCP C, SCP A, and SCP N on the severity of inflammation and colon morphology in UC induced by DSS ( Figure 2G). DSS treatment caused crypt loss, blurred goblet cells, and inflammatory cell infiltration, resulting in histopathological scores that increased significantly compared to the NCD group. After the intervention with SCP_C, SCP_A, and SCP_N, the DSS-induced pathological alterations are lessened. In particular, SCP_N reduced inflammatory cells' infiltration and alleviated goblet cell disappearance. Although SCP_A alleviated symptoms to some extent, there was still local inflammatory cell infiltration and goblet cell loss. Histological score analysis also revealed that SCP_C and SCP_N decreased the histopathologic damage ( Figure 2F).

Effect of Polysaccharide on Inflammatory Cytokine Levels
Cytokines are closely related to the progression of UC, and studies have shown that disease severity is relevant to the balance between pro/anti-inflammatory cytokines. TNFα and IL-1β, which are primarily released by macrophages and monocytes upon activation have numerous pro-inflammatory properties. Both cytokines are essential for the expansion of intestinal inflammation. TNF-α, in particular, is an important promoter of tissue damage, increasing the production of IL-6 and IL-1β and inducing mucosal inflammation [26,27]. SCP_C, SCP_A, and SCP_N significantly decreased the levels of TNF-α, IL-6, and IL-1β compared with the DSS group ( Figure 3F-H). Furthermore, IL-10, an anti-inflammatory cytokine, ameliorates intestinal damage by inhibiting the immune response to autoantigens or foreign antigens and limiting TNF-α, IL-6, and IL-1β secretion [28]. SCP_C, SCP_A, and SCP_N remarkably increased the level of IL-10 as compared with the DSS group ( Figure 3I). Accumulating evidence suggests that polysaccharides containing rhamnose and galactose have distinct anti-inflammatory activities, blocking pro-inflammatory cytokines in cell and mouse models [29]. In DSS-induced UC mice, SCP_A and SCP_N composed of Rha and Gal exhibited obvious anti-inflammatory activities. However, the specific molecular mechanism needs to be further investigated. These data indicated that polysaccharide alleviates DSS-induced intestinal inflammation.

Effect of Polysaccharide on the Diversity of Gut Microbiota
The pan/core curves, rarefaction curves, and Shannon index of OTU level analyses showed that the completeness of the sample and the sequencing depth covered rare new phenotypes and diversity ( Figure S3). As can be seen from Table S2, no noticeable difference in microbial diversity was observed between the NCD group and treatment group. Supplementation with SCP_N increased community diversity (Shannon index), compared to the DSS group. Markedly, the Chao1 and ACE indices in the SCP_C, SCP_A, and SCP_N groups were higher than those of the DSS group (Table S2). Bacterial β-diversity, as assayed by nonmetric multidimensional scaling (NMDS) of the weighted UniFrac index, was then analyzed to depict the structural variability of gut bacterial communities among different groups. The results showed that the gut microbiota of SCP_N group was significantly different from that of the DSS group ( Figure 4A). Increasing evidence has indicated that an undesirable alteration of gut microbial diversity and structure is a crucial factor con-tributing to the occurrence of colonic inflammation [30]. Studies have shown that microbial diversity and richness was decreased in UC patients compared to healthy individuals [12], which is in line with our results. Taken together, the results indicated that polysaccharide significantly increased the gut microbial diversity in DSS-treated mice, which might be partially responsible for its inhibitory effect against UC.
inflammation. Moreover, SCP_A and SCP_N increased the growth of Lachnospiraceae in DSS-induced mice ( Figure 4H). It was reported that Lachnospiraceae is a probiotic that can induce an accumulation of T reg cells in the colon, which is involved in the generation of butyrate and reduces inflammation [39]. As shown in Figure 4I,J, compared with the DSS group, the relative abundance of Muribaculaceae in the SCP_N group significantly increased, and that of Deferribacteraceae significantly decreased. SCP_N drastically elevated the growth of the Muribaculaceae in DSS-induced mice. Specific bacteria of the Muribaculaceae, which belongs to the Bacteroidetes, is related to the intestinal barrier function, which regulates immune cells to decrease the production of pro-inflammatory cytokines [40,41]. Muribaculaceae has been detected as the dominant gut microbiota in healthy humans and animals and is linked to the production of acetate and propionate [42].

Effect of Polysaccharide on the Structure of Gut Microbiota
Lachnospiraceae_NK4A136_group, norank_f_Muribaculaceae, Parabacteroides, norank_f_ Lachnospiraceae, norank_f_Ruminococcaceae, unclassified_f_Lachnospiraceae, Blautia, Akkermansia, and Oscillibacter were the most dominant genera in each group ( Figure 4B). Next, we assessed the differences in microbiota composition and relative abundance among different groups at phyla, family, and genus levels.
As shown in Figure 4C, Firmicutes, Bacteroidetes, Verrucomicrobia, and Deferribacteres were major phyla in the colonic microbiota. Firmicutes and Bacteroidetes were found to play an important role in the development of UC [31]. A decline in the phyla Firmicutes and Bacteroidetes is a predominant signature for dysbiosis in UC patients [32]. Remarkably, Firmicutes play an important role in gut homeostasis via the metabolites production, and their abundance promotes the anti-inflammatory and anti-tumorigenic properties [33]. SCP_N treatment effectively increased the abundance of Firmicutes and Bacteroidetes ( Figure 4D,E). One homogeneous polysaccharide from Scutellaria baicalensis Georgi, which is mainly composed of mannose, ribose, rhamnose, glucuronic acid, glucose, xylose, arabinose, and fucose, improved colitis by increasing the growth of Firmicutes, Bifidobacterium, Lactobacillus, and Roseburia [34]. Additionally, polysaccharides from soybean residue fermented by Neurospora crassa, which mainly contained rhamnose, arabinose, fucose, mannose, glucose, and galactose, increased the growth of Firmicutes in DSS-induced colitis [35]. Consistent with previous findings, the current results showed that SCP_N, which primarily contained fucose, rhamnose, glucose, galactose, and xylose, prevented gut dysbiosis in DSS-induced UC mice. As shown in Figure 4F, at the family level, the gut microbiota was mainly composed of Lachnospiraceae, Deferribacteraceae, Muribaculaceae, Akkermansiaceae, and Ruminococcaceae. In this study, SCP_A and SCP_N significantly decreased the abundance of Akkermansiaceae in UC mice ( Figure 4G). Akkermansiaceae, which belongs to Verrucomicrobia, one of the mucin-degrading specialists in the gut, was reported to be increased in mice with DSS-induced colitis and in patients suffering from colorectal cancer [36]. Intestinal mucins are degraded after A. muciniphila overgrowth, eventually leading to colitis deterioration [37]. Its potential role in the development of colitis remains controversial. A. muciniphila was found to be a potent anti-inflammatory intestinal bacterium that adhered to intestinal enterocytes, strengthened the integrity of the epithelial cell layer, and was inversely correlated with inflammation [38]. Considering its mucin-degrading properties, we speculated that A. muciniphila invaded and degraded the colon mucosa, which led to an increased permeability and access to other microbes that cause inflammation. Moreover, SCP_A and SCP_N increased the growth of Lachnospiraceae in DSS-induced mice ( Figure 4H). It was reported that Lachnospiraceae is a probiotic that can induce an accumulation of T reg cells in the colon, which is involved in the generation of butyrate and reduces inflammation [39]. As shown in Figure 4I,J, compared with the DSS group, the relative abundance of Muribaculaceae in the SCP_N group significantly increased, and that of Deferribacteraceae significantly decreased. SCP_N drastically elevated the growth of the Muribaculaceae in DSS-induced mice. Specific bacteria of the Muribaculaceae, which belongs to the Bacteroidetes, is related to the intestinal barrier function, which regulates immune cells to decrease the production of pro-inflammatory cytokines [40,41]. Muribaculaceae has been detected as the dominant gut microbiota in healthy humans and animals and is linked to the production of acetate and propionate [42].
At the genus level, compared to the DSS group, the relative abundance of Lach-nospiraceae_NK4A136_group, Blautia, norank_f_Muribaculaceae, Erysipelatocostridium, and Rikenellaceae_RC9_gut_group significantly increased in SCP_A and SCP_N, while that of Eubacterium_fissicatena_group, norank_f_Clostridiales_vadinBB60_group, norank_f_Ruminococcaceae, Oscillibacter, Mucispirillum, and Lactobacillus significantly decreased ( Figure 5). SCP_A and SCP_N treatment significantly increased the abundance of norank_f_Muribaculaceae, and the genera have beneficial effects on the prevention of IBD [43]. In addition, as a kind of key probiotic, the Lactobacillus genera may be beneficial during DSS-induced UC [44]. In the present study, the amount of Lactobacillus on colonic mucosa was decreased ( Figure 5), and similar results have been obtained in other studies [45]. Notably, SCP_C, SCP_A, and SCP_N administration drastically inhibited the growth of Oscillibacter in DSS-induced mice. Oscillibacter has been recognized as a newly discovered genus associated with digestive diseases and the severity of DSS-induced UC [46]. The genus Lachnospiraceae_NK4A136_group, which has anti-inflammatory properties [47], increased significantly after SCP_A and SCP_N treatment, and is negatively correlated with MPO, DAI, and TNF-α. Meanwhile, SCP_A and SCP_N significantly increased the abundance of Blautia in UC mice. Blautia is one of the major intestinal microbes. The richness of Blautia has been linked to colorectal cancer and IBDs [48]. Together, these findings demonstrated that SCP_C, SCP_A, and SCP_N intervention played a key role in the regulation of the gut microbiota in DSS-induced colonic inflammation.

Correlation between UC-Related Parameters and Key Phylotypes of Microbiota
Results of db-RDA showed a significant correlation between microbiota and several pro-inflammatory cytokines, NO, MPO, MPA, and IL-10. The degree of correlations between UC-related biochemical parameters and microbiota was as follows: MDA > TNF-α > IL-1 β > IL-6 > NO > MPO ( Figure 6A).

Conclusions
In summary, different polysaccharides, namely, SCP_C, SCP_A, and SCP_N from Smilax china L., were isolated and purified, and their chemical structures were determined by HPGPC, HPAEC-PAD, UV -Vis, and FTIR. The in vivo anti-inflammatory effect of polysaccharide was investigated, and an acute colitis model was induced by 3% DSS. The basic symptoms observed in this model, including body weight loss, diarrhea, and bloody stools reflect the degree of severity. Supplementation with SCP_C, SCP_A, and SCP_N decreases DAI scores, alleviates colon inflammation and oxidative stress, and modulates the gut microbiota, thereby ameliorating DSS-induced colitis. However, the structure-activity relationship of SCP_C, SCP_A, and SCP_N in the treatment of UC needs further study. Our findings indicate that SCP_C, SCP_A, and SCP_N have strong potential for preventing and treating UC.
Author Contributions: Data curation, X.L. and G.Q.; writing-original draft preparation, X.L.; writing-review and editing, X.L. and G.Z.; visualization, G.Q. and L.C.; supervision, L.C. and L.L.; project administration, G.Z.; funding acquisition, G.Z. All authors have read and agreed to the published version of the manuscript. Informed Consent Statement: Not applicable.

Data Availability Statement:
The data presented in this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.