Plant Polyphenols Attenuate DSS-induced Ulcerative Colitis in Mice via Antioxidation, Anti-inflammation and Microbiota Regulation

The pathogenesis of ulcerative colitis (UC) is associated with inflammation, oxidative stress, and gut microbiota imbalance. Although most researchers have demonstrated the antioxidant bioactivity of the phenolic compounds in plants, their UC-curing ability and underlying mechanisms still need to be further and adequately explored. Herein, we studied the antioxidation–structure relationship of several common polyphenols in plants including gallic acid, proanthocyanidin, ellagic acid, and tannic acid. Furthermore, the in vivo effects of the plant polyphenols on C57BL/6 mice with dextran-sulfate-sodium-induced UC were evaluated and the action mechanisms were explored. Moreover, the interplay of several mechanisms was determined. The higher the number of phenolic hydroxyl groups, the stronger the antioxidant activity. All polyphenols markedly ameliorated the symptoms and pathological progression of UC in mice. Furthermore, inflammatory cytokine levels were decreased and the intestinal barrier was repaired. The process was regulated by the antioxidant-signaling pathway of nuclear-erythroid 2-related factor 2. Moreover, the diversity of the intestinal microbiota, Firmicutes-to-Bacteroides ratio, and relative abundance of beneficial bacteria were increased. An interplay was observed between microbiota regulation and oxidative stress, immunity, and inflammatory response. Furthermore, intestinal barrier repair was found to be correlated with inflammatory responses. Our study results can form a basis for comprehensively developing plant-polyphenol-related medicinal products.


Introduction
Ulcerative colitis (UC) is a common non-specific inflammatory bowel disease (IBD) characterized by chronic inflammation and ulceration of the rectal and colon mucosa. Clinical symptoms include pain, weight loss, blood in the stool, and intestinal mucosal ulcers [1][2][3][4][5]. At present, there is still no cure available for UC in clinical settings. Furthermore, recurrence causes immense economic and mental distress to patients and their families. Therefore, it is extremely important to study UC pathogenesis and identify effective drugs to treat this condition. UC pathogenesis includes inflammation, to the colon barrier damage, and oxidative stress imbalance and the intestinal microbial environment imbalance [1].
Oxidative stress is critical in UC pathogenesis. Organisms continuously produce reactive nitrogen (RNS) and reactive oxygen species (ROS) via mitochondrial bioenergetics and oxidative metabolism, and excessive production of ROS will lead to oxidative damage [6]. On the other hand, the disruption of the microbial barrier, including an imbalance in the intestinal microbial environment, decreased bacterial diversity [7], and excessive proliferation of Gram-negative bacteria, can lead to the production of large amounts of lipopolysaccharides (LPSs), resulting in an excessive production of ROS or RNS [8]. Excess ROS/RNS affects colon epithelial cells and immune cells, thereby disrupting the immune barrier and exacerbating the inflammatory response [9,10]. Intestinal microbiota disorders are characterized by an imbalance between beneficial and pathogenic bacteria. Beneficial bacteria play immune barrier roles by regulating host immune cells, whereas some pathogenic bacteria induce inflammatory cytokines via immune cell interactions or their metabolites, aggravating intestinal damage [11]. The mechanical barrier of the intestinal tract mainly includes tight junction (TJ) proteins. TJ proteins are essential for maintaining gut homeostasis [12]. TJ proteins are responsible for regulating water, electrolyte, and nutrient uptake by the cells [13]. Destruction of the mechanical barrier of the intestinal tract will cause bacteria and antigens in the intestinal cavity to enter the intestinal mucosa lamina propria, aggravating the intestinal damage. Intestinal antigens translocate to mucosal lamina propria and activate lamina propria immune cells, producing a significant amount of inflammatory cytokines and inflammatory mediators [14]. Therefore, UC pathogenesis is a complex process, with close involvement of the mechanical, immune, and microbial barriers of the intestinal tract. Therefore, treatment strategies for UC should consider the regulation of multiple mechanisms.
Polyphenols are widely found in tea, fruits, and medicinal plants [15][16][17], including chlorogenic acid, hydrolytic tannins, and flavonoids [18]. Gallic acid (GA), as an active component, has been found in many plants [19]; proanthocyanidins (PC), known as condensed tannins, are widely available in the bark of many plants [20]; ellagic acid (EA) is a natural polyphenol found in many fruits and vegetables [21,22]. Tannic acid (TA) is found mainly in the seeds of legumes and usually acts as a defense against oxidative damage to the seeds [23]. A study has reported that polyphenols exhibit excellent antioxidant and anti-inflammatory activities [24]. Moreover, the antioxidant activity of polyphenols depends largely on the presence of structural groups and the number of hydroxyl groups in a molecule [25]. Furthermore, polyphenols are sensitive to some intestinal pathogenic bacteria and can affect host immune response by causing changes in microbiota. The long-term consumption of foods rich in polyphenols can reduce the risk of various diseases [26,27], including the prevention and relief of IBD [19]. Most researchers have made efforts to elucidate the antioxidant bioactivity of the plant phenolic compounds [28][29][30]; only a few have adequately explored phenolic compounds as agents to cure UC and elucidated the relationship of between antioxidation and gut microbiota regulation. Therefore, the present study aimed to elucidate the antioxidant effects of polyphenols in plants including GA, PC, EA and TA and the structure-function relationship, and to explore the mechanism of relieving UC, including alleviating oxidative stress, intestinal barrier repair, and intestinal microbiota regulation ( Figure 1). Simultaneously, the relationship between microbiota imbalance and oxidative stress, microbiota and immune cells, and inflammatory cytokines in intestinal barrier was elucidated. Figure 1. The potential mechanism of polyphenols in plants in alleviating ulcerative colitis (U Nuclear-factor-erythroid 2-related factor 2 (Nrf2); heme oxidase-1 (HO-1); NAD(P)H dehydrog ase [quinone] 1 (NQO1); interleukin-6 (IL-6); and interleukin-10 (IL-10).

Antioxidant Performance
The antioxidant activity of the polyphenols was evaluated by measuring the scaven ing ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH·) and 2, 2′-azino-bis (3-ethylbenzot azoline-6-sulfonic acid) (ABTS) free radicals and the total antioxidant capacity. Figur presents the results. The DPPH· radical scavenging rate of four polyphenols at the co centration of 10-0.1563 mM was >90%. The DPPH· radical scavenging rate of GA, TA a PC were significantly higher than EA at the concentration of 0.078 mM. The DPPH· radi scavenging rates of TA were significantly better than those of the other three groups, G and PC were better than EA, and PC was better than GA at the concentration of 0.039 m The scavenging ability of DPPH· radical was TA > PC > GA and EA at the concentrati of 0.0195 mM ( Figure 2A).
As shown in Figure 2B, the ABTS scavenging rates of four polyphenols were >8 even at concentrations of 10-0.625 mM. The ABTS scavenging rates of GA, TA, and were significantly higher than that of EA at 0.3125 mM. With a decrease in concentrati (0.1563 mM~0.0195 mM), the ABTS scavenging ability of the four polyphenols exhibite general decreasing trend. The ABTS scavenging rate of TA was significantly higher th that of the other compounds, followed by PC, GA and EA.

Antioxidant Performance
The antioxidant activity of the polyphenols was evaluated by measuring the scavenging ability of 1,1-diphenyl-2-picrylhydrazyl (DPPH·) and 2, 2 -azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) free radicals and the total antioxidant capacity. Figure 2 presents the results. The DPPH· radical scavenging rate of four polyphenols at the concentration of 10-0.1563 mM was >90%. The DPPH· radical scavenging rate of GA, TA and PC were significantly higher than EA at the concentration of 0.078 mM. The DPPH· radical scavenging rates of TA were significantly better than those of the other three groups, GA and PC were better than EA, and PC was better than GA at the concentration of 0.039 mM. The scavenging ability of DPPH· radical was TA > PC > GA and EA at the concentration of 0.0195 mM ( Figure 2A).
As shown in Figure 2B, the ABTS scavenging rates of four polyphenols were >80% even at concentrations of 10-0.625 mM. The ABTS scavenging rates of GA, TA, and PC were significantly higher than that of EA at 0.3125 mM. With a decrease in concentration (0.1563 mM~0.0195 mM), the ABTS scavenging ability of the four polyphenols exhibited a general decreasing trend. The ABTS scavenging rate of TA was significantly higher than that of the other compounds, followed by PC, GA and EA.

Polyphenols Alleviate Dextran-Sulfate-Sodium-Salt (DSS)-Induced Colitis
To determine whether polyphenols can ameliorate colon injury and inflammation, we assessed the effect of the polyphenols on 3% DSS-induced UC mice. In the established model, the weight of the mice decreased in the DSS group. After administration, the weight of the mice in all groups increased in varying degrees ( Figure 3A), the weight gain in the PC group, EA group and GA group were higher than that of in the model group. Disease activity index (DAI) score of the PC, EA, and GA groups was lower than that of the model group ( Figure 3B). Figure 3C,D demonstrate that GA, EA, TA alleviate colon  To determine whether polyphenols can ameliorate colon injury and inflammation, we assessed the effect of the polyphenols on 3% DSS-induced UC mice. In the established model, the weight of the mice decreased in the DSS group. After administration, the weight of the mice in all groups increased in varying degrees ( Figure 3A), the weight gain in the PC group, EA group and GA group were higher than that of in the model group. Disease activity index (DAI) score of the PC, EA, and GA groups was lower than that of the model group ( Figure 3B). Figure 3C,D demonstrate that GA, EA, TA alleviate colon shortening. Furthermore, serum IL-6 and IL-10 levels were detected using enzyme-linked immunosorbent assay; GA, EA, and PC significantly decreased IL-6 levels ( Figure 3E) and PC, EA increased IL-10 levels ( Figure 3F). shortening. Furthermore, serum IL-6 and IL-10 levels were detected using enzyme-linked immunosorbent assay; GA, EA, and PC significantly decreased IL-6 levels ( Figure 3E) and PC, EA increased IL-10 levels ( Figure 3F).

Effects of Polyphenols on Colon Histopathology
Hematoxylin-eosin (H&E) staining results in the colon revealed distortion of the crypt architecture and hyperplasia, mucosal epithelial cell degeneration/necrosis, and large amounts of inflammatory cell infiltration in the model group ( Figure 4). In the

Effects of Polyphenols on Colon Histopathology
Hematoxylin-eosin (H&E) staining results in the colon revealed distortion of the crypt architecture and hyperplasia, mucosal epithelial cell degeneration/necrosis, and large amounts of inflammatory cell infiltration in the model group ( Figure 4). In the treatment groups, the epithelial cells were mild or non-degenerative/necrotic and a small number of inflammatory cells were present. Among them, the histological characteristics of the GA and TA groups were similar to those of the healthy control group. treatment groups, the epithelial cells were mild or non-degenerative/necrotic and a small number of inflammatory cells were present. Among them, the histological characteristics of the GA and TA groups were similar to those of the healthy control group.

Effects of Polyphenols on Regulatory T (Treg) Cell Expression
The expression of Treg cells was detected using immunohistochemical (IHC)-staining-labeled FOXP3 antigen. The results are shown in Figure 5. Brown particles represent positive staining results, and semi-quantitative analysis was performed on them ( Figure  5G). Treg cell expression in the model group was significantly decreased compared with the control group. Furthermore, Treg cell expression was significantly increased in the GA, PC, and EA groups when compared with the model group, implying that GA, PC, and EA could reverse the decreased expression of Treg cells in ulcerative colitis.

Effect of Polyphenols on the Expression of Tight Junction
The intact intestinal epithelial TJ plays an essential role in preventing inflammatory reactions [31]. IHC staining was performed to elucidate the levels of the TJ proteins, zonula occludens 1 (ZO-1), claudin-2, and occludin. The results are presented in Figure 6. Compared with the control group, occludin levels in the model group were significantly decreased ( Figure 6A,D). Furthermore, occludin levels were significantly increased in the treatment groups compared to the model group. Compared with the control group, ZO-1 levels were significantly decreased in the model group ( Figure 6B,E). Furthermore, ZO-1 levels in the administration groups were significantly increased compared the model group. Compared with the control group, claudin-2 levels were significantly increased in

Effects of Polyphenols on Regulatory T (Treg) Cell Expression
The expression of Treg cells was detected using immunohistochemical (IHC)-staininglabeled FOXP3 antigen. The results are shown in Figure 5. Brown particles represent positive staining results, and semi-quantitative analysis was performed on them ( Figure 5G). Treg cell expression in the model group was significantly decreased compared with the control group. Furthermore, Treg cell expression was significantly increased in the GA, PC, and EA groups when compared with the model group, implying that GA, PC, and EA could reverse the decreased expression of Treg cells in ulcerative colitis. treatment groups, the epithelial cells were mild or non-degenerative/necrotic and a smal number of inflammatory cells were present. Among them, the histological characteristics of the GA and TA groups were similar to those of the healthy control group.

Effects of Polyphenols on Regulatory T (Treg) Cell Expression
The expression of Treg cells was detected using immunohistochemical (IHC)-stain ing-labeled FOXP3 antigen. The results are shown in Figure 5. Brown particles represen positive staining results, and semi-quantitative analysis was performed on them ( Figure  5G). Treg cell expression in the model group was significantly decreased compared with the control group. Furthermore, Treg cell expression was significantly increased in the GA, PC, and EA groups when compared with the model group, implying that GA, PC and EA could reverse the decreased expression of Treg cells in ulcerative colitis.

Effect of Polyphenols on the Expression of Tight Junction
The intact intestinal epithelial TJ plays an essential role in preventing inflammatory reactions [31]. IHC staining was performed to elucidate the levels of the TJ proteins, zon ula occludens 1 (ZO-1), claudin-2, and occludin. The results are presented in Figure 6 Compared with the control group, occludin levels in the model group were significantly decreased ( Figure 6A,D). Furthermore, occludin levels were significantly increased in the treatment groups compared to the model group. Compared with the control group, ZO-1 levels were significantly decreased in the model group ( Figure 6B,E). Furthermore, ZO-1 levels in the administration groups were significantly increased compared the mode group. Compared with the control group, claudin-2 levels were significantly increased in

Effect of Polyphenols on the Expression of Tight Junction
The intact intestinal epithelial TJ plays an essential role in preventing inflammatory reactions [31]. IHC staining was performed to elucidate the levels of the TJ proteins, zonula occludens 1 (ZO-1), claudin-2, and occludin. The results are presented in Figure 6. Compared with the control group, occludin levels in the model group were significantly decreased ( Figure 6A,D). Furthermore, occludin levels were significantly increased in the treatment groups compared to the model group. Compared with the control group, ZO-1 levels were significantly decreased in the model group ( Figure 6B,E). Furthermore, ZO-1 levels in the administration groups were significantly increased compared the model group. Compared with the control group, claudin-2 levels were significantly increased in the model group ( Figure 6C,F). Moreover, claudin-2 levels were significantly decreased in the GA, TA, and PC groups compared with the model group. The increased claudin-2 level was not improved in the EA group. the model group ( Figure 6C,F). Moreover, claudin-2 levels were significantly decreased in the GA, TA, and PC groups compared with the model group. The increased claudin-2 level was not improved in the EA group.  ; ns -no significance; * represents p < 0.05; ** represents p < 0.005; *** represents p < 0.0005; **** represents p < 0.0001.

Antioxidant Mechanism of Polyphenols
To determine the antioxidant effect of polyphenols, we determined nuclear-factorerythroid 2-related factor 2 (Nrf2), heme oxidase-1 (HO-1), and NAD(P)H dehydrogenase [quinone] 1 (NQO1) levels in the colon tissue. The IHC staining results for Nrf2, HO-1, and NQO1 are presented in Figure 6. The expression levels of Nrf2 ( Figure 7A,D) and HO-1 ( Figure 7B,E) in model group was decreased compared with the control group. However, Nrf2 and HO-1 levels were significantly increased in the treatment groups compared with the model group. Lastly, NQO1 levels in the TA and EA groups were also significantly increased compared with model group (Figure 7C,F).

Antioxidant Mechanism of Polyphenols
To determine the antioxidant effect of polyphenols, we determined nuclear-factorerythroid 2-related factor 2 (Nrf2), heme oxidase-1 (HO-1), and NAD(P)H dehydrogenase [quinone] 1 (NQO1) levels in the colon tissue. The IHC staining results for Nrf2, HO-1, and NQO1 are presented in Figure 6. The expression levels of Nrf2 ( Figure 7A,D) and HO-1 ( Figure 7B,E) in model group was decreased compared with the control group. However, Nrf2 and HO-1 levels were significantly increased in the treatment groups compared with the model group. Lastly, NQO1 levels in the TA and EA groups were also significantly increased compared with model group (Figure 7C,F).

Effect of Polyphenols on the Gut Microbiota
Changes in the intestinal microbiome are one of the characteristics of UC pathogenesis [32]. To further evaluate the protective effect of polyphenols on UC, we elucidated microbiota changes induced by the polyphenols via 16s rRNA sequencing. The Venn diagram shows three overlapping sets of operational taxonomic units (OTUs), suggesting that there is a significant difference in OTUS between the DSS group and the other two groups ( Figure 8A). In addition, compared with the control group, the alpha diversity of the microbiota in the model group was decreased, as indicated by a decrease in Chao index, Shannon, observed species, and PD whole-tree indices. However, the addition of polyphenols reversed the decline in the bacterial community richness index ( Figure 8B-E). The histogram in Figure 9 reveals the species and relative abundance of the intestinal microbiota at the phylum level. Compared with the control group, the abundance of Firmi-cutes decreased, and that of Bacteroidetes increased in the model group. After polyphenol treatment, Bacteroidetes decreased significantly and Firmicutes increased significantly. At the family level, the relative abundance of Staphylococcaceae was increased and that of Erysipelotrichaceae was decreased in the model group compared to that in the control group. The use of polyphenols reversed the increase of Staphylococcaceae, and TA and PC reversed the decrease of Erysipelotrichaceae. In addition, treatment with GA and EA increased the relative abundance of Lactobacillaceae, and treatment with GA increased the relative abundance of Bacteroidales. However, treatment with GA and EA decreased the relative abundance of Rikenellaceae, and treatment with GA and PC increased the relative abundance of Bifldobacteriaceae (Figure 10). At the genus level, the relative abundance of Staphylococcus was markedly increased in the model group compared with the control group. Treatment with polyphenols decreased the relative abundances of Staphylococcus compared with the model group. Interestingly, treatment with GA and PC increased the relative abundances of Lactobacillus (Figure 11).

Correlation Analysis
The correlation between intestinal microbiota and Nrf2, HO-1, NQO1, intestinal microbiota and Treg cells, inflammatory factors (IL-6, IL-10), immune cell (Treg cells), and tight junction proteins was analyzed. As shown in Figure 12A, UC pathogenesis includes changes in inflammation levels, breakdown of the intestinal barrier, and imbalance in the intestinal microbiome. The indicators involved in the three mechanisms were correlated with each other. Firmicutes and Proteobacteria were positively correlated with Nrf2, HO-1, and NQO1 levels; Bacteroidetes were negatively correlated with Nrf2, HO-1, and NQO1 levels; Bifidobacterium and Lactobacillaceae were positively correlated with Nrf2 and HO-1 levels ( Figure 12B). Furthermore, Firmicutes, Bifidobacterium, and Lactobacillus were positively correlated with Treg cells, whereas Bacteroidetes were negatively correlated with Treg cells ( Figure 12C). The proinflammatory, cytokine IL-6, was negatively correlated with ZO-1 and occludin and positively correlated with claudin-2, whereas the anti-inflammatory cytokine IL-10 and Treg cells were positively correlated with ZO-1 and occludin and negatively correlated with Cluadin2 ( Figure 12D).

Polyphenols Relieve Colitis-Associated Splenomegaly
We observed that the polyphenols significantly prevented the increase in spleen weight when compared with the model group ( Figure 13G). H&E staining results of th spleen are presented in Figure 13. The mice in the control group had more white pulp with the central artery located in the white pulp, and the marginal zone at the junction o

Polyphenols Relieve Colitis-Associated Splenomegaly
We observed that the polyphenols significantly prevented the increase in spleen weight when compared with the model group ( Figure 13G). H&E staining results of the spleen are presented in Figure 13. The mice in the control group had more white pulp, with the central artery located in the white pulp, and the marginal zone at the junction of the white and red pulp was obvious. In mice in the model group, the red pulp continued to extend, and the spleen was almost entirely composed of hyperplastic hematopoietic tissue and dominated by red tissue. The white pulp was significantly reduced. Furthermore, the marginal zone at the junction of white pulp and red pulp was inconspicuous. In GA, PC and TA group, there was a small degree of hematopoietic tissue hyperplasia, and the marginal zone at the junction of white pulp and red pulp was obvious. In EA group, there was no red pulp expansion and the marginal zone at the junction of white pulp and red pulp was obvious. The histological structure of the spleen in the EA group was similar to that of the spleen in the control group.

In Vivo Safety Evaluation
H&E staining results of the liver and kidney are shown in Figure 14. Histology ex amination found no damaging effects of polyphenol administration on the kidney and liver.

In Vivo Safety Evaluation
H&E staining results of the liver and kidney are shown in Figure 14. Histology examination found no damaging effects of polyphenol administration on the kidney and liver.

Discussion
In this study, we analyzed the antioxidant activity and the antioxidant structural relationship of the four polyphenols, and studied their alleviating effects on UC. Studies have shown that polyphenols inhibit the production of free radicals by inhibiting the ac-

Discussion
In this study, we analyzed the antioxidant activity and the antioxidant structural relationship of the four polyphenols, and studied their alleviating effects on UC. Studies have shown that polyphenols inhibit the production of free radicals by inhibiting the activities of the oxidoreductive enzymes and/or chelating to prevent the production of metals via free radicals [33][34][35]. The phenol group accepts electrons to form phenoxy, interrupting the chain oxidation reaction. Conjugated aromatic systems can delocalize unpaired electrons. The antioxidant capacity of polyphenols is related to the number of hydroxyl groups and characteristic structural groups such as the o-phenol group, in which o-phenol can make free radicals form with higher stability [36]. In the present study, TA had twenty-five phenolic hydroxyl groups, including five catechol groups and five pyrogallol groups; PC had eight phenolic hydroxyl groups, including two catechol groups; GA had three phenol hydroxyl groups, which are the pyrogallol group; and EA had four phenol hydroxyl groups, with two catechol groups. The antioxidant activity of polyphenols was measured via determining the DPPH radical scavenging activity, ABTS radical scavenging activity, and total antioxidant capacity. All four polyphenols exhibited good antioxidant capacity. At lower concentrations, TA still exhibited the strongest antioxidant properties, owing to the highest number of phenol hydroxyl and o-phenol groups, followed by PC. PC exhibited stronger ABTS free radical scavenging ability than GA, and EA ranks last. Among them, the scavenging ability of GA was better than that of EA; this suggests that when the number of phenol hydroxyl groups is similar, compounds with more pyrogallol groups exhibit better antioxidant activity than those with catechol groups [36].
UC is one of the most common gastrointestinal disorders characterized by chronic, recurrent inflammation that causes damage to the colon mucosa [31]. It can occur at all ages, leading to lifetime morbidity and even death [37,38]. Several studies have shown that polyphenols help relieve colitis [39,40]. In the present study, we elucidated the effects of four polyphenols in a DSS-induced UC mouse model. We observed that the polyphenols significantly improved colitis based on weight recovery, colon length recovery, and decreased DAI. In addition, several potential mechanisms were described, including ameliorating intestinal barrier damage, regulating inflammatory responses and oxidative stress, and reshaping of the intestinal microbiota of mice.
TJs include the transmembrane proteins, occludin and claudin family members, and proteins such as claudin-2 and ZO-1 [23,41,42]. ZO-1, a major TJ protein, is associated with epithelial integrity and can be used as a marker of the intestinal barrier. Occludin plays an important role in TJ stability and barrier function [42,43]. Claudin-2 is a typical pore-forming claudin that forms active gated channels, with selectivity for water and small cations. Studies have reported that claudin-2 levels are increased in IBD and that claudin-2 degradation can enhance the intestinal barrier of TJs [41,44,45]. In the present study, we demonstrated that occludin and ZO-1 levels were decreased and claudin-2 levels were increased after DSS administration and that the four polyphenols can increase ZO-1 and occludin levels, whereas GA, PC, and TA can decrease claudin-2 levels; these results indicate that the four polyphenols can relieve UC by repairing the intestinal barrier.
Increasing evidence suggests that proinflammatory cytokines are critical in UC pathogenesis. Elevated IL-6, a proinflammatory cytokine, levels are a major feature of UC [46][47][48]. In the present study, proinflammatory cytokine levels were increased in the model group. However, GA, PC, and EA treatment significantly decreased proinflammatory cytokine secretion. A study reported that the anti-inflammatory cytokine IL-10 can improve intestinal damage caused by UC [49]. Consistent with these findings, we observed that IL-10 levels were increased in the PC and EA groups. These results suggest that polyphenols play important roles in regulating intestinal inflammation in DSS-induced UC. In addition, Treg cells can play an anti-inflammatory role in various immune diseases by secreting anti-inflammatory cytokines, including IL-10, and inhibiting immune cell activity [50,51]. In the present study, we observed that GA, PC, and EA could significantly improve the acti-vation level of Treg cells; this was consistent with the tendency of polyphenols to regulate inflammatory cytokines.
Nrf2 is an important anti-inflammatory and antioxidant signaling pathway molecule that is closely associated with UC occurrence. It plays an antioxidant role by promoting the expression of the downstream molecules HO-1 and NQO-1 [31,52]. We demonstrated that polyphenols can increase the protein levels of HO-1 and Nrf2 in UC mice. Furthermore, EA and TA can increase NQO1 protein levels. These results suggest that polyphenols play an anti-inflammatory role by enhancing antioxidant activity.
Many studies have reported that intestinal microbial environment disorders play an important role in UC pathogenesis. Compared with healthy individuals, intestinal microbial disturbances in UC patients have demonstrated reduced diversity and richness, and changes in the intestinal microbiota composition [53,54]. Consistent with the findings of a previous study [2,23,55], we found that Firmicutes, Bacteroidetes, Actinobacteria, and Proteobacteria are the dominant phyla, and their compositions changed. After polyphenol intervention, these changes were reversed to varying degrees. Staphylococcus aureus is a kind of intestinal pathogenic bacteria [7,23]. Polyphenol intervention reduced the relative abundance of Staphylococcus aureus. Simultaneously, treatment with polyphenols decreased the relative abundance of Staphylococcus at the genus level. Lactobacillus and Bifidobacteria secrete inflammatory inhibitors by downregulating NF-κB-dependent gene expression, IL-8 secretion, and macrophage chemokine levels. Furthermore, they downregulate effector T-cell-mediated inflammatory responses but upregulate the expression of anti-inflammatory Treg cells in mice [7,56]. Some byproducts of short-chain fatty acids (SCFAs) exhibit anti-inflammatory effects and may enhance the intestinal barrier [7]. Bifidobacteria is an SCFA-producing bacteria [56]. GA and PC significantly increased the relative abundance of Lactobacillus and Bifidobacteriaceae. Erysipelotrichaceae is a potential probiotic [57], and a study has shown that it is a butyrate producing bacteria, which is a SCFAs [58]. In the present study, TA and PC significantly increased the relative abundance of Erysipelotrichaceae. A study clarified that the Bacteroidales S24-7 group can reduce pro-inflammatory activity and play an immunomodulatory role [59]. In our study, GA increased the enrichment of the Bacteroidales S24-7 group. Based on these results, polyphenols can relieve UC by regulating the imbalance of microbiota, increasing the abundance of beneficial bacteria, and reducing the abundance of harmful bacteria in the intestine.
In addition, studies have shown that gut microbes influence the gut's antioxidant response, which is essential for gut health [60,61]. Oxidative stress during the inflammatory response can reduce intestinal microbial diversity [62], whereas an imbalance in intestinal microbiota can increase oxidative damage and decrease antioxidant capacity by destroying the intestinal barrier [63]. Studies have reported that probiotics play beneficial roles in regulating oxidative stress responses. For example, lactic acid bacteria can improve the antioxidant capacity of the body, Bifidobacteriaceae can remove free radicals and superoxide anions, and enhance the activity of antioxidant enzymes [64]. Meanwhile, SCFAs can activate the Nrf2 pathway [60]; Bifidobacteriaceae is an SCFA-producing bacteria ( Figure 11A). Intestinal microbiota analysis revealed that GA, PC, and EA can improve the relative abundance of Lactobacillaceae and Bifidobacteriaceae. In addition, we determined the correlation between the abundance of intestinal microbiota and the expression of oxidative stress biomarkers. At the phylum level, when the relationship between oxidative stress biomarkers and intestinal microbial abundance was analyzed, Nrf2, HO-1, and NQO1 levels were positively correlated with the relative abundances of Firmicutes and Actinobacteria and negatively correlated with the relative abundance of Bacteroides. At the family level, when the relationship between oxidative stress biomarkers and intestinal microbial abundance was analyzed, Nrf2, HO-1, and NQO1 levels were positively correlated with Lactobacillaceae and Bifidobacteriaceae ( Figure 11B). The byproducts of SCFAs are butyrate, propionic acid, acetate, etc. Imbalance in the intestinal microbiota and an increase in intestinal inflammatory cells in patients with IBD are associated with a decrease in SCFA levels, which can regulate intestinal immunity and play an anti-inflammatory role by acti-vating the differentiation and expansion of Treg cells [65]. Studies have reported that some bacteria in Firmicutes can produce butyrate; lactic acid bacteria have butyrate-producing capacity; and Bifidobacterium can produce acetic acid, propionic acid, butyric acid, lactic acid, and other SCFAs [66]. In the present study, correlation analysis between the intestinal microbiota and Treg cells revealed that Firmicutes, Lactobacillus, and Bifidobacterium were positively correlated with Treg cells ( Figure 11C). Barrier integrity is mostly due to the normal functioning of the TJs between epithelial cells. TJs comprise transmembrane proteins that regulate intestinal barrier permeability by controlling the diffusion of water, ions, and nutrients while limiting pathogen entry. The intestinal barrier dysfunction can exacerbate inflammation throughout the body [67]. In the present study, we determined the correlation between inflammatory cytokine levels and intestinal TJ protein levels. We observed that ZO-1 and occludin, which exhibit protective effects on intestinal permeability, were negatively correlated with proinflammatory cytokines and positively correlated with anti-inflammatory cytokines and Treg cells. Claudin-2 protein levels, which increase intestinal permeability, were positively correlated with proinflammatory cytokines and negatively correlated with anti-inflammatory cytokines and Treg cells ( Figure 11D).
In summary, four plant polyphenols were shown to alleviate UC through multiple mechanisms. Many plants which are traditionally used in China contain these polyphenols. Lycium ruthenicum Murray (LR), for example, is a kind of medicinal and edible plant widely distributed in the salinized desert of Northwestern China. LR has been widely used in food, medicine, and other fields, as is of great economic value [68]. Studies have found that LR contains the four polyphenols [69][70][71]. Hence, the potential therapeutic mechanism of LR for ulcerative colitis can be explored.

Materials
TA and GA were purchased from Sigma Aldrich Chemie (St. Louis, Missouri, USA). EA and PC were purchased from Xi'an Green Biotechnique Co., Ltd. (Xi'an, China).

DPPH·-Radical-Scavenging Assay
The antioxidant activity of the four polyphenols were evaluated by measuring their stable-DPPH·-free-radical-scavenging ability [72,73]. A series of polyphenol compound solutions were prepared, (10, 5, 2.5, 1.25, 0.625, 0.3125, 0.1563, 0.0781, 0.625, 0.0391, and 0.0195 mM). Then, 0.006 g of DPPH· powder (Shanghai Macklin Biochemical Co., Ltd., Shanghai, China) was dissolved in 50 mL of anhydrous ethanol and stored in the dark. The samples and DPPH alcohol solution were mixed in 96-well plates and incubated for 30 min at 25 • C in the dark. The absorbance was measured at 517 nm using an enzyme-labeled instrument. DPPH· degradation was calculated using the equation from the previous study [74].

ABTS-Scavenging-Assay
The antioxidant activity of the four polyphenols was evaluated by measuring their scavenging ability on ABTS free radicals and preparing polyphenolic compound solutions of a series of concentrations, followed by the determination of the ABTS scavenging rates by using an ABTS free radical clear-ability assay Kit (Beijing Solarbio Science & Technology Co., Ltd., Beijing, China).

Total Antioxidant Capacity
The antioxidant activity of the four polyphenols was evaluated by calculating the total antioxidant capacity. The polyphenolic compounds solutions were prepared in a series of concentrations. The sample solution was added to the 96-well plate, to which the FRAP working solution (Beyotime, Shanghai China) was added and the solution was incubated at 37 • C for 3 min. The absorbance was measured at 593 nm wavelength by using an enzyme-labeled instrument. The total antioxidant capacity is expressed as the concentration of the FeSO 4 standard solution.

Experimental Animals
Forty-two male C57BL/6 mice (9-10 weeks of age) were purchased from SPF (Beijing, China) Biotechnology Co., Ltd. The mice were accommodated for 1 week. All the plans were approved by the Animal experiment Committee of Peking Union Medical College.

Animal Experimental Design
Mice were randomly divided into a control group (n = 6) and a DSS group (n = 36). The control group received normal water and the DSS group received 3% DSS (35~50 kD, MP Biologicals, Solon, OH, USA) solution (w/v) to induce acute UC. After 7 days, the mice in the DSS group were divided into the model group and GA, TA, PC, and EA groups, with an equal number of mice in each group. The control and model groups were administered normal water via gavage every morning for 6 days. Conversely, the treatment groups were administered a 100 mg/kg polyphenol solution.
The weight of the mice was recorded daily throughout the experiment. DAI combined weight loss score, stool diarrhea score, and fecal blood levels were used to evaluate UC severity [42,75]. On the 14th day, the mice were sacrificed, and the length of their colons was photographed and measured. Then, 5 mm of the colons was removed and cleaned with normal saline and fixed with 4% paraformaldehyde for subsequent sections and staining. The remaining colon tissues were washed with normal saline and quickly frozen with liquid nitrogen for subsequent analysis. The spleen tissue was flushed with physiological saline and then weighed. The spleen, liver, and kidney tissues were fixed in 4% paraformaldehyde for subsequent sectioning and staining. Fecal samples from mice were collected and stored at −80 • C for subsequent analysis. Serum was obtained via centrifugation (1200× g, 4 • C, 15 min) for measuring cytokine levels. The concentrations of IL-6 and IL-10 were measured by using an enzyme-linked immunoassay kit (Thermo Fisher Scientific, Massachusetts, MA, USA).

Histological Analysis
Colon tissue sections were fixed with paraformaldehyde and stained with H&E for morphological measurements. All sections were analyzed and photographed under a microscope (Leica, Wetzlar, Germany).

Impact on Gut Microbiota
The collected fecal samples were analyzed by 16S rRNA sequencing at Beijing Yike Baide Technology Co., Ltd. (Beijing, China) for intestinal microbiota analysis. Samples processing includes whole genome DNA extraction, PCR amplification, amplicon quantization, and high throughput sequencing. The sequencing results were analyzed on the company's interactive platform.

Statistical Analysis
GraphPad Prism 8.0.2 and origin 2021 software were used for mapping and statistical analysis, and the results were expressed by mean ± SD. One-way ANOVA and Tukey's test were used to analyze the differences between groups.

Conclusions
We elucidated the antioxidant effects of four polyphenols in plants and on DSS-induced colitis. Our study results indicate that the antioxidant capacity of polyphenols is associated with the number of phenolic hydroxyl groups and the structure of o-phenol groups. The studied polyphenols can alleviate UC, including decreasing DAI and improving histological alteration. The mechanism involves regulation of inflammatory factors, intestinal barrier repair, regulation of oxidative stress, and regulation of intestinal microbiota. The functions of several mechanisms are interrelated and influence each other. Moreover, the polyphenols have good biosafety. The results suggest that polyphenols play an important role in UC remission, may provide a basis for the development of products containing plant polyphenols in the prevention and treatment of colitis.