**3. Mechanisms**

Natural polysaccharides regulate the expression of cytokines engaged in the inflammatory responses of IBD through multiple signaling pathways (Figure 1). Polysaccharides could ameliorate IBD symptoms via decreasing the neutrophil infiltration and regulation of oxidative stress. The following is a discussion of the potential mechanism of the effect of polysaccharides on therapy for IBD in Table 2.

**Figure 1.** The effects of natural polysaccharides on IBD.

**Table 2.** The mechanisms of natural polysaccharides involved in colon inflammation amelioration.


### *3.1. TLRs-MAPK/NF-κB Mediated Signal Transduction Pathway*

TLRs belong to the pattern recognition receptors and they are widely found on the external sides of immune cells such as macrophages, neutrophils, and lymphocytes. In the immunity signaling pathway, the process of TLR-recruiting adaptors enhances the production of numerous pro-inflammatory cytokines, which is followed by the activation of pro-inflammatory signaling pathways and host defense pathogens by participating in the signal transducer and activator [87]. After binding to polysaccharide ligands, TLRs motivate TNF receptor-associated factor 6 (TRAF6), which consequently activates MAPK and NF-κB. The anti-inflammatory impact of polysaccharide from *Sarcodon aspratus* was checked into LPS induced-RAW264.7 macrophage cells and it turned out to increase the production level of TLR4 as well as the activation of macrophage cells. It revealed that SAP exerts its protective effect on the intestine by enhancing the immunity reaction. Otherwise, when treating DSS-induced colitis mice with FCPS, it not only decreased the TLR4 expression but also down-regulated the manufacturing of pro-inflammatory cytokines including IL-1β, TNF-α, and IL-6 [82]. The outcome explains that the mechanism of FCPS alleviates IBD by inhibiting the immunity reaction.

MAPK is a family of serine/threonine protein kinases that are comprised of c-jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase 1/2 (ERK 1/2) [88]. NF-κB is a transcription factor that is involved in the management of the expression of critical immunity and inflammation regulatory genes [89]. NF-κB always keeps an inactive complex form binding with inhibitory protein IκB in the normal condition [38]. As the cells are stimulated, NF-κB is activated and IκB is phosphorylated by IKK. Then, the activated complex form is transferred into the nucleus and regulates the expression of inflammatory cytokines and proteins. The underlying mechanism of polysaccharide- regulated intestine protective effects is considerably connected with the governed expression of MAPK and NFκB. For example, when either of the polysaccharides obtained from *Ganoderma lucidum* and *G. sinense* or *Pleurotus eryngii* was administrated with LPS-induced RAW264.7 macrophage cells, the results revealed that all of the phosphorylation level of ERK, JNK, p38, and NF-κB were up-regulated [38,84], which indicated that all of these polysaccharides enhance the immune regulation effects. In another study, treating a high dose of DIP could significantly reduce the phosphorylation level of ERK and NF-κB and pro-inflammatory cytokines in DSS-induced mice [23]. These results are consistent with the previous results that polysaccharides have immunomodulatory activities on the immune cells, and further confirm that polysaccharides could ameliorate IBD via modulating the MAPK and NF-κB signaling pathway.

#### *3.2. G-Protein-Coupled Receptors*

G-protein-coupled receptors (GPRs) are a family of receptors for different second messengers. It has been demonstrated that GPRs highly expressed in the gastrointestinal tract could ameliorate IBD by promoting the colon epithelium repair [90] and improving the glucagon-like peptide-2 production [91]. In the previous study, GPR43 has been found in the neutrophils and colon [92]. GPR43 is vital to neutrophil recruitment in intestinal inflammation and their deficiency induces severe inflammation. SCFAs would bind to GPR41/43 and influence anti-inflammation activity in the cells [85]. SCFAs metabolized from natural polysaccharides also stimulate the GPR43 on colonic T cells followed by up-regulating the expression of Foxp3 [90] and facilitating peripheral T-reg cells, which finally inhibits the inflammatory response in the intestinal mucosa [35]. Otherwise, activated GPR43 involves the NF-κB pathway, which inhibits the expression of pro-inflammatory cytokines such as IL-6 and IL-1β. It also indicates that GPR43 is beneficial to attenuate the inflammation responses in the gut [93]. β-arrestin1, one of the important regulators of the GPR signaling pathway [94], makes a crucial contribution in the activation of nucleotide leucine repeat pyrin 3 (NLRP3) inflammasome [95]. NLRP3 inflammasome exerts importance in the inflammation and immunity responses via the caspase-1 activation and IL-1β maturation. The motivation of NLRP3 inflammasome could aggravate gut inflammation [96]

and the variant of NLRP3 in the cells would trigger severe IBD [86]. Therefore, the proinflammatory effects of β-arrestin1 and NLRP3 were mediated through the cytokines in the inflammatory intestine like the DSS-induced colitis models. However, the researchers have offered evidence that administration of *Dendrobium officinaleon* polysaccharides (DOPS) ameliorated the injury of the colon and moderated the symptoms of colitis including the rapid body weight loss and the somatotype decreased in DSS-induced mice. The inhibition effect of DOPS to the NLRP3 inflammasome pathway was showed by down-regulation of the IL-1β, IL-18, and caspase-1 expression. The results also displayed that DOPS could suppress the β-arrestin1 signaling pathway. Moreover, in vitro experiments indicated that DOPS significantly inhibit their expressions in the LPS-stimulated NCM 460 cells, the results were the same as before in vivo [97]. Above all the results have indicated that natural polysaccharides could regulate the expression of GPRs on behalf of alleviating the IBD.

#### *3.3. Increase Intestinal Integrity via Up-regulating Tight Junction Proteins*

Gut integrity is vital to maintaining the host's innate immunity, which could prevent intestine damage from lipopolysaccharides (LPS) and toxins [98]. The tight junction proteins such as the ZO family, claudins, and occludins are responsible for the diffusion of epithelium and among cells. Numerous inflammatory responses would affect the tight junction proteins expressions and since colitis makes the levels change, the expression of tight junction proteins is always decreased in IBD [99]. TNF-α plays an important role in the mediation of occludin internalization, and the occludin has a positive relationship with gut permeability. Although the cytokines increase the gut permeability, overexpression of occludin could ameliorate it [100]. Moreover, the other mechanism involved in gut integrity regulation is TNF-α activation of the NF-κB signal transduction pathway. The inhibition of NF-κB could prevent colitis mice from having diarrhea and serious loss of water, which demonstrated the modulatory function of NF-κB in barrier capability [101]. In the colitis patient, TNF-α and IL-1β not only increase the infiltration of neutrophils, but also cause damage to the intestinal barrier, which may induce diarrhea symptoms in patients [102]. Otherwise, TNF-α attracts the myosin light-chain phosphorylation, which damages the tight junction protein. Then the risky factors and microbes like LPS, the signals from gram-negative bacteria, could enter the intestine through the mucous membrane leak, which would bind to toll-like receptors and stimulate the TLR-mediated signaling pathway [103], further provoking the inflammatory response. IFN-γ diminishes the ZO-1 and occluding expression through the adenosine monophosphate-activated protein kinase dependent pathway without regard to the energy level of the cell. Therefore, it also could increase barrier permeability [104]. FCPS treatment could ameliorate colitis symptoms by restoring the expression of light junction protein claudin-1 and down-regulating the level of TNF-α and IL-1β in the DSS-induced colitis mice [82]. The animal models' results proved that FCPS could facilitate the repair of damaged intestinal barriers, thus blocking the penetration of endangering molecules and microbes through the mucous membrane [105]. In addition, the gastrointestinal protective effect of DIP has been reported to recover the expression level of claudin-1, occludin, and ZO-1 in the DSS-induced colitis mice [27]. These studies indicated that despite the fact that these inflammatory cytokines are always presented in the intestine at the same time during inflammation and harmful to the patient due to the disconnection of the tight junction proteins [106], the natural polysaccharides could restrain the response of colon inflammation and promote IBD recovery via increasing the tight junction protein expression in the gut and thereby protect the colon structure of the IBD patient (Figure 2).

**Figure 2.** The summary of mechanisms about polysaccharides involved in IBD patient intestine.

#### *3.4. Regulation of Oxidative Stress*

In the intestine, immunity plays a critical role in the pathogenesis of IBD. As the inflammation induces the T-cell response through the secretion of inflammatory cytokines, the other activated immune cells, including macrophages and neutrophils [107], induce significantly increased production of oxygen free radicals, which cause further damage in colon tissue. The synthesis of oxygen free radicals and the antioxidase in the intestine, including ROS, myeloperoxidase (MPO), nitric oxide (NO), malondialdehyde (MDA), plasmic diamine oxidase (DAO), and glutathione (GSH). ROS have various physiological functions but under the condition of oxidative stress, overproduction of it is harmful to intestine cell membrane lipids [108], DNA, and proteins. ROS also are responsible for causing diarrhea, one of the most common symptoms in IBD, due to the excessive secretion of water and electrolytes [109]. MPO, as one of the reactive oxygen species, is always regarded as the inflammatory marker of neutrophil infiltration. However, polysaccharides might ameliorate damage through inhibiting neutrophil infiltration in the gut. Han et al. have treated DSS-induced mice with SP and found the expression level of MPO significantly decreased in the gut [44].

After being stimulated by oxidative stress, iNOS belonging to reactive oxygen/nitrogen species (ROS/RNS)-generating systems catalyzes the production of NO. As one of the markers of inflammation, NO is released by the neutrophils in damaged tissues and induces an immunity inflammatory response, whose generation has a positive relationship with the level of oxygen free radicals [26]. Prior research has shown that administrated FVP would lead to a decline in NO content in the gut of DSS-induced colitis models [34]. Except for promoting the production of NO, iNOS also improves the expression of COX-2 in the meanwhile. COX-2 could catalyze prostaglandin production and finally induce the inflammation response. Shao et al. administrated acetic acid-induced mice with EP-1 and found both the expression of COX-2 and related mRNA were down-regulated, which might prove that polysaccharides could balance the immunity in the intestinal system via regulating the oxidative stress, following subsequently moderate IBD [14]. The above results have indicated that polysaccharides make a critical contribution to the down-regulation

of reactive oxygen species and therefore exert an essentially protective effect on the colon of patients.

The different concentration of MDA and GSH in the tissues has revealed that diverse levels of oxidants caused by the epithelial cell rupture. Dutra et al. have confirmed that *G. caudata* PLS suppresses the acetic acid-induced UC mice [43], which induces the restoration of the GSH and down-regulation of MDA level in the cells. In the pathogenesis of IBD, above all the results have proved that polysaccharides could decrease the production of reactive oxygen species and their metabolites as well as increase the level of anti-oxidant molecules meanwhile to ameliorate IBD that is caused by prooxidant substances.

#### **4. Future Outlooks and Conclusions**

Natural polysaccharides have the potential ability to be used in IBD treatment. Many types of polysaccharides from mushroom, seaweed, herbs, and plants can be fermented in the colon, which not only changes the diversity of gut microbiota, but also recovers gut health via stimulating various types of immune cells and motivating numerous immunityrelated signaling pathways. The main mechanism of polysaccharides on inflammatory bowel disease relies on immune regulation, anti-oxidation, and regulation of probiotics in the intestine.

Natural polysaccharides have attracted considerable attention because of their low side effects, nontoxicity to individuals, and easy availability in diet. However, further examination is necessary to explore the link between natural polysaccharides and immune regulation in diseases, together with analyzing the variety of microbiota in the intestine. Systemic studies would help create better understanding of the specific mechanism that the function of polysaccharides exert on diseases. Thus, complementary alternative treatments could be provided to IBD patients. Additionally, for the purpose of better applying polysaccharides to normal immunity recovery, more clinical work is needed on the related foods and drugs to ensure the safety of dosage and half-life time. More innovative polysaccharide-based foods and medicines for IBD can be expected under a comprehensive understanding of the structure, biological activities, and underlying mechanism of polysaccharides.

**Author Contributions:** Conceptualization, Y.W.; Writing—original draft preparation, Y.W.; Validation, H.Z.; Investigation, Y.Y.; Visualization, Writing—review, X.W.; Writing—review, supervision and editing, J.X. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Studies not involving humans or animals.

**Informed Consent Statement:** Patient consent was waived.

**Data Availability Statement:** Data is contained within the article.

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

#### **References**

