The pumpkin (C. moschata) is one of the most important vegetables in traditional agricultural systems worldwide [23] and has considerable nutritional and health-protective values. Some pumpkin polysaccharide components may play a role in the treatment of IBD [24]. Polysaccharides are the major chemical constituents of C. moschata and possess important pharmacological activities. However, currently, there are various extraction methods with no unified or standardized methodologies. The uncertainty and complexity of PP compositions limit their further development. Many extraction and purification methods have been developed to improve the total yield and purity of PPs. Several studies have isolated a series of polysaccharides from pumpkins and preliminarily determined their molecular weight and monosaccharide composition, including glucose, galactose, rhamnose, arabinose, galacturonic acid, glucuronic acid, mannose, xylose, and fucose, with Mw ranging from 103 to 106 Da [7]. Based on the methods of extraction, separation, and purification reported in other studies [25], we improved upon and designed a simple and efficient extraction method with high yield, high purity (> 97%), and relatively simple steps. We extracted high-purity PPs using an improved method and found that PPs are mainly composed of mannose, rhamnose, galacturonic acid, galactosamine, glucose, and xylose with Mw of 3.10 × 105 Da. These results are consistent with those obtained for polysaccharides isolated from C. moschata [26,27].
Natural plant polysaccharides always have low toxicity and possess immune and prebiotic activities, with positive effects in alleviating IBD by regulating intestinal flora imbalance, repairing intestinal barrier injury, and improving immunity [3,28]. Previous studies regarding polysaccharides from pumpkin fruit have mainly focused on their anti-oxidant, anti-diabetic, anti-tumor, immune regulatory, anti-coagulant, and other properties [7,26]. In addition to its repeatedly-proven antioxidant activity, PPs have also been shown to regulate intestinal microbiota disorders [27,29]. Dehydrodiconiferyl alcohol, a lignan compound isolated from C. moschata, display anti-UC effects on a DSS-induced colitis model by suppressing the production of TNF-α and IL-1β, as well as preventing weight loss and colon shrinkage [30]. These preliminary studies suggest that PPs exhibit anti-oxidant, anti-inflammatory, and intestinal microbiota effects, which may contribute to its therapeutic potential against UC. Although PP did not show significant anti-inflammatory activity in vitro, reasonable changes of many inflammatory factors (TNF-α, IFN-γ, IL-1β, IL-4, IL-6, IL-10, and IL-18) were still detected in the colon tissue of UC mice after PPs treatment. These results suggest that PPs might play an anti-inflammatory role through some mechanism after the metabolism of gut microbiota (Fig. S2E-K). The correlation between the relative abundance of gut microbiota and these inflammatory levels were analyzed using correlation heatmap analysis (Fig. S3E). IL-4 was significantly positively correlated with the increased abundance of Mailhella and Lachnospiraceae_NK4A136_group after PPs treatment. These results expand the application range of PPs’ anti-inflammatory activity. The repair of the intestinal mucosa and the regulation of intestinal microbiota are at least the two anti-UC mechanisms of PPs.
The diversity and abundance of intestinal microbiota are important indicators that reflect the composition of the intestinal microbiota and intestinal health status [31]. At the phylum level, Firmicutes and Bacteroides were the dominant phyla in the intestinal microbiota of the control mice, and Firmicutes/Bacteroidetes ratio is always regarded as a hallmark of UC. Our results are consistent with those of other studies [32,33]; the abundance of Firmicutes/Bacteroidetes ratio in mice was always reduced after DSS administration, whereas PPs could reverse this ratio and make it closer to the control group. In addition, the enrichment of Deferribacteres [34], and Proteobacteria [35,36] have been reported to associate with colitis and intestinal barrier injury. The relative abundance of Campylobacterota often shows a decreasing trend after DSS intervention [37]. These results were consistent with the increase of their relative abundance after DSS administration in our results and PPs could reverse these changes. As for the main genus-level changes, Bacteroides and Escherichia-Shigella commonly exhibit a higher relative abundance in IBD individuals and further led to severe colitis [38]. We also detected an increase in the relative abundance of them in the model group. In addition, two probiotics, the Faecalibaculum_rodentium and Oscillospiraceae play important roles in the regulation and stabilization of the intestinal microbiota, and the abundance of these two bacteria always decrease in UC mice after DSS stimulation [15,16]. Our results showed that PPs and SASPs could significantly reverse the changes in these bacteria after DSS treatment, suggesting that variations in the abundance of these bacteria may have mainly contributed to the anti-UC effects of PPs.
Tryptophan metabolism is dysregulated in IBD and can be metabolized by commensal microbiota to produce multiple indole metabolites, including 5-HIAA, with diverse effects on mucosal immunity and homeostasis [39]. Serotonin signaling, as one of the metabolic pathways of tryptophan, may influence the intestinal immune response by modulating the gut microbiota composition, resulting in increased susceptibility to colitis [40]. Serotonin (5-HT) is produced by the EC cells and by the serotonergic neurons of the myenteric plexus within the bowel. Upon release, 5-HT is inactivated by the serotonin reuptake transporter and is broken down into 5-HIAA. 5-HT influences the immune response and intestinal inflammation [41]. Although enhanced levels of 5-HIAA have been reported in several studies of various colon inflammatory conditions [40,42], research on the effect of 5-HIAA in UC mice is scarce. In this study, we found for the first time that 5-HIAA alone had a significant anti-UC effect during the validation of the activity of a large number of metabolites obtained from the metabolomics results. In addition, we also discovered and validated multiple metabolites with similar anti-UC activities, such as propionic acid, 2-hydroxyacetic acid, indole-2-carboxylic acid, 5-hydroxyindole-3-acetic acid, L-alanyl-L-proline, cholic acid, and phenylbutyric acid — among others.
With the exception of the ability to adjust the abundance and diversity of intestinal flora, natural plant polysaccharides usually have the ability to regulate the abnormal expression of tight junction proteins so as to repair intestinal barrier damage [3]. After careful analysis of the colon tissue transcriptome, we found that, with the exception of some common classical signaling pathways, Rap1 related pathways may be most closely related to the anti-UC effect of PPs [43]. Rap1 plays a role in diverse processes such as cell proliferation, adhesion, differentiation, and embryogenesis. After the activation of Rap1, Rap1GTP is transferred to the cell membrane and exerts various pharmacological effects by affecting multiple downstream signaling pathways, such as activating the common Raf-1/B-Raf-MEK pathway to promote intestinal epithelial cell proliferation and that activating the GSK3β/β-catenin pathway plays a role in repairing the mucosal barrier [44–46]. Therefore, the upregulation of Rap1GTP is considered a key factor in achieving various therapeutic effects, including the treatment of UC. Some reports have suggested that serotonin activates Rap1 related pathways to promote cell adhesion and other cellular activities [47]. As a metabolite and structural analog of serotonin, we speculated and preliminary confirmed that 5-HIAA may also have similar activities. cAMP is an endogenous mechanism that downregulates the inflammatory response and prevents the progression of the acute inflammatory response to chronic inflammation and associated tissue destruction [48]. We preliminarily confirmed, for the first time, that 5-HIAA activates the Rap1 pathway, which may be related to an increase in cAMP concentration.
We found and preliminarily confirmed the therapeutic potential of PP in UC and other disorders related to intestinal microbiota disturbances, which has great originality and expands the scope of C. moschata for the treatment of inflammatory diseases. Plant polysaccharides usually have low toxicity and C. moschata is a common food widely consumed worldwide, suggesting the potential value of PPs for further development [7]. In addition, we identified a new active metabolite (5-HIAA) and a new therapeutic modality (Rap1 activation) for UC after improving gut microbiota disorders. This study provides a new explanation for the mechanism of action of gut microbiota regulation in the treatment of UC. However, there are still many unsolved problems and shortcomings present in this study. For example, no systematic toxicological studies have evaluated the toxicity of PPs. Moreover, there are various extraction methods for PPs, and there remains a lack of unified and fixed extraction processes. PPs obtained by our extraction method showed the advantages of high yield, high purity, and process stability. PPs showed significant anti-UC activity, which warrants further study of its active monomer components and pharmacological mechanisms. Finally, the mechanism of action has not yet been fully elucidated upon and more data from transgenic animal or human studies are needed to fully confirm these findings.
In conclusion, we extracted, isolated, and identified the main polysaccharide components of Pumpkin (PPs). PPs protect against DSS-induced colitis by improving intestinal barrier function and microbial dysbiosis. Meanwhile, PPs promotes the enrichment of the microbiota-derived 5-HT metabolite 5-HIAA, which induces the elevation of cAMP and activation of the Epac/Rap1 signaling pathway, contributing to the effects of PPs on the improvements of intestinal functions. These findings suggest that PPs, 5-HIAA, and Epac/Rap1 activators might be used to treat UC and other intestinal dysfunctions.