Discussion
S. ningpoensi s is a traditional Chinese medicine that has been utilized for an extended period in the treatment of T2DM ( Yan et al., 2022 ). In T2DM rats, S. ningpoensis polysaccharide (SNP) can enhance the metabolism of lipid and glucose ( Ma et al., 2019 ). Although previous research has shown that SNP is anti-diabetic, the potential effect mechanisms of SNP on gut microbiota composition and metabolism in T2DM patient remain to be clarified. On the basis of the aforementioned theoretical background, SNP probably shows a probiotic effect and thus improves T2DM through regulation of composition structure and metabolite profile of gut microbiota. In the present study, we extracted crude polysaccharide from S. ningpoensis and conducted in-vitro fermentation to detect differences in gut microbiota and metabolites associated with T2DM after treatment of SNP.

Our results found that SNP is predominantly indigestible in the stomach and small intestine, which enhances its interaction and utilization by gut microbiota. SNP significantly reduced microbial community diversity, probably due to the insufficient carbohydrates present during in-vitro fermentation, which restricted the proliferation of gut microbiota ( Wu et al., 2023 ). It is widely acknowledged that T2DM is associated with a low ratio of F/B ( Fassatoui et al., 2019 ; Hamasaki-Matos et al., 2021 ). The increase in the F/B ratio observed in SNP indicates its potential therapeutic relevance in the management of T2DM.

In addition, SNP selective modulation of the growth of specific bacterial genera has the potential to be beneficial for host health, such as Parabacteroides , Faecalibacterium, Lachnospira. Butyricicoccus, Bifidobacterium , and Lactobacillus. Phascolarctobacterium is also favorable to the human body considering the metabolic characteristics of succinic acid and it also represents a new biomarker in the early diagnosis of T2DM ( Li, Hu & Xiong, 2023 ). Furthermore, Lachnospiraceae, Butyricicoccus, Parabacteroides , Bifidobacterium , and Lactobacillus can ferment plant polysaccharides to produce SCFAs, which exert anti-inflammatory and immune-enhancing properties ( Ragavan & Hemalatha, 2024 ; Wei et al., 2018 ; Wu et al., 2019b ). Seemingly, Bifidobacterium is a genus with the highest consistent literature support and including microbes with a potential protection effect against T2DM. Almost all related reports suggest that this genus is negatively associated with T2DM ( Gao et al., 2018 ; Gurung et al., 2020 ). Bifidobacterium is able to enhance synthesis of glycogens and down-regulate expressions of genes related to hepatic gluconeogenesis ( Kim et al., 2014 ). Among potentially probiotic bacteria, the highly diverse genus Lactobacillus includes most OTUs in the human guts. Patient-control cross-sectional research shows that abundance of Lactobacillus is positively associated with that of T2DM ( Candela et al., 2016 ; Ni et al., 2018 ). Researchers reported that Bifidobacterium and Lactobacillus may operate synergistically for improvement of symptoms related to human T2D ( Gurung et al., 2020 ). Lactobacillus and Bifidobacterium generate bile salt hydrolases that enable conversion of primary conjugated bile salts into deconjugated bile acids (BAs), which are then transformed into secondary BAs. Secondary BAs allow activation of the membrane bile acid receptor (TGR5), thus inducing production of GLP-1 ( Allin, Nielsen & Pedersen, 2015 ). SNP inhibited the excessive production of harmful bacteria. The influence of pathogenic Escherichia - Shigella on the host is significant. These pathogens affect the host through various virulence factors, including enterotoxins, Shigella-like toxins, and adhesin fimbriae. Additionally, they can cause damage to the intestinal mucosa, disrupt the actin cytoskeleton of the host cell, and stimulate the secretion of pro-inflammatory cytokines, which can in turn modify the expression of intestinal tight junction proteins ( Zhang et al., 2022a ). As gram-negative bacteria, Escherichia-Shigella and Desulfovibrionaceae are significantly related to the inflammation, LPS level, IR, and T2DM ( Pedersen et al., 2016 ; Thingholm et al., 2019 ; Wu et al., 2019a ). SNP supplements suppressed abundances of Escherichia-Shigella and Desulfovibrionaceae , in which bacteria served as conditional pathogens in the previous statement.

The elevated level of pro-inflammatory cytokines is a significant factor in T2DM. Gut microbiota enhanced release of inflammatory cytokines through the production of LPS. LPS can promote low-grade inflammation and endotoxemia. Conversely, certain microbiota are recognized to cause increased levels of anti-inflammatory cytokines, such as IL-22 and IL-10, which can enhance the insulin sensitivity, particularly those from the Bacteroides and Lactobacillus ( He et al., 2023 ; Qiu et al., 2017 ). Lactobacillus has been shown to decrease the effects of LPS-related pathology, particularly by diminishing β-cell dysfunction ( Tian et al., 2016 ). Prior study indicated that Faecalibacterium is negatively correlated with the IL-6 level, likely attributable to its production of butyrate, which serves to inhibit the activation of NF-B ( Di Sabatino et al., 2005 ). In summary, SNP can modulate the diversity and structure of gut microbiota, and increase abundances of beneficial bacteria while decreasing those of pathogenic bacteria. This is the probably the underlying mechanism of hypoglycemia, enhancing insulin resistance and inflammatory responses.

As SNP cannot be digested in the human small intestine, they are able to reach the colon and promote selective growth of beneficial bacteria. These bacteria can generate metabolites conducive to human body, of which the most significant are gas and SCFAs ( Tsukuda et al., 2021 ). Microbiota and chemical interactions in the distal small intestine and colon enable the production of CO 2 , CH 4 , H 2 , and H 2 S. H 2 and CO 2 are released during carbohydrate fermentation, while CH 4 and H 2 S are generated by metabolism of H 2 and CO 2 under the action of methanogens and sulphate reducers ( Zhao et al., 2023 ). The level of total gas production, CO 2 , CH 4 , H 2 , and H 2 S reduced in the T2DM group ( vs . HC group), which was reversed by SNP (T2DM + SNP group vs . T2DM group). The findings are consistent with previous research suggesting that the concentration of H 2 in exhaled air is lower in T2DM compared to the healthy group ( Misnikova et al., 2024 ). Prior study indicated that molecular hydrogen can reduce the content of fasting blood glucose, enhance the insulin sensitivity and improve the hepatic glycogen synthesis. Moreover, H 2 can reduce the pathological changes of kidney and pancreatic islets associated with T2DM ( Ming et al., 2020 ). H 2 S reduced fasting blood glucose level, increased insulin sensitivity, and improved glucose tolerance, accompanied by enhanced phosphorylation of Akt and PI3K in muscle ( Xue et al., 2013 ). The volume of gas produced affects the expansion of colonic walls and in turn influences the material transition rate via the colon ( Mutuyemungu et al., 2023 ). Moreover, the great discrepancy between individuals in terms of the gas production pattern is found to be related both to the characteristics of gut microbiota and the available substrates ( Kalantar-Zadeh et al., 2019 ).

SCFAs, also called volatile fatty acids, are organic acids that contain between one and six carbon atoms. They are main metabolites attained by bacterial fermentation of prebiotic polysaccharides in the gut and vital for the health of human intestine ( Bai et al., 2023 ). SCFAs absorbed in the cecum and colon, contributing for about 5–10% to the energy of host supply. They can enter systemic circulation through the portal vein, thereby affecting hepatic and peripheral tissues by directly regulating metabolism or tissue functionality ( Fagundes et al., 2024 ). SCFAs not only serve as essential energy substrates but also play an important role as signaling molecules ( Liu et al., 2024a ). They have significant physiological effects on various organs, including the pancreas and liver, through multiple complementary pathways. This action enhances insulin sensitivity and glucose metabolism on different levels and influences the progression of T2DM ( Pham et al., 2024 ; Xie et al., 2025 ).

The levels of total SCFAs (acetic acid, propionic acid, butyric acid, isobutyric acid, and valeric acid) reduced inT2DM group ( vs . HC group), which were reversed by SNP (T2DM + SNP group vs . T2DM group), thereby corroborating the findings of prior study such that the levels of total SCFA, acetic acid and butyric acid reduced in T2DM and pre-diabetic states ( Wu et al., 2020 ; Yang et al., 2024 ). The phenomenon may be explained by a significant increase in the Firmicutes , which can degrade polysaccharides to oligosaccharides and monosaccharides. The majority of butyric acid producers belong to the Clostridium cluster within the phylum Firmicutes ( Singh et al., 2022 ). SNP significantly increased the level of SCFAs, likely due to the enrichment of the Dorea species. Our data showed that the Dorea was positively correlated with butyric acid. The levels of butyric acid and the butyrate-producing bacteria in the gut frequently decreased in pre-diabetes and T2DM. Reversal of this trend can improve disturbances in glycometabolism ( Zhang et al., 2022a ). Butyric acid, an anti-inflammatory metabolite, is acknowledged for its role in inhibiting pathways that drive the production of pro-inflammatory cytokines ( Hodgkinson et al., 2023 ). It also rescues palmitate-induced insulin resistance by enhancing insulin signaling ( Rios-Morales et al., 2022 ). Additionally, butyric acid has been demonstrated to limit the translocation of LPS in the intestines, thereby alleviating LPS-related effects ( Bakshi & Mishra, 2025 ). In contrast to butyric acid, acetic acid has been associated with increased insulin resistance, enhanced insulin secretion, and elevated ghrelin secretion in response to glucose. These effects are primarily mediated through the activation of the parasympathetic nervous system ( Petersen et al., 2019 ). SCFAs enhance metabolic functions of T2DM through FFAR3 (or GPR41) and FFAR2/GPR43 ( e.g ., controlling the blood glucose level, as well as IR and GLP-1 secretion) ( Puddu et al., 2014 ). GPR43 shows particular responses to nutrients derived from intestinal flora (such as butyrate, propionate, and acetate) ( Yang et al., 2020 ). SCFAs play a significant role in stimulating the secretion of GLP-1, which increases the expression of hepatic peroxisome proliferator-activated receptor α (PPARα) and activates the AMPK pathway. This activation contributes to promote IR and fat oxidation by inhibiting gluconeogenesis and hepatic adipogenesis ( Zhang et al., 2019 ). Furthermore, GLP-1 interacts with its receptor on pancreatic islet beta cells to facilitate increased insulin secretion ( Zaïmia et al., 2023 ). Besides, SCFAs also as histone deacetylase (HDAC) inhibitors, can up-modulate the expression of metabolism-related genes, such as AMPK, PPARα, and uncoupled protein 1 (UCP-1) by epigenetic modifications ( Chen et al., 2024 ; Zhang et al., 2023 ).

Gut microbial metabolites were investigated in the present study. SNP have the potential to regulate the gut metabolites especially amino acid dysbiosis. Amino acid has emerged as a novel biomarker for T2DM prediction. Amino acid is the basic unit of protein and the main repository of glucose production and it also shows influences on glucagon and insulin secretion. Reports show that branched chain amino acids (BCAAs, such as leucine, isoleucine, and valine) are signaling molecules that regulate glucose, lipid, and protein metabolism, and have the potential to elevate the risks of T2DM and IR ( Lynch & Adams, 2014 ; Nie et al., 2018 ). Isoleucine and leucine are insulinotropic, while isoleucine and valine exert gluconeogenic effects. Leucine influences the function of insulin receptors by activating mammalian targets of rapamycin complex 1 (mTORC1) ( Ding, Wang & Lu, 2023 ). The correlation revealed that Parabacteroides was positively correlated with L-leucine, L-valine, and L-isoleucine. Here we found that SNP increased the abundance of Parabacteroides and enhanced BCAA catabolism ( Qiao et al., 2022 ). By profiling responses of the above data to SNP, functions and products of gut microbiota and their influences upon T2DM can be revealed.