How does Astragalus polysaccharide-D1 enrich Staphylococcus tarda and improve the effect of low-dose metformin?

Abstract

To explore adjunctive therapy for low-dose metformin, a homogeneous polysaccharide, designated APS-D1, was purified by DEAE-52 cellulose and Sephadex G-100 column chromatography. Its chemical structure was characterized by molecular weight distribution, monosaccharide composition, infrared spectroscopy, methylation analysis, and nuclear magnetic resonance spectroscopy. The results showed that APS-D1 (7.36 kDa) is composed of glucose, galactose, and arabinose (97.51%:1.56%:0.93%). It consists of a →4)-α-D-Glcp-(1→ residue backbone and →3)-β-D-Galp-(1→ residue and terminal-α/β-D-Glcp-(1→ side chains). APS-D1 can significantly improve inflammation (TNF-α, LPS, and IL-10). APS-D1 improves the efficacy of low-dose metformin without adverse reactions. APS-D1 combined with low-dose metformin regulates several intestinal bacteria, among which APS-D1 enriches Staphylococci to produce L-carnitine (one of the 136 metabolites of Staphylococcus lentus). Staphylococcus lentus and L-carnitine can improve diabetes, while reducing the production of L-carnitine by Staphylococcus lentus impairs the improvement of diabetes. The combination of Staphylococcus lentus and L-carnitine can promote fatty acid oxidation (CP T1), inhibiting gluconeogenesis (PCK and G6Pase). The results indicate that APS-D1 enhances the efficacy of low-dose metformin in improving diabetes by enriching Staphylococcus lentus, with the effect of S. lentus being mediated by L-carnitine. Taken together, these findings support that low-dose metformin supplementation with APS-D1 may be a beneficial therapeutic strategy for type 2 diabetes.

Introduction

Polysaccharides are natural polymers that have attracted widespread attention for their diverse bioactivities, including antitumor and immunomodulatory activities. The bioactivity of polysaccharides is directly related to their structure, including monosaccharide composition and glycosidic bonds. Astragalus polysaccharide is one of the main active components of the tonic Chinese herbal medicine Astragalus. Astragalus polysaccharide (Astragalus) is extracted from the roots and stems of Astragalus membranaceus. Polysaccharide (APS) has multiple pharmacological effects, including immune regulation, anti-inflammatory, blood sugar lowering, and lipid lowering. For example, APS has shown potential anti-inflammatory activity by inhibiting the production of inflammatory factors. APS can promote the growth of Desulfovibrio vulgaris and produce acetic acid. This inhibits the expression of fatty acid synthase, which helps prevent non-alcoholic fatty liver disease. APS can lower blood sugar in rats with type 2 diabetes (T2D) by reducing endoplasmic reticulum stress. In addition, APS can increase AMPK expression and improve insulin sensitivity.

Metformin is recommended as a first-line treatment for T2D. However, approximately 92.2% of T2D patients experience adverse events when using metformin clinically, of which approximately 22.8% experience serious adverse events such as cardiovascular disease and stroke. Adverse events reduce the efficacy of metformin, and its clinical strategy is to combine it with other blood sugar-lowering drugs. Combination antidiabetic drugs, such as dapagliflozin and rosiglitazone, are being used. Clinical studies have shown that the incidence of adverse events associated with rosiglitazone exceeds 92.0%, and the incidence of serious adverse events associated with dapagliflozin exceeds 34.0%. While these strategies can enhance glucose-lowering effects, they cannot prevent the occurrence of adverse events. The incidence of adverse events associated with metformin is positively correlated with dose. Previous studies have found that newly diagnosed T2D patients experiencing no adverse events after taking low-dose metformin have poor glycemic control. To improve efficacy and reduce adverse events, adjuvant drugs and biofunctional materials have become important strategies. From the perspectives of cost and application, exploring adjuvant therapies for low-dose metformin is valuable.

This study aims to elucidate the structure and function of polysaccharides and explore the mechanism of action of low-dose metformin as an adjuvant therapy. This study used a DEAE-52 column and Sephadex Crude APS polysaccharide was purified using a G-100 column to obtain a homogeneous polysaccharide, APS-D1. The structure of APS-D1 was characterized by monosaccharide composition analysis, Fourier transform infrared spectroscopy (FT-IR), ultraviolet-visible spectroscopy (UV-vis), methylation, and nuclear magnetic resonance (NMR). In vivo results confirmed that APS-D1 exhibits significant anti-inflammatory activity. Furthermore, it was hypothesized that APS-D1 combined with low-dose metformin could improve diabetes. Subsequently, an insulin resistance mouse model was established to evaluate the antidiabetic effects and pharmacodynamic mechanisms of APS-D1 combined with low-dose metformin. These findings will help establish the structure-function relationship of APS and provide new insights into therapeutic strategies for type 2 diabetes.

Results

Structural Analysis of APS-D1

1.Mw, Homogeneity of APS-D1, and Monosaccharide Composition

HPGPC analysis revealed a broad Mw distribution for APS. After purification, the HPGPC spectrum of APS-D1 exhibited a single, symmetrical peak (retention time 14.64). min), indicating that APS-D1 is relatively homogeneous and highly pure, with an average Mw of 7.36 kDa (Figure 1C).

HPLC analysis of APS-D1 and a standard (Figure 1D) revealed that APS-D1 is composed of glucose, galactose, and arabinose in a molar ratio of 104.38:1.67:1 (97.51%:1.56%:0.93%) (Table S2). Clearly, glucose is the predominant monosaccharide in the isolated APS-D1.

2.FT-IR and UV-Vis Analysis

The FT-IR spectrum of APS-D1 in the 4000-400 cm-1 range exhibits characteristic absorption peaks typical of polysaccharides (Figure 1E). Specifically, the broad, intense peak near 3436 cm-1 is attributed to O-H stretching vibrations, while the peak at 2931 cm-1 is attributed to C-H stretching vibrations. The absorption band at 1638 cm-1 is due to bound water, and the bands at 1400 and 1203 cm-1 are attributed to bound water. The absorption peak at 1 cm-1 is attributed to carbon-hydrogen bending vibrations. The three characteristic absorption peaks between 1200 and 1000 cm-1 are attributed to pyranose stretching vibrations. Furthermore, weak peaks near 859 cm-1 and 764 cm-1 confirm the presence of α- and β-glycosidic bonds.

UV-visible spectroscopy revealed that APS-D1 contained neither nucleic acids nor proteins, as no distinct absorption peaks were observed at 260 nm and 280 nm (Figure 1F).

Figure 1 Purification, physicochemical analysis, and spectral analysis of APS-D1

3.NMR spectroscopy

NMR spectroscopy was used to further characterize the structural characteristics of APS-D1. In the NMR spectrum (Figure 2A), the ectopic proton signals at δ5.32, 5.30, 4.89, 4.56, and 4.55 represent five different sugar residues, which are labeled with letters A to E, respectively. Correspondingly, in the 13C NMR spectrum (Figure 2B), based on the cross peaks of the HSQC spectrum (Figure 2D), the ectopic carbon signals were captured at δ99.87/5.32 (A1), 99.66/5.30 (B1), 98.97/4.89 (C1), 96.11/4.56 (D1), and 104.73/4.55 (E1). According to the 1H-1H The adjacent correlations in the COSY spectrum (Figure 2C) revealed three cross-peaks at δ5.32/3.58, 3.58/3.89, and 3.89/3.57, indicating that the H1-H4 signals of residue A were located at δ5.32, 3.58, 3.89, and 3.57, respectively. Subsequently, the C1-C4 chemical shifts corresponding to δ99.87, 99.66, 98.97, and 96.11 were easily deduced from the 1H-13C HSQC spectrum (Figure 2D). The cross-coupling resonances detected at δ3.78/71.21 and 3.75/60.5 in the 1H–13C HSQC spectrum were then assigned to H5/C5 and H6/C6 of residue A. These shifts are consistent with literature data, suggesting that residue A belongs to (→4)-α-D-Glcp-(1→). The 1H NMR spectrum (Figure 2A) shows a distinct telomeric proton resonance at δ4.56 for residue D, indicating a β-telomeric configuration. Signals at δ1.8-2.0 in the 1H NMR spectrum, combined with signals at δH 1.91 in the 1H NMR spectrum, δC 178.40 and 20.95 in the 1D and 2D NMR spectra, and signals at δ1.91/20.95 in the HSQC spectrum, indicate that APS-D1 contains an O-acetyl group. The cross-peak at δ1.91/178.40 in the HMBC spectrum also confirms the presence of the O-acetyl group. Furthermore, the integrated ratio of residue E to the O-acetyl methyl proton H1 is close to 1:3, indicating that residue E is present in equal numbers as residue E, thus representing an O-acetylated galactose residue.

Figure 2 Structural Analysis of APS-D1

Based on the cross-peaks observed in the HMBC spectrum (Figure 2E), the linkage sequence between these residues in APS-D1 was determined. Signals from A-H1 and A-C4 (δ5.32/76.75) and A-H4 and A-C1 (δ3.57/99.87) indicate the presence of a (→4)-α-D-Glcp-(1→4)-α-D-Glcp-(1→) structure. Signals from B-H1 and A-C4 (δ5.30/76.75) and B-H4 and A-C1 (δ3.55/99.87) indicate a (→4,6)-α-D-Glcp-(1→4)-α-D-Glcp-(1→) structure. -α-D-Glcp-(1→) C-1 and C-4 are both connected to →4)-α-D-Glcp-(1→. Compared with the literature report, the chemical shift of C-2 in residue E shifted downward, indicating that the acetyl group may be attached to O-2 of residue E. The δ3.72/99.87 (AC1-EH3) signal showed that C-3 of →3)-β-D-Galp-(1→) was attached to →4)-α-D-Glcp-(1→). The signal at δ4.89/104.73 (CH1-EC1) indicates that (3)-β-D-Galp-(1→) is attached to the terminal glucose in an α/β configuration. In summary, the structure of APS-D1 consists of a (4)-α-D-Glcp-(1→) residue backbone with (3)-β-D-Galp-(1→) residues and a terminal (α/β-D-Glcp-(1→) side chain. Therefore, the predicted repeating unit of APS-D1 was proposed, as shown in Figure 2F.

Pharmacological Activity and Mechanism of Action

1.APS-D1 enhances the efficacy of low-dose metformin by promoting fatty acid oxidation and inhibiting gluconeogenesis.

To avoid potential adverse events associated with commonly used metformin doses, the antidiabetic effects of APS-D1 combined with low-dose metformin were evaluated. After four weeks of administration, high-dose metformin (300 mg/kg) was significantly associated with diabetic renal failure. mg/kg, equivalent to a clinical dose of 2.0 g/day for patients) improved body weight, IGT, insulin dysfunction, and insulin resistance in HFD mice, while adverse events occurred (Figure 3A). The above results indicate that although high-dose metformin provides good blood sugar control, it is prone to adverse events, which is consistent with clinical results. Low-dose metformin (150 mg/kg) had no effect on body weight. Similarly, metformin had no effect on IGT, but combined with APS-D1 it could improve IGT (Figure 3A). Consistent with IGT, combined medication improved insulin dysfunction (Figure 3B). Similarly, APS-D1 combined with metformin improved insulin resistance (Figure 3C). The above results indicate that APS-D1 combined with metformin not only achieved the goal of controlling blood sugar but also prevented the occurrence of adverse events. In addition, combined medication promoted the secretion of immunomodulatory factors (IgA, IgG, IFN-γ) and anti-inflammatory factors (IL-10), and inhibited the secretion of pro-inflammatory factors (IL-1β, TNF-α, and LPS) (Figure 3D- F), suggesting that APS-D1 can enhance metformin's improvements in immune disorders and inflammation. Correspondingly, combination therapy can regulate liver function. APS-D1 combined with metformin improved pancreatic tissue damage (Figure 3G). Furthermore, combination therapy significantly improved liver tissue damage and hepatic fat accumulation (Figure 3H, I). Combination therapy promoted GLP-1 secretion from intestinal L cells, leading to elevated serum GLP-1 concentrations (Figure 3J). In liver tissues of mice treated with combination therapy, CPT1 protein expression was significantly upregulated, while PCK and G6Pase protein expression was significantly downregulated (Figure 3K). In summary, APS-D1 improves the efficacy of low-dose biguanides by promoting fatty acid oxidation and inhibiting gluconeogenesis.

Figure 3 APS-D1 reduces the effective dose of metformin by promoting fatty acid oxidation and inhibiting gluconeogenesis.

2.APS-D1 enriches Staphylococcus tarda and improves diabetes.

It has been previously demonstrated that oral administration of bioactive components from traditional Chinese medicines first alters the composition of the gut microbiota and then affects the production of its metabolites. It is speculated that the gut microbiota may play a key role in the anti-diabetic effects of PS-D1 combined with low-dose metformin. The Shonnon index and Chao1 index increased after treatment (Figures 4A, B), indicating that APS-D1 can enhance the α-diversity of the gut microbiota, which is improved by metformin. HFD-induced insulin resistance causes The intestinal microbiota of mice differed (Figure 4C). The intestinal microbiota of each group was clearly separated (Figure 4D), indicating that the differences caused by HFD can be reversed after combined treatment. Consistent with previous reports, the ratio of Firmicutes to Siderophores increased and then decreased after combined treatment (Figure 4E). The species-level abundance, particularly of Staphylococcus tarda and Lactobacillus murinus, increased significantly after treatment (Figure 4F). Among them, APS-D1 had a significant regulatory effect on Staphylococcus tarda. In summary, APS-D1 combined with metformin treatment significantly altered the composition and function of the intestinal microbiota, leading to a significant enrichment of Staphylococcus tarda.

Figure 4 APS-D1 enriches Staphylococcus lentus

3.Staphylococcus lentus treatment can improve diabetes

Staphylococcus lentus, a Gram-positive bacterium belonging to the Staphylococcus genus, is a major cause of hospital-acquired infections. To investigate the role of S. lentus in diabetes, mice fed a high-fat diet (HFD) were used as a model. It was found that mice treated with S. lentus significantly lost weight despite consuming the same diet (Figure 5A). Furthermore, S. lentus treatment reduced postprandial 2-3-4-8-12-24-18-19 (Figure 5A). h blood glucose concentration (Figure 5B), indicating that Staphylococcus lentus improved IGT. In addition, Staphylococcus lentus can improve insulin dysfunction (Figure 5C) and insulin resistance (Figure 5D). In addition, Staphylococcus lentus can promote the secretion of immunomodulatory factors (IgA, IgG, IFN-γ) and anti-inflammatory factors (IL-10), and inhibit the secretion of proinflammatory factors (LPS) (Figure 5E-I), suggesting that Staphylococcus lentus can improve immune disorders and inflammation. Histological analysis showed that Staphylococcus lentus improved pancreatic and liver tissue damage and liver fat accumulation (Figure 5J-L). In addition, Staphylococcus lentus can also promote the secretion of GLP-1 (Figure 5M). Correspondingly, in the liver tissue of mice treated with Staphylococcus lentus, CPT1 protein expression was significantly upregulated and G6Pase protein expression was significantly downregulated (Figure 5N). Therefore, these results strongly indicate that Staphylococcus lentus promotes  APS-D1 improves diabetes by inhibiting fatty acid oxidation and gluconeogenesis.

Figure 5: Treatment with Staphylococcus tarda improves impaired glucose tolerance and insulin resistance.

4.L-carnitine is enriched in serum, and administration improves diabetes.

The potential mechanism by which APS-D1 enhances the therapeutic effects of low-dose metformin may be through its effects on host metabolism and the production of secondary metabolites during gut microbial fermentation. Therefore, it is intriguing to investigate whether treatment alters serum metabolites. Therefore, untargeted metabolomics analysis of serum samples was performed. To further investigate the potential antidiabetic effects of L-carnitine, HFD-fed mice were used as a model. Although L-carnitine had no effect on body weight, it significantly reduced fasting blood glucose, 2-hour postprandial blood glucose concentrations, and OGTT AUC (Figure 6A,B). Furthermore, L-carnitine improved insulin dysfunction (Figure 6C) and insulin resistance (Figure 6D). Although L-carnitine did not affect IFN-γ, it significantly promoted the secretion of immunomodulatory factors (IgA, IgG) and the anti-inflammatory factor IL-10, and inhibited the secretion of pro-inflammatory factors (IL-1β, TNF-α, and LPS) (Figures 6E-K), indicating that L-carnitine can improve immune disorders and inflammation. Although L-carnitine did not affect the activities of alanine aminotransferase and aspartate aminotransferase, it improved pancreatic and liver tissue damage and hepatic fat accumulation (Figures L6-N). In addition, L-carnitine promoted GLP-1 secretion (Figure 6O). L-carnitine promoted fatty acid oxidation by enhancing CPT1 activity rather than protein expression (Figures 6P,Q). Furthermore, PCK and G6Pase protein expression levels were significantly downregulated in the liver tissue of L-carnitine-treated mice (Figure 6Q), indicating that L-carnitine treatment inhibits gluconeogenesis. Taken together, these results strongly suggest that L-carnitine improves diabetes by promoting fatty acid oxidation and inhibiting gluconeogenesis.

Figure 6 L-Carnitine Improves Impaired Glucose Tolerance and Insulin Resistance

5.Reducing L-Carnitine Production in Staphylococcus lentus Impairs Diabetes Improvement

Previous reports have shown that electron transfer inhibitors such as nitrates can inhibit bacterial L-carnitine synthesis. To elucidate the importance of L-carnitine in the anti-diabetic effects of S. lentus, potassium nitrate was added to the culture medium. The results showed that potassium nitrate inhibited L-carnitine synthesis in S. lentus (Figure 7A). Six weeks after oral administration of S. lentus with L-carnitine synthesis inhibited (KD S. lentus), serum L-carnitine concentrations were significantly reduced (Figure 7B). Clearly, inhibition of L-carnitine synthesis in S. lentus did not affect its antidiabetic pharmacological effects (Figure 7C). KD S. lentus did not improve insulin resistance, but exogenous L-carnitine supplementation did (Figure 7D). Similarly, KD S. lentus did not improve insulin dysfunction in HFD-fed mice (Figure 7E). Consistent with this, supplementation with exogenous L-carnitine prevented KD S. lentus from losing its ability to improve insulin resistance (Figure 7F). Treatment with S. lentus enhanced IgA secretion, which was suppressed by HFD, whereas KD S. lentus did not. Exogenous L-carnitine supplementation significantly enhanced IgA and IgM secretion in KD S. lentus (Figure 7G, H). Similarly, S. lentus failed to suppress the secretion of proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and did not promote the secretion of the anti-inflammatory cytokine IL-10, indicating that S. lentus treatment failed to improve inflammation (Figure 7I-M). In short, reduced L-carnitine production in S. lentus impairs its antidiabetic capacity.

Figure 7. Reduction of L-carnitine production in S. lentus impairs improvements in glucose tolerance and insulin resistance.

Conclusion

This study purified a novel polysaccharide, APS-D1 (7.36 kDa). Structural analysis showed that APS-D1 is composed of a main chain of (→4)-α-D-Glcp-(1→ residues, a →3)-β-D-Galp-(1→ residues, and a terminal -α/β-D-Glcp-(1→ side chain. In addition to its anti-inflammatory effects, it was confirmed for the first time that APS-D1 promotes fatty acid oxidation and inhibits gluconeogenesis by enriching Staphylococcus tarda, thereby enhancing the efficacy of low-dose metformin in improving diabetes. The effect of Staphylococcus tarda is mediated by L-carnitine. In summary, this study elucidates the chemical structure and pharmacological activity of APS-D1. Therefore, it is believed that low-dose metformin supplementation with APS may be a favorable strategy for the clinical treatment of T2D.

anna@hihealthbio.com