Effects of Polymannuronic Acid on the Intestinal Microbiota in Mice after Long-Term Intragastric Administration.

Polymannuronic acid (PM) is an alginate oligosaccharide derived from brown algae with a characterized structure and excellent biological activities. Herein, mice were given different doses of PM through 30-day-long-term intragastric administration, and the contents of the jejunum, ileum, and colon were analyzed by 16S rRNA gene sequencing technology for microbial diversity, and relevant experiments were verified according to the analysis results so as to comprehensively evaluate the effects of PM on the intestinal flora. The PM (400 mg/kg and 100 mg/kg) could regulate the microflora balance at the phylum level and increase the microflora richness in the jejunum, ileum, and colon of the mice. The PM could induce more strains that are negatively correlated with Escherichia, thereby reducing the relative abundance of Escherichia. Analysis of bacterial function showed that high and low doses of PM could promote lipid metabolism in the bacterial communities. Moreover, the PM could reduce serum total cholesterol and cholesterol ester levels in a concentration-dependent manner. High-dose PM could lead to colonic intestinal inflammation by increasing the relative abundance of multiple bacterial groups in the jejunum, ileum, and colon. Moreover, high-dose PM could increase lipopolysaccharide-binding protein and interleukin-1ß levels. Therefore, the dose of PM plays an important role in its efficacy, and its biological activity is dosedifferent.

Fluorescent Labeling of Polymannuronic Acid and Its Distribution in Mice by Tail Vein Injection.

Polymannuronic acid (PM) possesses more pharmacological activities than sodium alginate, but there have been few studies on its absorption mechanism, tissue distribution, and pharmacokinetics. Studies of pharmacokinetics and tissue distribution are necessary to elucidate the pharmacological effects of PM. Thus, we used fluorescein isothiocyanate (FITC) to produce fluorescently labeled PM (FITC-PM) and detected the distribution and pharmacokinetics of PM in vivo via tail vein injection. The results demonstrate that the FITC-PM showed high stability in different pH solutions. After the tail vein injection, FITC-PM tended to be distributed in the kidney, followed by the liver and in the heart, spleen, and lungs at lower concentrations. Pharmacokinetic analysis showed that the elimination rate constant of FITC-PM was 0.24, the half-life time was 2.85 h, the peak concentration was 235.17 µg/mL, the area under the curve was 631.48 µg/mL·h, the area under the curve by statistical moment was 1843.15 µg/mL·h2, the mean residence time was 2.92 h, and the clearance rate was 79.18 mL/h. These results indicate that FITC-PM could be used for PM distribution and pharmacokinetic studies, and the studies of pharmacokinetics and tissue distribution provided basic information that can be used to further clarify PM pharmacodynamic mechanisms.

A Novel PTP1B Inhibitor-Phosphate of Polymannuronic Acid Ameliorates Insulin Resistance by Regulating IRS-1/Akt Signaling.

Protein tyrosine phosphatase 1B (PTP1B) is a critical negative modulator of insulin signaling and has attracted considerable attention in treating type 2 diabetes mellitus (T2DM). Low-molecular-weight polymannuronic acid phosphate (LPMP) was found to be a selective PTP1B inhibitor with an IC50 of 1.02 ± 0.17 µM. Cellular glucose consumption was significantly elevated in insulin-resistant HepG2 cells after LPMP treatment. LPMP could alleviate oxidative stress and endoplasmic reticulum stress, which are associated with the development of insulin resistance. Western blot and polymerase chain reaction (PCR) analysis demonstrated that LPMP could enhance insulin sensitivity through the PTP1B/IRS/Akt transduction pathway. Furthermore, animal study confirmed that LPMP could decrease blood glucose, alleviate insulin resistance, and exert hepatoprotective effects in diabetic mice. Taken together, LPMP can effectively inhibit insulin resistance and has high potential as an anti-diabetic drug candidate to be further developed.

Polymannuronic acid ameliorated obesity and inflammation associated with a high-fat and high-sucrose diet by modulating the gut microbiome in a murine model.

Polymannuronic acid (PM), one of numerous alginates isolated from brown seaweeds, is known to possess antioxidant activities. In this study, we examined its potential role in reducing body weight gain and attenuating inflammation induced by a high-fat and high-sucrose diet (HFD) as well as its effect on modulating the gut microbiome in mice. A 30-d PM treatment significantly reduced the diet-induced body weight gain and blood TAG levels (P2·0). PM also had a profound impact on the microbial composition in the gut microbiome and resulted in a distinct microbiome structure. For example, PM significantly increased the abundance of a probiotic bacterium, Lactobacillus reuteri (log10 LDA score>2·0). Together, our results suggest that PM may exert its immunoregulatory effects by enhancing proliferation of several species with probiotic activities while repressing the abundance of the microbial taxa that harbor potential pathogens. Our findings should facilitate mechanistic studies on PM as a potential bioactive compound to alleviate obesity and the metabolic syndrome.

Alginate and its Two Components Acted Differently Against Dopaminergic Neuronal Loss in Parkinson's Disease Mice Model.

SCOPE: This study aims to investigate and compare the potentially neuroprotective effects and underlying mechanisms for brown seaweed polysaccharides (PS) of Alginate (Alg) and its two components, including polymannuronic acid (PM) and polyguluronic acid (PG), against Parkinson's disease (PD) pathogenesis. METHODS AND RESULTS: Model mice of PD are pretreated with Alg or PM or PG, separately via oral gavage once per day for four weeks. Our results found PM improved motor functions of PD mice, but Alg or PG did not. PM or PG, but not Alg, can prevent dopaminergic neuronal loss by increasing tyrosine hydroxylase (TH) expressions in midbrain of PD mice. The neuroprotective effects of PM rely on its anti-inflammation effects and its ability to improve striatal neurotransmitters (serotonin (5-HT) and 5-hydroxyindole acetic acid (5-HIAA)) levels in PD mice. PM inhibits inflammation, but PG or Alg induces inflammation in systemic circulation of PD mice. The neuroprotection provided by PG might be related to its ability to increase striatal neurotransmitter of 5-hydroxyindole acetic acid levels in PD mice. CONCLUSION: PM plays better than PG to provide neuroprotection, but Alg did not show any neuroprotection against PD. Alg and its two components acted differently in preventing dopaminergic neuronal loss in PD mice.

Polymannuronic acid prebiotic plus Lacticaseibacillus rhamnosus GG probiotic as a novel synbiotic promoted their separate neuroprotection against Parkinson's disease.

Gut microbiota (GM) dysbiosis plays key roles in aggravating Parkinson's disease (PD) and discovery of agents targeting GM may open new avenues for PD therapy. This study aims to investigate the potentially neuroprotective effects and underlying mechanisms of polymannuronic acid (PM) or Lacticaseibacillus rhamnosus GG (LGG), or their combination in a chronic PD mice model. Our results found oral administration of prebiotic PM or LGG separately or in combination for 5 weeks could prevent dopaminergic neuronal loss via improving reduced walking distance and activity or weakened muscle strength in behavior tests by enhancing tyrosine hydroxylase (TH) gene and/or protein expressions in the midbrain and striatum of PD mice. Strikingly, PM and LGG in combination had a much better neuroprotective effects than separate PM or LGG. PM provided neuroprotection via a short chain fatty acids (SCFAs)-mediated anti-inflammation and anti-apoptosis mechanism. The neuroprotective effects of LGG might be associated with its ability to improve the expression of striatal glial cell-derived neurotrophic factor (GDNF) and to increase bacteria abundance of Clostridiales. When PD mice were administered with PM + LGG, PM as prebiotic favored bacterial growth (from Bacilli class to Lactobacillus genus) in the colon, which helped to improve blood brain barrier (BBB) integrity and increase brain-derived neurotrophic factor (BDNF) and GDNF expressions, thereby inhibiting apoptosis in the striatum. In conclusion, PM and LGG in combination promoted their separate neuroprotection against PD. Our study discovered and testified a novel synbiotic that might be one of the ideal oral agents for PD therapy.

Specificities and Synergistic Actions of Novel PL8 and PL7 Alginate Lyases from the Marine Fungus Paradendryphiella salina.

Alginate is an anionic polysaccharide abundantly present in the cell walls of brown macroalgae. The enzymatic depolymerization is performed solely by alginate lyases (EC 4.2.2.x), categorized as polysaccharide lyases (PLs) belonging to 12 different PL families. Until now, the vast majority of the alginate lyases have been found in bacteria. We report here the first extensive characterization of four alginate lyases from a marine fungus, the ascomycete Paradendryphiella salina, a known saprophyte of seaweeds. We have identified four polysaccharide lyase encoding genes bioinformatically in P. salina, one PL8 (PsMan8A), and three PL7 alginate lyases (PsAlg7A, -B, and -C). PsMan8A was demonstrated to exert exo-action on polymannuronic acid, and no action on alginate, indicating that this enzyme is most likely an exo-acting polymannuronic acid specific lyase. This enzyme is the first alginate lyase assigned to PL8 and polymannuronic acid thus represents a new substrate specificity in this family. The PL7 lyases (PsAlg7A, -B, and -C) were found to be endo-acting alginate lyases with different activity optima, substrate affinities, and product profiles. PsAlg7A and PsMan8A showed a clear synergistic action for the complete depolymerization of polyM at pH 5. PsAlg7A depolymerized polyM to mainly DP5 and DP3 oligomers and PsMan8A to dimers and monosaccharides. PsAlg7B and PsAlg7C showed substrate affinities towards both polyM and polyG at pH 8, depolymerizing both substrates to DP9-DP2 oligomers. The findings elucidate how P. salina accomplishes alginate depolymerization and provide insight into an efficient synergistic cooperation that may provide a new foundation for enzyme selection for alginate degradation in seaweed bioprocessing.

Polymannuronic acid prevents dopaminergic neuronal loss via brain-gut-microbiota axis in Parkinson's disease model.

The study aims to investigate the potentially neuroprotective effects and underlying mechanisms for brown seaweed polysaccharide of polymannuronic acid (PM) against Parkinson's disease (PD) pathogenesis. PD model mice were pretreated with PM via oral gavage once per day for 4 weeks and the preventative effects of PM against neuronal loss together with its modulation on brain-gut-microbiota axis were systematically explored. The results showed PM administration improved motor functions by preventing dopaminergic neuronal loss in the substantia nigra pars compacta (SNpc) and enhanced contents of striatal homovanillic acid (HVA), serotonin (5-HT), 5-hydroxyindole acetic acid (5-HIAA) and γ-aminobutyric acid (GABA) in PD mice. PM significantly alleviated inflammation in gut, brain and systemic circulation as shown by reduced levels or expressions of pro-inflammatory cytokines concurrently and inhibited mitogen-activated protein kinases (MAPK) signaling pathway in mice colon. Meanwhile, PM greatly improved integrity of intestinal barrier and blood brain barrier (BBB) as indicated by increased expressions of tight junction associated proteins in both mice colon and SNpc. Further studies indicated PM treatment resulted in changes of gut microbial compositions, together with great alterations of digestion and metabolism of dietary proteins and fats, which led to surge increase of fecal short chain fatty acids (SCFAs) in the colon of PD mice. In conclusion, pre-administration of PM could provide neuroprotective effects against PD pathogenesis by suppressing inflammation in gut, brain and systemic circulation, and by improving integrity of intestinal barrier and BBB. PM might modulate brain-gut-microbiota axis, at least in part, via gut microbiota derived SCFAs as mediators.

Transport Mechanisms of Polymannuronic Acid and Polyguluronic Acid Across Caco-2 Cell Monolayers.

Detailed knowledge of the intestinal transport of polymannuronic acid (PM) and polyguluronic acid (PG) is critical for understanding their biological activities. To investigate the transport in the gastrointestinal tract, PM and PG were chemically modified with tyramine and conjugated with fluorescein isothiocyanate (FITC) to synthesize FITC-PM (F-PM) and FITC-PG (F-PG) successfully. The transport mechanisms of F-PM and F-PG across the intestinal epithelial cell monolayers (Caco-2 cell monolayers) were then investigated. The results demonstrated that the transport of F-PM and F-PG into epithelial cells was time- and energy-dependent, which was mediated by the macropinocytosis pathway and the clathrin- and caveolae (or lipid raft)-mediated endocytic pathway. The transport process of F-PM and F-PG in Caco-2 cells depended on the acidification of endosomes and involved lysosomes. Tubulin mediated the transport of F-PM, but not of F-PG. Moreover, the absorption enhancer chitosan (CS) promoted the transport of F-PM and F-PG, increasing the apparent permeability coefficient (Papp) by 1.9-fold and 2.6-fold, respectively, by reversibly opening the tight junction (TJ). In summary, this study provided a comprehensive understanding of the transport of PM and PG in the small intestinal epithelial cells, which will provide a theoretical basis for the development of PM and PG with good intestinal absorption.

Novel oleyl amine-modified polymannuronic acid micelle loading tacrolimus for therapy of allergic conjunctivitis.

Marine polysaccharide-based nanomaterial attracted considerable attention due to its biocompatibility and biodegradability. Here, a novel polymannuronic acid derivative (PM-C18) was synthesized as nanocarrier modified with hydrophobic oleyl amine. Its structure was confirmed by Fourier-transformed infrared spectrometry and nuclear magnetic resonance. To investigate its function, we developed tacrolimus (FK506) loaded nanoparticle by self-assembling PM-C18 and studied its drug loading, cytotoxicity and mechanism of in vitro release. Furthermore, we established a mouse model of allergic conjunctivitis (AC) to evaluate the efficacy of FK506 loaded PM-C18. The results indicated that PM-C18 carrier can encapsule FK506 with high drug loading (18%) and average particle size of 110.34±1.6nm. The release mechanism was diffusion initially followed a lasting release induced by swelling and collapse of PM-C18. One-week treatment with FK506 loaded PM-C18 efficiently relieved the symptoms of AC, which indicated it could serve as a potential ophthalmic drug for patients with AC. Compared with FK506 ophthalmic suspension, FK506 loaded PM-C18 improved medication adherence. Most importantly, we developed a novel versatile nanomaterial of PM-C18 which could encapsule other active agents for more applications.

Preparation, characterization and antioxidant activities of polymannuronic acid phosphate, H-phosphonate and sulfate.

Three derivatives of polymannuronic acid (PM) were prepared by chemically polyanionic modification. PM was substituted with phosphate (OPO3(2-)), H-phosphonate (OPO2H(-)) and sulfate (OSO3(-)) groups with the degree of substitution (DS) of 0.41, 1.42 and 1.04, respectively. The structures of all PM derivatives were characterized by FT-IR, (31)P NMR and (13)C NMR spectroscopy. The weight average molecular weight (Mw) was determined by high performance gel permeation chromatography (HPGPC). The antioxidant activities of PM and its derivatives were evaluated in vitro. The results indicated that both phosphate and H-phosphonate groups improved the hydroxyl radical scavenging activity, while sulfate group enhanced the superoxide radical scavenging activity. However, all of the derivatives scavenged DPPH less effectively than PM. The mechanism for how the different anionic substituent groups influenced the antioxidant activities was discussed.