Anti-hepatoma immunotherapy of Pholiota adiposa polysaccharide-coated selenium nanoparticles by reversing M2-like tumor-associated macrophage polarization.

Targeting macrophages to regulate the tumor microenvironment is a promising strategy for treating cancer. This study developed a stable nano drug (PAP-SeNPs) using Se nanoparticles (SeNPs) and the Pholiota adiposa polysaccharide component (PAP-1a) and reported their physical stability, M2-like macrophages targeting efficacy and anti-hepatoma immunotherapy potential, as well as their molecular mechanisms. Furthermore, the zero-valent and well-dispersed spherical PAP-SeNPs were also successfully synthesized with an average size of 55.84 nm and a negative ζ-potential of -51.45 mV. Moreover, it was observed that the prepared PAP-SeNPs were stable for 28 days at 4 °C. Intravital imaging highlighted that PAP-SeNPs had the dual effect of targeting desirable immune organs and tumors. In vitro analyses showed that the PAP-SeNPs polarized M2-like macrophages towards the M1 phenotype to induce hepatoma cell death, triggered by the time-dependent lysosomal endocytosis in macrophages. Mechanistically, PAP-SeNPs significantly activated the Tlr4/Myd88/NF-κB axis to transform tumor-promoting macrophages into tumor-inhibiting macrophages and successfully initiated antitumor immunotherapy. Furthermore, PAP-SeNPs also enhanced CD3+CD4+ T cells and CD3+CD8+ T cells, thereby further stimulating anti-hepatoma immune responses. These results suggest that the developed PAP-SeNPs is a promising immunostimulant that can assist hepatoma therapy.

Core fucose-specific Pholiota squarrosa lectin (PhoSL) as a potent broad-spectrum inhibitor of SARS-CoV-2 infection.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike protein (S protein) is highly N-glycosylated, and a "glycan shield" is formed to limit the access of other molecules; however, a small open area coincides with the interface to the host's receptor and also neutralising antibodies. Most of the variants of concern have mutations in this area, which could reduce the efficacy of existing antibodies. In contrast, N-glycosylation sites are relatively invariant, and some are essential for infection. Here, we observed that the S proteins of the ancestral (Wuhan) and Omicron strains bind with Pholiota squarrosa lectin (PhoSL), a 40-amino-acid chemically synthesised peptide specific to core-fucosylated N-glycans. The affinities were at a low nanomolar level, which were ~ 1000-fold stronger than those between PhoSL and the core-fucosylated N-glycans at the micromolar level. We demonstrated that PhoSL inhibited infection by both strains at similar submicromolar levels, suggesting its broad-spectrum effect on SARS-CoV-2 variants. Cryogenic electron microscopy revealed that PhoSL caused an aggregation of the S protein, which was likely due to the multivalence of both the trimeric PhoSL and S protein. This characteristic is likely relevant to the inhibitory mechanism. Structural modelling of the PhoSL-S protein complex indicated that PhoSL was in contact with the amino acids of the S protein, which explains the enhanced affinity with S protein and also indicates the significant potential for developing specific binders by the engineering of PhoSL.

Rheological properties of polysaccharides from Pholiota nameko with different temperature extraction: Concentration, pH, temperature, and saltion.

Cold and hot water extracted polysaccharides (CW-PNPs and HW-PNPs) were isolated from Pholiota nameko. The rheological properties of PNPs were investigated by steady shear and oscillatory rheological measurements. The PNPs exhibited typical non-Newtonian and shear-thinning behavior, which are affected by PNP concentration, temperature, pH value, salt ion, and concentration. Specifically, the apparent viscosity of the two PNPs solutions at concentration of 1% (w/w) was shown as HW-PNPs > CW-PNPs. The apparent viscosity of PNPs decreases under acidic and alkaline conditions and when the temperature rises; K+ and Na+ cause the apparent viscosity of CW-PNPs to decrease, while Ca2+ and Al3+ are opposite. The addition of four different salt ions all caused the apparent viscosity of the HW-PNPs to decrease. The results of dynamic rheological experiments show that G' and G″ showed slightly frequency dependency with G' exceeding G″ throughout the accessible range of frequency for CW-PNPs and HW-PNPs.

Study on the physicochemical properties and immunomodulatory anti-tumor effect of the Pholiota adiposa polysaccharide.

In this study, the extraction, purification, physical and chemical properties, and biological activity of the Pholiota adiposa (PAP) polysaccharide were investigated. One fraction (PAP-1a) of Pholiota adiposa polysaccharides was isolated using DEAE Sepharose™ Fast Flow and Sephacryl™ S-300 High-Resolution columns. The HPLGPC results revealed that the molecular weight of PAP-1a was 16.453 kDa. PAP-1a was composed of mannose, ribose, rhamnose, glucuronic acid, galacturonic acid, glucose, galactose, xylose, arabinose, and fucose and their molar % was 33.41, 0.53, 1.33, 0.07, 0.27, 5.28, 38.31, 0.83, 18.04 and 2.23, respectively. PAP-1a could activate macrophages to secrete NO and cytokines such as TNF-a, IL-6, and IL-12p70. When hepatocellular carcinoma cells (HCCs) and macrophages were co-cultured, it was observed that PAP-1a inhibited the growth of Hep-G2, Hep-3B, and Huh7 via immunoregulation. It triggered cell apoptosis by blocking the cell cycle in the G0/G1 stage. Furthermore, PAP-1a had no direct cytotoxicity against the hepatocyte cell line L02 and macrophages RAW264.7.

Chemical properties and anti-fatigue effect of polysaccharide from Pholiota nameko.

The aim of this study was to explore the primary chemical properties and anti-fatigue effect in vivo of Pholiota nameko polysaccharide (PNP). Through UV-visible spectrum, the absorption peaks of proteins, nucleic acids and pigments were not found. The organic functional groups of polysaccharides (3,289.97, 1,584.72, and 1,045.23 cm-1 so on) were measured by IR spectroscopy. The PNP was a semi-crystalline or non-crystalline substance, possessed a three-dimensional lump structure with a smooth, dense surface and amorphous structure according to the scanning electron microscopy and XRD images. Moreover, the PNP was chain or bright-spot structures formed by the entanglement of multiple polysaccharide fibers on the basis of atomic force microscopy. The results of anti-fatigue suggested the PNP could significantly extend the forced swim time from 121.58 ± 18.48 and 101.91 ± 14.27 min to 154.95 ± 24.26 and 134.13 ± 25.71 min in male and female mice respectively. The LDH activity was up to 31.68 ± 4.60 U/ml in male mice and 29.49 ± 5.12 U/ml in female mice. Meanwhile, the Ca2+ -Mg2+ -ATPase activity was reached to 2.49 ± 0.41 µmol/(mg·h) in male mice and 2.44 ± 0.29 µmol/(mg·h) in female mice. The SOD activity was increased to 5.92 ± 1.19 U/ml in male mice and 5.89 ± 0.98 U/ml in female mice, while the MDA content was decreased to 2.24 ± 0.34 nmol/mg in male mice and 2.02 ± 0.41 nmol/mg in female mice. These results showed a theoretical basis for application of the PNP in food and pharmacy as a natural physical strengthening substance. PRACTICAL APPLICATIONS: Fatigue affects physical and mental health in vivo, which resulted in negative effects on everyday tasks, leisure activities, cognitive and behavioral performances and is very common in modern life. Therefore, this study was designed to explore the primary chemical properties and research the anti-fatigue effects of Pholiota nameko polysaccharide (PNP) in mice. And then, it would be a reference for the development and utilization of PNP as a kind of healthy food on sub-health.

Increased antioxidant activity and improved structural characterization of sulfuric acid-treated stepwise degraded polysaccharides from Pholiota nameko PN-01.

The aim of this study was to investigate sulfuric acid degradation of the Pholiota nameko polysaccharide (AIPS-1). Three stepwise degraded polysaccharides (AIPS-2, AIPS-3, and AIPS-4) were obtained by sequentially increasing the strength of sulfuric acid treatment. Structural characterization showed that sulfuric acid treatment significantly decreased molecular weight, increased the content of uronic acid and changed the molar ratio of monosaccharide composition, while the major functional groups and the triple helical conformation of polysaccharides did not change significantly. In vitro experiments proved that the antioxidation ability of the stepwise degraded polysaccharides gradually increased (AIPS-1 < AIPS-2 < AIPS-3 < AIPS-4). An oxidative stress zebrafish model was established, which demonstrated that the ability of AIPS-3 and AIPS-4 to scavenge free radicals in zebrafish was significantly improved compared to AIPS-1. In conclusion, sulfuric acid treatment is an effective method for improving the antioxidant activity of polysaccharides, and increased antioxidant activity was closely related to the changes in their structural characteristics.

An ultrasmall chitosan nanosphere encapsulating carbon dots and rhodamine B as a ratiometric probe for the determination of Hg2.

Hg2+-sensitive carbon dots (CDs) were synthesized by microwave-assisted pyrolysis of citric acid, sodium fluoride, and urea. The CDs as a signal report unit and rhodamine B (RhB) as a reference were then encapsulated in a nanosphere of chitosan assembled by a nonsolvent-induced chitosan colloidal formation and in situ cross-linking to construct a ratiometric probe for Hg2+ (chitosan-CDs-RhB). Interestingly, without any assistance from acids to improve the solubility of chitosan, the nanosphere containing CDs and RhB had an ultrasmall size of 9.7 nm with only approximately 1.1-nm-thick layers of chitosan enclosing one dot. In order to keep the residual functional groups on the nanosphere from compromising the fluorescence response of CDs to Hg2+, Co2+ was used as a fluorescently intact metal ion to saturate the functional groups. The saturated chitosan-CDs-RhB was thus potentially developed for determining Hg2+ in the fruit bodies and mycelia of edible and medicinal fungi. Limits of detection (LODs) of 2.24, 5.29, and 2.03 µM and recoveries in the ranges 98.3 to 101.8%, 99.5 to 104.6%, and 97.4 to 100.9% were estimated for the determination of Hg2+ in the fruit bodies of Pleurotus ostreatus, Lentinus edodes, and Hypsizygus marmoreus, respectively. Chitosan-CDs-RhB was further developed as a fluorescent ratiometric probe for quantitatively determining intracellular Hg2+ in fungal mycelia with a linear calibration curve of RIgreen/Ired = - 0.145c + 1.69 within the range 0.013 to 0.356 µg g-1. Graphical abstract An ultrasmall chitosan nanosphere encapsulating carbon dots and rhodamine B as a ratiometric probe for the determination of Hg2+.

Pholiotone A, a new polyketide derivative from Pholiota sp.

Pholiotone A (1), a new polyketide derivative, with tetrahydrobenzofuran-4(2H)-one skeleton, together with four known compounds, trichodermatides A (2) and B (3) and koninginins B (4) and E (5), were isolated from the crude extract of Pholiota sp. The structures of all the isolated compounds were determined mainly by NMR experiments, the modified Mosher method and electronic circular dichroism (ECD) calculations. The antifungal and cytotoxicity of all isolates were evaluated.

Effect of ultrasound-assisted extraction on physicochemical properties and TLR2-affinity binding of the polysaccharides from Pholiota nameko.

The effect of the ultrasonic treatment on the physicochemical properties, oil-holding capacities, foaming capacities, and TLR2-affinity of the polysaccharides from Pholiota nameko (PNPS), was evaluated. Compared with the protein content of PNPS before ultrasonic treatment, the protein content of PNPS all presented a decrease under different ultrasonic conditions. The viscosity of PNPS showed a decrease when the ultrasonic intensity was strong enough, as well as the molecular weight. The oil-holding capacity and the foaming capacity of PNPS showed a continuous increasing trend with the increase of the ultrasonic treatment time under a set ultrasonic power of 400 W. Further, the ultrasonic operation could induce the decrease of the affinity binding between PNPS and the receptor proteins TLR2. These data confirmed that applying the ultrasonic treatment could obtain PNPS with high carbohydrate contents, low viscosity and low TLR2-affinity under proper ultrasonic condition.

Chemical analysis, moisture-preserving, and antioxidant activities of polysaccharides from Pholiota nameko by fractional precipitation.

This study explored Pholiota nameko (P. nameko) polysaccharide fractions, PNP-40, PNP-60, and PNP-80, purified by gradient concentrations of ethanol (40%, 60%, and 80% (v/v)). The physicochemical properties, functional group composition, moisture-preserving, and antioxidant ability were determined. The results indicate that the polysaccharide contents of PNP-40, PNP-60, and PNP-80 are 45.12%, 78.04%, and 72.22%, respectively, while the ß-glucan, protein, and uronic acid contents are 20.20%, 12.20%, and 10.15%, respectively; 11.24%, 14.53%, and 26.94%; and 5.99%, 7.73%, and 3.78%. Furthermore, PNP-60 has better moisture absorption, while PNP-80 has better antioxidant ability and H2O2-injury resistance activity. Monosaccharide composition analysis shows that P. nameko belongs to heteropolysaccharides, which consists of galactose, glucose, and mannose with different types and ratios, and the molecular weight are distributed at 4.40-333.49kDa. It was found that different polysaccharide fractions have the potential to be a moisturizer and an antioxidant, and their active ingredients could be used in the development of cosmetic ingredients.

The structural characterization of a polysaccharide exhibiting antitumor effect from Pholiota adiposa mycelia.

PAP80-2a, purified from Pholiota adiposa mycelia, is a polysaccharide exhibiting prominent antitumor effects. However, the yield of PAP80-2a was low and its structure has not been characterized, impeding the exploration of its structure-function relationship, thus influencing the development of oral drugs for antitumor therapy and immunomodulation. In order to improve the yield of PAP80-2a, response surface methodology along with Box-Behnken design was applied to optimize the ultrasonic-assisted extraction conditions for polysaccharides. Then, the structure of PAP80-2a exhibiting antitumor activity was determined from different angles. The results showed that the extraction yield of P. adiposa polysaccharides increased by 11.5% under optimized ultrasonic extraction conditions. Structural analysis showed that PAP80-2a was mainly composed of glucose, rhamnose, xylose, and galactose in a ratio of 10.00: 2.09: 4.09: 1.13. The total amino acid content in the sugar chain was 69.92 µg/mL. The sugar chain structure was [α-Rha (1 → 3)-]n, and rhamnose was located at the non-reducing end of the sugar chain, while glucose was located at the non-reducing end or in the sugar chain in 1,2,6- and 1,3,6-linked forms. Our study clearly illuminates the primary structure of PAP80-2a, but 3D structure of PAP80-2a and its structure-function relationship is a future challenge.

Two new sesquiterpenes from cultures of the higher fungus Pholiota nameko.

Two new sesquiterpenes (1-2), along with one known sesquiterpene (3), were isolated from cultures of the higher fungus Pholiota nameko. The structures of new compounds were elucidated by extensive spectroscopic methods. The known compound was identified by comparing its spectroscopic data with those reported in the literature. All compounds were evaluated for their cytotoxicities against five human cancer cell lines.

Structural properties and antioxidant activities of polysaccharide from fruit bodies of Pholiota nameko.

A polysaccharide named PNP was extracted and purified from Pholiota nameko. The total sugar content of PNP was 95.29% and the molecular weight was 1.89 × 103 kDa. The structural features of PNP were investigated by the combination of chemical and instrumental analysis such as UV spectrophotometer, specific rotation determination, FT-IR, methylisation analysis and Congo red. The results showed that the optical rotation of PNP was +120° and that it had a triple-helical structure. Besides, PNP was mainly composed of glucose and mannose at the molar ratio of 4.24:1.00. The backbone of PNP was composed of (1→3)-linked-Glc and (1→3)-linked-Man whereas the branches of (1→3,6)-linked- Glc, (1→3,6)-linked-Man and T- Glc. Consistenting with the results of UV-Vis spectra, FT-IR spectroscopy and 1H NMR, indicated that PNP was a complex of polysaccharides and polyphenols. In vitro antioxidant results suggested that PNP was processed with certain scavenging capacity.

Gelling functional property, anti-inflammatory and antinociceptive bioactivities of ß-D-glucan from the edible mushroom Pholiota nameko.

A ß-D-glucan was obtained from the edible mushroom Pholiota nameko by hot aqueous extraction and purification. NMR and methylation analyses of the purified fraction (GHW-PN, 1.46% yield) indicated the presence of a (1 → 3)-linked ß-D-glucan, highly substituted (~27%) at O-6 by single units of ß-D-Glcp or by (1 → 6)-ß-D-Glcp fragments. The ß-glucan (at 0.5, 1, and 2%) showed shear thinning behavior and when the concentration of the solution increased, there was an increase in apparent viscosity. The ß-D-glucan presented gel-like behavior and thermal stability under a simulated pasteurization process, suggesting its potential as a thickening and gelling agent in products submitted to temperature variations. The ß-D-glucan at 0.3, 1.0 and 3.0 mg kg-1 significantly inhibited the inflammatory pain in 24.8, 56.9 and 82.3%, respectively, in the formalin-induced nociception in mice. The results pointed out that the ß-D-glucan (GHW-PN) isolated from P. nameko presents potential application for the food industry or for medical purposes.

Chemical and structural characterization of Pholiota nameko extracts with biological properties.

Edible mushrooms including Pholiota nameko are excellent sources of extractable bioactive compounds with much to explore. Enzymatic extractions with Cellulase and Viscozyme were responsible for highest extraction yields (67-77%). No strong antioxidant activity was observed although extracts were able to scavenge ABTS(+) and OH(+). Potential prebiotic activity was observed in all extracts, some increasing 1.4-2 Log cycles of Lactobacillus acidophilus La-5 and Bifidobacterium animalis BB12. 30-50% α-glucosidase inhibition was observed in ultrasound, Flavourzyme and Cellulase extracts. Flavourzyme and Cellulase extracts are richer in macro (Mg, K and P) and micro elements (Zn, Mn and Fe) in comparison to mushroom. Monosaccharides content and profile varied slightly among both extracts with predominance of glucose, galactose and mannose with no uronic acids detection; Flavourzyme extract reported higher free amino acids content. Presence of α and ß-glycosidic structures such as glucans and glucan-protein complexes are among the polysaccharides found in both extracts.

A Polysaccharide from the Culinary-Medicinal Mushroom Pholiota nameko (Agaricomycetes) Inhibits the NF-κB Pathway in Dendritic Cells Through the TLR2 Receptor.

A polysaccharide purified from Pholiota nameko (PNPS-1) was found to have anticancer and anti-inflammatory activity. This study investigated the effect of PNPS-1 on the nuclear factor (NF)-κB signaling pathway of TLR2 small interfering RNA-silenced murine bone marrow-derived dendritic cells (BMDCs) and relevant mechanisms. The expression of messenger RNA of 4 NF-κB-related genes, including MyD88, IKBKB, RelA(p65), and CCL2, was determined by real-time polymerase chain reaction; the expression of the phenotype molecule intercellular adhesion molecule-1 (ICAM-1) by flow cytometry; the protein expression of IKKß and p65 by Western blot; the production of p65 by enzyme-linked immunosorbent assay; and the expression of p65 by immunocytochemistry. The results showed that TLR2-specific small interfering RNA could effectively inhibit the decrease in the expression of MyD88, IKBKB, CCL2, p65, and ICAM-1 in BMDCs induced by PNPS-1, and thus the transcription inactivation of NF-κB, which obviously suggests that PNPS-1 could downregulate the NF-κB signaling pathway via the TLR2 receptor.

Target-selective photo-degradation of AFP-L3 and selective photo-cytotoxicity against HuH-7 hepatocarcinoma cells using an anthraquinone-PhoSL hybrid.

A purposefully-designed anthraquinone-Pholiota squarrosa lectin (PhoSL) hybrid effectively degraded α-fetoprotein-L3 (AFP-L3) associated with liver cancer. Degradation was achieved under light irradiation in the absence of any additives and under neutral pH conditions. Moreover, the hybrid effectively exhibited selective photo-cytotoxicity against HuH-7 hepatocarcinoma cells upon photo-irradiation.

Effects of Pholiota nameko polysaccharide on NF-κB pathway of murine bone marrow-derived dendritic cells.

This study investigated the effect of a polysaccharide purified from Pholiota nameko (PNPS-1) on the NF-κB signaling pathway of murine bone marrow-derived dendritic cells (BMDCs) and relevant mechanisms. The results showed that PNPS-1 could decrease the expression of maturation markers CD40 and CD80 on BMDCs. PNPS-1 also could decrease the mRNA expression of Myd88, TRAF6, TIRAP, IRAKI, IKBKB, NFKB1, NFKB2 and RelA in immature BMDCs determined by RT-PCR, and decreased the IKKß and P65 production in BMDCs determined by Western blot, and decreased the NF-кB P65 production determined by ELISA. In addition, the effects of PNPS-1 on BMDCs were significantly impaired by treating the cells with anti-TLR2 antibody prior to PNPS-1 treatment, implying direct interaction between PNPS-1 and TLR2 on cell surface. These results indicate that PNPS-1 regulates BMDCs through TLR2 and downstream NF-кB signalings.

Antioxidant and anti-ageing activities of mycelia zinc polysaccharide from Pholiota nameko SW-03.

BACKGROUND: Edible fungi polysaccharides usually exhibit antioxidant activity, and zinc has been shown to have antioxidant properties. In the present work, Pholiota nameko SW-03 was used as a vector of zinc biotransformation in order to obtain mycelia zinc polysaccharide (MZPS), and the structural characterization and anti-ageing activity of MZPS were investigated. RESULTS: Pholiota nameko SW-03 could accumulate zinc in the form of zinc-enriched polysaccharide, and the zinc content in MZPS was 16.39 ± 0.72 mg g(-1) . Three fractions (MZPS-1, MZPS-2 and MZPS-3) were successfully isolated. The main fraction (MZPS-2) with the highest antioxidant activity in vitro was composed of glucose, mannose, glucuronic acid, galactose, galacturonic acid and arabinose in a molar ratio of 172.59:5.29:4.61:4.20:1.01:1.00, with a weight-average molecular weight of 13.63 kDa. The anti-ageing capability was measured in d-galactose-induced ageing mice, and the results showed that MZPS could improve antioxidant status (superoxide dismutase, total antioxidant capability, malondialdehyde and lipid peroxide), indicating that MZPS had strong anti-ageing capability in vivo. CONCLUSION: This study suggested that organification of zinc through edible fungi liquid fermentation provided a novel method to produce MZPS, which might be used as a natural antioxidant to slow the progression of ageing.

Recognization of receptors on bone marrow-derived dendritic cells bound with Pholiota nameko polysaccharides.

Three major active polysaccharides isolated from Pholiota nameko (PNPS), including PNPS-1, PNPS-2 and PNPS-3, had been proved to inhibit the maturation of the murine bone marrow-derived dendritic cells (BMDCs). This paper recognized the affinity bind between PNPS and the five receptors (TLR2, TLR4, CD14, Dectin-1 and Mannose receptor) on BMDCs, using the bio-layer interferometry (BLI)-based biosensor technology developed by ForteBio on Octet RED system (Fortebio, Inc.). From the primary binding experiment, the gradient binding experiment and the inhibition binding experiment between the receptor proteins and PNPS, combined with the binding experiment between PNPS and the BMDCs membranes, we found that PNPS-1, PNPS-2 and PNPS-3 presented strong affinity bind with both TLR2 and Dectin-1 on BMDCs, only PNPS-3 with Mannose receptor. These data confirmed that PNPS could interact with TLR2, Dectin-1 and Mannose receptor that were very important for the affinity bind of these receptors and PNPS, which triggered the further stimulation on BMDCs.