The effect of Schizophyllan on the differentiation of osteoclasts and osteoblasts.

Schizophyllan (SPG), a ß-glucan from Schizophyllum commune, is recognized for its antioxidant, immunoregulatory, and anticancer activities. In this study, its effects on bone cells, particularly osteoclasts and osteoblasts, were examined. We demonstrated that SPG dose-dependently inhibited osteoclastogenesis and reduced gene expression associated with osteoclast differentiation. SPG also decreased bone resorption and F-actin ring formation. This inhibition could have been due to the downregulation of transcription factors c-Fos and nuclear factor of activated T cells 1 (NFATc1) via the MAPKs (JNK and p38), IκBα, and PGC1ß/PPARγ pathways. In coculture, SPG lowered osteoclastogenic activity in calvaria-derived osteoblasts by reducing macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor-κB ligand (RANKL) expression. In addition, SPG slightly enhanced osteoblast differentiation, as evidenced by increased differentiation marker gene expression and alizarin red staining. It also exhibited antiresorptive effects in a lipopolysaccharide-induced calvarial bone loss model. These results indicated a dual role of SPG in bone cell regulation by suppressing osteoclastogenesis and promoting osteoblast differentiation. Thus, SPG could be a therapeutic agent for bone resorption-related diseases such as osteoporosis, rheumatoid arthritis, and periodontitis.

Schizophyllan from Schizophyllum commune BRM 060008: Potential application as an inhibitor of lipase.

ß-D-glucan has significant implications in regulating lipid metabolism and preventing diseases associated with lipid accumulation. Schizophyllan (SPG) from Schizophyllum commune fungus is a commercially important ß-glucan with applications in the health food industry, pharmacy, and cosmetics. However, SPG was obtained by submerged culture of the wood-rotting and filamentous fungus S. commune BRM 060008, which may have been isolated from the Cerrado Biome of Brazil. In this study, to confirm that the polysaccharide produced by BRM 060008 strain fermentation was indeed (1→3)(1→6)-ß-D-glucan, it was purified and characterized using Fourier transform infrared spectroscopy, thermogravimetric analysis, high-performance size exclusion chromatography, nuclear magnetic resonance, and methylation analysis. The polysaccharide produced was identified as the ß-D-glucan expected with a high molecular weight (1.093 × 106 g/mol) and the thermogravimetric analysis indicated a maximum degradation temperature of ~324 °C and a 60 % residual weight, lower than commercial SPG. The molecular structure and thermal properties of the ß-D-glucan were similar to the commercial sample. Additionally, the in vitro pancreatic lipase inhibitory activity was evaluated, investigating anti-obesity and anti-lipidemic properties. The results showed unprecedented lipase inhibition activity to SPG prepared using the S. commune strain BRM 060008, making it promising for food and pharmaceutical applications.

Structural analysis of polysaccharide/antisense DNA complexes during cytoplasmic target mRNA hybridization.

We previously demonstrated antisense oligonucleotides (AS-ODNs) delivery system based on the complex formed with poly (dA) and schizophyllan, a type of ß-1,3-glucan. This complex enables efficient intracellular delivery of AS-ODNs. In this communication, we investigated the cytoplasmic translocation of the complex itself and its mechanism of action on mRNA. As a result, we found that the complex moved into the cytoplasm while keeping its structure, and AS-ODN hybridized with the target mRNA. This result encourages pharmaceutical applications of the complex.

Efficient production and skincare activity evaluation of schizophyllan, a ß-glucan derived from Schizophyllum commune NTU-1.

Schizophyllan (SPG), a ß-glucan produced by the fungus Schizophyllum commune, possesses a ß-(1 â†’ 3)-linked backbone with single ß-(1 â†’ 6)-linked glucose side chains at approximately every third residue. In this study, we screened SPG-producing strains of S. commune from different provinces in China. A candidate strain (NTU-1) with a high SPG yield was chosen, and the fermentation conditions were optimized. The optimal carbon and nitrogen sources were sucrose (40 g/L) and yeast extract (20 g/L), respectively. The optimal conditions for pH and temperature were 5.0 and 28 °C, respectively. Inclusion of 0.2 mg/L of 2,4-Dichlorophenoxyacetic acid in the medium further increased the SPG concentration. In a 5-L bioreactor, the fermentation cycle was reduced from the initial seven days to five days, and the concentration of SPG obtained was 21.3 g/L, which is the highest reported to date. In addition, we evaluated the bioactivity of the SPG prepared using strain NTU-1. The results showed that SPG had certain characteristics of anti-oxidation, anti-photoaging, and inhibition of melanin production, making it a promising reagent for skin care.

Production and Anti-Inflammatory Performance of PVA Hydrogels Loaded with Curcumin Encapsulated in Octenyl Succinic Anhydride Modified Schizophyllan as Wound Dressings.

Amphiphilic polysaccharides can be used as wall materials and applied to encapsulate hydrophobic active chemicals; moreover, there is significant demand for novel medical high-molecular-weight materials with various functions. In order to prepare amphiphilic schizophyllan (SPG), octenyl succinic anhydride (OSA) was chosen to synthesize OSA-modified schizophyllan (OSSPG) using an esterified reaction. The modification of OSSPG was demonstrated through FT-IR and thermal analysis. Moreover, it was found that OSSPG has a better capacity for loading curcumin, and the loading amount was 20 µg/mg, which was 2.6 times higher than that of SPG. In addition, a hydrogel made up of PVA, borax, and C-OSSPG (OSSPG loaded with curcumin) was prepared by means of the one-pot method, based on the biological effects of curcumin and the immune-activating properties of SPG. The mechanical properties and biological activity of the hydrogel were investigated. The experimental results show that the dynamic cross-linking of PVA and borax provided the C-OSSPG/BP hydrogel dressing with exceptional self-healing properties, and it was discovered that the C-OSSPG content increased the hydrogel's swelling and moisturizing properties. In fibroblast cell tests, the cells treated with hydrogel had survival rates of 80% or above. Furthermore, a hydrogel containing C-OSSPG could effectively promote cell migration. Due to the excellent anti-inflammatory properties of curcumin, the hydrogel also significantly reduces the generation of inflammatory factors, such as TNF-α and IL-6, and thus has a potential application as a wound dressing medicinal material.

Schizophyllum commune ß-glucan: Effect on interleukin-10 expression induced by lipopolysaccharide from periodontopathic bacteria.

ß-glucans are potent immunomodulators, with effects on innate and adaptive immune responses via dectin-1 as the main receptor. In this study, we investigated the biological effect of ß-glucan from Schizophyllum commune, called Schizophyllan (SPG) on Interleukin-10 (IL-10) expression induced by a lipopolysaccharide (LPS) from Aggregatibacter actinomycetemcomitans in murine macrophages (J774.1). SPG and dectin-1 interaction up-regulates LPS-induced IL-10 expression. The regulative effect of SPG on IL-10 expression is dependent on prolongation of nuclear translocation activity of nuclear factor-kappa B (NF-κBα) pathway induced by LPS. We also found that LPS-induced phosphorylation of mitogen- and stress-activated protein kinase 1 (MSK1) and cAMP-responsive-element-binding protein (CREB), followed by up-regulation of IL-10, was stimulated by SPG priming via activation of the spleen tyrosine kinase (Syk). Our data indicate that SPG augments the anti-inflammatory response in murine macrophages which can be useful to create an intervention for periodontal disease treatment.

Induction of potent cell growth inhibition by schizophyllan/K-ras antisense complex in combination with gemcitabine.

Antisense oligonucleotides (AS-ODNs) specifically hybridize with target mRNAs, resulting in interference with the splicing mechanism or the regulation of protein translation. In our previous reports, we demonstrated that ß-glucan schizophyllan (SPG) can form a complex with AS-ODNs attached with oligo deoxyadenosine dA40 (AS-ODN-dA40/SPG), and that this complex can be recognized by ß-glucan receptor Dectin-1 on antigen presenting cells and lung cancer cells. In many types of cancer cell, activating K-ras mutations related to malignancy are frequently observed. In this study, we first designed 78 AS-ODNs for K-ras to optimize the sequence for highly efficient gene suppression. The selected AS-ODN (K-AS07) having dA40 made a complex with SPG. The resultant complex (K-AS07-dA40/SPG) showed an effect of silencing the ras gene in the cells (PC9: human adenocarcinoma differentiated from lung tissue) expressing Dectin-1, leading to the suppression of cell growth. Furthermore, the cytotoxic effect was enhanced when used in combination with the anticancer drug gemcitabine. Gemcitabine, a derivative of cytidine, was shown to interact with dA40 in a sequence-dependent manner. This interaction did not appear to be so strong, with the gemcitabine being released from the complex after internalization into the cells. SPG and the dA40 part of K-AS07-dA40 play roles in carriers for K-AS07 and gemcitabine, respectively, resulting in a strong cytotoxic effect. This combination effect is a novel feature of the AS-ODN-dA40/SPG complexes. These results could facilitate the clinical application of these complexes for cancer treatment.

Dietary schizophyllan reduces mitochondrial damage by activating SIRT3 in mice.

Schizophyllan (SPG), produced by Schizophyllum commune, is an exopolysaccharide with multiple academic and commercial uses, including in the food industry and for various medical functions. We previously demonstrated that SPG conjugated with c-Src peptide exerted a significant therapeutic effect on mouse models of the acute inflammatory diseases polymicrobial sepsis and ulcerative colitis. Here we extended these results by investigating whether SPG exerted a protective effect against mitochondrial damage in the liver via sirtuin 3 (SIRT3) induction, focusing on the deacetylation of succinate dehydrogenase A (SDHA) and superoxide dismutase 2 (SOD2). Liver damage models induced by alcohol or conjugated linoleic acid (CLA, which simulates lipodystrophy) in SIRT3-/-, SOD2-/-, and SDHA-/- mice were used. Results showed that dietary supplementation with SPG induced SIRT3 activation; this was involved in mitochondrial metabolic resuscitation that countered the adverse effects of alcoholic liver disease and CLA-induced damage. The mitochondrial SIRT3 mediated the deacetylation and activation of SOD2 in the liver and SDHA in adipose tissues, suggesting that SPG supplementation reduced ethanol-induced liver damage and CLA-induced adverse dietary effects via SIRT3-SOD2 and SIRT3-SDHA signaling, respectively. Together, these results suggest that dietary SPG has a previously unrecognized role in SIRT3-mediated mitochondrial metabolic resuscitation during mitochondria-related diseases.

Inflammatory responses of macrophage-like RAW264.7 cells in a 3D hydrogel matrix to ultrasonicated schizophyllan.

The water-soluble ß-glucan schizophyllan has diverse immunomodulatory activities. However, its biological activities have only been explored using cells grown in two-dimensional (2D) culture condition, which does not replicate the three-dimensional (3D) microenvironment of actual tissue, resulting in mismatches between in vitro and in vivo findings. In this study, we investigated the immunomodulatory effects of ultrasonicated schizophyllan (uSPG) on RAW264.7 cells encapsulated in 3D poly(ethylene glycol) hydrogel. Cells grown in 3D were less sensitive to uSPG than those grown in 2D, although uSPG upregulated M1 macrophage phenotype markers in both conditions. This might be due to the low availability of uSPG recognition receptors of cells grown in 3D. Conversely, uSPG promoted gene expressions of M2 macrophage phenotype markers in 3D, suggesting uSPG-induced immune-regulation of macrophages in real tissues. These findings imply that the immunomodulatory effects of uSPG on macrophages should be carefully interpreted in terms of the microenvironment.

A novel double-network antibacterial hydrogel based on aminated bacterial cellulose and schizophyllan.

In this study, a novel hydrogel composed of bacterial cellulose (BC) and schizophyllan (SPG) biopolymers with improved mechanical, swelling and antibacterial properties was developed. BC was firstly functionalized using 3-aminopropyl triethoxysilane (APTES) to provide better interaction with SPG. A diffraction peak at 5.7° in the XRD pattern of amine-grafted BC/SPG membrane revealed the intercalation of SPG into BC network. SEM images of amine-grafted BC/SPG showed that the fibrillar network of BC was totally covered with schizophyllan. Two distinct stages of weight loss in TGA thermo-gram of amine grafted BC/SPG verified the successful entrance of SPG into BC fibrils. Tensile strength of the amine-grafted BC/SPG considerably increased to 42.19 ±â€¯7.16 MPa as compared to neat BC (4.41 ± 0.38 MPa) and amine-grafted BC (7.90 ±â€¯0.71 MPa). It also showed the highest swelling degree (800 ±â€¯80%). Amine-grafted BC/SPG exhibited moderate antibacterial activity. MTT assay showed that amine-grafted BC/SPG stimulated the proliferation of normal human fibroblast cells.

Electrospun Schizophyllan/polyvinyl alcohol blend nanofibrous scaffold as potential wound healing.

In this study, aqueous Schizophyllan (SPG) (1.5 w/v%) was mixed with aqueous polyvinyl alcohol (PVA) (10 w/v%) at various volume ratios and electrospun to prepare nanofibers. The fiber diameter was decreased by increasing the SPG content. A reliable linear relationship (p < 0.01) was established between the solution properties and fiber diameter. Contiguous, bead-free, and smooth fibers were obtained when the SPG/PVA ratios were 20:80, 30:70, and 40:60. FT-IR spectra, SEM images, tensile testing, swelling ratios, and water contact angle were utilized to characterize this glutaraldehyde (Glu) vapor cross-linked nanofibers in order to analyze the morphology, functional groups, mechanical attributes, hydrophilicity, and humidity of the nanofibers for skin tissue regeneration. Furthermore, the cell culture that was studied by the use of fibroblast (L929) cells showed that these SPG-based nanofibrous scaffolds could generate the improved cell adhesion. In conclusion, it was observed that SPG/PVA nanofiber mat in a volume ratio of 20:80 after 3 day was a suitable material for improving the wound healing, as it could increase cell proliferation and migration that possessed fiber diameter. The characteristics of this nanofiber were 267 nm, contact angle of 47.75°, good swelling behavior (289%), the ultimate strength of 6.513 MPa, Young modulus of 2.665 MPa, and cell viability of 150%.

Circulating nano-particulate TLR9 agonist scouts out tumor microenvironment to release immunogenic dead tumor cells.

Recent evidence suggest that a ß-glucan derived from mushroom Schizophyllan(SPG) complexed with a humanized TLR9 agonistic CpG DNA, K3 (K3-SPG) is a promising vaccine adjuvant that induces robust CD8 T cell responses to co-administered antigen. However, it has not been investigated whether K3-SPG alone can act as an anti-cancer immunotherapeutic agent or not. Here, we demonstrate that intravenous injection of K3-SPG, but not CpG alone, is accumulated in the tumor microenvironment and triggered immunogenic cell death (ICD) of tumor cells by local induction of type-I interferon (IFN) as well as IL-12. Resultant innate immune activation as well as subsequent tumor-specific CD8 T cell responses were contributed the tumor growth suppression. This anti-tumor effect of K3-SPG monotherapy was also confirmed by using various tumor models including pancreatic cancer peritoneal dissemination model. Taken together, nano-particulate TLR9 agonist injected intravenously can scout out tumor microenvironment to provoke local innate immune activation and release dead tumor cells into circulation that may induce broader and protective tumor antigen-specific CD8 T cells.

Immunoregulatory and antitumor activity of schizophyllan under ultrasonic treatment.

Aim of this study was to investigate the effect of ultrasonic treatment on the biological activities of schizophyllan (SPG) from Schizophyllum commune. The immunoregulatory and antitumor activity in vitro and in vivo of SPG and ultrasonic-treated SPG (USPG) were evaluated by splenic lymphocytes, macrophages RAW264.7 and human breast carcinoma T-47D cells. Compared with SPG, USPG fractions had small molecular weight and narrow distribution. Meantime, more enhancement of NO production in macrophages RAW264.7, lymphocytes proliferation rates, IL-2 and TNF-α level from spleen lymphocytes and T-47D cells inhibition rates were observed in USPG fractions groups. This result indicated that the immune-enhancing and antitumor activity of SPG was significantly improved after ultrasonic treatment. USPG60 exhibited the highest biological activity in this study. In conclusion, application of ultrasonic technology on SPG preparation is an efficient approach to get high biological polysaccharide, and USPG60 might be a potential functional component for immunoregulatory and cancer treatment.

Rheological properties and antitumor activity of schizophyllan produced with solid-state fermentation.

Schizophyllan (SPG) was produced by the fungus Schizophyllum commune under solid-state fermentation conditions in this study. SPG was physically characterized and its rheological properties and antitumor effects on S180 tumors in vivo were evaluated. SPG is a neutral polysaccharide with three main fractions, and the major fraction comprises 55.5%, with an average molecular weight 4.65 × 10(7) Da. Steady shear rheological measurements showed the typical pseudoplastic flow behavior of SPG at the experimental concentrations. The power law model was used to fit the shear curves of SPG and both its viscosity and consistency indices changed regularly as the concentration increased. SPG solution showed different viscoelastic behaviors at different concentrations: typical viscoelastic behavior was observed at lower concentrations, whereas solid-like behavior was observed at higher concentrations. Experimental doses of SPG exerted extreme antitumor effects in vivo, and the maximum inhibition rate was almost 70%.

Schizophyllan inhibits the development of mammary and hepatic carcinomas induced by 7,12 dimethylbenz(α)anthracene and decreases cell proliferation: comparison with tamoxifen.

BACKGROUND: Breast cancer is one of the leading causes of cancer mortality among women. Some anticancer compounds have been isolated from mushrooms. The aim of the present work was to study the anticancer effects of schizophyllan (SCH), a ß-D: -glucan extracted from the mushroom Schizophyllum commune alone or in combination with tamoxifen (TAM) on 7, 12 Dimethylbenz(α)anthracene (DMBA)-induced carcinomas in mice. METHODS: We isolated SCH from S. commune. Female mice received DMBA, SCH, DMBA+SCH, DMBA+TAM or DMBA+TAM+SCH or vehicles. We studied mice survival, tumour incidence, histopathology, oestrogen receptor (ER) expression, cell proliferation by immunohistochemical detection of proliferating cell nuclear antigen (PCNA), apoptosis by TUNEL assay, as well as caspase-3 expression. RESULTS: DMBA treatment resulted in mammary and hepatocellular carcinomas (HCC). Both SCH and TAM reduced the incidence of DMBA-induced mammary tumours by 85 and 75 %, respectively, and equally decreased the PCNA labelling index relative to DMBA. TAM treatment increased the incidence of- and PCNA index in HCCs relative to DMBA, while SCH suppressed these effects. TAM was more effective than SCH in the induction of apoptosis in both mammary and hepatic carcinomas. Caspase-3 levels correlated with the apoptotic index in most experimental groups. CONCLUSIONS: Only one dose of SCH had similar therapeutic effects against DMBA-induced mammary carcinomas as 4 weeks of TAM treatment. This coupled with the ability of SCH to suppress hepatic lesions associated with TAM treatment provides the rationale for further investigating the combined therapeutic effects of TAM+SCH in preclinical models of ER-positive breast cancer, as well as in liver cancer.

ß-1,3-Glucan/antisense oligonucleotide complex stabilized with phosphorothioation and its gene suppression.

Most of antisense oligonucleotides (ASOs) subjected to current clinical evaluation belong to phosphorothioate (PS) analogues. Although PS has great advantage in DNase resistance, it can induce nonspecific side-effects. Thus it is important to investigate the influence of ASOs with different PS contents. In this paper, we prepared the complex consisting of schizophyllan (SPG) and ASOs attached a dA40 tail with different PS contents to the 3' end of the ODN, which is introduced to stabilize the complex with SPG. With increase of PS content in the dA40, its complexation ability with SPG was improved and the complex showed high thermal stability. The thermal stability of the fully phosphorothioated ASOs was obtained by only replacing 20% of the oxygen of the phosphodiester moiety. The ability of gene suppression between PS and phosphodiester for antisense sequences was almost the same, indicating that the antisense sequences need not to be PS backbone. These data may provide new insight for the interaction between ß-1,3-glucan and DNA and help to deliver therapeutic ODNs.

Effect of SPG/indomethacin treatment on sepsis, interleukin-6 production, and expression of hepatic cytochrome P450 isoforms in differing strains of mice.

We previously reported that a combination of beta-glucan and indomethacin (IND), a non-steroidal anti-inflammatory drug, was lethal to mice. This lethality was strongly related to translocation of enterobacterial flora to various organs and the development of a systemic inflammation. In this study, we examined expression of microsomal cytochrome P450 (CYP), a drug-metabolizing enzyme mostly found in the liver. Normal ICR mice and endotoxin-low responder C3H/HeJ mice were employed to assess effects of endotoxin on impairment of CYP. In the ICR mice, CYP3A11 expression was decreased by beta-glucan or IND. In the early stage of beta-glucan + IND-treatment, 3A11 expression decreased more significantly; when shock was induced, CYP was dramatically decreased. 3A11 expression was also decreased in C3H/HeJ mice, but the effect was milder. In contrast, in both strains, CYP2E1 expression did not vary due to beta-glucan or IND, but decreased during sepsis. To clarify the molecular mechanisms of induced sepsis in C3H/HeJ mice, the reactivity of other pathogen-associated molecular patterns (PAMPs) was assessed. Those studies showed cooperative effects between Pam(3)CSK(4) (Pam(3)) and CpG ODN (CpG-oligodeoxynucleotide) on the induction of IL-6 synthesis by C3H/HeJ spleen cells. The findings here suggest that the beta-glucan + IND combination influenced hepatic cytochrome P450 expression, particularly in the late stage of sepsis. The results also indicate that this change may be associated with not only endotoxin but other PAMPs as well, and could be affected by the integrity of a host's drug metabolism function.

Blocking effect of anti-Dectin-1 antibodies on the anti-tumor activity of 1,3-beta-glucan and the binding of Dectin-1 to 1,3-beta-glucan.

Schizophyllan (SPG) is used to treat cervical cancer in combination with irradiation to enhance the immunological surveillance system. Dectin-1 is a cell surface receptor for 1,3-beta-glucan. In this study, we prepared two anti-Dectin-1 monoclonal antibodies, 4B2 and SC30 having a K(D) of 7.04 x 10(-8) M and 1.55 x 10(-7) M, respectively, and evaluated the role of Dectin-1 in SPG-induced anti-tumor activity in mice. Expression of Dectin-1 on peritoneal macrophages and binding of SPG to the cells were decreased by administration of 4B2 and SC30. SPG-mediated anti-tumor activity was inhibited by 4B2 and SC30. 4B2 and SC30 inhibited the binding of SPG to splenocytes from mice. The binding of SPG-biotin to Dectin-1-transfected HEK293 cells was inhibited by 4B2, but not SC30. 4B2 and SC30 differ in their influence on Dectin-1 between primary cells and transduced cells, and Dectin-1 effects 1,3-beta-glucan-mediated anti-tumor activity in mice by binding to SPG.

Galactose-PEG dual conjugation of beta-(1-->3)-D-glucan schizophyllan for antisense oligonucleotides delivery to enhance the cellular uptake.

Antisense oligonucleotides (AS ODNs) are applied to silence a particular gene, and this approach is one of the potential gene therapies. However, naked oligonucleotides are easy to be degraded or absorbed in biological condition. Therefore, we need a carrier to deliver AS ODNs. This paper presents galactose moieties that were conjugated to the side chain of SPG to enhance cellular ingestion through endocytosis mediated by asialoglycoprotein receptor specifically located on parenchymal liver cells. We introduced galactose with two types of chemical bonds; amide and amine, and the amine connection showed lower ingestion and more toxicity than the amide one. Since PEG was known to induce endocytosis escape, we combined PEG and galactose aiming to provide both cellular up-take and subsequent endocytosis escape. We designed lactose or galactose moieties to attach to the end of the PEG chain that connects to the SPG side chain. When the PEG had the molecular weight of 5000-6000, the antisense effect reached the maximum. We believe that this new type of galactose and PEG dual conjugation broaden the horizon in antisense delivery.

Schizophyllan-folate conjugate as a new non-cytotoxic and cancer-targeted antisense carrier.

Schizophyllan having folate-appendages was synthesized from native schizophyllan through NaIO(4)-oxidation and the subsequent reductive amination in aqueous ammonia followed by amido-coupling with folic acid. The resulting folate-appended schizophyllan can form stable complex with poly(dA), show specific affinity toward folate binding protein, and mediate effective antisense activity in cancer cells.