Hispidin rescues palmitate­induced insulin resistance in C2C12 myotubes.

Skeletal muscle serves an important role in the utilization of glucose during insulin­stimulated conditions. Excessive saturated fatty acids are considered to be a major contributing factor to insulin resistance in skeletal muscle cells. The present study investigated the effects of hispidin on palmitate­induced insulin resistance in C2C12 skeletal muscle myotubes via an MTT assay, glucose uptake assay, Oil­Red­O staining and western blot analysis. Hispidin reversed the palmitate­induced inhibition of glucose uptake, and inhibited palmitate­induced intracellular lipid accumulation. Hispidin suppressed insulin receptor substrate­1 Ser307 phosphorylation, and significantly promoted the activation of phosphatidylinositol­3­kinase and Akt, via inhibition of protein kinase C theta. Furthermore, hispidin treatment of C2C12 muscle cells increased glucose uptake via activation of adenosine monophosphate­activated protein kinase. These findings indicated that hispidin may improve palmitate­induced insulin resistance in skeletal muscle myotubes, and therefore hispidin treatment may be beneficial for patients with diabetes.

Effects of polysaccharides isolated from Inonotus obliquus against hydrogen peroxide-induced oxidative damage in RINm5F pancreatic ß-cells.

The purpose of the present study was to elucidate the cytoprotective effects of polysaccharides isolated from Inonotus obliquus. The polysaccharides were extracted from the fruiting body of I. obliquus (PFIO) and the liquid culture broth of I. obliquus (PLIO). The effects of PFIO and PLIO on hydrogen peroxide (H2O2)­induced oxidative damage of RINm5F pancreatic ß­cells were comparatively investigated using an MTT assay, immunofluorescent staining, flow cytometry, and western blot analyses in vitro. The results of the present study demonstrated that treatment with PFIO and PLIO decreased DNA fragmentation and the rate of apoptosis. In addition, pretreatment of cells with PFIO and PLIO prior to H2O2 exposure resulted in increased insulin secretion and scavenging activity for intracellular reactive oxygen species, as compared with treatment with H2O2 alone. The results of the present study suggested that PFIO and PLIO may exert protective effects against H2O2­induced oxidative stress via the regulation of mitogen­activated protein kinases, nuclear factor­κB and apoptotic proteins. Therefore, PFIO and PLIO may have potential merit as a medicinal food for the prevention of diabetes.

Polysaccharide isolated from the liquid culture broth of Inonotus obliquus suppresses invasion of B16-F10 melanoma cells via AKT/NF-κB signaling pathway.

A number of polysaccharides exhibit pharmacological activities. Polysaccharides derived from Inonotus obliquus (PLIO) appear to have various potential pharmacological properties, including anti­tumor activity. However, the molecular mechanisms underlying these properties remain to be elucidated. The present study investigated the anti­metastatic potential of PLIO and the underlying signaling pathways in B16­F10 murine melanoma cells using the MTT colorimetric assay, in vitro migration and invasion assays, and flow cytometric and western blot analyses. PLIO inhibited the invasion of B16­F10 cells and suppressed the expression of matrix metalloproteinases. PLIO treatment inhibited nuclear factor­κB (NF­κB) nuclear translocation in B16­F10 cells. In addition, PLIO treatment inhibited the phosphorylation of c-Jun N­terminal kinases and AKT. These results suggest that PLIO may suppress the invasion of highly metastatic melanoma cells via inhibition of the AKT/NF-κB signaling pathways.

Cytoprotective effect of hispidin against palmitate-induced lipotoxicity in C2C12 myotubes.

It is well known that Phellinus linteus, which produces hispidin and its derivatives, possesses antioxidant activities. In this study, we investigated whether hispidin has protective effects on palmitate-induced oxidative stress in C2C12 skeletal muscle cells. Our results showed that palmitate treatment in C2C12 myotubes increased ROS generation and cell death as compared with the control. However, pretreatment of hispidin for 8 h improved the survival of C2C12 myotubes against palmitate-induced oxidative stress via inhibition of intracellular ROS production. Hispidin also inhibited palmitate-induced apoptotic nuclear condensation in C2C12 myotubes. In addition, we found that hispidin can suppress cleavage of caspase-3, expression of Bax, and NF-κB translocation. Therefore, these results suggest that hispidin is capable of protecting C2C12 myotubes against palmitate-induced oxidative stress.

Mechanism of macrophage activation induced by polysaccharide from Cordyceps militaris culture broth.

Mushroom-derived polysaccharides have been shown to stimulate immune responses. Our previous report showed that the novel polysaccharide PLCM isolated from the culture broth of Cordyceps militaris could induce nitric oxide production in the murine macrophage-like cell line RAW264.7. In this study, we show that PLCM enhances immunostimulatory activities such as the release of toxic molecules (nitric oxide and reactive oxygen species), secretion of the cytokine tumor necrosis factor (TNF)-α, and phagocytic uptake in RAW264.7 macrophages. In addition, all the specific inhibitors against the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) (SN50, BAY11-7082, PD98059, SP600125 and SB203580) markedly suppressed the nitric oxide production and phagocytic uptake induced by PLCM. Moreover, antibodies specific to the extracellular domain of Toll-like receptor-2, Toll-like receptor-4 or the macrophage receptor Dectin-1 significantly attenuated PLCM-induced secretion of TNF-α. Our results indicate that the C. militaris polysaccharide activates macrophages through the MAPKs and NF-κB signaling pathways via Toll-like receptor 2, Toll-like receptor 4, and Dectin-1.

Cyanidin-3-glucoside isolated from mulberry fruit protects pancreatic ß-cells against oxidative stress-induced apoptosis.

The extract obtained from berries contains high amounts of anthocyanins, and this extract is used as a phytotherapeutic agent for different types of diseases. In this study, we examined the cytoprotective effects of cyanidin-3-glucoside (C3G) isolated from mulberry fruit against pancreatic ß-cell apoptosis caused by hydrogen peroxide (H2O2)-induced oxidative stress. The MIN6 pancreatic ß-cells were used to investigate the cytoprotective effects of C3G on the oxidative stress-induced apoptosis of cells. Cell viability was examined by MTT assay and lipid peroxidation was assayed by thiobarbituric acid (TBA) reaction. Immunofluorescence staining, flow cytometry and western blot analysis were also used to determine apoptosis and the expression of proteins associated with apoptosis. Our results revealed that H2O2 increased the rate of apoptosis by stimulating various pro-apoptotic processes, such as the generation of intracellular reactive oxygen species (ROS), lipid peroxidation, DNA fragmentation and caspase-3 activation. However, C3G reduced the H2O2-induced cell death in the MIN6N pancreatic ß-cells. In addition, we confirmed that H2O2 activated mitogen-activated protein kinases (MAPKs), such as extracellular signal-regulated kinase (ERK), c-Jun NH2-terminal kinase (JNK) and p38 MAPK. C3G inhibited the phosphorylation of ERK and p38 without inducing the phosphorylation of JNK. Furthermore, C3G regulated the intrinsic apoptotic pathway-associated proteins, such as proteins belonging to the Bcl-2 family, cytochrome c and caspase-3. Taken together, our results suggest that C3G isolated from mulberry fruit has potential for use as a phytotherapeutic agent for the prevention of diabetes by preventing oxidative stress-induced ß-cell apoptosis.

Neuroprotective effect of prenylated arylbenzofuran and flavonoids from morus alba fruits on glutamate-induced oxidative injury in HT22 hippocampal cells.

A prenylated arylbenzofuran and six flavonoids were isolated from the fruits of Morus alba L. through silica gel, octadecyl silica gel, and Diaion HP-20 column chromatography. Based on the nuclear magnetic resonance, mass spectrometry, and infrared spectroscopic data, the chemical structures of the compounds were determined to be artoindonesianin O (1), isobavachalcone (2), morachalcone A (3), quercetin (4), astragalin (5), isoquercetin (6), and rutin (7). The isolated compounds were evaluated for protection of HT22-immortalized hippocampal cells against glutamate-induced oxidative stress. Compounds 1 and 3 exhibited protective effects with EC(50) values of 19.7±1.2 and 35.5±2.1 µM, respectively. The major compounds 1-3 and 7 were quantified using liquid chromatography/mass spectrometry analysis and were determined to be 1.88±2.1, 1.90±1.8, 0.78±1.5, and 37.29±2.2 mg/kg, respectively, in the ethanol extract of M. alba L. fruits.

Cyanidin-3-glucoside isolated from mulberry fruits protects pancreatic ß-cells against glucotoxicity-induced apoptosis.

The present study investigated the cytoprotective effects of cyanidin­3­glucoside (C3G), isolated from mulberry fruits, on the glucotoxicity­induced apoptosis of pancreatic ß­cells to evaluate the antidiabetic effects of this compound. MIN6N pancreatic ß­cells were used to investigate the cytoprotective effects of C3G. In addition, the effects of C3G on the glucotoxicity­induced apoptosis of pancreatic ß­cells was evaluated using MTT assay, immunofluorescent staining, flow cytometric and western blot analyses. The pancreatic ß­cells cultured under high glucose conditions exhibited distinct apoptotic features. C3G decreased the generation of intracellular reactive oxygen species, DNA fragmentation and the rate of apoptosis. C3G also prevented pancreatic ß­cell apoptosis induced by high glucose conditions by interfering with the intrinsic apoptotic pathways. In addition, C3G treatment resulted in increased insulin secretion compared with treatment with high glucose only. In conclusion, the results of the present study suggested that C3G obtained from mulberry fruits may be a potential phytotherapeutic agent for the prevention of diabetes.

Inonotus obliquus-derived polysaccharide inhibits the migration and invasion of human non-small cell lung carcinoma cells via suppression of MMP-2 and MMP-9.

Polysaccharides isolated from the fruiting body of Inonotus obliquus (PFIO) are known to possess various pharmacological properties including antitumor activity. However, the anti-metastatic effect and its underlying mechanistic signaling pathway involved these polysaccharides in human non-small cell lung carcinoma remain unknown. The present study therefore aimed to determine the anti-metastatic potential and signaling pathways of PFIO in the highly metastatic A549 cells. We found that PFIO suppressed the migration and invasive ability of A549 cells while decreasing the expression levels and activity of matrix metalloproteinase (MMP)-2 and MMP-9. Furthermore, PFIO decreased the phosphorylation levels of mitogen-activated protein kinases (MAPKs) and phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) as well as the expression level of COX-2, and inhibited the nuclear translocation of nuclear factor κB (NF-κB) in A549 cells. These results suggested that PFIO could suppress the invasion and migration of human lung carcinoma by reducing the expression levels and activity of MMP-2 and MMP-9 via suppression of MAPKs, PI3K/AKT, and NF-κB signaling pathways.

Cordyceps militaris extract protects human dermal fibroblasts against oxidative stress-induced apoptosis and premature senescence.

Oxidative stress induced by reactive oxygen species (ROS) is the major cause of degenerative disorders including aging and disease. In this study, we investigated whether Cordyceps militaris extract (CME) has in vitro protective effects on hydrogen peroxide-induced oxidative stress in human dermal fibroblasts (HDFs). Our results showed that the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging activity of CME was increased in a dose-dependent manner. We found that hydrogen peroxide treatment in HDFs increased ROS generation and cell death as compared with the control. However, CME improved the survival of HDFs against hydrogen peroxide-induced oxidative stress via inhibition of intracellular ROS production. CME treatment inhibited hydrogen peroxide-induced apoptotic cell death and apoptotic nuclear condensation in HDFs. In addition, CME prevented hydrogen peroxide-induced SA-ß-gal-positive cells suggesting CME could inhibit oxidative stress-induced premature senescence. Therefore, these results suggest that CME might have protective effects against oxidative stress-induced premature senescence via scavenging ROS.

Mulberry fruit extract protects pancreatic ß-cells against hydrogen peroxide-induced apoptosis via antioxidative activity.

Among the many environmental stresses, excessive production of reactive oxygen species (ROS) and the ensuring oxidative stress are known to cause significant cellular damage. This has clinical implications in the onset of type 1 diabetes, which is triggered by the destruction of pancreatic ß-cells and is associated with oxidative stress. In this study, we investigated the protective and antioxidative effects of mulberry extract (ME) in insulin-producing pancreatic ß-cells. We found that ME protects pancreatic ß-cells against hydrogen peroxide (H2O2)-induced oxidative stress and the associated apoptotic cell death. ME treatment significantly reduced the levels of H2O2-induced 2-diphenyl-1-picrylhydrazyl (DPPH) radicals, and lipid peroxidation and intracellular ROS accumulation. In addition, ME inhibited DNA condensation and/or fragmentation induced by H2O2. These results suggest that ME protects pancreatic ß-cells against hydrogen peroxide-induced oxidative stress.

Polysaccharide from Inonotus obliquus inhibits migration and invasion in B16-F10 cells by suppressing MMP-2 and MMP-9 via downregulation of NF-κB signaling pathway.

Polysaccharides derived from Inonotus obliquus (PIO) are known to possess multiple pharmacological activities including antitumor activity. However, the possible molecular mechanisms of these activities are unknown. In the present study, we determined the anti-metastatic potential and signaling pathways of PIO in the highly metastatic B16-F10 mouse melanoma cell line in vitro. We found that PIO suppressed the migration and invasive ability of B16-F10 cells and decreased the expression levels and activities of matrix metalloproteinase (MMP)-2 and MMP-9. In addition, PIO decreased the phosphorylation levels of extracellular signal-regulated protein kinase (ERK), c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK); PIO also decreased the expression level of cyclooxygenase (COX)­2 and inhibited the nuclear translocation of nuclear factor κB (NF-κB) in B16-F10 melanoma cells. These results suggest that PIO could suppress the invasion and migration of B16-F10 melanoma cells by reducing the expression levels and activities of MMP-2 and MMP-9 through suppressing MAPK, COX-2 and NF-κB signaling pathways.

Effects of mulberry ethanol extracts on hydrogen peroxide-induced oxidative stress in pancreatic ß-cells.

Reactive oxygen species (ROS) are key mediators of mammalian cellular damage and are associated with diseases such as aging, arteriosclerosis, inflammation, rheumatoid arthritis and diabetes. Type 1 diabetes develops upon the destruction of pancreatic ß-cells, which is partly due to ROS activity. In this study, we investigated the cytoprotective and anti-oxidative effects of fractionated mulberry extracts in mouse insulin-producing pancreatic ß-cells (MIN6N cells). Treatment with hydrogen peroxide (H2O2) induced significant cell death and increased intracellular ROS levels, lipid peroxidation and DNA fragmentation in the MIN6N cells. Fractionated mulberry extracts significantly reduced the H2O2-dependent production of intracellular ROS, 2,2-diphenyl-1-picrylhydrazyl (DPPH) radicals and lipid peroxidation. In addition, mulberry extracts inhibited DNA fragmentation induced by H2O2. Thus, the antioxidant properties of mulberry extracts in pancreatic ß-cells may be exploited for the prevention or treatment of type 1 diabetes.

Culture conditions and medium components for the production of mycelial biomass and exo-polysaccharides with Paecilomyces japonica in liquid culture.

In this study, the liquid culture conditions were optimized for maximal production of mycelial biomass and exo-polysaccharide by Paecilomyces japonica. The effects of medium composition, C/N ratio and physical parameters were investigated. From these experiments, 30 g glucose, 20 g yeast extract, 0.5 g KH2PO4, and 0.1 g CuCl2 2H2O in 1-l distilled water were found to be the most suitable carbon, nitrogen, and mineral sources, respectively. The optimal temperature, initial pH, agitation, and aeration were determined to be 27°C, uncontrolled pH, 400 rpm, and 1.0 vvm, respectively. Under these optimal conditions, the maximum mycelial growth and polysaccharides production were 23.1 g/l and 2.5 g/l, respectively.

Hispidin isolated from Phellinus linteus protects against hydrogen peroxide-induced oxidative stress in pancreatic MIN6N ß-cells.

Reactive oxygen species (ROS) have been shown to cause DNA damage, protein denaturation, loss of antioxidative enzyme activity, and lipid peroxidation. Thus, ROS are associated with tissue damage and are considered to be prime contributing factors in inflammation, diabetes, aging, and cancer. In this study, we investigated whether or not hispidin protects pancreatic MIN6N ß-cells from oxidative stress caused by hydrogen peroxide. Treatment of MIN6N ß-cells with 0.5 mM hydrogen peroxide for 4 hours caused significant loss of cell viability and an increase in the number of apoptotic cells. However, pretreatment of MIN6N ß-cells with hispidin for 24 hours reduced loss of cell viability and decreased the number of apoptotic cells. In addition, 70 µM hispidin significantly scavenged intracellular ROS and inhibited apoptosis and caspase-3 induced by hydrogen peroxide. Furthermore, the generation of thiobarbituric acid-reactive substances was inhibited in the presence of hispidin in a dose-dependent manner. Also, 70 µM hispidin significantly increased insulin secretion in hydrogen peroxide-treated MIN6N ß-cells. These results suggest that hispidin may be effective for protecting MIN6N ß-cells from ROS toxicity in diabetes.

Immunostimulating activity of the polysaccharides isolated from Cordyceps militaris.

In this study, the signaling mechanism of the polysaccharides isolated from fruiting body of Cordyceps militaris (CM) was investigated in macrophages to evaluate its immuno-stimulating properties. We found that CM was capable of upregulation of NO, ROS, TNF-α and phagocytic uptake in mouse peritoneal macrophages and RAW264.7 macrophages. Macrophages activation by CM seemed to occur via activation of NF-κB and all three MAPKs pathways through dectin-1 and TLR2 macrophage receptors. Additionally, we showed that CM suppressed in vivo growth of melanoma in an experimental mouse model. Based on these data, we suggested that CM may potentially regulate the immune response.

Inonotus obliquus protects against oxidative stress-induced apoptosis and premature senescence.

In this study, we investigated the cytoprotective effects of Inonotus obliquus against oxidative stress-induced apoptosis and premature senescence. Pretreatment with I. obliquus scavenged intracellular ROS and prevented lipid peroxidation in hydrogen peroxide-treated human fibroblasts. As a result, I. obliquus exerted protective effects against hydrogen peroxide-induced apoptosis and premature senescence in human fibroblasts. In addition, I. obliquus suppressed UV-induced morphologic skin changes, such as skin thickening and wrinkle formation, in hairless mice in vivo and increased collagen synthesis through inhibition of MMP-1 and MMP-9 activities in hydrogen peroxide-treated human fibroblasts. Taken together, these results demonstrate that I. obliquus can prevent the aging process by attenuating oxidative stress in a model of stress-induced premature senescence.

Agaricus blazei Murill enhances doxorubicin-induced apoptosis in human hepatocellular carcinoma cells by NFκB-mediated increase of intracellular doxorubicin accumulation.

It has been demonstrated that the Agaricus blazei Murill (ABM) mushroom, which primarily consists of polysaccharides, possesses anti-tumor activities. However, the mechanisms by which ABM inhibits human hepatocellular carcinoma growth remain unknown. Our study demonstrates that ABM acts as an enhancer to sensitize doxorubicin (Dox)-mediated apoptotic signaling, and this sensitization can be achieved by enhancing intracellular Dox accumulation via the inhibition of NFκB activity. These findings suggest that ABM, when combined with low doses of Dox, has the potential to provide more efficient therapeutic effects against drug-resistant human hepatocellular carcinoma.

Immunostimulating activity by polysaccharides isolated from fruiting body of Inonotus obliquus.

In this study, we investigated the immunostimulating activity of polysaccharides isolated from fruiting body of Inonotus obliquus (PFIO). Additionally, the signaling pathway of PFIO-mediated macrophage activation was investigated in RAW264.7 macrophage cells. We found that PFIO was capable of promoting NO/ROS production, TNF-α secretion and phagocytic uptake in macrophages, as well as cell proliferation, comitogenic effect and IFN-γ/IL-4 secretion in mouse splenocytes. PFIO was able to induce the phosphorylation of three MAPKs as well as the nuclear translocation of NF-κB, resulting in activation of RAW264.7 macrophages. PFIO also induced the inhibition of TNF-α secretion by anti-TLR2 mAb, consequently, PFIO might be involved in TNF-α secretion via the TLR2 receptor. In addition, our results showed that oral administration of PFIO suppressed in vivo growth of melanoma tumor in tumorbearing mice. In conclusion, our experiments presented that PFIO effectively promotes macrophage activation through the MAPK and NF-κB signaling pathways, suggesting that PFIO may potentially regulate the immune response.

Study of macrophage activation and structural characteristics of purified polysaccharide from the fruiting body of Cordyceps militaris.

Cordyceps militaris, an entomophathogenic fungus belonging to the class Ascomycetes, has been reported to have beneficial biological activities such as hypoglycemic, anti-inflammatory, anti-tumor, anti-metastatic, hypolipidemic, immunomodulatory, and antioxidant effect. In this study, the crude water-soluble polysaccharides CMP, which was obtained from the fruiting body of C. militaris by hot water extraction and ethanol precipitation, was fractionated by DEAE cellulose and Sepharose CL-6B column chromatography. This process resulted in three polysaccharide fractions, termed CMP Fr I, CMP Fr II, and CMP Fr III. Of these fractions, CMP Fr II, with an average molecular weight of 127 kDa, was able to upregulate effectively the phenotypic functions of macrophages such as NO production and cytokine expression. The chemical property of the stimulatory polysaccharide, CMP Fr II, was determined based on monosaccharide composition, which consisted of glucose (56.4 %), galactose (26.4 %), and mannose (17.2%). Its structural characteristics were investigated by a combination of chemical and instrumental analyses, including methylation, reductive cleavage, acetylation, Fourier transform infrared spectroscopy (FT-IR), and gas chromatography-mass spectrometry (GC-MS). Results indicated that CMP Fr II consisted of the (1-->4) or (1-->2) linked glucopyranosyl or galactopyranosyl residue with a (1-->2) or (1-->6) linked mannopyranosyl, glucopyranosyl or galactopyranosyl residue as a side chain. The configuration of the beta-linkage and random coil conformation of CMP Fr II were confirmed using a Fungi Fluor kit and Congo Red reagent, respectively.