Hongping Yin

Protection of chronic renal failure by a polysaccharide from Cordyceps sinensis.

A water-soluble polysaccharide (CPS-2), isolated from the cultured Cordyceps sinensis, was obtained by hot-water extraction, anion-exchange and gel permeation chromatography. Its structural characteristics were investigated by PMP pre-column derivation, periodate oxidation, methylation analysis, FTIR and NMR spectroscopy. CPS-2 was found to be mostly of alpha-(1-->4)-D-glucose and alpha-(1-->3)-D-mannose, branched with alpha-(1-->4,6)-D-glucose every twelve residues on average. CPS-2 had a molecular weight of 4.39x10(4) Da. The protective effect of CPS-2 on the model of chronic renal failure was established by fulgerizing kidney. The changes in blood urea nitrogen and serum creatinine revealed that CPS-2 could significantly relieve renal failure caused by fulgerizing kidney.

Structural Determination and Antioxidant Activity of a Polysaccharide from the Fruiting Bodies of Cultured Cordyceps sinensis

A water-soluble polysaccharide named CPS1 had been isolated from C. sinensis mycelium by hot water extraction, ethanol precipitation, anion-exchange, and gel-permeation chromatography. UV spectra, FTIR spectra, partial acid hydrolysis, PMP precolumn derivation, periodate oxidation and Smith degradation studies were conducted to elucidate its structure. The results indicated that CPS1 was a glucomannogalactan with the monosaccharide composition of glucose: mannose: galactose = 2.8: 2.9: 1. The total carbohydrate content of CPS1 was 99.0%. The weight-average molecular weight was 8.1 x 10(3) Da. The results predicted (1-->2) and (1-->4)-linkage of mannose, (1-->3)-linkage of galactose, (1--> ) and (1-->3, 6)-linkage of glucose composed the backbone of CPS1. CPS1 was also evaluated for its antioxidant activity in vitro, including scavenging effects on the hydroxyl radicals, the reducing power, Fe(2+)-chelating activity, scavenging effect on superoxide radicals, as well as the inhibition of hydrogen peroxide induced haemolysis. CPS1 showed a high antioxidant effect, especially scavenging effect of hydroxyl radicals, the reducing power and Fe(2+)-chelating activity. The results provide scientific support for the antioxidant activity and indicated a connection between antioxidant activity and reparation of renal failure.

Purification, Characterization and Immuno-Modulating Properties of Polysaccharides Isolated from Flammulina velutipes Mycelium

To investigate the immuno-modulating activity of Flammulina velutipes mycelium, three different Flammulina velutipes polysaccharides (FVPs) were isolated by fractionation using gel filtration and were identified as the immunomodulators of murine peritoneal macrophages. Based on the results of fourier transform infrared spectroscopy (FTIR), high performance liquid chromatography (HPLC), NMR spectroscopy, methylation analysis and gas chromatography-mass spectrogram (GC-MS), FVP2C was identified as glucose, galactose, mannose and fucose in molar ratio of 100: 14: 7: 4. FVP2C, molecular weight of 27.3 x 10(3) Da, was characterized as alpha-D-(1-->4)-glucan which was highly branched with alpha-D-(1-->6)-glucosyl residues, a single galactose or small amounts of mannoses and fucose at the C-6 position every twelve residues, on average, along the main chain. In the present study, it was found that three FVPs induced a significant increase in cellular nitric oxide formation, interleukin-1 production and tumor necrosis factor-alpha secretion in macrophages in vitro. The immuno-modulating activity of FVP2A, FVP2B and FVP2C was dose-dependent.

Cordyceps sinensis polysaccharide inhibits PDGF-BB-induced inflammation and ROS production in human mesangial cells.

CPS-F, a polysaccharide derived from Cordyceps sinensis, is a potential anti-inflammatory and anti-oxidative agent. We demonstrated that CPS-F not only inhibits platelet-derived growth factor BB (PDGF-BB)-induced intracellular reactive oxygen species (ROS) generation, and up-regulation of tumor necrosis factor-α (TNF-α), TNF-α receptor 1 (TNFR1), and monocyte chemotactic protein-1 (MCP-1), but also acts synergistically in combination with MAPK/ERK inhibitor U0126 and PI3K/Akt inhibitor LY294002. Additionally, up-regulation of pro-inflammatory factors was reversed by use of a combination of CPS-F and NADPH oxidase (NOX) inhibitor diphenyleneiodonium chloride (DPI) or silencing of NOX1. Furthermore, CPS-F prevents the PDGF receptor β (PDGFRβ) promoter activity induced by PDGF-BB in transfected cells and ameliorates increased levels of TNF-α, TNFR1, and MCP-1 when PDGFRβ is silenced, thereby suggesting that CPS-F possesses a bidirectional regulatory function. Our findings suggest CPS-F may exert its therapeutic effect for the treatment of glomerulonephritis related to human mesangial cells (HMCs) through the ERK1/2/Akt pathways.

Bletilla striata polysaccharide inhibits angiotensin II-induced ROS and inflammation via NOX4 and TLR2 pathways.

In the current study, we analyzed the functions and mechanisms of Bletilla striata polysaccharide b (BSPb) against Angiotensin II (Ang II)-induced oxidative stress and inflammation in human mesangial cells (HMCs). It was found that BSPb could inhibit generation of Ang II-induced reactive oxygen species (ROS) and activation of proinflammatory cytokines interleukin 6 (IL-6) and tumor necrosis factor α (TNF-α) in a dose-dependent manner. Further studies revealed that BSPb effectively blocked upregulation of NADPH oxidase 4 (NOX4). Moreover, knockdown of NOX4 significantly impaired the anti-oxidative function of BSPb. In addition, BSPb decreased overexpression of Toll-like receptor 2 (TLR2) induced by Ang II. Blocking TLR2 expression impaired the anti-inflammatory effects of BSPb. In conclusion, BSPb was found to possess anti-oxidative stress and anti-inflammatory functions against Ang II-induced ROS generation and proinflammatory cytokines activation. The NOX4 and TLR2 pathways played important roles in the biological effects mediated by BSPb.

Fengycin inhibits the growth of the human lung cancer cell line 95D through reactive oxygen species production and mitochondria-dependent apoptosis.

To investigate the antitumor activity and action mechanism of fengycin using the human lung cancer cell line 95D. The antitumor activity of fengycin was tested in vitro and in vivo. Reactive oxygen species production, Ca2+ uptake, and mitochondrial membrane potential loss induced by fengycin in 95D cells were measured by flow cytometry and a laser confocal microscope. Lactate dehydrogenase release and caspase activity in fengycin-treated 95D cells were assayed using cytotoxicity detection kits. Apoptosis triggered by fengycin was identified by 4,6-diamidino-2-phenylindole (DAPI) staining and flow cytometry. The effects of fengycin on cell-cycle and apoptosis-related proteins were evaluated by quantitative reverse-transcription PCR and western blot. Treatment with fengycin not only significantly decreased cell proliferation in various cancer cell lines including 95D but inhibited the growth of xenografted 95D cells in nude mice. Fengycin also induced reactive oxygen species production and Ca2+ uptake, as well as lactate dehydrogenase release and mitochondrial membrane potential loss. Further experiments showed that fengycin could trigger apoptosis in 95D cells and cause cell-cycle arrest at the G0/G1 stage by downregulating cyclin D1 and cyclin-dependent kinase 4 (CDK4). While investigating caspase activity and the expression of apoptosis-related proteins, fengycin was found to induce apoptosis in 95D cells through the mitochondrial pathway, evidenced by increased caspase activity, Bax expression, and cytochrome c release into the cytoplasm, as well as decreased Bcl-2 levels. Fengycin can inhibit the growth of the cancer cell line 95D by regulating the cell cycle and promoting apoptosis, suggesting that it may have potential as an anticancer treatment.

Cordyceps sinensis polysaccharide CPS-2 protects human mesangial cells from PDGF-BB-induced proliferation through the PDGF/ERK and TGF-β1/Smad pathways.

CPS-2, a Cordyceps sinensis polysaccharide, has been demonstrated to have significant therapeutic activity against chronic renal failure. However, little is known about the underlying molecular mechanism. In this study, we found that CPS-2 could inhibit PDGF-BB-induced human mesangial cells (HMCs) proliferation in a dose-dependent manner. In addition, CPS-2 notably suppressed the expression of α-SMA, PDGF receptor-beta (PDGFRβ), TGF-β1, and Smad 3 in PDGF-BB-treated HMCs. Furthermore, PDGF-BB-stimulated ERK activation was significantly inhibited by CPS-2, and this inhibitory effect was synergistically potentiated by U0126. CPS-2 could prevent the PDGFRβ promoter activity induced by PDGF-BB, and return expression of PDGFRβ, TGF-β1, and TGFβRI to normal levels while cells were under PDGFRβ and ERK silencing conditions and transfected with DN-ERK. Taken together, these findings demonstrated that CPS-2 reduces PDGF-BB-induced cell proliferation through the PDGF/ERK and TGF-β1/Smad pathways, and it may have bi-directional regulatory effects on the PDGF/ERK cellular signaling pathway.

Coordinated age-dependent and pancreatic-specific expression of mouse Reg2Reg3α, and Reg3β genes

Reg family proteins such as Reg1 and islet neogenesis-associated protein (INGAP) have long been implicated in the growth and/or neogenesis of pancreatic islet cells. Recent reports further suggest similar roles to be played by new members such as Reg2, Reg3α, and Reg3β. We have studied their age-, isoform-, and tissue-specific expressions. RNA and protein were isolated from C57BL/6 mice aged 7, 30, and 90 days. Using real-time polymerase chain reaction, the levels of Reg gene expression in the pancreas were 20–600-fold higher than that in other tissues (≫duodenum>stomach>liver); gene expression of Reg2, Reg3α, and Reg3β was age dependent as it was hardly detectable at day 7, increased drastically at day 30, and significantly decreased at day 90; the levels of pancreatic proteins displayed similar age-dependent variations. Using dual-labeled immunofluorescence, Reg2, Reg3α, and Reg3β were abundantly expressed in most acinar cells of the pancreas, in contrast to INGAP which exhibited stepwise increases from day 7 to day 90 and colocalized with the α-cells. These new Reg genes were mainly expressed in the pancreas, with clear age-dependent and isoform-specific patterns.

Transcriptional activation of Reg2 and Reg3β genes by glucocorticoids and interleukin-6 in pancreatic acinar and islet cells

Reg family proteins are expressed in the pancreas and involved in pancreatitis and islet β-cell growth. In order to explore transcriptional control, we transfected luciferase reporter genes driven by Reg promoters into acinar AR42J and islet MIN6 cells. Dexamethasone (DEX) significantly increased the promoter expression of Reg2 and Reg3β genes and the levels of endogenous Reg3β mRNA and protein in AR42J cells. DEX-induced promoter activation was inhibited by the inhibitor of poly(ADP-ribose) polymerase, nicotinamide. In MIN6 cells, DEX moderately stimulated the transcription of Reg3β but not Reg2 promoter. While IL-6 alone had no effect, coculture with DEX produced a remarkable synergism on Reg3β gene transcription, which was abolished by nicotinamide. Our results demonstrated a significant and direct stimulation of Reg2 and Reg3β genes by glucocorticoids, all three were activated in response to inflammation such as in pancreatitis. Prominent stimulation of specific Reg genes by glucocorticoids may constitute a functional synergism.

The mAb against adipocyte fatty acid-binding protein 2E4 attenuates the inflammation in the mouse model of high-fat diet-induced obesity via toll-like receptor 4 pathway.

Adipocyte fatty acid-binding protein (A-FABP) plays an important role in fatty acid-mediated processes and related metabolic and inflammatory responses. In this study, we prepared a novel monoclonal antibody against A-FABP, designated 2E4. Our data showed that 2E4 specifically binded to the recombinant A-FABP and native A-FABP of mice adipose tissue. Furthermore, we investigated the effect of 2E4 on metabolic and inflammatory responses in C57BL/6J obese mice fed on a high fat diet. 2E4 administration improved glucose response in high-fat-diet induced obese mice. The 2E4 treated groups exhibited lower free fatty acids, cholesterol, and triglycerides in a concentration-dependent manner. These changes were accompanied by down-regulated expression of pro-inflammatory cytokines in adipose tissue, including tumor necrosis factor α, monocyte chemotactic protein-1, and interleukin-6. Meanwhile, our data demonstrated that 2E4 significantly decreased the mRNA and protein levels of A-FABP in adipose tissue of mice. Further experiments showed that 2E4 notably suppressed the phosphorylation of IκBα and jun-N-terminal kinase through toll-like receptor 4 signaling pathway. Taken together, 2E4 is an effective monoclonal antibody against A-FABP, which attenuated the inflammatory responses induced in the high-fat-diet mice. These findings may provide scientific insight into the treatment of chronic low-grade inflammation in obesity.