Immunomodulation Through Beta-D-glucan in Chemically-induced Necrotizing Pancreatitis.

BACKGROUND: Although the ability of ß-D-glucan and monophosphoryl lipid A (MPLA) to modulate immune responses has been studied in human primary cells, their effect on sterile inflammation models such as necrotizing pancreatitis has never been investigated. MATERIALS AND METHODS: 85 male New Zealand rabbits were assigned into following groups: A: control, B: pretreatment with ß-D-glucan 3 d before pancreatitis, C: pretreatment with MPLA 3 d before pancreatitis, D: pretreatment with ß-D-glucan and laminarin 3 d before pancreatitis, E: treatment with ß-D-glucan 1 d after pancreatitis, and F: MPLA 1 d after pancreatitis. Pancreatitis was induced by sodium taurocholate injection into the pancreatic duct and parenchyma. Survival was recorded for 21 d. On days 1, 3, and 7, blood was collected for amylase measurement. Peripheral blood mononuclear cells were isolated and stimulated for tumor necrosis factor alpha and interleukin 10 production. Pancreatic necrosis and tissue bacterial load were assessed. RESULTS: 21-d survival was prolonged after pretreatment or treatment with ß-D-glucan; this benefit was lost with laminarin administration. At sacrifice, pancreatic inflammatory alterations were more prominent in the control group. Bacterial load was lower after pretreatment or treatment with ß-D-glucan and MPLA. Tumor necrosis factor alpha production from stimulated peripheral blood mononuclear cells was significantly decreased, whereas interleukin 10 production remained unaltered after pretreatment or treatment with ß-D- glucan. CONCLUSIONS: ß-D-glucan reduces mortality of experimental pancreatitis in vivo. This is mediated through attenuation of cytokine production and prevention of bacterial translocation.

Proteoglycan-4 is correlated with longer survival in HCC patients and enhances sorafenib and regorafenib effectiveness via CD44 in vitro.

Sorafenib and regorafenib administration is among the preferential approaches to treat hepatocellular carcinoma (HCC), but does not provide satisfactory benefits. Intensive crosstalk occurring between cancer cells and other multiple non-cancerous cell subsets present in the surrounding microenvironment is assumed to affect tumor progression. This interplay is mediated by a number of soluble and structural extracellular matrix (ECM) proteins enriching the stromal milieu. Here we assess the HCC tumor expression of the ECM protein proteoglycan 4 (PRG4) and its potential pharmacologic activity either alone, or in combination with sorafenib and regorafenib. PRG4 mRNA levels resulted strongly correlated with increased survival rate of HCC patients (p = 0.000) in a prospective study involving 78 HCC subjects. We next showed that transforming growth factor beta stimulates PRG4 expression and secretion by primary human HCC cancer-associated fibroblasts, non-invasive HCC cell lines, and ex vivo specimens. By functional tests we found that recombinant human PRG4 (rhPRG4) impairs HCC cell migration. More importantly, the treatment of HCC cells expressing CD44 (the main PRG4 receptor) with rhPRG4 dramatically enhances the growth-limiting capacity of sorafenib and regorafenib, whereas not significantly affecting cell proliferation per se. Conversely, rhPRG4 only poorly potentiates drug effectiveness on low CD44-expressing or stably CD44-silenced HCC cells. Overall, these data suggest that the physiologically-produced compound PRG4 may function as a novel tumor-suppressive agent by strengthening sorafenib and regorafenib effects in the treatment of HCC.

Extracellular Polysaccharopeptides from Fermented Turkey Tail Medicinal Mushroom, Trametes versicolor (Agaricomycetes), Mitigate Oxidative Stress, Hyperglycemia, and Hyperlipidemia in Rats with Type 2 Diabetes Mellitus.

The antihyperglycemic activity of extracellular polysaccharopeptides (ePSP) obtained from Trametes versicolor (TV) strain LH-1 has been reported to increase cellular glucose uptake in HepG2 cells in an insulin-independent manner. Evidence indicates that oxidative stress plays a pivotal role in the development of diabetic complications. We aimed to use an in vivo model to investigate the effects of TV-ePSP on oxidative stress and glucose homeostasis in type 2 diabetes mellitus (T2DM). Male Wistar rats fed with a high fat diet followed by a streptozotocin injection to induce T2DM were orally administered water or 0.1, 0.5, or 1.0 g/kg of TV-ePSP per day. After a 4-week administration of TV-ePSP, T2DM rats had attenuated elevations in blood glucose levels, areas under the curve in oral glucose tolerance tests, insulin resistance indices, and serum fructosamine and triglyceride in a dose-dependent manner (P < 0.05, one-way ANOVA). In addition, TV-ePSP significantly alleviated oxidative stress in T2DM rats, as shown by the decreased lipid peroxidation and the increased activity of superoxide dismutase in the plasma, and by the elevated glutathione levels in the plasma and erythrocytes. The antihyperglycemia and antihypertriglyceridemia activities of TV-ePSP may be associated with the improved oxidative stress, suggesting the beneficial effects of TV-ePSP in preventing the development of diabetic complications in T2DM patients.

Bioactive structural basis of proteoglycans from Sarcandra glabra based on spectrum-effect relationship.

ETHNOPHARMACOLOGICAL RELEVANCE: Proteoglycans are one of the active ingredients of great importance in Sarcandra glabra. The biological activities of proteoglycans extracted from Sarcandra glabra including suppressing tumor growth and antioxidant activity were studied. However, raw materials from different regions may cause differences in the activity of natural extracts, especially for bioactive biomacromolecules. Conventional identification of S.glabra cannot accurately reflect the distinguishing relationship between internal components and the pharmacological activity. The identification of biologically active structures was obtained by constructing multiple fingerprint and spectrum-effect relationship. AIM OF THE STUDY: To evaluate the bioactive structural basis of proteoglycans from S.glabra based on spectrum-effect relationship and chemometric methods. MATERIALS AND METHODS: Multiple fingerprinting including HPSEC, PMP-HPLC, and FT-IR of proteoglycans was established from 18 batches of samples based on the structural characteristics. Both antitumor activity and antioxidant activity were determined. Mathematical analysis was used to analyze the spectrum-effect relationship. RESULTS: PCA results showed monosaccharides including Xly, Rha, and GlcA, carboxyl group in acidic sugars, peptide bond in proteins, and methylene groups could be used as markers for distinguishing the samples from different sources. The results of the spectrum-effect relationship analysis indicated that the bioactive markers of inhibitory activity on MG63 and U2OS cells by PLS-DA were related to GlcA, Xyl, Fuc, ß-glycosidic bonds, peptide linkage, and methylene groups. Markers composing monosaccharide for antioxidant activity were Xyl, GlcA, and GlcN. Meanwhile, the group markers were pyranose ring, carboxyl group, peptide linkage, and methylene structure. CONCLUSIONS: The material basis that affects the pharmacological efficacy could be found according to the spectrum-effect relationship analysis. This study could lay a foundation for further exploring the relationship between structural characteristics and pharmacodynamics of macromolecular glycoconjugates in Traditional Chinese Medicine.

Proteoglycan isolated from Corbicula fluminea exerts hepato-protective effects against alcohol-induced liver injury in mice.

Corbicula fluminea (Asian clam), a freshwater bivalve mollusk, has been consumed in China for centuries as a health food and traditional Chinese medicine for treating liver diseases and alcoholism. This study aimed to evaluate the hepato-protective effects and potential mechanisms of a proteoglycan (PSP) from C. fluminea on alcohol-induced liver injury in mice. Results showed that PSP pretreatment significantly antagonized the increases in serum alanine aminotransferase, aspartate aminotransferase, triacylglycerides, and hepatic malondialdehyde levels; elevated the antioxidant enzyme activities and hepatic glutathione levels; and suppressed the levels of hepatic inflammatory cytokines in alcohol-induced liver injury in mice (P < 0.05). Histopathological observation further revealed the potential hepato-protective effect of PSP against alcohol damage. Particularly, PSP pretreatment resulted in significantly decreased expression of cytochrome P450 2e1 (CYP2E1) while significantly upregulating the expression of hemeoxygenase-1 (HO-1) (P < 0.05). These results suggested that PSP could protect the liver from hepatocyte injury induced by alcohol possibly by alleviating hepatic lipid metabolism, elevating antioxidant-enzyme activity, suppressing the immune inflammatory response, and reversing the expression levels of CYP2E1 and HO-1. Therefore, PSP may be developed as a food supplement that can be used to prevent liver diseases.

Optimization of Canalicular ABC Transporter Function in HuH-7 Cells by Modification of Culture Conditions.

Human hepatoma cell lines are useful for evaluation of drug-induced hepatotoxicity, hepatic drug disposition, and drug-drug interactions. However, their applicability is compromised by aberrant expression of hepatobiliary transporters. This study was designed to evaluate whether extracellular matrix (Matrigel) overlay and dexamethasone (DEX) treatment would support cellular maturation of long-term HuH-7 hepatoma cell cultures and improve the expression, localization, and activity of canalicular ATP-binding cassette (ABC) transporters, multidrug resistance protein 1 (MDR1/P-glycoprotein/ABCB1), multidrug resistance-associated protein 2 (MRP2/ABCC2), and bile salt export pump (BSEP/ABCB11). Matrigel overlay promoted the maturation of HuH-7 cells toward cuboidal, hepatocyte-like cells displaying bile canaliculi-like structures visualized by staining for filamentous actin (F-actin), colocalization of MRP2 with F-actin, and by accumulation of the MRP2 substrate 5(6)-carboxy-2',7'-dichlorofluorescein (CDF) within the tubular canaliculi. The cellular phenotype was rather homogenous in the Matrigel-overlaid cultures, whereas the standard HuH-7 cultures contained both hepatocyte-like cells and flat epithelium-like cells. Only Matrigel-overlaid HuH-7 cells expressed MDR1 at the canaliculi and excreted the MDR1 probe substrate digoxin into biliary compartments. DEX treatment resulted in more elongated and branched canaliculi and restored canalicular expression and function of BSEP. These findings suggest that hepatocyte polarity, elongated canalicular structures, and proper localization and function of canalicular ABC transporters can be recovered, at least in part, in human hepatoma HuH-7 cells by applying the modified culture conditions. SIGNIFICANCE STATEMENT: We report the first demonstration that proper localization and function of canalicular ABC transporters can be recovered in human hepatoma HuH-7 cells by modification of cell culture conditions. Matrigel overlay and dexamethasone supplementation increased the proportion of hepatocyte-like cells, strongly augmented the canalicular structures between the cells, and restored the localization and function of key canalicular ABC transporters. These results will facilitate the development of reproducible, economical, and easily achievable liver cell models for drug development.

Endocan regulates acute lung inflammation through control of leukocyte diapedesis.

Acute respiratory distress syndrome is a severe form of respiratory failure, occurring in up to 20% of patients admitted to the intensive care unit with sepsis. Dysregulated leukocyte diapedesis is a major contributor to acute respiratory distress syndrome. Endocan is a circulating proteoglycan that binds to the leukocyte integrin leukocyte functional antigen-1 and blocks its interaction with its endothelial ligand, ICAM-1. The objective of this study was to evaluate the role of endocan in the control of acute lung inflammation. In vitro, endocan inhibited human leukocyte transendothelial migration as well as ICAM-1-dependent migration but had a very mild effect on ICAM-1-dependent adhesion. Endocan also acted as an inhibitor of transendothelial migration of mouse leukocytes. The effect of systemic administration of recombinant human endocan was assessed in a model of acute lung inflammation in BALB/c mice. Treatment with endocan 1 h after intratracheal LPS challenge reduced the alveolar inflammatory response, diminished histological features of acute lung injury, and improved respiratory function. These results highlight the anti-inflammatory role of human endocan and its protective effect against acute lung injury.NEW & NOTEWORTHY We show here that endocan inhibits ICAM-1-dependent human leukocyte transendothelial migration and ICAM-1-dependent adhesion. We also found that in BALB/c mice with tracheal LPS-induced acute lung injury treatment with recombinant human endocan reduces lung inflammation, notably through reduction of neutrophilic recruitment, and restores normal lung function. These results confirm the hypothesis that human endocan may have a protective effect against acute lung inflammation.

Andrographolide binds to ATP-binding pocket of VEGFR2 to impede VEGFA-mediated tumor-angiogenesis.

Vasculogenesis and angiogenesis are process of formation of blood vessels. Blood vessels are evolved to distribute nutrients and oxygen to distant organs. These vessels are crucial for growth and repair of wounded tissue. During tumor condition there occurs imbalance in the growth of blood vessels which leads to neo-angiogenesis. Neo-angiogenesis is major perpetrator behind the establishment of tumor. Tumor cells secrete pro-angiogenic factor VEGFA which binds to VEGFR2 present over surface of endothelial cells and triggers formation of new blood vessels. To inhibit tumor-angiogenesis, a physiologically-safe small molecule inhibitor was screened which can potentially interact with kinase domain of VEGFR2 and inhibit its activity. Molecular-docking module and biochemical analysis identified andrographolide as one of the best docking molecules that binds to ATP-binding pocket of VEGFR2 and inhibits its kinase activity. Thus, for a more radical approach towards safe VEGFR2 inhibitor, andrographolide was repurposed to inhibit tumor-angiogenesis and reduce tumor burden.

Alveolus-like organoid from isolated tip epithelium of embryonic mouse lung.

Embryonic lungs were obtained from embryonic day 13.5 ICR mice. The lung-tip epithelium isolated using dispase treatment was embedded in low-growth factor Matrigel, cultured in DMEM/F12 medium containing 0.1% bovine serum albumin, supplemented with insulin, transferrin, and selenium (ITS), with or without fibroblast growth factor 7 (FGF7), and were observed for 14 days. With the addition of FGF7, the tip epithelium grew to form a cyst by culture day 7. Then, tubular tufts-like alveolus appeared around the cyst surface. Reverse transcription-polymerase chain reaction revealed that, with the addition of FGF7, the cultured lung explants expressed alveolar-type 1 cell markers, such as HopX and Aquaporin5, and type 2 cell markers, such as Lamp3 and Surfactant apoproteins (Sftp) C and D. Paraffin-embedded sections were stained with hematoxylin and eosin, and alveolar structures at culture day 14 were composed of squamous and cuboidal epithelial cells. Immunohistochemical studies showed that the squamous epithelial cells were positive for HopX, and the cuboidal epithelial cells were positive for pro-SftpC. Furthermore, transmission electron microscopic observation confirmed that the squamous epithelial cells were alveolar-type 1 cells and the cuboidal cells were type 2 cells, because they had many lamellar inclusion bodies. Embryonic lung-tip epithelium forms an alveolus-like organoid through the self organization with the aid of Matrigel, ITS, and FGF7. This method to make alveolus-like organoid in vitro is easy, reproducible, and economical. This method could have potential to solve many issues in alveolar epithelial cells in normal and pathological conditions.

Biomimetic proteoglycans can molecularly engineer early osteoarthritic cartilage in vivo.

Biomimetic proteoglycans (BPGs) have the potential to treat osteoarthritis (OA) given that these molecules mimic the structure and properties of natural proteoglycans, which are significantly reduced in OA. We examined the effects of BPGs injected into the intra-articular space in an in vivo OA rabbit knee model and evaluated the effect on histological response, joint friction, and BPG distribution and retention. Rabbits underwent ACL transection to create an arthritic state after 5 weeks. OA rabbits were treated with BPGs or Euflexxa® (hyaluronic acid) intra-articular injections. Non-OA rabbits were injected similarly with BPGs; contralateral joints served as controls. The progression of OA and response to injections were evaluated using Mankin and gross grading systems indicating that mild OA was achieved in operated joints. The coefficient of friction (COF) of the intact knee joints were measured using a custom pendulum friction apparatus, showing that OA joints and OA + Euflexxa® joints demonstrated increased COF than non-operated controls, while BPG-injected non-OA and OA + BPGs were not significantly different from non-OA controls. Injected fluorescently labeled BPGs demonstrated that BPGs diffused into cartilage with localization in the pericellular region. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:403-411, 2019.

Potential activities and mechanisms of extracellular polysaccharopeptides from fermented Trametes versicolor on regulating glucose homeostasis in insulin-resistant HepG2 cells.

Polysaccharides derived from mushrooms have potential to control blood sugar, reduce insulin resistance and prevent diabetic complications. The intracellular polysaccharopeptides of Trametes versicolor (TV) have been used as immunologic and oncologic adjuvants. The aim of this study was to investigate the potential activities and mechanisms of extracellular polysaccharopeptides (ePSP) obtained from TV strain LH-1 on regulating glucose homeostasis. Human hepatoma HepG2 cells incubated with normal glucose (5.5 mM, NG model), high glucose (33 mM, HG model), or high glucose (33 mM) plus high insulin (10-7 M, HGI model) concentrations were administered with TV LH-1 ePSP (50, 100, and 1000 µg/ml) for 24 hr. Glucose uptake of HepG2 cells, determined by flow cytometry, was significantly decreased in the HG and HGI models with insulin stimulation, suggesting insulin resistance of these cells; however, ePSP reversed this decrease in a dose-dependent manner (one-way ANOVA, p<0.05). In the HG and HGI models, ePSP significantly increased glycogen content, insulin receptor substrate-2 protein and phosphorylated AMP-activated protein kinase (AMPK), as determined by western blot analysis. In addition, ePSP significantly increased glucokinase in the NG and HG models, increased membrane glucose transporter-1 and decreased glycogen synthase kinase-3ß in the HGI model, and increased glucose-6-phosphatase in the NG and HGI models (one-way ANOVA, p<0.05). In summary, TV LH-1 ePSP may elevate cellular glucose uptake to regulate glucose homeostasis via the activation of AMPK and glycogen synthesis in an insulin-independent manner. These results suggest that TV LH-1 ePSP may be a nutraceutical with anti-hyperglycemic activity.

Preclinical Animal Studies of Intravesical Recombinant Human Proteoglycan 4 as a Novel Potential Therapy for Diseases Resulting From Increased Bladder Permeability.

OBJECTIVE: To test in an animal model the hypothesis that recombinant human proteoglycan 4 (rhPRG4; lubricin), a highly O-glycosylated mucin-like glycoprotein, may be a novel surface-active therapeutic for treating bladder permeability with comorbid bowel permeability. Previously we showed that inducing bladder permeability in rats with dilute protamine sulfate (PS) produced colonic permeability and visceral hypersensitivity, suggesting increased bladder permeability could represent an etiologic factor in both interstitial cystitis-bladder pain syndrome and irritable bowel syndrome. METHODS: We used an animal model of catheterized ovariectomized female rats instilled intravesically with 1 mg/mL PS for 10 minutes that after 24 hours were treated with 1.2 mg/mL lubricin or with vehicle alone. After 24 hours the bladder and colon were removed and permeability assessed electrophysiologically with the Ussing chamber to measure the transepithelial electrical resistance. A second set of rats was treated identically, except permeability was assessed on day 3 and on day 5 using contrast-enhanced magnetic resonance imaging with gadolinium diethylenetriamine penta-acetic acid instilled into the bladder. RESULTS: Intravesical lubricin reversed bladder permeability induced by PS and prevented the concomitant increase in permeability induced in the bowel (organ crosstalk). The protective effect was confirmed with magnetic resonance imaging, and because individual rats could be followed over time, the impermeability of the bladder restored by rhPRG4 remained for 5 days. CONCLUSION: These data indicate that instillation of rhPRG4 into a permeable bladder can restore its normally impermeable state, and that the effect lasts for 5 days and also prevents bowel symptoms often comorbid with interstitial cystitis-bladder pain syndrome.

Brucine suppresses breast cancer metastasis via inhibiting epithelial mesenchymal transition and matrix metalloproteinases expressions.

OBJECTIVE: To examine the effect of brucine on the migration, invasion, adhesion and expressions of epithelial-to-mesenchymal transition (EMT) markers and matrix metalloproteinases (MMPs) in the highly metastatic breast cancer cell lines MDA-MB-231 and Hs578-T. METHODS: MDA-MB-231 and Hs578-T cells were divided to 4 groups: the control group (0.1% DMSO), and 25, 50 and 100 µmol/L brucine groups. The cell viability was determined using a CellTiter-Glo® luminescent cell viability. The scratch wound healing assay and tanswell migration assay were used to determine the migration ability of these cells treated by different concentrations of brucine. The proliferation rate, invasive potential and adhesive ability were respectively performed by colony formation assay, transwell invasion assay and adhension assay. The protein and mRNA expressions of EMT biomarkers, MMP-2 and MMP-9 were investigated by real-time reverse transcription polymerase chain reaction and Western blot. RESULTS: Compared with the control group, brucine had little effect on cell viability or proliferation (P>0.05), but led to a dose-dependent decrease on migration, invasion, adhension of MDA-MB-231 and Hs578-T cells (P<0.01). Furthermore, brucine increased the protein and mRNA levels of EMT markers such as E-cadherin and ß-catenin in MDA-MB-231 and Hs578-T cells, and decreased the protein and mRNA levels of mesenychmal markers such as vimentin and fibronectin, as well as the expressions of MMP-2 and MMP-9 (all P<0.01). CONCLUSION: Brucine inhibited triple negative breast cancer cells metastasis potentially through EMT reversion and MMP-2 and MMP-9 inhibition.

Immunomodulatory effects of herbal formula of astragalus polysaccharide (APS) and polysaccharopeptide (PSP) in mice with lung cancer.

OBJECTIVE: This study is to investigate the immunomodulatory effects of the herbal formula of astragalus polysaccharide (APS) and polysaccharopeptide (PSP) in mouse models of immunosuppression and lung cancer. METHODS: Immune parameters were recorded for these model mice. Peripheral white blood cells (WBC) were detected with the automatic blood cell analyzer. Spleen and thymus indices, and tumor inhibition ratio were obtained. Percentage of peripheral blood CD4+ and CD8+ T lymphocytes were detected by flow cytometry. Serum levels of Th1 (IL-2, TNF, and IFN-γ), Th2 (IL-4, IL-6, and IL-10), and Th17 (IL-17A) were detected with the BD cytometric bead array (CBA) mouseTh1/Th2/Th17 cytokine kit. RESULTS: Compared with the NS group, the PSP and APS herbal formula significantly improved the WBC, thymus index, spleen index, CD4+/CD8+ ratio, TNF, IFN-γ, IL-2, andIL-17Ainimmunosuppressivemice and lung cancer mice (P<0. 05). On the contrary, IL-10 was relatively low in the PSP+APS herbal formula group (P<0. 05). Besides, the PSP+APS herbal formula group induced comparable tumor inhibiting effect with the AMD group (23.3% and 24.1%, respectively). CONCLUSION: The PSP+APS herbal formula have immunomodulatory effects and anti-tumor activity in mice with of lung cancer.

Triterpenoids and polysaccharide peptides-enriched Ganoderma lucidum: a randomized, double-blind placebo-controlled crossover study of its antioxidation and hepatoprotective efficacy in healthy volunteers.

CONTEXT: Ganoderma lucidum (Leyss: Fr) Karst. (Polyporaceae) is an oriental medicinal fungus, commonly used in traditional Chinese medicine (TCM) for treating various condition or diseases such as hypertension, hyperglycaemia, hepatitis and cancer. OBJECTIVE: The current study examines whether triterpenoids and polysaccharide-enriched G. lucidum (GL) influence antioxidation and hepatoprotective efficacy by suppressing oxidative stress. MATERIALS AND METHODS: Forty-two healthy subjects (22 male and 20 female) were recruited and segregated into two groups as experimental or placebo and requested to intake GL (n = 21) or placebo (n = 21) capsule (225 mg; after lunch or dinner) for six consecutive months and vice versa with one month washout period in between. The anthropometric analysis and biochemical assays, as well as abdominal ultrasonic examination were performed. RESULTS: Consumption of GL substantially improved (p < 0.05) the total antioxidant capacity (TEAC; 79.33-84.04), total thiols and glutathione content (6-8.05) in plasma as well as significant (p < 0.05) enhanced the activities of antioxidant enzymes. Whereas, the levels of thiobarbituric acid reactive substances (TBARS; 3.37-2.47), 8-hydroxy-deoxy-guanosine (8-OH-dG; 15.99-11.98) and hepatic marker enzymes (glutamic-oxaloacetic transaminase; GOT and glutamic-pyruvic transaminase; GPT) were concomitantly reduced (42 and 27%) on treatment with GL. Furthermore, the abdominal ultrasonic examination in GL subjects displayed a notable alteration on hepatic condition by reversing from mild fatty liver condition (initial) to normal condition. DISCUSSION AND CONCLUSION: The outcome of the present intervention demonstrated the antioxidation, anti-aging and hepatoprotective nature of GL by effectively curbing oxidative stress.

Recent Advances and Challenges in Studies of Control of Cancer Stem Cells and the Gut Microbiome by the Trametes-Derived Polysaccharopeptide PSP (Review).

The medicinal mushroom Trametes versicolor has been well recognized for its activity in maintaining the general health of the population and in managing and treating human diseases in various cultures. Its use has been recently gaining acceptance and popularity in Western countries. The reported health benefits of T. versicolor led to a search for the identity of its bioactive ingredients. These efforts have resulted in the isolation of the polysaccharopeptide PSP from cultured mycelia of strain Cov-1, which expresses large amounts of PSP. The availability of highly purified PSP was followed by studies of its biological activities using tissue culture models and limited human clinical trials. In this review we summarize recent advances in the antitumorigenic and immunomodulatory effects of PSP, elimination of prostate cancer stem cells and control of the intestinal microbiome, and its interplay with host cells as a prebiotic. These findings may have implications for widening and repurposing the use of PSP.

Pro-angiogenic activity of notoginsenoside R1 in human umbilical vein endothelial cells in vitro and in a chemical-induced blood vessel loss model of zebrafish in vivo.

OBJECTIVE: This study aimed at investigating whether notoginsenoside R1 (R1), a unique saponin found in Panax notoginseng could promote angiogenic activity on human umbilical vein endothelial cells (HUVECs) and elucidate their potential molecular mechanisms. In addition, vascular restorative activities of R1 was assessed in a chemically-induced blood vessel loss model in zebrafish. METHODS: The in vitro angiogenic effect of R1 was compared with other previously reported angiogenic saponins Rg1 and Re. The HUVECs proliferation in the presence of R1 was determined by cell proliferation kit II (XTT) assay. R1, Rg1 and Re-induced HUVECs invasion across polycarbonate membrane was stained with Hoechst-33342 and quantified microscopically. Tube formation assay using matrigelcoated wells was performed to evaluate the pro-angiogenic actions of R1. In order to understand the mechanism underlying the pro-angiogenic effect, various pathway inhibitors such as SU5416, wortmannin (wort) or L-Nω-nitro- L-arginine methyl ester hydrochloride (L-NAME), SH-6 were used to probe the possible involvement of signaling pathway in the R1 mediated HUVECs proliferation. In in vivo assays, zebrafish embryos at 21 hpf were pre-treated with vascular endothelial growth factor (VEGF) receptor kinase inhibitor II (VRI) for 3 h only and subsequently post-treated with R1 for 48 h, respectively. The intersegmental vessels (ISVs) in zebrafish were assessed for the restorative effect of R1 on defective blood vessels. RESULTS: R1 could stimulate the proliferation of HUVECs. In the chemoinvasion assay, R1 significantly increased the number of cross-membrane HUVECs. In addition, R1 markedly enhanced the tube formation ability of HUVECs. The proliferative effects of these saponins on HUVECs were effectively blocked by the addition of SU5416 (a VEGF-KDR/Flk-1 inhibitor). Similarly, pre-treatment with wort [a phosphatidylinositol 3-kinase (PI3K)-kinase inhibitor], L-NAME [an endothelial nitric oxide synthase (eNOS) inhibitor] or SH-6 (an Akt pathway inhibitor) significantly abrogated the R1 induced proliferation of HUVECs. In chemicallyinduced blood vessel loss model in zebrafish, R1 significantly rescue the damaged ISVs. CONCLUSION: R1, similar to Rg1 and Re, had been showed pro-angiogenic action, possibly via the activation of the VEGF-KDR/Flk-1 and PI3K-Akt-eNOS signaling pathways. Our findings also shed light on intriguing pro-angiogenic effect of R1 under deficient angiogenesis condition in a pharmacologic-induced blood vessels loss model in zebrafish. The present study in vivo and in vitro provided scientific evidence to explain the ethnomedical use of Panax notoginseng in the treatment of cardiovascular diseases, traumatic injuries and wound healing.

The TLR2 agonist in polysaccharide-K is a structurally distinct lipid which acts synergistically with the protein-bound ß-glucan.

Protein-bound polysaccharide-K (Krestin; PSK) is a hot-water extract of Trametes versicolor with immune stimulatory activity. It has been used for the past 30 years and has demonstrated anti-tumor efficacy in multiple types of cancer. The ability of PSK to activate dendritic cells and T cells is dependent on its ability to stimulate Toll-like receptor 2 (TLR2), yet it remains unknown which structural component within PSK activates TLR2. The purpose of this study was to identify the TLR2 agonist within PSK and understand its role in the overall mechanism of PSK's immunogenic activity. TLR2 activity was eliminated by treatment with lipoprotein lipase but not by trypsin or lyticase. Rapid centrifugation of PSK can separate the fraction with TLR2 agonist activity from the soluble ß-glucan fraction. To study the potential interaction between the ß-glucan component and the lipid component, we labeled the soluble ß-glucan with fluorescein. Uptake of the labeled ß-glucan by J774A macrophages and JAWSII dendritic cells was inhibited by anti-Dectin-1 antibody but not by anti-TLR2 antibody, confirming that Dectin-1 is the receptor for ß-glucan. Interestingly, pre-treatment of JAWSII cells with the TLR2-active lipid fraction significantly enhanced the uptake of the soluble ß-glucan, indicating the synergy between the TLR2 agonist component and the ß-glucan component. Altogether, these results present evidence that PSK has two active components-the well-characterized protein-bound ß-glucan and a previously unreported lipid-which work synergistically via the Dectin-1 and TLR2 receptors.

A study to investigate the biological activity of proteoglycan mixture extract from Convolvulus arvensis.

BACKGROUND: Convolvulus arvensis L. (Convolvulaceae), bindweeds, is inhabitant to Iran and its proteoglycan mixture (PGM) has been reported to possess different biological activities. In the present study, we aimed to investigate different properties of PGM including anti-tumor, anti-angiogenesis and immunostimulatory activities. METHODS: PGM was prepared from the roots of C. arvensis. Various cancer cell lines were treated with PGM and the cytotoxicity was assessed after 24 h of incubation using MTT assay. In addition, J774A.1 macrophages were stimulated with LPS (1 µg/mL) and then with PGM. Then, production of nitric oxide (NO) as a marker of inflammation was measured using Griess reagent. Moreover, PGM was subjected to cultivated Leishmania major promastigotes and leishmanicidal activity was determined using MTT assay. More importantly, human umbilical vein endothelial cells (HUVEC) cultured on matrigel basement matrix and tube formation after treatment with PGM was considered microscopically for the determination of angiogenesis. RESULTS: Obtained results revealed that PGM significantly inhibited the formation of vascular-like tubes by HUVECs without any effect on their viability. Furthermore, PGM significantly exhibited leishmanicidal activity by the mechanism of suppressing L. major promastigotes developmental growth in vitro. However, PGM was shown to have no effect on the growth of cancer cells and production of NO by LPS-stimulated macrophages. CONCLUSIONS: The present study provides some new evidence on remarkable leishmanicidal and anti-angiogenic activities of PGM. These findings also afford the scientific basis for the use of C. arvensis as a candidate medicinal plant for further thoroughly phytochemical investigations toward discovering leishmanicidal and anti-angiogenic compounds.