Fluorescent Labeling of Polymannuronic Acid and Its Distribution in Mice by Tail Vein Injection.

Polymannuronic acid (PM) possesses more pharmacological activities than sodium alginate, but there have been few studies on its absorption mechanism, tissue distribution, and pharmacokinetics. Studies of pharmacokinetics and tissue distribution are necessary to elucidate the pharmacological effects of PM. Thus, we used fluorescein isothiocyanate (FITC) to produce fluorescently labeled PM (FITC-PM) and detected the distribution and pharmacokinetics of PM in vivo via tail vein injection. The results demonstrate that the FITC-PM showed high stability in different pH solutions. After the tail vein injection, FITC-PM tended to be distributed in the kidney, followed by the liver and in the heart, spleen, and lungs at lower concentrations. Pharmacokinetic analysis showed that the elimination rate constant of FITC-PM was 0.24, the half-life time was 2.85 h, the peak concentration was 235.17 µg/mL, the area under the curve was 631.48 µg/mL·h, the area under the curve by statistical moment was 1843.15 µg/mL·h2, the mean residence time was 2.92 h, and the clearance rate was 79.18 mL/h. These results indicate that FITC-PM could be used for PM distribution and pharmacokinetic studies, and the studies of pharmacokinetics and tissue distribution provided basic information that can be used to further clarify PM pharmacodynamic mechanisms.

Fucoidan reduces lipid accumulation by promoting foam cell autophagy via TFEB.

Atherosclerotic cardiovascular disease became one of the major causes of morbidity and mortality worldwide. As a sulfated polysaccharide with anti-inflammatory and hypolipidemic activities, fucoidan can induce autophagy. We show here that fucoidan reduces lipid accumulation in foam cells, which is one of the causes of atherosclerosis. Further studies show that fucoidan promotes autophagy showed by the expression of p62/SQSTM1 and microtubule-associated protein light chain 3 (LC3) II, which can be blocked by autophagy inhibitors 3-MA and bafilomycin A1. In addition, the expression of transcription factor EB (TFEB), master regulator of autophagy and lysosome function, is upregulated after the treatment with fucoidan. Moreover, the knockout of TFEB with small interfering RNA suppressed the effect of fucoidan. Together, fucoidan reduces lipid accumulation in foam cells by enhancing autophagy through the upregulation of TFEB. In view of the role of foam cells in atherosclerosis, fucoidan can be valuable for the treatment of atherosclerosis.

Discovery of lysosome-targeted covalent anticancer agents based on isosteviol skeleton.

Covalent drugs play corresponding bioactivities by forming covalent bonds with the target, which possess many significant pharmacological advantages including high potency, ligand efficiency, and long-lasting effects. However, development of covalent inhibitors is a challenge due to their presumed indiscriminate reactivity. Here, we report the discovery of series of lysosome-targeting covalent anticancer agents by introducing nitrogenous bases to the modified isosteviol skeleton in order to minimize the toxicity and increase the selectivity. By introducing the electrophilic α, ß-unsaturated ketones into the A- and D-rings of isosteviol, the cytotoxicity of the obtained compounds were greatly increased. Further nitrogen-containing modifications to the D-ring led to the discovery of novel molecules that targeted lysosomes, and of which, compound 30 was the most potent and selective antiproliferative one to kill A549 cells in vitro and in vivo. Mechanism investigation revealed that compound 30 was trapped into lysosomes and damaged lysosomes to cause cell death.

Advances in Studies on the Pharmacological Activities of Fucoxanthin.

Fucoxanthin is a natural carotenoid derived mostly from many species of marine brown algae. It is characterized by small molecular weight, is chemically active, can be easily oxidized, and has diverse biological activities, thus protecting cell components from ROS. Fucoxanthin inhibits the proliferation of a variety of cancer cells, promotes weight loss, acts as an antioxidant and anti-inflammatory agent, interacts with the intestinal flora to protect intestinal health, prevents organ fibrosis, and exerts a multitude of other beneficial effects. Thus, fucoxanthin has a wide range of applications and broad prospects. This review focuses primarily on the latest progress in research on its pharmacological activity and underlying mechanisms.

Current Research Landscape of Marine-Derived Anti-Atherosclerotic Substances.

Atherosclerosis is a chronic disease characterized by lipid accumulation and chronic inflammation of the arterial wall, which is the pathological basis for coronary heart disease, cerebrovascular disease and thromboembolic disease. Currently, there is a lack of low-cost therapeutic agents that effectively slow the progression of atherosclerosis. Therefore, the development of new drugs is urgently needed. The research and development of marine-derived drugs have gained increasing interest from researchers across the world. Many marine organisms provide a rich material basis for the development of atherosclerotic drugs. This review focuses on the latest technological advances in the structures and mechanisms of action of marine-derived anti-atherosclerotic substances and the challenges of the application of these substances including marine polysaccharides, proteins and peptides, polyunsaturated fatty acids and small molecule compounds. Here, we describe the theoretical basis of marine biological resources in the treatment of atherosclerosis.

Advances in the Study of Marine Products with Lipid-Lowering Properties.

With twice the number of cancer's deaths, cardiovascular diseases have become the leading cause of death worldwide. Atherosclerosis, in particular, is a progressive, chronic inflammatory cardiovascular disease caused by persistent damage to blood vessels due to elevated cholesterol levels and hyperlipidemia. This condition is characterized by an increase in serum cholesterol, triglycerides, and low-density lipoprotein, and a decrease in high-density lipoprotein. Although existing therapies with hypolipidemic effects can improve the living standards of patients with cardiovascular diseases, the drugs currently used in clinical practice have certain side effects, which insists on the need for the development of new types of drugs with lipid-lowering effects. Some marine-derived substances have proven hypolipidemic activities with fewer side effects and stand as a good alternative for drug development. Recently, there have been thousands of studies on substances with lipid-lowering properties of marine origin, and some are already implemented in clinical practice. Here, we summarize the active components of marine-derived products having a hypolipidemic effect. These active constituents according to their source are divided into algal, animal, plant and microbial and contribute to the development and utilization of marine medicinal products with hypolipidemic effects.

Fucoidan Induces Apoptosis of HT-29 Cells via the Activation of DR4 and Mitochondrial Pathway.

Fucoidan has a variety of pharmacological activities, but the understanding of the mechanism of fucoidan-induced apoptosis of colorectal cancer cells remains limited. The results of the present study demonstrated that the JNK signaling pathway is involved in the activation of apoptosis in colorectal cancer-derived HT-29 cells, and fucoidan induces apoptosis by activation of the DR4 at the transcriptional and protein levels. The survival rate of HT-29 cells was approximately 40% in the presence of 800 µg/mL of fucoidan, but was increased to 70% after DR4 was silenced by siRNA. Additionally, fucoidan has been shown to reduce the mitochondrial membrane potential and destroy the integrity of mitochondrial membrane. In the presence of an inhibitor of cytochrome C inhibitor and DR4 siRNA or the presence of cytochrome C inhibitor only, the cell survival rate was significantly higher than when cells were treated with DR4 siRNA only. These data indicate that both the DR4 and the mitochondrial pathways contribute to fucoidan-induced apoptosis of HT-29 cells, and the extrinsic pathway is upstream of the intrinsic pathway. In conclusion, the current work identified the mechanism of fucoidan-induced apoptosis and provided a novel theoretical basis for the future development of clinical applications of fucoidan as a drug.

A peptide inhibitor of macrophage migration in atherosclerosis purified from the leech Whitmania pigra.

ETHNOPHARMACOLOGICAL RELEVANCE: Atherosclerosis has become a worldwide public health problem that seriously threatens human health. Leech is traditional Chinese medicine that can be utilized to treat cardiovascular disease. Based on the anti-atherosclerosis activity of leech hydrolysate, we separated and purified the leech peptide capable of inhibiting macrophage migration and studied the pathways of the anti-migration leech peptide. MATERIALS AND METHODS: The leech peptide capable of inhibiting macrophage migration that measured by cell migration assays from the leech Whitmania pigra was separated and purified by Q Sepharose FF strong alkaline anion exchange column chromatography, Superdex 30, Superdex peptide and G10 gel column chromatography. And the purity, molecular weight of the leech peptide was determined by high-performance liquid chromatography and high-resolution mass spectrometry. The pathways of anti-migration to macrophages of the leech peptide were studied by inhibitors, Western blotting and RT-PCR. RESULTS: We obtained a purified leech peptide with a sequence of EAGSAKELEGDPVAG from the leech Whitmania pigra. We also showed that the anti-migration to macrophages of the leech peptide was blocked by c-Jun N-terminal kinase (JNK) inhibitor and p38 mitogen-activated protein kinase (p38 MAPK) inhibitor. Moreover, the result of RT-PCR and Western blotting revealed that the leech peptide induced an increase in JNK, p38 phosphorylation and the transcription of mitogen-activated protein kinase kinase kinase 4 (MEKK4) and apoptosis signal-regulating kinase 2 (ASK2). These data indicated that the anti-migration to macrophages of the leech peptide occurred through JNK and p38 MAPK pathways. In addition, the results demonstrated that the leech peptide had no significant effect on the immunological activity of macrophages including phagocytic ability, lysozyme activity, and levels of expression of inflammatory factors. CONCLUSION: A sequence peptide was obtained from the hydrolysate of leech Whitmania pigra that inhibits macrophage migration.

Advances in Research on the Bioactivity of Alginate Oligosaccharides.

Alginate is a natural polysaccharide present in various marine brown seaweeds. Alginate oligosaccharide (AOS) is a degradation product of alginate, which has received increasing attention due to its low molecular weight and promising biological activity. The wide-ranging biological activity of AOS is closely related to the diversity of their structures. AOS with a specific structure and distinct applications can be obtained by different methods of alginate degradation. This review focuses on recent advances in the biological activity of alginate and its derivatives, including their anti-tumor, anti-oxidative, immunoregulatory, anti-inflammatory, neuroprotective, antibacterial, hypolipidemic, antihypertensive, and hypoglycemic properties, as well as the ability to suppress obesity and promote cell proliferation and regulate plant growth. We hope that this review will provide theoretical basis and inspiration for the high-value research developments and utilization of AOS-related products.

Study on Absorption Mechanism and Tissue Distribution of Fucoidan.

Fucoidan exhibits several pharmacological activities and is characterized by high safety and the absence of toxic side effects. However, the absorption of fucoidan is not well-characterized. In the present study, fucoidan were labeled with fluorescein isothiocyanate (FITC) and their ability to traverse a monolayer of Caco-2 cells was examined. The apparent permeability coefficients (Papp × 10-6) of FITC-labeled fucoidan (FITC-fucoidan) were 26.23, 20.15, 17.93, 16.11 cm/sec, respectively, at the concentration of 10 µg/mL at 0.5, 1, 1.5 and 2 h. The absorption of FITC-fucoidan was suppressed by inhibitors of clathrin-mediated endocytosis, chlorpromazine, NH4Cl, and Dynasore; the inhibition rates were 84.24%, 74.61%, and 63.94%, respectively. This finding suggested that clathrin-mediated endocytosis was involved in fucoidan transport. Finally, tissue distribution of FITC-fucoidan was studied in vivo after injection of 50 mg/kg body weight into the tail vein of mice. The results showed that FITC-fucoidan targeted kidney and liver, reaching concentrations of 1092.31 and 284.27 µg/g respectively after 0.5 h. In summary, the present work identified the mechanism of absorption of fucoidan and documented its tissue distribution, providing a theoretical basis for the future development of fucoidan applications.

Determination of fucoidan in rat plasma by HPLC and its application in pharmacokinetics.

This is a new expanded method of determining the characterisation of fucoidan from Laminaria japonica (kelp) in rat plasma by high-performance liquid chromatography (HPLC) with fluorescence detection. We tagged fucoidan by fluoresce in isothiocyanate (FITC) for tracking and treated the plasma samples via protein precipitation with 10% trichloroacetic acid and methanol. Column chromatography separation was on a TSK-G4000sw column (7.8 mm × 300 mm, 5 mm) by elution with 0.15 M NaCl. The quantification of fucoidan was performed by HPLC with fluorescence detection. The results suggested that the calibration curve for fucoidan concentration was linear dependent in the limits of 0.5-100µg/mL. The lower limit of quantitation (LLOQ) was 0.5µg/mL and the lower limit of detection (LLOD) was 0.15µg/mL. The intra-day and inter-day precision values were less than 13%and the accuracy ranged from 96.83 to 100.03% at 3 different concentrations. The fucoidan stability of rat plasma at different temperatures and time-points was estimated. The extraction efficiencies ranged from 93.33 to 96.53%and the matrix effect ranged from 92.67 to 95.83%. Method selectivity was evaluated as well. We successfully studied the pharmacokinetic of fucoidan in rat plasma after oral by the validated method. Fucoidan was administered to rats intravenously at a dose of 6 mg/kg and orally at a dose of 20 mg/kg. The Cmax was 7.33µg/mL within 2 h by oral administration; The initial Cmax was 75.59µg/mL. The bioavailability of fucoidan after oral administration to rats was 8.91%.

Transport Mechanisms of Polymannuronic Acid and Polyguluronic Acid Across Caco-2 Cell Monolayers.

Detailed knowledge of the intestinal transport of polymannuronic acid (PM) and polyguluronic acid (PG) is critical for understanding their biological activities. To investigate the transport in the gastrointestinal tract, PM and PG were chemically modified with tyramine and conjugated with fluorescein isothiocyanate (FITC) to synthesize FITC-PM (F-PM) and FITC-PG (F-PG) successfully. The transport mechanisms of F-PM and F-PG across the intestinal epithelial cell monolayers (Caco-2 cell monolayers) were then investigated. The results demonstrated that the transport of F-PM and F-PG into epithelial cells was time- and energy-dependent, which was mediated by the macropinocytosis pathway and the clathrin- and caveolae (or lipid raft)-mediated endocytic pathway. The transport process of F-PM and F-PG in Caco-2 cells depended on the acidification of endosomes and involved lysosomes. Tubulin mediated the transport of F-PM, but not of F-PG. Moreover, the absorption enhancer chitosan (CS) promoted the transport of F-PM and F-PG, increasing the apparent permeability coefficient (Papp) by 1.9-fold and 2.6-fold, respectively, by reversibly opening the tight junction (TJ). In summary, this study provided a comprehensive understanding of the transport of PM and PG in the small intestinal epithelial cells, which will provide a theoretical basis for the development of PM and PG with good intestinal absorption.

Development of double network gels based on soy protein isolate and sugar beet pectin induced by thermal treatment and laccase catalysis.

Soy protein isolate (SPI) and sugar beet pectin (SBP) were adopted to fabricate double network (DN) gels via thermal treatment and laccase-catalysis. The concentrations of SBP (0.5%-2.5%, w/w) and SPI (4%-8%, w/w) were evaluated. DN gels showed higher holding water capacity (WHC, above 83%), compared with SBP single gel (75.96%). The presence of SPI improved the mechanical properties of gels significantly. Apparent phase separation could be observed when SPI concentration was 4%. Moreover, interpenetrating networks gradually formed with the increase of SPI concentration. The favorable structural heterogeneity and mechanical integrity derived from these polymers might be mainly responsible for the enhancement of the mechanical properties. The presence of SBP and laccase could improve the ß-sheet amounts of SPI and make it form more rigid structure according to the results of circular dichroism (CD) and fluorescence spectra. The excellent performance of DN gels could enable the delivery of various components.

Biological Activities of Fucoidan and the Factors Mediating Its Therapeutic Effects: A Review of Recent Studies.

The marine acid polysaccharide fucoidan has attracted attention from both the food and pharmaceutical industries due to its promising therapeutic effects. Fucoidan is a polysaccharide that mainly consists of L-fucose and sulphate groups. Its excellent biological function is attributed to its unique biological structure. Classical activities include antitumor, antioxidant, anticoagulant, antithrombotic, immunoregulatory, antiviral and anti-inflammatory effects. More recently, fucoidan has been shown to alleviate metabolic syndrome, protect the gastrointestinal tract, benefit angiogenesis and bone health. This review focuses on the progress in our understanding of the biological activities of fucoidan, highlighting its benefits for the treatment of human disease. We hope that this review can provide some theoretical basis and inspiration for the product development of fucoidan.

SKP2 promotes breast cancer tumorigenesis and radiation tolerance through PDCD4 ubiquitination.

BACKGROUND: S-phase kinase-associated protein 2 (SKP2) is an oncogene and cell cycle regulator that specifically recognizes phosphorylated cell cycle regulator proteins and mediates their ubiquitination. Programmed cell death protein 4 (PDCD4) is a tumor suppressor gene that plays a role in cell apoptosis and DNA-damage response via interacting with eukaryotic initiation factor-4A (eIF4A) and P53. Previous research showed SKP2 may interact with PDCD4, however the relationship between SKP2 and PDCD4 is unclear. METHODS: To validate the interaction between SKP2 and PDCD4, mass spectrometric analysis and reciprocal co-immunoprecipitation (Co-IP) experiments were performed. SKP2 stably overexpressed or knockdown breast cancer cell lines were established and western blot was used to detect proteins changes before and after radiation. In vitro and in vivo experiments were performed to verify whether SKP2 inhibits cell apoptosis and promotes DNA-damage response via PDCD4 suppression. SMIP004 was used to test the effect of radiotherapy combined with SKP2 inhibitor. RESULTS: We found that SKP2 remarkably promoted PDCD4 phosphorylation, ubiquitination and degradation. SKP2 promoted cell proliferation, inhibited cell apoptosis and enhanced the response to DNA-damage via PDCD4 suppression in breast cancer. SKP2 and PDCD4 showed negative correlation in human breast cancer tissues. Radiotherapy combine with SKP2 inhibitor SMIP004 showed significant inhibitory effects on breast cancer cells in vitro and in vivo. CONCLUSIONS: We identify PDCD4 as an important ubiquitination substrate of SKP2. SKP2 promotes breast cancer tumorigenesis and radiation tolerance via PDCD4 degradation. Radiotherapy combine with SKP2-targeted adjuvant therapy may improve breast cancer patient survival in clinical medicine.

The effect of leech extracts on endothelial cell coagulation-related factors and endothelial dysfuction-related molecules.

OBJECTIVE: This study was focused on screening leech extracts to identify those with little or no anti-coagulation effect or with significant anti-endothelial dysfunction activity. METHODS: Different leech extracts were prepared by enzymolysis and microbial transformation and their cytotoxicity were measured by MTT assay. The effect of different leech extracts on mRNA expression of coagulation-related factors (PAI, vWF, tPA, PS, TFPI, TM) was quantified by RT-PCR. After identifying a leech extract with little anti-coagulatory effect, RT-PCR was then used to assess the effect of this extract on the mRNA expression of endothelial dysfunction-related molecules (ET-1, iNOS, MCP-1, IL-6). RESULTS: 8 leech extracts were obtained, including 4 enzymatic extracts (LP, PHL, PTHL, CEHL) and 4 Lactobacillus metabolites (MRS, MRS-1, MRS-2, and MRS-3). Following optimization of conditions using MTT assays, we treated EA.hy926 cells with 0, 12.5, 25, 50 µg/mL of LP, PTHL, CEHL, MRS, MRS-1 or MRS-3 extract for 24 h. We found that PHL and MRS-1 had no significant effect on coagulation-related factors. Furthermore, treatment with 50 µg/mL PHL resulted in significant decreases in ET-1, iNOS, MCP-1, and IL-6 mRNA expression by 28.06%, 33.30%, 19.80%, and 52.34%, respectively. CONCLUSIONS: In the present study, we found that PHL, a pepsin hydrolysate of leech with little anti-coagulatory effect, could significantly suppress TNF-α induced mRNA overexpression of endothelial dysfunction-related molecules (ET-1, iNOS, MCP-1, and IL-6). These results provide a reliable experimental basis for identifying new anti-atherosclerosis therapeutics for long term use and with minimal bleeding side effects.

Efflux pump-mediated resistance to antifungal compounds can be prevented by conjugation with triphenylphosphonium cation.

Antifungal resistance due to upregulation of efflux pumps is prevalent in clinical Candida isolates. Potential efflux pump substrates (PEPSs), which are active against strains deficient in efflux pumps but inactive against wild-type strains, are usually missed in routine antifungal screening. Here we present a method for identification of PEPSs, and show that conjugation with mitochondria-targeting triphenylphosphonium cation (TPP+) can enhance or restore the compounds' antifungal activity. The screening method involves co-culturing a wild-type C. albicans strain and a Cdr efflux pump-deficient strain, labelled with different fluorescent proteins. We identify several PEPSs from a library of natural terpenes, and restore their antifungal activity against wild-type and azole-resistant C. albicans by conjugation with TPP+. The most active conjugate (IS-2-Pi-TPP) kills C. albicans cells, prevents biofilm formation and eliminates preformed biofilms, without inducing significant resistance. The antifungal activity is accompanied by mitochondrial dysfunction and increased levels of intracellular reactive oxygen species. In addition, IS-2-Pi-TPP is effective against C. albicans in a mouse model of skin infection.

Characterization of a Novel PolyM-Preferred Alginate Lyase from Marine Vibrio splendidus OU02.

Alginate lyases are enzymes that degrade alginate into oligosaccharides which possess a variety of biological activities. Discovering and characterizing novel alginate lyases has great significance for industrial and medical applications. In this study, we reported a novel alginate lyase, AlyA-OU02, derived from the marine Vibrio splendidus OU02. The BLASTP searches showed that AlyA-OU02 belonged to polysaccharide lyase family 7 (PL7) and contained two consecutive PL7 domains, which was rare among the alginate lyases in PL7 family. Both the two domains, AlyAa and AlyAb, had lyase activities, while AlyAa exhibited polyM preference, and AlyAb was polyG-preferred. In addition, the enzyme activity of AlyAa was much higher than AlyAb at 25 °C. The full-length enzyme of AlyA-OU02 showed polyM preference, which was the same as AlyAa. AlyAa degraded alginate into di-, tri-, and tetra-alginate oligosaccharides, while AlyAb degraded alginate into tri-, tetra-, and penta-alginate oligosaccharides. The degraded products of AlyA-OU02 were similar to AlyAa. Our work provided a potential candidate in the application of alginate oligosaccharide production and the characterization of the two domains might provide insights into the use of alginate of this organism.

Use of fluorescein isothiocyanate isomer I to study the mechanism of intestinal absorption of fucoidan sulfate in vivo and in vitro.

A new method to label fucoidan sulfate was established with tyramine and fluorescein isothiocyanate isomer I (FITC). Fluorescence spectrophotometry and high performance liquid chromatography verified the successful labelling of fucoidan by FITC. The results of the single-pass intestinal perfusion indicated that the jejunum and ileum are the main absorption sites, and there was carrier saturation. In addition, fucoidan sulfate at 1 mg/ml had no inhibitory effect on Caco-2 cell proliferation. Studies on the transmembrane transport mechanism showed that fucoidan can be absorbed because the apparent permeability coefficient of the drugs (Papp ) A â†’ B was 3.78 + 0.03 ×10-6 and that of B â†’ A was 1.42 + 0.19 ×10-6 . The peak absorption of fucoidan occurred at 120 min after administration; moreover, the higher the concentration used, the worse the absorption was, suggesting the saturation of transport carriers. The absorption was temperature dependent: the absorption at 37°C was much better than that at 4°C. Further, the absorption of fucoidan sulfate might rely on clathrin endocytosis as chlorpromazine (10 µg/ml) significantly inhibited it.

Carbon-Nanotube-Wrapped Spider Silks for Directed Cardiomyocyte Growth and Electrophysiological Detection.

The combination of nanostructures with biomaterials offers great opportunities in constructing innovative functional devices such as biosensors and actuators. Here, we create a multifunctional fiber by wrapping a thin film of carbon nanotubes (CNTs) on naturally found spider silks, which shows great flexibility and conductivity. The hybrid CNT-silk fiber demonstrates intimate contact with cardiomyocytes and can direct the cell growth and simultaneously record potential signals evoked from cell beating. Cell activities reflected in the form of potential signals have been monitored clearly and reliably through the CNT-silk fibers without degradation over the long term.