Octanoyl esterification of low molecular weight sulfated galactan enhances the cellular uptake and collagen expression in fibroblast cells.

Low molecular weight sulfated galactan (LMSG) supplemented with octanoyl ester (Oct-LMSG) demonstrated superior wound healing activity compared to the unsupplemented LMSG in a fibroblast wound model. To test the hypothesis that the increased bioactivity of Oct-LMSG may depend on its penetration into the plasma membrane, its cellular uptake was investigated and collagen production in fibroblast cells was assessed for the first time. The cellular uptake of Oct-LMSG was examined using indirect immunofluorescence and a confocal laser scanning microscope. In addition, the degree of fibroblast activation associated with this uptake was evaluated. The results indicated increased LMSG internalization in fibroblasts treated with Oct-LMSG. Transmission electron micrographs revealed the ultrastructure of active protein production in fibroblasts upon treatment with Oct-LMSG. In addition, Oct-LMSG upregulated the expression of type I collagen mRNA and proteins, as well as related signaling molecules involved in collagen synthesis, including collagen type I α1 chain (Col1A1), Col1A2, phosphorylated (p)-Smad2/3 and p-Smad4. The current findings support the notion that the supplementation of LMSG with octanoyl enhanced its cellular uptake into fibroblasts and, as a result, regulated the expression of type I collagen in fibroblasts via the activation of the Smad signaling pathway. This study demonstrates the therapeutic potential of Oct-LMSG in promoting tissue regeneration.

Sulfated Galactans from Gracilaria fisheri with Supplementation of Octanoyl Promote Wound Healing Activity In Vitro and In Vivo.

Sulfated galactans (SG) isolated from Gracilaria fisheri is partially degraded (DSG), and subsequentially supplemented with octanoyl (DSGO) and sulfate (DSGS) groups. The molecular weights of DSG, DSGO, and DSGS are 7.87, 152.79, and 97.07 kDa, respectively. The modification is confirmed using FTIR and NMR, while in vitro wound healing activity is assessed using scratched wound fibroblasts. The results reveal that DSGO exhibits highest percentage of wound closure in scratched fibroblast L929 cells. Furthermore, DSGO is able to promote proliferation and accelerate migration of scratched fibroblasts, which correspond to the regulation of proteins and mRNA (Ki67, p-FAK, vimentin, and E-cadherin) determined by Western blotting and qPCR analysis. The superior wound healing activity of DSGO is also confirmed in excision wound of rats. The results demonstrate that DSGO significantly enhances the percentage of wound closure, re-epithelialization, and collagen arrangement, increases α-smoth muscle actin (α-SMA) and vimentin expression, and decreases that of tumor necrosis factor-α (TNF-α) at the wound site. The results suggest that degraded SG supplemented with medium-chain fatty acids of octanoyl group may pass through the membrane, subsequently activating the mediators associated with proliferation and migration of fibroblasts, which can potentially lead to the promotion of wound healing activity.

Depolymerized Fractions of Sulfated Galactans Extracted from Gracilaria fisheri and Their Antibacterial Activity against Vibrio parahaemolyticus and Vibrio harveyi.

Various seaweed sulfated polysaccharides have been explored for antimicrobial application. This study aimed to evaluate the antibacterial activity of the native Gracilaria fisheri sulfated galactans (NSG) and depolymerized fractions against the marine pathogenic bacteria Vibrio parahaemolyticus and Vibrio harveyi. NSG was hydrolyzed in different concentrations of H2O2 to generate sulfated galactans degraded fractions (SGF). The molecular weight, structural characteristics, and physicochemical parameters of both NSG and SGF were determined. The results revealed that the high molecular weight NSG (228.33 kDa) was significantly degraded to SGFs of 115.76, 3.79, and 3.19 kDa by hydrolysis with 0.4, 2, and 10% H2O2, respectively. The Fourier transformed spectroscopy (FTIR) and 1H- and 13C-Nuclear magnetic resonance (NMR) analyses demonstrated that the polysaccharide chain structure of SGFs was not affected by H2O2 degradation, but alterations were detected at the peak positions of some functional groups. In vitro study showed that SGFs significantly exerted a stronger antibacterial activity against V. parahaemolyticus and V. harveyi than NSG, which might be due to the low molecular weight and higher sulfation properties of SGF. SGF disrupted the bacterial cell membrane, resulting in leakage of intracellular biological components, and subsequently, cell death. Taken together, this study provides a basis for the exploitation and utilization of low-molecular-weight sulfated galactans from G. fisheri to prevent and control the shrimp pathogens.

Increased Sulfation in Gracilaria fisheri Sulfated Galactans Enhances Antioxidant and Antiurolithiatic Activities and Protects HK-2 Cell Death Induced by Sodium Oxalate.

Urolithiasis is a common urological disease characterized by the presence of a stone anywhere along the urinary tract. The major component of such stones is calcium oxalate, and reactive oxygen species act as an essential mediator of calcium oxalate crystallization. Previous studies have demonstrated the antioxidant and antiurolithiatic activities of sulfated polysaccharides. In this study, native sulfated galactans (N-SGs) with a molecular weight of 217.4 kDa from Gracilaria fisheri were modified to obtain lower molecular weight SG (L-SG) and also subjected to sulfation SG (S-SG). The in vitro antioxidant and antiurolithiatic activities of the modified substances and their ability to protect against sodium oxalate-induced renal tubular (HK-2) cell death were investigated. The results revealed that S-SG showed more pronounced antioxidant activities (DPPH and O2- scavenging activities) than those of other compounds. S-SG exhibited the highest antiurolithiatic activity in terms of nucleation and aggregation, as well as crystal morphology and size. Moreover, S-SG showed improved cell survival and increased anti-apoptotic BCL-2 protein in HK-2 cells treated with sodium oxalate. Our findings highlight the potential application of S-SG in the functional food and pharmaceutical industries.

Structural characterization, antioxidant activity, and protective effect against hydrogen peroxide-induced oxidative stress of chemically degraded Gracilaria fisheri sulfated galactans.

Sulfated polysaccharides (SPs) possess an extensive range of biological activities, such as the inhibition of oxidation, correlated with their molecular weight (MW) and chemical structure. In this study, we used the trifluoroacetic acid (TFA) controlled degradation method to degrade sulfated galactans (SG) isolated from Gracilaria fisheri and evaluated the antioxidant and protective effects of the low molecular weight SG (LMSG) against H2O2 on fibroblast cells for the first time. Degradation of native SG (NSG) with an initial MW of 217.45 kDa using different concentrations of TFA resulted in five degraded NSG with MW of 97.23, 62.26, 30.74, 2.63, and 2.59 kDa. The reduction in MW was positively correlated with TFA concentrations. Chemical structure analyses using FTIR and NMR indicated that the TFA degradation process did not significantly change the LMSG polysaccharide main chain but did change the functional groups. LMSG exhibited higher scavenging activities and enhanced the cellular activities of GSH, CAT, and SOD enzymes. Moreover, LMSG activated Nrf-2/ARE signaling and increased expression of antioxidant genes CAT and SOD, which corresponded to increased protective effects against H2O2-induced ROS generation in fibroblast cells. The study reveals modification of NSG by acid TFA degradation resulted in the creation of LMSG, which showed greater antioxidant activity.

Probing the Anti-Cancer Potency of Sulfated Galactans on Cholangiocarcinoma Cells Using Synchrotron FTIR Microspectroscopy, Molecular Docking, and In Vitro Studies.

Sulfated galactans (SG) isolated from red alga Gracilaria fisheri have been reported to inhibit the growth of cholangiocarcinoma (CCA) cells, which was similar to the epidermal growth factor receptor (EGFR)-targeted drug, cetuximab. Herein, we studied the anti-cancer potency of SG compared to cetuximab. Biological studies demonstrated SG and cetuximab had similar inhibition mechanisms in CCA cells by down-regulating EGFR/ERK pathway, and the combined treatment induced a greater inhibition effect. The molecular docking study revealed that SG binds to the dimerization domain of EGFR, and this was confirmed by dimerization assay, which showed that SG inhibited ligand-induced EGFR dimer formation. Synchrotron FTIR microspectroscopy was employed to examine alterations in cellular macromolecules after drug treatment. The SR-FTIR-MS elicited similar spectral signatures of SG and cetuximab, pointing towards the bands of RNA/DNA, lipids, and amide I vibrations, which were inconsistent with the changes of signaling proteins in CCA cells after drug treatment. Thus, this study demonstrates the underlined anti-cancer mechanism of SG by interfering with EGFR dimerization. In addition, we reveal that FTIR signature spectra offer a useful tool for screening anti-cancer drugs' effect.

Bioencapsulation efficacy of sulfated galactans in adult Artemia salina for enhancing immunity in shrimp Litopenaeus vannamei.

Live food organisms like Artemia have been used for delivery of different substances such as nutrients, probiotics and immune-stimulants to aquatic animals. Previously, we reported that sulfated galactans (SG) from the red seaweed Gracilaria fisheri (G. fisheri) increased immune activity in shrimp. In the present study we further investigated the capacity and efficiency of bioencapsulation of SG in adult Artemia for delivery to tissues and potentially boosting the expression of immune genes in post larvae shrimp. SG were labelled with FITC (FITC-SG) for in vivo tracking in shrimp. Bioencapsulation of adult Artemia with FITC-SG (0-100 µg mL-1) was performed and the fluorescence intensity was detected in the gut lumen after enrichment periods of 30 min, 1 h, 2 h, 6 h and 24 h. The results showed the Artemia took up SG over time in a concentration-dependent manner. Shrimp were fed with the bioencapsulated Artemia (FITC-SG, 20 µg mL-1) and the shrimp were evaluated under a stereo-fluorescent microscope. At 24 h after administration, FITC-SG was located in gills and hepatopancreas and also bound with haemocytes. With daily SG administration, the genes IMD, IKKß were up-regulated (after 1 day) while genes dicer and proPO-I were up-regulated later (after 7 days). Moreover, continued monitoring of shrimp fed for 3 consecutive days only with SG at the dose of 0.5 mg g-1 BW showed increases in the expression of IMD, IKKß genes on day 1 and which gradually declined to normal levels on day 14, while the expression of dicer and proPO-I was increased on day 3 and remained high on day 14. These results demonstrate that bioencapsulation of SG in adult Artemia successfully delivers SG to shrimp tissues, which then bind with haemocytes and subsequently activate immune genes, and potentially increase immunity in shrimp. In addition, the present study suggests that a 3-consecutive-day regimen of SG supplemented in Artemia (0.5 mg g-1 BW) may boost and sustain the enhanced immune functions in post larvae shrimp.

Assessment of the effects of sulfated polysaccharides extracted from the red seaweed Irish moss Chondrus crispus on the immune-stimulant activity in mussels Mytilus spp.

Seaweeds contain a number of health enhancing and antimicrobial bioactive compounds including sulfated polysaccharides (SP). In the present study, SP extracted from a European red seaweed Irish moss Chondrus crispus was chemically analyzed, SP content extracted and the immune-response effect on wild Irish mussels Mytilus spp. investigated for the first time. A high percent yield of SP was extracted from C. crispus and the immune-stimulant activity of SP was assessed in a laboratory trial with mussels exposed to three different treatments of low (10 µg mL-1), medium (20 µg mL-1) and high (50 µg mL-1) SP dose concentrations and a control mussel group with no exposure to SP. An initial mussel sample was processed prior to the trial commencing and mussels were subsequently sampled on Days 1, 2, 3, 4, 7, and 10 post SP exposure. Both cell, humoral and immune related gene responses including haemocyte cell viability, haemocyte counts, lysozyme activity and expression of immune related genes (defensin, mytimycin and lysozyme mRNA) were assessed. No mussel mortalities were observed in either the treated or non-treated groups. Mussels exposed with SP showed an increase in haemocyte cell viability and the total number of haemocytes compared to control mussels. Lysozyme activity was also higher in treated mussels. Additionally, up-regulated expression of defensin, mytimycin and lysozyme mRNA was observed in SP treated mussels shortly after exposure (on Days 1, 2, and 3) to SP. These results indicate that a high quality yield of SP can be readily extracted from C. crispus and more importantly based on the animal model used in this study, SP extracted from C. crispus can rapidly induce health enhancing activities in Mytilus spp. at a cellular, humoral and molecular level and with a prolonged effect up to ten days post treatment.

A sulfated galactans supplemented diet enhances the expression of immune genes and protects against Vibrio parahaemolyticus infection in shrimp.

A sulfated galactans (SG) supplemented diet was evaluated for the potential to stimulate immune activity in shrimp Penaeus vannamei (P. vannamei). Shrimp given the SG supplemented diet (0.5, 1 and 2% w/w) for 7 days showed enhanced expression of the downstream signaling mediator of lipopolysaccharide and ß-1,3-glucan binding protein (LGBP) and immune related genes including p-NF-κB, IMD, IKKß and IKKε, antimicrobial peptide PEN-4, proPO-I and II. Following immersion with Vibrio parahaemolyticus (V. parahaemolyticus) for 14 days, the shrimp given the SG supplemented diet (1 and 2% w/w) showed a decrease in bacterial colonies and bacterial toxin gene expression, compared to shrimp given a normal diet, and they reached 50% mortality at day 14. However, shrimp given the normal diet and challenged with the bacteria reached 100% mortality at day 6. SG-fed shrimp increased expression of immune genes related to LGBP signaling at day 1 after the bacterial immersion compared to control (no immersion), which later decreased to control levels. Shrimp on the normal diet also increased expression of immune related genes at day 1 after immersion which however decreased below control levels by day 3. Taken together, the results indicate the efficacy of the SG supplemented diet to enhance the immune activity in shrimp which could offer protection from V. parahaemolyticus infection.

Sulfated galactans from Gracilaria fisheri bind to shrimp haemocyte membrane proteins and stimulate the expression of immune genes.

Previous studies demonstrated that sulfated galactans (SG) from Gracilaria fisheri (G. fisheri) exhibit immunostimulant activity in shrimp. The present study was conducted to test the hypothesis that SG stimulates signaling molecules of the immune response of shrimp by binding to receptors on the host cell membrane. Accordingly, we evaluated the ability of SG to bind to shrimp haemocytes and showed that SG bound to the shrimp haemocyte membrane (SHM), potentially to specific receptors. Furthermore, this binding was associated with an activation of immune response genes of shrimp. Data from confocal laser scanning micrographs revealed that FITC-labeled SG bound to haemocytes. Far western blot analysis demonstrated that SHM peptides, with molecular sizes of 13, 14, 15, 17, and 25 kDa, were associated with SG. Peptide sequence analysis of the isolated bands using LC-MS/MS and NCBI blast search revealed the identity of the 13, 14, and 17 kDa peptides as lipopolysaccharide and ß-1,3-glucan binding protein (LGBP). SG induced the expression of immune related genes and downstream signaling mediators of LGBP including IMD, IKKs, NF-κB, antimicrobial peptides (crustin and PEN-4), the antiviral immunity (dicer), and proPO system (proPO-I and proPO-II). A LGBP neutralizing assay with anti-LGBP antibody indicated a decrease in SG-induced expression of LGBP downstream signaling mediators and the immune related genes. In conclusion, this study demonstrated that the SG-stimulated immune activity in haemocytes is mediated, in part, through the LGBP, and IMD-NF-κB pathway.

Sulfated galactans isolated from the red seaweed Gracilaria fisheri target the envelope proteins of white spot syndrome virus and protect against viral infection in shrimp haemocytes.

The present study was aimed at evaluating an underlying mechanism of the antiviral activity of the sulfated galactans (SG) isolated from the red seaweed Gracilaria fisheri against white spot syndrome virus (WSSV) infection in haemocytes of the black tiger shrimp Penaeus monodon. Primary culture of haemocytes from Penaeus monodon was performed and inoculated with WSSV, after which the cytopathic effect (CPE), cell viability and viral load were determined. Haemocytes treated with WSSV-SG pre-mix showed decreased CPE, viral load and cell mortality from the viral infection. Solid-phase virus-binding assays revealed that SG bound to WSSV in a dose-related manner. Far Western blotting analysis indicated that SG bound to VP 26 and VP 28 proteins of WSSV. In contrast to the native SG, desulfated SG did not reduce CPE and cell mortality, and showed low binding activity with WSSV. The current study suggests that SG from Gracilaria fisheri elicits its anti-WSSV activity by binding to viral proteins that are important for the process of viral attachment to the host cells. It is anticipated that the sulfate groups of SG are important for viral binding.

Immunostimulatory activity of sulfated galactans isolated from the red seaweed Gracilaria fisheri and development of resistance against white spot syndrome virus (WSSV) in shrimp.

Sulfated galactans (SG) were isolated from the red seaweed Gracilaria fisheri (G. fisheri). Chemical analysis revealed SG contains sulfate (12.7%) and total carbohydrate (42.2%) with an estimated molecular mass of 100 kDa. Structure analysis by NMR and FT-IR spectroscopy revealed that SG is a complex structure with a linear backbone of alternating 3-linked ß-D-galactopyranose and 4-linked 3,6-anhydrogalactose units with partial 6-O-methylate-ß-D-galactopyranose and with sulfation occurring on C4 of D-galactopyranose and C6 of L-galactopyranose units. SG treatment enhanced immune parameters including total haemocytes, phenoloxidase activity, superoxide anions and superoxide dismutase in shrimp Penaeus monodon. Shrimp fed with Artemia salina enriched with SG (100 and 200 µg ml(-1)) and inoculated with white spot syndrome virus (WSSV) showed a significantly lower mortality rate and lower viral VP 28 amplification and expression than control. The results suggest that SG from G. fisheri exhibits immune stimulatory and antiviral activities that could protect P. monodon from WSSV infection.