κ-Carrageenan Oligosaccharides Protect Nerves by Regulating Microglial Autophagy in Alzheimer's Disease.

κ-Carrageenan is a linear sulfated polysaccharide extracted from the cell wall of marine red algae, and its enzymatically digested oligosaccharides (KOS) can inhibit microglial hyperactivation. Alzheimer's disease (AD) is a common chronic neurodegenerative disease, characterized by cognitive and memory impairment accompanied by nerve cell damage. Microglia activation causing enhancement of proinflammatory effects and neurotoxicity is one of the early events in AD disease. In this study, whether KOS have therapeutic or preventive effects in the AD model prepared from APP/PS1 transgenic mice was determined. Learning and memory of AD mice were detected by water maze experiments, and microglial activation-related protein expression and deposition of APP and Aß1-42 in the brain were examined. The effects of KOS on expressed inflammatory factors and inflammation-related proteins by microglia were tested by cell experiments. Transwell coculture was used to investigate the effect of microglia on neural cell activity after KOS treatment. The results showed that KOS could relieve the clinical symptoms in AD mice, and a decrease in the expression of inflammatory factors and inflammation-related proteins in brain tissue was detected. KOS alleviated nerve cell apoptosis by inhibiting the overactivation of microglia, thus exhibiting neuroprotective effects. Exploring the protective effect of KOS inhibition of microglia inflammation is expected to provide a theoretical basis for KOS as a therapeutic drug for neurodegenerative diseases.

κ-Carrageenan Oligosaccharides Inhibit the Inflammation of Lipopolysaccharide-Activated Microglia Via TLR4/NF-κB and p38/JNK MAPKs Pathways.

Microglial inflammation plays an essential role in neurodegenerative disease. Our previous studies had shown that κ-carrageenan oligosaccharides (KOS) could inhibit the excessive activation of microglia that induced by LPS, while the interrelated mechanisms were still indistinct. Therefore, we detected the inflammatory signaling pathway on LPS-activated microglia that pretreat by different content of KOS to reveal the mechanism on KOS's inhibition of microglia inflammatory response. ELISA was used to detect the effects of KOS on the secretion of interleukin-1 (IL-1ß), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6) and prostaglandin E2 (PG-2) by LPS-activated microglia, respectively. The production of reactive oxygen species (ROS) and nitric oxide (NO) in microglia cells was detected by flow cytometry, and the protein expression of immunoinflammation-related signaling pathways were detected by Western Blot. The results showed that KOS could significantly protected the microglia from the over-activated inflammatory by inhibiting the release of inflammatory cytokines and the oxidative stress response. And KOS could reduce the expression of the protein that related to the TLR4/NF-κB and p38/JNK MAPKs pathways activated by LPS in microglia. However, there may be no specific target of KOS in cells. Therefore, KOS, a natural algal source oligosaccharide, has immunomodulatory effects and can be used as a potential intervention therapy for inflammatory related neurodegenerative diseases.

κ-Carrageenan oligosaccharides induce microglia autophagy through AMPK/ULK1 pathway to regulate their immune response.

Microglia are the main effector cells of immune response in central nervous system and are important targets for disease prevention and treatment. Κ-carrageenan Oligosaccharide (KOS), obtained by enzymatic hydrolysis from carrageenan of marine red algae, can inhibit the release of inflammatory factors from the over-activated microglia. The mechanism of microglia autophagy induced by KOS and its relationship with inflammation were studied to explore the development prospect of KOS in the research and treatment of inflammatory related diseases. The effect of KOS on inducing autophagy was detected by the secretion of cytokines by lipopolysaccharide (LPS)-activated microglia, respectively. The protein expression of autophagy-related signaling pathways were detected by Western Blot. The results showed that KOS could significantly protect the microglia from over-activated inflammatory by inducing the autophagy and inhibiting the release of inflammatory cytokines. And KOS could reduce the expression of the protein that related to the AMPK/ULK1 pathways in microglia, so as to regulate the autophagy pathway, and inhibit the inflammatory response of over-activated microglia. The study on the effect of KOS on microglia autophagy and excessive inflammatory response will provide a theoretical basis for further studies on the inhibition of nerve injury by regulating microglia autophagy and inflammatory response.

Autophagy is essential for the endothelial differentiation of breast cancer stem­like cells.

Blood vessels serve an important role in tumor growth and metastasis, and recent studies have shown that certain tumor cancer stem cells may differentiate into endothelial cells and contribute to angiogenesis. In the present study, vascular endothelial growth factor (VEGF) was used to induce endothelial differentiation of breast cancer stem­like cells (BCSLCs), and methods including flow cytometry, western blotting and immunofluorescence were used to study the relationship between autophagy and the endothelial differentiation of BCSLCs. The results showed that BCSLCs could differentiate into endothelial cells under the induction of VEGF in vitro. Subsequently, the role of autophagy in the endothelial differentiation of BCSLCs was examined. Autophagic activity was measured during endothelial differentiation of BCSLCs, and the association between autophagy and endothelial differentiation was investigated using autophagy activators, autophagy inhibitors and autophagy related 5 (Atg5)­knockdown BCSLCs. Autophagy was increased during endothelial differentiation of BCSLCs, and there was a positive association between autophagy and endothelial differentiation. The ability of cells to undergo endothelial differentiation was reduced in BCSLCs with Atg5 knockdown. Therefore, autophagy was essential for endothelial differentiation of BCSLCs, and the findings of the present study may highlight novel potential avenues for reducing angiogenesis and improving treatment of breast cancer.

Efficacy of chitosan and sodium alginate scaffolds for repair of spinal cord injury in rats.

Spinal cord injury results in the loss of motor and sensory pathways and spontaneous regeneration of adult mammalian spinal cord neurons is limited. Chitosan and sodium alginate have good biocompatibility, biodegradability, and are suitable to assist the recovery of damaged tissues, such as skin, bone and nerve. Chitosan scaffolds, sodium alginate scaffolds and chitosan-sodium alginate scaffolds were separately transplanted into rats with spinal cord hemisection. Basso-Beattie-Bresnahan locomotor rating scale scores and electrophysiological results showed that chitosan scaffolds promoted recovery of locomotor capacity and nerve transduction of the experimental rats. Sixty days after surgery, chitosan scaffolds retained the original shape of the spinal cord. Compared with sodium alginate scaffolds- and chitosan-sodium alginate scaffolds-transplanted rats, more neurofilament-H-immunoreactive cells (regenerating nerve fibers) and less glial fibrillary acidic protein-immunoreactive cells (astrocytic scar tissue) were observed at the injury site of experimental rats in chitosan scaffold-transplanted rats. Due to the fast degradation rate of sodium alginate, sodium alginate scaffolds and composite material scaffolds did not have a supporting and bridging effect on the damaged tissue. Above all, compared with sodium alginate and composite material scaffolds, chitosan had better biocompatibility, could promote the regeneration of nerve fibers and prevent the formation of scar tissue, and as such, is more suitable to help the repair of spinal cord injury.

Cloning, identification and characterization of a novel κ-carrageenase from marine bacterium Cellulophaga lytica strain N5-2.

A novel κ-carrageenase gene (Cly-κ-car) was cloned and heterologously expressed from marine bacterium Cellulophaga lytica strain N5-2. The gene comprised an open reading frame of 1488bp encoding 495 amino acid residues. The deduced protein had a calculated molecular weight of 55.24kDa with an estimated isoelectric point of 9.90. Multiple alignment analysis revealed that Cly-κ-CAR shared identity with κ-carrageenases from Zobellia sp. M-2 (46%), Zobellia galactanivorans (42%) and Rhodopirellula islandica (38%). Recombinant Cly-κ-CAR (R-Cly-κ-CAR) had maximum specific activity of 620.08U/mg at 35°C, pH 7.0, 0.7% κ-carrageenan and in the presence of 0.6% NaCl. It retained >75% of its initial activity after heat treatment below 35°C for 2h. More than 50% of its activity was maintained after incubation at pH 5.0-8.0 and 4°C for 6h. The Km and Vmax values for κ-carrageenan were 0.94mg/ml and 13.42mM/min/mg, respectively. Thin layer chromatographic analysis of the R-Cly-κ-CAR hydrolysis products revealed that the enzyme hydrolyzed κ-carrageenan into neo-κ-carraoctaose and neo-κ-carrahexaose. R-Cly-κ-CAR is a novel κ-carrageenase enzyme and could be a valuable tool to produce high degree of polymerization κ-carrageenan oligosaccharides with various biological activities.

Constitutive Activation of mTORC1 in Endothelial Cells Leads to the Development and Progression of Lymphangiosarcoma through VEGF Autocrine Signaling.

Angiosarcoma/lymphangiosarcoma is a rare malignancy with poor prognosis. We generated a mouse model with inducible endothelial-cell-specific deletion of Tsc1 to examine mTORC1 signaling in lymphangiosarcoma. Tsc1 loss increased retinal angiogenesis in neonates and led to endothelial proliferative lesions from vascular malformations to vascular tumors in adult mice. Sustained mTORC1 signaling was required for lymphangiosarcoma development and maintenance. Increased VEGF expression in tumor cells was seen, and blocking autocrine VEGF signaling abolished vascular tumor development and growth. We also found significant correlations between mTORC1 activation and VEGF, HIF1α, and c-Myc expression in human angiosarcoma samples. These studies demonstrated critical mechanisms of aberrant mTORC1 activation in lymphangiosarcoma and validate the mice as a valuable model for further study.

Immunomodulatory function of κ-carrageenan oligosaccharides acting on LPS-activated microglial cells.

The major neurodegenerative diseases are characterized by increasing of activated-microglial cells and inflammatory cytokines in the central nervous system. Carrageenan extracted from red algae is a kind of polysaccharide with sulfate groups. The oligosaccharides were obtained from carrageenan by enzymatic degradation. To detect the immunomodulatory activity of κ-carrageenan oligosaccharides (KOS) on microglial cells and the relationship to the sulfate group content, the desulfated derivatives of KOS (DSK) were obtained by dimethyl sulfoxide-methanol-pyridine method. KOS was labeled with fluorescein isothiocyanate. The effect of KOS and DSK on lipopolysaccharide (LPS)-activated microglial cells was detected. Hematoxylin-eosin staining and flow cytometric were used to detect the cell viability. The "scratch" migration assay, ornithine analysis and RT-PCR were used to determine the cell migration, arginase and TNF-α released by microglial cells, respectively. The effect of LPS and KOS on microglial cells was determined by flow cytometry and laser scanning confocal microscopy. The results showed that KOS and DSK could inhibit the cell viability, arginase and TNF-α released by LPS-activated microglia cell with concentration dependent manner. But the effect of DSK was weaker than that of KOS. KOS aggregated on the cell surface firstly, and then they enter into the cell to the nucleus, spread over the entire cell finally. But the exist of LPS could prevent the entrance of KOS. It could be concluded that KOS could protect microglial cells from being activated by LPS, and its inhibition function had relationship to the sulfate group content of KOS, while there were competition between LPS and KOS.

Enzymatic preparation of κ-carrageenan oligosaccharides and their anti-angiogenic activity.

The mixture of κ-neocarrabiose-sulfate, κ-neocarrahexaose-sulfate and κ-neocarraoctaose-sulfate were prepared from κ-carrageenan with enzyme. The anti-tumor and anti-angiogenic activity of obtained κ-carrageenan oligosaccharides (KOS), were explored. The results showed that KOS could inhibit the proliferation, migration and tube formation of ECV304 cells, and could inhibit the growth of new vessels in CAM model. KOS displayed strong anti-tumor activity in both S180 and MCF-7 xenograft models. Only human CD105 was detected in MCF-7 xenograft tumor, moreover KOS could decrease the growing of new blood vessels derived from tumor cell. Real-time PCR results showed that KOS could suppress the mRNA expression of human VEGF, bFGF, bFGFR and CD105 in MCF-7 xenograft tumor. All these results indicated that KOS has anti-tumor and anti-angiogenic activity in vivo and in vitro. Especially K has the potency to inhibit the differentiation of tumor cell to blood vessel endothelial cell.

Characterization of a κ-carrageenase from marine Cellulophaga lytica strain N5-2 and analysis of its degradation products.

A carrageenan-degrading marine Cellulophaga lytica strain N5-2 was isolated from the sediment of carrageenan production base. A κ-carrageenase (EC 3.2.1.83) with high activity was purified to electrophoretic homogeneity from the culture supernatant by a procedure of ammonium sulfate precipitation, dialyzing and gel filtration on SephadexG-200 and SephadexG-75. The purified enzyme was verified as a single protein on SDS-PAGE, and whose molecular weight was 40.8 kDa. The κ-carrageenase yielded a high activity of 1170 U/mg protein. For κ-carrageenase activity, the optimum temperature and pH were 35 °C and pH 7.0, respectively. The enzyme was stable at 40 °C for at least 2.5 h. The enzyme against κ-carrageenan gave a Km value of 1.647 mg/mL and a Vmax value of 8.7 µmol/min/mg when the reaction was carried out at 35 °C and pH 7.0. The degradation products of the k-carrageenase were analyzed by thin layer chromatography (TLC), high performance liquid chromatography (HPLC), electrospray ionization time-of-flight mass spectroscopy (ESI-TOF-MS) and 13C-NMR spectroscopy, and the results indicated that the enzyme was specific of the ß-1,4 linkage and hydrolyzed κ-carrageenan into κ-neocarraoctaose-sulfate and κ-neocarrahexaose-sulfate first, and then broke κ-neocarraoctaose-sulfate into κ-neocarrabiose-sulfate and κ-neocarrahexaose-sulfate.

The immune regulation of κ-carrageenan oligosaccharide and its desulfated derivatives on LPS-activated microglial cells.

Neurodegenerative disease involves an inflammatory response in the central nervous system characterized by an increase in inflammatory cytokines and activation of microglial cell. To reveal the immune regulation activity of κ-carrageenan oligosaccharides (KOS) on microglia cell activated by LPS and the relationship between the sulfate group content of KOS and its immune regulation activity, KOS was prepared by enzymatic hydrolysis. The degradation products of κ-carrageenan were analyzed by high performance liquid chromatography (HPLC), ESI-TOF-MS and (13)C NMR spectroscopy, and the results indicated that the hydrolyzed products of the κ-carrageenase were κ-neocarrabiose-sulfate, κ-neocarrahexaose-sulfate and κ-neocarraoctaose-sulfate, respectively. Then desulfated derivatives of KOS (DSK) were obtained with DMSO-methanol-pyridine method. The effect of KOS and DSK on the viability of microglia cell activated by LPS was determined with MTT method. Griess assay and ELISA method were used to determine the contents of NO/NO(2-), TNF-α and IL-10 released by activated microglia cell, respectively. The results showed that KOS could inhibit the viability and content of NO, TNF-α and IL-10 released by LPS-activated microglia cell dose dependently. Compared with that of KOS, the inhibiting activity of DSK is weaker. So it could be concluded that KOS could protect microglial cell from being activated by LPS, and there was a positive relationship between the sulfate group content of KOS and its protection function.

Inhibition of angiogenesis by chitooligosaccharides with specific degrees of acetylation and polymerization.

Chitooligosaccharides (CHOS) inhibit angiogenesis and may be used in the treatment of cancer tumors. We have studied the effect of the fraction of acetylation (FA) and the degree of polymerization (DP) on CHOS anti-angiogenic activity. We tested enzymatically produced CHOS-mixtures with FA0.15, FA0.3 and FA0.6, and DP≤12 in initial experiments with chorioallantoic membranes. All of the samples reduced the formation of new blood vessels, CHOS with FA0.3 giving the best effect. Single-DP fractions from the FA0.3 sample purified by size-exclusion chromatography (DP3-DP12) were then tested for inhibition of migration of human endothelial cells, which is an important element of the angiogenesis process. All of the fractions inhibited migration, meaning that, within the DP area tested in this study, FA is more important than DP for the effect. Generally, the results reveal that DP3-DP12 CHOS have considerable potential as anti-angiogenic compounds.

Potent angiogenic inhibition effects of deacetylated chitohexaose separated from chitooligosaccharides and its mechanism of action in vitro.

This study was performed to demonstrate the effects of deacetylated chitohexaose (hexamer) separated from a chitooligosaccharide (COS) mixture on tumor angiogenesis and its mechanism of action. Five fractions from dimer to hexamer were separated by a linear gradient solution of HCl on a cation-exchange resin. Then HCl was removed from the fractions by a charcoal column. The purity of the five fractions was analyzed by HPLC and the molecular masses were analyzed by MALDI-TOFMS. The hexamer expressed an inhibitory influence on CAM angiogenesis in a dose-dependent manner at concentrations of 6.25-50microg/egg. On further investigation, we found that the hexamer had no toxic effect on normal ECV304 cells, but could inhibit the proliferation and migration of tumor-induced ECV304 cells in a dose-dependent manner. The mechanism was demonstrated through the detection of mRNA expression of VEGF, MMP-9, TIMP-1, TIMP-2, and uPA by RT-PCR, which showed that the hexamer down-regulated the VEGF and uPA mRNA expressions in ECV304 cells, but up-regulated the TIMP-1 mRNA expression.

[The effect of the degree of deacetylation of chitosan on the biocompatibility of chitosan membrane with corneal stromal cells].

Some different membranes were prepared by Chitosan with the degree of deacetylation (DD) of 63.7%, 73.7%, 83% and 97% respectively. To study the biocompatibility of Chitosan membrane toward corneal stromal cells, the rabbit cells were cultured on the surface of different DD chitosan membranes. The morphological characteristics, the cell-adhesion, the cell proliferation and the activity of LDH in the medium were investigated. The results of experiment shows that the DD of Chitosan has very significant effect on the biocompatibility of Chitosan membrane toward corneal stromal cells. The more DD of Chitosan, the less injury was made to corneal stromal cells by the chitosan membrane, which is favor of the growing and adhesion of corneal stromal cells. The biocompatibility of the membrane made with low DD Chitosan with corneal stromal cells became worse.

The antithrombotic action of propylene glycol mannite sulfate (PGMS).

In order to reveal the antithrombotic mechanism of propylene glycol mannite sulfate (PGMS) on gene level and explore the correlation between PGMS and fibrinolytic system, the urokinase-type plasminogen activator (uPA) mRNA expression and uPA activity were detected in vitro and in vivo with semi-quantitative reverse transcription (RT)-PCR and chromogenic method. The results showed that PGMS was able to increase uPA activity in dosage-dependent manner in rat plasma, which resulted in the increase of rat fibrinolytic activity. The change of rat uPA mRNA expression with PGMS was similar to that of uPA activity. The results of experiments with cultured cells were similar to those results of experiments in vivo. The uPA mRNA expression and activities of cultured cells showed increases with PGMS in a concentration-dependent manner. All of these results indicated that PGMS may induce the expression of uPA mRNA. The increase of uPA mRNA expression may therefore reflect, in part, an increase in the stability of uPA mRNA and/or processing of nuclear uPA transcription, which, in turn, may increase the uPA activity in rat plasma and trigger the fibrinolytic system.