Synthesis of chitosan oligosaccharide selenium and its antitumor activity

Background:Polysaccharides have various biological activities;the complexation of polysaccharides with trace element ions can produce synergistic effects,improving the original biological activities of sugars and trace elements.Methods:The preparation process of chitosan oligosaccharide selenium(COSSe)was optimized by the response surface method,followed by a detailed analysis of the resultant compound’s characteristics.The anti-cancer activity of COSSe was studied using the human ovarian cancer cell line SKOV3 as a cell model.Results:The prepared COSSe response surface was well predicted,indicating successful chitosan oligosaccharide binding with selenium.Response surface method analyses identified the optimal synthesis conditions for COSSe:the reaction time of 5.08 h,the reaction temperature of 71.8°C,and mass ratio(Na2SeO3:chitosan oligosaccharide)of 1.02.Under the optimal conditions,the final product,the selenium content,reached 1.302%.The results of cell experiments showed that COSSe significantly inhibited SKOV3 proliferation in a concentration-dependent manner.RNA-seq results showed that chitosan oligosaccharide and COSSe significantly modulated the expression of genes’DNA metabolic processes and cell cycle in SKOV3 cells.Gene enrichment analysis showed the inhibition of the cell cycle,and the results of flow cytometry showed that SKOV3 cells increased in the S phase and decreased in the G2/M phase,with a noted suppression in the protein expression of cyclin-dependent kinase 2(CDK2)and cyclin A1(CCNA1).Conclusion:COSSe has a stronger effect than chitosan oligosaccharide,leading to the arrest of the cell cycle in the S phase.Thus,COSSe may be an effective candidate for the treatment of ovarian cancer.

Chitosan Oligosaccharide-Ca Complex Accelerates the Depuration of Cadmium from Chlamys ferrari

This study investigated the effect of a chitosan oligosaccharide-Ca complex (COS-Ca) on the depuration of cadmium (Cd) from Chlamys ferrari. After exposure to 0.5 mg L-1 CdCl2 for 3 or 7 d, the scallops were treated by COS-Ca prior to determina-tion of Cd, calcium (Ca) and zinc (Zn) contents, Cd distribution in organs, malondialdehyde (MDA) content and antioxidant variables. Results showed that COS-Ca reduced Cd content in the viscera of the scallops, with highest Cd depuration rate (47%) observed on day 3. The COS-Ca concentration substantially affected Cd depuration, and the exposure to 8.75 mg L-1 COS-Ca led to significantly higher Cd depuration rate compared with those of lower COS-Ca concentrations (1.75, 3.5, 5.25, and 7.00 mg L-1). Distribution analysis of Cd in scallop organs indicated that COS-Ca significantly reduced Cd content in the kidney throughout the 5-d experiment, as well as in the gill during the early stage of Cd depuration. In addition, COS-Ca treatment decreased glutathione peroxidase (GSH-Px) activity and MDA content while increasing superoxide dismutase (SOD) and catalase (CAT) activities on different days. Our work suggested COS-Ca complex treatment as an effective method for acceleration of Cd depuration from Cd-contaminated bivalves.

Specific Targeting MRI of Chitosan Oligosaccharide Modified Fe_(3)O_(4) Nanoprobe on Macrophage and the Inhibition of Macrophage Foam­ing Induced by ox-LDL

Atherosclerosis(AS)is a primary cause of morbidity and mortality all over the world.Molecular imaging techniques can enable early localization and diagnosis of atherosclerosis plaques.Recent newly developed chitooligosaccharides(CSO)is considered to be capable of target mannose receptors on the surface of macrophages and to inhibit foam cell formation.Here we present a targeting magnetic resonance imaging(MRI)nanoprobe,which was successfully constructed with polyacrylic acid(PAA)modified nanometer iron oxide(Fe_(3)O_(4))as the core,and coating with CSO molecules,possessing the abilities of targeted MRI and specifically inhibition of the formation of foamy macrophages in the atherosclerotic process.The experimental results showed that the distributions of PAA-Fe_(3)O_(4) and CSO-PAA-Fe_(3)O_(4) were uniform and the corresponding sizes were about 5.93 nm and 8.15 nm,respectively.The Fourier transform infrared spectra(FTIR)testified the CSO was coupled with PAA-Fe_(3)O_(4) successfully.After coupled with CSO,the r1 of PAA-Fe_(3)O_(4) was increased from 5.317 mM s-1 to 6.147 mM s-1,indicating their potential as MRI contrast agent.Oil Red O staining and total cholesterols(TC)determination showed that CSO-PAA-Fe_(3)O_(4) could significantly inhibit the foaming process of RAW264.7 cells induced by oxidatively modified low density lipoprotein(ox-LDL).In vitro cellular MRI displayed that,compared with PAA-Fe_(3)O_(4),CSO-PAA-Fe_(3)O_(4) could lower the T1 relaxation time of RAW264.7 cells better.In summary,construction of CSO-PAA-Fe_(3)O_(4) nanoprobe in this study could realize the targeted MRI of macrophages and inhibition of ox-LDL induced macrophage foaming process.This will provide a new avenue in the diagnosis and treatment of AS.

Effects of Five Chitosan Oligosaccharides on Nuclear Factor-kappa B Signaling Pathway

The effects of five chito-oligomers, from dimer to hexamer （chitobiose, chitotriose, chitotetraose, chitopentaose, chitohexaose） separated from chitosan oligosaccharides, on nuclear factor -kappaB （NF-rd3） signaling pathway were investigated by using luciferase assay and laser scanning microscopy. The expression of NF-rd3 downstream genes （cyclin DI, TNFa and IL-6） were tested by real time PCR. We found that all five chitosan oligosaccharides increased NF-KB-dependent luciferase gene expression and NF-KB downstream genes transcription, and the most significant were chitotetraose and chitohexaose. In addition, laser scanning microscopy experiments showed that chitotetraose and chitohexaose also activated the p65 subunite of NF-kB translocating from cytoplasm to nucleus, which suggested that they were the most potent activators of NF-kB signaling pathway.

Fibroblast exosomal TFAP2C induced by chitosan oligosaccharides promotes peripheral axon regeneration via the miR-132-5p/CAMKK1 axis

Chitosan and its degradation product,oligosaccharides,have been shown to facilitate peripheral nerve regeneration.However,the underlying mechanisms are not well understood.In this study,we analyzed the protein expression profiles in sciatic nerves after injury using proteomics.A group of proteins related to exosome packaging and transport is up-regulated by chitosan oligosaccharides(COS),implying that exosomes are involved in COS-induced peripheral nerve regeneration.In fact,exosomes derived from fibroblasts(f-EXOs)treated with COS significantly promoted axon extension and regeneration.Exosomal protein identification and functional studies,revealed that TFAP2C is a key factor in neurite outgrowth induced by COS-f-EXOs.Furthermore,we showed that TFAP2C targets the pri-miRNA-132 gene and represses miR-132-5p expression in dorsal root ganglion neurons.Camkk1 is a downstream substrate of miR-132-5p that positively affects axon extension.In rats,miR-132-5p antagomir stimulates CAMKK1 expression and improves axon regeneration and functional recovery in sciatic nerves after injury.Our data reveal the mechanism for COS in axon regeneration,that is COS induce fibroblasts to produce TFAP2C-enriched EXOs,which are then transferred into axons to promote axon regeneration via miR-132-5p/CAMKK1.Moreover,these results show a new facet of fibroblasts in axon regeneration in peripheral nerves.

The neuroprotective efects of peracetylated chitosan oligosaccharides againstβ‑amyloid‑induced cognitive defcits in rats

Chitosan oligosaccharides(COSs)have been reported to possess a broad range of activities such as antitumor,antioxidant and neuroprotective activities.In this study,the protective efects and mechanisms of peracetylated chitosan oligosaccharides(PACOs)against Aβ-induced cognitive defcits were investigated in Sprague–Dawley(SD)rats.PACOs treatment signifcantly improved the learning and memory function of Alzheimer’s disease(AD)rats and attenuated the neuron cell damage caused by Aβ.PACOs also markedly reduced the levels of lactate dehydrogenase(LDH)and Malondialdehyde(MDA)and decreased the phosphorylation of Tau protein to inhibit oxidative injury and infammatory responses in AD rats.Further studies indicated that PACOs may promote the repair of Aβinduced nerve damage and inhibit neuronal apoptosis mainly through regulating PI3K/Akt/GSK3βsignaling pathway.Consistently,the transcriptome analysis verifed that the diferentially expressed genes(DEGs)were mainly involved in neuron development and the PI3K-Akt signaling pathway.Taken together,peracetylated chitosan oligosaccharides(PACOs)have the potential to be developed into novel anti-AD agents targeting the cellular PI3K/Akt/GSK3βsignaling pathway.

Maternal chitosan oligosaccharide intervention optimizes the production performance and health status of gilts and their offspring

Chitosan oligosaccharides(COS)are the hydrolyzed product of chitosan and have multifunctional health benefits.The objective of this study was to elucidate the effect of COS as a dietary supplement to gilts on their productivity and health and that of their litters.Gilts were randomly assigned to either a treatment(n=30)or control group(n=30).The treatment gilts were fed a standard dry sow ration supplemented with COS at 0.12 and 0.24 g/gilt per d during gestation and lactation,respectively,and the control group was fed the standard dry sow ration only.The body weight,reproductive performance,milk production and litter size for each gilt and body weight of corresponding litters were recorded.The serum immunoglobulins(IgA,IgG,IgM)and secretory immunoglobulin A(sIgA)concentrations of gilts and piglets and fecal sIgA concertation of gilts were measured by Enzyme-linked immunosorbent assay(ELISA).Our study showed that maternal COS supplementation 1)significantly increased gilt body weight in late pregnancy(P<0.05),2)significantly increased milk production of gilts at different stages(d 1,3,7 and19)of lactation(P<0.05),3)significantly increased body weight gain of piglets at weaning(P<0.05),4)significantly increased the serum concentrations of IgM and sIgA in piglets,and slgA in fecal sample of gilts(P<0.05),and 5)tended to increase the pregnancy success rate(P>0.05)in the treatment group compared to the control group.These results suggest that maternal COS intervention in gilts can improve gilt milk production,piglet pre-weaning growth and immunity parameters in both gilts and piglets.

Effects of chitosan oligosaccharide-nisin conjugates formed by Maillard reaction on the intestinal microbiota of high-fat diet-induced obesity mice model

Objectives:The goal of this study was to evaluate the modulatory effect of chitosan oligosaccharide-nisin conjugate(CON-C)on intestinal microbiota of human flora-associated(HFA)mice and also reveal its effect towards the high-fat diet(HFD)-induced obesity.Both Chitosan oligosaccharides and nisin showed great potential in modulating the intestinal microbiota,so it is worth to explore whether the modulation effect of chitosan oligosaccharide could be improved by covalently binding with nisin.Materials and Methods:CON-C was prepared by heating the mixed solution of chitosan oligosaccharide and nisin at 80℃ and pH 2.0 for 24h.The structure of CON-C were analyzed by Fourier transform-infrared spectroscopy(FT-IR)and X-ray diffraction(XRD).The CON-C’s anti-obesity effect and modulatory effect toward intestinal microbiota were analyzed using human flora-associated(HFA)mice model.Results:CON-C could alleviated HFD-induced gut dysbiosis,by significantly decreasing the numbers of Bifidobacterium and Lactobacillus/Enterococcus spp.,and increasing the numbers of Bacteroides-Prevotella and Clostridium groups.CON-C could also enriched the most differentially expressed genes through KEGG pathways of biosynthesis of amino acids,two-component system,and ATP binding cassette(ABC)transporters.Conclusions:The improved therapeutic effect of CON-C against HFD-induced obesity has been approved,and hence,CON-C has a great potential to be utilized as a functional food ingredient in reducing body weight.

Synthesis and in vitro Evaluation of Manganese（Ⅱ）-porphyrin Modified with Chitosan Oligosaccharides as Potential MRI Contrast Agents

Mn-TCPP-CSn（n=6,1 1,20） as a type of potential magnetic resonance imaging（MRI） contrast agents were synthesized via manganese（Ⅱ） meso-tetra（4-carboxyphenyl） porphyrin（Mn-TCPP） modified with chitosan oligosaccharides（CSn）.Experimental data of infared（IR）,UV-Vis,MS,inductively coupled plasma-atomic emission spectrometer（ICP-AES） and size exclusion chromatography evidenced the formation of Mn-TCPP-CSn-The stability results show that Mn-TCPP-CSn in aqueous solution was stable enough to prevent Mn（Ⅱ） ions from leaking.The magnetic properties in vitro indicate that Mn-TCPP-CS20 possesses higher longitudinal relaxivity（r1=10.38 L·mmol^-1·s^-1） in aqueous solution than unmodified porphyrin Mn-TCPPNa4[manganese（Ⅱ） meso-tetra（4-carboxyphenyl） porphyrin,tetrasodium salt]（r1=5.10 L·mmol^-1·s^-1） and the commercial contrast agent Gd-DTPA（r1=4.05 L·mmol^-1·s^-1）.The preliminary T1-weighted flash image studies in vitro show that the contrast and the imaging signal of Mn-TCPP-CSn were superior to those of Mn-TCPPNa4 and Gd-DTPA under the same conditions.The 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assay shows that Mn-TCPP-CSn has a good biocompatibility.In addition,the thermodynamical parameters（ΔH〈0,ΔS〈0,ΔG〈0） of Mn-TCPP-CSn bound to bovine serum albumin（BSA） show that Mn-TCPP-CSn could bind to BSA spontaneously,where the binding complex was stabilized mainly by van der Waals interactions and hydrogen bonds.These results suggest that Mn-TCPP-CSn have the advantage of becoming a potential MRI contrast agent.

Preparation,characterization,and antibacterial activity of oleic acid-grafted chitosan oligosaccharide nanoparticles

An oleic acid-grafted chitosan oligosaccharide(CSO-OA)with different degrees of amino substitution(DSs)was synthesized by the 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide(EDC)-mediated coupling reaction.Fourier transform infrared spectroscopy(FT-IR)suggested the formation of an amide linkage between amino groups of chitosan oligosaccharide and carboxyl groups of oleic acid.The critical aggregation concentrations(CACs)of CSO-OA with 6%,11%,and 21%DSs were 0.056,0.042,and 0.028 mg·mL^(-1),respectively.Nanoparticles prepared with the sonication method were characterized by means of transmission electron microscopy(TEM)and Zetasizer,and the antibacterial activity against Escherichia coli and Staphylococcus aureus was investigated.The results showed that the CSO-OA nanoparticles were in the range of 60-200 nm with satisfactory structural integrity.The particle size slightly decreased with the increase of DS of CSO-OA.The antibacterial trial showed that the nanoparticles had good antibacterial activity against E.coli and S.aureus.

Antoioxidant Activity of Carboxymethyl Chitosan with Different Substituted Degrees

[Objective] In order to study the relations among different positions, degrees of substitution and antioxidant ability. [Method] N, O-carboxymethyl chitosan （NOA, NOB and NOC）with various degrees of substitution （DS）were obtained by etherizing chito-oligosacchaside. Their structure and substituted degree were characterized and their antioxldant activity to·OH was evaluated. [ Result] The IC50 s of NOA ,NOB and NOC were 0.15 ,0. 29 ,0. 23 mg/ml while their DSs of -NH2 position（DSN） were 0.51,0.29 and 0.38 and DSo were 0. 74 ,0. 84 ,0. 97respectively.[ Conclusion] With the increase of DSN ,antioxidant activity of N,O-carboxymethyl chitosan oligosaccharide to·OH was up.

饲料中添加壳寡糖对异育银鲫非特异性免疫功能的影响

为了研究饲料中添加不同水平的壳寡糖(Chitosan oligosaccharide,β-1,4-寡糖-葡萄糖胺)对异育银鲫(Carassius auratus gibelio)非特异性免疫功能的影响,试验选择体重约为50 g健康的异育银鲫480尾,随机分成4个处理,每个处理3个重复,每个重复40尾异育银鲫。各组异育银鲫分别投喂含有0%(对照组)、0.25%、0.50%和0.75%的壳寡糖的试验饵料。试验周期为60 d。结果显示,与对照组相比,当异育银鲫养殖中添加0.50%和0.75%的壳寡糖可以使血清中丙二醛水平显著降低(P<0.05),血清溶菌酶活性和超氧化物歧化酶活性显著高于对照组和0.25%壳寡糖组(P<0.05),血清过氧化氢酶活性显著高于对照组(P<0.05)。研究表明,在异育银鲫养殖生产中添加适量的壳寡糖可以显著提高机体的非特异性免疫功能。综合养殖成本考虑,壳寡糖的最适添加量为0.50%~0.75%。

Combined effects of chitosan and microencapsulated Enterococcus faecalis CG1.0007 probiotic supplementation on performance and diarrhea incidences in enterotoxigenic Escherichia coli K88^+ challenged piglets

The aim of this study was to investigate the combined effects of chitosan oligosaccharide(COS) and a microencapsulated Enterococcus faecalis CG1.0007 probiotic(PRO) on growth performance and diarrhea incidences in enterotoxigenic Escherichia coli(ETEC) K88^+ challenged piglets in a 14-d study. Thirty piglets,7.19 ± 0.52 kg initial BW weaned at 21 ± 1 d.were allotted to 5 treatment groups(n = 6)consisting of a corn-soybean meal diet with no additive(negative control, NC), NC + 0.25% chlortetracycline(positive control, PC), NC + 400 mg/kg COS(COS), NC + 100 mg/kg PRO(PRO) and NC + a combination of COS and PRO(CPRO). Pigs were individually housed in cages, acclimated to treatments for a 7-d period and had ad libitum access to feed and water throughout the study, On d 8, pigs were weighed, blood samples were collected, and then orally challenged with 6 mL(1 ×10^(11) cfu/mL) of freshly grown ETEC inoculum. During post-challenge period, blood was sampled at 24 and 48 h to determine plasma urea nitrogen(PUN), and diarrhea incidences and fecal consistency scores were recorded from d 9 to 12. On d 14, all pigs were weighed and then euthanized to obtain intestinal tissue samples for histomorphometric measurements. Growth performance responses were similar among treatments during the pre-and post-challenge periods. There were no significant differences in PUN content, incidences of diarrhea, and fecal consistency scores among treatments. The intestinal histomorphology results did not differ significantly among treatments except for PC with increased(P = 0.0001) villus:crypt ratio compared with the NC. Under the conditions of the present study, it can be concluded that supplementation of piglet diets with 400 mg/kg COS, 100 mg/kg microencapsulated PRO or their combination did not significantly improve piglet growth performance both during the pre-and post-ETEC K88+ oral inoculation. Also, there were no significant reduction of incidences and severity of diarrhea after challenge compared with the control group.