Studies On The Effects And Mechanisms Of Low Molecular Weight Chondroitin Sulfate In Improving The Cognitive Function Of 5XFAD Mice And The Relationships Between Its Molecular Weights And Activities

Alzheimer's disease(AD)is a neurodegenerative disease developing in people's later years.Its clinical manifestations are progressive memory loss and multiple dysfunctions,including cognition,language,motoring and so on.These symptoms develop into changes in personality and abnormal behavior,and at last the loss of life ability.One of pathological characteristics of AD is senile plaques(SPs)in brains of patients.SPs are composed of amyloid ? protein(A?),which are the enzyme-digested products of amyloid precursor protein(APP).A? and A? aggregates disturb energy metabolism and calcium homeostasis of neurons,contribute to the production of reactive oxygen species(ROS),interfere with nerve transmission and induce chronic inflammation.A? and A? aggregates also persistently activate glial cells to damage nearby neurons.Moreover,A? and A? aggregates contribute to the hyperphosphorylation of tau protein.In previous work of our group,chondroitin sulfate(CS)from shark cartilage was degraded by hydrogen peroxide oxidation and fractionated with ultrafiltration membranes,and a low molecular weight chondroitin sulfate(LMWCS)with average molecular weight of 3928 Da was obtained.It was found that this LMWCS inhibited A?-induced oxidative stress,improved mitochondrial dysfunction and suppressed A?-induced apoptosis of human neuroblastoma cells SH-SY5Y and rat pheochromocytoma cells PC-12.As for the mice injected with A? in cerebral ventricles,oral administration of LMWCS improved oxidative stress,maintained normal energy metabolism and promoted cholinergic function.Collecctively,the abilities of learning and memory were improved.Based on these findings,5XFAD transgenic mice were used to further evaluate the anti-AD activity and effects of LMWCS on pathological changes in this project.Furthermore,the relationships between the molecular weight of CS oligosaccharides and its anti-AD activity and the mechanism were also studied to facilitate the medical application of LMWCS in AD treatment.The research contents were as follows.(1)Studies on the effects and mechanisms of LMWCS in improving the cognitive function of 5XFAD miceTwo-month-old 5XFAD transgenic mice were gavaged with LMWCS which was used in previous research.There were 3 doses in the experiment,namely,low,middle and high dose,which is 50 mg/kg,150 mg/kg and 450 mg/kg5 respectively.The administration duration was 3 months.Morris water maze(MWM)test and open field test(OFT)were used to evaluate the changes of behavioral indicators of the mice,including excitability,vitality,spatial learning and memory.Then the brain tissues were obtained,hippocampus and cortexes were separated from the brain tissue,and the proteins were extracted.ELISA was used to assess the amounts of A?1-42 in the hippocampus of mice,and Western blot was used to evaluate the expressions of APP and enzymes related with the digestion of APP,including presenilin 1(PS 1),?-site APP cleaving enzyme 1(BACE1),a disintegrin and metalloproteinase family 10(ADAM10)in the hippocampus and cortex of mice.ELISA was used to measure the amounts of interleukin-1?(IL-1?),tumor necrosis factor a(TNF-?)and IL-6 in the hippocampus of mice,and Western blot was used to measure the expressions of glial fibrillary acidic protein(GFAP),toll-like receptor 2(TLR-2)and phospho-extracellular regulated protein kinases 1/2(pERK 1/2).The chromogenic substrate assay was used to measure the concentrations of glutathione(GSH)and the activity of glutathione peroxidase(GSH-Px).The color reaction between malondialdehyde(MDA)and thiobarbituric acid(TBA)was used to measure the concentrations of MDA.And nitroblue tetrazolium chloride was used to measure the activities of total superoxide dismutase(SOD).Western blot was used to assess the expressions of SOD2,phospho-tau and enzymes related with hyperphosphorylation of Tau,including phospho-glycogen synthase kinase 3?(phospho-GSK3?)cyclin-dependent kinase 5(CDK5),p35 and p25.The results showed that,in MWM test,LMWCS administrations modestly decreased the escape latency,increased the times crossing the platform and the target quadrant and extended the distance and time in the target quadrant for 5XFAD mice.In OFT,LMWCS administrations moderately increased the distance in the central area and total distance of 5XFAD mice.Conclusively,LMWCS administrations moderately improved excitability,vitality,spatial learning and memory ability of 5XFAD mice.LMWCS administrations blocked the expressions of APP and PS 1,and moderately inhibited the expressions of ADAM 10 and BACE1 in the hippocampus and cortex of 5XFAD mice,which decreased the amount of A?1-in the brain and the neurotoxicity of A?1-42.LWCS administrations suppressed the expressions of GFAP and TLR2 in the hippocampus and cortex,as well as the amounts of IL-1?,TNF-? and IL-6 in the brain of 5XFAD mice.It was indicated that LMWCS blocked the production of proinflammatory cytokines through the inhibition of inflammatory pathways and activation of glial cells,which facilitated the inhibition of neuroinflammation.LMWCS administrations increased the activity of SOD and GSH-Px to varying degrees,reduced the amount of MDA and increased the amount of GSH in the brain,which indicated the improvement of oxidative stress.LMWCS administrations inhibited the hyperphosphorylation of tau protein,which might be related with the regulation of enzymes that catalyze the phosphorylation of tau protein.In summary,LMWCS administrations inhibited the production of A?,neuroinflammation,oxidative stress and hyperphosphorylation of tau protein in the brain of 5XFAD mice,which all resulted in the improvement of behavior indicators of 5XFAD mice,including excitability,vitality,spatial learning and memory ability.(2)Preparation and characterization of CS oligosaccharides with different molecular weightsThe gene for Chondroitinase ABC I(CHSase ABC I)was cloned from the genome of Proteus vulgaris by PCR method.And the gene was ligated with E.coli expression vector pET-28a(+),producing a recombinant plasmid pET-28a(+)-CHSase ABC I.This recombinant plasmid was transformed into Exoli BL21 for expression.The expression was induced by IPTG with a final concentration of 0.2 mg/L for 20 h at 20?,and CHSase ABC I with a 6×His tag(His-CHSase ABC 1)was expressed.His-CHSase ABC I was separated by Ni2+-Sepharose column.The catalysis characteristics of His-CHSase ABC I was studied to confirm the reaction condition.CS from shark cartilage was degraded by His-CHSase ABC I.The enzymolysis products was fractioned by Bio-gel P10 column and then purified by high performance liquid size exclusion chromatograph(HPSEC),producing CS disaccharide(CS DP2),tetrasaccharide(CS DP4),hexasaccharide(CS DP6),octasaccharide(CS DP8),decasaccharide(CS DP10),and dodecasaccharide(CS DP 12).HPSEC and electrospray ionization mass spectrometry(ESI-MS)were used to measure and characterize the products.The results showed that,His-CHSase ABC I had good substrate specificity.The specific activity to CS was 2600.7 ?mol/min·mg protein,and the specific activities to hyaluronan and heparin were fairly low.The optimal pH,temperature and NaCl concentration of the reaction condition of the degradation of CS catalyzed by His-CHSase ABC I were 7.5,37? and below 0.5 mol/L,respectively.Therefore,the reaction condition used in this project was as follows:1 unit enzyme was added in 1 mL aqueous solution of CS(80 mg/mL),and the reaction system was kept at 30? for 10 h.After a series of separation and purification,the peak purities of all CS oligosaccharide were 95%or above.The results of ESI-MS indicated the success of the preparation of CS oligosaccharides.(3)Study on the relationships between molecular weights of CS oligosaccharides and the activities of anti-Ap-induced oxidative tress and mitochondrial dysfunctionA?-injured human neuroblastoma cells SH-SY5Y were used as a model to study the effects of CS oligosaccharides on A?-injured neurons.MTT and Annexin-FITC/PI staining were used to assess SH-SY5Y apoptosis.For the change of oxidative stress,Griess reagent was used to assess the change of NO.The fluorescent probe DCFH-DA was used to evaluate the change of ROS.Relevant kits were used to measure the amount of MDA and the activities of SOD and GSH-Px.And Western blot was used to measure the expressions of enzymes related with oxidative stress.For the change of energy metabolism of SH-SY5Y cells,the fluorescent probe JC-1 was used to measure the change of mitochondrial membrane potential(MMP),and relevant kits were used to measure the change of the activity of Na+/K+-Pase.For the mitochondrial apoptotic pathway of SH-SY5Y cells,Western blot was used to measure the expressions of proteins related with apoptosis,including Bcl-2,Bax and Caspase-3.For cellular uptake of A? aggregates,Alexa Fluor 647 succinimidyl ester labeled A? aggregates were reacted with CS oligosaccharides and then incubated with the cells,and flow cytometry analysis was used to measure the median fluorescence intensity per cell to evaluate cellular uptake of AP aggregates.The results showed that,for A?-injured SH-SY5Y cells,CS oligosaccharides blocked A?-induced cell apoptosis via suppressing A?-induced oxidative stress and mitochondrial dysfunction.The effect against A? toxicity towards SH-SY5Y cells was increased with the increase of molecular weights of CS oligosaccharides,therefore,CS DP 12 had the best protective activity.This might be due to that CS inhibited cellular uptake of A? aggregates through binding with A? aggregates.(4)Study on the relationships between molecular weights of CS oligosaccharides and anti-Ap-induced neuroinflammationA?-injured mice microglia cells BV2 were used as a model to study the effects of CS oligosaccharides on A?-activated glial cells.CCK-8 was used to measure the change of cell viability.ELISA was used to assess the amounts of IL-1? and TNF-?.For the change of inflammatory pathways of BV2 cells,Western blot was used to measure the change of the expressions of TLR2 and NF-?B.For cellular uptake of A? aggregates,Alexa Fluor 647 succinimidyl ester labeled A? aggregates were reacted with CS oligosaccharides and then incubated the cells,and flow cytometry analysis was used to measure the median luorescence intensity per cell to evaluate cellular uptake of A?aggregates.The results showed that,for A?-incubated BV2 cells,CS oligosaccharides inhibited A?-induced activation of BV2 cells,and suppressed the secretion of proinflammatory cytokines and NF-?B.The effect against A?-induced activation of BV2 cells was decreased with the increase of molecular weight of CS oligosaccharide and CS DP2 had the best inhibitory activity.This might be due to that CS oligosaccharides inhibited the binding between A? aggregates and TLR2 to block the cellular uptake of A? aggregates,and CS DP2 had the best inhibitory effects.In summary,in this project,anti-AD activity of LMWCS was further confirmed with 5XFAD transgenic mice as models,and it was found that anti-AD activity was related with the regulation of APP metabolism,anti-oxidant and anti-inflammatory effts.Study on the relationships between molecular weights of CS oligosaccharides and anti-AD activity revealed that CS oligosaccharides inhibited cellular uptake of A?aggregates through binding with A? aggregates,blocked A?-induced oxidative stress and mitochondrial dysfunction,and finally protected SH-SY5Y cells.Besides,the effect against A? toxicity was increased with the increase of molecular weight of CS oligosaccharide.Study on the relationship between molecular weights of CS oligosaccharides and the inhibitory activity of A?-induced neuroinflammation revealed that,CS oligosaccharides inhibited the activation of BV2 cells through blocking the interaction between A? aggregates and TLR2,and decreasing the secretions of pro-inflammatory cytokines.Conclusively,the anti-AD activity of LMWCS was further supported by this study,and the relationship between molecular weights of CS oligosaccharides and their anti-AD activities was proved for the first time.More importantly,these findings may give some guidance for the studies on anti-AD functions of glycosaminoglycans and their derivatives and lay the foundation for the development of LMWCS as anti-AD drugs.