| Parkinson’s disease(PD)is a neurodegenerative disease common in the elderly,affecting nearly 6 million people over 65 years old worldwide.At present,PD drugs used in clinic can only alleviate the clinical symptoms of patients to partly ensure the quality of patients’ daily life;however,long-term use of drugs will lead to a series of motor complications in the late stage of PD,which will bring greater inconvenience to patients’ daily life.Therefore,it is particularly important to search natural neuroprotective agents with low toxic and strong neuroprotective effect for preventing or alleviating the development of PD.Chitosan oligosaccharide(COS)has a variety of biological activities and has been proved to prevent or alleviate the development of a variety of neurodegenerative diseases.It is a potential neuroprotective agent;it has been approved as a new food raw material by National Health and Family Planning Commission of the People’s Republic of China,showing a good application prospect in the field of functional food and biomedicine.The degree of polymerization(DP)of COS is closely related to its biological activity,but most of the previous studies were carried out in mixtures containing a variety of DP,so it is impossible to evaluate which DP of COS molecules play a role.Besides,there is not enough data to support whether COS can cross the blood-brain barrier and enter the brain to play a neuroprotective role.Therefore,this paper separated COS monomers with different DPs.Then,we explored the permeability of blood-brain barrier of COS and the absorption and distribution in vivo.Furthermore,we evaluated the prevention and intervention effect of COS with different DPs on PD in mouse model and explored the mechanisms.So as to provide a theoretical basis for the rational use of COS in the prevention and intervention of PD.The specific work and conclusions of the paper are as follows:1)Separation of COS monomers with different DPs.In this paper,the D75 macroporous strong acid cation exchange resin with large adsorption quantity was selected to separate and prepare the COS monomers of single DP.Finally,the purity and structure of the separated monomers were detected and identified by thin layer chromatography(TLC),high performance liquid chromatography,mass spectrometry and nuclear magnetic resonance hydrogen spectroscopy.The results showed that after precipitation pretreatment with 80%ethanol,the content of DP1～5 in COS increased from 59.36% to 83.87%.We obtained four monomers which are chitobiose(COS2),chitotriose(COS3),chitotetraose(COS4)and chitopentaose(COS5).The purity of COS2,COS3,COS4,and COS5 is 93.44%,93.30%,91.48% and 93.11% respectively.The m/z of molecular ion peak of COS2,COS3,COS4,and COS5 is 341,502,664 and 825,respectively.The ratios of α-/β-configuration are 2:1,1:2,10:1 and 5:6,respectively.In addition,the recoveries of COS2,COS3 and COS4 were increased to more than 50% due to the phased isocratic elution.2)Study on blood brain barrier permeability and absorption and distribution of COS monomers with different DPs in vivo.In this study,fluorescein isothiocyanate(FITC)fluorescence labeling technique combined with in vivo imaging technology and fluorescence quantitative analysis were used to detect the absorption and distribution of COS with different DP in vivo.With the help of in vivo imaging,COS2,COS3,COS4 and COS5 can be visually observed to be distributed to the brain tissue through the blood-brain barrier.Fluorescence spectrophotometer was used for quantitative analysis.The results showed that COS2,COS3,COS4 and COS5 could be absorbed into the blood,and could be distributed across the blood-brain barrier to the brain tissue,which was mainly distributed in the kidney and liver,followed by the heart and spleen,and a small amount in the lung and brain.The distribution of COS monomers with different DPs in kidney and liver was positively correlated with DP,and the peak distribution in serum,heart,spleen,lung and brain was COS2 > COS4 >COS3 > COS5.3)Neuroprotective effect of COS monomers with different DPs on PD model mice.In this study,C57BL/6J mice were intragastrically administered with the isolated COS monomers at a dose of 65 mg/kg for 14 days.On the last five days,1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine(MPTP)was intraperitoneal injected at a dose of 30 mg/kg to induce PD model.Through behavioral experiments and pathological indicators to evaluate its effect and explore its potential mechanism.The results showed that the four COS monomers could reduce the climbing time of PD mice,increase the score of traction test and the motor ability of PD mice,suggesting that all momomers can alleviate the movement disorder of PD mice;and the four COS monomers could increase the expression of TH protein in striatum and protect TH positive cells in substantia nigra(dopaminergic neurons)of PD mice;besides,the effect of COS3 and COS5 was better than that of COS2 and COS4.In addition,after preventive intervention with four COS monomers,the overexpression ofα-synuclein could be alleviated,and the effect of COS2 was better than that of the other three COS monomers.Four COS monomers could also increase the expression of DAT in striatum;it also could reduce the release of inflammatory factors TNF-α,IL-1β and IL-6 in striatum,and the activation of microglia and astrocytes to alleviate the neuroinflammation in PD mice after preventive intervention.Moreover,the four COS monomers could also up-regulate the phosphorylation of PI3 K and Akt,increase the expression of Bcl-2 protein,and down-regulate the expression of Bax and c-caspase3 protein to activate PI3K/Akt/Bcl-2 pathway,reducing apoptosis and protecting dopaminergic neurons.In summary,it was also confirmed that COS2,COS3,COS4 and COS5 could enter the brain tissue through the blood-brain barrier;moreover,COS with different DPs had significant preventive and palliative effects on PD,and COS3 and COS5 had better protective effects on dopaminergic neurons than COS2 and COS4. |
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