Protective Effect Of Ketogenic Diet On Myelin Sheath Induced By Cuprizone-induced Demyelination In Mouse Model And The Preparation Of Ketogenic Emulsion

The ketogenic diet(KD)is a high-fat and low-carbohydrate formula that has been used as early as 1921 to treat treatment-resistant epilepsy in children.In recent years,more and more studies have confirmed that KD also plays an important neuroprotective role in other neurological diseases,such as multiple sclerosis(MS),Alzheimer’s disease,Parkinson’s disease and so on.MS is a chronic neurodegenerative disease characterized by central nervous system demyelination,glial cell lesions,inflammatory cell infiltration and neuronal damage.The incidence of MS in China is increasing year by year,which has aroused widespread concern.However,the mechanism by which KD improves MS demyelination remains unclear,and the possible neuroprotective effects involved include regulation of oxidative stress,anti-inflammation,maintenance of energy supply and regulation of deacetylase activity.On the other hand,KD has been used in clinical treatment for nearly a hundred years,mainly in the form of nutritional meals,solid milk powder and emulsions.At present,the existing KD terminal products are relatively scarce,and the domestic market of KD products is almost monopolized by multinational companies(including joint ventures),such as the ketogenic milk produced by Nutrica and Abbott.Because the formulation of ketogenic emulsion is complex,the technical barrier is high and the process is difficult.Therefore,the preparation of highly stable ketogenic emulsions can further promote the application of KD in clinical treatment.In this study,the demyelinating mice induced by cuprizone(CPZ)were used as the disease model to explore the protective effect of KD on myelin and its mechanism,and the ketogenic emulsion with high stability was prepared by high pressure homogenization method.The specific research contents are as follows:Part Ⅰ: Study on the mechanism of KD protecting myelin sheath of corpus callosum and inhibiting inflammatory cytokines in CPZ mice.The mouse model of demyelination induced by CPZ was established and treated with KD.Blood glucose and blood ketones were measured regularly during the experiment to determine that KD can effectively induce and maintain ketosis.The loss of myelin and myelin basic protein in the corpus callosum of mice was evaluated by LFB staining and MBP immunohistochemical staining,which showed that KD could protect the myelin sheath,and the glial cells were stained by immunohistochemistry and immunofluorescence to determine that KD inhibited the overactivation of glial cells.The effect of KD on the expression of pro-inflammatory factors and chemokines was evaluated by q RT-PCR.Finally,the method of Western blot was used to explain the correlation between the inhibition of inflammatory response by KD and the promotion of myelination of corpus callosum.Part II: Protective effect of KD on hippocampus of CPZ mice and its mechanism.Through open field,rotating stick,water maze and other behavior experiments,it is proved that KD can improve the behavior abnormalities such as decreased exploration behavior,motor coordination ability and memory impairment of CPZ model mice.The expression of CNPase protein was detected by Western Blot to evaluate the demyelination of mouse hippocampus.Through HE and Nissl staining of hippocampus,it is confirmed that KD intervention can improve the shrinkage of hippocampal neurons and make them arrange more tightly and orderly.GSH-Px,GSH and MDA enzyme-linked immunosorbent assay were used to determine that KD reduced oxidative stress.Finally,the method of Western blot was used to explain that KD protects the hippocampal myelin sheath by up-regulating the expression of SIRT1/PPAR-γ/P-Akt/m TOR protein.Part Ⅲ: Preparation technology and quality evaluation of ketogenic emulsion.The results of dynamic light scattering showed that the ketone emulsion prepared with WPI(3%)-Tween 80(2.0%)composite emulsifier showed a smaller particle size.At the same time,through the measurement of zeta-potential,it is found that the emulsifier can effectively increase the absolute value of zeta potential.In addition,the anti-ionic strength experiment confirmed that the non-ionic surfactant can be more effective against the effects of salt ions.By evaluating the emulsification index of the prepared emulsion after placing,it is found that the ketogenic emulsion prepared by Tween 80 and WPI is not easy to settle and more stable.