| Sea cucumber cerebrosides (SCC) are unique structure sphingolipids, which areproved to exhibit various physiological activities, including anti-tumor,immunomodulatory, anti-bacteria. Recently, its neuroprotective effect has causedmuch attention, and it is expected to be used in drugs alleviating brain dysfunction inelderly. Alzheimer‘s disease is a common neurodegenerative disease, and its exactcauses and pathogenic mechanism are complex. Today, it has been a problem can notbe ignored because of its rapid growth rate and social threats. Publications suggestedthat abnormal sphingolipid levels and alterations in brain could be connected with AD.Therefore, this paper studies the influence of cerebroside from sea cucumbe on ADmouse brain‘s sphingolipids, and explores the improvement to AD in the aspects ofsphingolipidgenomics. This study provides a theoretical reference for theneuroprotection of cerebrosides from sea cucumber, and provides theoretical supportfor dietary assessment of sea cucumber and the development of sea cucumbercerebroside drugs. The content and results of this study are as follows:A new simple and accurate method is established for the determination of thesphingolipids content in brain tissues. Four kinds of sphingolipids are separated andquantified by high performance liquid chromatography-triple quadrupole massspectrometry (LC-QQQ). An Agilent RX-SIL (2.1mm×100mm,1.8μm) column isemployed for separation. And a gradient elution by using chloroform/methanol/ammonia (89.5/10/0.5, v/v) and chloroform/methanol/water/ammonia (55/39/5/0.5,v/v) as mobile phase is performed. Cerebroside, ceramide, sulfatide andsphingomyelin in mouse brain cortex and hippocampus belong to the normal group,model group and experimental group (mouse are fed with SCC) were analyzed by thismethod. The results show that, compared to control group, cerebroside content inhippocampus and cortex of model group are reduced by23.96%and24.23%respectively; Ceramide content in hippocampus and cortex increase67.96%and 10.15%respectively; Sulfatide levels in hippocampus and cortex are decreased by54.82%and23.39%respectively. And sphingomyelin do not change significantly.Compared to modle group, ceramide content of experimental group in hippocampusdecrease32.79%, sulfatide content increase45.38%, and sphingomyelin is nosignificant difference.An accurate method is established for the quantitative and qualitative analysis ofcerebroside，ceramide, sulfatide molecular species in mouse brain. By LC-QQQ andan Agilent Eclipse Plus C18(2.1mm×50mm,1.8μm) column, cerebroside andceramide molecular species were separated by the mobile phase of methanol/water(97/3, v/v, containing0.5%formic acid,5mmol/L acid); And sulfatide molecularwere separated by the mobile phase of methanol/isopropanol/water (85/10/5, v/v,containing0.5%formic acid,5mmol/L acid). In order to simplify analysis work andincrease the accuracy, the sphingolipid molecular specier are separated and identifiedon the basis of both mass and hydrophobicity. MRM mode is used to quantification ofcerebroside, sulfatide, ceramide molecule species which belong to the normal group,model group and experimental group. Potential sphingolipids biomarkers are selectedas follows: HFA-GalCer/NFA-GalCer, Cer-d18:1-C18:0, Sulf-d18:1-C24:1,HFA-Sulf/NFA-Sulf, VLFA-Sulf/LFA-Sulf. Sulf-d18:1-C24:1. Compared to controlgroup, the ratio of HFA-GalCer/NFA-GalCer in hippocampus and cortex of modelgroup are increased by25.13%and14.36%respectively; Cer-d18:1-C18:0content inhippocampus and cortex increase30.63%and10.49%respectively; Sulf-d18:1-C24:1levels in hippocampus and cortex are decreased by43.31%and30.26%respectively;HFA-Sulf/NFA-Sulf in hippocampus and cortex are decreased by34.16%and26.34%respectively; And VLFA-Sulf/LFA-Sulf are decreased by38.08%and18.33%respectively. At the same time, the sphingolipids in hippocampus of mouse fed withSCC also have different degrees of change compared with model group. The ratio ofHFA-GalCer/NFA-GalCer in hippocampus decreases12.24%; Cer-d18:1-C18:0decreases11.81%; Sulf-d18:1-C24:1increases19.39%; HFA-Sulf/NFA-Sulf increases26.34%，And VLFA-Sulf/LFA-Sulf increases22.44%. There are no significantchanges of sphingolipids in cortex but the ratio of VLFA-Sulf/LFA-Sulf is increased by15.30%. In addition, the molecular species of cerebroside and sulfatide inhippocampus of three groups can be clearly distinguished by the anlyisis of PLS-DA.A method is established for the determination of GlcCer in brain tissues. ByLC-QQQ, the Waters Atlantis HILIC Silica（4.6mm×150mm,5μm）for separation,acetonitrile/methanol/water (95/2.5/2.5, v/v, containing2%formic acid,2mMammonium formate) as mobile phase at8min to achieve the anylsis of GlcCermolecular species. MRM mode is used to analyze GlcCer in hippocampus andcerebral cortex of three groups. Results show that d18:1-C18:0of hippocampus andcerebral cortex increases45.05%and30.41%respectively, and GalCer/GlcCer ofhippocampus and cerebral cortex decreases50.68%and21.08%respectively. Butthere is no significant difference between experimental group and model group. |
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