Study On Lipid Histology Analysis And Its Application In The Study Of The Mechanism Of Disease - Related Potential Biomarkers And Traditional Chinese Medicine

Lipidomics, which belonged to an important embranchment of metabolomics, gained more and more attention. Meanwhile, development of new analytical technology facilitated the development of lipidomic platform, making it better and more comprehensive for profiling, identification and quantification of lipids. Uncovering the relationship between lipid metabolic disorders and disease and finding potential biomarkers are the hot spots of research currently. This thesis focus on the analytical technique of lipidomics and appilication to discover potential disease-related biomarkers and elucidate material basis and mechanism of traditional Chinese medicine (TCM).In this thesis based on lipidomics, a large-scaled targeted lipidomic method for human stratum corneum (SC) sphingolipids profiling and quantification was established. Normal-phase high-performance liquid chromatography combining with dynamic multiple reaction monitoring mass spectrometer (NP-HPLC-dMRM-MS) was used to profile and quantify SC sphingolipids based on characteristic fragmentation rules. The sample preparation was simple, only methanol that contain internal standard compounds was needed for extraction. Then the mixture was vortexed and extracted with ultrasonic. After that, the solution was dried under nitrogen and then redissolved for analysis. NPLC was used to separated SC ceramides by their subclasses so that the detected compounds could be illuminated which subclass they belong to. The atmosphere pressure chemical ionization (APCI) source and dynamic multiple reaction monitoring (dMRM) was used for data acquisition. Sphingolipids had good responses in the APCI source under NPLC conditions so that good detection sensitivity was ensured. The application of dMRM made it possible for large-scaled quantification in short analysis time. In total 483 sphingolipids were quantified in a single run within 20 minutes, covering 12 subclasses along with glycosylation ceramides which have not been reported. Each subclass had typical standard substances (if available) to establish standard curves and detected compounds could be quantified by structure-similar standard substances. Method validation was carried out. The linear range of standard substances were from 50-2000 pmol/mL and r>0.99. LOD for these compounds were 2-10 pmol/mL and LOQ were 5-20 pmol/mL. The recovery of each standard substance at each concentration level was between 83-117%. All compounds’RSD is less than 13.1% with accuracy between 81-112%. This indicated that the instrument had good precision. Accuracy results showed that quality control (QC) samples were stable when stored in 4℃ for 3 days. The matrix effects of these ceramides were between 97-119%. This method has been exemplarily applied in the study of SC samples from healthy and atopic dermatitis (AD) children.48 potential biomarkers were found to present different status of healthy and AD children, containing 7 subclasses (2 Cer[ADS],1 Cer[AH],15 Cer[AS],4 Cer[NH],18 Cer[NS],4 Cer[NDS] and 4 Cer[NP]). This method has also been applied in the study of human’s SC of different age groups.193 potential biomarkers were found to represent age differences between children and adults, containing all 12 subclasses and the glycosylation ceramide (12 Cer[ADS],47 Cer[AH],13 Cer[AP],33 Cer[AS],4 Cer[EODS],12 Cer[EOH],4 Cer[EOP],19 Cer[EOS],17 Cer[NDS],6 Cer[NH],20 Cer[NP],1 Cer[NS] and 5 glycosylation ceramides). In this thesis, we analyzed children’s SC samples for the first time, providing new evidence for the evaluation of the differences of skin barrier function between healthy and AD children and uncovering the characteristics of sphingolipids separation in different age groups.In this thesis, the previously established sphingolipidomic analytical method was optimized, increasing the number of quantified compounds from 47 to 86. Then the lipidomic analytical platform was formed, together with our previous analytical method for other lipids with high-abundance (glyceride, glycerophospholipid and sphingolipids with high-abundance). HPLC coupled with triple quadrupole MS was used for the optimized sphingolipidomic analytical method. Peeke C8 SR (150×3.0 mm,3μm) column was used for separation. The mobile phase was water (containing 0.1% formic acid (FA),1 mmol/L ammonium formate) and methanol (containing 0.1% FA,1 mmol/L ammonium format) with a flow rate of 0.5 mL/min for gradient elution. MRM was used for data acquisition and segments were set to increase the number of detected compounds and the sensitivity. Based on the established lipidomic analytical platform, our research investigated the therapeutic treatment of the XXMD for chronic cerebral ischemia (CCI) and uncovered the material basis and the therapeutic mechanism of the XXMD from the perspective of lipid metabolic network. A chronic cerebral ischemia (CCI) rat model established by the permanent occlusion of bilateral common carotid arteries (2VO) method was used in the present study. Three different groups were studied:a sham operation, a model, and a model with dose group (XXMD,0.15 g/kg, i.g., for one month). A Morris water maze was used to investigate memory behavior. Biochemical criteria, superoxide dismutase (SOD) activity, and malondialdehyde (MDA) content were used to evaluate the CCI rat model. Nissl’s staining was carried out to observe the structures of the hippocampus and cortex neurons. Kluver-Barrera (KB) staining was used to observe the white matter. All the results provided the success of the modeling and effects of XXMD. A targeted comprehensive lipidomic platform was used to characterize the lipid profile of the target organ brain and the plasma in three groups. The lipid profiles changed statistically after the 2VO model and improved significantly after the oral administration of the active fraction of the XXMD. A total of 89 lipids (9 classes) in brain tissue and 53 lipids (8 classes) in plasma changed significantly in the model group compared to the sham operation group. A total of 116 lipids (11 classes) in brain tissue and 113 lipids (12 classes) in plasma changed significantly after dosing. Multivariate statistical modeling (orthogonal partial least squares discriminant analysis, OPLS-DA) was performed to identify potential biomarkers related to injury in the CCI model and the improvement upon treatment with the active fraction of the XXMD. A total of 52 potential biomarkers (8 classes) in brain tissue and 38 potential biomarkers (7 classes) in plasma were associated with the model injury. A total of 100 potential biomarkers (10 classes) in brain tissue and 73 potential biomarkers (10 classes) in plasma indicated the regulation effect of the XXMD. The components and metabolites of the active fraction of the XXMD in rat brains and plasma were identified by an HPLC-HRMS/MS" method combined with the mass spectral trees similarity filter (MTSF) technique. A total of 28 compounds (6 parent compounds from XXMD and 22 metabolites) in plasma and 2 compounds (1 parent compound from XXMD and 1 metabolite) in the brain were identified. The correlation between the concentration of potential lipid biomarkers and the peak areas of XXMD material basis were analyzed by Person correlation analysis and compound pairs were found. There were 9 pairs of compounds (corresponding to 2 exogenous compounds) in brain tissue and 95 pairs of compounds (corresponding to 17 exogenous compounds) in plasma exhibiting good correlations. This thesis investigated the mechanisms of CCI injury and therapeutic treatment of the XXMD from a lipidomic perspective. The exploratory research investigated the mechanisms of CCI injury and therapeutic treatment of XXMD combining material basis and lipidomics, providing insight into the active compounds detection and efficacy evaluation in TCM from a lipidomic perspective.Saussurea involucrata herba (Saussurea involucrata (Kar. et Kir.) Sch.-Bip) is a rare TCM in Xinjiang province that displays anti-tumor and protecting skin effects. In this thesis, an HPLC-LTQ/FTICRMS" qualitative method was established for targeted identification of sphingolipids in Saussurea involucrate based on lipidomic analytical platform.75% ethanol was used for extraction. A silica gel column was used for enrichment of low abundant sphingolipids using a step-wise system of chloroform/methanol under gradient elution. The elutes were evaporated to dryness and redissolved for analysis. A Spectra C8 SR column (150x3.0 mm,3μm) was used for separation and the mobile phase was acetonitrile/isopropanol (5:2, v/v) and a 0.1% acetic acid aqueous solution containing 2 mmol/L ammonium acetate, with a flow rate of 0.3 mL/min under gradient elution. FTICRMS was used for data acquisition. Mass spectra data were recorded by full-scan mass analysis at a resolving power of 50,000 with data-dependent MS2 analysis triggered by the two most abundant ions from full-scan mass analysis, followed by MS3 analysis of the most abundant ions of MS2. The characteristic fragmentation rules of known compounds were summarized. The characteristics of the unknown compounds were speculated according to the summarized rules and then these compounds were identified. In this thesis, we also established an HPLC-LTQ/FTICRMSn method for the identification of other compounds (flavonoids and phenylpropanoids) in Saussurea involucrata.75% ethanol was used for extraction. An ODS gel column was used for enrichment of low abundant compounds using a step-wise system of methanol/water under gradient elution. The elutes were evaporated to dryness and redissolved for analysis. A Thermo Accucore C18 column (4.6x100 mm, 2.6μm) was used for separation. The mobile phase consisted of acetonitrile and 0.2% acetic acid aqueous solution containing 10 mmol/L ammonium acetate with a flow rate of 1.0 mL/min under gradient elution. FTICRMS was used for data acquisition. Mass spectra data were recorded by full-scan mass analysis at a resolving power of 50,000 with data-dependent MS2 analysis triggered by the two most abundant ions from full-scan mass analysis, followed by MS3 analysis of the most abundant ions of MS2. The MTSF technique was used for compounds identification. Via combined pre-treatment and post-acquisition techniques,38 compounds were identified, including 19 flavonoids,11 phenylpropanoids and 8 sphingolipids. Among them,7 flavonoids,8 phenylpropanoids and 8 sphingolipids were reported in Saussurea involucrate for the first time. This thesis provides evidence to clarify the pharmacological activities and enrich the material basis of Saussurea involucrate, providing evidence for the function analysis of peculiar ceramides in plants. We investigated pre-treatment methods for enrichment of low abundant compounds in TCM, combining with chromatographic technique, high resolution mass spectrum technique and data post-processing technique. This novel strategy provides new insights for the fast identification of compounds in TCM.