Basic Research On The TCM Incompatibility Theory Based On Eighteen Incompatible Medicament

This work was supported by the foundation of 2010’National Basic Research Program of China ("973 Program") (No.2011CB505300, No.2011CB505303), and the part of topic "Basic research on the TCM incompatibility theory based on Eighteen Incompatible Medicament".This dissertation is divided into five chapters and the contents are summarized as follows:Chapter 1. Literature researchThe relevant literatures were summarized including the development of modern research on drug induced liver injury (DILI). A detailed review was given to components basis research on hepatotoxicity of Sargassum, Euphorbiae Pekinensis Radix, Kansui Radix, Genkwa Flos.Chapter 2. Hepatocyte platform and technology systemSection 1 Hepatocyte platformOn the basis of literature analysis, research ideas were summarized and experimental program and technology roadmap were designed. The literature research, platform and design were provided too much reference for the following research.Section 2 Technology thoughts and designThe thesis draws the Eighteen Incompatible Medicament of Glycyrrhizae Radix et Rhizoma, Sargassum, Euphorbiae Pekinensis Radix, Kansui Radix, Genkwa Flos to be the research object. Reveal material basis from three levels:Incompatible Combinations, extracts and components with hepatocyte platform by the modern analytical technology; To analyse hepatic injury biomarkers based on metabonomics; Apply the computer to fit toxicity mechanism, combined with proteomics, mechanism was further explained.Chapter 3. Effects of Eighteen Incompatible Medicament on L02 cells viability Section 1 Effects of Incompatible Combination of Glycyrrhizae Radix et Rhizoma with Sargassum, Euphorbiae Pekinensis Radix, Kansui Radix or Genkwa Flos on L02 cells viabilityMTT assay was applied to assess human liver cell line L02 proliferation after incubation with single decoction and co-decoction. Single Glycyrrhizae Radix et Rhizoma decoction had no markedly toxic activity, while each single decoction of Euphorbiae Pekinensis Radix, Kansui Radix, Genkwa Flos decoction could inhibit the growth of L02 cells to different degrees. According to Webb coefficients, the inhibition effect of each co-decoction was stronger than the single decoction.Section 2 Effects of the extracts of Sargassum, Euphorbiae Pekinensis Radix, Genkwa Flos on L02 cells viabilitySargassum, Euphorbiae Pekinensis Radix, Genkwa Flos were extracted by different solvents and MTT assay was applied to assess human liver cell line L02 proliferation, preliminary result identifies that the active fraction could inhibit of cell proliferation in vitro. The active fraction of Sargassum is Water-fraction. The active fraction of Euphorbiae Pekinensis Radix mainly concentrated in petrol-ether and dichloromethane fraction. The active fraction of Genkwa Flos is dichloromethane fraction.Section 3 Effects of diterpenes constituents on the necrosis and apoptosis of human liver cell line L02Part 1 Effects of diterpenes constituents on L02 cells viabilityMTT assay was adopted to analyze the inhibitory effects of yuanhuacine and pekinenal on the proliferation of L02. With the yuanhuacine concentration increased, cell inhibition rate increased gradually, showed the dose-effect relationship. With the pekinenal concentration increased, cell inhibition rate gradually increased, but overall toxicity was not strong.Part 2 Effects of diterpenes constituents on L02 by Hoechst 33342 fluorescence staining methodUsing high connotation imaging system to observe apoptotic body by scanning within the scope of 384 nm exciting light, living cells showed homogeneous fluorescence, while yuanhuacine group and pekinenal group showed typical characteristics of apoptosis, which is dose related.Part 3 Effects of diterpenes constituents on L02 for biochemical indexesThe ALT and AST levels in L02 Cells Supernatant were detected by kits. After treated with yunahuacin, ALT and AST levels increased significantly, while in pekinenal group, AST levels increased significantly. ALT level was no statistical significance in the low dose group.Part 4 Detection of the apoptosis with flow cytometryAfter the treatment of yunahuacine and pekinenal with Annexin V-FITC/PI double staining by flow cytometry analysis, the apoptosis rate significantly increased to 21.13% and 25.98%, late apoptosis is the main death model.Chapter 4. Metabolomics study on diterpenes constituents-induced toxicity in human liver cell line L02Section 1 Metabolomics study on diterpenes constituents-induced toxicity in L02 cellsThe cellular metabolites were analyzed by UPLC-QTOF/MS. Normalized peak area of five metabolite were significantly decreased in yuanhuacine treated groups, correlation coefficient was -0.53～-0.96. Five metabolite normalized peak area significantly increased, and correlation coefficient was 0.72-0.99. In pekinenal treated groups, ten metabolite normalized peak area significantly decreased, and correlation coefficient was -0.672～-0.931.Normalized peak area of tow metabolite were significantly increased, correlation coefficient was 0.978～0.984. These markers were phospholipids, fatty acids and the other intermediate products, mainly involved in glycerophospholipid metabolism, sphingolipid metabolism, fatty acid oxidation, arachidonic acid metabolism, PPAR, MAPK cell apoptosis signaling pathways.Section 2 Studys on oxidative damage and inflammation-causing effects of diterpenes constituents-induced toxicityPart 1 The effect of Oxidative StressThe fluorescence intensity was determined by laser scanning confocal microscope, showed that intracellular ROS levels significantly increased with the drug concentration(P < 0.01). It may be associated with oxidative injury.Part 2 The study of inflammation-causing effectsThe content change of PGE2, PGF2a in cell supernatant was detected by UPLC-TQ/MS, the results showed that PGE2, PGF2a content increases with drug concentration, and compared to the control group with significant difference (P<0.01), revealing that drug-induced inflammatory cells provide the basis.Chapter 5. Proteomics study on diterpenes constituents-induced toxicity in L02 cells Section 1 Prediction of multi-target of diterpenes constituents and their network pharmacologyTo predict the targets of yuanhuacine and pekinenal according to public databases, establish the specific network model, which could predict action mechanism.Section 2 Effects of diterpenes constituents on the expression of proteins in L02The proteins were seperated in two-dimensional gel electrophoresis(2-DE). The differentially expressed protein spots were measured by MADI-TOF/MS, the data obtained from peptide mass fingerprinting (PMF) were used for protein database search. Four proteins in yuanhuacine treated groups were identified as glutathione S-transferase, triosephosphate isomerase, alpha-enolase isoform 1, protein disulfide isomerase-related protein 5. Eight proteins in pekinenal treated groups were identified as Tubulin, beta, galectin-1, MYL6, Rho GDI1, chloride intracellular channel protein 1, tropomyosin alpha-3, GAPDH, HSPA5/BiP/GRP78. These proteins mainly belong to the cytoskeleton family and molecular chaperone, and most of these have anti-apoptotic effect in cells.