Study On The Components And Mechanism Of Liver Injury In Polygonum Multiflorum Thunb

Objective:Polygonum multiflorum is commonly used in clinical medicine, while the adverse reactions of Polygonum multiflorum and its preparation are reported increasingly in recent years, especially the liver injury. But the constituent causing liver injury is not clear and study also exist many contradictions; and the mechanism study about Polygonum multiflorum and the different parts of Polygonum multiflorum leading to the hepatic injury is not comprehensive, so this study uses correlation analysis and other means to speculate and verify which ingredients in Polygonum multiflorum induce liver damage. The iTRAQ quantitative proteomics and the non-targeted metabonomics are employed to research the mechanism of different parts of Polygonum multiflorum leading to the liver injury. This project is expected to provide the references for the reasonable application of Polygonum multiflorum.Method:1) A UHPLC-Q-TOF-MS method is established to test the chemical compositions from Polygonum multiflorum water extracted (PMW), Polygonum multiflorum ethanol extracted (PME), Polygonum multiflorum preparata water extracted (PMPW) and Polygonum multiflorum preparata ethanol extracted (PMPE). The obtained data was analyzed by the Progenesis QI and Makerlynx XS software, and found out the differences by PCA and OPLS-DA software. L-02 cell experiments were done to estimate the hepatotoxicity of Polygonum multiflorum. The toxicity differences and composition differences were linked to analyze to find out which composition has the same changing trend with the toxicity. And this composition may lead to the liver injury.2) Dichloromethane extraction and HPD-300 macroporous separation was employed to isolate the composition of Polygonum multiflorum to four parts, that is hydrophilic part(Tannins and polysaccharides),30% ethanol part (Polyhydroxy stilbenes),70% ethanol part (Combined anthraquinones) and dichloromethane part (Free anthraquinones). Expect for them, there were non-isolated total extract. Firstly, CCK-8 and high-content screening was done in vivo to estimate the toxicity level of five parts. Then, SD rats were orally administered with extracts for 90 days and were sacrificed. The plasma and serum were used for the analysis of biochemical index including ALT、AST、TBIL、DBIL、ALP、GGT by non-targeted metabonomics and livers were used for the analysis of pathological conditions, antioxidant index such as SOD, MDA and by iTRAQ quantitative proteomics.3) Different parts of Polygonum Multiflorum were orally administered to rats once. LC-MS/MS method was established to analyze the plasma concentration and corresponding pharmacokinetic parameters of main constituents in the rats’plasma. Linked to the results of proteomics, the transaction of main ingredients and compositions were determined.Results:1) The toxicity level of four kinds is presented by the L-02 cell toxicity experiments, that is the toxicity of PME>PMW>PMPE>PMPW. Ten compounds were found out to be associating with toxicity, the seven of them were anthraquinones and five of seven were emodin derivatives. It showed the hepatotoxicity of Polygonum Multiflorum was related to the anthraquinones.2) The experiments in vitro showed that total extract and 30% ethanol extract possessed the higher cell proliferation inhibition by the results of CCK-8 and cell numbers. Although the hydrophilic part, the 70% ethanol extract and the dichloromethane extract showed the weak cell toxicity under the same amount of crude herbs, the GSH level of five kinds of extract was positive which explained that these five extract presented the toxicity to liver cell. The reason of it was related to the diminished ability of cell scavenging free radical. In addition, the MMP test results for the total extract, hydrophilic part and dichloromethane extract were positive. The above results illustrated that the cell toxicity generated from three kinds of extracts is related to the change of MMP. The liver tissue pathological examination in vivo showed liver cells can appear different degree of edema, fatty degeneration and spotty necrosis, balloon like degeneration, vacuolar degeneration and may of inflammatory cells in the central vein focal infiltration, liver tissue with focal necrosis phenomenon after feeding different sites of Polygonum Multiflorum. It illustrated five kinds of extracts of Polygonum multiflorum can lead to liver damage. The results from SOD and other antioxidant indices determined that liver injury mechanisms may be related to oxidative damage, which coincided with the evaluation in vitro. Serum biochemical indexes showed that The AST, ALT, LDH values in the rats serum of the water soluble part,30% ethanol group,70% ethanol group and dichloromethane group were significantly higher than that of the blank control group, and 30% ethanol group increased obviously. But the AST, ALT ALP and LDH of total extract group had no obvious increase, but TBIL, DBIL, ALP, GGT significantly increased compared with the blank control group, indicating rats liver injury mechanisms in this group may exist difference. This difference may associate with the categories and content differences of components in the extract.3) The iTRAQ quantitative proteomic technology compared two pieces of proteinograms, proteinogram of injured liver caused by different sites of Polygonum multiflorum, and normal rats liver proteinogram. The peptide number marked by iTRAQ labeling quantitative information was 23619, and the protein number was 2040. The results of Pathway analysis showed that the oxidative phosphorylation pathway, Parkinson’s disease, Huntington’s disease, Alzheimer’s disease and the alternative splicing pathway were contained in 5 groups, and 31 co expressed proteins were screened out. The PCA and OPLS-DA analysis were used in the study of the metabolism, using VIP and S-plot to select the variables which have great correlation. These 5 groups of different metabolites were identified to find 15 metabolic pathway including glycerophospholipid metabolism, unsaturated fatty acid biosynthesis, linoleic acid and alpha linolenic acid metabolism, peanut four acid metabolism, phenylalanine metabolism by the Metabolic Pathway, and twenty-two associated potential biological markers including glycerol phospholipids, such as lysophosphatidylcholine (lysoPC (15:0) and lysoPC (16:0) and lysoPC (17:0) and lysoPC (18:0), lysoPC (P-18:1)), phosphatidyl choline (PC 16:0/16:0); unsaturated fatty acids, flowery life arachidonic acid, DHA,; steroids and bile acids, such as cholesterol sulfate, bezoar deoxycholic acid, chenodeoxycholic cholic acid sulfate; except for the above, there are compounds such as hydroxy acid, benzene, and palm acid.4) The simultaneous determination of stilbene glycoside, emodin-8-O-beta-D-and emodin in SD rats plasma was built in LC-MS/MS. Due to the water soluble content of extract from three main components are very low, so it is not measured in the rat plasma, the remaining four extracts parts was used to calculate pharmacokinetic parameters according to the blood concentration of corresponding components. The content of stilbene glycoside in the total extract group and the 30% ethanol group is similar, but the AUC, Cmax of 30% ethanol group were higher than that of the total extraction group; MRT, t1/2 of 30% ethanol group were lower than that of the total extraction group. The emodin dosages of the total extract group,30% ethanol group and 70% ethanol group and dichloromethane group were respectively 4.91, 0.11,0.23 and 3.67mg/Kg. From the blood concentration of each emodin group, and the Cmax of 70% ethanol group was higher than that of the total extract group and dichloromethane group. The emodin dosages of dichloromethane fractions were similar with that of total extract group, but the Cmax and AUC parameters of dichloromethane fractions were significantly less than that of total extract group.Conclusion:The components in Polygonum multiflorum inducing liver injury is related to the anthraquinone compounds (especially of emodin and its derivatives), but due to a part of components are not currently isolated to the monomer compounds, so the verification work is needed for the further study.Proteomics and metabolomics research results show that, Polygonum multiflorum causing liver injury is mainly associated with mitochondrial function of oxidative phosphorylation, TCA cycle pathway conduction abnormalities. The abnormal expression of NADH dehydrogenase family and the metabolism of phenylalanine lead to disorders of oxidative phosphorylation, mitochondrial dysfunction, the rise of the electronic leakage in respiratory chain increased, and then the increase in ROS production and the release of Cyt C. Tthe caspase-9 that activated by Cyt C can continue to activate Caspase-3 and induce cell apoptosis. Thyroid hormone signaling pathway is also associated with the expression of Caspase-3, caspase-9, the low expression of transporter proteins Slc16a2 causes that PI3K/Akt signaling pathway and Akt cannot be successfully activated, furthermore p53 protein and the expression of caspase-9 cannot be suppressed and result in cell apoptosis. The abnormal metabolism of glycerol phospholipids is a common problem in 5 groups of rats with liver injury.The lysophosphatidylcholine such as LPC(18:2), LPC (22:0), LPC (22:6) that is produced by the glycerol phospholipids metabolic pathway abnormalities can be used as a marker of liver injury induced by Polygonum multiflorum. At the same time, the low expression of ABP can make the ectopic expression in PPAR-α、β/δ、γ, PPAR-α regulates many physiological functions by regulating the expression of target genes, including fatty acid uptake, binding, oxidation and lipid transport, growth and development, which plays a key role in the regulation of lipid metabolism; Increased lysophosphatidylcholine concentration in blood made the red blood cell membrane lysis, cause hemolytic disease. The increased level of plasma bilirubin in total extract group may be related to this, but the specific reasons need to be further study. In addition to the above mechanism, the arachidonic acid metabolism pathways, the synthesis of bile acid and steroid compounds, the steroidogenic pathway related to the metabolism and the steroid hormone biosynthesis pathway are all related to the liver injury induced by Polygonum multiflorum.The pharmacokinetic study results show that the Cmax and AUC of stilbene glycoside in the 30% ethanol group are higher than that in the total extract group, the MRT, T1/2 of stilbene glycoside in the 30% ethanol group are lower than that in the total extract group, the MRT of emodin glycoside in the 30% ethanol group and 70% ethanol group are lower than that in the total extract group. The above differences may caused by the expression of the total extract metabolism enzyme. Meanwhile, the study found that emodin-8-O-β-D-glucoside rapidly removes of sugar and generates emodin after administration in vivo, which leads to the increase of Cmax and AUC of emodin. Thus, different extracts may lead to differences in the expression of metabolic enzymes and the in vivo behavior of different components, it may lead to liver injury.