| Background and objective:Alcohol-induced liver injury caused by long-term or heavy alcohol consumption is a major cause of liver disease and related diseases worldwidely, which is threatening human health. Given the universality and potentially harm of alcohol consumption, it’s of great significance to seek effective functional factors that can prevent alcoholic liver injury from food resources and to study their action mechanism in order to develop functional food for liver protection.Polysaccharide DHP is extracted and purified from an edible plant Dendrobium huoshanense Tang et Cheng. Previous studies have shown that DHP possesses high antioxidative, anti-tumor, hypoglycemic, immunoregulating and hepatoprotective activities.Based on the establishment of subacute alcoholic liver injury model in mice and the oral administration of DHP, this dissertation aims at evaluating the protective efficacy of DHP on subacute alcoholic liver injury in mice, comparing the differences of liver proteins by proteomics techniques and metabolites in serum and liver tissues by metabolomic techniques, and probing the possible molecular mechanisms of DHP against subacute alcoholic liver injury in order to provide a theoretical basis for exploiting functional factors and functional food from Dendrobium huoshanense to prevent alcoholic liver injury. Methods:1. Establishment of subacute alcoholic liver injury model in mice and intervention of DHP:96healthy adult male Kunming mice were randomly divided into control group, ethanol group, DHP groups (100,200,400mg/kg), DHP complete hydrolysate group (200mg/kg), starch group (200mg/kg), silymarin group (50mg/kg)(n=12per group). Except the control group and ethanol group, all the other6groups were orally given corresponding prevention once daily for30days. Control group and ethanol group mice were given an equal volume of solvent. From the31th day, the treatment groups and model group were suffered to intragastric administration with ethanol (2400mg/kg) for consecutive15days to induce subacute alcoholic liver injury. During the experiment, the control mice were intragastrically administered with equal solvent.2. Evaluation of the protective efficacy of DHP on subacute alcoholic liver injury in mice:evaluating indicators included body weight changes, liver index, serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities, levels of serum total cholesterol (TC), triglyceride (TG), total bilirubin (TBIL), low density lipoprotein (LDL), high density lipoprotein (HDL) and uric acid (UA) as well as liver pathological alteration. Among them, serum ALT and AST were determined by rate method; serum TC was determined by COD-PAP method; serum TG was determined by GPO-PAP method; serum LDL and HDL were measured by homogeneous method; serum TBIL was determined by modified J-G method; serum UA was determined by enzymatic colorimetric method; liver pathological alteration were observed by HE staining using a light microscope.3. Proteomics analysis:two-dimensional difference gel electrophoresis (2D-DIGE) coupled with matrix-assisted laser desorption/ionization-time of flight/time of flight mass spectrometry (MALDI-TOF-TOF) technology was employed to identify the differentially expressed protein spots. Western blot was used to verify the selected proteins.4. Metabolomics analysis:UHPLC/LTQ Orbitrap XL MS-based metabolomics approach was employed to investigate the metabolic profiles of serum and liver tissue extracts from control, ethanol-treated and DHP plus ethanol co-treated (DHP\ethanol) mice. By pattern recognition methods, non parametric test was used to analyze the data and to find the differences of serum and liver tissue metabolites.5. Bioinformation analysis:Biological pathway analysis tool-Ingenuity Pathway Analysis (IPA) was applied to analyze the experimental data from our metabonomics and proteomics and to establish a multi-level biological molecular interaction networks from the related protein and small molecule metabolite informations. Results:1. Evaluating indicators of liver function showed that as compared with ethanol group, DHP groups improved living conditions of mice and significantly reduced liver index (P<0.01, P<0.01, P<0.01). DHP groups (200,400mg/kg) reduced the levels of AST (P<0.05, P<0.01), TBIL (P<0.05, P<0.05) and UA (P<0.01, P<0.05) in serum; DHP (400mg/kg) group also reduced the elevated levels of TC (P<0.05) and LDL (P<0.05) in serum. Pathological examination showed that as compared with ethanol group, DHP groups alleviated liver fatty degeneration and inflammation in mice. The above results hinted that DHP possesses protective effects on sub-acute alcoholic liver injury in mice. 2. The hepatic proteomics analysis by the combined application of2D-DIGE and MALDI-TOF-TOF mass spectrometry revealed that18protein spots were differentially expressed between ethanol group and control group (9proteins up-regulated,9proteins down-regulated), and7proteins differentially expressed between DHP/ethanol group and ethanol group (4proteins up-regulated,3proteins down-regulated). By the mass spectrum analysis,18proteins were successfully identified:Serotransferrin(Trf), cystathionine beta-synthase (Cbs), D-lactate dehydrogenase (Ldhd), hydroxymethylglutaryl-CoA synthase (Hmgcs2), S-adenosylmethionine synthase isoform type-1(Matla), keratin8(Krt8), Acetyl-CoA acetyltransferase (Acat2), fructose-bisphosphate aldolase B (Aldob), putative deoxyibonuclease TatD DNase domain containing1(TATDN1), glyoxalase domain-containing protein4(Glod4), NADH dehydrogenase (ubiquinone) Fe-S protein3(Ndufs3), carbamoyl-phosphate synthase (Cpsl), fructose-1,6-bisphosphatase (Fbpl), carbonic anhydrase3(Car3), GTP-Binding protein SARI a (Sarla), glutathione S-transferase P1(Gstpl), adenine phosphoribosyltransferase (Aprt). Searched in ExPASy (http://www.expasy.org/) and UniProtKB (http://www.uniprot.org/) for protein functional classification and found that those proteins were involved in glucose metabolism, amino acid metabolism, lipid metabolism, energy metabolism, nucleotide metabolism and oxidative stress. Selected differentially expressed proteins Cbs, Matla and Ldhd were used to methodologically verify the applied proteomics approach. For each protein, the regulation pattern previously observed by2D-DIGE was successfully reproduced.3. The metabolic profiles of serum and liver tissues from control, ethanol-treated and ethanol-treated with DHP (DHP\ethanol) mice were investigated using a UHPLC/LTQ Orbitrap XL MS combined with SIMCA-P11software analysis. The results showed that compared with control group, there were35metabolites changed significantly in serum in ethaol group, mainly involving amino acids, glycerol phospholipids, sphingolipids, prostaglandins, leukotrienes, purine derivatives, fatty acid metabolism, carbohydrate; there were11metabolites significantly changed in liver tissues, mainly involving vitamins, glycerol phospholipids, fatty amide, prostaglandins and organic phosphate esters. Notably, the levels of phosphocholine and PC (13:0) changed in the same trendency in serum and liver tissues in mice. Those metabolites may be potential characteristic biomarkers for alcoholic liver injury. Compared with ethanol group, DHP (400mg/kg) significantly changed16metabolites in serum, mainly involving amino acids, glycerol phospholipids, carnitine, steroid, fatty amide, peptides and nitro quinoline;3metabolites [namely choline phosphate phosphocholine, LysoPC (20:3) and acetyl phosphate] were significant changed in liver tissues. Notably, DHP significantly lowered increased levels of serum L-proline and carnitine.4. Using Ingenuity Pathway Analysis (IPA) software, protein-and small molecule metabolite-networks regulated by DHP treatment were constructed.Conclusions:1. DHP (200,400mg/kg) can protect the liver function during ethanol-induced subacute injury in mice to different degree. DHP possesses good hepatoprotection action on subacute alcoholic liver injury.2. The proteomics results revealed that the protection action of DHP against sub-acute alcoholic liver injury involved complicated mechanisms and multiple targets, which included MG pathway and methionine pathway.3. The metabolomics results revealed that the metabolic profiles of serum and liver tissues regulated by DHP included fatty acid metabolism, glycerophospholipid metabolism, amino acid metabolism and cytochrome P450metabolism.4. Based on the above two omics techniques, the changed proteins and small molecule metabolites by DHP would provide evidence at molecular level for the effects of DHP on subacute alcoholic liver injury, which will help to systematically explain DHP’s intervention mechanism on subacute alcoholic liver injury and lay the foundation for the further mechanism study on how DHP intervenes in subacute alcoholic liver injury. |
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