Isobaric Tags For Relative And Absolute Quantitation(iTRAQ)-Based Proteomics For The Investigation Of The Effect Of Human Qingzhi On Non-alcoholic Fatty Liver Disease In Rats

Background and objective:Nonalcoholic fatty liver disease(NAFLD)is characterized by hepatic fat accumulation in the absence of significant ethanol consumption.In our country,the incidence of NAFLD has markedly increased in recent years;it has become a main cause of liver injury following the virus hepatitis.However,there are no valid therapies for NAFLD in clinical.Thus,there is an urgent need to develop medications for treatment of NAFLD.Hugan Qingzhi tablet(HQT),a Chinese herbal formula,has a long history of use to alleviate NAFLD in clinical practice.It consists of five traditional Chinese medicines,alisma,notoginseng,cattail pollen,the lotus leaf and hawthorn.Our previous study showed that HQT reduced the liver index,serum TG and inflammation response in a NAFLD rat model.Further exploration revealed that HQT ameliotates hepatic steatosis and increases antioxidant capacity by activating adiponectin and downstream targets in HepG2 cells.However,traditional Chinese medicine(TCM)is comprised of multiple components,single pathway is not enough to explain its therapeutic mechanism.The development of proteomics offers a protocol to study the mechanism of traditional Chinese medicine.Isobaric tags for relative and absolute quantification(iTRAQ),developed by Applied Biosystems Incorporation of USA.This technique had shown huge advantaged in through put,specificity,and sensitivity compares with traditional proteomic techniques.To better understand the anti-NAFLD mechanisms of HQT,an iTRAQ-based proteomics approach was performed to study the expression profiles of proteins in high fat diet-induced NAFLD rats following HQT treatment.MethodsRats were randomly divided into 6 groups:HFD(high-fat diet),control(normal diet),fenofibrate(high-fat diet,0.1g/kg)Hugan Qingzhi low/moderate/high dosage(high-fat diet,0.54g/1.08g/2.16g/kg).The corresponding treatments were administered to each group daily and last 12 weeks.After 12 weeks,biochemical markers,liver histology,oxidative stress/antioxidant biomarkers and proinflammatory cytokines were assayed to evaluate the effect of HQT in HFD-induced NAFLD rats.Furthermore,the techniques of iTRAQ labeling,together with strong cation exchange-non-liquid chromatography-tandem mass spectrometry(SCX-non-LC-MS/MS)analysis were used to study the expression profiles of proteins and explore the mechanisms of HQT protective effect against NAFLD rats.Proteomic results were verified by western blotting.Results1.HQT exhibited an obvious therapeutic effect for the prominently decrease of serum ALT,AST,TG and TC as compared to the HFD group.Besides,levels of liver T-AOC,SOD and GSH-Px were significantly increased after treated by HQT.Additionally,HQT decreased the levels of TNF-α,IL-6 and IL-1β compared with HFD group.The histopathological data clearly showed a reduction in lipid accumulation in the liver with HQT treatment.2.After being analyzed by iTRAQ,we identified 275 differentially expressed proteins in HFD group compared to a control group;207 altered proteins in the HQT high dose+HFD group,compared to HFD group;and 316 altered proteins in the HQT high dose+HFD group,compared to the control group.Based on the Kyoto Encyclopedia of Gene and Genomes pathway enrichment analysis,we concluded that several pathways including microbial metabolism in diverse environments,fatty acid metabolism,bile secretion,and peroxisome proliferator activated receptor signaling are closely associated with the effects of HQT in HFD-induced NAFLD rats.3.Several differentially expressed proteins,including PHYH,ACSL1,hemopexin,ORM1,FABP4,Sult2a1 and ASS1 were confirmed by western blotting,and the results were consistent with the data from the proteomics results.ConclusionsHQT has excellent protective effects in HFD-induced NAFLD rats.These effects were associated with reduced lipid and bile acid accumulation,oxidative stress,and inflammation,and the inhibition of ER stress and endotoxin induced hepatic damage.Subsequent validation experiments proved that PHYH,ACSL1,Hemopexin,ORM,FABP4,Sult2a1 and ASS1 might be the targets of HQT,which should be developed as candidates against NAFLD in the future.