The Proteomics And Multi-Parametric MR Characterization Of Liver Fibrosis Mouse Model

Objective:To investigate the growth kinetics,histological changes,protein changes and the main involved pathways of mice with cholestatic liver fibrosis induced by partial bile duct ligation(pBDL).In addition,to investigate the differences of multi-MRI parameters between two different liver fibrosis mouse models(pBDL mouse model and CCl4 mouse model)and sham liver and to determine the diagnostic potential of multiparametric MRI in diagnosing and staging liver fibrosisMethods:Localized(left and middle hepatic lobe)cholestatic liver fibrosis mouse model was established by ligating the confluence of the left and the central hepatic bile duct in mice.The mice in pBDL group were randomly divided into the pure pBDL group and the pBDL+MR group in which mice received repeated MR scans after surgery.Pure pBDL group were randomly selected every 2weeks after operation to evaluate their body weight(g),liver body weight(g)and ligated liver weight percentage(%)to describe growth kinetics.After that,liver tissue sections were taken for H&E,Masson and F4/80 immunohistochemical staining(IHC).Liver fibrosis and inflammation were staged according to the METAVIR and the active inflammation scoring system.Behavioral experiments,including open field test and rotarod test,were performed among all 3 groups.Parameters including the latency to fall(s)from rotarod test and the total distance traveled(mm),velocity in center(mm/s)and duration in center(s)from open field test were recorded.ANAOVA or nonparametric tests were performed to compare theses parameters between 3 groups.The ligated and non-ligated liver from pBDL mice and the liver from Sham mice at the 8 weeks after surgery were used for the proteomics analysis using LC-MS/MS labeled by TMT.GO function annotation enrichment analysis and IPA analysis including: disease function annotation;differential expression protein(DEP);canonical pathways;protein-protein networks and top expression proteins were used for the proteomics.The DEPs of ligated and non-ligated liver were compared to reveal the characteristic changes in cholestatic liver fibrosis.DEPs of nonligated liver and Sham liver were compared to understand the protein changes characteristics in non-ligated liver that under the same system effect with the ligated liver.Key proteins associated with cholestatic liver fibrosis were found using top expression analysis of IPA and were validated using the enzyme-linked adsorption assay(ELISA)technique.BALB/C mice were included and randomly divided into pBDL group,CCl4 group and sham group.pBDL group was randomly divided into 4subgroups based on the postoperative time(2w,4w,6w and 8w).Mice in CCl4 group were divided into 3 subgroups according to the injection time(3w,5w and 7w).The mice in the Sham group were fed until the end of 8weeks.All mice received clinical 3.0 T MR scanning at the end of their prescribed modeling time.Multiparametric sequences including: T1-WI,T2-WI,T1 mapping,T2mapping,T2~*mapping,and multib-value diffusion-weighted imaging.MR parameters were obtained after postprocessing and analysis,including: 1)T1,T2 and T2~* relaxation time from T1 mapping,T2mapping and T2~*mapping images respectively;2)The apparent diffusion coefficient(ADC)from DWI model fitting of diffusion images;3)D,D~* and f values obtained from the diffusion image using the in vivo incoherent motion diffusion-weighted model(IVIM);4)Four different compartmental models(2-compartmentals,3-compartmentals,4-compartmentals and 5-compartmentals)and the signal contributions for each compartments in each models were obtaind from the diffusion images which were analyzed using restricted spectral imaging(RSI)algorithm.ANOVA or nonparametric tests were used to compare the differences of MR parameters between ligated,non-ligated and sham liver in pBDL mouse.T-test or Mann Whitney test were performed to compared MR parameters between CCl4 and Sham liver.Receiver operating curve(ROC)was used to evaluate the diagnostic potential of MR parameters in diagnosing liver fibrosis;Spearman correlation analysis was performed between MR parameters with liver fibrosis stage.Results:None of mouse died during the experiment.The body weight of mice,the total liver weight and the proportion of the ligated liver weighted decreased with the modeling time.Masson staining showed that liver fibrosis increased in the ligated liver and reached to the F3 stage at the end of 8 weeks.From 2w to 8w after surgery,between the pure pBDL group,the pBDL+MR group and the Sham group,the behavioral parameters lack of statistically significance(P>0.05).Totally 5871 proteins were identified in this study by proteomics.Total of 423 DEPs(364 up-regulated,129 down-regulated,P<0.001)between the ligated and non-ligated liver,50 DEPs(20 up-regulated and30 down-regulated,P<0.001)between non-ligated and Sham liver were screened and analyzed.GO functional annotation enrichment showed that DEPs in the ligated liver were mainly related to the dysregulation of various biological processes such as amino acid metabolism and redox processes.IPA analysis supported the above results.In IPA disease function analysis,it was found that the differential proteins of ligated liver were mainly related to diseases such as cancer,endocrine system disorders,and cell dysfunctions such as amino acid and lipid metabolism disorders.The IPA canonical pathways suggest that the ligated liver mainly involved in the activation of Rho GTPases pathway(Z score=5.43),integrin pathway(Z score=5.40)and cholesterol biosynthesis pathway(Z score=3.31),and the inhibition of Rho GTD enzyme pathway(Z score=-4.81)and fatty acid β-oxidation pathway(Z score=-3.63).Among all the differentially expressed proteins in ligated liver,various collagen fibrin(CO15A2,CO124A1,Col1A2 and Col1a1),DCN,OGN,LUM and SPRR1 A were the top up-regulated expression proteins and could be used as the potential key molecules(P<0.001 for all).ELISA test confirmed that the above proteins were significantly up-regulated in cholestatic liver(P<0.05 for all).Regarding to the comparison between non-ligated and sham liver,IPA disease function analysis showed the non-ligated liver were mainly related to the inflammatory-related disesase and processes and were mainly involving the activation of acute phase response pathway(Z sore=2.83)and the inhibition of cholesterol biosynthesis signaling pathway(Z sore=-3.60).T1 and T2 relaxation time were significantly increased(P<0.001 for all)and T2~* relaxation time(P<0.001 for all)were dramatically decreased in both pBDL-ligated liver and CCl4 liver.In the CCl4 liver,the ADC,D and f values were significantly decreased than Sham liver(P <0.05 for all).Regarding to the RSI models,the changes of signal contribution distribution of each compartment was considered to be the characteristics of liver fibrosis.In the CCl4 liver,the signal contribution of the RSI head compartments(C1,or C2)increased significantly while the contribution of tail compartments(C3-C5 component)decreased dramatically.ROC curve showed that the diagnostic performance(AUC,sensitivity and specificity)obtained by the combined the T1,T2 and T2~* relaxation times was significantly higher than that of the three alone.RSI compartmental models obtained higher area under the curve(AUC)than ADC and IVIM parameters(D,D~* and f).The results of correlation analysis showed that T1,T2,T2~* and ADC values were moderately correlated with liver fibrosis,and RSI multi-compartment model was highly correlated with liver fibrosis.Conclusion:pBDL mouse model was confirmed to be a stable and reproducible animal model that can well withstand repeated MRI scanning.Cholestatic hepatic fibrosis involves the dysregulation of various complex biological processes,such as fatty acid and amino acid metabolism disorders,and is associated with significant activation of the Rho pathway and cholesterol biosynthesis pathway,and significant inhibition of the fatty acid β-oxidation pathway.Col1a2 and SPRR1 A may act as the potential key molecules for cholestatic liver fibrosis.T1,T2 and T2~* relaxation times can distinguish liver fibrosis with a moderate correlation with liver fibrosis stage,but the combination of these three can significantly improve the diagnostic efficacy.Each of RSI compartmental models performed better diagnosis potential and relationship than ADC and IVIM parameters.