Comparative Proteomics Analysis Of Primary Biliary Cirrhosis: Basic And Clinical Research

【Background and Objective】Primary biliary cirrhosis (PBC) is a slowly progressive autoimmune disease of the liver that primarily affects women. Histopathologically, PBC is characterized by portal inflammation and immune-mediated destruction of the intrahepatic bile ducts. The loss of bile ducts leads to decreased bile secretion and the retention of toxic substances within the liver, resulting in further hepatic damage, fibrosis, cirrhosis, and eventually, liver failure. Serologically, PBC is characterized by the presence of antimitochondrial antibodies (AMA), which are present in 90 to 95 percent of patients. But some of the AMA-negative PBC patients will be easily missed diagnosis. Currently, ursodeoxycholic acid (UDCA) is the only drug approved for the treatment of PBC, while a meta-analysis showed no difference between UDCA and placebo in the incidence of liver related death, liver transplantation, and in the development of complications of liver disease.Early diagnosis and early effective intervention of PBC are critical. Proteins are the most important executors to realize versatile functions of life activity, which will dynamically change in disease status. In recent years, proteomics has been expansively applied in many areas, ranging from basic research, various disease and malignant tumors diagnostic and biomarker discovery to therapeutic applications. However, the technology of proteomics has not been applied in the study of PBC so far. Therefore in our study we used the technology of isotope labeling relative and absolute quantification (iTRAQ) with LC/MS or LC-MS/MS to analyze the sera from PBC, HBV related liver fibrosis, HBV related liver cirrhosis and normal control groups, and to screen, identify and verify the differentially expressed proteins of PBC. It may help to illustrate the pathogenesis of PBC and to explore the effective treatments.Establishment of animal model is an important method to study diseases. Several animal models have been set up for PBC study recently, including antigen-induced, chemical-induced and gene knockout-induced models. There are some differences between the models and human PBC on the aspects of the appearance of autoantibody or the changes of serology or pathology in liver tissues. Clinically, we found several hepatitis B patients occurred PBC during the treatment with IFN-αand some studies reported that the level of IFN-αin sera increased in some other autoimmune diseases, such as Rheumatoid Arthritis and Graves'disease. It is supposed that IFN-αmay induce an animal model for PBC. So we applied Poly I:C (a strong inducer of IFN-α) by intraperitoneal injection to set up a PBC mouse model. At the same time, we also applied iTRAQ and LC/MS (LC-MS/MS) to analyze, screen and identify the differentially expressed proteins of sera and liver tissues of the model on different time points.【Methods】1. Establishment of the PBC model by Poly I:C1.1 Eighty adult 6-8 week-old female C57BL/6 mice were maintained separately, under controlled conditions (22℃, 55% humidity, and 12 h day/night).1.2 Poly I: C injection was dissolved in sterilized phosphate-buffered saline (PBS) at a concentration of 1mg/ml and stored at -20℃until needed. Female C57BL/6 mice were injected with poly I: C 5mg/kg of body weight intraperitoneally twice a week for 16 consecutive weeks. As controls, a group of female C57BL/6 mice was injected with PBS according to the poly I: C injection protocol.1.3 Mice were killed by cervical dislocation at different times (4w, 8w, 12w, 16w) after administration of poly I:C or PBS. Liver tissues were collected. Part of liver tissue was fixed in buffered formalin (10%) and Paraffin-embedded, followed by HE staining and CK19 immunohistochemical staining for histological evaluation. Part of serum specimens were examined for biochemistry and autoantibody. The remaining serum and tissue specimens were stored at -80℃until used.2. Proteomics analysis of the serum and liver tissue specimens of the model2.1 Serum specimens were divided into 4 groups (4w, 12w, 16w and PBS control), with the same of liver tissue specimens.2.2 Grinded liver tissues into powder form at liquid nitrogen and then added lysate (7M urea, 2M thiourea, 0.1% PMSF,0.5% DTT) with the concentration of lysate vol: tissue weight (v:w)=5:1 to the samples. Resuspended them fully followed with lysating on the ice for half an hour.2.3 Mix samples in each group respectively and quantitated total proteins in each group by means of Bradford.2.4 Added reducing reagent to 100μg samples and reacted 1 h at 60℃. Cysteine block at room temperature 10 min. Added precooling acetone (V_acetone: V_sample=5:1)and precipitated at -20℃1 h, then samples were centrifugated 12000 rpm 20 min at 4℃to get precipitation. Added dissolution buffer 20μl and resuspended. Trypsin digested overnight. Each group sample was labeled with a different iTRAQTM Reagent(Reagent114,115,116 and 117).Added 70μl ethanol in each tube,incubated 1 h at room temperature and then added 3 times volume H2O to resolve label reagent.Mixed the labeled samples of all thegroups and to freeze drying in vacuum.2.5 The first dimension was to separate the labeled protein samples by SCX. RPLC-MS was the second dimension.The samples were separated by RPLC for 70 min and then scanned by MS(sweeping range was m/z 400-1800)or MS/MS(sweeping range was m/z 100-2000).3.Proteomics analysis of the sera of human PBC3.1 Collect blood from fasting patients and healthy volunteers in the morning. Standing 1h at temperature,then centrifugate at 4℃1400g for 5min to derive the serum.3.2 Serum specimens were divided into 4 groups(control,PBC,HBV related liver fibrosis and HBV related liver cirrhosis).3.3 Mix samples in each group respectively and quantitate total proteins in each group by means of Bradford.3.4 After reduction,cysteine block and digestion,each group sample was labeled with a different iTRAQTM Reagent.3.5 Combine the iTRAQ Reagent-labeled the 4 groups samples digests into one sample mixture.3.6 Clean up or(optionally)perform high resolution fractionation.3.7 Analyze the mixture by LC-MS/MS for protein identification and quantitation.3.8 Verify the differentially expressed proteins of human PBC in serum by ELISA or Western blot.【Results】1.The effects of poly I:C induced PBC model1.1 We examined ALP among the 4 groups and found that the level of ALP in modelgroups increased with time and at 16th week it had significant difference between the model and control group.1.2 We examined AMA among the 4 groups and found that AMA appeared in 4 mice in 4th week model group and its positive rate increased with time.In 16 th week model group there were 16 mice with AMA positive(positive rate=80%)in serum.While it last negativity in control group.1.3 The level of ANA in serum was examined in the model group and control group at different time point sequentially.At 4th week,ANA was detected positively in either model group or control group with lower titer.From 8th week to 16th week,the positive rate of ANA in model group maintained 100%and the titer at each time point was higher than the corresponding control group,respectively.We also found that the titer of ANA decreased after 8th week in model group.1.4 With light microscope detection of liver pathology,we found that lymphocytes infiltration in the liver tissues was low in 4th week model roup;however,considerablenumbers of infiltrating cells were detected around intrahepatic bile ducts in 8th week model group.After this time point,the extent of the infiltrating cells progressively increased.In 16th week model group,numerous of inflammatory cells were observed not only around intrahepatic bile ducts but also in the portal areas.Some infiltrating cells accumulated around the damaged bile ducts.Contrast to model groups,we found little inflammatorycells infiltration around intrahepatic bile ducts or in the portal areas.Meanwhile,we found no characteristic of liver fibrosis.1.5 With light microscope detection after CK19 immunohistochemical staining,wefound that CK19 positive expression rate in the liver tissues of model group was significant higher than it in the control group.And the trend of the expression of CK19 was increased with the time.2.Proteomics analysis of the serum and liver tissue specimens of the model2.1 We identified 86 proteins in the serum in the model of PBC.Contrast to the normal group,there were 5 proteins up-regulated significantly,while 5 proteins down-regulated significantly in the 16th week model group.The differentially expressed proteins are related to lipid metabolism,immune response,cytoskeleton and enzymes.2.2 519 proteins were identified in the tissues of the PBC model.Contrast to the normal group,there were 35 proteins up-regulated significantly,while 29 proteins down-regulated significantly in the 16th week model group.The differentially expressed proteins are related to carrier protein,immune response,energy etabolism,enzymes,cell growth,cell adhesion and movement.2.3 Through a comparative analysis of the differential expression proteins in the liver tissues of the different time points model groups with the control group,we have identified 34 proteins had a certain degree of correlation with the modeling time.Of these, 17 proteins in the model group were persistently high expression,and with the modeling time,and their expression level gradually increased.The functions included energy metabolism, cell growth and differentiation regulation, transportation and ion channels, etc. From the expression of proteins in the cell location of view, they were mainly located in mitochondria, followed by plasma membrane, nucleus and cytoplasm. 17 proteins in the model group were persistently low expression, and with the modeling time, and their expression level gradually decreased. The functions included enzymes, cytoskeleton, energy metabolism, cell growth and differentiation regulation, etc. From the expression of proteins in the cell location of view, they were mainly located in the cytoplasm, followed by plasma membrane, extracellular matrix, mitochondria and nuclei.2.4 We found that (i) most of the significant high expression proteins in the serum of model groups were related to the immune function closely; (ii) most of the significant low expression proteins in the serum of model groups were related to lipid transportation and metabolism; (iii) most of the significant high expression proteins in the liver tissues of model groups were located in the mitochondria and related to energy metabolism; (iv) most of the significant low expression proteins in the liver tissues of model groups were located in the cytoplasm and related to enzymes. These results were consistent with the histopathological and serum biochemical test results we carried out before, and were similar to the performance of human PBC.3. Proteomics analysis of the sera of human PBC3.1 We identified 93 proteins successfully in serum of people with the function of immune response, transportation, lipid metabolism, cell adhesion and movement, coagulation, enzyme inhibitors, enzymes, cell growth and differentiation regulation, cytoskeleton, energy metabolism. 3.2 30 of them were significantly different between PBC and healthy people. 14 proteins of them, such as LYVE1, IGHM, RBP4 and AZGP1, were up-regulated significantly in PBC patients, while the other 16 proteins, such as ApoB, ApoA2, ApoA1, ApoC-III, SERPINF2 and ApoM, were down-regulated significantly. These differential expression proteins were related to lipid metabolism, transportation and storage, cell adhesion and movement, immune response, coagulation, enzymes and enzyme inhibitors.3.3 Comparison of PBC patients and HBV related liver fibrosis and liver cirrhosis patients, we found a total of 9 marked differential expression proteins. Five of them, such as FN1, GC, LYVE1, A2M and AGT, were lower expressed in PBC patients, while LYVE1 was higher expressed than it was in healthy control. The remaining four proteins, including A1BG, ApoC-III, ApoC-II and IGHM, were higher expressed in PBC patients, while A1BG and ApoC-III were lower than they were in healthy control.3.4 By using the technology of western blot and ELISA, we validated the level of LYVE-1, AZGP-1 and RBP-4, and got the accordant results with the iTRAQ and LC-MS/MS.【Conclusion】1. By intraperitoneal injection of poly I:C, we established an animal model of PBC, which was similar to the human PBC on the characteristic of live pathology, autoantibody and biochemistry of serum.2. The technology of iTRAQ combining with LC-MS/MS is an efficient way to distinguish and identify the differential expression proteins in diseases. 3. There were significant differences on the aspect of protein expression in serum and liver tissues between PBC model group and control group. We identified that most of the differential expression proteins were involved with immune response, lipid metabolism, energy metabolism, cell adhesion and movement. The level of some differential expression proteins showed a significant positive correlation or negative correlation with the modeling time. It needs a further study of functional proteomics for these findings to explain the mechanism of this PBC model.4. Comparison with healthy people, patients of HBV related liver fibrosis or HBV related liver cirrhosis, we found that there were significant differentially expressed proteins in the serum of PBC, most of which were involved with lipid transportation, lipid metabolism, extracellular matrix, and so on. Some of the proteins we found might be related to the pathogenesis of PBC and should be explored further. 5. In the candidate proteins we found, the apolipoprotein family seems to be important to PBC, because of its decrease in both of PBC and its model.6. In this study, by comparing the physiological and different pathological conditions of different protein components of the change in expression level, we detected and identified some disease-related proteins (group), especially Ig family and apolipoprotein family. It may be explored to the search for potential drug targets, as well as early diagnosis, treatment, molecular markers, but also for the study of the pathogenesis of PBC.