Inhibiting Hepatocyte Uric Acid Synthesis and Reabsorption Ameliorates Acetaminophen-Induced Acute Liver Injury in Mice.

BACKGROUND & AIMS: Acetaminophen (APAP) overdose is the most common cause of drug-induced liver injury worldwide. Uric acid (UA) is involved in sterile inflammation in many organs, but its role in APAP-induced liver injury remains elusive. METHODS: We quantified the concentration of UA in the serum and liver tissues of APAP-overdosed mice and explored the changes in proteins involved in UA synthesis, absorption, and degeneration on APAP stimulation. We also examined the effects of inhibiting hepatocyte UA synthesis or reabsorption on APAP-induced liver injury in mice. Furthermore, we explored the process of UA clearance by peripheral macrophages. RESULTS: APAP overdose significantly increased intrahepatic UA contents, which occurred earlier than apparent hepatocyte injury in APAP-overdosed mice. APAP overdose induced significant DNA leakage and may thereby increase the substrate of UA synthesis. APAP overdose also significantly increased the enzymatic activity of xanthine oxidase and urate oxidase and decreased the expression of the UA reabsorption transporter GLUT9 in hepatocytes. Inhibiting hepatocyte UA synthesis by febuxostat or reabsorption by hepatic-specific knockout of GLUT9 alleviated APAP-induced liver injury. Further experiments showed that monosodium urate but not soluble UA may be a major form of UA mediating hepatocyte injury. Additionally, monosodium urate further recruited circulating macrophages into the liver and then aggravated inflammation by increasing the levels of inflammatory factors and reactive oxygen species. Deletion of macrophages significantly ameliorated APAP-induced liver injury in mice. CONCLUSIONS: APAP overdose induces excessive UA production and leads to local high concentrations in the liver, which further injures cells and induces liver inflammation. Inhibiting the production of UA may be a potential therapeutic option for treating APAP-induced liver injury.

Gastroduodenal strongyloidiasis infection causing protein-losing enteropathy: A case report and review of the literature.

Strongyloides stercoralis is an intestinal nematode in which adult worms in the host small intestine can cause strongyloidiasis. Symptoms in immunocompromised patients might range from diarrhea and bleeding to sepsis and even death. A 56-year-old patient presented with a 2-month history of weight loss, vomiting, and diarrhea. The patient had type 2 diabetes mellitus (T2DM) and was on long-term prednisone for chronic kidney disease. The results of the gastric emptying test and head magnetic resonance (MRI) were normal. A blood test revealed increased IgE levels, eosinophilia, and hypoalbuminemia. Parasitic larvae were not discovered during stool analysis. Gastroscopy revealed chronic nonatrophic gastritis with erosions and dilation of lymphatic vessels of the duodenum. Small-bowel capsule endoscopy suggested dilation of lymphatic vessels of the small intestine. Colonoscopy revealed no abnormalities. Finally, a histopathology examination identified S. stercoralis pervasion in the gastric antrum and duodenum. The patient was treated with albendazole and discharged successfully. In conclusion, we discovered S. stercoralis as a cause of protein-losing enteropathy in a patient with a long-term oral corticosteroid therapy and T2DM. The diagnosis was made through histopathology, once parasitological examination was negative. Therefore, health professionals should stay alert to S. stercoralis infection in immunocompromised patients with vague gastrointestinal symptoms. More sensitive methods should be applied in the diagnosis.

Endoscopic mucosal resection-precutting vs conventional endoscopic mucosal resection for sessile colorectal polyps sized 10-20 mm.

BACKGROUND: The optimal method to remove sessile colorectal lesions sized 10-20 mm remains uncertain. Piecemeal and incomplete resection are major limitations in current practice, such as endoscopic mucosal resection (EMR) and cold or hot snare polypectomy. Recently, EMR with circumferential precutting (EMR-P) has emerged as an effective technique, but the quality of current evidence in comparative studies of conventional EMR (CEMR) and EMR-P is limited. AIM: To investigate whether EMR-P is superior to CEMR in removing sessile colorectal polyps. METHODS: This multicenter randomized controlled trial involved seven medical institutions in China. Patients with colorectal polyps sized 10-20 mm were enrolled and randomly assigned to undergo EMR-P or CEMR. EMR-P was performed following submucosal injection, and a circumferential mucosa incision (precutting) was conducted using a snare tip. Primary outcomes included a comparison of the rates of en bloc and R0 resection, defined as one-piece resection and one-piece resection with histologically assessed clear margins, respectively. RESULTS: A total of 110 patients in the EMR-P group and 110 patients in the CEMR group were finally evaluated. In the per-protocol analysis, the proportion of en bloc resections was 94.3% [95% confidence interval (CI): 88.2%-97.4%] in the EMR-P group and 86% (95%CI: 78.2%-91.3%) in the CEMR group (P = 0.041), while subgroup analysis showed that for lesions > 15 mm, EMR-P also resulted in a higher en bloc resection rate (92.0% vs 58.8% P = 0.029). The proportion of R0 resections was 81.1% (95%CI: 72.6%-87.4%) in the EMR-P group and 76.6% (95%CI: 68.8%-84.4%) in the CEMR group (P = 0.521). The EMR-P group showed a longer median procedure time (6.4 vs 3.0 min; P < 0.001). No significant difference was found in the proportion of patients with adverse events (EMR-P: 9.1%; CEMR: 6.4%; P = 0.449). CONCLUSION: In this study, EMR-P served as an alternative to CEMR for removing nonpedunculated colorectal polyps sized 10-20 mm, particularly polyps > 15 mm in diameter, with higher R0 and en bloc resection rates and without increasing adverse events. However, EMR-P required a relatively longer procedure time than CEMR. Considering its potential benefits for en bloc and R0 resection, EMR-P may be a promising technique in colorectal polyp resection.

Silencing of functional p53 attenuates NAFLD by promoting HMGB1-related autophagy induction.

BACKGROUND AND AIM: Nonalcoholic fatty liver disease (NAFLD) is a common chronic liver disease worldwide, but its pathogenesis remains imprecisely understood and requires further clarification. Recently, the tumor suppressor p53 has received growing attention for its role in metabolic diseases. In this study, we performed in vivo and in vitro experiments to identify the contribution of p53-autophagy regulation to NAFLD. METHODS: Livers from wild-type and p53 knockout mice as well as p53-functional HepG2 cells and p53-dysfunctional Huh7 cells were examined for autophagy status and HMGB1 translocation. In vivo and in vitro NAFLD models were established, and steatosis was detected. In the cell models, autophagy status and steatosis were examined by p53 and/or HMGB1 silencing. RESULTS: First, the silencing of p53 could induce autophagy both in vivo and in vitro. In addition, p53 knockout attenuated high-fat diet-induced NAFLD in mice. Similarly, knockdown of p53 could alleviate palmitate-induced lipid accumulation in cell models. Furthermore, high mobility group box 1 (HMGB1) was proven to contribute to the effect of silencing p53 on alleviating NAFLD in vitro as an autophagy regulator. CONCLUSION: The anti-NAFLD effect of functional p53 silencing is associated with the HMGB1-mediated induction of autophagy.

Convolutional neural network for the diagnosis of early gastric cancer based on magnifying narrow band imaging.

BACKGROUND: Magnifying endoscopy with narrow band imaging (M-NBI) has been applied to examine early gastric cancer by observing microvascular architecture and microsurface structure of gastric mucosal lesions. However, the diagnostic efficacy of non-experts in differentiating early gastric cancer from non-cancerous lesions by M-NBI remained far from satisfactory. In this study, we developed a new system based on convolutional neural network (CNN) to analyze gastric mucosal lesions observed by M-NBI. METHODS: A total of 386 images of non-cancerous lesions and 1702 images of early gastric cancer were collected to train and establish a CNN model (Inception-v3). Then a total of 341 endoscopic images (171 non-cancerous lesions and 170 early gastric cancer) were selected to evaluate the diagnostic capabilities of CNN and endoscopists. Primary outcome measures included diagnostic accuracy, sensitivity, specificity, positive predictive value, and negative predictive value. RESULTS: The sensitivity, specificity, and accuracy of CNN system in the diagnosis of early gastric cancer were 91.18%, 90.64%, and 90.91%, respectively. No significant difference was spotted in the specificity and accuracy of diagnosis between CNN and experts. However, the diagnostic sensitivity of CNN was significantly higher than that of the experts. Furthermore, the diagnostic sensitivity, specificity and accuracy of CNN were significantly higher than those of the non-experts. CONCLUSIONS: Our CNN system showed high accuracy, sensitivity and specificity in the diagnosis of early gastric cancer. It is anticipated that more progress will be made in optimization of the CNN diagnostic system and further development of artificial intelligence in the medical field.

Expression of factors and key components associated with the PI3K signaling pathway in colon cancer.

The pathophysiology of colorectal cancer (CRC) has not been fully elucidated. The dysregulation of the phosphatidylinositol 3-kinase (PI3K) signaling pathway frequently contributes to the tumorigenesis and progression of human cancer. The aim of the present study was to explore the expression and clinical significance of a number of associated factors and key components of the PI3K signaling pathway, including phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (p110α), phosphorylated protein kinase B (p-Akt) Ser473, p-mammalian target of rapamycin (mTOR) Ser2448, cyclin D1, cyclin dependent kinase (CDK)4, RELA proto-oncogene, nuclear factor-κß subunit (p65), Ras and extracellular signal-regulated kinase (ERK)1/2 in human CRC. The expression of target proteins was detected using immunohistochemistry (IHC) in 65 CRC cases and 15 colonic adenoma cases. The association between the expression of target proteins and clinical pathological parameters was analyzed using a χ2 test. IHC results revealed that the expression of all target proteins was significantly increased in CRC tissues compared with in colonic adenoma tissues (P<0.05). No significant associations were observed between the expression of p110α, p-Akt Ser473, p-mTOR Ser2448 and sex, age, differentiation, lymph node metastasis or Tumor-Node-Metastasis staging (P>0.05). Cyclin D1, CDK4 and Ras were revealed to be expressed significantly higher in poorly differentiated CRC compared with moderately differentiated CRC (P<0.05). Expression of p65 and ERK1/2 were significantly increased in cancer tissues with lymph node metastasis compared with cancer tissues without lymph node metastasis (P<0.05). These results suggest that the target proteins may all participate in the tumorigenesis of CRC. Furthermore, cyclin D1, CDK4, Ras, p65 and ERK1/2 may be important in the progression of CRC. The results of the present study may provide novel predictive factors and therapeutic targets for CRC.

Targeted inhibition of the phosphoinositide 3-kinase impairs cell proliferation, survival, and invasion in colon cancer.

BACKGROUND: Colon cancer is the third most common cancer in the world, and its metastasis and drug resistance are challenging for its effective treatment. The PI3K/Akt/mTOR pathway plays a crucial role in the pathogenesis of colon cancer. The aim of this study was to investigate the targeting of PI3K in colon cancer cells HT-29 and HCT-116 in vitro. METHODS: In HT-29 and HCT-116 cells, BEZ235, a dual inhibitor of PI3K/mTOR, and shRNAtarget to PI3KCA were used to inhibit PI3K/Akt/mTOR pathway. The inhibition efficiency of PI3K/Akt/mTOR pathway was detected by RT-PCR and Western blot. Cell proliferation, migration, invasion, and apoptosis were evaluated by Cell Counting Kit-8, Transwell, and flow cytometry assays. The expression of apoptosis-related proteins (cleavage caspase 3, Bcl-2, Bax, and Bim) were also detected. RESULTS: We found that in HT-29 and HCT-116 cells, the treatment of BEZ235 (1 µM) and PI3KCA knockdown inhibited the activation of PI3K/Akt/mTOR pathway and significantly suppressed cell proliferation, migration, and invasion of HT-29 and HCT-116 cells. In addition, we confirmed that knockdown of BEZ235 and PI3KCA induced cell apoptosis through the upregulated levels of cleavage caspase 3 and Bax and downregulated expression of Bcl-2 and Bim. CONCLUSION: Our results indicated that targeted inhibition of the PI3K/Akt/mTOR pathway impaired cell proliferation, survival, and invasion in human colon cancer.

PDIA3 Knockdown Exacerbates Free Fatty Acid-Induced Hepatocyte Steatosis and Apoptosis.

Nonalcoholic fatty liver disease (NAFLD) has emerged as one of the most common chronic liver disease over the past decades. Endoplasmic reticulum stress (ERS) plays a pivotal role during the development of NAFLD. This study aims to analyze the potential role of protein disulfide isomerase A3 precursor (PDIA3), one of the ER chaperones, in free fatty acid-induced cell model of NAFLD. Human liver L02 cell line was treated with sodium palmitate for 24 hours, which developed severe intracellular lipid accumulation. The increased protein level of PDIA3 was detected via immunoblotting analysis in the fat loaded cell models of NAFLD. siRNA-mediated knockdown of PDIA3 in L02 cells not only increased the cellular lipid accumulation, but also exacerbated hepatocytes apoptosis induced by sodium palmitate. Further investigation revealed that knockdown of PDIA3 up-regulated protein expression of fatty acid synthase (FAS), a key enzyme involved in fatty acid synthesis. PDIA3 knockdown also up-regulated key molecules of ERS pathway, including glucose-regulated protein 78 (GRP78), phospho-PKR-like ER kinase (p-PERK), and C/EBP homologous protein (CHOP). Our results suggested that ER chaperone PDIA3 plays a pivotal role in FFA-induced hepatocyte steatosis and apoptosis.

A role of microRNA-370 in hepatic ischaemia-reperfusion injury by targeting transforming growth factor-ß receptor II.

BACKGROUND & AIMS: MicroRNAs (miRNAs) are a group of small non-coding RNAs with modulator activity of gene expression. The role of miRNAs in hepatic ischaemia-reperfusion (IR) injury is currently largely unknown. The aim of this study was to investigate the potential role of miR-370 in hepatic IR injury. METHODS: The expression levels of hepatic miR-370 in male C57BL/6 mice subjected to hepatic IR injury or ischaemia preconditioning were assessed by quantitative real-time PCR. The effect of miR-370 on hepatic IR injury was investigated by serum enzyme analysis and histological examination of liver following treatment of mice with antagomir-370 or control. The levels of proinflammatory cytokines and apoptosis- and proliferation-related genes were also determined by quantitative real-time PCR. Furthermore, the potential targets of miR-370 in this injury were studied by bioinformatics analysis, luciferase assays, quantitative real-time PCR and Western blot. RESULTS: The results showed that miR-370 expression was significantly upregulated in the mice subjected to hepatic IR injury as compared with the sham-operated mice. Inhibition of miR-370 led to the downregulation of serum aminotransferase and proinflammatory cytokines, as well as the improvement of hepatic histological damage. Reporter assays confirmed that miR-370 directly targeted the 3' untranslated region of transforming growth factor-ß receptor II (TßRII). Inhibition of miR-370 was sufficient to reinstate the expression of TßRII and its downstream target phosphorylated Smad3. CONCLUSION: Our data suggest that miR-370 acting via TßRII might play a potential role in hepatic IR injury, and inhibition of miR-370 efficiently attenuated the damage to the liver.

Proteomic analysis of liver mitochondria from rats with nonalcoholic steatohepatitis.

AIM: To explore mitochondrial dysfunction in nonalcoholic steatohepatitis (NASH) by analyzing the proteome of liver mitochondria from a NASH model. METHODS: The NASH rat model was established by feeding rats a fat-rich diet for 24 wk and was confirmed using hematoxylin and eosin staining of liver tissue and by changes in the levels of serum alanine transaminase, aspartate aminotransferase, triglyceride, total cholesterol and other markers. Liver mitochondria from each group were isolated using differential centrifugation. The mitochondrial samples were lyzed, purified and further analyzed using two-dimensional electrophoresis combined with matrix-assisted laser desorption/ionization tandem time-of-flight mass spectrometry. Bioinformatic analyses of assigned gene ontology and biological pathway was used to study functional enrichments in the abundant proteomic data. RESULTS: Eight up-regulated and sixteen down-regulated proteins were identified that showed greater than 1.5-fold differences between the controls and the NASH group. These dysregulated proteins were predicted to be involved in different metabolic processes including fatty acid ß-oxidation processes, lipid metabolic processes, cell-cycle arrest, cell polarity maintenance, and adenosine triphosphate/sex hormone metabolic processes. Novel proteins that may be involved in NASH pathogenesis including the trifunctional enzyme Hadha, thyroxine, prohibitin, aldehyde dehydrogenase ALDH1L2, UDP-glucuronosyltransferase 2B31, and carbamoyl-phosphate synthase were identified using bioinformatics tools. The decreased expression of Hadha in NASH liver was verified by Western blotting, which was used as a complementary technique to confirm the proteomic results. CONCLUSION: This novel report on the liver mitochondrial proteome of a NASH model may provide a reservoir of information on the pathogenesis and treatment of NASH.

Role of endoplasmic reticulum stress in the pathogenesis of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD) has emerged as a common public health problem in recent decades. However, the underlying mechanisms leading to the development of NAFLD are not fully understood. The endoplasmic reticulum (ER) stress response has recently been proposed to play a crucial role in both the development of steatosis and progression to nonalcoholic steatohepatitis. ER stress is activated to regulate protein synthesis and restore homeostatic equilibrium when the cell is stressed due to the accumulation of unfolded or misfolded proteins. However, delayed or insufficient responses to ER stress may turn physiological mechanisms into pathological consequences, including fat accumulation, insulin resistance, inflammation, and apoptosis, all of which play important roles in the pathogenesis of NAFLD. Therefore, understanding the role of ER stress in the pathogenesis of NAFLD has become a topic of intense investigation. This review highlights the recent findings linking ER stress signaling pathways to the pathogenesis of NAFLD.

Association between serum growth hormone levels and nonalcoholic fatty liver disease: a cross-sectional study.

Growth hormone (GH) is an important regulator of metabolism and body composition. GH deficiency is associated with increased visceral body fat and other features of the metabolic syndrome. Here we performed a cross-sectional study to explore the association of GH levels with nonalcoholic fatty liver disease (NAFLD), which is considered to be the hepatic manifestation of the metabolic syndrome. A total of 1,667 subjects were diagnosed as NAFLD according the diagnostic criteria, and 5,479 subjects were defined as the controls. The subjects with NAFLD had significantly lower levels of serum GH than the controls. Those with low GH levels had a higher prevalence of NAFLD and the metabolic syndrome. A stepwise logistic regression analysis showed that GH levels were significantly associated with the risk factor for NAFLD (OR = 0.651, 95%CI = 0.574-0.738, P<0.001). Our results showed a significant association between lower serum GH levels and NAFLD.

Functional proteomic analysis of nonalcoholic fatty liver disease in rat models: enoyl-coenzyme a hydratase down-regulation exacerbates hepatic steatosis.

UNLABELLED: Nonalcoholic fatty liver disease (NAFLD) has emerged as a common public health problem that can progress to end-stage liver disease. A high-fat diet (HFD) may promote the development of NAFLD through a mechanism that is poorly understood. We adopted a proteomic approach to examine the effect of HFD on the liver proteome during the progression of NAFLD. Male Sprague-Dawley rats fed an HFD for 4, 12, and 24 weeks replicated the progression of human NAFLD: steatosis, nonspecific inflammation, and steatohepatitis. Using two-dimensional difference gel electrophoresis (DIGE) combined with matrix-assisted laser desorption ionization time of flight/time of flight analysis, 95 proteins exhibiting significant changes (ratio > or = 1.5 or < or =-1.5, P < 0.05) during the development of NAFLD were identified. Biological functions for these proteins reflected phase-specific characteristics during the progression of the disease. The potential role of enoyl-coenzyme A hydratase (ECHS1), an enzyme that catalyzes the second step of mitochondrial fatty acid beta-oxidation, received further investigation. First, the reduced protein level of ECHS1 was validated both in rat models and in patients with biopsy-proven hepatic simple steatosis via immunoblotting or immunohistochemical analysis. Then the small interfering RNA (siRNA)-mediated knockdown of ECHS1 in the murine hepatocyte cell line alpha mouse liver 12 (AML12) demonstrated increased cellular lipid accumulation induced by free fatty acid (FFA) overload. Furthermore, using a hydradynamic transfection method, the in vivo silencing effect of siRNA duplexes targeting ECHS1 was further investigated in mice. Administering ECHS1 siRNA specifically reduced the expression of ECHS1 protein in mice liver, which significantly exacerbated the hepatic steatosis induced by an HFD. CONCLUSION: Our results revealed that ECHS1 down-regulation contributed to HFD-induced hepatic steatosis, which may help clarify the pathogenesis of NAFLD and point to potential targets for therapeutic interventions.

Liverbase: a comprehensive view of human liver biology.

The Liverbase ( http://liverbase.hupo.org.cn ) integrates information on the human liver proteome, including the function, abundance, and subcellular localization of proteins as well as associated disease information. The overall objective of the Liverbase is to provide a unique public resource for the liver community by providing comprehensive functional annotation of proteins implicated in liver development and disease. The central database features are manually annotated proteins localized in or functionally associated with human liver. In this first version of Liverbase, the associated data includes the human liver proteome (6788 proteins) and transcriptome (11205 significantly expressed genes: 10224 from CHIP and 5422 from MPSS, respecively) from the Chinese human liver proteome project (CNHLPP). As a database made publicly available through the Web site, Liverbase provides browsing and searching capabilities and a compilation of external links to other databases and homepages. Liverbase enables (i) the establishment of liver GO slim with 51 nonredundant items; (ii) systematic searches for proteins within specific functional or metabolic pathways; (iii) systematic searches that aim to find the proteins that underlie common and rare liver diseases; and (iv) the integration of detailed protein annotations derived from the literature. Liverbase also contains an external links page with links to other biological databases and homepages, including GO, KEGG, pfam, SWISS-PROT, and GNF databases. Liverbase users can utilize all these information to conduct systems biology research on liver.

Relationships between hematopoiesis and hepatogenesis in the midtrimester fetal liver characterized by dynamic transcriptomic and proteomic profiles.

In fetal hematopoietic organs, the switch from hematopoiesis is hypothesized to be a critical time point for organogenesis, but it is not yet evidenced. The transient coexistence of hematopoiesis will be useful to understand the development of fetal liver (FL) around this time and its relationship to hematopoiesis. Here, the temporal and the comparative transcriptomic and proteomic profiles were observed during the critical time points corresponding to the initiation (E11.5), peak (E14.5), recession (E15.5), and disappearance (3 ddp) of mouse FL hematopoiesis. We found that E11.5-E14.5 corresponds to a FL hematopoietic expansion phase with distinct molecular features, including the expression of new transcription factors, many of which are novel KRAB (Kruppel-associated box)-containing zinc finger proteins. This time period is also characterized by extensive depression of some liver functions, especially catabolism/utilization, immune and defense, classical complement cascades, and intrinsic blood coagulation. Instead, the other liver functions increased, such as xenobiotic and sterol metabolism, synthesis of carbohydrate and glycan, the alternate and lectin complement cascades and extrinsic blood coagulation, and etc. Strikingly, all of the liver functions were significantly increased at E14.5-E15.5 and thereafter, and the depression of the key pathways attributes to build the hematopoietic microenvironment. These findings signal hematopoiesis emigration is the key to open the door of liver maturation.

Proteomic analysis of hepatic ischemia/reperfusion injury and ischemic preconditioning in mice revealed the protective role of ATP5beta.

Hepatic ischemia/reperfusion (I/R) injury is an inevitable consequence during liver surgery. Ischemic preconditioning (IPC) has been shown to protect the livers from I/R injury, partially mediated by preservation of hepatic ATP contents. However, the precise molecular mechanisms of these events remain poorly elucidated. In this study, liver proteomes of the mice subjected to I/R injury pretreated with or without IPC were analyzed using 2-DE combined with MALDI-TOF/TOF mass analysis. Twenty proteins showing more than 1.5-fold difference were identified in the livers upon I/R injury. Among these proteins, four proteins were further regulated by IPC when compared with nonpretreated controls. One of these proteins, ATP synthase beta subunit (ATP5beta) catalyzes the rate-limiting step of ATP formation. The expression level of ATP5beta, which was further validated by Western blot analysis, was significantly decreased upon I/R injury while turned over by IPC pretreatment. Change pattern of hepatic ATP corresponded with that of ATP5beta expression, indicating that increasing hepatic ATP5beta expression might be a reason for ATP-preserving effect of IPC. In summary, this study provided new clues for understanding the mechanisms of IPC against I/R injury. The protective role of ATP5beta might give evidences for developing new therapeutic approaches against hepatic I/R injury.

Proteomic analysis of individual variation in normal livers of human beings using difference gel electrophoresis.

Normal Chinese Liver Proteome Expression Profile is one of the major parts of Human Liver Proteome Project. Before starting the studies, it is necessary to examine the interindividual variation of normal liver proteome and evaluate the minimal size of samples for proteomic analysis. In this study, normal liver samples from ten individual volunteers were collected and the proteome profiles of these samples were analyzed using 2-D difference gel electrophoresis (DIGE) combined with MALDI-TOF/TOF MS. The individual liver tissue lysates were labeled with Cy3 and Cy5 while the pooled sample was labeled with Cy2 as an internal standard, which minimized gel-to-gel variation. After analysis by the DeCyder software, up to 2056 protein spots were detected on the master gel. The CV of standardized abundance was calculated for the protein spots that were matched across all ten gels. The CV values of these protein spots ranged from 6.4 to 108.5% and the median CV was approximately 19%, which demonstrated that the protein expression of normal liver among different individuals was relatively stable. The eight proteins with CV values over 50% were identified which would be a caveat when considering these proteins as potential disease-related markers. Moreover, the one-way ANOVA feature showed a correlation between sample size and individual variations. The results showed that when the sample size exceeded 7, the individual variations were not significant to the whole pool. Our results are an important basis for liver protein expression profiles and comparative proteomics of liver disease.