We prospectively studied 58 biopsy-proven NAFLD patients (30 with NASH and 28 with simple steatosis) at the Third Affiliated Hospital of Hebei Medical University between July 2008 and October 2010. NAFLD was diagnosed by ultrasound, alanine aminotransferase (ALT) and biopsy, the diagnostic criteria was the guidelines for the diagnosis and treatment of NAFLD suggested by the Chinese National Consensus Workshop on Nonalcoholic Fatty Liver Disease . Exclusion criteria were as follows: a history of excessive alcohol consumption (>140 g/wk for male and >70 g/wk for female), viral hepatitis (hepatitis B and C), alcoholic liver disease, autoimmune hepatitis, primary biliary cirrhosis, primary sclerosing cholangitis, Wilson’s disease, hemochromatosis, α-1 antitrypsin deficiency, biliary obstruction, secondary causes of steatosis, and drug-induced liver disease. Forty-three healthy blood donor volunteers matched for age and sex were recruited as controls: normal liver function, no signs of fatty liver on ultrasound and negative serology for viral hepatitis. Seven liver samples from patients with cavernous hemangioma were also involved in this study. None of the participants had history of primary hypertension, cardiovascular disease, malignancy, hypo- or hyperthyroidism and the use of thiazolidinediones. Previously diagnosed type 2 diabetes patients were also excluded from this cohort.
Written informed consent was obtained from all participants prior to enrollment. And the study protocol was reviewed and approved by the Ethics Committee of the Third Affiliated Hospital of Hebei Medical University.
Clinical and biochemical evaluations
Anthropometric parameters including weight, height, waist circumference, and hip circumference were measured. Body mass index (BMI) was calculated as the body weight (kg) divided by the square of the height (m). Waist-hip ratio (WHR) was calculated as the waist circumference (cm) divided by the hip circumference (cm). A venous blood sample for determination of ALT, aspartate aminotransferase (AST), γ-glutamyl transferase (GGT), glucose, triglyceride (TG), total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C) was collected in each participant following a 12-hour overnight fasting. Serum insulin was determined by a radioimmunoassay technique. The homeostasis model assessment of insulin resistance (HOMA-IR) score was calculated as the product of fasting glucose (mmol/L) and fasting insulin (μIU/mL) divided by 22.5.
Liver tissue collection
Percutaneous liver biopsies were performed in 58 NAFLD patients. A sample was considered valid for the study if it was at least 1.5 cm in length. Since insufficient amount of liver tissues by percutaneous liver biopsies, a few representative liver tissues for western blotting were also obtained in 13 patients with NAFLD (5 with NASH and 8 with simple steatosis) during laparoscopic cholecystectomy due to gallstone. For further comparison, seven samples of relatively normal liver tissue were collected from patients with cavernous hemangioma of the liver during surgical hepatectomy. One third of each specimen was immediately stored in RNA fixer (GENEray Biotechnology, Shanghai, China) and then stored at -80°C until RNA or protein extraction. The remains were fixed in 10% formalin for histology and immunohistochemistry.
The biopsies containing at least six portal tracts were considered appropriate for evaluation. The specimens were stained with hematoxylin-eosin and Masson trichrome. Histology was reviewed by a single hepatopathologist who was blind to the clinical data. The histological scoring of NAFLD followed the NAFLD Activity Score (NAS) proposed by The Pathological Committee of the NASH Clinical Research Network . The score was composed of steatosis (0 = <5%, 1 = 5% - 33%, 2 = 34% - 66%, 3 = >66%), lobular inflammation (0 = no foci, 1 = <2 foci per 200 × field, 2 = 2–4 foci per 200 × field, 3 = >4 foci per 200 × field), and ballooning (0 = none, 1 = rare or few, 2 = many or prominent). Fibrosis staging was recorded as following criteria: 0 = none, 1 = perisinusoidal or periportal fibrosis, 2 = perisinusoidal and portal/periportal fibrosis, 3 = bridging fibrosis, and 4 = cirrhosis. The score of NAS ≥ 5, 2 < NAS <5, NAS ≤ 2 were defined as NASH, “borderline”, and simple steatosis, respectively. The “borderline” patients with NAS 3 or 4 were also identified as NASH under further investigation by original Brunt criteria  (i.e. steatosis together with ballooning and/or Mallory-Denk bodies or fibrosis ≥ 2), while the remainder were regarded as simple steatosis.
Real-time polymerase chain reaction (PCR)
Total RNA was extracted from the RNA fixer-stored specimen using Trizol reagent (Invitrogen, Carlsbad, CA) according to the protocol. The extracted RNA was reverse transcribed to first strand cDNA using the PrimeScript RT reagent Kit (Fermentas, Burlington, ON, Canada). Quantification of the gene expressions were carried out in a thermal cycler (ABI 7500; Applied Biosystems, Foster City, CA) using a SYBR-green RealMaster Mix kit (TianGen Biotech, Beijing, China). The PCR parameters were an initial denaturation at 94°C for 5 min; followed by 30 cycles (94°C for 45 s, 56°C for 45 s, and 72°C for 1 min). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control. The sequences of oligonucleotide primers for PCR included: resistin: forward: 5′-CCA TGG AAG AAG CCA TCA AT-3′and reverse: 5′-CTG GCA GTG ACA TGT GGT CT-3′ (product size: 209 bp); and GAPDH: forward: 5′-ACC ACA GTC CAT GCC ATC ACT-3′ and reverse: 5′-TCC ACC ACC CTG TTG CTG TA-3′ (product size: 452 bp). For each sample, PCR was performed twice in triplicates, and all data were analyzed by the thermal cycler’s software to calculate the △Ct value (△Ct = Ct value of the target gene minus Ct value of the internal control gene). The level of resistin mRNA in healthy controls was assigned as a reference value of 1. Relative expression of resistin to the internal control was calculated using a 2-△△Ct method .
Liver tissues were lysed in RIPA lysis buffer with protease inhibitors according to manufacturer’s instructions (BestBio, China). The concentration of protein was measured by the Bradford method. Equal amounts of proteins (100 μg) were loaded onto 5% condensed gel and 12% SDS-PAGE gel for electrophoresis, followed by western blotting onto polyvinylidene difluoride membranes by semi-dry transfer method (Hoefer TE70X Semi-dry Bloters, USA). The membranes were blocked with 1×TBST containing 5% nonfat milk, and then incubated with both anti-resistin monoclonal antibody (1:2000; Epitomics) and anti-β-actin monoclonal antibody (1:800; Santa Cruz Biotechnology) overnight at 4°C. After washing three times, the blots were detected with Odyssey infrared imaging system (LI-COR, USA). The levels of protein were calculated as the ratio of the intensity of resistin to that of β-actin.
Five μm paraffin-embedded liver sections were digested with trypsinase for 30 min at 37°C, followed by the antigen retrieval via pressure cooking for 2 min in 0.01 mol/L citrate buffer (PH 6.0). After blocking the activity of endogenous peroxidase with 3% methanol-H2O2, all sections were incubated with monoclonal antibody against human resistin (1:100 dilution; R&D Systems, USA) overnight at 4°C. After washing with 0.01 mol/L phosphate-buffered saline (PBS) (PH 7.2), Two-Step IHC Detection Reagent (Zhongshan Golden Bridge Biotech, Beijing, China) containing secondary rabbit anti-mouse antibody, and 3, 3′-Diaminobenzidine tetrahydrochloride Substrate Kit (Zhongshan Golden Bridge Biotech, Beijing, China) were applied according to the manufacturer’s protocols. The slides incubated with PBS instead of primary antibody were used as negative controls. All sections were counterstained with hematoxylin following immunochemical staining. Semi-quantitative (SQ) analysis of the average density of resistin expression was performed using a CMIAS-II morphometric analysor (Beijing University of Aeronautics and Aerospace, China). The average density was calculated by positive staining areas/total areas × 100% in a 400× high-power field (hpf) .
Serum resistin and cytokeratin-18 (CK-18) levels by enzyme-linked immunosorbent assays (ELISA)
Serum levels of resistin and CK-18 fragment were measured by ELISA using commercially available kits (resistin: Rapidbio, West Hills, CA, USA; CK-18: PEVIVA, Alexis, Grunwald, Germany) according to the manufacturer’s instructions. All serum samples were analyzed in duplicates.
All analyses were performed by SPSS version 13.0 (Chicago, IL, USA). All statistical tests were two-tailed, and P < 0.05 was considered to be statistically significant.
Continuous variables were expressed as means ± standard deviation (SD). Differences between groups were analyzed by One-Way ANOVA, followed by Student-Newman-Keuls test for multiple comparisons. Logarithmic transformation of data was performed when appropriate. Pearson Chi-square test or Fisher’s exact test were used to analyze categorical variables. Bivariate correlation analysis was assessed by Pearson correlation test or Spearman correlation test.
Logistic regression analysis with forward stepwise variables selection was used to demonstrate the independent predictors for the histological severity of steatosis, necroinflammation, and fibrosis. The covariates included age, gender, BMI, WHR, ALT, TG, HOMA-IR score, serum resistin, serum CK-18 fragment and positive area of hepatic resistin expression.