Expression And Significance Of Adiponectin And Its Receptor In Liver Tissue Of Patients With Different Types Of Chronic Hepatitis B

Objective: Hepatitis B virus(HBV)infection was prevalent worldwide. HBV infection related liver cirrhosis, liver failure and hepatocelluler carcinoma are responsible for over one million death per year. It was suggested that there are a variety of inflammatory processes characterized by the imbalance of pro-inflammatory and anti-inflammatory cytokines in the chronic hepatitis B (CHB) progression. Adiponectin, which was discovered recently, is the only known adipocytokine with protective effect. Its mainly role is to regulate energy metabolism, to increase insulin sensitivity, anti-inflammatory and anti-hepatic fibrosis. It was confirmed that serum adiponectin levels reduced in non-alcoholic steatohepatitis patients, and associated with liver inflammation, steatotis and fibrosis. However, adiponectin with chronic HBV infection, adiponectin with progression or outcome of CHB patients have no precise reports.The levels of adiponectin in peripheral blood, adiponectin and adiponectin receptor 2 in liver tissues of various types chronic hepatitis B patients were monitored,to explore the changes and clinical significance of adiponectin and adiponectin receptor 2 in patients with different types of chronic hepatitis BMethods: Patients in our hospital from January 2009 to December 2010 were enrolled in this study, including 20 patients with CHB, 20 patients with liver cirrhosis(LC), 20 patients with chronic severe hepatitis B(CSHB). The diagnoses complied with the Chinese Management of Chronic Hepatitis B in 2005 and Diagnostic and treatment guidelines for liver failure in 2006. 20 healthy individuals were chosen as controls.Serum fasting blood glucose (FBG), cholesterol(TC), triglyceride(TG), albumin, alanine aminotransferase(ALT), total bilirubin(TBiL) and prothrombin activity(PTA)were recorded by using an Olympus AU2700 auto-biochemical analyzer. HBV markers: HBsAg, HBsAb, HBeAg, HBeAb, HBcAb, anti-HAV, anti-HCV, anti-HEV and anti-HIV were detected by various commercial enzyme immunoassay kits. To exclude other hepatotropic viruses and HIV infections, the expression of HBVDNA was checked by RT-PCR.Peripheral heparinized blood samples were collected from all the enrolled individuals. The levels of adiponectin were recorded by Fnzyme Linked Immunosorbent Assay(ELSA).Haematoxylin-eosin (HE) and Masson staining were used for the general pathologic changes of liver and hepatic fibrosis respectively. Immunohistochemical staining (Power VisionTM method) was used to measure the expression of adiponectin and adiponectin receptor 2. Positive expression was brownish yellow. The expression of adiponectin and adiponectin receptor 2 were calculated by a multifunctional pathological image analyzer. Five areas from the center and periphery of each section were randomly chosen to calculate the average area density (the percentage of positive area to statistical one) observed under 20 power object lens.The expressions of adiponectin and adiponectin receptor 2 mRNA were detected by Reverse Transcription Polymerase Chain Reaction (RT-PCR).All data was analyzed by SPSS version 17.0 for Windows software.Results:1 The demograpgic information and biochemical indexes of every group: Age, Gender Proportion, BMI, TC, TG were similar in CHB, LC and CSHB groups(P>0.05). Cormpared with the control group, the levels of serum bilirubin, ALT increased and the level of albumin and PTA decreased in the order of CHB, LC and CSHB groups, significant changes in CSHB group.2 Changes of the levels of serum adiponectin: The levels of serum adiponectin increased significantly in the LC(0.718±0.090) and CSHB (0.821±0.149) groups compared with those in the control group(0.587±0.147), F=13.296, P<0.05. The levels of serum adiponectin in CHB group(0.616±0.126) were similar with those in control group(0.587±0.147) (P>0.05).3 Histopathological changes of the liver: Normal liver tissue mainly as follow: the normal hepatocyte arranged in radiation from the central veins with HE staining. There was no fibrous tissue deposition in the hepatic sinusoids,central veins and portal areas through Masson staining; The liver tissue of CHB patients with HE staining mainly as follow: Various degrees of hepatic cell degeneration and necrosis were seen in hepatic lobules. Hepatic sinusoid and necrosis were surrounded by inflammatory cells with lymphocyte. The portal tracts were expanded and some hepatic limiting plates were damaged. Masson staining: there were different degrees of fibrosis deposition in portal tracts expanded and partly formed in fibrous septa; The liver tissue of LC patients with HE staining mainly as follow: Hepatic plates were disordered and the pseudo-live plates were formed. Central veins were not uniform and showed various degrees of inflammatory cell infiltration and interface hepatitis. Masson staining: Fibrous septa crossed or connected with each other, surrounded and separated from normal hepatocytes to form pseudo-live plates; The liver tissue of CSHB patients with HE staining mainly as follows: Massive or sub-massive necrosis of hepatocytes was seen, hepatocyte structures disappeared with a large amount of inflammatory cell infiltration. The hepatocyte islands that remained and/or were regenerated were observed in parts of the necrotic areas. The bile-duct proliferation occurred and bile thrombi appeared. Masson staining: Hepatic lobules were disordered, fibrous scaffolds collapsed in necrotic areas and there was a large amount of proliferation of collagen fibers around the necrotic areas.4 Changes of adiponectin and adiponectin receptor 2 mRNA expression in liver tissue: Adiponectin and adiponectin receptor 2 mRNA expression was significantly higher in patients with different types of chronic hepatitis B than in the control group (0.091±0.018)(0.075±0.016), and increased in the order of CHB(0.123±0.019)(0.106±0.018), LC (0.196±0.017) (0.167±0.016)and CSHB (0.245±0.028)(0.223±0.020)groups. There was statistical difference between CHB, LC and CSHB groups. F=84.493 P<0.05 and F=96.108, P<0.05. 5 Changes of adiponectin and adiponectin receptor 2 protein expression position and strength in liver tissue: The was only a small positive staining of adiponectin in liver cells of the control group. The expression of adiponectin gradually increased in patients with different types of chronic hepatitis B. Positive staining of adiponectin was principally in the hepatocytes cytoplasm in the CHB and LC groups, whereas positive staining of adiponectin was principally pronounced in the endothelial cells of liver sinusoids and only a small positive staining of adiponectin in the hepatocytes cytoplasm in the CSHB group. Positive staining of adiponectin receptor 2 was principally in the liver cells in all groups. Adiponectin receptor 2 expression was consistent with the expression of adiponectin in patients with different types of chronic hepatitis B. Adiponectin and adiponectin receptor 2 protein expression in liver tissue in healthy control (4.044±0.815) (2.804±0.622), CHB (5.545±0.613) (4.775±0.458), LC (6.553±0.614) (5.678±0.539) and CSHB (8.482±0.772) (7.654±0.272) groups was analyzed by SPSS. There was statistical difference between four groups, F=45.675, P<0.05 and F=63.386, P<0.05.6 Correlation analysis: In CHB group, the hepatic adiponectin mRNA and adiponectin protein levels correlated positively with the liver inflammation score (r=0.745, r=0.891, P<0.05). In CSHB group, the hepatic adiponectin mRNA and adiponectin protein levels correlated positively with the levels of ALT(r=0.702, r=0.583, P<0.05), correlated negatively with the levels of Albumin(r=-0.683, r=-0.641, P<0.05). The serum adiponectin levels, the hepatic adiponectin mRNA and adiponectin protein levels in patients with different types of chronic hepatitis B have not correlated with the serum HBV DNA levels, r=-0.094, r=-0.131, r=-0.014, r=-0.254, r=-0.349, r=0.380, r=0.189, r=-0.302, r=-0.195,P>0.05.Conclusions:1 Adiponectin increased in order of CHB, LC and CSHB patients, suggesting adiponectin can be a predictor of the severity of liver disease.2 Hepatic adiponectin mRNA and protein were positively correlated with liver inflammation score of CHB patients, liver fibrosis score of LC patients and ALT levels of CSHB patients, negatively correlated with Albumin levels of CSHB patients, implying adiponectin play important roles in various types of chronic hepatitis B progression.3 The was only a small positive staining of adiponectin in liver cells of the healthy control. Positive staining of adiponectin was principally in the hepatocytes cytoplasm in the CHB and LC patients. Positive staining of adiponectin was principally pronounced in the endothelial cells of liver sinusoids in the CSHB patients, only a small positive staining of adiponectin in the hepatocytes cytoplasm.4 The circulating and hepatic adiponectin frequency did not correlate with the HBV DNA level of CHB, LC and CSHB patients, indicating that adiponectin might have no effect on HBV DNA copying and clearance.