Expression Of Inducible Nitric Oxide Synthase And Interferon Alpha Receptor 2 In Chronic Hepatitis B Patients

Objective To investigate the intrahepatic expression of inducible nitric oxide synthase (iNOS)in patients with chronic hepatitis B(CHB)and its relation to liver histopathology.Methods Seventy-four patients with CHB from the Department of Hepatology,Qilu Hospital of Shandong University were enrolled in this present study.The diagnosis of all the cases was made according to the criteria established on the Viral Hepatitis Conference held in Xi'an in 2000.Liver biopsy specimens,obtained by a percutaneous route,were fixed in 10%buffered formaline,embedded in paraffin and stained with haemotoxylin-eosin,periodic acid-Schiff,reticulin and evaluated by the same pathologist,recording the conventional histological diagnoses and assessing the grading of liver inflammation and the staging of fibrosis.Seven liver biopsies with normal liver histology,from the Pathology Department of Shandong University Qilu Hospital,taken during partial hepatectomy for benign lesions such as hepatic echinococcosis or liver hemangiomas were used as controls for the immunohistochemical analysis.The intensity and distribution of the immunohistochemical staining of intrahepatic iNOS were studied in the liver sections by using non-biotin polymer detection system kit.Considering cytoplasmic and/or membranous staining as positive,immunoreative cells were counted and expressed as a percentage.Each section was evaluated semiquantitatively according to the staining intensity and the percentage of the positive cells.All data were presented as Mean±Standard Deviation and were analyzed by SPSS 13.0.Comparisons were performed by using the non-parametric Mann-Whitney U-tests and the correlation of the iNOS expression vs.ALT,grading of liver inflammation and staging of fibrosis was tested by using Spearman's rank correlation. Statistical significance was established at a P value＜0.05.Results Immunohistochemistry showed that iNOS positive staining was found in 71.6%of liver sections from patients with chronic hepatitis B and iNOS immunoreactivity was observed mainly in hepatocytes,showing a predominant cytoplasmic staining,with the positive liver cells distributed diffusely throughout the hepatic lobule.Immunopositive staining could also be detected in Kupffer cells, sinusoidal lining cells and vascular endothelial cells.In contrast,in seven normal liver tissues,the expression of iNOS was negative.Statistical analysis showed that the mean iNOS staining score was significantly high in elevated ALT patients(1.50±0.97)than in normal ALT patients(0.73±0.82) (P=0.001).Intrahepatic expression of iNOS positively correlated with the serum level ofALT(r=0.601,P=0.000),the grading of liver inflammation and the staging of fibrosis(r=0.660,P=0.000;r=0.506,P=0.000)in CHB patients.Furthermore the intrahepatic iNOS immunopositivity was not correlated with the serum level or hepatic level of HBV DNA(r=0.07,P＞0.05;r=0.03,P＞0.05)and it was not different between the HBeAg positive and negative group(1.00±0.87 vs.1.20±0.99, P＞0.05).Conclusions Our results showed that the intrahepatic expression of iNOS is elevated in chronic hepatitis B patients and correlated well with the severity of the disease, which indicated that inducible nitric oxide synthase may have a critical role in the pathogenesis of chronic virus hepatitis. Objective To investigate the expression of interferon a receptor 2(IFNAR2)mRNA in Peripheral Blood Mononuclear Cells(PBMCs)in chronic hepatitis B patients and its relation to liver damage,virus load and intrahepatic expression of inducible nitric oxide synthase(iNOS).Methods Sixty-six patients with CHB from the Department of Hepatology,Qilu Hospital of Shandong University were enrolled in this present study.The diagnosis of all the cases was made according to the criteria established on the Viral Hepatitis Conference held in 2000.A percutaneous liver biopsy was performed on each patient for diagnostic purpose,recording the conventional histological diagnoses and assessing the grading of liver inflammation and the staging of fibrosis.Patients who had a history of IFN,antiviral agent treatment or other immunomodulators were excluded.Other exclusion criteria included coinfection with hepatitis C virus, hepatitis D virus or HIV;history of hepatocellular carcinoma(HCC);other causes of liver disease.Venous blood samples from another 10 healthy donors were also used as normal controls.All of these controls were negative for HBsAg,anti-HCV,anti-HIV and other liver disease.The PBMCs were separated from 4 ml of heparinized blood by density gradient centrifugation with Ficill-Cinray density centrifugation,washed three times with phosphate buffered saline and stored at -80℃until use.Total RNA was extracted from homogenized PBMCs(10~6-10~7cells)using RNAiso Reagent.Levels of IFNAR2 andβ-actin mRNA expression were quantified using reverse-transcriptase polymerase chain reaction assay.PCR products were electrophoresed on 1.5% agarose gel,stained with ethidium bromide,and bands of the expected size were measured visually under ultraviolet light.IFNAR2 mRNA level in PBMC was assessed as the IFNAR2-mRNA/β-actin-mRNA ratio.The result reported for each RNA sample is the mean of at least three RT-PCR assays.The intrahepatic expression of iNOS was studied in the liver sections by immunohistochemistry using non-biotin polymer detection system kit.All data were presented as Mean±Standard Deviation and were analyzed by SPSS 13.0.Comparisons were performed by using the non-parametric Mann-Whitney U-tests and the correlation of the IFNAR2 mRNA level vs.ALT,HBV DNA,grading of liver inflammation,staging of fibrosis and intrahepatic expression of iNOS was tested by using Spearman's rank correlation.Statistical significance was established at a P value＜0.05.Results Among 66 CHB patients,the mRNA level of IFNAR2 was above the detection limit in 53 patients(80.3%),while it was detectable in 7 subjects of 10 healthy controls(70%).The mean amount of IFNR2 mRNA was higher in CHB patients than in healthy controls(0.54±0.45 and 0.27±0.17,respectively),but it was not reached statistical difference(P=0.143).Statistical analysis showed that the mean IFNR2 mRNA level was similar between elevated ALT patients(0.50±0.42)and normal ALT patients(0.60±0.50)(P＞0.05). The amount of PBMC IFNAR2 mRNA has no association with the serum level of ALT(r=-0.06,P=0.66).Among 66 CHB patients,IFNR2 mRNA was above the detection limit in 19 of the 20 HBeAg negative patients and in 34 of the 46 HBeAg positive patients,respectively, while it was detectable in 20 of the 22 serum HBV DNA negative patients and in 33 of 44 serum HBV DNA positive patients,respectively.It was interesting to notice that the expression level of IFNR2 mRNA in HBeAg negative group was significantly higher than in HBeAg positive group(0.83±0.57 vs.0.37±0.25,P=0.004),so was in the serum HBV DNA negative group higher than in serum HBV DNA positive group(0.74±0.55 vs.0.41±0.33,P=0.011).The levels of serum and hepatic HBV DNA were expressed as the logarithm and correlated with the level of IFNR2 mRNA.But there was no significant correlation between the amount of IFNAR2 mRNA and the serum level or hepatic level of HBV DNA(r=0.06,P=0.74;r=-0.25,P=0.08,respectively)in CHB patients.Liver tissue damage,represented by the grading of liver inflammation and the staging of fibrosis was not correlated with the IFNAR2 mRNA expression(r=-0.044,P=0.76; r=-0.182,P=0.19,respectively).The study also showed that the intrahepatic expression of iNOS is elevated in CHB patients but there was no significant correlation between the hepatocelluar iNOS expression and the amount of PBMC IFNAR2 mRNA(r=0.147,P=0.294).Conclusions Our results demonstrated that the expression level of IFNR2 mRNA in HBeAg negative and serum HBV DNA negative group was significantly higher than HBeAg positive and serum HBV DNA positive group,respectively,which may suggested that hepatitis b virus might attenuate interferon response by downregulate the expression the interferon receptor.In chronic hepatitis B patients PBMCs IFNR2 mRNA was not correlated with ALT,the grading of liver inflammation and the staging of fibrosis,or the intrahepatic iNOS expression,indicated that the severity of liver disease and elevated expression of iNOS-generated NO may have no correlation with the IFNAR2 mRNA expression.