Noninvasive Diagnosis And Mechanism Of Liver Fibrosis Induced By Chronic Hepatitis B

Background and ObjectiveHepatitis B virus infection is a global publi c health issues, and about 400 million people totally are chronic infected. Most patients with chronic hepatitis B(CHB) patients have different degrees of liver fibrosis. Liver fibrosis is a chronic and progressive liver injury and may lead to liver cirrhosis or even hepatocellular carcinoma without treatment. According to various guidelines, antiviral treatment should be administered as soon as possible when pathological evidence of significant liver fibrosis(S ≥ 2) is found in patients with CHB. Therefore, early detection of liver fibrosis or cirrhosis is very critical. Liver biopsy is gold standard but with a lot of limitations or defections. Noninvasive methods of liver fibrosis, including Fibro Scan, serum fibrosis biomarkers and combined models, have been developed and showed good diagnostic performance in patients with CHB. This study was to assess the diagnostic value of these liver fibrosis models(Fibro Scan, AAR, API, APRI, GPRI, S index, FIB-4 and Fibro-Q) in diagnosis of significant liver fibrosis to screen for several simple serum models for primary care. And we also analysed the influence of hepatic inflammation on liver stiffness measurement. In addition, we intended to find out novel and fibrosis-related serum markers to predicted significant liver fibrosis and investigate the role of macrophage inflammatory protein-3α(MIP-3α) in the pathogenetic process of liver fibrosis.MethodsPart I:Patients with CHB in the Department of Infectious Diseases, So uthwest Hospital, who were received liver biopsy, Fibro Scan and laboratory tests within two days, were recruited in this study. Liver fibrosis stage(S) and inflammation grade(G) were determined according to “Guideline of Prevention and Treatment of Chronic Hepatitis B” of China. Significant liver fibrosis was defined as S2 or higher levels. Performance of Fibro Scan, AAR, API, APRI, GPRI, S index, FIB-4 and Fibro-Q in diagnosing significant liver fibrosis, and influence of inflammation on liver stiffness m easurement was assessed.Part II:Four patients with chronic hepatitis B(CHB) and 4 with CHB related cirrhosis were enrolled. After screening by protein arrays containing 507 cytokines, cytokines that were differentially expressed were obtained.Serum levels of MIP-3α, PDGF-BB and TGF-β were assessed by enzyme-linked immunosorbent assays(ELISA) in healthy volunteers and patients(significant fibrosis and non-significant liver fibrosis) dating from March 2012 to November 2014 from the outpatient department of infectious diseases, southwest hospital, who were received liver biopsy, Fibro Scan, and laboratory test within 2 days.Part III:Hepatic stellate cells(LX-2) were cultured in vitro. And they were divided into blank control group, and groups with different concentrations of MIP-3α. After 24 hours, PDGF-BB levels in culture supernatants were measured by ELISA; the cell proliferation was measured by MTT colorimetry.ResultsPart I:1. AUROCs of Fibro Scan、S index、GPRI、FIB-4、APRI、API、Fibro-Q and AAR in diagnosing significant liver fibrosis were 0.757(P < 0.001) 、 0.726(P < 0.001)、0.726(P < 0.001)、0.621(P = 0.001)、0.619(P = 0.001)、0.580(P = 0.033)、0.569(P = 0.066) and 0.495(P = 0.886), respectively.2. AUROCs of Fibro Scan combined with S index or GPRI in the diagnosis of significant liver fibrosis were 0.753(P < 0.001)、0.740(P < 0.001), respectively.3. Among different inflammatory grades, AUROCs of Fibro Scan for predicting significant fibrosis were 0.8267(P < 0.001)、0.6956(P < 0.001)、0.7092(P = 0.0012) and 0.6947(P = 0.137).4. In patients with non-significant liver fibrosis, liver stiffness measurement values increased significantly(G0 vs. G1, P = 0.051; G1 vs. G2, P = 0.005; G2 vs. G3, P < 0.001); however, there were no significant difference in patients with significant fibro sis(G0 vs. G1, P = 0.135; G1 vs. G2, P = 0.294; G2 vs. G3, P = 0.260; G3 vs. G4, P = 0.056).Part II:1. There were 12 cytokines showed significant difference between patients with cirrhosis and patients without cirrhosis(P < 0.05) as follows: β-catenin, CD27, CD163, endothelin, GCP-2(CXCL6), IL-12 P40, MIF, TFPI, u-PA, VEGF and WISP-1(CCN4).2. MIP-3α showed a good performance in predicting significant liver fibrosis in patients with CHB(AUROC = 0.724, P < 0.001).Part III:When the concentrations of MIP-3α were 0 ng/ml, 250 ng/ml, 500 ng/ml, 1 μg/ml, 2 μg/ml, respectively; the average values of optical density of PDGF-BB in culture supernatants were 0.0608, 0.2533, 0.1112, 0.0561 and 0.0581 detected by ELISA; corresponding average values of optical density were 1.6550, 0.9670, 1.1319, 1.1405 and 1.2157.Conclusion1. Fibro Scan showed a good performance in assessing the degree of liver fibrosis in patients with CHB, but was influenced by hepatic inflammation.2. S index and GPRI were the simple and most useful and simple of the seven models we evaluated for prediction of significant fibrosis in patients with CHB, which is of particular importance in primary care where Fibro Scan is unavailable.3. Inflammation has no significant influence on the accuracy of Fibro Scan in patients with significant fibrosis. Elevation of histological inflammation can decrease performance of Fibro Scan for predicting significant liver fibrosis.4. Serum MIP-3α levels have showed a good performance in predicti ng significant fibrosis in patients with CHB.5. MIP-3α could activate hepatic stellate cells.