| Objective: In the present study, using the ratio of IFN-γ and IL-4torepresent the balance of Th1and Th2cells, we investigated the frequencies ofcirculating Th1cells and Th2cells, the expressions of interferon-γ (IFN-γ) andinterleukin4(IL-4) mRNA in peripheral blood mononuclear cells (PBMCs), aswell as the expressions of serum and intrahepatic IFN-γ and IL-4in patientswith chronic hepatitis B (CHB) fibrosis, and we further explored the role ofTh1/Th2cells and its cytokines in the occurrence and development of chronichepatitis B fibrosis.Methods: A total of46chronic hepatitis B patients, who underwent liverbiopsy, including31men and15women in department of infectious disease ofThe Third Hospital of Hebei Medical University from March2011to October2011were enrolled in the study. The diagnoses of CHB were according to theGuidelines on Prevention and Treatment for Chronic Hepatitis B (2010) issuedby the Chinese Society of Infectious Disease and the Chinese Society ofHepatology. The diagnoses of the stage of liver fibrosis were complied withthe diagnostic criteria of the2000Viral Hepatitis Management Scheme.Among the46patients,15patients were found with fibrosis stage0-1(S0-1),20patients with S2-3and11patients with S4. Moreover,10healthy bloodvolunteers and liver tissues from10liver donors during liver transplantations.Liver function parameters were measured using Olympus AU2700anto-biochemical analyzer. HBV markers: HBsAg, HBsAb, HBeAg, HBeAb,HBcAb were detected by commercial enzyme immunoassay kits. Theexpressions of HBV DNA were checked by real-time quantitative polymerasechain reaction (real-time PCR).The frequencies of circulating Th1cells and Th2cells were detected byflow cytometry. IFN-γ and IL-4mRNA expressions in peripheral blood mononuclear cells (PBMCs) were detected by real-time PCR. The serumIFN-γ and IL-4concentrations were determined by enzyme-linkedimmunosorbent assays. Intrahepatic expressions of IFN-γ and IL-4weredetected by immunohistochemical staining.All data were analyzed by SPSS version13.0for windows software. Alldata were showed by mean±standard deviation. Differences between groupswere analyzed using non-parametric Kruskal-Wallis H test, followed byMann-Whitney U test for multiple comparisons. Bivariate correlation analysistest was used to analyze categorical variables. Logistic regression was used tomultivariate analysis.Results:1Demographic and clinical characteristics of the studied subjectsAll studied subjects were comparable for age and sex. Routine laboratorymarkers: age, male, AST, TB, DB, TBA, ALKP, PT, INR were positivelycorrelated with hepatic histological fibrosis stage; Alb, CHE, PTA, PLT werenegatively correlated with hepatic fibrosis stage; no correlation was foundbetween ALT, HBV DNA, HBeAg and liver fibrosis stage (P=0.074, P=0.244,P=0.172, respectively).2Peripheral Th1cells frequencies and Th2cells frequencies in patients withCHB fibrosisThe frequencies of peripheral Th1cells were no significant change inhealthy controls, S0-1group, S2-3group and S4group (P>0.05). There was asignificant difference in circulating proportion of Th2cells between CHBpatients and healthy controls (P<0.05), with the degree of liver fibrosisexacerbations, peripheral Th2cells frequencies were increased gradually, thedifference in S0-1group, S2-3group and S4group was statistically significant(all P<0.05), but there was no statistically significance between S0-1group andhealthy controls (P>0.05). Compared with healthy controls, the ratio of Th1and Th2cells frequencies in three groups of CHB fibrosis patients had varyingdegrees of decline, and the decline of Th1/Th2ratio in S2-3group and S4groupwere more obvious, but the ratio had no difference between S0-1group and control group, and the patients of S2-3group and S4group had a significantlylower Th1/Th2ratio than that of S0-1group (all P<0.05).3The expressions of IFN-γ and IL-4mRNA in peripheral bloodmononuclear cells of CHB fibrotic patientsIFN-γ and IL-4mRNA expressions in PBMCs had significant differencebetween healthy controls, S0-1group, S2-3group and S4group (all P<0.05), theexpressions of IFN-γ and IL-4mRNA were increased in the course of hepaticfibrosis (all P<0.05), and the increased expressions of IL-4mRNA was moreobvious, but the IFN-γ and IL-4mRNA expressions had no difference in S0-1group and control group (P>0.05). The ratio of IFN-γ mRNA and IL-4mRNAlevels of S0-1group, S2-3group and S4group were0.96±0.11,0.71±0.16and0.44±0.14, which were decreased with the aggravation of hepatic fibroticdegree, the difference between each two groups had statistical significance (allP<0.05), but the IFN-γ mRNA and IL-4mRNA ratio had no differencebetween S0-1group and healthy controls.4The serum IFN-γ and IL-4levels in CHB fibrotic patientsThe serum IFN-γ levels in CHB patients were higher in different degreethan healthy controls, and increased IFN-γ levels of S2-3group and S4groupwere more significant (all P<0.05), but there was no difference between S0-1group and control group, the serum IFN-γ levels in S2-3group and S4groupwere also significantly higher than that in S0-1group (all P<0.05), while therewere no significant differences between S2-3group and S4group. The serumIL-4levels in CHB patients were increase gradually with the degree of liverfibrosis exacerbations, the differences between CHB patients and healthycontrols were statistically significant (all P<0.05), but there was no significantdifference between S0-1group and control group (P>0.05). The ratio of serumIFN-γ and IL-4levels from healthy controls, S0-1group, S2-3group, and S4group were2.05±0.10,2.13±0.28,2.09±0.62,0.80±0.08respectively, andIFN-γ/IL-4ratio of S4group was obvious lower than that of S0-1group and S2-3group (all P<0.05), but there was no difference between S0-1group and S2-3group, and the comparisons between S0-1group, S2-3group and healthy controls had no statistical significance. The serum IFNγ/IL-4ratio wasnegatively associated with the stage of liver fibrosis (r=–0.616, P<0.01).5The expressions of intrahepatic IFN-γ and IL-4in CHB fibrosis patientsThe level of intrahepatic IFN-γ from patients with CHB was much higherthan that of healthy controls (P<0.05), and the expressions of IFN-γ in S4group were obviously decreased compared with S0-1group and S2-3group(Z=–3.815, P=0.000; Z=–4.418, P=0.000, respectively), and comparisonsbetween S0-1group and S2-3group had significant difference (Z=–3.367,P=0.000). The level of intrahepatic IL-4from healthy controls, S0-1group, S2-3group, and S4group were30322.3±7260.3,791094.5±60882.8,1291570.0±355263.9, and1910577.0±156621.1, respectively (all P<0.05for comparisonsbetween groups). The ratio of intrahepatic IFN-γ and IL-4levels from CHBpatients S0-1group, S2-3group, and S4group were5.13±0.53,4.39±0.50,1.64±0.12, which was decreased with aggravation of hepatic fibrosis degree,the difference between groups had statistical significance (P<0.001). LiverIFNγ/IL-4ratio were negatively associated with the stage of liver fibrosis(r=–0.531, P<0.01). IFN-γ and IL-4expressions were exclusive mainly tolymphocytes in CHB patients, occasionally scattered in the hepatic cells, andthe distribution of IFN-γ and IL-4positive cells were predominantly seen inthe portal areas. Very few scattered IFN-γ positive lymphocytes were found inhealthy controls, but there were almost no IL-4positive expression in thehealthy controls.6Assess the risk factors of significant liver fibrosis by Logistic regressionstatistical analysisThere were14variables, which were age, sex, ALB, AST, TB, TBA,GGT, ALKP, CHE, PT, PTA, INR, PLT, that were significantly differentbetween patients with mild fibrosis (S0-1) and significant fibrosis (S2-4) byunivariate analysis. The serum IFNγ/IL-4ratio was one of risk factors for theoccurrence of significant liver fibrosis by Logistic regression analysis(OR=4.755,95%CI:1.034～21.862, P=0.045).Conclusions: 1The patients with CHB exhibited decreased circulating and intrahepaticTh1/Th2ratio compared with healthy controls. CHB fibrosis patients showsimbalanced Th1/Th2ratio, and Th1/Th2cytokines drift into Th2lymphocytesub-cluster with the degree of liver fibrosis exacerbations.2Liver and serum IFN-γ/IL-4ratio were negatively associated with the stageof liver fibrosis, and the serum IFNγ/IL-4ratio was one of risk factors for theoccurrence of significant liver fibrosis (S2-4) by Logistic regression analysis.3Th1/Th2imbalance may be involved in the pathogenesis of CHB fibrosis. |
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