Correlation Between Endogenous Steroid Hormone Receptor And Liver Histopathology In Peripheral Blood Mononuclear Cells And Liver Tissues Of Patients With Chronic

ObjectiveWe aim to investigate the relationships between the ESRa mRNAor AR mRNA in the PBMC and liver tissue and the liver histopathology state,and the expression of HBsAg, HBcAg in the liver tissue in patients who suffer from chronic hepatitis B.Method1Investigated subject1.1Inclusion criteria:With reference to the diagnostic criteria of the"Guideline on prevention and treatment of chronic hepatitis B in China (2010)", we selected162cases of hepatitis B patients with liver biopsy, without gender and age limitation.1.2Exclusion criteria:(1) Alcoholic liver disease, non-alcoholic fatty liver disease, hepatitis C, drug-induced hepatitis, autoimmune liver disease, cholestatic liver disease, congestive liver disease, liver degeneration, hemochromatosis and other non-hepatitis B virus infection caused by liver disease;(2) Patients who had used hormone replacement drugs, hormone antagonist drugs,or antifibroticin drugs the nearly six months;(3)Patients who suffered from decompensated liver disease;(4) Patients mixed with gonadal disease,endocrine diseases or diseases that may affect the body testosterone and estradiol level.2The collection and preservation of specimen2.1The collection of liver tissue specimen and storage:We used one second percutaneous transhepatic method to extract liver tissue,and the liver tissue was divided into two sections,one segment was less1.0cm,another was less0.2cm.They were placed in an empty plastic tube and tube stored RNA later, were frozen immediately and were placed in-80℃refrigerator.2.2Separation and preservation of PBMCs:Using Ficoll density gradient centrifugation obtained PBMCs; adding a small amount of PBS made the cells suspended,and the cells adding300μL RNAlater were placed in-80℃refrigerator.3Experiment methods3.1The liver pathological diagnosis:the paraffin embedded liver sections were stained with Masson and Hematoxylin and Eosin. The diagnosis of liver tissues with reference to the revised standards of the2000National Xi’an viral hepatitis and liver disease conferences, Pathological inflammatory grade including the G0-G4, fibrosis including S0-S4.3.2The expressions of HBsAg and HBcAg in the liver tissue:the expressions of HBsAg and HBcAg in the the paraffin embedded liver sections were measured by immunohistochemistry(S-P method).3.3The determination of liver collagen proportionate area:the paraffin embedded liver sections stained with Masson,using slice analysis system from PerkinElmer company measured liver collagen proportionate area.3.4The levels of ESRα mRNA and AR mRNA in the PBMCs and liver tissue were measured by real-time RT-PCR.4Statistical analysisThe results were analyzed using SPSS13.0software. The relationships with the ESRα mRNA and AR mRNA in the PBMCs and liver tissue and liver histopathology status and the expression of HBsAg, HBcAg in the liver tissue were measured by the Spearman rank correlation analysis. The relationships with the ESRα mRNA and AR mRNA in the PBMC and liver tissue and liver histopathology status and liver collagen proportionate area were measured by the Pearson linear correlation analysis. The difference between the ESRα mRNA and AR mRNA in the PBMCs and liver tissue and the liver pathological inflammatory grading and fibrosis staging was measured by the One-Way ANOVA.Gender,age, the ESRα mRNA and AR mRNA in the PBMCs and liver tissue predicted the liver pathological inflammatory grading and fibrosis staging,which was measured by the Bayes stepwise discriminant analysis. The ESRa mRNA and AR mRNA in the PBMCs and liver tissue predicted liver collagen proportionate area,which was measured by the curve estimation.Results1The correlations with the ESRa mRNA and AR mRNA in the PBMCs and liver tissue,age,gender,the liver pathological inflammatory grading and fibrosis staging,and liver collagen proportionate area.ESRα mRNA,AR mRNA in the liver tissue had a significant negative correlation with gender (rs=-0.250,P=0.001、rs=-0.206,P=0.008;Men assignment was1,Women assignment was0);and had a significant negative correlation with age (r=-0.298, P=0.000、r=-0.240, P=0.002); and had a significant negative correlation with the liver pathological inflammatory grading and fibrosis staging (ESRa mRNA:rs=-0.484, P=0.000, rs=0.477, P=0.000; ARmRNA; rs=-0.591, P=0.000, rs=-0.523, P=0.000); and had a significant negative correlation with liver collagen proportionate area (r=0.421, P=0.000、r=-0.299, P=0.002); and had no correlation with the expression of HBsAg in the liver tissue (rs=0.053, P=0.502、rs=0.081, P=0.053), and had no correlation and a positive correlation with the expression of HBcAg in the liver tissue (rs=0.101, P=0.202、 rs=0.170, P=0.031) ESRa mRNA and AR mRNA in the PBMCs had no correlation with gender (rs=0.014, P=0.894、rs=-0.172,P=0.098; Men assignment was1,Women assignment was0); and had no correlation with age (r=-0.099, P=0.357、r=-0.032, P=0.771);and had no correlation and a negative correlation with the liver pathological inflammatory grading and fibrosis staging (ESRa mRNA:rs=-0.085, P=0.436and rs=-0.012, P=0.913; AR mRNA:rs=-0.243, P=0.023and rs=-0.326, P=0.002); and had no correlation and a negative correlation with liver collagen proportionate area (r=-0.051,P=0.702、 r=-0.108,P=0.051);and had no correlation with the expression of HBsAg in the liver tissue (rs=0.143,P=0.184、rs=0.199,P=0.063),and had no correlation with the expression of HBcAg in the liver tissue (rs=-0.169, P=0.1、rs=-0.085, P=0.430)2The levels of ESRa mRNA and AR mRNA in liver tissue and PBMCs in the difference between the liver pathology inflammatory grading and fibrosis staging of liver tissueThe levels of ESRa mRNA and ARmRNA in the liver tissue had a difference between the different pathological inflammatory grade,which was statistically significant (F=30.056, P=0.000, F=45.819, P=0.000);The levels of ESRa mRNA and AR mRNA in the liver tissue had a difference between the different pathological fibrosis stage,which was statistically significant (F=17.796, P=0.000、F=20.570, P=0.000)The levels of ESRa mRNA and AR mRNA in the PBMCs had a difference between the different pathological inflammatory grade,which was no statistically significant (F=0.180, P=0.836、F=2.577, P=0.082). The levels of ESRa mRNA in the PBMCs had a difference between the different pathological fibrosis stage,which was no statistically significant (F=0.052, P=0.984).The level of AR mRNA in the PBMCs had a difference between the different pathological fibrosis stage,which was significant (F=3.434, P=0.021)3The prediction effect of gender,age,the levels of ESRa mRNA and AR mRNA in the PBMCs and liver tissue on the liver tissue inflammatory grade and fibrosis stage were done by Bayes stepwise discriminant analysisGender, age, the levels of ESRa mRNA and AR mRNA in the liver tissue were set as independent variable,liver tissue inflammatory grade and fibrosis stage as dependent variable,by Bayes stepwise discriminant analysis,ARmRNA was the only data which was in accordance with the entering variable of the model.AR mRNA based on Fisher discriminant function of different liver tissue inflammatory activity grade were as follows:G1=-0.298+2.852×the content of AR mRNA,G2=-1.208-0.098×the content of ARmRNA, G3=-1.892-2.925×the content of AR mRNA, correct rate of AR mRNA based Fisher discriminant function of G1,G2,G3was88.9%,12.2%,49.0%respectively.AR mRNA based on Fisher discriminant function of different liver tissue fibrosis stage were as follows:S1=-1.413+3.148×the content of AR mRNA,S2=-1.028+0.931×the content of ARmRNA,S3=-2.542-2.215×the content of ARmRNA,S4=-1.685-1.855×the content of AR mRNA,correct rate of AR mRNA based Fisher discriminant function of S1,S2,S3,S4was48.0%、59.5%、0%and28.9%respectively.Gender,age,the levels of ESRamRNA and ARmRNA in the PBMC were set as independent variable,liver tissue inflammatory grade and fibrosis stage as dependent variable,by Bayes stepwise discriminant analysis,there was no data in accordance with the entering variable of the model.4Curve estimates about the prediction effect of ESRamRNA and AR mRNA in the PBMC and liver tissue on liver collagen proportionate areaSet ESRa mRNA and AR mRNA content in the liver tissue as independent variable, liver collagen proportionate area as dependent variable to do curve fitting, the coefficient of determination of predictive regression model of ESRa mRNA content-based liver collagen proportionate area,the maximum was coefficient of determination of the cubic model (R2=0.094),liver collagen proportionate area ratio=0.187-0.128xcontent of ESRa mRNA+0.081×content of ESRa mRNA2+0.073×content of ESRa mRNA3.The coefficient of determination of predictive regression model of AR mRNA content-based liver collagen proportionate area, the maximum was coefficient of determination of the cubic model (R2=0.202), liver collagen proportionate area=0.311-2.692xcontent of AR mRNA+7.818xcontent of AR mRNA2-9.765xcontent of AR mRNA3.Take ESRa mRNA and AR mRNA content in the PBMC as independent variable,liver collagen proportionate area as dependent variable to do curve fitting,the result as ESRa mRNA and AR mRNA based prediction of liver collagen proportionate area regression model did not established.Conclusions1The levels of ESRa and AR in the liver tissue have relationships with gender and age.The level of AR in male patients is lower than female patients.For puberty and adolescence, The levels of ESRa and AR in the liver tissue were gradually reduced as age increasing.ESRa and AR in the liver tissue may take part in the pathogenesis and progression of chronic hepatitis B,the degree of inflammatory and fibrosis of the liver tissue gets more seriously as the the expression of ESRa and AR in the liver tissue gets lower.AR may contribute to the expression of HBcAg in liver tissue.2The levels of ESRa and AR in the PBMCs have nothing to do with gender and age.The expression of AR in PBMCs takes part in the progression of chronic hepatitis B.The lower the level of AR in the PBMCs express,the more serious liver fibrosis will be.AR level in PBMCs have no relationship with expression of HBcAg in liver tissue.3ESRa and AR in the PBMCs and liver tissue are not parallel; For puberty and adolescence,the decrease of ESRa and AR in the liver tissue may take part in the pathogenesis and progression of chronic hepatitis B. The mechanism which the decrease of ESRa and AR in the liver tissue may take part in pathogenesis and progression of chronic hepatitis B don’t depend on the hepatitis B virus replication and antigen expression.