The Expression Of Glucocorticoid Receptor In Patients With Type I Autoimmune Hepatitis And Its Significance

Background and ObjectivesAutoimmune hepatitis is an autoimmune disease affecting the parenchyma,which is associated with hypergammaglobulinaemia,high titers of a wide range of circulating auto-antibodies,the characteristic histological picture of an interface hepatitis and lobular hepatitis.It is the interactive result of complicated factors,which include causative factors,autoantigen respondence,hereditary susceptibility,immunological reglulation disorders and cytokine network.Glucocortocoid(GC) is extenxively used to treat autoimmune hepatitis,however it has been found that clinical patients of autoimmune hepatitis have different response to GC and the part of patients are insensitive to the GC therapy,Even if given large dose of Glucocortocoid,they can not get expectant therapeutic effect,or resistant to GC.At present,the mechanism of insensitivity to GC or resistant to GC is unclear,it need to explore the mechanism of resistant to GC futheremore.Glucocorticoid receptor(GR) is the mesomerism object of GC to educe its physiolgical and phamacolofical functions,different patients have different responces to GC,this is relevant to the GR conditions.The previous study found that GR messenger Ribonucleic Acid(GRmRNA) undergoes different splicing and it can generate two kinds of high homologic subtypes:GRamRNA and GRβmRNA,GRais a kind of ligand-activated transcription factor,in the homone bound state,it can bind specific glucocorticoid responsive element(GRE) DNA sequence modulate the expression of glucocorticoid responder gene.In contrast,Grβmay be endogenous inhibiting factor of glucocorticoid effect,which makes transcription devitalized,and it is well known that Grβprotein is correlated to the homonal resistance.GR maintain invariable quantity and function,to make endogenous GC function achieve invariable level in nerve-endocrine-immunolgic system network,wich is benefit for relieving the diseases.On the basis of reading large amount of internal and abroad documents, combining the clinical therapy and observation,We can detect the level of GR mRNA in the patients with typeⅠautoimmune hepatitis who have different responses to glucocorticoid,and compare the clinical features of GRβ-positive and GRβ-negative patients with typeⅠautoimmune hepatitis.Our purpose is understanding the mechanism of hormonal resistance in order to provide reference for clinical therapy.Materials and MethodsThe specimens are obtained from the patients with typeⅠautoimmune hepatitis who were out-patient and inpatient department in the first affiliated hospital of ZhengZhou University from June 2007 to September 2008,the total number was fifty-five instances which contained forty ones that were hormone-sensitive and fifteen ones that were hormone-resistant,all of them were female,their ages range from twenty-one to fifty-nine years old.The diagnostic criteria is consistent with the criteria of the international autoimmune hepatitis group in 1998.All of the specimens need liver puncturation pathological examination to help diagnosis before glucocorticoid therapy,it should remove the patients with liver cirrhosis,hepatic cell carcinoma and gravis type hepatitis,in the past six months all of them have never use the glucocorticoid,the normal controls contain twenty healthy examinationers,no significant difference is observed in gender and age in both study groups and control groups.At eight in the morning,we draw off three milliliter of blood,and then extract the peripheral blood mononuclear cells(PBMCs) in one hour,then continue the subsequent experiment operations.The total RNA are extracted using Trizol-A~+ Total RNA Reagen(Qiagen),according to the manufacture's recommended protocols.And then the intensity ratio of 28s to 18s rRNA in total RNA samples is examined by 2% agarose gel electrophoresis(80V,0.5h) to estimate the total RNA integrity.The absorptance is examined at 260/280nm by spectrophotometer.And then reverse transcription polymerase chain reaction(RT-PCR) is performed according to the protocol provided by Promega.Amplication product is tested by 2%agarose gel electrophoresis which contains 0.5mmol/L,scanned and semiquantitative analyzed by DBT-08 automic gel imaging system(America,Syngene).Meanwhile,the clinical and laboratory examination datas are analyzed.The data is treated statitically with SPSS10.0,F test is used in two samples homogeneity test for variance to measurement data,then continue with t or t'test;x~2 test,fisher's test is used in numeration data;correlation analysis adopt Pearson(double-variance normal distribution),Spearman(nonnormal distribution) test,get coefficient correlation r; and the test criteria is 0.05(α=0.05).Results1.The integrity of RNA isolated in each group is fine.The ratio of absorbance of total RNA at OD 260/280nm is between 1.8～2.0.The band of amplication product is clear and the band of keephousing gene is bright.2.The expression of GRa and GRβmRNAGRamRNA is detected in all patients with typeⅠautoimmune hepatitis and all normal controls,no significant difference is observed among hormone-sensitive patients,hormone-resistant patients and normol controls(P＞0.05);Four normol controls are GRβmRNA positive expression(20%),eleven of hormone-sensitive patients(27.5%),twelve of hormone-resistant patients(80%),and the expression of GRβmRNA in the hormone-resistant patients is significantly higher than the other groups(P＜0.01).3.Alanine aminotransferase(ALT) and total bilirubin(Tb) from the group of positive GRβmRNA expression is higher than that from the group of negetive GRβmRNA(P＜0.05);And the jaundice,debilitation,anepithymia from the group of positive GRβmRNA expression are higher than that from the group of negetive GRβmRNA(P＜0.05);The correlation between the hormone- resistant patients and GRβmRNA expression is obvious(OR=10.545,P＜0.05).4.The correlation analysis between the expression level of GRamRNA and that of GRβmRNA:with the increasing expression of GRβmRNA,GRamRNA is descending(r=-0.593,P＜0.05).The correlation analysis between the expression level of GRβmRNA and the measure value of ALT:with the increasing expression of GRβmRNA,the measure value of ALT is ascending(r=0.512,P＜0.05)Conclusions1.The increasing expression of GRβmRNA in the patients with typeⅠautoimmune hepatitis may be relevant with GC-resisitant;and GRβmRNA may be an endogenous inhibitory factor of GRa mRNA.2.The rates of relapse of in the GRβ-positive and GRβ-negetive groups is significantly different from each other,indicating a close relationship between glucocorticoid unresponsiveness and GRβmRNA expression level in PBMCs,in other words,the expression of GRβmRNA has the negative correlation with hormone sensitivity.3.The expression of GRβmRNA in PBMCs is likely to serve as a novel predictor of glucocorticoid response in patients with AIH.