Activator protein 1 (ap1) and interferon regulatory factor 3 (irf3) are transcription factors that play important roles induction of immune responses, including production of inflammatory cytokines, against viral infections. therefore, the aim of this study was to examine expression levels of activator protein 1 in chronic hbv infected (chb) patients.
In this study, peripheral blood samples were obtained from 40 chb patients and 40 healthy controls in 5.5 ml tubes with and without anti-coagulant for mrna extraction and separation of serums, respectively. chb patients with hiv and hcv, cytomegalovirus, hepatitis a, c, d, and e viruses and epstein-barr virus co-infection, patients with pregnancy or breastfeeding, age younger than 18 years or older than 55 years; features suggestive of other coexistent liver disease including previous liver transplantation, alcoholic liver disease, autoimmune liver diseases, cirrhosis, evidence of hepatocellular carcinoma, and antiviral and immunosuppressive drugs users were excluded from the study. the “guide of prevention and treatment in viral hepatitis” and previous clinical and experimental records was used for diagnosis of chb (15). the controls were selected with the same age and sex. the protocols for isolation of pbmcs from peripheral blood samples were described elsewhere (14). the protocol of this study was approved by the ethical committee of the azad university pharmacy branch and all of participants filled out the written informed consent prior to sample collection.
detection of serological hbv markers: the status of hbsag and hepatitis b e antigen (hbeag) in participants were determined using the elisa technique
(behring, marburg, germany) according to the manufacture’s guidelines. hbv-dna extraction and real-time pcr condition: 200 μl of plasma was used for hbv-dna purification using a commercial kit (cinnaclon, tehran, iran) according to manufacturer’s instructions. a commercial kit from the primer design
company (london, uk) was used for hbv-dna quantification.
rna extraction, reverse transcription and quantitative real-time pcr:
total rna purification from pbmcs was performed using a trizol ls extraction kit (invitrogen). the quality of extracted rna was examined by either electrophoresis on an ethidium bromide pretreated agarose gel or by measuring absorption at 260/280 nm. genes were quantified by qrt-pcr using beta actin as control. the qrt-pcr was carried out using a sybr premix kit (kit ampliqon master mix, denmark) the following program was programmed on a abi real time pcr (applied biosystems 7500 real-time pcr system, usa). primer sequences are
presented in the table 1.
data analysis and statistical methods
the t-test under the spss software version 18 was used for data analysis and a p value less than 0.05 was considered as significant
The results demonstrated that expression of ap1 was significantly increased in pbmcs of chb patients in comparison to healthy controls. while, expression level of irf3 was not differ between patients and controls
Based on the results presented here, over expression of the ap1 in the patient which it may lead to approve immune responses against hbv infection which seem impaired in the patients.