A total of 178 patients with CHC were recruited from the Infection Department of the Second Affiliated Hospital of Wenzhou Medical College from 2016 to 2019. This study conforms to the diagnostic criteria of the ‘Guidelines for Prevention and Treatment of Hepatitis C’ . Inclusion and exclusion criteria as our previous describe , in brief the inclusion criteria were: age 18–70 years, HCV infection for >6 months or epidemiological history of six months, positive for anti-HCV and HCV RNA, histopathology of liver, HCV1b, no antiviral treatment and use of immunomodulators within three months, no absolute contraindication of PR treatment, would receive PR treatment, and signed the informed consent. The exclusion criteria were as follows: a combination of chronic hepatitis B and other hepatitis, alcoholic liver disease, drug-induced liver injury (DILI), autoimmune hepatitis, and other liver diseases, HIV infection, malignant tumors, patients with severe cerebrovascular disease, hematological disease, thyroid disease, diabetes, and incomplete data, which might affect efficacy and safety.
A total of 82 health volunteers were enrolled from the Physical Examination Center at the Second Affiliated Hospital of Wenzhou Medical University as the control group. Exclusion criteria were as follows: accompanied with other viral hepatitis, such as chronic hepatitis B, alcoholic liver disease, DILI, autoimmune hepatitis and other liver diseases; HIV infection; Malignant tumor; Serious cardiovascular and cerebrovascular diseases; Blood disease or thyroid disease and diabetes. All the subjects were unrelated Han Chinese from China's Zhejiang province. This study was approved by the Medical Ethics Committee of the Second Affiliated Hospital of Wenzhou Medical University. Informed consent was obtained for all enrolled patients.
Determination of efficacy
All patients were treated with pegylated interferon α (Peg IFNα) combined with ribavirin (RBV) as follows: IFN-α-2a (subcutaneous injection, 180 μg each time, once a week) and ribavirin (oral, 800-1000 mg/d), with a basic course of 48 weeks. HCV RNA was monitored before treatment and at 4, 12, 24 and 48 weeks of treatment to assess the viral response and adjust the dose accordingly. Efficacy determination and grouping : according to the HCV RNA level at week 12 of treatment, the patients were classified as ① virological response group: HCV RNA decreased ≥ 2Log at week 12 of treatment compared with that before treatment; ② no virological response: HCV RNA decreased < 2Log at week 12 of treatment compared with that before treatment. According to the HCV RNA level at week 24 of follow-up after the end of treatment, the patients were classified as ① Sustained virological response (SVR): HCV RNA was not measurable at week 24 after treatment; ② Non-SVR: HCV RNA was measurable at week 24 after treatment.
About 5 mL of venous blood sample was collected from fasting patients into EDTA-K2 in the morning. Thereafter, plasma and blood cells were separated and stored at -80°C. Plasma was used to detect IRF-3, IFN-β, HCV RNA and other parameters, while blood cells were used to extract DNA and analyze IRF-3 gene polymorphisms.
Genomic DNA extraction and amplification
Blood genomic DNA extraction kit (Shanghai Bioengineering Co., Ltd., SK8224) was used to extract human genomic DNA, according to the manufacturer’s instructions. The amplification was performed by polymerase chain reaction (PCR) amplification kit (SK2072; Sangon Biotech) in Veriti ® 96-well PCR instrument (Applied Biosystems Inc., Foster City, CA, USA). The PCR reaction conditions were as previously reported . Briefly, pre-denaturation at 95℃ for 3 min, denaturation at 94℃ for 30s, annealing at 58℃ for 30s, extension at 72℃ for 50s, total 35 cycles, repair extension at 72℃ for 10 min, storage at 4℃. The primers were synthesized by Sangon Biotech as follows: rs2304204-F 5'-CTCCACTCAACTTGCGGTCTC-3', rs2304204-R 5'-CGGGT AGCTCTCTCAAACTCGA-3', rs2304206-F 5'- GTTGGTTTTATTTCAAGAAGTCGAT-3', rs2304206-R 5'-GCTCCTTCCTTGCTCCACTTT-3'.
Product purification and sequencing
The PCR product purification and recovery kit (Shanghai Bioengineering Co., Ltd., SK1141) was used to purify the amplified products, and the NanoDrop 2000C UV spectrophotometer (Thermo, USA) was used for DNA quantitative analysis. Sequencing was completed on a 3730XL sequencing instrument (ABI, USA), and the sequencing profiles were compared and analyzed using SeqMan software.
Determination of plasma IRF-3 and IFN-β levels
We used double-antibody sandwich ELISA to detected the levels of plasma IRF-3 and IFN-β, according to the manufacturer’s instructions (SuJingmei Biotechnology Co., Ltd.). The OD values of the samples and standards were detected with Anthos 2010 enzyme standardizer (Shanghai Bosai Si Technology Co., Ltd.). The standard curve was drawn to calculate the IRF-3 and IFN-β levels of the samples. If the OD value of the sample exceeds the upper limit of the standard curve, the sample would be diluted and repeated.
Quantitative analysis of HCV
We performed real-time quantitative fluorescence (FQ)-PCR to extracted the peripheral blood RNA according to the protocol of the kit (Piji Bioengineering Co., Ltd., Shenzhen, China), and detected by the ABI 7500 FQ-PCR instrument (Applied Biosystems Inc.). The normal reference ranges were defined as < 5.0 × 10 2 copies/mL for HCV RNA.
All statistical analyses were performed using SPSS23.0. Comparison of counting data between groups was performed by Pearson Chi-square test or Fisher's exact test, normal distribution data were described by mean and standard deviation, and comparison of counting data between groups was performed by T test or analysis of variance. Non-normally distributed data were represented by median (M), 25% (P25), and 75% (P75), and rank-sum test was used for comparison between groups. Significant differences were noted in genotype and allele frequency. Hardy-Weinberg equilibrium test, odds ratio (OR) and 95% confidence interval (95% CI), linkage imbalance analysis, and haplotype construction were performed using SHEsis (http://analy sis.biox.cn/myana lysis. PHP). CHWE website (https://www.genecalculators.net/pq-chwe-polypicker.html) is applied to the analysis of all the team of heterozygosity and polymorphism information content (PIC), to determine whether the SNP has enough polymorphism. All tests were bilateral and p value < 0.05 was considered statistically significant.