Patients
From January 2017 to December 2019, a total of 53 patients diagnosed with HEV induced liver failure, including 21 HEV-ACLF patients on the background of CHB, 15 HEV-ACLF patients on the background of ALD, and 17 HEV-ALF patients, were enrolled at the First Affiliated Hospital of Xi’an Jiaotong University, Shaanxi, China. All participants provided written informed consent, depending on the patient’s altered mental status, and the study was approved by the Research Ethics Committee of the First Affiliated Hospital of Xi’an Jiaotong University.
Patients were diagnosed with ACLF based on the following criteria of the Asian Pacific Association for the Study of the Liver (APASL): (1) serum bilirubin ≥ 85 mol/L; (2) international normalized ratio (INR) ≥ 1.5 or prothrombin activity ≤ 40%; (3) any degree of encephalopathy and/or clinical ascites within 4 weeks; (4) and an evidence of ongoing chronic liver disease.
Patients were diagnosed with ALF based on the following criteria of APASL: (1) coagulation abnormalities, typically with an INR ≥ 1.5 or prothrombin activity ≤ 40%; (2) any degree of encephalopathy; (3) no preexisting cirrhosis, and with an illness duration of < 26 weeks.
Patients diagnosed with ACLF or ALF were all aged from 18 to 75 years old.
A total of 66 patients in our cohort were excluded for the following reasons: (1) manifestation of decompensated liver cirrhosis prior to liver failure diagnosis, such as ascites and variceal hemorrhage; (2) trans-jugular intrahepatic portosystemic shunt (TIPS) in patients with portal hypertension; (3) pathological diagnosis or clinical susception of hepatocellular carcinoma; (4) other malignancies such as gastric cancer; (5) pregnancy; (6) HIV or hepatotropic virus infection.
We calculated the MELD score using the standard formula: 11.2 × ln (INR) + 9.57 × ln (creatinine, mg/dL) + 3.78 × ln (bilirubin, mg/dL), with a lower limit of 1 for all variables.
During the same period, age- and sex-matched 30 healthy participants and 30 HEV-AVH patients were recruited as controls.
Detections
Serum SOD levels were measured using an ELISA commercial kit (#EIASODC, Thermo Fisher Scientific, Waltham, MA, USA) in according with the manufacturer’s protocol at hospital administration. Samples and standards were run in duplicate, and the sensitivity of the assay was 0.044 U/mL.
HMGB1 was measured by a commercially available ELISA kit (Cusabiotech, Hubei, China). This assay recognizes recombinant as well as natural human HMGB1 without significant cross-reactivity or interference.
The diagnosis of acute HEV infection was based on the detection of HEV-RNA by polymerase chain reaction (PCR) and anti-HEV IgM with a commercial kit (Genelabs Technologies, Singapore). All of the recruited subjects had the typical profile of positive HEV-RNA and anti‐HEV IgM before the onset of liver failure. HBsAg, HBsAb, HBeAg, HBeAb, and HBcAb were detected with an automatic rapid immunoassay system (AxSYM, Abbott, USA).
Cell culture
HL-7702 cells were cultured in DMEM medium supplemented with 10% fetal bovine plasma and 2 Mm L-glutamine at 37 °C in a 95% air, 5% CO2-humidified atmosphere. Cells were trypsinized, and 5 × 105 cells were seeded onto plastic dishes and then treated with serum from HEV-AVH, HEV-ALF and HEV-ACLF patients, and 10 mM N-Acetyl-L-cysteine (NAC).
Quantitation of apoptosis
To quantify apoptosis, flow cytometry was used. A total of 2.0 × 104 HL-7702 cells were seeded into each well of a 12-well plate. The following day, the cells were washed with a new medium and exposed to various reagents, as indicated. At harvest, the cells were washed and harvested with PBS and fixed with 3.7% paraformaldehyde in PBS for 10 min at room temperature. Annexin V-PI double-staining was performed in according with the manufacturer’s instructions (#640932, BD Biosciences, Franklin Lakes, NJ, USA). Briefly, 5 μL of Annexin V conjugate and PI were added to 100 μL of cell suspension for 15 min, and then, 400 μL of binding buffer was added and mixed gently while the samples were kept on ice. Flow cytometric analyses were performed with BD Jazz (BD FACS Jazz).
Statistical methods
Results are presented as means and standard deviations (SDs). Demographics were compared for categorical variables using a chi-squared test or Fisher’s exact test as appropriate, and for continuous variables using a Wilcoxon rank sum test. ROC curves were generated using logistic regression where the event was death within 90 days post-enrollment. Cut-offs for continuous variables were determined as the maximum of the sum of sensitivity and specificity. Kaplan–Meier survival curves to 90 days post-admission were compared using log-rank tests. Data were analyzed using SPSS version 16.0 software (IBM Corporation, Somers, NY, USA). Differences were considered to be of statistical significance when the P value < 0.05.