Table 1.
Clinical and virological characteristics of the subjects enrolled in the study.
Fig 1.
Secretion concentrations of IL-34 in HBV patients and HBV-expressing cells.
(A) The serum levels of IL-34 in chronic HBV patients and healthy controls were determined by ELISA kits, * p<0.0001. (B) The peripheral blood mononuclear cells (PBMCs) in chronic HBV patients and healthy controls were isolated from full blood and total RNA were extracted. Then the mRNA level of IL-34 were determined by quantitative real-time PCR (qPCR), * p<0.05. The mRNA level of β-actin was used as an internal control. (C) The levels of serum IL-34 in chronic HBV patients with or without cirrhosis were determined by ELISA kits, ns: no significance. (D) QPCR were used to determine the HBV copy numbers in HepAD38 cells with tetracycline (no HBV replication) and without tetracycline (HBV replication) and Huh-7 cells transiently transfected with plasmid pCH9/3091 or control vector pCH9. (E-F) The supernatants of HepAD38 without tetracycline (HBV replication) and Huh-7 cells transiently transfected with plasmid pCH9/3091 post seeded or transfected 2 days, 3 days, 4 days, 5 days, 6 days and 7 days were collected and then the HBV DNA or IL-34 secretion level were detected by qPCR or ELISA kits. (G) The mRNA levels of IL-34 in HepAD38 cells (With or without tetracycline) and Huh-7 cells transiently transfected with plasmid pCH9/3091 or pCH9 were determined by quantitative real-time PCR (qPCR), * p<0.05. The mRNA level of β-actin was used as an internal control. (H) Supernatants of HepAD38 cells (with or without tetracycline) and Huh-7 cells transiently transfected with plasmid pCH9/3091 or pCH9 were collected and secretion concentrations of IL-34 were determined by ELISA kits, * p<0.05.
Table 2.
Correlative analysis of serum IL-34 level with clincopathologic features in chronic HBV patients.
Fig 2.
Correlation of clinical parameters with IL-34 levels in chronic HBV patients.
The clinicopathologic and outcomes data of those patients were collected retrospectively. The correlations of HBV DNA (A), ALT (B), AST (C), TB (D), DB (E) and AFP (F) with IL-34 levels were further analyzed by Spearman’s rank test.
Fig 3.
IL-34 inhibited HBV replication in vitro.
HepAD38 and HepG2.2.15 cells were seeded into 6-well plates and treated with different concentrations of rhIL-34. After 3 or 5 days of treatment, total RNA, protein, or HBV replicative intermediates were extracted. (A-B) rhIL-34 treatment inhibited the production of HBV replicative intermediates. The absolute quantification PCR and Southern blot were performed to determine the copies of HBV replicative intermediates after 5 days treatment, *p<0.01. (C-D) Total RNA was extracted after 3 days treatment. Relative real-time PCR was subjected to detect the HBV total RNA and 3.5kb mRNA levels, * p<0.05. The mRNA level of β-actin was used as an internal control. (E) Northern blot was applied to determine the HBV total RNA and 3.5kb mRNA levels. The rRNA level of 28s/18s were used as an internal control. (F) Western blotting analysis of HBc expression after 3 days treatment.
Fig 4.
IL-34 inhibited HBV replication in transgenic mice.
The mice were randomly assigned to two groups of 6–7 individuals per group. RhIL-34 was diluted in 1 ml PBS contained 0.1% BSA and injected through the tail vein at a dose of 1ug/25g. (A-B) Serum samples at 6 days were collected to detected ALT and AST by Reitman-Frankel methods. (C) Serum samples were collected at the indicated time points (0 day, 2 days, 4 days, 6 days) after injection via tail vein. The serum level of HBV DNA was extracted and analyzed by absolute quantification PCR, * p<0.001. (D-G) The mice were sacrificed at 6 days after injection, and the liver was isolated for extraction of HBV DNA, total RNA and total protein. (B) Liver HBV DNA was analyzed by absolute quantification PCR, * p<0.05. (E-F) Relative real-time PCR was subjected to detect the HBV total RNA and 3.5kb mRNA levels, * p<0.01. The mRNA level of β-actin was used as an internal control. (G) The expression of HBc was analyzed by Western blotting.