Hepatic ROS levels and Jak2/Stat3 activity were increased simultaneously at the early stage of liver inflammation in a persistent HBV-replication mouse model (IMAGE)
Caption
(A) Schematic representation of experiment design and procedures. A persistent HBV replication mouse model was established by HDI of 6 μg pAAV-HBV 1.2 plasmid into 5 to 6-week-old male C57BL/6J mice. As normal controls, mice were injected hydrodynamically with the same volume of phosphate-buffered saline (PBS). Serum and liver samples from HBV-replication mice and normal controls were collected at 2, 3, 4, and 5 weeks after HDI for later experiments. (B) Dynamic changes in serum ALT levels of HBV-replication mice and normal controls collected at 2, 3, 4, and 5 weeks after HDI, or HBsAg, HBeAg, and HBV-DNA levels in HBV-replication mice. (C) Representative images of hematoxylin-eosin staining of liver tissue sections from normal controls (top row) and HBV-replication mice (bottom row). Ishak scoring system was used to evaluate the degrees of liver inflammation and fibrosis. (D) Detection of hepatic ROS on frozen section of liver tissue using DCFH-DA dye by fluorescence confocal microscope. The ROS levels were expressed as MFI of DCFH-DA dye and compared among these groups. (E) Measurement of GSH or GSSG in fresh liver tissues obtained from HBV-replication mice or controls. Reduced ratio of GSH to GSSG indicated increased oxidative stress. (F) Protein expression of p-Stat3, p-Jak2, Nrf2, Stat3, and Jak2 in liver tissues from the 2-week group (normal ALT levels and no obvious liver inflammation) and the 4-week group (increased ALT levels and early stage of significant liver inflammation) of HBV-replication mice and their controls. The relative protein expression was shown as fold-change in comparison with the 2-week group of controls. (G) Serum levels of IL-6 and IL-8 in the 2-week and 4-week groups of HBV-replication mice and their controls were assessed by ELISA. (H) Relative mRNA expression of downstream target genes of p-Stat3 (Saa1, S100a9, Icam1, and Socs3) in liver tissues from the 4-week group of HBV-replication mice and their controls was detected by real-time PCR. All data were shown as mean ± standard error of the mean. Each dot represented an individual mouse, and each group consisted of 5 or 6 mice. P-values < 0.05 were considered statistically significant (∗P < 0.05, ∗∗P < 0.01, or ∗∗∗P < 0.001 between two indicated groups). HDI, hydrodynamic injection; MFI, mean fluorescence intensity; GSH, reduced glutathione; GSSG, oxidized glutathione; ROS, reactive oxygen species; HBV, hepatitis B virus.
Credit
Rui Song, Shasha Yu, Xueyan Chen, Ning Ling, Dachuan Cai, Hong Ren, Min Chen
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