Figure 4.

Mitochondrial apoptotic signaling was activated during HEV infection. HEV-RNA positive brain tissues collected on 14, 21, and 28 dpi were used for the immunohistochemistry study of caspase-9 (rabbit polyclonal IgG), caspase-3 (rabbit polyclonal IgG) and PCNA (rabbit polyclonal IgG). Goat anti-rabbit IgG was chosen as secondary antibody. The positive signal was measured via the Motic Med 6.0 CMIAS Image Analysis System. (A–D) Immunohistochemistry study showed that expression levels of activated caspase-9 and caspase-3 were expressed in neuronal cells and vascular endothelial cells of the brain tissue, with higher amount in HEV infected animals compared with mock animals (*p < 0.05). (E,F) For western blot, HBMVECs were inoculated with 300 MOI HEV for 48 h and HEV-negative homogenate served as control. Data showed that expression levels of cleaved caspase-9 and caspase-3 were significantly increased in HBMVECs infected with HEV compared with mock group (**p < 0.01). (G,H) Positive signal of PCNA was detected in glial cells and vascular endothelial cells, with significantly higher level in HEV infected brain sections compared with mock group (**p < 0.01). For western blot, gray value was analyzed with ImageJ to quantitatively analyze the expression levels of targeted proteins according to the level of exposure gray (resolution). Data were finally normalized to the expression of anti-β-actin.