Hepatic autophagy after severe burn in response to endoplasmic reticulum stress

Abstract

Background: Previous studies showed that liver dysfunction develops soon after severe burn, and this is associated with activation of endoplasmic reticulum (ER) stress. Autophagy is a catabolic process to maintain cellular organelle balance; ER stress is associated with autophagy signaling cascades. We thus sought to determine whether autophagy signals were associated with damage in the liver after burn, and further whether burn-associated ER stress activates autophagy signals in hepatocytes.

Methods: C57BL/6 male mice received a 25% total body surface area full-thickness scald burn, and liver was harvested at 24 h after burn. HepG2 cells were stimulated with an ER stress inducer thapsigargin (TG) for 24 h to mimic ER stress in vitro. Terminal deoxyuridine nick-end labeling staining was performed on histologic sections of liver. Autophagy was assessed by immunoblotting. Statistical analysis was performed using the Student t-test and significance was accepted at P < 0.05.

Results: Terminal deoxyuridine nick-end labeling positive-stained hepatocytes increased in burned animals with a significant elevation of caspase 3 activity (P < 0.05). Hepatic autophagy-related (ATG) protein 3, ATG5 and light chain (LC) 3B elevated significantly in burn animals as well (P < 0.05). Expression of Beclin-1, LC3A, and LC3B increased in HepG2 cells in response to TG, similar to the response seen in vivo. Cytosolic adenosine triphosphate dropped significantly, and adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) were phosphorylated as well in response to TG (P < 0.05).

Conclusions: ER stress, which occurs in hepatocytes after severe injury, is associated with autophagy and liver damage after severe burn. In response to ER stress, activated autophagy is associated with adenosine monophosphate-activated protein kinase and mammalian target target of rapamycin pathway.

Keywords: Apoptosis; Autophagy; Endoplasmic reticulum (ER); Mouse liver; Thermal injury.

Figure 1

(A) TUNEL immunofluorescent staining showed positive apoptotic cells stained with green nuclei in liver tissue from non-burn (left) and burn animals (right); (B) liver tissue caspase 3 level was measured with 5 µM of Z-DEVD-R110 reaction and quantified as the change in fluorescence per minute per microgram protein. Data are mean ± SEM. * p<0.05, burned vs. non-burn.

Figure 2

Western blot data and statistical analysis results showed expression of autophagy signals including (A) ATG3, (B) ATG5, (C) ULK1, and (D) LC3B in mouse liver 24 hours after burn, β-actin as loading control. Data are mean ± SEM. * p<0.05, burned vs. non-burn.

Figure 3

Western blot data and statistical analysis results showed expression of ER stress protein GRP78 in liver homogenate from burn animals. Data are mean ± SEM. * p<0.05, burned vs. non-burn.

Figure 4

In vitro, western blot data showing the expression of (A) autophagy makers LC3A and LC3B, Beclin-1, (B) phosphorylation of AMPK, and (C) mTOR in TG treated HepG2 cells. (D) Cytosolic ATP level alteration in HepG2 cell with TG challenge. Data are mean ± SEM. * p<0.05, TG treated group vs. vehicle treated group.