Simvastatin protects hepatocytes from apoptosis by suppressing the TNF-α/caspase-3 signaling pathway in mice with burn injury

Abstract

Objective: To investigate the liver cellular apoptosis in response to burn injury and find out if statin treatment can ameliorate this process. The hypothesis is that statin may modulate apoptosis-related gene expression and thereby reduce hepatocytic apoptosis after burn injury.

Methods: Mice were subjected to 30% full-thickness burn injury and then treated either with or without simvastatin. The livers were harvested for histological assessment and determinations of gene expression. To investigate the mechanism involved, tumor necrosis factor (TNF)-α and caspase-3 knockout (KO) mice were also used to evaluate the effects of burn injury and simvastatin treatment on burn-induced liver injury. The effects of simvastatin on TNF-α and caspase-3 expressions were also evaluated in cultured mouse hepatocytes.

Results: Burn injury induced significant liver damage, which was indicated by striking levels of apoptosis. Simvastatin reduced the apoptotic index in the livers of mice with burn injury and this effect could be abrogated by TNF-α or caspase-3 inhibitors. Simvastatin also decreased burn-induced TNF-α and caspase-3 expression in the liver. TNF-α and caspase-3 KO mice demonstrated lower levels of apoptotic hepatocytes in response to burn, and simvastatin did not further decrease hepatocyte apoptosis in either strain of KO mice. An in vitro study demonstrated that simvastatin suppresses TNF-α and caspase-3 expression in primary cultures of mouse hepatocytes.

Conclusions: Simvastatin reduces mouse hepatocyte apoptosis by suppressing expression of the TNF-α/caspase-3 pathway.

Figure 1. Burn induced remote liver injury

A and B: Representative images of H&E – stained liver sections. A. Sham-treated mice exhibited normal hepatic structure. B. Burn injury produced significant damage to the liver with broad areas of cellular destruction (CV: central vein; PV: portal vein). C and D: Representative histological images of TUNEL-stained liver sections. C. Occasional apoptotic cells were detected in the livers of sham treated mice. D. Increased numbers of TUNEL-positive hepatocytes were observed in the livers of burn injured mice. Apoptotic cells were present in a diffuse distribution pattern. Examples of apoptotic cells are indicated by arrows (5 mice in each time point).

Figure 2. Time course of burn induced hepatocellular apoptosis

Representative histological images of TUNEL stained liver sections. A. Sham-treated mice. B. Mice at one day after burn injury. C. Mice at three days after burn injury. Examples of apoptotic cells are indicated by arrows. D. Percent apoptotic cells at various times after injury. Mean ± SEM, n=5 animals per group (sham vs. burn: *p<0.05, **p<0.01, ***p<0.001; within sham group: p>0.05; within burn group: p<0.01 except burn/6hr vs. burn/12hr: p>0.05).

Figure 3. Time course of TNF-α and caspase-3 expression in liver of mice with peripheral burn injury

A. Western blot detection of TNF-α and caspase-3 expression, sham group: n=4, other groups: n=6(the experiment was repeated once). B. Level of TNF-α expression normalized to the expression level of GAPDH. C. Level of caspase-3 expression normalized to the expression level of GAPDH.

Figure 4. Effects of simvastatin on the burn-induced hepatic cellular apoptosis

Representative histological images of TUNEL stained liver sections. A. burned mice without simvastatin treatment. B. burned mice treated with simvastatin. C. Percent apoptotic cells with various treatments. Mean ± SEM, n=5 animals per group. Burn injury produced significant apoptosis in mouse liver which was significantly reduced by treatment with simvastatin (*p<0.001 vs. sham, and simvastatin/burn; burn vs. burn/saline: p>0.05).

Figure 5. Effects of simvastatin on TNF-α and Caspase-3 expression in livers of mice with burn injury and in cultured mouse hepatocytes

A. Western blots analysis of the effect of simvastatin treatment on TNF-α expression in mouse liver. The graph indicates mean ± SEM for n=5 animals with each treatment. B. Western blots analysis of the effect of simvastatin treatment on caspase-3 expression in mouse liver. The graph indicates mean ± SEM for n=5 animals with each treatment. Burn injury increased expression of TNF-α and caspase-3 in mouse liver and this effect was abolished by treatment with simvastatin. C. Western blots analysis of the effect of simvastatin treatment on TNF-α expression in primary cultures of mouse hepatocytes. The experiments were repeated three times with 5 wells per study). D. Western blots analysis of the effect of simvastatin treatment on caspase-3 expression in primary cultures of mouse hepatocytes. The experiments were repeated three times with 5 wells per study). Simvastatin clearly reduced expression of both TNF-α and caspase-3 in these cultured cells.

Figure 6. Effects of TNF-α and caspase-3 inhibitors on hepatic apoptosis in burned mice

A. Effects of the TNF-α inhibitor, Pentoxifyline (50 μg/kg, IP), on apoptosis. Each value is the mean ± SEM for n=5 animals with each treatment (*p<0.01 vs. all other groups; **p<0.01 vs. sham, burn; **p>0.05: burn/inhibitor vs. burn/inhibitor/statin). B. Effects of the caspase-3 inhibitor, Ac-DEVD-CHO (50 μg/kg, IP), on apoptosis. Each value is the mean ± SEM for n=5 animals with each treatment. Burn injury induced marked hepatocellular apoptosis which was partially reversed by treatment with simvastatin, TNF-α or caspase-3 inhibitors (*p<0.01 vs. all other groups; **p<0.01 vs. sham, burn; **p>0.05: burn/inhibitor vs. burn/inhibitor/statin).

Figure 7. Effects of TNF-α and caspase-3 knockout on burn-induced liver apoptosis

A. Effects of TNF-α knockout on apoptosis in mouse liver. Each value is the mean ± SEM for n=5 animals with each treatment (ANOVA: *p<0.001; **p>0.05) B. Effects of caspase-3 knockout on apoptosis in mouse liver (ANOVA: *p<0.001; **p>0.05). Each value is the mean ± SEM for n=5 animals with each treatment. Representative histological images of TUNEL stained liver sections are also illustrated with examples of apoptotic cells indicated by arrows. Compared with the wild-type mice, TNF-α−/− and Caspase-3−/− mice demonstrated much lower levels of apoptosis in the liver after burn injury. The levels of apoptosis in the knockout mice treated with simvastatin were not significantly different from levels in knockout mice without statin treatment, indicating that statin might reduce apoptosis in burn injured mice, at least partially, through inhibition of the TNF-α/caspase-3 pathway.