Simvastatin reduces burn injury-induced splenic apoptosis via downregulation of the TNF-α/NF-κB pathway

Abstract

Objective: Recent studies have suggested that epidermal burn injuries are associated with inflammation and immune dysfunction. Simvastatin has been shown to possess potent anti-inflammatory properties. Thus, we hypothesized that simvastatin protects against burn-induced apoptosis in the spleen via its anti-inflammatory activity.

Methods: Wild-type, tumor necrosis factor alpha knockout (TNF-α KO) and NF-κB KO mice were subjected to full-thickness burn injury or sham treatment. The mice then were treated with or without simvastatin, and the spleen was harvested to measure the extent of apoptosis. Expression levels of TNF-α and NF-κB were also determined in spleen tissue and serum.

Results: Burn injury induced significant splenic apoptosis and systemic cytokine production. Simvastatin protected the spleen from apoptosis, reduced cytokine production in the serum, and increased the survival rate. Simvastatin decreased burn-induced TNF-α and NF-κB expression in the spleen and serum. TNF-α and NF-κB KO mice demonstrated lower levels of apoptosis in spleen in response to burn injury. Simvastatin did not further decrease burn-caused apoptosis and mortality in either strain of KO mice.

Conclusions: Simvastatin reduces burn-induced splenic apoptosis via downregulation of the TNF-α/NF-κB pathway.

Figure 1

Burn induced splenic apoptosis. A. Occasional apoptotic cells were detected in the spleens of sham-treated mice. B. Significantly increased numbers of TUNEL-positive cells were found in the splenic white pulp of the burned mice. C. Burn injury-induced apoptosis was found in the spleen as early as 8 hours after burn, and peaked at 24 hours (n=6 animals per group; sham vs. *: P<0.05, sham vs. **: P<0.001; within burn group: P>0.05 by one-way ANOVA test). D. Burn injury caused significant IL-1α, IL-6, MCP-1 and CRP production in the serum (* P<0.01).

Figure 2

Time course of TNF-α and NF-κB expression in the spleen and serum of burned mice. A. Western blot detection of TNF-α and NF-κB expressions (n=6 in each group). B. Levels of TNF-αexpression in splenic tissues normalized to the expression levels of GAPDH (no difference exists among ** groups: P>0.05; * vs. **: P<0.01; * vs. ***: P<0.05). C. Levels of NF-κB expression in splenic tissues normalized to the expression levels of GAPDH (no difference exists ** groups: P>0.05; * vs. **: P>0.05; ** vs. ***: P<0.01). D. Time course study on the expressions of TNF-α in the serum (ELISA; no difference exists among ** groups: P>0.05; * vs. **: P<0.05; * vs. ***: P<0.01; ** vs. ***: P<0.05). E. Time course study on the expressions of NF-κB in the serum (ELISA; no difference exists among **groups or *** groups: P>0.05; * vs. **: P<0.05; * vs. ***: P<0.01; ** vs. ***: P<0.05). The statistics above are based on one-way ANOVA tests.

Figure 3

Effects of simvastatin on burned mice. Representative histological images of TUNEL stained spleen sections: A. burned mice; B. Burned mice treated with simvastatin. C. Apoptotic index in each group: burn injury induced significant apoptosis in the spleen, and simvastatin reduced the apoptotic index (n=6 animals per group. * vs. **: P<0.001; no difference exists among * groups or ** groups: P>0.05, two-way ANOVA tests). D. Kaplan-Meier survival curves demonstrated simvastatin-treated mice had a survival advantage over untreated and saline-treated mice (burn vs. burn/saline: P>0.05; sham vs. burn or burn/saline vs. burn/statin: P< 0.01). Simvastatin reduced IL-1α, IL-6, MCP-1 and CRP production in the serum of burned mice (* P>0.05; * vs. **: P<0.01).

Figure 4

Effects of simvastatin treatment on TNF-α and NF-κB expressions in burned mice. A. Western blots analysis of the effects of simvastatin on TNF-α expression in mouse spleen (no difference exists within in * groups or ** groups, but significant difference was demonstrated between * and **groups: P<0.05). B. Western blots analysis of the effects of simvastatin on NF-κB expression in mouse spleen (no difference exists within * or ** groups, but significant difference was found between * and **groups, P<0.05). C. ELISA test of TNF-α in serum. Burn increased remarkably the expression of TNF-α in serum (No difference exists among * or ** groups: P>0.05; * vs. **: P<0.005 (n=6 animals per group). D. ELISA test of NF-κB in serum. Burn induced remarkably increased expression of NF-κB in serum (n=6 animals per group. No difference exists among * or ** groups: P>0.05; * vs. **: P<0.005).

Figure 5

Effects of TNF-α KO and NF-κB KO on simvastatin treatment and survival rate. A. simvastatin exhibited no additive decrease in spleen apoptosis in TNF-α KO mice after burn injury (no difference within * or *** groups: P>0.05; * vs. ** vs. ***: P<0.01; by two-way ANOVA test). B. simvastatin treatment produced no additive survival benefit in burned TNF-α KO mice (Burn/ TNF-α KO vs. burn/ TNF-α KO/statin: P>0.05; sham vs. burn/ TNF-α KO or burn/ TNF-α KO/statin vs. burn/WT: P<0.01). C. simvastatin exhibited no additive reduction in spleen apoptosis in burned NF-κB KO mice (no difference among * or *** groups: P>0.05; * vs. ** vs. ***: P<0.01 by two way ANOVA test). D. simvastatin treatment produced no additive survival benefit to burned NF-κB KO mice (Burn/ NF-κB KO vs. burn/ NF-κB KO/statin: p>0.05; sham vs. burn/NF-κB KO or burn/ NF-κB KO/statin vs. burn/WT: P<0.01).