Burns, inflammation, and intestinal injury: protective effects of an anti-inflammatory resuscitation strategy

Abstract

Background: Intestinal barrier breakdown after severe burn can lead to intestinal inflammation, which may act as the source of the systemic inflammatory response. In vitro intestinal cell studies have shown that mitogen-activated protein kinase (MAPK) signaling is an important modulator of intestinal inflammation. We have previously observed that pentoxifylline (PTX) attenuates burn-induced intestinal permeability and tight junction breakdown. We hypothesized that PTX would limit intestinal barrier breakdown and attenuate inflammatory signaling via the MAPK pathway.

Methods: Male balb/c mice underwent 30% total body surface area full-thickness steam burn. Immediately after burn, animals received an intraperitoneal injection of PTX (12.5 mg/kg) in normal saline or normal saline alone. In vivo intestinal permeability to 4 kDa fluorescein isothiocyanate-dextran was measured. Intestinal extracts were obtained to measure interleukin-6 by enzyme-linked immunosorbent assay, and phosphorylated p38 MAPK, p38 MAPK, phosphorylated extracellular signal-related kinase (1/2) (ERK (1/2)), and ERK (1/2) by immunoblotting. Acute lung injury was assessed by histology at 24 hours after burn.

Results: Administration of PTX immediately after injury attenuated burn-induced intestinal permeability. PTX also decreased the burn-induced phosphorylation of p38 MAPK and decreased phosphorylation of ERK (1/2) at 2 hours and 24 hours after injury. Animals given PTX had decreased intestinal interleukin-6 levels. A single dose of PTX also decreased histologic lung injury at 24 hours after burn.

Conclusion: PTX attenuates burn-induced intestinal permeability and subsequent intestinal inflammation. Use of PTX after burn was also associated with decreased acute lung injury. Because of its compelling anti-inflammatory effects, PTX may be an ideal candidate for use as an immunomodulatory adjunct to resuscitation fluid.

Figure 1

Effects on PTX on intestinal IL-6 levels after 30% TBSA full-thickness burn. IL-6 levels measured from intestinal extracts using enzyme-linked immunosorbent assay. (A) PTX limits the burn-induced increase in intestinal IL-6 concentration at 2 hours after burn. (B) Intestinal IL-6 remains elevated at 24 hours after burn, whereas animals given PTX immediately after burn have intestinal IL-6 levels similar to sham. *p < 0.001 versus sham, †p < 0.01 versus burn at 2 hours, and **p < 0.05 versus burn at 24 hours.

Figure 2

Pentoxifylline prevents burn-induced intestinal permeability. Intestinal permeability assay to intraluminal 4 kDa FITC-dextran measured 2 hours after severe burn (n ≥ 3 animals per group). Burn injury causes a significant increase in intestinal permeability. Injection with PTX (12.5 mg/kg) immediately after injury attenuates this burn-induced increased in intestinal permeability. *p < 0.01 versus sham and †p < 0.05 versus burn.

Figure 3

Burn-induced phosphorylation of intestinal p38 MAPK is attenuated by PTX. p38 MAPK from intestinal extracts evaluated using Western blot (n ≥ 3 animals per group). (A) Burn injury results in an 8-fold increase in phosphorylated p38 MAPK at 2 hours after burn injury. Treatment with PTX after injury results in phosphorylated p38 MAPK levels similar to sham. (B) Phosphorylated p38 MAPK protein expression remains increased at 24 hours after burn. PTX continues to prevent the burn-induced phosphorylation of intestinal p38 MAPK. There is no difference in total p38 MAPK expression in either group. *p < 0.001 versus sham and †p < 0.001 versus burn at 2 hours. **p < 0.01 versus sham and ‡p < 0.01 versus burn at 24 hours.

Figure 4

Activation of intestinal ERK ½ after severe burn is decreased by PTX. Phosphorylated ERK ½ measured from intestinal extracts using Western blot (n ≥ 3 animals per group). (A) PTX prevents the burn-induced phosphorylation of intestinal ERK ½ at 2 hours after 30% TBSA burn. (B) Phosphorylated ERK ½ decreases somewhat by 24 hours after injury but remains significantly higher than sham. Treated with PTX continues to limit intestinal inflammation, with phosphorylated ERK ½ expression similar to sham. There is no difference in total ERK ½ protein expression in either group. *p < 0.01 versus sham and †p < 0.01 versus burn at 2 hours. **p < 0.001 versus sham and ‡p < 0.01 versus burn at 24 hours.

Figure 5

Pentoxifylline attenuates burn-induced distant organ injury. The lung sections harvested 24 hours after burn-injury stained with hematoxylin-eosin and viewed at ×20 magnification (n = 3 animals per group). (A) Normal appearance of the lung tissue from sham animal. (B) Evidence of acute lung injury in animals 24 hours after burn characterized by cellular infiltration, presence of hyaline membranes, and intra-alveolar hemorrhage. (C) Appearance of the lung tissue from animals given PTX immediately after burn is noticeably improved at 24 hours compared with burn with only minimal cellular infiltration. Bar = 100 μm.