Melatonin plays a protective role in postburn rodent gut pathophysiology

Abstract

Melatonin is a possible protective agent in postburn gut pathophysiological dynamics. We investigated the role of endogenously-produced versus exogenously-administered melatonin in a major thermal injury rat model with well-characterized gut inflammatory complications. Our rationale is that understanding in vivo melatonin mechanisms in control and inflamed tissues will improve our understanding of its potential as a safe anti-inflammatory/antioxidant therapeutic alternative. Towards this end, we tested the hypothesis that the gut is both a source and a target for melatonin and that mesenteric melatonin plays an anti-inflammatory role following major thermal injury in rats with 3rd degree hot water scald over 30% TBSA. Our methods for assessing the gut as a source of melatonin included plasma melatonin ELISA measurements in systemic and mesenteric circulation as well as rtPCR measurement of jejunum and terminal ileum expression of the melatonin synthesizing enzymes arylalkylamine N-acetyltransferase (AA-NAT) and 5-hydroxyindole-O-methyltransferase (HIOMT) in sham versus day-3 postburn rats. Our melatonin ELISA results revealed that mesenteric circulation has much higher melatonin than systemic circulation and that both mesenteric and systemic melatonin levels are increased three days following major thermal injury. Our rtPCR results complemented the ELISA data in showing that the melatonin synthesizing enzymes AA-NAT and HIOMT are expressed in the ileum and jejunum and that this expression is increased three days following major thermal injury. Interestingly, the rtPCR data also revealed negative feedback by melatonin as exogenous melatonin supplementation at a dose of 7.43 mg (32 micromole/kg), but not 1.86 mg/kg (8 micromole/kg) drastically suppressed AA-NAT mRNA expression. Our methods also included an assessment of the gut as a target for melatonin utilizing computerized immunohistochemical measurements to quantify the effects of exogenous melatonin supplementation on postburn gut mucosa barrier inflammatory profiles. Here, our results revealed that daily postburn intraperitoneal melatonin administration at a dose of 1.86 mg/kg (8 micromole/kg) significantly suppressed both neutrophil infiltration and tyrosine nitrosylation as revealed by Gr-1 and nitrotyrosine immunohistochemistry, respectively. In conclusion, our results provide support for high mesenteric melatonin levels and dynamic de novo gut melatonin production, both of which increase endogenously in response to major thermal injury, but appear to fall short of abrogating the excessive postburn hyper-inflammation. Moreover, supplementation by exogenous melatonin significantly suppresses gut inflammation, thus confirming that melatonin is protective against postburn inflammation.

Keywords: AA-NAT; ELISA; GR1.; HIOMT; Melatonin; burn; extravasation; gut barrier; ileum; immunohistochemistry; inflammation; jejunum; mesenteric; neutrophils; nitrotyrosine; rtPCR; sepsis.

Figure 1

The gut is a source of melatonin and levels of mesenteric melatonin and gut melatonin synthesizing enzymes are influenced by major themal injury as well as exogenous melatonin. A. In sham rats, ELISA melatonin measurements revealed that melatonin levels in mesenteric blood were around 9 times higher than those found in the general circulation (Far left bars). Contrariwise, plasma melatonin levels were profoundly increased in both mesenteric and general circulation in rats with a 3rd degree burn over 30% TBSA 3 days after thermal injury (TI, far right, p<0.01). Interestingly, these mesenteric and general circulation plasma melatonin levels were a very close match to circulating and mesenteric plasma melatonin levels found in sham rats 2 hours after exogenous melatonin (Sham+mel; 2mM, 1 ml/250 g b.wt., i.p.) injection. B. Gut AANAT and HIOMT mRNA expression levels were higher (^^) in thermally injured (TI) than sham control (*) ileum.C. AANAT mRNA expression in TI subjects was more suppressed by a higher (8 mM) than a lower (2 mM) dose of exogenous melatonin administered by IP injection. rtPCR products appear at 109 bp (AANAT), 260bp (HIOMT) and 222 bp (housekeeping GAPDH). All measurements were performed on blood samples and tissues obtained at ~ZT 8 of a 12:12 light cycle at resting conditions with free access to chow feed.

Figure 2

Melatonin decreases intestinal tissue inflammation as visualized by hematoxylin and eosin (H&E) staining as well as Gr-1 and nitrotyrosine immunohistrochemistry (IHC). Inflammation was highest in ileum tissue of thermally injured rats (TI, column 3) as evidenced by tissue infiltration with extravasated neutrophils (H&E, top row; and Gr-1 IHC, middle row; see insets) as well nitrotyrosine immunohidtrovhemistry (bottom row). These markers were markedly decreased in melatonin treated rats, be they sham with melatonin (sham+Mel; second column) or thermally inured (TI+Mel; 4^th column). All measurements were performed on samples obtained ~ZT 8 of a 12:12 light cycle at resting conditions with free access to chow feed.

Figure 3

Levels of postburn inflammation in the terminal ileum. Tissue inflammation as assessed using both relative Gr-1 immunofluorescence intensity (open circles) and Gr-1 immunopositive cell counting was significantly higher in thermally injured (TI) subjects relative to sham controls (p<0.01). The signal was significantly decreased in both control (p<0.01) and burn (p<0.05) subjects treated with daily IP melatonin injections (Sham+Mel and TI+Mel). Notably, variability in labeling intensity was least evident among melatonin treated sham subjects. All measurements were performed on samples obtained ~ZT 8 of a 12:12 light cycle at resting conditions with free access to chow feed.

Figure 4

Levels of postburn nitrosylation in the terminal ileum. Tissue nitrosylation as assessed using quantitative nitrotyrosine immunohistochemistry appeared significantly higher in thermally injured (TI) subjects relative to sham controls (p<0.01). The signal was significantly decreased in both control (p<0.01) and burn (p<0.05) subjects treated with daily IP melatonin injections (Sham+Mel and TI+Mel). Notably, variability in labeling intensity was least evident among melatonin treated sham subjects. All measurements were performed on samples obtained ~ZT 8 of a 12:12 light cycle at resting conditions with free access to chow feed.