Fig. 1. Chromatographic profile obtained on purification of the crude peptide material on an analytical Vydac C₁₈ column (250 mm × 4.6 mm, 300 Å pore size, 5 μm particle size) using a gradient of 20–60%B over 40 min at a flow rate of 1.0 ml min⁻¹ (A = 0.1%TFA–H₂O, B = 0.1%TFA in 90% CH₃CN–10% H₂O). The major peak eluting at 25 min contained octanoylated-ghrelin.

Fig. 2. Ghrelin (30 nmol/rat, i.p.) significantly enhanced food intake in both sham-burn and burn rats. (A) Effects of ghrelin or saline injections on 2 h food intake in freely feeding sham-burn and burn rats on day 3, 5, 7, 9 and 11 days after 30% TBSA burn. (B) Averaging 2 h food intake over the five test days revealed that the orexigenic effect of ghrelin was significantly greater than that of saline in both sham and burn groups.

Fig. 3. Investigation of the dose–response relationship of ghrelin on overnight (A) and 1 h (B) food intake in burn rats. (A) Burn injury significantly reduced food intake on post-burn day 3 compared to sham controls, and this was restored to normal by ghrelin at all doses used. ^*p < 0.01 vs. Burn (0 nmol ghrelin); ^†p < 0.05 vs. Burn (0 nmol ghrelin). (B) Ghrelin dose-dependently stimulated 1 h food intake on post-burn day 10. ^*p < 0.01 vs. Burn (0 nmol ghrelin) or Sham-burn. In both studies, rats were injected with saline alone or saline containing increasing doses of ghrelin (0.50 ml, i.p.).

Fig. 4. Effects of saline or ghrelin (10 nmol/rat, i.p.) on plasma GH levels in sham and burn rats on day 15 after 30% TBSA burn. Plasma was collected 30 min after the treatment.

Fig. 5. (A) Burn injury attenuated the expression of rat stomach ghrelin. Upper panel: semiquantitative data on the expression levels of ghrelin mRNA in rat stomach after burn injury or sham manipulations. Relative expression levels were obtained in each sample by normalization of absolute OD of the ghrelin signal to that of GAPDH signal. Values are mean ± S.E.M. of five independent determinations for each one of the test days. *p < 0.05 vs. Sham Control for that day. Lower panel: representative RT-PCR assay of the expression levels of ghrelin mRNA in rat stomach after 1, 3, 5, 7 or 10 days of burn injury (B) or sham manipulations (C). Amplification of GAPDH mRNA served as an internal control. The amplified products for ghrelin and GAPDH were 196 base pair (bp) and 86 bp, respectively. M denotes molecular weight markers. (B) Total plasma levels of ghrelin (octanoylated-ghrelin + non-octanoylated-ghrelin) were significantly lower on day 1 after 30% TBSA burn compared to sham-burn controls.

Fig. 6. Possible beneficial effects of ghrelin in attenuating burn-induced cachexia. Peripherally administered ghrelin could counteract these dysfunctions via increasing synthesis and release of: (1) central NPY/AGRP, hormones which promotes food (energy) intake, while reducing energy expenditure [23] and [38]; and (2) the anabolic hormones, GH and IGF-I, which could directly attenuate muscle catabolism and promote muscle protein synthesis [8], [27] and [31]. Increased levels of GH and IGF-I may also improve gut barrier function [18], and promotes rapid closure of burn wounds [27]. Ghrelin could also enhance immune response through direct inhibition of Th1, Th2 and other proinflammatory cytokines [9] and [42].

Scheme 1.