Acute effect of Ghrelin on ischemia/reperfusion injury in the rat spinal cord

Abstract

Ghrelin, a 28-amino acid peptide, is mainly secreted by the stomach. Ghrelin has been shown to have neuroprotective effects. However, whether ghrelin protects the spinal cord from ischemia/reperfusion (I/R) injury is unknown. To investigate this, 60 rats were randomly divided into three different groups: the sham group (n = 20), the vehicle group (n = 20), and the Ghrelin group (100 μg/kg, n = 20). Rats were sacrificed 12, 24, 48 and 72 h after ischemia. After the evaluation of neurologic function (48 h), the spinal cords were immediately removed for the determination of myeloperoxidase (MPO) activity (12-72 h). Apoptosis was quantitatively measured using the terminal transferase UTP nick end-labeling (TUNEL) method (24 h). The expression of bax and bcl-2 were evaluated by Western blot analysis (1 h), and GHSR-1a mRNA expression was detected using reverse transcriptase polymerase chain reaction (24 h). The neurological motor function was evaluated by 'Tarlov's score'. The neurologic outcomes in the ghrelin-group were significantly better than those in the vehicle group (p < 0.05). Serum tumor necrosis factor (TNF-α) levels were assessed in the peripheral venous blood. Ghrelin decreased the serum TNF-α levels and ameliorated the down regulation of spinal cord MPO activity. The expression of ghrelin receptors (GHSR-1a) in the rat spinal cord was decreased by I/R injury and increased by ghrelin. Ghrelin reduced the TUNEL-positive rate. Greater bcl-2, HSP27, HSP70, and attenuated bax expression were observed in the ghrelin-treated rats. Our results suggest that ghrelin administration may inhibit spinal I/R injury. Moreover, the improvement of neurologic function in rats was increased after the ghrelin treatment.

Keywords: ghrelin; ischemia/reperfusion injury; spinal cord.

Figure 1

Effects of ghrelin on motor function of hind limbs. The numbers of animals in each group was 20. One rat each died in vehicle group and sham group. The degree of motor disturbance was assessed by Tarlov’s score evaluated during a 48 h observation period. Rats subjected to I/R injury showed significant effects on hindlimb movement (* p < 0.05). Ghrelin (100 μg/kg) reduced the degree of motor disturbances induced by I/R injury, the neurologic function was significantly better than in the vehicle group (** p < 0.05).

Figure 2

Expression of ghrelin receptor GHSR-1a in the spinal cord. Expressions of GHSR-1a were presented in the treatment groups by RT-PCR at 24 h after I/R. The GHSR-1a were 165 bp. Compared with the sham group, the I/R group showed significantly reduced GHSR-1a mRNA expression (* p < 0.01) that is reversed by ghrelin (** p < 0.05). GAPDH mRNA was also detected as an internal control (n = 5 per group).

Figure 3

Effects of ghrelin on TUNEL staining. Ghrelin attenuated cell apoptosis in rat spinal cord ischemia/reperfusion injury by TUNEL staining at 24 h ((a) Sham group; (b) vehicle group; (c) ghrelin group). TUNEL-staining positive cells are brown in (A). The number of TUNEL-staining positive cells in the ghrelin group is less than in the I/R group, also, the positive cells in both the vehicle and ghrelin groups are significantly different compared with sham group (* p < 0.05, (B)) (n = 5 per group).

Figure 4

Effects of ghrelin on expression of HSP70 and HSP27. Heat shock protein 27,70 (HSP27,70) in spinal cord after 1h of ischemia followed by different times of reperfusion by Western blot. ((A) HSP70; (B) HSP27) Relative density given by the ratio of target band to GADPH band. * p < 0.05 vs. corresponding NS group. (n = 5 per group and per time point).

Figure 5

Effects of ghrelin on expression of Bax and Bcl-2. Bcl-2/Bax protein in spinal cord after 1 h of ischemia followed by different times of reperfusion by Western blot. ((A) Bax; (B) bcl-2) Relative density given as the ratio of target band to GADPH band. * p < 0.05 vs. corresponding NS group. (n = 5 per group and per time point).