Hemin offers neuroprotection through inducing exogenous neuroglobin in focal cerebral hypoxic-ischemia in rats

Abstract

Objective: To investigate the inducible effect of hemin on exogenous neuroglobin (Ngb) in focal cerebral hypoxic-ischemia in rats.

Methods: 125 healthy SD rats were randomly divided into five groups: sham-operation control group, operation group, hemin treatment group, exogenous Ngb treatment group, and hemin and exogenous Ngb joint treatment group. Twenty-four hours after focal cerebral hypoxic-ischemia, Ngb expression was evaluated by immunocytochemistry, RT-PCR, and western blot analyses, while the brain water content and infarct volume were examined.

Results: Immunocytochemistry, RT-PCR, and western blot analyses showed more pronounced Ngb expression in the hemin and exogenous Ngb joint operation group than in the hemin or exogenous Ngb individual treatment groups, thus producing significant differences in brain water content and infarct volume (p<0.05).

Conclusions: Hemin may be beneficial in protecting against focal cerebral hypoxic-ischemia through inducing the expression of exogenous Ngb.

Keywords: Hemin; focal cerebral hypoxic-ischemia; neuroglobin; neuroprotection.

Figure 1

Hemin, exogenous Ngb, and Hemin plus exogenous Ngb decrease infract volume by cerebral ischemia-induced injury. Five rats of each group were treated as indicated and sacrificed 24 h after a permanent focal right cerebral middle artery cerebral ischemia operation. The brains were frozen at -20°C and a total of six consecutive coronal sections were taken at 2-mm intervals and submitted for TTC stain. Photos of the brain sections were taken and the volume of the brain infarct were calculated. A: Sham-operation group; B: Operation group; C: Hemin treatment group; D: Exogenous Ngb treatment group; E: Hemin and exogenous Ngb joint treatment group.

Figure 2

Hemin increases expression of exogenous Ngb in rats under focal cerebral Hypoxic-Ischemic conditions. Cerebral cortex Ngb immunohistochemical examinations of indicated group were shown. A: Sham-operation group; B: Operation group; C: Hemin treatment group; D: Exogenous Ngb treatment group; E: Hemin and exogenous Ngb joint treatment group (The brown positive substances in the cytoplasm were Ngb immune-positive neuron cells; the neuron nuclei were blue; the light blue or colorless cytoplasm was the negative control).

Figure 3

The expression of Ngb mRNA in cerebral cortex of different treatments. Rat cerebral cortex of indicated groups were obtained and total RNA were extracted from tissue homogenate and were analyzed by RT-PCR using primers appropriate for the Ngb transcripts. GAPDH were used as the internal control. P+H: Hemin and exogenous Ngb joint treatment group; P: Exogenous Ngb treatment group; H: Hemin treatment group; M: Operation group; B: Blank; GAPDH: internal control.

Figure 4

Variation in the expression of Ngb mRNA in the rat cerebral tissues of each group. Rat cerebral cortex of indicated groups were obtained and total RNA were extracted and analyzed by RT-PCR using Ngb primers. P+H: Hemin and exogenous Ngb joint treatment group; P: Exogenous Ngb treatment group; H: Hemin treatment group; M: operation group; B: Blank control.

Figure 5

Hemin, exogenous Ngb, and Hemin plus exogenous Ngb increases the expression of Ngb protein in cortex of MCAO rat. Rat cerebral cortex of indicated groups were obtained and proteins were extracted from tissue homogenate and analyzed by Western blots using antibodies against Ngb. β-actin was used as the internal control. P+H: Hemin and exogenous Ngb joint treatment group; P: Exogenous Ngb treatment group; H: Hemin treatment group; M: Operation group; B: Blank control; β-actin: internal control.