Molecular analysis of endoplasmic reticulum stress response after global forebrain ischemia/reperfusion in rats: effect of neuroprotectant simvastatin

Abstract

Simvastatin is a cholesterol-lowering agent whose functional significance and neuroprotective mechanism in ischemic brain injury is not yet solved. The purpose of this study is to evaluate the effect of simvastatin on ischemic brain injury. We examined the endoplasmic reticulum stress response (UPR/unfolded protein response), by measuring the mRNA and protein levels of specific genes such as ATF6, GRP78, and XBP1 after 15 min 4-VO ischemia and different times of reperfusion (1, 3, and 24 h). The results from the group of naïve ischemic rats were compared with results from the group of pre-treated animals with simvastatin. The results of the experiments showed significant increase in all genes at the mRNA level in ischemic phase (about 43% for XBP1, 58% for GRP78, and 39% for ATF6 more than control). The protein level of XBP1 was decreased in pre-treated animals at ischemic phase and first hour of reperfusion (about 15% less), and did not reach control levels. The protein levels of GRP78 were maximal at third hour of reperfusion in statin group with a small decrease at 24 h of reperfusion in both groups. The levels of ATF6 mRNA in statin-treated animals was higher in comparison to non-statin animals at the ischemic phase and the third hour of reperfusion (about 35% higher), which was also translated into the higher protein level. This could indicate that one of the main proteins targeted to enhance neuroprotective effect to ER during the first two hours of reperfusion was ATF6 protein, the levels of which were 60% higher than in non-treated animals. These data suggest that simvastatin, in addition to the proposed neuroprotective effect, exerts a neuroprotective role in the attenuation of ER stress response after acute ischemic/reperfusion insult.

Fig. 1

Ponceau staining after Western blot (a) non-treated group of animals and (b) simvastatin pre-treated group of animals

Fig. 2

mRNA levels of XBP1 in rat forebrain after 15 min. ischemia and 1, 3, and 24 h reperfusion, comparison between groups with and without simvastatin pre-treatment, respectively. Results are presented as mean ± SEM for n = 6, normalized to control levels. * P < 0.05; *** P < 0.001 significantly different as compared to naïve and pre-treated controls, respectively. ⁺ P < 0.05 and ⁰⁰ P < 0.01 mean significantly different between naïve ischemic and pre-treated animals in the same time points

Fig. 3

Protein levels of XBP1 in rat forebrain after 15 min. ischemia and 1, 3, and 24 h reperfusion (a) comparison between groups with and without simvastatin pre-treatment, respectively. Results are presented as mean ± SEM for n = 6, normalized to control levels. ** P < 0.01 significantly different as compared to pre-treated control animals. ^{+++, ###} P < 0.001 mean significantly different between naïve ischemic and pre-treated animals in the same time points (b) detected bands for protein XBP1 in naïve ischemic animals. (c) detected bands for protein XBP1 in simvastatin pre-treated group of animals

Fig. 4

mRNA levels of GRP78 in rat forebrain after 15 min. ischemia and 1, 3, and 24 h reperfusion, comparison between groups with and without simvastatin pre-treatment, respectively. Results are presented as mean ± SEM for n = 6, normalized to control levels. * P < 0.05; ** P < 0.01; *** P < 0.001 significantly different as compared to naïve and pre-treated controls, respectively. ^{+++, 000} P < 0.001 mean significantly different between naïve ischemic and pre-treated animals in the same time points

Fig. 5

Protein levels of GRP78 in rat forebrain after 15 min. ischemia and 1, 3, and 24 h reperfusion (a) comparison between groups with and without simvastatin pre-treatment, respectively. Results are presented as mean ± SEM for n = 6, normalized to control levels. ** P < 0.01 significantly different as compared to naïve and pre-treated controls, respectively. ⁺⁺ P < 0.01; ^{0, X} P < 0.5 mean significantly different between naïve ischemic and pre-treated animals in the same time points. (b) Detected bands for protein GRP78 in naïve ischemic animals. (c) detected bands for protein GRP78 in simvastatin pre-treated group of animals

Fig. 6

mRNA levels of ATF6 in rat forebrain after 15 min. ischemia and 1, 3, and 24 h reperfusion, comparison between groups with and without simvastatin pre-treatment, respectively. Results are presented as mean ± SEM for n = 6, normalized to control levels. * P < 0.05; *** P < 0.001 significantly different as compared to naïve and pre-treated controls, respectively. ^# P < 0.05; ⁰⁰  P < 0.01; ^XXX P < 0.001 mean significantly different between naïve ischemic and pre-treated animals in the same time points

Fig. 7

Protein levels of ATF6 in rat forebrain after 15 min. ischemia and 1, 3, and 24 h reperfusion (a) comparison between groups with and without simvastatin pre-treatment distinctively. Results are presented as mean ± SEM for n = 6, normalized to control levels. * P < 0.05 significantly different as compared to naïve and pre-treated controls, respectively. ⁺⁺ P < 0.01; ^{000, ***, ###} P < 0.001 mean significantly different between naïve ischemic and pre-treated animals in the same time points. B) detected bands for protein ATF6 in naïve ischemic animals. C) detected bands for protein ATF6 in simvastatin pre-treated group of animals