Epigallocatechin gallate alleviates neuronal cell damage against focal cerebral ischemia in rats

Abstract

Cerebral ischemia is a neurological disorder that causes permanent disability and is sometimes fatal. Epigallocatechin gallate (EGCG) is a natural polyphenol that exerts beneficial antioxidant and anti-inflammatory effects. The aim of this study was to investigate the neuroprotective effects of EGCG against cerebral ischemia. Middle cerebral artery occlusion was surgically initiated to induce focal cerebral ischemia in adult male rats. EGCG (50 mg/kg) or vehicle was intraperitoneally injected just prior to middle cerebral artery occlusion (MCAO) induction. Neuronal behavior tests were performed 24 hr after MCAO. Brain tissues were isolated to evaluate infarct volume, histological changes, apoptotic cell death, and caspase-3 and poly ADP-ribose polymerase (PARP) levels. MCAO injury led to serious functional neurological deficits and increased infarct volume. Moreover, it induced histopathological lesions and increased the numbers of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in the cerebral cortex. However, EGCG improved MCAO-induced neurological deficits and reduced infarct volume, alleviated histopathological changes, and decreased TUNEL-positive cells in the cerebral cortex of MCAO rats. Western blot analysis showed increases of caspase-3 and PARP expression levels in MCAO rats with vehicle, whereas EGCG administration alleviated these increases after MCAO injury. These results demonstrate that EGCG exerts a neuroprotective effect by regulating caspase-3 and PARP proteins during cerebral ischemia. In conclusion, we suggest that EGCG acts as a potent neuroprotective agent by modulating the apoptotic signaling pathway.

Keywords: caspase-3; epigallocatechin gallate; neuroprotection; poly ADP-ribose polymerase.

Fig. 1.

Neurobehavioral scores (A) and edema measurements (B) in vehicle+sham, epigallocatechin gallate (EGCG)+sham, vehicle+middle cerebral artery occlusion (MCAO), and EGCG+MCAO animals. EGCG attenuated the functional neurological deficits and edema caused by ischemic stroke. Data (n=4) are represented as mean ± S.E.M. *P<0.01, **P<0.05 vs. vehicle+sham animals, ^#P<0.05 vs. vehicle+MCAO animals.

Fig. 2.

Representative photograph of Triphenyltetrazolium chloride (TTC) staining (A) and infarct volume (B) in vehicle+sham, epigallocatechin gallate (EGCG)+sham, vehicle+middle cerebral artery occlusion (MCAO), and EGCG+MCAO animals. Intact areas stained red, while ischemic areas remained white in color (A). Infarct volume was calculated as the ratio of infarct area to total brain area (B). EGCG attenuated MCAO-induced infarction. Data (n=4) are represented as mean ± S.E.M. *P<0.01, **P<0.05 vs. vehicle+sham animals, ^#P<0.05 vs. vehicle+MCAO animals.

Fig. 3.

Representative photograph of hematoxylin and eosin staining (A–D) in vehicle+sham, epigallocatechin gallate (EGCG)+sham, vehicle+middle cerebral artery occlusion (MCAO), and EGCG+MCAO animals. EGCG alleviated histopathological changes caused by MCAO. Filled arrows indicate shrunken and condensed nuclei, and open arrows indicate swelled and vacuolated forms. Scale bar=100 µm.

Fig. 4.

Representative photos of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining (A–D) in the cerebral cortex in vehicle+sham, epigallocatechin gallate (EGCG)+sham, vehicle+middle cerebral artery occlusion (MCAO), and EGCG+MCAO animals. The number of TUNEL positive cells was markedly increased in vehicle+MCAO animals, while EGCG decreased the number of these positive cells (A and B). Arrows indicate cells with positive TUNEL staining. Scale bar: 100 µm. Apoptotic index (E) indicates the percentage of TUNEL-positive cells to total cells. Data (n=4) are shown as mean ± S.E.M. *P<0.01, **P<0.05 vs. vehicle+sham animals, ^#P<0.05 vs. vehicle+MCAO animals.

Fig. 5.

Western blot analysis of poly ADP-ribose polymerase (PARP) and caspase-3 proteins in vehicle+sham, epigallocatechin gallate (EGCG)+sham, vehicle+middle cerebral artery occlusion (MCAO), and EGCG+MCAO animals. EGCG prevents MCAO-induced increases of PARP and caspase-3 expressions. Densitometric analysis is represented as a ratio of PARP (B) and caspase-3 (D) staining intensity to actin intensity. Data (n=4) are shown as mean ± S.E.M. *P<0.01, **P<0.05 vs. vehicle+sham animals, ^#P<0.05 vs. vehicle+MCAO animals.