Cerebral Cortex Apoptosis in Early Aged Hypertension: Effects of Epigallocatechin-3-Gallate

Abstract

This study aimed to investigate cerebral cortex apoptosis on the early aged hypertension and the effects of green tea flavonoid epigallocatechin-3-gallate (EGCG). Twenty-four rats were divided into three groups: a control Wistar-Kyoto group (WKY, n = 8), a spontaneously early aged hypertensive group (SHR, n = 8), and an early aged hypertension with EGCG treatment group (SHR-EGCG, n = 8; daily oral EGCG 200 mg/kg-94%, 12 weeks). At 48 weeks old, blood pressures (BPs) were evaluated and cerebral cortexes were isolated for TUNEL assay and Western blotting. Systolic, diastolic, and mean blood pressure levels in the SHR-EGCG were reduced compared to the SHR. The percentage of neural cell deaths, the levels of cytosolic Endonuclease G, cytosolic AIF (Caspase-independent apoptotic pathway), Fas, Fas Ligand, FADD, Caspase-8 (Fas-mediated apoptotic pathway), t-Bid, Bax/Bcl-2, Bak/Bcl-xL, cytosolic Cytochrome C, Apaf-1, Caspase-9 (Mitochondrial-mediated apoptotic pathway), and Caspase-3 (Fas-mediated and Mitochondria-mediated apoptotic pathways) were increased in the SHR relative to WKY and reduced in SHR-EGCG relative to SHR. In contrast, the levels of Bcl-2, Bcl-xL, p-Bad, 14-3-3, Bcl-2/Bax, Bcl-xL/Bak, and p-Bad/Bad (Bcl-2 family-related pro-survival pathway), as well as Sirt1, p-PI3K/PI3K and p-AKT/AKT (Sirt1/PI3K/AKT-related pro-survival pathway), were reduced in SHR relative WKY and enhanced in SHR-EGCG relative to SHR. In conclusion, green tea flavonoid epigallocatechin-3-gallate (EGCG) might prevent neural apoptotic pathways and activate neural survival pathways, providing therapeutic effects on early aged hypertension-induced neural apoptosis.

Keywords: EGCG; caspase-independent apoptosis; hypertension; neural apoptosis; neural survival.

FIGURE 1

The line chart of systolic blood pressure (SBP), diastolic blood pressure (DBP), mean blood pressure (MBP), and pulse pressure (PP) levels with measurement time points in a control Wistar Kyoto (WKY) group, a spontaneously early aged hypertensive (SHR) group and an early aged hypertension with Epigallocatechin-3-gallate (EGCG) treatment (SHR-EGCG) group. Data were expressed as means ± SEM. ^∗∗∗p < 0.001 in the comparison between the SHR group or SHR-EGCG group with the WKY rat group. #p < 0.05 in the comparison between the SHR-EGCG group and the SHR group.

FIGURE 2

The TUNEL-positive neural cells in a control Wistar Kyoto (WKY) group, a spontaneously early aged hypertensive (SHR) group, and an early aged hypertension with Epigallocatechin-3-gallate (EGCG) treatment (SHR-EGCG) group. (A) Representative photomicrographs of DAPI staining (top, blue spots) and TUNEL assay (bottom, green spots) in cerebral cortex sections from the WKY, SHR, and SHR-EGCG groups (400× magnification). (B) The bar chart represents the percentage of TUNEL-positive neural cells relative to total DAPI-stained cells and expresses as mean values ± SEM (n = 6 in each group). ***p < 0.001 in the comparison between the SHR group and WKY rat group; ##p < 0.01 in the comparison between SHR-EGCG group and SHR group.

FIGURE 3

The Caspase-independent Endonuclease G (EndoG) and Apoptosis-inducing factor (AIF) apoptotic pathway in a control Wistar Kyoto (WKY) group, a spontaneously early aged hypertensive (SHR) group and an early aged hypertension with Epigallocatechin-3-gallate (EGCG) treatment (SHR-EGCG) group. (A) Representative Western blotting images of cytosolic AIF, mitochondrial AIF, cytosolic EndoG and mitochondrial EndoG in the cerebral cortex tissues from WKY, SHR, and SHR-EGCG groups with α-tubulin as an internal control. (B) Bars represent the protein quantification of cytosolic EndoG to mitochondrial EndoG as well as cytosolic AIF to mitochondrial AIF normalized by α-tubulin and COX IV, expressed as fold changes relative to the WKY group and mean values ± SEM (n = 6 in each group). **p < 0.01 and ***p < 0.001 in the comparison between the SHR group and WKY rat group; ##p < 0.01, in the comparison between the SHR-EGCG group and SHR group.

FIGURE 4

The upstream components of the Fas-mediated Caspase-dependent apoptotic pathway in a control Wistar Kyoto (WKY) group, a spontaneously early aged hypertensive (SHR) group and an early aged hypertension with Epigallocatechin-3-gallate (EGCG) treatment (SHR-EGCG) group. (A) Representative Western blotting images of Fas ligand (FasL), Fas receptor (Fas), and Fas-associated death domain (FADD) in the cerebral cortex tissues from the WKY, SHR, and SHR-EGCG groups with α-tubulin as an internal control. (B) Bars represent the protein quantification of FasL, Fas, and FADD normalized by α-tubulin, expressed as fold changes relative to the WKY group and mean values ± SEM (n = 6 in each group). **p < 0.01 and ***p < 0.001 in the comparison between the SHR group and the WKY rat group. #p < 0.05 and ##p < 0.01, in the comparison between SHR-EGCG group and SHR group.

FIGURE 5

The upstream components of the mitochondrial-mediated Caspase-dependent apoptotic pathway in a control Wistar Kyoto (WKY) group, a spontaneously early aged hypertensive (SHR) group and an early aged hypertension with Epigallocatechin-3-gallate (EGCG) treatment (SHR-EGCG) group. (A) Representative Western blotting images of the truncated BH3 Interacting Domain Death Agonist (t-Bid), Bcl-2 associated protein X (Bax), Bcl-2 homologous antagonist/killer (Bak), Bcl-2 associated agonist of cell death (Bad), B-cell lymphoma 2 (Bcl-2), B-cell lymphoma-extra large (Bcl-xL), p-Bad, and 14-3-3 in the cerebral cortex tissues from the WKY, SHR, and SHR-EGCG groups with α-tubulin as an internal control. (B,C) Bars represent the protein quantification of t-Bid, Bax/Bcl-2, Bak/Bcl-xL, and Bad, as well as Bcl-2, Bcl-xL, pBad and 14-3-3 normalized by α-tubulin, expressed as fold changes relative to the WKY group and mean values ± SEM (n = 6 in each group). **p < 0.01 and ***p < 0.001, in comparison between the SHR and WKY rat group; #p < 0.05, ##p < 0.01, and ###p < 0.001 in comparison between the SHR-EGCG group and SHR group.

FIGURE 6

The downstream components of the Caspase dependent-apoptotic pathway in a control Wistar Kyoto (WKY) group, a spontaneously early aged hypertensive (SHR) group and an early aged hypertension with Epigallocatechin-3-gallate (EGCG) treatment (SHR-EGCG) group. (A) Representative Western blotting images of Caspase-8 (Fas downstream), cytosolic Cytochrome C, mitochondrial Cytochrome C, Apaf-1, Caspase-9 (Mitochondrial downstream), Caspase-3 (Fas and Mitochondrial downstream) in the cerebral cortex tissues from the WKY, SHR, and SHR-EGCG groups with α-tubulin as an internal control. (B) Bars represent the protein quantification of Caspase-8, cytosolic Cytochrome C to mitochondrial Cytochrome C ratio, Apaf-1, Caspase-9, Caspase-3 normalized by α-tubulin, expressed as fold changes relative to the WKY group and mean values ± SEM (n = 6 in each group). **p < 0.01 and ***p < 0.001 in comparison between the SHR group and WKY rat group; ##p < 0.01, ###p < 0.001 in comparison between the SHR-EGCG group and SHR group.

FIGURE 7

The Sirt1/PI3K/AKT-related survival pathway in a control Wistar Kyoto (WKY) group, a spontaneously early aged hypertensive (SHR) group, and an early aged hypertension with EGCG treatment (SHR-EGCG) group. (A) Representative Western blotting images of Sirt1, PI3K, p-PI3K, AKT, and p-AKT in the cerebral cortex tissues from the WKY, SHR, and SHR-EGCG groups with α-tubulin as an internal control. (B) Bars represent the protein quantification of Sirt1, p-PI3K/PI3K, and pAKT/AKT normalized by α-tubulin, expressed as fold changes relative to the WKY group and mean values ± SEM (n = 6 in each group). **p < 0.01, in comparison between the SHR group and WKY rat group; ##p < 0.01, in the comparison between the SHR-EGCG group and SHR group.

FIGURE 8

The hypothesized diagram. We proposed that the cerebral cortex EndoG and AIF-related Caspase-independent, Fas-mediated Caspase-dependent and mitochondrial-mediated Caspase-dependent apoptotic pathways were promoted in early aged hypertension, whereas those pathways were suppressed after EGCG treatment. This hypothesis was evidenced by which early aged hypertension increased the levels of EndoG, and AIF-related Caspase-independent apoptotic proteins (EndoG, AIF), Fas-mediated Caspase-dependent apoptotic proteins (FasL, Fas, FADD, Caspase-8, Caspase-3), and mitochondrial-mediated Caspase-dependent apoptotic proteins (Bax, Bak, t-Bid, Cytochrome C, Apaf-1, Caspase-9, Caspase-3), whereas EGCG treatment reduced those levels in the early aged hypertensive brain. On the contrary, the Bcl-2 family-related and Sirt1/PI3K/AKT related pro-survival pathways were inhibited in the early aged hypertensive brain, while those pathways were enhanced by EGCG treatment. This hypothesis was evidenced in which early aged hypertension suppressed Bcl-2 family-related pro-survival proteins (Bcl-2, Bcl-xL, pBad, 14-3-3) and Sirt1/PI3K/AKT related pro-survival proteins (Sirt1, p-PI3K/PI3K, p-AKT/AKT), whereas EGCG treatment enhanced those levels in the early aged hypertensive brain.