Anti-Apoptotic and Pro-Survival Effect of Alpinate Oxyphyllae Fructus (AOF) in a d-Galactose-Induced Aging Heart

Abstract

Aging, a natural biological/physiological phenomenon, is accelerated by reactive oxygen species (ROS) accumulation and identified by a progressive decrease in physiological function. Several studies have shown a positive relationship between aging and chronic heart failure (HF). Cardiac apoptosis was found in age-related diseases. We used a traditional Chinese medicine, Alpinate Oxyphyllae Fructus (AOF), to evaluate its effect on cardiac anti-apoptosis and pro-survival. Male eight-week-old Sprague-Dawley (SD) rats were segregated into five groups: normal control group (NC), d-Galactose-Induced aging group (Aging), and AOF of 50 (AL (AOF low)), 100 (AM (AOF medium)), 150 (AH (AOF high)) mg/kg/day. After eight weeks, hearts were measured by an Hematoxylin-Eosin (H&E) stain, Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-assays and Western blotting. The experimental results show that the cardiomyocyte apoptotic pathway protein expression increased in the d-Galactose-Induced aging groups, with dose-dependent inhibition in the AOF treatment group (AL, AM, and AH). Moreover, the expression of the pro-survival p-Akt (protein kinase B (Akt)), Bcl-2 (B-cell lymphoma 2), anti-apoptotic protein (Bcl-xL) protein decreased significantly in the d-Galactose-induced aging group, with increased performance in the AOF treatment group with levels of p-IGFIR and p-PI3K (Phosphatidylinositol-3' kinase (PI3K)) to increase by dosage and compensatory performance. On the other hand, the protein of the Sirtuin 1 (SIRT1) pathway expression decreased in the aging groups and showed improvement in the AOF treatment group. Our results suggest that AOF strongly works against ROS-induced aging heart problems.

Keywords: ">d-galactose-induced aging; AOF; SIRT1; apoptosis.

Figure 1

Echocardiography findings. Representative echocardiographic M-mode images from rats with d-galactose and Alpinate Oxyphyllae Fructus (AOF) treatment. AL (AOF low), AM (AOF medium), AH (AOF high) represent the doses of 50, 100 and 150 mg of Alpinate Oxyphyllae Fructus per kg BW (Body weight). The long yellow arrow indicates cardiac diastole, and the short white arrow shows cardiac systole.

Figure 2

Morphological changes of rat cardiac tissue with Hematoxylin–Eosin (H&E) staining. Cardiac tissue sections stained with hematoxylin and eosin. The images of cardiac architecture were magnified ×400. The scale bar is 50 μm.

Figure 3

AOF treatment inhibits d-galactose-induced aging and apoptosis in Sprague–Dawley (SD) rat cardiac tissue. Cardiac tissue sections were stained with 4,6-diamidino-2-phenylindole (DAPI) (blue, nucleus) and TUNEL assay (green, double-stranded DNA break or single-stranded DNA nicks.), respectively. The statistical results were shown from three independent experiments; mean ± S.D.; *** p < 0.001, represent a significant difference versus the control; ^### p < 0.001, represent a significant difference versus the aging group.

Figure 4

Effect of AOF on d-galactose -induced apoptosis in SD rat cardiac tissue. (A) Immunohistochemical analysis for cleaved Caspase-3 in sections from the SD rat cardiovascular tissue. The protein levels decreased with AOF treatment and are shown in a brown color. Final magnifications: ×400 (bar, 50 μm). The total protein of SD rat cardiac tissue extracts was separated by 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, and immunoblotted with antibodies against Fas, Fas-associated death domain (FADD), caspase-8, cleaved caspase-3, B-cell lymphoma 2-associated X protein (Bax), cytochrome c and caspase-9 antibody to detect apoptotic markers expression (B,C); Levels of cytochrome c were determined from cytoplasmic and from the mitochondria (D). Equal loading was assessed with an anti-α-tubulin antibody. These blots were quantified by densitometry. α-tubulin served as a loading control. Data are presented as means ± S.D. Bars indicate averages, * p < 0.05; ** p < 0.01; *** p < 0.001, represent a significant difference versus the control; ^# p < 0.05; ^## p < 0.01; ^### p < 0.001, represent a significant difference versus the aging group. n = three independent experiments for each data point.

Figure 4

Effect of AOF on d-galactose -induced apoptosis in SD rat cardiac tissue. (A) Immunohistochemical analysis for cleaved Caspase-3 in sections from the SD rat cardiovascular tissue. The protein levels decreased with AOF treatment and are shown in a brown color. Final magnifications: ×400 (bar, 50 μm). The total protein of SD rat cardiac tissue extracts was separated by 12% SDS polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene difluoride (PVDF) membranes, and immunoblotted with antibodies against Fas, Fas-associated death domain (FADD), caspase-8, cleaved caspase-3, B-cell lymphoma 2-associated X protein (Bax), cytochrome c and caspase-9 antibody to detect apoptotic markers expression (B,C); Levels of cytochrome c were determined from cytoplasmic and from the mitochondria (D). Equal loading was assessed with an anti-α-tubulin antibody. These blots were quantified by densitometry. α-tubulin served as a loading control. Data are presented as means ± S.D. Bars indicate averages, * p < 0.05; ** p < 0.01; *** p < 0.001, represent a significant difference versus the control; ^# p < 0.05; ^## p < 0.01; ^### p < 0.001, represent a significant difference versus the aging group. n = three independent experiments for each data point.

Figure 5

Effect of AOF on d-galactose-induced survival in SD rat cardiac tissue. The total protein of SD rat cardiac tissue extracts was separated by 12% SDS-PAGE, transferred to PVDF membranes, and immunoblotted with antibodies against IGF1R, p-PI3K, AKT, Bcl-xL and Bcl2 antibody to detect survival markers expression. Equal loading was assessed with an anti-α-tubulin antibody. These blots were quantified by densitometry. α-tubulin served as a loading control. Data are presented as means ± S.D. Bars indicate averages, * p < 0.05, represent a significant difference versus the control; ^# p < 0.05; ^## p < 0.01; ^### p < 0.001, represent a significant difference versus the aging group. n = three independent experiments for each data point.

Figure 6

Effect of AOF on d-galactose-induced Sirtuin 1 (SIRT1) pathway related protein in SD rat cardiac tissue. The total protein of SD rat cardiac tissue extracts was separated by 12% SDS-PAGE, transferred to PVDF membranes, and immunoblotted with antibodies against p-AMPK, SIRT1, PGC-1α and PPARα antibody to detect survival markers expression. Equal loading was assessed with an anti-α-tubulin antibody. These blots were quantified by densitometry. α-tubulin served as a loading control. Data are presented as means ± S.D. Bars indicate averages, *** p < 0.001, represent a significant difference versus the control; ^# p < 0.05; ^### p < 0.001, represent a significant difference versus the aging group. n = three independent experiments for each data point.