Flos Puerariae extract prevents myocardial apoptosis via attenuation oxidative stress in streptozotocin-induced diabetic mice

Abstract

Background: Diabetic cardiomyopathy (DCM) suggests a direct cellular insult to myocardium. Apoptosis is considered as one of the hallmarks of DCM. Oxidative stress plays a key role in the pathogenesis of DCM. In this study, we explored the prevention of myocardial apoptosis by crude extract from Flos Puerariae (FPE) in experimental diabetic mice.

Methods: Experimental diabetic model was induced by intraperitoneally injection of streptozotocin (STZ, 50 mg/kg/day) for five consecutive days in C57BL/6J mice. FPE (100, 200 mg/kg) was orally administrated once a day for ten weeks. Cardiac structure changes, apoptosis, superoxide production, NADPH oxidase subunits expression (gp91phox, p47phox, and p67phox), and related regulatory factors were assessed in the heart of mice.

Results: Diabetic mice were characterized by high blood glucose (≥11.1 mmol/L) and reduced body weight. In the end of the experiment, aberrant myofilament structure, as well as TUNEL positive cardiac cells coupled with increased Bax/Bcl-2 ratio and Caspase-3 expression was found in diabetic mice. Moreover, ROS formation, the ratio of NADP+/NADPH and NADPH oxidase subunits expression of gp91phox and p47phox, lipid peroxidation level was significantly increased, while antioxidant enzyme SOD and GSH-Px activity were reduced in the myocardial tissue of diabetic mice. In contrast, treatment with FPE resulted in a normalized glucose and weight profile. FPE administration also preserved myocardial structure and reduced apoptotic cardiac cell death in diabetic mice. The elevated markers of oxidative stress were significantly reversed by FPE supplementation. Further, FPE treatment markedly inhibited the increased Bax/Bcl-2 ratio and Caspase-3 expression, as well as suppressed JNK and P38 MAPK activation in the heart of diabetic mice.

Conclusions: Our data demonstrate for the first time that FPE may have therapeutic potential for STZ-induced diabetic cardiomyopathy through preventing myocardial apoptosis via attenuation oxidative stress. And this effect is probably mediated by JNK and P38 MAPK signaling pathway.

Figure 1. FPE ameliorated myocardial injury in experimental diabetic mice.

A: Representative pictures of myocardial tissue sections stained with hematoxylin and eosin (magnification = 400×), n = 6 per group. Arrow a indicates perinuclear vacuolization, bar is 20 µm. B: Representative transmission electron micrographs of left ventricular specimens, n = 4 per group. Arrow b indicates swollen mitochondria. C: Representative pictures of the TUNEL assay, n = 4 per group, bar is 20 µm. HFPE: high dose, 200 mg/kg; LFPE: low dose, 100 mg/kg.

Figure 2. FPE decreased blood glucose and normalized body weight in experimental diabetic mice.

A: FPE decreased blood glucose in the experimental diabetic mice. B: FPE increased body weights in the experimental diabetic mice. HFPE: high dose, 200 mg/kg; LFPE: low dose, 100 mg/kg. Blood glucose and body weight were measured in the basal fasting state on the day the mice were killed. Data are mean ± SD. ^* P<0.05 vs control group; ^# P<0.05 vs DM group, n = 10–12 per group.

Figure 3. FPE decreased ROS production in experimental diabetic mice.

Representative fluorescence images of heart sections stained with DHE(magnification = 400×), n = 3 per group, bar is 20 µm. HFPE: high dose, 200 mg/kg; LFPE: low dose, 100 mg/kg.

Figure 4. FPE inhibited NADPH oxidase activity and subunits expression in experimental diabetic mice.

A: FPE decreased the ratio of NADP⁺/NADPH in experimental diabetic mice, n = 6 per group. B: western blots analysis of gp91^phox, n = 3 per group. C: western blots analysis of p47^phox, n = 3 per group. D: western blots analysis of p67^phox, n = 3 per group. HFPE: high dose, 200 mg/kg; LFPE: low dose, 100 mg/kg. Data are mean ± SD. ^* P<0.05 vs control group; ^# P<0.05 vs DM group.

Figure 5. FPE reduced oxidative stress in experimental diabetic mice.

A: FPE effected SOD activity in heart tissue. SOD: superoxide dismutase. B: FPE increased GSH-Px activity in heart tissue. GSH-Px: glutathione peroxidase. C: FPE decreased MDA content in heart tissue. MDA: malondialdehyde. HFPE: high dose, 200 mg/kg; LFPE: low dose, 100 mg/kg. Data are mean ± SD, ^* P<0.05 vs control group; ^# P<0.05 vs DM group, n = 10–12 per group.

Figure 6. FPE regulated the expression of Bcl-2, Bax and Caspase-3 proteins in experimental diabetic mice.

A–C: Representative immunohistochemical staining of Bax, Bcl-2 and Caspase-3 expression, respectively (magnification = 400×), n = 5 per group, bar is 20 µm. D: Representative immunohistochemical staining quantitative analysis of Bax, Bcl-2 and Caspase-3 expression. E: western blots analysis of Bcl-2 and Bax expression, n = 3 per group. HFPE: high dose, 200 mg/kg; LFPE: low dose, 100 mg/kg. Data are mean ± SD. ^* P<0.05 vs control group; ^# P<0.05 vs DM group.

Figure 7. FPE attenuates mitogen activated protein kinases (MAPK) activation in experimental diabetic mice.

A: western blots analysis of p-P38MAPK and P38MAPK. B: western blots analysis of p-JNK and JNK. C: western blots analysis of p-ERK1/2 MAPK and ERK1/2 MAPK. HFPE: high dose, 200 mg/kg; LFPE: low dose, 100 mg/kg. Data are mean ± SD, ^* P<0.05 vs control group; ^# P<0.05 vs DM group, n = 3 per group.