| [Objective]Advanced glycation end products (AGEs), the final products of nonenzymatic glycation and oxidation of proteins, are found in the plasma and accumulated in the tissues during aging and at an accelerated rate in diabetes, renal failure and atherosclerosis. However, their effects on cardiac myocytes have not been elucidated. In this study, we investigated the effects of AGEs on cell cycle distribution, the magnitude of apoptosis and the expression of the related protein in cultured neonatal rat cardiac myocytes (CMs). We also studied the interfering action of Losartan on CMs apoptosis induced by AGEs. An integral membrane protein, termed receptor for AGE (RAGE), forms a central part of the cell surface binding site for AGEs. Using cell immunohistochemistry, we detected the distribution of RAGE. In order to elucidate the cell signal transduction pathway of AGEs on CMs, we examined the activation of p38 mitogen-activated protein kinases (p38-MAPK) and extracellular signal regulated kinase (ERK) in CMs treated with AGEs.[Methods]CMs cultured by trypsin digestion method for 3-5 days in vitro were continuously cultured with DMEM including 1% FBS or 10% FBS for 24 hours. These cells were then exposed to vehicle (control), AGEs-HAS (50μg/ml, 100μg/ml) for 12, 24 and 48 hours. All cells were collected to analyze cell cycle distribution by Flow Cytometer(FC), the magnitude of apoptosis by FC and the number of apoptotic body/nucleus of CMs by In Situ Cell Death Detection kit (Fluorescein). The expressions of p57, p27, p21,hhLIM and the activation of p38-MAPK and ERK1/2 were measured by western blotting. Cell immunohistochemistry was applied in observation of the distribution of RAGE.[Results]1. Receptors for advanced glycation end products were visualized in CMs. They distributed widely at cell membrane, cytoplasm and nuclear membrane.2. AGEs had no effects on cell cycle distribution in CMs.3. AGEs could increase the number of apoptotic body/nucleus of CMs in a time-dependent manner to up to 0.55, 1.23 times at 24h, 48h respectively, as compared with the number at 12h in control cells. AGEs can significantly increase the number of apoptotic body/nucleus of CMs in a time- and dose-dependent manner. AGEs at 50μg/ml and 100μg/ml increased apoptotic body/nucleus number of CMs at different time points: 0.74 and 1.21 times respectively at 12h, 1.15 and 1.78 times at 24h, 0.83 and 1.19 times at 48h. The average number of apoptotic body/nucleus in the groups of 100μg/ml AGEs at 12, 24, 48 hours were 0.27, 0.29, 0.20 times than the AGEs 50μg/ml treatment (P<0.001).4. Losartan could inhibit AGEs induced apoptosis in CMs. AGEs 50μg/ml + Losartan10-5 mol/L(M), 100μg/ml AGEs + Losartan10-5 M reduced the magnitude of apoptosis of CMs at different time points: 0.26 and 0.26 times respectively at 12h, 0.14 and 0.23times at 24h, 0.13 and 0.23 times at 48h(P<0.05).5. Compared with the control group, AGEs at 50μg/ml and 100μg/ml increased the expression of hhLIM at different time points: 103.8% and 177.8% at 12h; 27.1% and 32.6% at 24h. 48 hours after treatment with AGEs, its expression was not obvious again (p>0.05). The intervention of Losartan could not decrease this trend.6. The expression of p21 in CMs with AGEs treatment was weak and not obvious compared with control group (p>0.05). AGEs at 50μg/ml and100μg/ml increased the expression of p27 85.5%, 20.7% respectively at 12h while had no effects at 24 h and 48 h. The expression of p57 showed a fluctuant trend which decreased at 12h but increased at 24, 48h. AGEs at 50μg/ml and 100μg/ml decreased the expression of p57 at 54.3%, 73.7%(P<0.001)at 12h while increased 136.2%(P<0.001), 23.4%(P<0.05)at 48h. The intervention of Losartan could not change these trends.7. Stimulation of CMs with AGEs enhanced the activity of p38 and p44/42 MAPK. The activity of nonphosphorylated-p44/42 and non-phosphorylated-p38 did not change at each time point. AGEs-mediated p38 MAPK activity peaked at 15min (1.41 fold vs co...
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