Mechanism of carvedilol against myocardial ischemia/reperfusion injury / 医学研究生学报

Objective Carvedilol (Cvd) has a potential cardioprotective effect against myocardial ischemia/reperfusion (I/R) injury but its molecular mechanism is not yet clarified. The present study aimed to investigate whether the mechanism of Cvd against myocardial I/R injury-induced cardiomyocyte apoptosis is associated with its protection of the myocardium by modulating the unfolded protein response (UPR).Methods Forty male SD rats were randomly divided into five groups of equal number, sham operation, I/R, I/R + UPR agonist dithiothreitol (I/R+DTT), I/R + DTT with Cvd pretreatment at 5 mg/kg (I/R+DTT+Cvd), and I/R with Cvd pretreatment at 5 mg/kg (I/R+Cvd). The myocardial infarct size (infarct area / area-at-risk, IA/AAR) was measured by TCC & Evans blue double staining, the left ventricular ejection fraction (LVEF) and left ventricular fractional shortening (LVFS) detected by echocardiography, the apoptosis of the myocardiocytes determined by TUNEL staining, the activation of the UPR signaling pathway and the expressions of Caspase-12 and Casoase-3 detected by Western blot, and the concentrations of LDH and CK-MB assayed with the detection kit, followed by a comprehensive evaluation of the effects of Cvd on myocardial I/R injury.Results Myocardial IA/AAR was significantly increased in the I/R+DTT group as compared with the sham operation control (\[54.1±3.28\] % vs \[24.25±3.19\] %, P<0.05), higher in the I/R+DTT and I/R+DTT+Cvd (P<0.05) but lower in the I/R+Cvd than in the I/R group (P<0.05), and lower in the I/R+DTT+Cvd than in the I/R+DTT group (P<0.05). In comparison with the sham operation control, all the other four groups showed significantly decreased LVEF and LVFS (P<0.05), both remarkably lower in the I/R+DTT (\[44.5±1.56\] % and \[19.2±2.23\] %) than in the I/R group (\[61.5±2.63\] % and \[28.4±1.42\] %) (P<0.05), but higher in the I/R+DTT+Cvd and I/R+Cvd groups (P<0.05). The apoptosis rate of the cardiomyocytes was markedly increased in the I/R, I/R+DTT, I/R+DTT+Cvd, and I/R+Cvd groups as compared with that in the sham operation control (\[24.4±2.65\]%, \[48.3±1.62\]%, \[32.6±1.28\] % and \[13.2±2.21\]% vs \[6.2±1.27\]%, P<0.05), higher in the I/R+DTT and I/R+DTT+Cvd (P<0.05) but lower in the I/R+Cvd than in the I/R group (P<0.05). Compared with the sham operation control, the other four groups exhibited significantly elevated levels of expressions of the GRP78, CHOP and ATF6 proteins (P<0.05), all markedly higher in the I/R+DTT (P<0.05) but lower in the I/R+Cvd (P<0.05) and that of GRP78 lower in the I/R+DTT+Cvd than in in the I/R group (P<0.05), and all lower in the I/R+DTT+Cvd than in the I/R+DTT group (P<0.05).Conclusion Carvedilol can significantly alleviate the apoptosis of cardiomyocytes, and its molecular mechanism is related to its inhibitory effect on the UPR pathway.

Membrane testosterone receptors in cultured vascular smooth muscle cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To determine the presence of membrane testosterone receptors in cultured vascular smooth muscle cells (VSMC), and investigate their relationship with classical intracellular androgen receptors (iAR).</p><p><b>METHODS</b>VSMCs were cultured from the thoracic aorta of male Sprague-Dawley rats by the explant method. Subconfluent VSMCs were incubated with serum-free medium for 24 h to obtain quiescent non-dividing cells, and then treated with the indicated agents. The aliquots of VSMCs were labeled with testosterone-BSA-FITC (T-BSA-FITC) and analyzed by flow cytometry. Classical iARs in intact- and permeabilized-cells were detected with anti-iAR antibodies and FITC-labeled secondary antibodies by immunofluorescence, followed by flow cytometry analysis.</p><p><b>RESULTS</b>Incubation of VSMCs with T-BSA-FITC obviously increased their relative fluorescence intensity at 10 sec as compared with the untreated controls (P < 0.01), and so did it at 10 min in comparison with the treatment with BSA-FITC alone or together with free testosterone (P < 0.01). Pretreatment with iAR antagonist flutamide exhibited no significant influence on the relative fluorescence intensity of VSMCs (P = 0.318). Traditional iARs were not detectable on the surface of intact VSMCs, although permeabilized cells contained iARs.</p><p><b>CONCLUSION</b>VSMCs contain testosterone receptors in the plasma membrane, and these membrane receptors are not identical to classical iARs.</p>

Testosterone at physiological level inhibits PGF2alpha-induced increase in intracellular Ca2+ in cultured vascular smooth muscle cells / 中华男科学杂志

<p><b>OBJECTIVE</b>To explore the acute effects of testosterone at the physiological level on PGF2alpha-induced increase in intracellular Ca2+ in cultured vascular smooth muscle cells (VSMCs).</p><p><b>METHODS</b>VSMCs from the thoracic aorta of male Sprague-Dawley rats were cultured using the explant method. The subconfluent VSMCs were incubated with serum-free medium for 24 hours to obtain quiescent non-dividing cells and then treated with the indicated agents. For the measurement of [Ca2+]i, the VSMCs were loaded with fura-2. Changes of [Ca2+]i were determined ratiometrically with a Nikon TE-2000E system.</p><p><b>RESULTS</b>The resting level of [Ca2+]i was around 100 nmol/L in the VSMCs. Exposing cells to perfusate containing 10 micromol/L PGF2alpha triggered an immediate and transient peak in [Ca2+]i, which gradually decreased afterwards. Interference at the peak with the physiological concentration (40 nmol/L) of testosterone significantly decreased the peak-to-baseline time of [Ca2+]i, compared with ethanol vehicle (104.9 +/- 27.0 s vs 153.5 +/- 40.4 s, P < 0.01). Pretreatment with testosterone at 40 nmol/L for 2 minutes also reduced the peak-to-baseline time of [Ca2+]i significantly in comparison with the ethanol control (120.6 +/- 32.0 s vs 151.4 +/- 27.4 s, P < 0.01), but it had no significant effect on the peak level of PGF2alpha-induced intracellular Ca2+ (390.0 +/- 126.0 nmol/L vs 403.4 +/- 160.7 nmol/L, P > 0.05).</p><p><b>CONCLUSION</b>Testosterone at physiological concentration inhibits PGF2alpha-induced Ca2+ fluxes, probably via receptor-operated calcium channels by a non-genomic mechanism in VSMCs, which may be involved in the vasodilatory effect of testosterone.</p>