As a central protein of the PI3K family, mTOR is an upstream transmission and plays an important role in the regulation of autophagy. of calcium overload by blocking ventricular myocyte calcium channels and suppressing parameter. Recently, we found that F2 could ameliorate H/R-induced apoptosis [15]. In this study, we used a well-established H/R injury model that causes cardiomyocyte death in the H9c2 culture line, and tested the hypothesis that this protective effects of F2 are associated with inhibiting autophagy to reduce cardiomyocyte apoptosis. Open in a separate window Physique 1 F2 promotes cell survival and reduces cell damage after H/R in myocardial H9c2 cellsA. Chemical structure of haloperidol (Hal). B. Chemical structure of < 0.05 vs. control, #< 0.05 vs. H/R. Ctrl: control; H/R: hypoxia/reoxygenation. RESULTS F2 alleviates hypoxia/reoxygenation injury We assessed cell viability in every group via MTT assay. F2 (10?5-10?7 mol/L) ameliorated cell viability in a concentration dependent manner (Physique ?(Physique1C).1C). Since lactate dehydrogenase (LDH) leakage is usually widely used as a marker of cellular damage, cardiomyocyte cells injury was assessed by determining LDH activity in AUY922 (Luminespib, NVP-AUY922) culture medium at the end of reoxygenation. LDH leakage increased in the H/R group compared AUY922 (Luminespib, NVP-AUY922) with the control group, but was significantly decreased by F2 treatment (Physique ?(Figure1D).1D). These findings indicated that F2 could promote cell survival and reduce cell AUY922 (Luminespib, NVP-AUY922) damage in H9c2 cells subjected to H/R. F2-mediated protection entails inhibition of autophagy in cardiomyocytes following H/R Activation of autophagy occurs in cardiomyocytes following H/R. To identify the role of F2 in regulating H/R-mediated autophagy in cardiomyocytes, we examined whether F2 could inhibit autophagy in cardiomyocytes, following H/R, by MDC staining and transmission electron microscopy (TEM). The autofluorescent material MDC has been shown to be a specific marker for autophagic vacuoles (AVs). When cells are viewed with a fluorescence microscope, AVs stained by MDC appear as unique dot-like structures distributed within the cytoplasm or localized to the perinuclear regions. In the H/R group, an increase in MDC-labeled vesicles was observed, as indicated by punctuate MDC fluorescence (Physique ?(Physique2A2A and ?and2B),2B), suggesting an induction of AV formation after H/R. In the F2-treated groups, the number of MDC-labeled vesicles declined in a dose-dependent manner. Autophagy was further confirmed by TEM. H9c2 cells after H/R showed common autophagic vacuoles, including accumulation of numerous autophagic vesicles with a distinct double membrane, compared with no or few autophagic vacuoles in control cells. As above, F2 treatment reduced autophagic vacuoles in a dose-dependent manner (Physique ?(Physique2C2C and ?and2D2D). Open in a separate window Physique 2 Effect of F2 on H/R-induced autophagy in H9c2 cellsA. Autophagic vacuoles were stained with MDC. B. Quantification of mean fluorescent intensity in panel A. C. Ultrastructure features were examined by transmission electron microscopy (TEM), detected with magnification of 25, 000. D. Quantification of the number of autophagosomes in panel C. E. Protein expression of p62. F. Quantification of panel E with densitometry. -actin was used as a loading control. The data shown are represented as the means SD confirmed in three individual experiments. *< 0.05 vs. control, #< 0.05 vs. H/R. Ctrl: control; H/R: hypoxia/reoxygenation. SQSTM1 (p62) is usually associated with mature autophagic vesicles and is degraded within autophagosomes. Western blot analysis revealed that p62 protein levels were reduced after H/R, and F2 treatment inhibited the reduction of p62 protein in a dose-dependent manner (Physique ?(Physique2E2E and ?and2F2F). F2 inhibits the expression of autophagy markers in H9c2 cells subjected to H/R Microtubule-associated protein light chain 3 (LC3) is usually a specific marker for autophagy initiation. LC3-II is an accepted marker for autophagosome formation, although higher autophagosome accumulation may result from either increased autophagosome formation (autophagy initiation) or interrupted autophagosome degradation (autophagosome clearance). Western blot analysis revealed that LC3-II was up-regulated in H9c2 cells exposed to H/R (Physique ?(Figure3A).3A). And F2 could inhibit the expression of LC3-II in a dose-dependent manner. To further investigate the effect of F2 on autophagy, we used qRT-PCR and western blot to determine the expression levels of the autophagy-related genes, Atg5 and Beclin-1. Expression of Atg5 or Beclin-1 mRNA and protein were increased in H9c2 cells subjected to H/R, and F2 reduced the expression of Atg5 and Beclin-1 in a dose-dependent manner (Physique ?(Physique3B3B and ?and3C).3C). The above data clearly indicate PRP9 that F2 could inhibit autophagy induced.
