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To investigate the delagyed protection of TanshinoneIIA against myocardial hypoxia/reoxgenation (H/R) injury and the mechanism of autophagy during the cardioprotection on cardiomyocytes.
In this study, H9C2 cardiomyocytes were randomly divided into five groups: Control group; H/R group, cardiomyocytes was exposed in the airtight container for 2 h followed by 1 h of reoxgenation; TanshinoneIIA group, cardiomyocytes was exposed to 1 h of 1μM TanshinoneIIA 24 h before H/R; The inhibitor of autophagy 3-methyladenine (3-MA) was added to culture medium 15 min before TanshinoneIIA exposure (3-MA+ TanshinoneIIA group) or cells were treated by 3-MA alone (3-MA group). Myocardiocytes were collected after different treatments. Cell proliferation was detected by MTT assay. Apoptosis rate was analyzed by flow cytometry. Autophagosomes and apoptosis were observed with transmission electron microscopy (TEM). The expression of autophagy protein microtubule associated protein light chain3-II (LC3-II, Beclin 1 and anti-apoptosis factor B-cell lymphoma-2 (Bcl-2) was determined by western blot.
The cell proliferation of TanshinoneIIA group, 3-MA group and 3-MA+ TanshinoneIIA group were significantly increased compared with the H/R group (P<0.05), and the apoptosis rate was decreased (P<0.05). TEM releaved that the formation of autophagosome and apoptosis was decreased in the TanshinoneIIA preconditioning groups compared with the H/R groups. The expression of LC3-II and Beclin 1 were upregulated in H/R and TanshinoneIIA groups compared with control group (P<0.05), which were lower in TanshinoneIIA group, 3-MA group and 3-MA+ TanshinoneIIA group than in H/R group, but Bcl-2 expression was upregulated.
The results of our study indicated that TanshinoneIIA preconditioning 24 h before H/R produced delayed myocardial protection against H/R injury by increasing cell proliferation and decreasing apoptosis rate in cardiomyocytes. TanshinoneIIA may inhibit autophagic cell death and apoptosis by down-regulation of autophagy protein LC3-II and Beclin 1 expression and up-regulation of Bcl-2 expression during reoxgenation.