Propofol Protects Rat Cardiomyocytes from Anthracycline‐Induced Apoptosis by Regulating MicroRNA‐181a In Vitro and In Vivo

Figures

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  Figure 1. Antiproliferative effect ADM alone or combination treatment with propofol in cardiomyocytes. Morphological changes of H9c2 cells (a) and primary cardiomyocytes (b) in different treatment (control, prop 1 μg/ml, ADM 10 μM, and prop 1 μg/ml + ADM 10 μM) by microscope. Compared with the group treated with the combination of ADM with propofol, the antiproliferative effect of ADM alone on cardiomyocytes was significantly greater in H9c2 cells (P = 0.0253) (c) and primary cardiomyocytes (P = 0.0182) (d). The inhibition concentration of propofol in the combination treatment was maintained at 1 μg/ml, while the concentrations of ADM were 100 nM, 500 nM, 1 μM, and 10 μM. Each column or data point represents the mean ± S.D. (n = 3). The statistical analysis was performed with two-way ANOVA using Tukey's test for pairwise comparisons. NS: not significant; ∗P ≤ 0.05 compared with the ADM group.

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  Figure 2. Effect of the combination of propofol and ADM induction of early apoptosis in cardiomyocytes by flow cytometry. After exposure to the different treatments for 24 h, the proportion of early apoptotic cells was analyzed using Annexin V-FITC and PI in H9c2 cells (a) and primary cardiomyocytes (b). Compared with the Adriamycin-alone group, combination treatment induced a decrease in the population of cells undergoing early apoptosis in H9c2 cells (P = 0.0037) (c). The phenomenon was also taken place in primary cardiomyocytes (P = 0.0272) (d). Each column represents the mean ± S.D. (n = 3). The statistical analysis was performed with Student's t-test. NS: not significant; ∗P ≤ 0.05 and ∗∗P < 0.01, compared with the ADM group.

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  Figure 3. Propofol reduced the upregulation of miR181a induced by ADM and inhibited ADM-induced STAT3 activating. The expression of miR-181a in different treatment on H9c2 (a) or primary cardiomyocytes (b) tested by RT-PCR. The combination treatment upregulation of miR-181a was reduced compared to the ADM-alone group. (c-d) Phospho-STAT3, Bcl-2, Bax, Caspase3, and Caspase9 protein levels were measured in different treatment in cardiomyocytes by Western blotting. (e-f) Semiquantitative data from densitometric analysis of phospho-STAT3, Bcl-2, Bax, Caspase3, and Caspase9 are presented as relative ratio of each protein to β-actin. Data are expressed as mean ± SD (n = 3). The statistical analysis was performed with Student's t-test. NS: not significant; ∗P ≤ 0.05 and ∗∗P < 0.01, compared with the ADM group.

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  Figure 4. miR-181a contributes to reducing ADM-induced cardiomyocyte apoptosis by repressing Bcl-2. (a) RNAs isolated from H9c2 cells transfected with miR-181a inhibitor or control were analyzed by RT-qPCR to access the levels of miR-181a. The expression of miR-181a was reduced by miR-181a inhibitor transfection. (b) The cell viability of H9c2 cells transfected with miR-181a inhibitor or control in different treatment was tested by MTT assay. (c-e) After 24 h exposure of ADM treatment, the proportion of apoptotic cells was decreased in miR-181a inhibitor cells compared with control cells (P = 0.006). (d-f) The silencing of miR-181a in H9c2 cell treatment with ADM resulted in increased Bcl-2 (P = 0.0007) and decreased Bax (P = 0.0495) protein levels. (g) Rat Bcl-2 has strong rno-miR-181a-binding sites at its 3′-UTR. MiR-181a seed pairing in the target regions is shown as vertical lines. (h) Relative luciferase activity was analyzed after wild-type or mutant Bcl-2-3′UTR reporter plasmids were cotransfected with miR-181a mimic in H9c2 cells as shown. Compared to the negative control, miR-181a repressed the activity of luciferase fused to the WT Bcl-2-3′UTR (P = 0.0299), while it failed to…

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  Figure 5. Propofol could reduce the apoptosis cells induced by ADM in cardiomyocytes in vivo. (a) Myocardial sections of different treatments were stained for TUNEL assay to measure apoptosis index (AI). The AI of rat heart tissue was significantly decreased in ADM + prop group compared with ADM-alone group (P = 0.0172). (b–d) RNAs or proteins isolated from heart tissues in different group were analyzed by RT-qPCR or Western blot to access the levels of miR-181a (b) or phospf-STAT3 (c-d). Both miR-181a and phospho-STAT3 were overexpressed in ADM group compared to ADM + prop group. The statistical analysis was performed with Student's t-test. NS: not significant; ∗P ≤ 0.05, ∗∗P ≤ 0.01 and ∗∗∗P < 0.001 compared with the control group.