Pterostilbene Attenuates Fructose-Induced Myocardial Fibrosis by Inhibiting ROS-Driven Pitx2c/miR-15b Pathway

Figure 6

Pterostilbene and allopurinol downregulate Pitx2c to upregulate miR-15b expression in fructose-fed rat hearts and fructose-exposed H9c2 cells. Pitx2c mRNA and protein levels were measured in fructose-fed rat hearts (a, b) and fructose-exposed H9c2 cells (c, d) (), respectively. The relative Pitx2c mRNA levels were normalized to GAPDH. To detect whether Pitx2c mediated miR-15b expression, cellular miR-15b expression was determined in H9c2 cells transfected with pEX1-Pitx2c plasmid (e) or Pitx2c siRNA (f) (). Pitx2c bounds to DNA region upstream of the miR-15b genetic loci, and there was an observed enrichment in Pitx2c binding to miR-15b in H9c2 cells by the ChIP assay (g). Cellular Pitx2c protein levels were determined in miR-15b mimic (h) or miR-15b inhibitor (i)-transfected H9c2 cells coincubated with 5 mM fructose, 10 μM pterostilbene, and 30 μM allopurinol (). The relative miR-15b expression levels were normalized to U6. Relative protein levels of Pitx2c were normalized to β-actin. Data are expressed as the ^#, ^##, and ^### vs. normal animal control group, normal cell control group, or negative control cell group; , , and vs. fructose-vehicle animal group, fructose-vehicle cell group, IgG-negative control group, or fructose-vehicle+miR-15b mimic or miR-15b inhibitor control cell group.

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