Plant Natural Product Formononetin Protects Rat Cardiomyocyte H9c2 Cells against Oxygen Glucose Deprivation and Reoxygenation via Inhibiting ROS Formation and Promoting GSK-3β Phosphorylation
Roles of PI3K, ERK1/2, and PKC in the regulation of GSK-3β phosphorylation and mitochondrial membrane integrity by formononetin. (a) Western blot analysis and quantification of GSK-3β phosphorylation. After the treatment with formononetin ± kinase inhibitors, H9c2 cells were lysed and analyzed for GSK-3β phosphorylation at Ser9 by Western blotting using specific antibodies. Western blots were determined by a densitometric method. Results are presented as means ± SD (). (drug versus OGD/R); , (drug + inhibitor versus drug). (b and c) Fluorescence images and quantification of TMRM and calcein sequestration in mitochondria. After the treatment with formononetin ± kinase inhibitors under OGD and reoxygenation condition, H9c2 cells were stained with TMRM and calcein-AM and imaged under a fluorescence microscope (Carl-Zeiss, Jena, Germany). TMRM and calcein fluorescence intensity were quantified by a densitometric method. (drug versus OGD/R), (drug + inhibitor versus drug). LY, PI3K inhibitor LY294002; GF, PKC inhibitor GF109203X; PD, ERK 1/2 inhibitor PD98059. Scale bar, 10 μm.