Mechanism of atractylenolide Ⅲ in alleviating H9c2 cell apoptosis through ROS/GRP78/caspase-12 signaling pathway based on molecular docking

Abstract

This study aims to investigate the effect of atractylenolide Ⅲ(ATL-Ⅲ) on hydrogen peroxide(H_2O_2)-induced endoplasmic reticulum stress and apoptosis of H9 c2 cells via the ROS/GRP78/caspase-12 signaling pathway.The binding activity of ATL-Ⅲ to GRP78 was determined by molecular docking.The result showed that ATL-Ⅲ had a good binding activity to GRP78, and the binding activity of ATL-Ⅲ was stronger than that of its specific inhibitor.The endoplasmic reticulum stress model of H9 c2 was established by H_2O_2(100 μmol·L~(-1)) treatment.Five groups were designed: blank control group, model group, and ATL-Ⅲ(15, 30, and 60 μmol·L~(-1)) groups.Apoptosis was detected by Hoechst/PI double staining and flow cytometry.The levels of superoxide dismutase(SOD), malondialdehyde(MDA), and lactate dehydrogenase(LDH) were measured by colorimetry.The levels of reactive oxygen species(ROS) and calcium(Ca~(2+)) in cytoplasm were determined by the fluorescence probe DCFH-DA and the calcium fluorescence probe Flou-4, respectively.The protein levels of GRP78, caspase-12, and caspase-3 were determined by Western blot, and the mRNA levels of GRP78 and caspase-12 by RT-qPCR.N-acetyl-L-cysteine(NAC) and 4-phenylbutyric acid(4-PBA) were respectively used to inhibit ROS and GRP78, and then the mechanism of ATL-Ⅲ in protecting the cells from endoplasmic reticulum stress induced by H_2O_2 were deduced.ATL-Ⅲ(15, 30, and 60 μmol·L~(-1)) decreased the apoptosis rate and ROS, MDA, and LDH levels(P<0.01), increased the SOD activity(P<0.01), and down-regulated the protein levels of GRP78, caspase-12, and caspase-3 and the mRNA levels of GRP78 and caspase-12(P<0.05).The addition of NAC decreased the apoptosis rate and ROS, MDA, GRP78, caspase-12, and caspase-3 levels(P<0.01), while it elevated the SOD level(P<0.01).The addition of 4-PBA also decreased the apoptosis rate and the levels of GRP78, caspase-12, caspase-3, and Ca~(2+)(P<0.01).The effect of inhibitors were consistent with that of ATL-Ⅲ.In conclusion, ATL-Ⅲ can protect H9 c2 cardiomyocytes by regulating ROS/GRP78/caspase-12 signaling pathway to inhibit H_2O_2-induced endoplasmic reticulum stress and apoptosis.