Ku70 silencing aggravates oxygen-glucose deprivation/reperfusion-induced injury by activation of the p53 apoptotic pathway in rat cortical astrocytes.

Abstract

Oxidative stress-induced DNA damage is an important mechanism that leads to the death of neuronal cells after ischemic stroke. Our previous study found that Ku70 was highly expressed in ischemic brain tissue of rats after cerebral ischemia-reperfusion injury. However, the role of Ku70 in glucose-oxygen deprivation/reperfusion (OGD/R) in astrocytes has not been reported. Therefore, we investigated the effect and mechanism of Ku70 on OGD/R-induced astrocyte injury in rats. Rat astrocytes were cultured in vitro to establish the OGD/R-induced injury model and transfected with small interfering RNA (siRNA) to disturb Ku70 expression. Real-time quantitative polymerase chain reaction (RT-qPCR), western blotting, and immunofluorescence were performed to assay the expression of mRNA and proteins. Cell viability, apoptosis, and ROS accumulation were determined by CCK-8 assay, flow cytometry, and fluorescence microscopy, respectively. Our results showed Ku70 can be expressed in both the nucleus and cytoplasm of astrocytes, although mainly in the nucleus. Ku70 expression showed a trend of first increasing and then decreasing after OGD/R, reaching its highest change at 24 h of reoxygenation. OGD/R induced ROS production and DNA damage in rat astrocytes, and Ku70 silencing further increased ROS production and DNA lesions, which aggravated astrocyte injury and apoptosis. Furthermore, the expression of p53, Bax, and caspase 3 proteins significantly increased after OGD/R in astrocytes, and downregulation of Ku70 further enhanced expression of the above proteins. These results indicate that Ku70 silencing promotes OGD/R-induced astrocyte apoptosis, which may be associated with p53 apoptotic pathway activation. Our study suggests that Ku70 may be a novel target for cerebral ischemia-reperfusion injury therapy.