Hypothermia prevents OGD/R induced injury by activating the PI3K/AKT/mTOR signaling pathway and enhancing autophagy

Abstract Objectives To evaluate the role of hypothermia in the injury of PC12 cells which induced by OGD/R. To investigate the mechanism of hypothermia intervention to alleviate OGD/R-induced injury of PC12 cells through phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway and autophagy, and to lay a foundation and provide theoretical basis for clinical application of hypothermia on protect nerve injury in the future. Methods Induce PC12 cells establish an Ischemia reperfusion injury model in vitro by OGD/R. Then, intervene PC12 cells with OGD/R and OGD/R treated with LY294002 inhibitor at 37℃, 32℃, 27℃. Valuate cell viability by CCK-8 assay, detect the phosphorylation levels of PI3K, AKT and mTOR proteins through western blotting, evaluate signaling pathway activation of PI3K/AKT/mTOR, detect protein expression of LC3I/II by immunofluorescence and western blotting, and observe the autophagosomes by fluoroscopic electron microscopy to evaluate autophagy. Detect apoptosis by flow cytometry, and evaluate the cell damage. Results Hypothermia treatment could effectively improve the cell viability of PC12 cells treated with OGD/R, and the cell viability increased with the decrease of temperature (P < 0.05). Hypothermia treatment also promoted the increase of the phosphorylation levels of PI3K, AKT and mTOR proteins in cells (P < 0.05). In addition, flow cytometry was used to detect the apoptosis rate of different treatments, and the results showed that hypothermia could inhibit the apoptosis that induced by OGD/R. In the control group, OGD/R group and LY294002 inhibitor group, autophagy specific protein LC3I/II which labeled with green fluorescently was increased significantly under the culture conditions of 32℃ and 27℃, and the fluorescence signal was enhanced. Meanwhile, the expression level of LC3I/II also increased. Autophagosome was increased under the electron microscopy. Conclusion Hypothermia can inhibit apoptosis by regulating the PI3K/AKT/mTOR axis and the level of autophagy, thus protecting PC12 cell damage that induced by OGD/R.