Role of Autophagy Mediated by AMPK/DDiT4/mTOR Axis in HT22 Cells Under Oxygen and Glucose Deprivation/Reoxygenation

Abstract

Background: cerebral ischemia/reperfusion (I/R) injury is an important complication of ischemic stroke, and autophagy is one of the mechanisms of it. In this study, we aimed to determine the role and mechanism of autophagy in cerebral I/R injury. Methods: the oxygen and glucose deprivation/reoxygenation (OGD/R) method was used to model cerebral I/R injury in HT22 cells. CCK-8 and LDH were conducted to detect viability and damage of the cells, respectively. Apoptosis was measured by flow cytometry and Tunel staining. Autophagic vesicles of HT22 cells were assessed by transmission electron microscopy. Western blotting analysis was used to examine the protein expression involving AMPK/DDiT4/mTOR axis and autophagy-related proteins. 3-Methyladenine and rapamycin were, respectively, used to inhibit and activate autophagy, compound C and AICAR acted as AMPK inhibitor and activator, respectively, and were used to control the starting link of AMPK/DDiT4/mTOR axis. Results: autophagy was activated in HT22 cells after OGD/R was characterized by an increased number of autophagic vesicles, the expression of Beclin1 and LC3II/LC3I, and a decrease in the expression of P62. Rapamycin could increase the viability, reduce LDH leakage rate, and alleviate cell apoptosis in OGD/R cells by activating autophagy. 3-Methyladenine played an opposite role to rapamycin in OGD/R cells. The expression of DDiT4 and the ratio of p-AMPK/AMPK were increased after OGD/R in HT22 cells. While the ratio of p-mTOR/mTOR was reduced by OGD/R, AICAR effectively increased the number of autophagic vesicles, improved viability, reduced LDH leakage rate, and alleviated apoptosis in HT22 cells which suffered OGD/R. However, the effects of compound C in OGD/R HT22 cells were opposite to that of AICAR. Conclusions: autophagy is activated after OGD/R; autophagy activator rapamycin significantly enhanced the protective effect of autophagy on cells of OGD/R. AMPK/DDiT4/mTOR axis is an important pathway to activate autophagy, and AMPK/DDiT4/mTOR-mediated autophagy significantly alleviates cell damage caused by OGD/R.

Ischemia reperfusion myocardium injuries in type 2 diabetic rats: Effects of ketamine and insulin on LC3-II and mTOR expression

“… 40 Furthermore, the expression of AMPK, BECLIN-1, and ATG5 proteins enhances and inhibits the mTOR protein involved in the induction of autophagy in cardiovascular diseases. 41 – 43 The study suggests that ketamine treatment and its combination with insulin ameliorated the altered expression of mTOR, AMPK, BECLIN-1, and ATG5 protein in myocardial tissues of I/R-induced myocardial injury in DM2. Our findings were further supported by the study report, 42 where autophagy-associated signaling pathways such as AMPK/mTOR were suggested to play a crucial role in protecting against myocardial ischemia-reperfusion injury in diabetes mellitus (DM).…”

Phenformin activates ER stress to promote autophagic cell death via NIBAN1 and DDIT4 in oral squamous cell carcinoma independent of AMPK

“…A previous study by Zhang et al reported that the expression of DDIT4 was increased significantly along with activated autophagy induced by oxygen and energy deprivation in HT22 cells (a neuronal line of mouse hippocampus), and could be further increased by treatment with the AMPK activator AICAR. 60 Therefore, we speculated that phenformin, another AMPK activator, induced DDIT4 and/or NIBAN1 through the activation of AMPK in OSCC cells. However, the inhibition of AMPK expression by the siRNA mediated knockdown of AMPK α1 and α2 did not block the increased expression of DDIT4 or NIBAN1 in phenformin-treated OSCC cells, indicating that phenformin promotes DDIT4 and NIBAN1 expression in an AMPK-independent manner.…”

Whole Transcriptome Analysis of Substantia Nigra in Mice with MPTP-Induced Parkinsonism Bearing Defective Glucocerebrosidase Activity

“…Supplementary Materials:The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/ijms241512164/s1. References[64,[86][87][88][89][90][91][92][93] are cited in the supplementary materials.…”