Knockdown of TRIM8 Protects HK-2 Cells Against Hypoxia/Reoxygenation-Induced Injury by Inhibiting Oxidative Stress-Mediated Apoptosis and Pyroptosis via PI3K/Akt Signal Pathway
Banghua Zhang, Hao Liu, Yan Yuan, Xiaodong Weng, Yang Du, Hui Chen, Zhiyuan Chen, Lei Wang, Xiuheng Liu
Abstract
Background: Acute kidney injury (AKI) emerges as an acute and critical disease. Tripartite motif 8 (TRIM8), one number of the TRIM protein family, is proved to participate in ischemia/reperfusion (I/R) injury. However, whether TRIM8 is involved in renal I/R injury and the associated mechanisms are currently unclear. Purpose: This study aimed to investigate the precise role of TRIM8 and relevant mechanisms in renal I/R injury. Materials and Methods: In this study, human renal proximal tubular epithelial cells (HK-2 cells) underwent 12 hours of hypoxia and 2 h, 3 h or 4 h of reoxygenation to establish an in vitro hypoxia/reoxygenation (H/R) model. The siRNAs specific to TRIM8 (si-TRIM8) were transfected into HK-2 cells to knockdown TRIM8. The cell H/R model included various groups including Control, H/R, H/R+DMSO, H/R+NAC, si-NC+H/R, si-TRIM8+H/R and si-TRIM8+LY294002+H/R. The cell viability and levels of reactive oxygen species (ROS), hydrogen peroxide (H 2 O 2 ), mRNA, apoptotic proteins, pyroptosis-related proteins and PI3K/AKT pathway-associated proteins were assessed. Results: In vitro, realtime-quantitative PCR and western-blot analysis showed that the mRNA and protein expression of TRIM8 were obviously upregulated after H/R treatment in HK-2 cells. Compared with the H/R model group, knockdown of TRIM8 significantly increased cell viability and reduced the levels of ROS, H 2 O 2 , apoptotic proteins (Cleaved caspasebase-3 and BAX) and pyroptosis-related proteins (NLRP3, ASC, Caspase-1, Caspase-11, IL-1β and GSDMD-N). Western-blot analysis also authenticated that PI3K/AKT pathway was activated after TRIM8 inhibition. The application of 5 mM N-acetyl-cysteine, one highly efficient ROS inhibitor, significantly suppressed the expression of apoptotic proteins and pyroptosis-related proteins. Moreover, the combined treatment of TRIM8 knockdown and LY294002 reversed the effects of inhibiting oxidative stress. Conclusion: Knockdown of TRIM8 can alleviate H/R-induced oxidative stress by triggering the PI3K/AKT pathway, thus attenuating pyropyosis and apoptosis in vitro. Keywords: TRIM8, renal I/R injury, oxidative stress, apoptosis, pyroptosis