Journal Article

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

Liu, Hao, Weng, Xiao-Dong, Chen, Hui, Liu, Xiu-Heng, Zhang, Bang-Hua, Yuan, Yan, Wang, Lei, Chen, Zhi-Yuan, Du, Yang

2021

Overview

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. This study aimed to investigate the precise role of TRIM8 and relevant mechanisms in renal I/R injury. 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 O ), mRNA, apoptotic proteins, pyroptosis-related proteins and PI3K/AKT pathway-associated proteins were assessed. 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 O , 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. Knockdown of TRIM8 can alleviate H/R-induced oxidative stress by triggering the PI3K/AKT pathway, thus attenuating pyropyosis and apoptosis in vitro.

• Publisher: Dove Medical Press Limited,Taylor & Francis Ltd,Dove Press,Dove,Dove Medical Press
• Subject: 1-Phosphatidylinositol 3-kinase / Acetylcysteine / Acute Kidney Injury - metabolism / AKT protein / Analysis / Antibodies / Apoptosis / Apoptosis - drug effects / Bax protein / Carrier Proteins - metabolism / Caspase-1 / Caspase-11 / Cell Survival / Cell viability / Cells, Cultured / Combined treatment / Epithelial cells / Epithelium / Experiments / Gene expression / Humans / Hydrogen peroxide / Hydrogen Peroxide - metabolism / Hypoxia / IL-1β / Injuries / Ischemia / Kidneys / mRNA / Nerve Tissue Proteins - metabolism / Original Research / Oxidative Stress / Peroxides / Phosphatidylinositol 3-Kinases - metabolism / Proteins / Proto-Oncogene Proteins c-akt - metabolism / Pyroptosis / Pyroptosis - drug effects / Reactive oxygen species / Reactive Oxygen Species - metabolism / Reagents / renal I/R injury / Reperfusion / Reperfusion Injury - prevention & control / RNA / Signal Transduction / siRNA / Software / Standard deviation / TRIM8