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Ketamine induces reactive oxygen species and enhances autophagy in SV‐HUC‐1 human uroepithelial cells
Author(s) -
Shan Zhengfei,
Wei Liqin,
Yu Shengqiang,
Jiang Shanling,
Ma Yue,
Zhang Chengjun,
Wang Jiantao,
Gao Zhenli,
Wan Fengchun,
Zhuang Guimin,
Wu Jitao,
Liu Dongfu
Publication year - 2019
Publication title -
journal of cellular physiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.529
H-Index - 174
eISSN - 1097-4652
pISSN - 0021-9541
DOI - 10.1002/jcp.27094
Subject(s) - tunel assay , viability assay , microbiology and biotechnology , apoptosis , western blot , reactive oxygen species , autophagy , terminal deoxynucleotidyl transferase , chemistry , biology , biochemistry , gene
This study was aimed at exploring the underlying mechanisms of ketamine in the SV‐40 immortalized human ureteral epithelial (SV‐HUC‐1) cells. The viability and apoptosis of SV‐HUC‐1 cells treated with 0.01, 0.1, and 1 mM ketamine were respectively detected via cell counting kit‐8 (CCK‐8) assay and terminal deoxynucleotidyl transferase‐mediated deoxyuridine triphosphate nick end labeling (TUNEL) staining. Reactive oxygen species (ROS) level was measured through ROS probe staining. Apoptosis‐related proteins (B‐cell lymphoma 2 [Bcl‐2] and Bax) and autophagy‐associated proteins (light chain 3‐I [LC3‐I] and LC3‐II) were determined by western blot or immunofluorescent assay. Additionally, transmission electron microscopy (TEM) was used to evaluate the formation of autophagosomes. After cotreatment of 3‐methyladenine (3‐MA) or N ‐acetyl‐ l ‐cysteine (NAC), the biological functions of SV‐HUC‐1 cells were analyzed to determine the association of ROS with cell viability and autophagy. CCK‐8 assay and TUNEL staining indicated that ketamine effectively decreased the viability of SV‐HUC‐1 cells and accelerated apoptosis of SV‐HUC‐1 cells through regulating the expression level of IKBα (phospho), nuclear factor кB (P65), Bcl‐2, and Bax proteins. Enhanced ROS production was also confirmed in ketamine‐treated SV‐HUC‐1 cells treated with ketamine. Ketamine‐induced autophagosomes in SV‐HUC‐1 cells were observed by means of TEM, and increased levels of LC3 II/I ratio and Beclin 1 were examined through western blot and immunofluorescent assay. Furthermore, ketamine exerted effects on SV‐HUC‐1 cells in a dose‐dependent and time‐dependent manner. Additionally, cotreatment of NAC with 3‐MA significantly attenuated the ROS level and suppressed the cell autophagy. Ketamine promoted SV‐HUC‐1 cell autophagy and impaired the cell viability of SV‐HUC‐1 cells by inducing ROS.