Department of Organ Transplantation, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China.
Department of Urology, The Affiliated Yantai Yuhuangding Hospital of Qingdao University, Yantai, China.
J Cell Physiol. 2019 Mar;234(3):2778-2787. doi: 10.1002/jcp.27094. Epub 2018 Aug 26.
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.
本研究旨在探索氯胺酮在 SV-40 永生化人输尿管上皮（SV-HUC-1）细胞中的作用机制。通过细胞计数试剂盒-8（CCK-8）检测法和末端脱氧核苷酸转移酶介导的脱氧尿苷三磷酸缺口末端标记（TUNEL）染色法分别检测了 0.01、0.1 和 1mM 氯胺酮处理的 SV-HUC-1 细胞的活力和凋亡情况。通过 ROS 探针染色测定活性氧（ROS）水平。通过 Western blot 或免疫荧光测定法测定凋亡相关蛋白（B 细胞淋巴瘤 2 [Bcl-2]和 Bax）和自噬相关蛋白（LC3-I [LC3-I]和 LC3-II）。此外，还使用透射电子显微镜（TEM）评估自噬体的形成。在用 3-甲基腺嘌呤（3-MA）或 N-乙酰-L-半胱氨酸（NAC）共处理后，分析 SV-HUC-1 细胞的生物学功能，以确定 ROS 与细胞活力和自噬的关系。CCK-8 检测法和 TUNEL 染色表明，氯胺酮通过调节 IKBα（磷酸化）、核因子κB（P65）、Bcl-2 和 Bax 蛋白的表达水平，有效降低了 SV-HUC-1 细胞的活力并加速了 SV-HUC-1 细胞的凋亡。氯胺酮处理的 SV-HUC-1 细胞中也证实了 ROS 产生增强。通过 TEM 观察到氯胺酮诱导的 SV-HUC-1 细胞中的自噬体，并通过 Western blot 和免疫荧光测定法检查了 LC3 II/I 比值和 Beclin 1 的水平升高。此外，氯胺酮对 SV-HUC-1 细胞的作用呈剂量和时间依赖性。此外，NAC 与 3-MA 共处理可显著降低 ROS 水平并抑制细胞自噬。氯胺酮通过诱导 ROS 促进 SV-HUC-1 细胞自噬并损害 SV-HUC-1 细胞的细胞活力。
