NADPH oxidase inhibitor, diphenyleneiodonium prevents necroptosis in HK‑2 cells

Dong,Wei; Li,Zhilian; Chen,Yuanhan; Zhang,Li; Ye,Zhiming; Liang,Huaban; Li,Ruizhao; Xu,Lixia; Zhang,Bin; Liu,Shuangxin; Wang,Weidong; Li,Chunling; Luo,Jialun; Shi,Wei; Liang,Xinling

doi:10.3892/br.2017.948

NADPH oxidase inhibitor, diphenyleneiodonium prevents necroptosis in HK‑2 cells

Authors:
- Wei Dong
- Zhilian Li
- Yuanhan Chen
- Li Zhang
- Zhiming Ye
- Huaban Liang
- Ruizhao Li
- Lixia Xu
- Bin Zhang
- Shuangxin Liu
- Weidong Wang
- Chunling Li
- Jialun Luo
- Wei Shi
- Xinling Liang
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Affiliations: Division of Nephrology, Guangdong General Hospital, Guangdong Academy of Medical Sciences, Guangzhou, Guangdong 510080, P.R. China, Institute of Hypertension, Zhongshan School of Medicine, Sun Yat‑sen University, Guangzhou, Guangdong 510080, P.R. China, Southern Medical University, Guangzhou, Guangdong 510515, P.R. China
Published online on: July 21, 2017 https://doi.org/10.3892/br.2017.948
Pages: 226-230

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Abstract

The aim of the present study was to investigate the protective effect of the NADPH oxidase inhibitor, diphenyleneiodonium (DPI) against necroptosis in renal tubular epithelial cells. A necroptosis model of HK‑2 cells was established using tumor necrosis factor‑α, benzyloxycarbonyl‑Val‑Ala‑Asp‑fluoromethylketone and antimycin A (collectively termed TZA), as in our previous research. The necroptosis inhibitor, necrostatin‑1 (Nec‑1) or the NADPH oxidase inhibitor, DPI were administered to the necroptosis model. Production of reactive oxygen species (ROS) was detected by dichlorodihydrofluorescein diacetate in the different groups, and the manner of cell death was identified by flow cytometry. Western blot analysis was used to determine the levels of phosphorylation of receptor‑interacting protein kinase 3 (RIP‑3) and mixed lineage kinase domain‑like (MLKL), which are essential to necroptosis. The results revealed that TZA increased the percentages of propidium iodide‑positive HK‑2 cells from 1.22±0.69 to 8.98±0.73% (P<0.001), and augmented the phosphorylation of RIP‑3 and MLKL. ROS levels were increased in the TZA group compared with the control group (27.74±1.60x104 vs. 18.51±1.10x104, respectively; P<0.001), and could be inhibited by Nec‑1 (TZA + Nec‑1 group, 22.90±2.22x104 vs. TZA group, 27.74±1.60x104; P=0.01). DPI decreased ROS production (TZA + DPI group, 22.13±1.86x104 vs. TZA group, 27.74±1.60x104; P<0.001) and also reduced the proportions of necrosis in the necroptosis model (TZA + DPI group, 4.40±1.51% vs. TZA group, 8.98±0.73%; P<0.001). Phosphorylated RIP‑3 and MLKL were also decreased by DPI treatment. The results indicate that ROS production increases in HK‑2 cells undergoing necroptosis, and that the NADPH oxidase inhibitor, DPI may protect HK‑2 cells from necroptosis via inhibition of ROS production.

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