NOX4/ROS mediate ethanol‑induced apoptosis via MAPK signal pathway in L‑02 cells

Yang,Cheng‑Fang; Zhong,Yu‑Juan; Ma,Zuheng; Li,Li; Shi,Lin; Chen,Li; Li,Chen; Wu,Dan; Chen,Qi; Li,Yong‑Wen

doi:10.3892/ijmm.2018.3390

NOX4/ROS mediate ethanol‑induced apoptosis via MAPK signal pathway in L‑02 cells

Authors:
- Cheng‑Fang Yang
- Yu‑Juan Zhong
- Zuheng Ma
- Li Li
- Lin Shi
- Li Chen
- Chen Li
- Dan Wu
- Qi Chen
- Yong‑Wen Li
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Affiliations: Department of Pharmacology, College of Pharmacy, Guilin Medical University, Guilin, Guangxi 541004, P.R. China, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm SE‑171 76, Sweden
Published online on: January 16, 2018 https://doi.org/10.3892/ijmm.2018.3390
Pages: 2306-2316

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Abstract

The aim of the present study was to assess the molecular mechanism of ethanol‑induced oxidative stress‑mediated apoptosis in L‑02 liver cells in order to elucidate novel pathways associated with alcoholic liver disease. L‑02 cells were treated with 400 mM ethanol with or without inhibitors. The cell viability was measured by an MTT assay. Cell apoptosis was assessed by flow cytometry and a single‑stranded DNA (ssDNA) assay. Intracellular reactive oxygen species (ROS) production of L‑02 cells was determined using the 2',7'‑dichlorofluorescein‑diacetate dye. The protein expression of c‑Jun N‑terminal kinase (JNK), phosphorylated (p)‑JNK, P38, p‑P38, NADPH oxidase (NOX)1, NOX4, p22phox, B‑cell lymphoma 2 (Bcl‑2) and Bcl‑2‑associated X protein were measured by western blot analysis. The mRNA expression of NOX1, NOX4 and p22phox was measured by reverse transcription polymerase chain reaction analysis. The results indicated that after treatment with various concentrations of ethanol for the indicated durations, L‑02 cells were displayed a significant decrease in cell viability in a dose‑and time‑dependent manner. Ethanol‑induced apoptosis and cell death of L‑02 cells was accompanied by the generation of ROS, elevated expression of NOX, as well as phosphorylation of JNK and P‑38. In addition, increased expression of Bcl‑2 was induced by 400 mM ethanol. Furthermore, treatment with NOX inhibitor attenuated the ethanol‑induced a decrease in cell viability, and an increase in apoptosis and Bcl‑2 expression. In conclusion, ethanol induced apoptosis in the L‑02 hepatocyte cell line via generation of ROS and elevated expression of NOX4. This indicated that activation of JNK and p38 in the mitogen‑activated protein kinase pathway promotes apoptosis in L‑02 cells.

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