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[Establishment and evaluation of an AAPH-induced oxidative stress model in mouse Leydig cells in vitro]
Xing Hou1,2, Xue Tang3, Bo-Nan Li4
1Department of Andrology, The First Affiliated Hospital of Hunan University of Traditional Chinese Medicine, Changsha, Hunan 410007.
Objective:
To establish a model of oxidative stress (OS) injury in mouse Leydig cells using α-α'-azodiisobutyramidine hydrochloride (AAPH) and evaluate the physiological function.
Methods:
In Experiment 1, we treated mouse TM3 Leydig cells with AAPH at 0, 1, 5, 10, 50 and 100 mmol/L for 4, 8 and 24 h respectively and measured the activity of the cells using MTS, their aging by β-galactoside staining, mitochondrial membrane potential by JC-1 fluorescence and mitochondrial DNA copy number by qPCR. In Experiment 2, we treated the TM3 cells selected in Experiment 1 according to the AAPH concentration range (≤10 mmol/L) with AAPH at 0, 0.1, 0.5, 1, 2, 4, 6, 8 and 10 mmol/L for 24, 48 and 72 h respectively, detected the activity and aging of the cells and the ROS positive rate, and determined the optimal concentration and time of AAPH in inducing OS injury in the TM3 cells.
Results:
Experiment 1 showed that the survival rate of the TM3 cells was ≥50% in the 4-h 50 mmol/L, 8-h 10 mmol/L and 24-h 5 mmol/L AAPH groups, the initial concentration of AAPH was ≤10 mmol/L, with the action time of ≥24 h. Experiment 2 manifested that in the 24-h 6 mmol/L AAPH group, the survival rate of the TM3 cells was ≥70%, with an ROS positive rate of 56.88%, normal mitochondrial membrane potential, increased number of mtDNA copies, but no senescence.
Conclusions:
Treatment with AAPH at the concentration of 6 mmol/L for 24 hours is suitable for induction of OS injury in the TM3 cells.
