Doxycycline Induces Mitophagy and Suppresses Production of Interferon-ß in IPEC-J2 Cells

Xing, Yang; Liqi, Zhu; Jian, Lin; Qinghua, Yu; Qian, Yang

Mitochondria are the major sites for the generation of ROS, including superoxide, hydroxyl radicals, and hydrogen peroxide (H 2 O 2 ), in non-phagocytic cells. Antibiotics that induce mitochondrial dysfunction could cause oxidative damage in mammalian cells (Kalghatgi et al., 2013) . DOX also affects mitochondrial morphology in cultured cells (Moullan et al., 2015) . We tested whether DOX induces production of intracellular ROS in IPEC-J2 cells. DCF fluorescence intensity, which indicates the intracellular level of ROS, increased significantly after a 24-h incubation with the positive control rotenone or DOX at all concentrations examined (Figure 2A) . The mitochondrial-specific ROS indicator MitoSox, which could selectively detects superoxide in mitochondria, was used to examine mitochondrial ROS levels. Once oxidized by superoxide, mitochondrial-targeted MitoSox generates red fluorescence. MitoSox fluorescence was enhanced in a dose-dependent manner ( Figure 2B) . We also measured the mRNA levels of Nrf2 and superoxide dismutase (SOD)2, which are involved in modulating ROS levels, to further examine oxidative stress status. According to the results, their expression were increased in a dose-dependent manner after a 24-h exposure to DOX (Figures 2C,D) . We speculated that DOX could lead to the accumulation of ROS and damaged mitochondria. The mitochondrial membrane potential was assessed by Rh123, but no changes were detected ( Figure 2E) . We then used two types of mitochondrialspecific label to distinguish respiring (MitoTracker Red) vs. total (MitoTracker Green) mitochondria. Dysfunctional non-respiring (MitoTracker green-positive, MitoTracker rednegative) mitochondria increased dramatically after DOX treatment (Figures 2F,G) . DOX could induce oxidative stress and increase the accumulation of dysfunctional mitochondria in IPEC-J2 cells.