miR-144 targets Nrf2 affecting the intestinal oxidative damage induced by oxidized fish oil in Megalobrama amblycephala with emphasis on autophagy and apoptosis

To explore the modulatory role of miR-144 in Nrf2 cascade reactions and assess its potential as a target for alleviating oxidative fish oil-induced intestinal damage in Megalobrama amblycephala , four experimental diets were formulated. A conventional diet was kept as the control diet (NC), while experimental diets consisted of the control diet supplemented with oxidized fish oil (OF, peroxide value: 323 ​mmol/kg), OF ​+ ​ miR-144 agomir and OF ​+ ​ miR-144 antagomir, respectively. In the oxidative stress model of M. amblycephala induced by oxidized fish oil, dihydroethidium (DHE) fluorescence probe and morphological results revealed that oxidized fish oil markedly elevated the level of reactive oxygen species (ROS) within intestinal cells and destroyed the intestinal barrier structure, such that the height of the intestinal villus and the thickness of the muscle layer were significantly reduced, and intestinal barrier-related genes ZO-1 , Occludin and Claudin-7α were significantly down-regulated. Additionally, oxidized fish oil treatment activated autophagy and apoptosis of intestinal cells, which was manifested by the significant up-regulation of autophagy-related genes Beclin-1 , VPS34 , P62 and ATG5 ( P ​< ​0.05), the increase of autophagosomes, and the significant up-regulation of apoptosis-related genes HSP27 , Caspase3 , Caspase8 and Caspase9 ( P ​< ​0.05). Further studies showed that miR-144 agomir significantly enhanced Keap1 expression and inhibited Nrf2 expression, while miR-144 antagomir had the opposite effect. Importantly, forced expression of miR-144 induced ROS accumulation by silencing Nrf2 , raised the expression of inflammatory factors ( P ​< ​0.05), and even further induced the development of autophagy and apoptosis, while miR-144 antagomir could offset this effect by reactivating Nrf2 expression. In summary, this study showed that silencing miR-144 could protect intestinal cells of M. amblycephala from oxidative damage by targeting the Nrf2 signaling pathway. Therefore, miR-144 is expected to be a regulatory target for ameliorating intestinal oxidative damage induced by oxidized oils. The hypothesis that miR-144 mediates Nrf2 to regulate apoptosis and autophagy in response to intestinal oxidative stress in M. amblycephala. Arrows in red color represent the regulation by oxidative stress, in blue color represent the regulation by miR-144 antagomir. • Oxidized fish oil and miR-144 overexpression induced ROS burst and impaired M. amblycephala intestinal barrier. • Oxidized fish oil and over-expression of miR-144 promoted autophagy and apoptosis in intestinal cells of M. amblycephala. • Silencing miR-144 reduced ROS production, and alleviated intestinal barrier damage, autophagy and apoptosis of cells.