MicroRNA-29a/b/c targets iNOS and is involved in protective remote ischemic preconditioning in an ischemia-reperfusion rat model of non-alcoholic fatty liver disease

Remote ischemic preconditioning (RIPC) protects against the injury that is incurred by ischemia and reperfusion (IR); however, the role of RIPC in liver IR injury in non-alco-holic fatty liver disease (NAFLD) remains unclear. In this study, a NAFLD rat model was utilized in a series of different surgical procedures and molecular experiments. Rats of the IR group and the RIPC+IR group exhibited more severe injury than NAFLD control rats (in which the liver was prodded following a median-incision laparotomy). The liver condition, measured by serum alanine transaminase and aspartate transaminase levels, of the RIPC+IR group was better than that of the IR group. In addition, alanine transaminase and aspartate transaminase levels were lower in the RIPC+IR group compared with the IR group (P<0.001). Flow cytometry revealed that the cell apoptosis ratio was significantly lower in the RIPC+IR group than in the IR group (P<0.001). Reverse transcription-polymerase chain reaction (RT-qPCR) was used to assess miR-29a/b/c levels, revealing that they were significantly reduced in the RIPC and RIPC+IR groups, but did not vary in the IR group compared with the control group. RT-qPCR also revealed that iNOS mRNA levels were not significantly different among any of the NAFLD groups; however, western blot analysis indicated that iNOS protein levels were increased in the RIPC group and the RIPC+IR group compared with the control and IR groups. A luciferase reporter assay demonstrated that transfection with miR-29a/b/c mimics significantly decreased the luciferase activities of plasmids containing the wild-type iNOS 3'-untranslated region (UTR) ( relative fluorescence intensity: 0.47 +/- 0.06 for miR-29a, 0.36 +/- 0.07 for miR-29b, 0.41 +/- 0.04 for miR-29c; P<0.001), whereas the activities of plasmids containing the mutant iNOS 3'-UTR sequence were not markedly affected [ relative fluorescence intensity: 0.99 +/- 0.08 for miR-29a (P=0.1349), 0.99 +/- 0.09 for miR-29b (P=0.1607), 0.97 +/- 0.07 for miR-29c (P=0.1824)]. This suggested that miR-29a/b/c downregulates iNOS by directly targeting its 3'-UTR. In summary, the results suggest that RIPC has a protective effect in NAFLD liver IR injury, which may be due to reduced miR-29a/b/c levels in the skeletal muscle, leading to increased iNOS and, therefore, nitric oxide.