| Objective: Hepatic ischemia reperfusion injury(IRI) represents an important clinical problem as related to liver resection or transplantation. Hepatic IRI represents an important factor with regard to the prognosis of surgical outcomes and patient survival, as well as the protection of hepatic cells. The study was designed to research mi R-30 b expression and autophagy activity responding to hepatic IRI, meanwhile, we attempted to determine whether mi R-30 b modulates autophagy and thus alleviate hepatic IRI, revealing potential mechanism of hepatic IRI and providing new ideas of prevention and treatment of hepatic IRI.Methods: The segmental(70%) hepatic ischemia model was performed according to that described previously, and serum AST and ALT levels of the mice were determined with use of a commercial assay kit. Histopathological features of IR injury were observed by HE and TUNEL staining. q RT-PCR was used to detected the expression of mi R-30b-5p, Atg12 and Atg5 m RNA. The expression of Atg12, Atg12-Atg5, LC3 and Caspase-3 were detected by the western blotting. Mice were transfected with mi R-30 b agomir or antagomir. In order to assess whether AML12 cells become more resistant to apoptosis in hypoxia/reoxygenation model simulating IRI model, we examined the effects of either activating or inhibiting autophagy with use of the rapamycin and 3-MA. The bioinformatics was used to predict the binding site on the 3?-UTR of mi R-30 b. We hypothesized that the mi R-30 b binding site was at the 3?-UTR of Atg12, and a luciferase reporter assay was performed to determine the effects of mi R-30 b on the Atg12 m RNA 3?-UTR. At the same time, AML12 cells were also treated with mi R-30b-5p mimics/inhibitor or Atg12 si RNA to investigate potential interactions between the mi R-30 b and Atg12 during IR. Autophagosomes changes were observed by confocal laser scanning microscope and transmission electron microscope.The expression of Atg12, Atg12-Atg5 and LC3 were detected by the western blotting analysis.Results: Serum AST and ALT levels gradually increased(P < 0.05) following reperfusion. Pathological analyses revealed considerable hepatocyte edema, congestion and apoptosis was observed at 6-24 h post-reperfusion as compared with the Sham group. mi R-30b-5p expression levels gradually decreased after reperfusion, however, levels of Atg12 and Atg5 m RNA increased thereafter(P < 0.001) as compared with the Sham group. Compared with Sham group, there was an significantly increasing of autophagosomes in IR group, meanwhile, apoptotic cells were significantly increasing. The expression of Atg12, Atg12-Atg5 conjugate and LC3 II was up-regulated following reperfusion. Cleave Caspase-3 expression showed a temporally dependent increase. Serum AST and ALT levels in the mi R-30b-5p agomir group mice decreased while that of the mi R-30b-5p antagomir group mice increased as a function of time following reperfusion(P < 0.001). The number of TUNEL-positive cells were significantly decreased in compared with that of the mi R-NC group, however, mi R-30b-5p antagomir increased the number of TUNEL-positive cells compared with that of the mi R-NC group. The mi R-30b-5p agomir resulted in a significant increase in PCNA expression but decrease in Caspase-3, Cleave Caspase-3 and PARP1 expression. In contrast, the mi R-30b-5p antagomir could decrease in PCNA expression but increase in Caspase-3, Cleave Caspase-3 and PARP1 expression. The luciferase reporter assay was performed to determine the effects of mi R-30 b on the Atg12 m RNA 3?-UTR. mi R-30 b could inhibit the induction of autophagosomes significantly. Rapamycin decreased the survival ratio of AML12 cells, but 3-MA inhibited the changes(P < 0.01). The viability of AML12 cells treated with mi R-30b-5p mimics or inhibitor responding to IR was enhanced by si RNA knockdown of Atg12. si RNA-mediated knockdown of Atg12 could decrease the levels of Atg12, Atg12-Atg5 conjugate and LC3 II.Conclusion: IR can induce down-regulation of mi R-30 b but up-regulation of Atg12, and increase autophagic activity. mi R-30 b can alleviate hepatic IRI by inhibiting apoptosis and repairing hepatic cell injury. |
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