RESUMO
Hepatic ischemia/reperfusion (I/R) injury contributes to morbidity and mortality during liver resection or transplantation, with limited effective treatments available. Here, we investigated the potential benefits and underlying mechanisms of pterostilbene (Pt), a natural component of blueberries and grapes, in preventing hepatic I/R injury. Male C57BL/6 mice subjected to partial warm hepatic I/R and human hepatocyte cell line L02 cells exposed to anoxia/reoxygenation (A/R) were used as in vivo and in vitro models, respectively. Our findings showed that pretreatment with Pt ameliorated hepatic I/R injury by improving liver histology, decreasing hepatocyte apoptosis, and reducing plasma ALT and AST levels. Likewise, cell apoptosis, mitochondrial membrane dysfunction, and mitochondrial ROS overproduction in L02 cells triggered by the A/R challenge in vitro were reduced due to Pt administration. Mechanistically, Pt treatment efficiently enhanced mitophagy and upregulated PINK1, Parkin, and LC3B expression. Notably, the protective effect of Pt was largely abrogated after cells were transfected with PINK1 siRNA. Moreover, Pt pretreatment promoted hepatocyte proliferation and liver regeneration in the late phase of hepatic I/R. In conclusion, our findings provide evidence that Pt exerts hepatoprotective effects in hepatic I/R injury by upregulating PINK1-mediated mitophagy.
Assuntos
Regulação da Expressão Gênica/efeitos dos fármacos , Infarto Hepático/genética , Infarto Hepático/prevenção & controle , Mitofagia/efeitos dos fármacos , Mitofagia/genética , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Traumatismo por Reperfusão/genética , Traumatismo por Reperfusão/prevenção & controle , Estilbenos/farmacologia , Estilbenos/uso terapêutico , Animais , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Modelos Animais de Doenças , Hepatócitos/fisiologia , Humanos , Regeneração Hepática/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
PURPOSE: To investigate the role and mechanism of melatonin-loaded polymer polyvinyl caprolactam-polyvinyl acetate-polyethyleneglycol graft copolymer micelles (Mel-Mic) in dry eye disease (DED). METHODS: In vitro, the apoptosis and reactive oxygen species (ROS) generation in human corneal epithelial cells (HCECs) were analyzed by immunostaining and flow cytometry. The effect of Mel-Mic on autophagy and mitophagy was evaluated by immunostaining and western blots. PINK1 knockdown was analyzed by small interfering RNA. In vivo, sodium fluorescein staining, tear secretion test, and periodic acid-Schiff staining were used to determine whether Mel-Mic can alleviate the severity of DED. Small molecule antagonists were pretreated to investigate whether melatonin type 1 and/or 2 receptors (MT1/MT2) mediate the effects of Mel-Mic. RESULTS: Mel-Mic improved the solubility and biological activities of Mel in aqueous solutions. Treatment with Mel-Mic decreased the apoptosis of HCECs exposed to hyperosmotic medium, accompanied by downregulation of cleaved Caspase-3 and upregulation of Bcl-2. In addition, Mel-Mic application suppressed ROS overproduction, rescued mitochondrial function, and decreased the level of oxidative stress associated biomarkers (COX-2 and 4-HNE) in HCECs. Interestingly, HCECs treated with Mel-Mic exhibited increased levels of mitophagy markers (PINK1, PARKIN, Beclin 1, and LC3B) and restored impaired mitophagic flux under hyperosmolarity. While PINK1 knockdown largely abolished its protective effects. In vivo, compared to vehicle group, topical Mel-Mic-solution-treated mice showed significantly improved clinical parameters, increased tear production, and decreased goblet cells loss in a dose-dependent manner. Also, transmission electron microscopic assay revealed increased autophagosome number in the corneal epithelium of Mel-Mic group. Moreover, luzindole, a nonselective MT1/MT2 antagonist, but not 4-P-PDOT, a selective MT2 antagonist, blocked the protective effect of Mel-Mic. CONCLUSIONS: Our findings demonstrated that Mel-Mic ameliorates hyperosmolarity-induced ocular surface damage via PINK1-mediated mitophagy and may represent an effective treatment for DED possibly through acting MT1 receptor.