Abstract
A rat HIRI model was constructed and treated with an intraperitoneal injection of agomir-miR-494 or agomir-NC (negative control) for 7 days after the surgery. The pathophysiological changes in sham-operated rats, HIRI, HIRI + agomir-miR-494, and HIRI + agomir-NC were compared. The effect of miR-494 was also assessed in an H2O2-induced apoptosis model. Hepatic AML12 cells were transfected with mimics NC or miR-494 mimics, followed by 6-h H2O2 treatment. Cell proliferation and apoptosis were detected by CCK8 assay and flow cytometry, respectively. Further, the miR-494 target gene was identified by luciferase reporter assay, and verified both in vitro and in vivo experiments. The activity of AKT pathway was further analyzed in vivo by Western blot. HIRI + agomir-miR-494 rats exhibited significantly higher miR-494 expression, lower serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and glutamate dehydrogenase (GLDH) level, lower hepatic MDA, TOA, and OSI, alleviated hepatic necrosis, reduced hepatocyte apoptosis, and decreased expression of apoptosis-related proteins, when compared with HIRI + agomir-NC rats (P<0.05 or 0.01). After H2O2 treatment, AML-12 cells transfected with miR-494 mimics had significantly higher proliferation and lower apoptosis rate compared with mimics NC group (P<0.01). PTEN was identified as an miR-494 target gene. PTEN expression was significantly down-regulated in AML12 cells transfected with miR-494 mimics, and was up-regulated by treatment of miR-494 inhibitor (P<0.01). Moreover, HIRI + agomir-miR-494 rats exhibited significantly lower PTEN expression, and higher p-AKT, p-mTOR, and p-p70S6K levels compared with HIRI + agomir-NC rats. Therefore, miR-494 protected rats against hepatic ischemia/reperfusion (I/R) injury through down-regulating its downstream target gene PTEN, leading to the activation of PI3K/AKT signaling pathway.
Highlights
Hepatic ischemia/reperfusion (I/R) injury is an inevitable complication occurring during surgical procedures such as partial hepatectomy and liver transplantation [1,2]
We further evaluated the extent of apoptosis in livers by transferase mediated dUTP nick-end labeling (TUNEL) assay, and found that the number of TUNEL-positive cells in HIRI group was notably higher compared with sham surgery group (Figure 1F)
To further explore the molecular mechanism underlining the protective effect of miR-494 in HIRI rats, we examined the expression of proteins in rat liver, including PTEN, p-AKT, AKT, and the downstream effectors of PI3K/AKT pathway (p-mTOR, mTOR, p-p70S6K, and p70S6K)
Summary
Hepatic ischemia/reperfusion (I/R) injury is an inevitable complication occurring during surgical procedures such as partial hepatectomy and liver transplantation [1,2]. Several studies have suggested the important roles of miRNAs in I/R injury, such as miR-122, miR-124, miR-146a, miR-223, miR-370 [11-15]. Expressed miR-494 has been reported in rats with cerebral I/R injury [16]. MiR-494 alleviates the I/R-induced myocardium injury in a mouse model through activating the AKT signaling pathway [17]. Overexpression of miR-494 exerts protective effects against hypoxia/ischemia-induced apoptosis in human hepatic L02 cells via the modulation of PI3K/Akt pathway [18]. The potential in vivo role of miR-494 in hepatic I/R injury remains unknown. The current study was undertaken to investigate the effect and relevant molecular mechanism of miR-494 in response to I/R-induced hepatic injury in a rat model. Our study may provide new insights into the development of novel therapeutic strategies for hepatic I/R injury
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