ABSTRACT
Objective To investigate the correlation of heme oxygenase-1 (HO-1) expression and autophagy in mice with liver ischemia reperfusion (IR) injury,and to study the influence of HO-1 on the hepatic function.Methods Control group,IR group,Hemin + IR group and Znpp + IR group were constructed.Mice in the Hemin + IR group and the Znpp + IR group were pretreated by Hemin (HO-1 inducer) and Znpp (HO-1 inhibitor) before IR.According to different reperfusion time,the IR group was divided into the 0,2,6,12,24,48 and 72 hours groups,and the Hemin + IR group and the Znpp + IR group were divided into the 0,2,6,12,24 hours groups.The levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were detected to evaluate the hepatic function.The autophagosome and pathological changes of liver were observed under electron microscope and hematoxylin and eosin (HE) staining,respectively.The expressions of HO-1 and autophagyassociated protein (LC3Ⅱ) in the protein level were detected by Western blot.All data were analyzed using the analysis of variance,t test and q test,respectively.Results The levels of ALT and AST of the IR group,Hemin + IR group and Znpp + IR group were significantly higher than those of the control group (F =96.17,85.53,P ＜0.05).The levels of ALT and AST of the IR 12 hours group and 24 hours group were significantly higher than those of the Hemin + IR 12 hours group and 24 hours group (qALT=--14.46,-7.85 ; qAST=-12.98,-5.26,P ＜0.05),but significantly lower than those of the Znpp + IR 12 hours group and 24 hours group (qALT=4.25,4.94;qAST=4.98,3.53,P ＜ 0.05).The results of HE staining showed that hepatic IR injury was significalty alleviated in the Hemin + IR group when compared with the IR group and the Znpp + IR group.The Suzuki degrees of hepatic IR injury in the control group,IR 12 hours group,Hemin + IR 12 hours group and the Znpp + IR 12 hours group were 0.5 ± 0.3,2.6 ± 0.5,1.1 ± 0.3,3.0 ± 0.4,respectively,with significant differences among the 4 groups (F =53.62,P ＜0.05).There were significant difference in the Suzuki degrees of hepatic IR injury between the IR 12 hours group and the Hemin + IR 12 hours group,and between the IR 12 hours group and the Znpp + IR 12 hours group (q =10.67,14.02,P ＜ 0.05).The results of electron microscopy showed that the number of autophagosome in the IR group was greater than the control group and Znpp + IR group,but lesser than the Hemin + IR group.The numbers of autophagosome in every 10 high power fields of the control group,IR 12 hours group,Hemin + IR 12 hours group and the Znpp + IR 12 hours group were 0.4 ±0.2,1.8 ±0.6,4.0 ± 1.8,0.7 ±0.5,respectively,with significant difference among the 4 groups (F =21.35,P ＜ 0.05).There were significant differences in the number of autophagosome between the IR 12 hours group and the Hemin + IR 12 hours group,and between the IR 12 hours group and the Znpp + IR 12 hours group (q =6.05,-3.03,P ＜ 0.05).The expressions of HO-1 and LC3Ⅱ in the IR group and the Hemin + IR group were significantly increased.The relative expressions of HO-1 protein in the Hemin + IR 6 hours group and the 12 hours group were 0.64 ±0.17 and 0.51 ±0.12,which were significantly higher than 0.45 ± 0.08 and 0.17 ± 0.03 of the IR 6 hours group and 12 hours group (t =4.03,4.69,P ＜ 0.05).The relative expressions of LC3Ⅱ protein in the Hemin + IR 6 hours group and the 12 hours group were 1.04 ± 0.20 and 1.20 ± 0.23,which were significantly higher than 0.58 ± 0.04 and 0.95 ±0.14 of the IR 6 hours group and 12 hours group (t =4.29,6.69,P＜0.05).The relative expressions of HO-1 protein in the Znpp + IR 6 hours group and 12 hours group were 0.23 ± 0.03,0.14 ± 0.02,respectively,and the relative expressions of LC3Ⅱ protein were 0.35 ± 0.04 and 0.49 ± 0.14,which were significantly lower than the HO-1 and LC3Ⅱ protein expressions in the IR 6 hours group and 12 hours group (tHO-1 =13.82,7.04; tLC3Ⅱ =7.21,4.03,P ＜0.05).Conclusion HO-1 is positively related to the amelioration of hepatic function,and it may be achieved by induction of autophagy.
ABSTRACT
Objective To study the expression and significance of miR-26a in intrahepatic cholangiocarcinoma.Methods The expression of miR-26a in 46 intrahepatic cholangiocarcinoma(ICC) tissues and peritumoral tissues was detected by quantitative real time polymerase chain reaction (qRT PCR).The intrahepatic eholangiocarcinoma cell line HCCC-9810 and RBE were transfected with miR 26a mimics and miR 26a inhibitors,respectively,by lipofectamine 2000.The growth curves were constructed by the CCK 8 method.The migration and invasion ability was demonstrated by wound healing and transwell assay.The cell cycle was analyzed by flow cytometry.The potential mechanism was illustrated by Western blotting.Results For the 46 ICC tissues and peritumoral tissues,miR 26a levels were significantly higher in the tumor tissues than in the peritumoral tissues (P＜0.05).Vascular invasion,TNM Ⅲ～Ⅳ stage and lymph node metastasis were significantly associated with high miR 26a expression levels (P＜0.05),but gender,age,tumor amounts,tumor encapsulation,tumor diameter and tumor differentiation showed no significant association (P＞0.05).Enhanced cell proliferation,migration and invasion ability,accelerated G0/G1 phase to S phase transition,activated AKT by PTEN suppression were observed in HCCC-9810 cells with up regulation of miR-26a.Conversely,cell proliferation,migration and invasion ability was inhibited,G0/G1 phase was blocked and AKT was restrained by PTEN increase wkh down regulation of miR-26a in RBE cells.PTEN mRNA in versely correlated with the miR-26a level (r=-0.8272,P＜0.01).Patients with a high miR-26a expression had significantly poorer overall survival (P＜0.05).A high miR 26a exprcssion,multiple tumors and lymph node metastasis were independent prognostic factors of overall survival (P＜0.01).Conclusion Overexpression of miR-26a in intrahepatic cholangiocarcinoma correlated with clinicopath ological features and overall survival,and it potentially promoted tumor proliferation and metastasis through the PTEN/AKT pathway.
