Hypoxia Preconditioning Induced HIF-1α Promotes Glucose Metabolism And Protects Mitochondria In Liver I/R Injury

BackgroundIn liver transplantation,transplanted liver suffers from a series of ischemia/reperfusion (I/R) injuries. Previous study showed hypoxic preconditioning (HP) could give protection to brain, which resulted in an increased tolerance towards organ hypoxia. The following research showed that this endogenous protection also existed in some other tissues and organs, such as in liver and kidney.For tumor cells,hypoxia inducible factor-la (HIF-la) plays a very important role in hypoxia condition, and HIF-la is also the critical transcription factor mediating cell hypoxia reaction. In order to decrease the damage of reactive oxygen species (ROS) on cell DNA during aerobic metabolism, the transcription of glycolysis regulated by HIF-1α can make tumor cells get adapted to the hypoxic environment.Our study previously found hypoxic-induced HIF-1α decreased liver I/R injury after orthotopic liver autotransplantation on a rat model. It shows that HP is sufficient to activate the B cell lymphoma-2 (Bcl-2) signaling pathway and up-regulated the expression of Bcl-2 protein, are gulatory factor restrains apoptosis, and it may regulate apoptosis by altering the configuration of mitochondria.Other researches find that HIF-1α, as a master transcription factor, regulates transcription of many genes involved in glycolysis and plays an important role in glucose metabolism of tumor cells. The effect of HIF-1α on the expression of the enzymes related to glucose metabolism and synthesis of lactate under aerobic and hypoxic conditions in cancer cells. Hexokinases (HK) effect the change of glucose metabolism by generating flucose-6-phosphate which can be used through glycolysis to produce energy by getting diverted into the pentose phosphate pathway (PPP) toproduce NADPH and anabolic intermediates or converted to glycogen for storage, and HK-2 is thought to play a key role in promoting anabolic pathways and is frequently over expressed in cancers.In this study, we promoted HIF-1α expression of rat liver tissues by hypoxia preconditioning, and detected the changes of glucose metabolism, NF-κB and extracellular regulated protein kinases (Erk) pathways. It showed hypoxia induced HIF-1α promoted HK-2 and Glut-1 expressions could decrease the liver inflammation and I/R injury after orthotopic liver transplantation.Part one Hypoxia preconditioning induced HIF-la in liver I/R injuryBackground.This study based on the rat model of Autologous orthotopic liver transplantation (AOLT). The rat should be dealt with hypoxic preconditioning (HP) through the appropriate preoperative. Then we studied the expression of Hypoxia-inducible Factor-1α(HIF-1α)。Methods.1.The cleansing inbred SD rats were randomly divided into 3 groups:The group of rat dealed with hypoxic preconditioning before autologous orthotopic liver transplantation(Group HP:n= 36):The rats dealed with HP before operative (to give rats 8%oxygen with nitrogen-oxygen gas mixture which flew 5 L/min of 90 min before operative), after 8 h have done the liver transplantation; The group of rat dealed with autologous orthotopic liver transplantation (Group AT:n= 36):rats were dealed with autologous orthotopic liver transplantation; The control group of normal rats (Group Ctrl:n=16). The autologous orthotopic liver transplantation in two groups chosed the way of portal vein perfusion.2.Comparison the changes of rats after autologous transplantation. Each group of rats were cut the middle hepatic. To use the RT-PCR detecting the expression changes of liver HIF-la mRNA. To use the Western blot detecting the changes of HIF-la results in liver tissue.Results.1. To determine hypoxia preconditioning increased HIF-la expression in rat liver tissue, the rats were treated under 90-minute hypoxic preconditioning environment. The level of HIF-1α expression was significantly increased in liver tissue after treatment 12h (HP vs. Ctrl, P< 0.001).Then, the rats with hypoxia-induced HIF-1α were underwent the autologous orthotopic liver transplantation to process an ischemia-reperfusion procedure. The mRNA expression of HIF-1α in liver tissue was detected to stay at an increased level at 12 h after operation compared with control group(HP vs. AT, P< 0.001). Meanwhile, we observed there was an increased level of HIF-1α mRNA expression in AT group, but not significantly (AT vs. Ctrl, P= 0.0597) in due to the ischemia-reperfusion injuries.2.For the protein level, the HIF-la expression of liver tissue in HP group was detected higher compared with AT group and control group at 24 h after operation.Conclusions.In Hypoxic preconditioning rat autologous liver transplantation, HIF-1α mRNA and HIF-1α protein are both significantly increased.Part two HIF-1α promotes glucose metabolism and protects mitochondria in liver I/R injuryBackground. On the base of previous research that HIF-1α mRNA and HIF-laprotein are both significantly increased In Hypoxic preconditioning rat autologous liver transplantation.We will research glucose metabolism and protects mitochondria in liver I/R injury.Methods.1.The cleansing inbred SD rats were randomly divided into 3 groups:The group of rat dealed with hypoxic preconditioning before autologous orthotopic liver transplantation(Group HP:n= 36):The rats dealed with HP before operative (to give rats 8%oxygen with nitrogen-oxygen gas mixture which flew 5 L/min of 90 min before operative), after 8 h have done the liver transplantation; The group of rat dealed with autologous orthotopic liver transplantation (Group AT:n= 36):rats were dealed with autologous orthotopic liver transplantation; The control group of normal rats (Group Ctrl:n= 16). The autologous orthotopic liver transplantation in two groups chosed the way of portal vein perfusion.2.Comparison the changes of rats after autologous transplantation. Each group of rats were cut the middle hepatic ant taken the blood from inferior vena as the specimen. And we observed the pathological changes of liver cells, the damage of liver mitochondrial in the light microscope, transmission electron microscope and tested the changes in liver function, detected the change of TNF-a and IL-6 in serum. To use the RT-PCR detecting the expression changes of liver Glut-1mRNA、HK-2mRNA andPDK-1.Results.1. HIF-1α induced the expression levels of NF-kB and Erk.To determine the protection of increased hypoxia-induced HIF-1α we observed the morphology changes of liver tissue in three groups. In HP group, the hepatocytes were observed to have slightly swollen, and liver sinusoids was no significantly narrowed changes compared with AT group, whose the morphology changes of liver tissues were observed ashepatocellular swollen, narrowed liver sinusoid with inflammatory cells at 12h after operation. Previous studies showed HIF-1α expression was considered to correlate with activation of the NF-kB pathway on carcinoma researches. Up-regulating HIF-la also can activate the Erk signaling pathway. Under the rat model with autologous orthotopic liver transplantation, we found that HP group had significantly increased expressions in phosphorylated NF-kB (HP vs. AT, P = 0.0078) and phosphorylated Erk (HP vs. AT, P= 0.0044) in liver tissues compared AT group and control group at 24h after operation.2. Glucose metabolism increased in hepatic cells with hypoxia-induced HIF-la expression.Hypoxia-induced HIF-1α lead to the increase of glucose metabolism through up-regulation of the enzymes related to glycolysis. In our rat model, we detected the mRNA level of Glut-1 in liver tissue was increased after operation (HP vs. AT,12h and 24 h, P< 0.001).And the HK-2 mRNA level was also increased (HP vs.AT,24h, P = 0.004). Lactate dehydrogenase (LDHA) mRNA level was significantly increased at 24h after operation(HP vs. AT,24 h, P= 0.003; 48 h, P= 0.031). Pyruvate dehydrogenase kinase 1 (PDK-1) was increased in HP group after operation (HP vs. AT,24 h, P= 0.007;48 h, P= 0.001). The expression of Glut-1 in hepatocellular lysates of HP group also were significantly higher than AT group at 24h after operation (HP vs. AT, P= 0.0476),the expression of HK-2 was also increased (HP vs. AT, p=0.0373).3. Hypoxia-induced HIF-1α protected hepatocellular mitochondria from damage in I/R injury.We observed the morphology changes of hepatocellular mitochondria under transmission electron microscopy(46000X). The morphology of mitochondria in HP grouphad less swollen compared with AT group. And there were some cristae ruptures, but still have a small amount of arrangement. The structures of the endoplasmic reticulum were survived. While in AT group, the mitochondria appeared different sizes, some of that were obviously swollen with round shapes,vacuolar degenerationand severe reduction of visible ridges which were broken or disappeared. We also checked the mitochondria complex and found complex Ⅰ of AT group had damage in according to the low expressions of complex Ⅰ. In HP group,there were no significant changes of complex Ⅰ and Ⅱ compared with control group. From the TUNEL assay,we also detected there were more apoptosis expressions of liver tissue in AT group at 12h after operation. The expression of cleaved-Caspase 3 in hepatocellular lysates of AT group also were significantly higher than HP group at 12h after operation (HP vs. AT, P= 0.0119) as the same as poly ADP-ribose polymerase (PARP) (HP vs. AT,P= 0.0134).4. The changes of liver function and inflammation in rat model.In order to investigate the effect of HP treatment protect liver from I/R injuries, we monitored the changes of Alanine Aminotransferase (ALT) and Aspartate Aminotransferase (AST) in rat serum samples. In HP group, both of ALT and AST serum expression levels were lower compared with AT group, in which ALT and AST levels stayed at a high level after operation (ALT:HP vs. AT,6 h, P= 0.003; 12h,P= 0.001; AST:HP vs. AT,12 h, P= 0.002; 24 h, P= 0.006).We detected the changed of tumor necrosis factor-α(TNF-α) and interleukin-6 (IL-6) in serum samples and found that HP treatment seemed to relieve some inflammatory reaction due to the lower TNF-α and IL-6 expression levels(TNF-α:HP vs. AT,48 h, P= 0.0008; IL-6:HP vs.AT,48 h, P= 0.0213), and the mRNA levels of rat liver tissue also showed TNF-α and IL-6 in HP group were significant lower than At group at 48h after operation. (TNF-α:HP vs. AT,48 h, P= 0.005; IL-6:HP vs. AT,48 h, p=1.016).Conclusions. In Hypoxic preconditioning rat autologous liver transplantation, HIF-1α protect the transplanted liver’s histological structure against ischemia-reperfusion injury,and increase of glucose metabolism.