Protective Effects And Mechanisms Of Ischemic Postconditioning Against Hepatic Warm Ischemia And Reperfusion Injury In Rats

Background and ObjectivesHepatic ischemia and reperfusion (I/R) is a pathophysiological phenomenon that occurs very frequently in clinical practice. More importantly, I/R is also responsible for liver injuries that often lead to poor prognosis. Many efforts are being made to investigate the pathophysiological mechanism of I/R injury and to search for strategies that will protect liver against I/R induced damage. One of the approaches that have gained widespread recognition involves the use of ischemic preconditioning (IpreC), which has displayed significant protective effects in hepatic I/R injury through its abilities to stimulate endogenous protective mechanisms. However, since IpreC must be performed prior to the sustained ischemia, its usefulness is limited in clinical practices. Recently, a new and alternative strategy focusing on ischemic postconditioning (IpostC) has attracted extensive interest largely due to its remarkable success in protecting heart from I/R injuries. Unlike IpreC, the IpostC applies the brief episodes of ischemia at the onset of reperfusion following a prolonged ischemia. Since IpostC can be easily applied with precisely controlled timing, this approach appears to have greater potential for clinical application. However, most of the current studies are concentrated on the I/R of heart, and it remained to be determined whether IpostC can provide similar effective protection to other I/R organs. This study was therefore designed to first explore the protective activities of IpostC against hepatic I/R injury, followed by investigating whether combined IpreC and IpostC could provide additive or synergistic protections. At the same time, we are also endeavored to define the underlying antioxidative mechanisms with which IpostC protects liver from I/R injuries.Apoptosis and necrosis are the final outcome for hepatocytes during hepatic I/R process. Although they have long been believed as two distinct death modes, recent studies have revealed that the two processes partly employ a common signaling pathway, namely necrapoptosis, in which the mitochondrial permeability transition (MPT) is established to play a critical role. Formation of MPT can lead to cell apoptosis or necrosis. There are two distinct phases of liver injury after warm I/R. The initial phase (<3 h after reperfusion) is characterized by oxidative stress, where production and release of ROS appears to directly result in hepatocellular injury. The late phase of liver injury, which occurs 6 to 48 h after hepatic reperfusion, is an inflammatory disorder mediated by recruited neutrophils. In addition, many proteases will also be activated during I/R, and one particulary family of these proteases, calpain, is often responsible for cell death. Since IpostC is designed to apply in the early phase of reperfusion, it may exert protective effects through modulating the molecular alterations in the first 3 h after reperfusion. Therefore, we further explored whether IpostC could activate the protective molecular pathoways against necrapoptosis and reverse the cell death process, especially those occur in the early reperfusion phase.Cells can spontaneously respond to injury insults through activating its defensive mechanisms. Among them, cell cycle regulation plays a key role in cell structure and function recovery. Unlike many other organs, the liver possesses extreme ability of regeneration. Although the majority of hepatocytes stay in quiescent phase (GO phase), they can be quickly activated into cell cycle and start rapid proliferation upon proper stimulation. There are many critical molecules regulating the process from priming to proliferating. Amongst these molecules the cell cycle promoting factors, cyclin D₁ and CDK4, and suppressing factors, p21, play a critical role through dynamic coordination with each other. It is unclear whether the protection afforded by IpostC is related with any of these molecular alterations. This has prompted us to examine the expression of cyclin D₁, CDK4 and p21 so as to determine the impact of IpostC on cell cycle regulation and provide experimental evidences to guide future research and clinical application.Methods1. Establishment of 70% hepatic ischemia and reperfusion models in SD rats. This is accomplished through two phases of experiments. For phase one study, rats were divided into 6 experimental groups, i.e. (1) sham operation (S) group, (2) ischemia only (60 min) (I), (3) ischemia and reperfusion (I/R), and 3 IpostC groups that are subjected to 3 different cycles of reperfusion and occlusion at the onset of reperfusion, including (4) IpostC 1 (30 sec on/off), (5) IpostC 2 ( 1 min on/off) and (6) IpostC 3 group ( 2 min on/off). Changes of alanine aminotransferase (ALT) and Aspartate aminotransferase (AST) activities were measured with automatic biochemistry analyzer, together with the pathological evaluation of liver damages with microscopy in all groups.In the second phase of experiment we divided the rats into 5 groups, i.e. S group, I/R group, IpreC group, IpostC group (3 cycles of 1 min on/off) and combined IpreC and IpbstC (IpreC/IpostC ) group. In addition to the parameters detected in the first phase of experiment we have also evaluated the 7 days of survival rate in I/R,IpreC,IpostC and IpreC/IpostC groups by using the total hepatic ischemia/reperfusion after 70% liver ischemia.2. To explore the role of antioxidative mechanisms in protection of IpostC against hepatic I/R injury, the tissue levels of MDA, GSH, SOD, GSH-P_X and MPO at 6 hour after reperfusion were analyzed by colorimetric method.3. To examine the changes of apoptosis and genes involved in apoptosis, three experimental groups were set up, including S group, I/R group and IpostC group. Tissue and plasma levels of TNF-αat 1 h and 3 h after reperfusion were quantified using ELISA. Expressions ofμ-Calpain,Talin,Bid,Cyt C and Caspase-3 proteins were also detected using Western blot analysis. The hepatic ultrastructure at 1 h after reperfusion were observed using transmission electron microscope (TEM). The survival rate of hepatocytes at 1 h and 3 h after reperfusion were assessed by flow cytometry (FCM) using Annexin V/PI double-stained method.4. To determine the role of cell cycle regulators, three groups were set up, including S group, I/R group and IpostC group. Expressions of cyclin D₁, CDK4 and p21 proteins and messenger RNA were detected using Western blot analysis and real-time PCR respectively. The cell cycle percentage of hepatocytes were detected by FCM using PI stained method.Results1. Protective effects of ischemic postconditioning against hepatic ischemia/reperfusion injury.In the first phase of experiment, our results showed increased plasma activities of ALT and AST in S group (68.30±11.12 U/L vs. 211.65±95.63 U/L), 30 min of ischemia (78.33±32.12 U/L vs. 260.33±45.12 U/L) and 60 min of ischemia (210.85±75.75 U/L vs. 340.93±108.52 U/L). With prolonged reperfusion time the plasma activities of ALT and AST further increased (P<0.05) . Compared with I/R group, the plasma activities of ALT and AST at 60 min and 6 h after reperfusion were lowered in IpostC 1 and IpostC 2 groups, with the later being more significant. No significant changes of the plasma activities of ALT and AST was found in IpostC 3 group. Histopatholoical examination revealed evidences of descreased cellular injuries in rat livers of IpostC 1 and IpostC 2 group, while rats in IpostC 3 groups suffered damages similar to those in the I/R group.In the second phase of experiment, significantly decreased activities of ALT and AST were detected in IpreC,IpostC and IpreC/IpostC groups as compared with I/R group. When IpreC and IpostC group were compared, no further decrease was observed in IpreC/IpostC group. Instead, there appears to be a slight increase in IpreC/IpostC group, although the differences was not significant. Similarly, combined IpreC and IpostC failed to minimize pathologic injuries compared with either IpreC or IpostC group. 2. The role of antioxidative mechanism in protection of IpostC against hepatic I/R injuryContrast to I/R group, decreased tissue MDA content and MPO activities, increased tissue GSH content and SOD and GSH-P_X activities at 6 h after reperfusion were found in IpostC 1 and IpostC 2 groups. The degree of alteration in IpostC 2 group was more pronounced than that in IpostC 1 group. No significant alteration was found in IpostC 3 group with the level comparable to I/R group. Similarly, combined IpreC and IpostC failed to produce improvement in the hepatic levels of MDA,SOD,GSH- PX,GSH and MPO.3. Effect of ischemic postconditioning on the necrapoptosis pathway against ischemia/reperfusion injuryCompared with S group the contents of tissue and plasma TNF-αat 1 h and 3 h after reperfusion were higher in both I/R and IpostC groups. After ischemic postconditioning, decreased level of TNF-α, albeit higher than that in S group, was produced. Compared with I/R group, IpostC inhibited the expression ofμ-Calpain, Bid, Cyt C and Caspase-3, enhanced Talin expression and lessened the injury on cellular ultrastructure. IpostC promoted cell survive and suppressed cell death at 1 h and 3 h after reperfusion.4. Effect of ischemic postconditioning on cell cycle molecules cyclin D₁,CDK4 and p21Compared with I/R group, IpostC increased the expression of cyclin D₁,CDK4 and p21 both at proteins and mRNA levels, with highest expression level in CDK4. Compared with S group, although a shift of cell population from G₀/G₁ phases to S phase was observed in both I/R, a more pronounced changes were produced in the IpostC group. Conclusions1. Ischemic postconditiomng is effective in protecting against hepatic ischemia and reperfusion injuries, resulting in preservation of liver function, decrease of tissue pathologic injuries and significant improvement of 7 days survival rate.2. Combined ischemic preconditioning with ischemic postconditiomng can not offer additive protection, only produced a weakened protection that is less effective than those produced by them acting alone.3. The protective role of ischemic postconditiomng against hepatic ischemia and reperfusion injury are related to its antioxidative mechanisms that include reducing the activities of lipid peroxidation, increaseing tissue GSH content and SOD and GSH-P_X activities and suppressing polymorphonuclear neutrophils infiltration.4. Ischemic postconditiomng can decrease the cellular injuries and promote cell survival through suppressing cytokine production and disturbing the protein expressions ofμ-Calpain, Bid, Cyt C, Caspase-3 and talin.5. Ischemic postconditiomng can prime and promote the cell cycle entry through increasing the mRNA and protein expressions of cyclin D₁, CDK4 and p21.4. Our study provided experimental evidences to support further study of the protective mechanism of ischemic postconditiomng against ischemia and reperfusion injury, which should not only guide future development of novel drugs mimicking the mechanisms of ischemic postconditiomng, but also provide new therapeutic targets in hepatic anti-ischemia and reperfusion injury researches.