Mechanisms Of Hepatocellular Damage Induced By Cardiopulmonary Bypass In Rats And The Protective Effects Of Growth Hormone Up-regulating STAT5 Signal Pathway

Background and objective:Cardiopulmonary bypass is a severe stress which can provoke a systemic inflammatory response syndrome (SIRS). Contact of the blood components with artificial surface of the bypass circuit, ischemia-reperfusion injury, operative trauma are mainly possible causes of SIRS. The excess inflammatory reaction may contribute to the development of postoperative complications and result in multiple organ dysfunction syndromes (MODS), which is one of the leading causes of death in cardiac surgery. Therefore, protection of organs during CPB is all along the emphasis in cardiovascular surgery. Over the past years, studies were mainly focused on the important organs such as heart, lung, brain and kidney. However, we know little about the liver. As we all know, liver is a central and important target organ responsing to the surgical stress and it is also one of the vulnerable organs for the attack of proinflammatory cytokines. It has long been recognized that early jaundice and liver damage could appear followed CPB surgery. Furthermore, critical hepatic lesion is still troublesome in clinical treatments and often can lead to MODS or mortality. For patients with preoperative chronic liver disease (CLD) or liver cirrhosis, mortality of CPB surgery can achieve 25-31%. So hepatosis is serious complication after CPB surgery. It is well known that liver disease is still a major health problem in China. As techniques of open-heart surgery and postoperative patient care improve, the number of patients with preoperative comorbidities who undergo CPB surgery is increasing. However, clinical outcome after CPB surgery in patients with CLD are still unsatisfactory. Due to the systemic and hepatic effects of CPB, the risk of further hepatic damage during CPB to an already compromised liver must also be a particular concern. But the research of CPB on the hepatocellular damage is absent and its accurate mechanism is still unknown.Clinical investigations showed that hyperbilirubinemia or jaundice followed CPB came about from an increase in both conjugated and unconjugated bilirubin. It indicates that both the impaired liver function for bilirubin transport and the increasing production of bilirubin from haemolysis lead to them. As we all know, hepatobiliary membrane transporters (HMTs) in liver cell are in main charge of bilirubin transporting. The expression anormaly of HMTs genes could reuslt in hepatocellular cholestasis and damage. However, the mechanisms of signal regulation of these genes expression are still unclear. Since CPB can provoke anormaly of bilirubin metabolism, the molecular mechanism is still unknown. We presume that it is concerned with the changes of HMTs expression.Combination of literature reports, promoter regions of major HMTs genes contain interferoid (IFN)-activated sequence. The transcription of HMTs can be driven by corresponding transcription factors binding to the sequence. Signal transducer and activator of transcription (STAT)-5 is the signal protein in endochylema. STAT5 activation is attributable to an increase in nuclear translocation of phosphorylated liver STAT5, which binds to IFN-activated sequence located in the target gene promoter region and thereby stimulates gene transcription. So we make a hypothesis that STAT5 pathway can regulate the expression of HMTs genes.STAT5 signal pathway have extensive biological effects, such as promote anabolic metabolism, stimulate cell regeneration, resist apoptosis, regulate acute phase reaction, stabilize mucosal barrier function, and etc. The axis of growth hormone (GH) and growth hormone receptor (GHR) is one of the main pathways to evoke the activation of STAT5. GHR locates mainly in liver cell. The activity of STAT5 will be depressed when GH secretes insufficiently or the expression GHR decreases. Therefore, it is feasible theoreticallythat up-regulation STAT5 by exogenous GH can regulate the expression of HMTs. This study will strengthen the recognition of molecular mechanisms of signal regulation of the hepatocellular bilirubin transporting.CPB is an expectable stress response. So building tolerance to perioperative damage during the unstressed condition may result in a more advanced method to assuage CPB-induced liver damage, and exploiting a natural defense mechanism called stress response may be a potential approach to overcome this problem. But the effect and mechanism of preconditioning with GH on the protection of liver function after CPB are still unknown. Consequently, this study based on the experimental model of CPB in rats is aimed to investigate the mechanisms of hepatocellular damage induced by CPB and the protective effect and mechanism of GH at cellular and molecular level, and to build a new strategy and provide a new target for the protection of liver function during and after CPB.Methods:1) Non-transthoracic CPB-2h model in rats was established via left carotid artery and right jugular vein cannulation for arterial perfusion and venous return respectively by using microsurgical instruments and miniature circuit devices. Adult male Sprague-Dawley rats, weighing 480±20g, were housed in wire-bottomed cages in a temperature-controlled room with a 12-hour light/dark cycle. Rats were acclimatized to the environment for 7 days. All animals received humane care. Rats were randomly divided into two groups according to the administration of GH before the initiation of CPB. Group G intramuscularly received 2.5mg/kg body weight of recombinant human GH (rhGH, Serono inc., Switzerland) at 8 AM every 24h for three days and just before the initiation of CPB. Group C (n=15) served as control and only saline was added in the same way. Sham-operated and normal control groups were assigned at the same time.2) To observe relationship between hepatocellular damage and SIRS, we detect indexes inferior: liver function; serum constitutive hepatic proteins and acute-phase proteins (APPs); serum cytokines such as tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), IL-6, and IL-10 by using a rat-specific enzyme linked immunosorbent assay (ELISA); serum GH, GH binding protein (GHBP), insulin-like growth factor (IGF)-I, and IGF binding protein (GHBP)-3 (radioimmunoassay, RIA); and liver changes: liver protein concentration, liver cell proliferation (immunohistochemical staining for proliferative cell nuclear antigen (PCNA)), liver cell apoptosis (TUNEL immunohistochemical method), and pathological and morphological changes.3) To observe the changes of liver function for bilirubin transport induced by CPB, we determine the expression of hepatic HMTs (NTCP,BSEP, and FXR) at the level of cells and molecules (mRNA and protein) by using immunofluorescence, RT-PCR and Western blot analysis.4) To study the influence of CPB on hepatocyte STAT5 and the mechanisms of protective effects of up-regulation STAT5 activity (by GH-GHR axis pathway) regulating HMTs, We determine the expression of hepatic GHR and STAT5 at the level of cells and molecules (mRNA and protein) by using immunofluorescence, hybridization in-situ, RT-PCR and Western blot analysis; furthermore, We also observe the influence of the changes of liver STAT5 activity on hepatocyte function.5) Statistical analysis: Values of continuous variables were expressed as mean±standard deviation. Comparisons between groups were analyzed by 2-way repeated-measures analysis of variance (ANOVA) and the unpaired Student t test. Correlation between data was analyzed with linear regression. Statistical analysises was performed with computerized statistical packages (SPSS 11.0 software, SPSS, Chicago, IL, USA). Significance was accepted at p<0.05.Results:The main results in this study were as follows:1) Non-transthoracic CPB model in rats, via left carotid artery and right jugular vein cannulation for arterial perfusion and venous return respectively by using miniature circuit devices, was successfully established and was associated with good recovery. The results of hemodynamics and blood gas analysis were within normal range during bypass.2) There were significant liver injuries at CPB termination and markedly at 3h after CPB termination in both groups. Administration of rhGH markedly increased serum IGF-I and IGFBP-3 compared with group C. Group G showed significantly lower serum concentrations of alanine aminotransferase (ALT) and total bilirubin (TB) after CPB termination. Those receiving rhGH demonstrated a significant increase in serum prealbumin and transferrin and a marked decrease in serum amyloid A and C-reactive protein. rhGH significantly decreased serum tumor necrosis factor (TNF)-αand interleukin (IL)-1β, whereas no changes were found for serum IL-6 and IL-10. rhGH significantly increased total liver protein content, hepatocyte proliferation and decreased hepatocyte apoptosis verus group C. Liver injury was positive correlation with the proinflammatory cytokine levels and serum GHBP level.3) There was significant hepatocellular bilirubin tansport dysfuntion after CPB. Hepatic HMTs were very sensitive to CPB and markedly depressed at 3h after CPB termination. In earlier period of CPB termination, the expression of HMTs at the levels of mRNA and protein was low and nonuniform to some extent, the expression of farnesoid X receptor (FXR) was also depressed obviously at the levels of mRNA and protein.4) Hepatic STAT5 and GHR were lower at the levels of mRNA and protein at CPB termination and markedly depressed at 3h after CPB termination. Administration of rhGH markedly increased the activity of STAT5 detected by EMSA compared with group C. There was marked positive correlation between the expression of STAT5 and GHR, they were all correlated with hepatocyte dysfunction negatively, which indicated resistance of GHR might contribute to the inhibition of STAT5 after CPB and they both influence the hepatocyte function.5) The effect of up-regulation of STAT5 activity on hepatocyte functions after CPB. Pretreatment with GH can up-regulate the expression of STAT5, which can control the expression of hepatic HMTs and elevate the expression of FXR. These results contribute to hepatic transportation of bilirubin and relieve the hepatocelluar damage directly.Conclusions:1) The nontransthoracic CPB model in rats is established successfully. The nontransthoracic CPB model in rats is easy to establish and is associated with good recovery, which can in principle simulate the clinical setting of human CPB. This reproducible model is fit to study on the pathophysiological process of CPB in vivo.2) CPB results in obvious hepatocellular damage. Possible patterns of manifestations are shown inferior: (1) CPB induces acute liver injury in a rat model via increases in serum ALT,serum TB, serum acute phase proteins, proinflammatory cytokines TNF-α,IL-1β, and hepatocyte apoptosis, which is associated with decreases in serum concentrations of constitutive hepatic proteins; (2)There exists significant hepatocellular bilirubin tansport dysfuntion followed CPB. Depressive and nonuniform expression of HMTs and FXR is one of the key points for hepatocellular damage induced by CPB. Bilirubin accumulation in hepatocyte, excessive inflammatory respose and APR, and down-regulation of STAT5 induced by GH resistance contribute to the Depressive and nonuniform expression of HMTs.3) GH can prevent CPB-induced acute liver injury in a rat model. This strategy of pretreatment with GH might be a prospective management for preventing acute liver injury and lessening SIRS when CPB is performed. Up-regulation of STAT5 pathway has obviously protective effects on hepatocyte after CPB. Possible mechanisms are shown inferior: (1) directly control the expression of hepatic HMTs, which can promote hepatic transportation of bilirubin and relieve the hepatocelluar damage directly; (2) elevate the expression of FXR, which can regulate the expression of HMTs; (3) relieve APR and proinflammatory cytokines TNF-αand IL-1β, which can lessen the inflammatory hepatocellular injury directly and remove the inflammatory inhibition of HMTs expression indirectly; (4) increase the synthesis of hepatic constitutive hepatic proteins decrease, decrease hepatocyte apoptosis, and stimulate hepatocyte proliferation.