| Ischemia/reperfusion injury is a phenomenon whereby tissues experience damage as a result of temporarily interrupted blood flow (ischemia) followed by its restoration (reperfusion). Clinically, liver I/R occurs in settings of elective liver surgery, trauma, and transplantation. Liver I/R is a pathophysiologic process whereby hypoxic organ damage is accentuated following return of blood flow and oxygen delivery. Liver IRI commonly results in liver failure after transplantation, delayed hepatic dysfunction and a variety of complications such as biliary injury, significantly impact on prognosis of liver transplantation. Ischemia-reperfusion injury (IRI) represents the major problem in clinical organ transplantation. Thus, understanding the pathophysiology and minimizing the effects of hepatic IRI would probably improve liver surgery and transplant outcomes.Heme oxygenase-1(heme oxygenase-1, HO-1) is also called heat shock protein32, mainly derived from kupffer cells, a rate-limiting stress-responsive enzyme, converts heme into biliverdin, free iron, and carbon monoxide. HO-1exerts potent cytoprotective defense functions against oxidative injury, including adaptive antiinflammatory and antiapoptotic effects in organ transplantation. Therefore, it is also called "graft survival genes". HO-1, the inducible form of HO, is induced by various stimuli, such as hypoxic, heavy metals, ischemia-reperfusion, and its expression is highly linked to fundamental adaptive cellular responses against oxidative stress. Indeed, HO-1overexpression by genetic engineering or by pharmacological agents exerts cytopro-tective effects in hepatic I/R injury transplant models, as evidenced by preserved organ function and prolonged. Moreover, HO-1possesses potent immunomodulatory properties through the regulation of pro-inflammatory pathways in hepatic I/R.Toll-like receptors systems (TLRs) function as pattern-recognition receptors (PRRs) that respond to a myriad of highly conserved ligands. These substrates include pathogen-associated molecular patterns (PAMPs) for the recognition of invading pathogens, as well as damage-associated molecular patterns (DAMPs) for the recognition of endogenous tissue injury. TLRs belong to the interleukin-1receptor (IL-1R) family, are expressed on various immune cells, such as hepatocytes, kupffer cells, macrophages, neutrophils, dendritic cells, and et al. Their activation results in an innate inflammatory response mediated by macrophages, neutrophils, and complement. TLRs were recently shown to participate in the recognition of endogenous proteins that are released from damaged tissues after I/R,such as heat shock protein, extracellular matrix degradation products,and thus initiat an innate immune system response,leading to the development of inflammation..A large body of evidence suggests that TLRs play central roles in sensing tissue damage and activating the innate immune system following I/R.Several recent studies reported cross talk between HO-1and the TLR system in the mechanism of hepatic I/R. Defective TLR4signaling increased HO-1expression in hepatic I/R, suggesting its role as a putative HO-1repressor. Interestingly, HO-1overexpression was capable of attenuating type1interferon (IFN) signaling triggered by I/R-mediated TLR4activation, aggravating liver tissue inflammation injury. TLR4gene defects can protect the mice from liver IRI heat, reduce the levels of ROS, cell factor, HMGB1expression and increase the protective HO-1.However, the complex mechanisms by which the link between the sentinel TLR4system and HO-1influence I/R injury sequel are still unclear. [Objective]In this study, we employed a rat model of partial liver warm IRI in an attempt to analyze the exact mechanismsof liver IRI, and dissect TLR4and heme oxygenase-1(HO-1) networks in liver IRI.[Method]1, Male Sprague-Dawley rats (200-250g) were obtained from Kunming Medical University.Rats were fasted for18hr before the experiments but were provided with tap water ad libitum.With reference to Nauta method, we employed a rat model of partial70%liver warm IRI.2, animal groups:36healthy adult male SD rats were randomly divided into four groups:(1) the sham operation group (group S)(n=9):no preoperative for injection, only laparotomy and anatomy of hepatic portal operation;(2) the ischemia reperfusion group (group Ⅰ)(n=9):no preoperative for injection, liver ischemia reperfusion;(3) cobalt protoporphyrin (CoPP) group (group C)(n=9):24hours after intraperitoneal injection of HO-1induced by CoPP in hepatic ischemia reperfusion;(4) zinc protoporphyrin (ZnPP) group (group Z)(n=9):reperfusion of ischemia for24hours before intraperitoneal injection of HO-1inhibitor ZnPP. Liver tissue and blood samples were taken at24h after reperfusion. The hepatocellular function (ALT, AST) was analyzed. ELISA to detect HMGB1level.(TNF-α, IL-6, HO-1and IL-10) mRNA expression levels of Liver tissue were examined by RT-PCR; Liver tissue protein (TLR4, MyD88, p/t-IRF3, TRIF), nucleoprotein (NF-kB) and cytoplasm protein I kB-a were examined by western blot; Immunohistochemical method to observe CD11b+macrophages infiltration of the liver portal area; The hepatocellular damage was evaluated by liver histology.[Results]1. The modified Nauta’s "local70%liver ischemia reperfusion model" is a stable, reliable, simple, convenient and practicable model, is suitable for research of ischemia-reperfusion injury.2. Regulation of expression of HO-1effect on liver.①Effect of HO-1expression.compared with S group, the levels of HO-1were higher (P< 0.05).Compared with I group, the levels of HO-1were significantly higher (P<0.05); in Z group the levels of HO-1were significantly lower (P<0.05).②Effect of liver function. Compared with group S, group I, group C, group Z transaminase (ALT, AST) increased significantly (P<0.05); compared with I group, C group of transaminase (ALT, AST) decreased significantly (P<0.05), group Z transaminase (ALT, AST) increased significantly (P<0.05).③The changes of liver morphological. S group:normal liver cells, no degeneration or necrosis, hepatic sinusoidal structure clear, only a little inflammatory cell infiltration. I group:liver cells in different degree edema, vacuolar degeneration, liver cells showed focal necrosis, nuclear dissolution, eosinophils increased, increased inflammatory cell infiltration. Compared with I group, C group, liver cell injury is lighter, the hepatic lobule structure basically normal, liver cells and portal area basically normal, only a few mild liver cell edema, infiltration of inflammatory cells reduced. Compared with I group, Z group, liver cell damage is more serious, liver cell edema, patchy necrosis of liver cells, nuclear dissolution, periportal inflammatory cell infiltration.3. TLRs is involved in hepatic ischemia-reperfusion injury, and have a talk with HO-1in hepatic ischemia-reperfusion injury and their possible mechanism:compared with S group, after ischemia/reperfusion, the levels of ALT and AST were significantly higher (P<0.05), the levels of serum HMGB-1were increased (P<0.05),the levels of HO-1were significantly higher (P<0.05), the inflammatory factors (TNF-α, IL-6) express were higher (P<0.05), the levels of liver tissue protein (TLR4, P-IRF3, TRIF, t-IRF3, MyD88) and nucleoprotein (nf-kappa B) were elevated (P<0.05), the levels of cytoplasm protein I kappa B-a were elevated (P<0.05), the macrophages CDllb+gathered at liver portal area were significantly increased,the injury in liver tissues aggravated. After CoPP interference, the levels of ALT and AST were significantly lower (P<0.05), the levels of serum HMGB-1were lower (P<0.05),the levels of HO-1were significantly higher (P<0.05), the inflammatory factors (TNF-α, IL-6) express were lower(P<0.05), the levels of liver tissue protein (TLR4, p-IRF3, TRIF, t-IRF3, MyD88) and nucleoprotein (nf-kappa B) were lower (P<0.05), the levels of cytoplasm protein I kappa B-α were elevated (P <0.05), the macrophages CD11b+gathered at liver portal area were significantly lower,the injury in liver tissues lessened. After ZnPP interference, the levels of ALT and AST were significantly increased (P<0.05), the levels of serum HMGB-1were increased (P<0.05),the levels of HO-1were significantly lowerr (P<0.05), the inflammatory factors (TNF-α, IL-6) express were increased(P<0.05), the levels of liver tissue protein (TLR4, p-IRF3, TRIF, t-IRF3,MyD88) and nucleoprotein (nf-kappa B) were increased (P<0.05), the levels of cytoplasm protein I kappa B-α were lower (P<0.05), the macrophages CD11b+gathered at liver portal area were significantly increased,the injury in liver tissues significantly aggravated.[Conclusion]1, The modified Nauta’s "local70%liver ischemia reperfusion model" is a stable, reliable, simple,convenient and practicable model.2, CoPP intervention can induce high expression of HO-1, reduce liver ischemia-reperfusion injury; ZnPP intervention can restrain the expression of HO-1, aggravate liver ischemia-reperfusion injury.4, TLR4play a role in liver I/R, and with HO-1close in hepatic ischemia-reperfusion injury.Speculating that TLR4as a putative HO-1repressor.The cross talk between heme oxygenase1and TLR4in the mechanism of hepatic I/R injury:CoPP exerts its immunom odulatory effects against hepatic I/R insult through suppression of the type1IFN pathway of the TLR4signaling cascade, and these effects are related to its HO-1upregulation. CoPP attenuated the increase in TLR4/TRIF/IRF3signals. Furthermore, ZnPP pretreatment reversed these inhibitory effects. |
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