| With the development of whole-organ transplantation, research in the area of ischemia/reperfusion (I/R)-mediated injury has extended to the most commonly transplanted organs, including the liver, kidney, pancreas and lung. I/R injury is closely related to primary non-function (PNF) and allograft survival in transplantations and occurs in a biphasic pattern:an initial acute phase that is characterized by cellular damage at 3-6 h and a subacute phase that is characterized by massive neutrophil infiltration at 18-24 h. Importantly, the acute generation of ROS subsequent to the re-oxygenation that occurs in I/R inflicts direct tissue damage and initiates a cascade of deleterious cellular responses, leading to inflammation, cell death, and organ failure, indicating that ROS and NADPH oxidase, which is the main source of ROS, may play an important role in this process.Monocytes and macrophages are unique cells of the immune system in that they move via multiple steps by adhering to the vascular endothelium, migrating through the vascular membrane, and recruiting at the inflammatory site. Multiple lines of evidence have suggested that Kupffer cells, as specialized macrophages that are located in the liver, are critical to the onset of liver injury. The activation of Kupffer cells directly or indirectly by endotoxin leads to the release of an array of inflammatory mediators and ROS, while the inactivation of Kupffer cells alleviates liver injury. The inflammatory response and acute generation of ROS are essential for the clearance of bacterial, viral and parasitic pathogens, whereas excessive and prolonged activation is harmful to the host.Previous studies have indicated that the surface receptor integrin CD1 lb/CD18, also known as macrophage-1 Ag (Mac-1), serves as a pathogen reorganization receptor (PRR) to recognize pathogen-associated molecular pattern molecules (PAMPs) and damage- associated molecular patterns (DAMPs), such as Gram-negative bacteria-derived Lipopolysaccharides (LPS), aggregated β-amyloid, and High Mobility Group Box 1(HMGB1). However, the ligand-binding ability of Mac-1 may be due to the intrinsic properties of the A domain in the CD11b subunit, suggesting that CD11b plays a pivotal role for Mac-1 in host defense. In vivo, CD1 lb is the major integrin that is expressed on phagocytic cells and mediates the extravasation of phagocytic cells to inflamed organs and the phagocytosis of serum-opsonized pathogens, including the production of ROS and the release of cytokines and cytotoxic granules. However, whether CD11b plays a role in Kupffer cells and its molecular mechanism remain to be investigated.In the present study, we studied the role of CD11b and Kupffer cells during liver I/R injury, their relationship and mechanisms. First, we established a stable and reliable 70% liver I/R injury in mice by observing physical and mental state of mice after surgery and comparing the survival time of different models. Second, we proved the role of CD11b in liver I/R and its effector cell by utilizing CDllb deficient mice (C57BL/6 background, CD11b-/-) and wide type mice (C57BL/6, WT). Finally, we explore the mechanisms of CD1 lb involving in liver I/R injury.Part Ⅰ. Establishment of a 70% liver I/R injury model in miceObjective:To establish a stale and reliable 70% liver I/R injury model in mice. Methods:C57BL/6 mice were divided into sham group and 3 groups with different ischemia time, each group included 5 mice. The blood stream of the left and median liver lobes of mice in each group was occluded with a microvascular clamp for 50,60 and 70 min respectively. After then, the clamp was removed and the abdominal incision was Sutured. Mice of each groups were assessed by comparing weight loss, anorexia, mental state, survival time and histopathological change in liver after surgery. Results:Mice in 50 min group lost the least body weight, showed no anorexia and good mental state, survived over 10 days, but showed slight histopathological damage of liver. Mice in 70 min group lost the most body weight, showed serious anorexia and worst mental state, all died in 3 days. Mice in 60 min group had a moderate loss of body weight, anorexia and mental state, survived for more than 5 days, and showed clear histopathological damage of liver. Conclusion:Ischemia for 60 min is most suitable in the 70% liver I/R model.Part Ⅱ. Role of CDllb in liver I/R injury and its mechanismsObjective:To explore the role of CD1 lb in liver I/R and its mechanisms. Methods: 10 similar weight (20-25 g) CDllb-/- mice were devided into CD11b-/- sham group (n=5) and CD11b-/- group (n=5). Also,10 similar weight (20-25 g) WT mice were devided into WT sham group (n=5) and WT group (n=5). At different time point post reperfusion (1、 3.6、12、24、48 h), serum ALT and AST were analyzed to assess the liver function. HE and TUNEL were performed to estimate the magnitude of liver damage and the mRNA expression of TNF-a, IL-1β, IL-6, IL-8 and IL-10 were assessed. MDA production and GSH/GSSG ratio were measured to examined oxidative stress in the liver of CD11b-/- mice and WT mice after I/R. GdCl3 was used to deplete Kupffer cells whose effect was proved by immunostaining with a macrophage-specific marker (F4/80). Then, serum ALT, serum AST and TNF-a were analyzed to prove the role of Kupffer cells. NADPH oxidase activity and ROS production were assessed to prove the way that CD1 lb involve in liver I/R. The expression of p47phox, Src kinase and its phosphorylation was assessed by Immunoblot analysis to explore the signal path of CD11b. The relationship between Src kinase and p47phox was verified by an immunoprecipitation assay. Finally, PP2 (an Src family tyrosine kinases inhibitor) was used to pretreat Kupffer cells in a simulated I/R model, then the NADPH oxidase activity, ROS production and expression of Src and p47phox were analyzed. Results:Serum ALT and AST reached their peak at 3 h post reperfusion in each group and decreased to normal levels after 48 h. CD11b-/- mice displayed a significantly preserved liver function as represented by lower ALT and AST level, less histological damage and apoptosis compared to WT mice. The mRNA expression of TNF-a, IL-1β, IL-6, IL-8 in CD11b-/- mice were lower than that in WT mice, while IL-10 mRNA expression was higher in CD11b-/- mice. MDA production was lower while GSH/GSSG ratio was higher in CD11b-/- mice. Immunostaining proved that GdCl3 can deplete Kupffer cells effectively, ALT, AST levels and TNF-αmRNA expression were higher in mice without GdCl3 pretreated. NADPH oxidase activity and ROS production were significant lower in CD11b-/- mice. Further experiments showed that the phosphorylation of p47phox in Kupffer cells of CD11b-/- mice 3 h post reperfusion was significantly lower compared to that in WT mice. Also, we found that Src kinase is banding p47phox in Kupffer cells. PP2 pretreated Kupffer cells also showed decreased NADPH oxidase activity and ROS production, the phosphorylation of p47phox and Src kinase were also decreased at the same time. Conclusion:The integrin CD11b of Kupffer cells can modulated liver I/R injury, especially in the early phase of reperfusion. CD11b promotes the production of ROS in Kupffer cells through a Src-p47phox-dependent mechanism in the early phase of liver I/R insults, suggesting that the inhibition of CD11b may be a potential therapeutic target for the prevention of I/R injury in liver transplantation. |
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