Protection And Mechanisms Of Lipoxin On Acute Liver Failure

[Objictive]To observe the Protection of LXA4 on rat liver cells injury in vitro.To observe the Protection of LXA4 on rat acute liver failure In vivo,and to probe the mechanisms preliminarily.[Methods]1. Kupffer cells (KCs) was isolated in situ by two ways liver perfusion. Using trypan blue dye method to measure activity of KCs, phagocytosis experiments to function,ED1 immunofluorescence cytochemistry to purity.2. Lipopolysaccharide (LPS) stimulated KCs for 12 hours,24 hours,36 hours, the supernatant were collected to detect the content of TNF-a. In another group,LXA4 and KCs were incubated half an hour before given LPS.3. Co-incultured with rat liver cells and KCs,and then gave D-galactosamine (D-GalN)/LPS stimulation for 24 hours. Measured alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels in supernatant. Collected liver cells to detect apoptosis with Annexin V-FITC method. In another group, LXA4 were incubated half an hour before given D-GalN/LPS.4. Rat model of acute liver failure was induced by intraperitoneal injection with D-GalN+LPS. Rats were divided into six groups:normal group, model group, LXA4 small, medium and high dose group, Pyrrolidinedithiocarbamic acid (PDTC) group. Pathology changes of liver were detected to evaluate liver tissue injury; ALT and AST were measured by automatic biochemical analyzer; The content of TNF-a and IL-6 in serum was examined by ELISA;Caspase 3 activity was detected by Caspase 3 Activity Assay Kit; TNF-a mRNA and IL-6 mRNA expression was assaied by Realtime-PCR; TUNEL assay Apoptosis of liver cells was detected by TdT-mediated dUTP nick end labeling(TUNEL); Rat hepatocytes and Kupffer cells were isolated, nuclear protein was extracted, liver cells and KCs nuclear NF-κB activity were detected by gel electrophoresis mobility shift assay (EMSA).[Results]1. KCs activity was measured:the number of alive cells was 90-96% with the average (92.3±2.12)%. Total amount of KCs isolated in each rat was (2.41±0.32)×107 in average.2. KCs phagocytic experiment showed that the number of cells (95.2±2.58)% had function.3. ED1 immunofluorescence cytochemistry staining showed cell purity was (96.3±1.46)%.4. After LPS stimulation to Kupffer cells, the supernatant of TNF-a levels increased significantly at 12 hours, and 24 hours to reach the peak. TNF-a levels in LXA4 preincubation group were significantly decreased (P<0.05).5. Co-incultured with KCs and liver cells,then stimulated with D-GalN/LPS for 24 hours, levels of AST in ernatant were significantly increased. Detection of liver cell apoptosis rate was increased, compared with preincubation with LXA4 group, P<0.05.6. There were severe liver inflammation and necrosis when induced acute liver failure,ALT and AST were significantly increased,;serum IL-6 and TNF-a levels were significantly increased; liver TNF-a mRNA and IL-6 mRNA expression was significantly increased, Caspase3 activity was significantly enhanced; TUNEL to detect apoptosis in liver tissue significantly increased, kupffer cells and liver nuclear protein NF-κB activity was significantly enhanced; lipoxin group and PDTC group were significantly improved the histological changes, liver transaminases, cytokines and NF-κB activity was significantly decreased (compared with model group, P<0.05,), and the effect of LXA4 was in a dose depend manner;Compared with LXA4 high-dose group and PDTC group, most of the above indexes was significantly different(P<0.05).[Conclusion]1.Rat KCs were successfully separated,The number,activity and purity were all meet experimental requirements.2. In vitro experiments, LXA4 can inhibit the secretion of TNF-a by KCs.It can also reduce liver cell apoptosis and necrosis in vitro.3. In vivo experiments, LXA4 was confirmed to protect Acute liver Failure in rats. It was able to improve survival, significantly improve liver histology and biochemical indicators. LXA4 decreased proinflammatory cytokines expression, inhibited hepatocyte apoptosis, its effect to liver protection was in a dose denpend manner.4. The effect of LXA4 to protect acute liver failure in rats was partly through blocking the activation of NF-κB in kupffer cells. When the activation of NF-κB in kupffer cells was blocked, the secretion of proinflammatory cytokines was reduced. At the same time, LXA4 can block liver cell apoptosis pathway.