The Protective Effect Of Isoflurane Pretreatment Against Ischemia-reperfusion Injury On Hepatic Sinusoidal Endothelium And The Related Mechanism

Part OneEffects of isoflurane pretreatment on warm ischemia-reperfusion injury in hepatic sinusoidal endothelium of ratsObjective: To investigate the effects of isoflurane pretreatment on warm ischemia-reperfusion injury (IRI) in hepatic sinusoidal endothelium of rats and related mechanisms of mitochondrial ATP-sensitive K~+ channels (MitoKATP).Methods: 1. Warm partial hepatic ischemia-reperfusion model of rat was established by clamping of the middle and left branches of liver arteries and portal vein which induced ischemia in about 70% portion of the liver for 60 minutes and reperfusion for 90 minutes.2. Forty-eight adult male Sprague-Dawley rats were randomly divided into six groups (n=8): (1) Sham group: Perform a similar operation without occlusion of liver blood flow; (2) Control group: Perform the operation of patial hepatic ischemia-reperfusion (I/R); (3) Iso group: Inhalation of 1.5% (1MAC) isoflurane for 30 minutes and washout for 10 minutes were performed before I/R; (4) 5HD+Iso group: Intravenous injection of 5-HD 10mg/kg 10 minutes before isoflurane pretreatment; (5) Dia group: Intravenous injection of diazoxide 5mg/kg 10 minutes before I/R; (6) 5-HD+Dia group: Intravenous injection of 5-HD 10mg/kg 20 minutes before I/R ,and 10 minutes later, Diazoxide 5mg/kg was injected intravenously.3. After 60 minutes of ischemia and 90 minutes of reperfusion, serum ALT and HA levels, NO and ET concentrations, viability of NOS and MPO, and NF-kB binding activity in liver were measured.The liver slices were also observed under microscope and electron microscope. At the same time, we used TUNEL staining for measuring the apoptosis cells and immunohistochemistry staining for measuring expression of ICAM-1 of liver tissues.Results: 1. The serum ALT and HA levels, liver ET concentration in Control groupwere all significantly higher than those in Sham group while liver NO concentration and NOS viability were lower (p<0.05) . Serum ALT and HA levels and liver NO concentration in Iso group and Dia group were all lower than those in Control group (p<0.05) . 5-HD can partially counteract the effect of isoflurane, but it can counteract the effect of diazoxide completely. Although the liver NO concentrations in Iso group and Dia group were both higher than those in Control group (p<0.05) , its concentration in Iso group was even higher. Although the effect of Iso group was counteracted by 5-HD in some dgree, the NO level in 5-HD+I group was also higher than that in Control group (p<0.05) . But the NO level in 5-HD+Dia group had decreased to the Control level (p>0.05) . The changes of NOS viability in each group was similar to the NO concentration expression.2. With the staining of HE, it can be found under microscope that the mainly pathologic changesof I/R liver tissue were sinusoidal stretch and congestion and hepatocytes degeneration while cells necrosis were relatively seldom. In Control group, there were obviously congestion and stretch in sinusoids and many neutrophils gathered. General hepatocytes degeneration and some necrosis were seen around the central veins, and the structure of hepatic cords was destroyed. Some sinusoidal endothelial cells exfoliated into hepatic sinusoids. Both in Iso group and Dia group, there were significant relieve of congestion and degeneration, and neutrophils gathering were few, and the basic structure of hepatic cords was reserved. The manifestation in 5-HD+Iso group was between the Iso group and Control group, but which in 5-HD+Dia was similar to Control group. Transmission electron microscope (TEM) examination showed the features of hydropic degeneration including endoplasmic reticulum's overwhelming expansion, the swelling of mitochondria, cracking of endo-crista. The formation of lipid droplets can been found in both sinusoidal endothelial cell(SEC) and hepatocyte. In some SECs, the evidence related to early stage apoptosis were seen. In Iso group and Dia group the injury of SEC and hepatocyte was diminished significantly. The manifestations in 5-HD+Iso group and 5-HD+Dia group were both between Iso group and Control group. With TUNEL staining we found a few positive staining cells around central veins which distributed mainly along with sinusoids in Control group. In Iso group, the positive staining cells were much fewer than in Control group (p<0.05) .The number of positive staining cells in Dia group was between the Control group and Iso group. 5-HD could counteract both the effects of isoflurane and diazoxide, which was more complete fordiazoxide.3. Results of ICAM-1 expression showed that no positive staining in Sham group. In Control group, it can be seen generally profound positive staining on SEC membrane. But in Iso group the positive area and intensity were much lower (p<0.05). The score of positive portion in Dia group was between Control group and Iso group. 5-HD can counteract the effects of isoflurane and diazoxide, and just like that in TUNEL, its effect was more complete for diazoxide. The results of EMSA showed that the DNA binding activity of NF-kB in Sham group was very low. It increased obviously in Control group. In Iso group and Dia group, the increase was diminished significantly. In 5-HD+Iso group and 5-HD+Dia group the manifestations were similar to that in Control group.Conclusions: 1. The model of 70% liver mass ischemia for 60 minutes and reperfusion for 90 minutes in rat can not only maintain the stability of systemic hemodynamics, but also induce the significant changes in liver structure and function, with hepatic sinusoidal endothelium excited. There was no general necrosis of SEC and hepatocyte. So it is suitable for study of early injury of hepatic ischemia-reperfusion.2. Pretreatment with 1.5% isoflurane for 30 minutes and washout for 10 minutes can provide significant protection against IRI of hepatic sinusoidal endothelium.3. The protective effects of isoflurane pretreatment against hepatic sinusoidal endothelium IRI are partially mediated through the pathway of MitoKATP.Part Two The protection of isoflurane pretreatment against anoxia-reoxygenation injury in endothelial cells in vitroObjective: To investigate the protective effects of isoflurane pretreatment against anoxia-reoxygenation injury in endothelial cells and its related mechanisms.Methods: 1. A model of anoxia-reoxygenation(A/R) of endothelial cells in vitro was established with anoxia for 3 hous and reoxygenation for 4 hours.2.Human umbilical vein endothelial cells (HUVEC, primary cultured, Cascade Biologies, USA) were subcultured in 6-well tissue culture plates to 90% confluence, and divided into 5 groups:(l)Normoxia (N) group: cultured in normal incubator without anoxia;(2)Anoxia/Reoxygenation(A/R) group: Anoxia for 3 hours and reoxygenation for 4 hous;(3)Isoflurane (I) group: 1.4% isoflurane pretreatment for 30 minutes before A/R; (4)5-HD+Iso(5+I) group: 10 minutes before isoflurane pretreatment, culture medium including lOOuM 5-HD was given; (5) NAC+I(N+I) group: 10 minutes before isoflurane pretreatment, culture medium containing 4mM NAC was given. After anoxia and reoxygenation, the appearance in each group was observed under microscope and the NO concentration in culture medium was measured. The expression of ICAM-1 and E-selectin of HUVEC was measured by flow cytometry with the FITC and PE-labeled antibodies.3. HUVEC were subcultured in 60mm culture dishes to 90% confluence, and divided into 5 groups as described above. After anoxia-reoxygenation, the DNA binding activity of NF-kB in each group was measured. We also measured the direct effects of isoflurane and other drugs as described above on HUVEC's DNA binding activity of NF-kB without A/R.Results: 1.Cells in group N appeared as polygonal, monolayer growth and closely apposed. Most cells in A/R group became round and stretched. The interspaces was widened with some cells exfoliated. Cells in group I still kept growing in confluence, some cells became round and stretched with interspaces a little widened. Group5+I was similar to group N+I: most cells became round and stretched. The interspaces was widened with some cells exfoliated.2. Compared with group N, NO concentrations in culture medium in other groups were lower after anoxia and reoxygenation (p<0.05) . However, it was much...