Kupffer cell mediated emulsified isoflurane preconditioning on rat liver ischemia-reperfusion injury in rats


【Abstract】 Objective To investigate the emulsified isoflurane preconditioning on rat liver ischemia-reperfusion injury in rats, and that this role by the Kupffer cell-mediated. Methods SD rats were made of hepatic ischemia-reperfusion model, were randomly divided into control group, fat emulsion (C), Kupffer cells, blocking the control group (CK), emulsified isoflurane preconditioning group (IPC), and Kupffer cells, after blocking emulsified isoflurane preconditioning group (IK), observation of hepatic ischemia-reperfusion 2h after 60min in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), liver cells, malondialdehyde (MDA), superoxide dismutase (SOD) and hepatic histopathological changes. Results C group, CK group and the IK group after hepatic ischemia-reperfusion levels of serum ALT and AST were significantly increased, a liver cell homogenate MDA levels increased, SOD levels decreased significantly increased liver histopathological damage; and IPC group hepatic ischemia - after reperfusion of serum ALT and AST levels compared with the three groups a significant reduction in liver homogenate MDA content decreased, SOD content increased, the liver histopathological damage significantly reduced. Conclusion emulsified isoflurane preconditioning on hepatic ischemia-reperfusion injury has a protective effect, and this role from Kupffer cell-mediated. 【Key Words】 Kupffer cells; emulsified isoflurane; ischemia-reperfusion injury Kepffer cells mediated emulsified isoflurane precondition against hepatic ischemia and reperfusion injury in the rats LV Hao, YANG Li-qun, YU Wei-feng, REN Hong-mei, ZHU Min, XU Li-ya.Department of Anesthesiology, Eastern Hepatobiliary Surgical Hospital, the Second Military Medical University, Shanghai 200438, China. Corresponding author: YU Wei-feng 【Abstract】 Objective To investigate the protective effect of pretreatment with emulsified isoflurane on liver ischemia and reperfusion injury in rats. Methods A total of 32 healthy adult SD rats, weighing between 250 g to 300 g, were randomly divided into four groups: preconditioning with intralipid group (C, n = 8), inhibition of Kupffer cell and preconditioning with intralipid group (CK, n = 8), preconditioning with emulsified isoflurane group (IPC, n = 8), inhibition of Kupffer cell and preconditioning with emulsified isoflurane group (IK, n = 8). Rats suffered from ischemia for 30 min and were followed by reperfusion for 2 hours. The amimals were killed at 2 hours after reperfusion. Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined.Hepatic cell homogenate levels of MDA and SOD were determined. The histopathologic changes in the liver were also observed. Results Compared with those in C, CK and IK groups, in IPC group serum levels of ALT and AST decreased significantly after liver ischemia and reperfusion. Hepatic cell homogenate levels of MDA decreased and SOD increased. The injury degrees of liver histopathology in IPC group was much less than that in C, CKand IK groups. Conclusion Pretreatment with emulsified isoflurane can protect liver from ischemia and reperfusion injury and this effect may mediated by Kupffer cells. 【Keywords:】 Kupffer cells; emulsified isoflurane; ischemia and reperfusion injury Recent studies have shown that inhaled anesthetics such as isoflurane preconditioning on rat heart, brain, kidney, lung, spinal cord ischemia-reperfusion injury in the early protective effect and mechanism of action revealed some of the [1 -- 5]. Effects of inhalation anesthetics on the liver, most of the previous studies focused on liver toxicity, however, volatile anesthetics can induce the liver protective effect. In rat cultured hepatic ischemia-reperfusion and hypoxia-reoxygenation injury model, isoflurane, sevoflurane and halothane can reduce the early ischemia-reperfusion or hypoxia-reoxygenation injury [6-7]. Isoflurane preconditioning on rat liver ischemia-reperfusion injury in rats is currently little research at home and abroad, its mechanism of study at an early stage. In this study, to be used in rat liver ischemia-reperfusion model, isoflurane preconditioning on hepatic ischemia-reperfusion injury in rats, with a view to provide a theoretical basis for clinical rational use of drugs. 1 Materials and methods 1.1 studied SD rats 32, male, quality, 250 ~ 300 g, by the Second Military Medical University Experimental Animal Center. The animals were divided into 4 groups: fat emulsion group (C group, n = 8), through the rat tail vein of fat emulsion using micro-pump for pre-treatment, a dose of 30% fat emulsion (SSPC Limited) 6ml / kg / h, the sustained delivery of half an hour, stopping for 15 minutes, and then ischemia-reperfusion; Kupffer cells, blocking the control group (CK group, n = 8), rats were injected intraperitoneally 24h before the experiment Kupffer cell-specific resistance breaking agent gadolinium chloride (10mg/kg), the other with the C group; emulsified isoflurane preconditioning group (IPC group, n = 8), through the tail vein of rats given emulsion using micro-pump of isoflurane pretreatment, a dose of 8% emulsified isoflurane 6ml/kg/h, the sustained delivery of half an hour, stopping for 15 minutes, and then ischemia-reperfusion; Kupffer cells after the blockade of emulsified isoflurane preconditioning group (IK group, n = 8), 24h before the experiment rats were injected Kupffer cell-specific inhibitor gadolinium chloride (10mg/kg), the other with the IPC group. 1.2 prepared emulsified isoflurane 8% emulsified isoflurane is prepared under sterile conditions in 20 ml sterile ampoule by adding 30% fat emulsion, respectively, and 18.4 ml of liquid isoflurane 1.6 ml, with alcohol blowtorch seal ampoule , was mixed spin oscillator oscillation 15 min, so that sufficient liquid emulsified isoflurane [8]. 1.3 hepatic ischemia-reperfusion model in the production of rat 12h preoperative fasting, free drinking water, intraperitoneal injection of 3% pentobarbital sodium (30 mg / kg) anesthesia, abdominal incision into the abdomen, separating ligaments around the liver exposed hepatic portal, free hepatic portal blood vessels, arteries folder with trumpet the left portal vein occlusion, middle branches (about 70% of the whole liver), complete folder camera vein and hepatic artery, completely blocked the left, in the liver blood flow, caused by the left, middle lobe completely ischemia, keep right liver (approximately 30% of the whole liver) blood supply, as the portal venous blood return channel, to prevent intestinal congestion. At the same time rat tail vein puncture 24G trocar placement, followed by pre-administration of micro-pump. The sustained delivery of micro-pump 30min, stop 15min, and then partially block the hepatic portal (left, middle) 30min, after the removal of Pringle folder, restore the liver perfusion, and reperfusion samples taken after 2h. 1.4 specimen collection and testing method of all animals at the end of the experiment the inferior vena venous blood collected 4 ml, centrifugal precipitation of serum in order to made in Japan Olympus AU 2700 automatic biochemical analyzer, measurement of serum alanine aminotransferase (ALT), aspartate aminotransferase (AST) levels. Take of fresh leaf tissue of the liver made of 10% of the left liver tissue homogenate, using Coomassie Brilliant Blue Protein Assay Kit (Nanjing Jiancheng Bio-agent Co., Ltd.) Determination of protein content in liver tissue and used MDA and SOD kits (Nanjing Jiancheng Bio-agent Co., Ltd. ) Determination of liver homogenate MDA and SOD levels. At the same time taking home the organization left lobe of liver in 10% formalin fixed, regular slicing and HE staining, light microscope observation of liver lesions. Select the most obvious reperfusion injury 24 h of the biopsy, light microscopy assessment of liver tissue injury classification, the grading criteria: 0: hepatic lobule, liver cells, liver cells, cord, the central vein, sinusoids were normal; Ⅰ Grade: individual liver cell degeneration, a small amount of inflammatory cells in periportal aggregation; Ⅱ grade: hepatic central vein, sinusoidal blood stasis and scattered within the liver cell degeneration and necrosis, inflammatory cell accumulation within the liver tissue are more structural integrity of the hepatic lobule; Ⅲ Class: hepatic central vein, sinusoidal blood stasis and extensive within the liver cell degeneration and necrosis, a large number of inflammatory cell accumulation, some of the organizational structure of the liver damage is incomplete. 1.5 Statistical treatment using SPSS11.0 statistical software, all the data to x

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