| Liver is the largest parenchymatous organ in the body. It has a reach blood supply, accept 25% of the cardiac output, portal vein and hepatic artery both supply blood to it. The time limit of hepatic ischemia and the differences of different intrahepatic cell groups in tolerance to hypoxia are widly concerned, since Pringle has reported the method of control the hepatoduodenal ligament to stop bleeding during the periods of hepatic surgery in 1908. Hepatic ischemia is common in trauma, shock, and many liver surgeries, especially for the resection of large intrahepatic lesions and liver transplant. After the reperfusion of the liver, a further attack arrives, and then hepatic ischemia-reperfusion injury(HIRI) will happen. The mechanisms of HIRI are complex, inflammatory response is considered to be the substance of HIRI. Liver metabolic dysfunction occurs, and a variety of inflammatory factors and oxygen free radicals are also generated during the period of HIR. At the same time, intestinal barrier dysfunction comes because of the congestion of the portal vein system. The portal vein with toxins cause a damage to the liver. A large number of inflammatory mediators are secreted by activate immune cells, while inflammatory mediators reverse to boost the immune cells activation. Inflammatory mediators and hypoxia act on liver cells may lead to a complex pathophysiological process. But the relationship of the inflammatory mediators and the hypoxic state in HIRI and the pathological changes of the gastrointestinal tract are seldom studied. In this paper, the pathological changes of the liver and intestin will be observed, the expressions of macrophage inflammatory protein-1a(MIP-la) and hypoxia inducible factor-la (HIF-1a) will be detected, and their significance and relevant mechanisms will be analyzed.ObjectiveThe hepatic ischemia-reperfusion injury model of SD rat was established to observe liver and small intestine pathological changes at different reperfusion time points after hepatic ischemia for 45min, the expressions of MIP-la and HIF-la in the liver and small intestine tissues were detected by immunohistochemistry. The role of MIP-la and HIF-la in HIRI was disscused for further understanding the effect of inflammatory mediators and oxidative stress in liver and gastrointestinal congestive injury after hepatic ischemia-reperfusion.Methords1. Grouping55 healthy adult male SD rats were randomly divided into three groups:normal control group (CO,n=5), ischemia-reperfusion group (IR,n=25), sham-operated group (SO,n= 25), then ischemia-reperfusion group and sham-operated group were randomly divided into five sub-groups according to different reperfusion time points (0h,3h,12h,24h,72h).2. AnimalsFasting for 12h, free drinking of water before operation. Took samples soon after broken neck executed in CO group. The preparation of preoperation in IR group was the same as normal group, took the middle abdominal incision about 3cm into the abdominal after intraperitoneal injection anesthesia (3%pentobarbital sodium 40mg/kg), then dissociated and blocked hepatic pedicle by non-invasive artery clamp for 45min, and released it. Directly took samples at Oh subgroup, but sew up the wound of rats to continue feeding in other subgroups, and took samples at the reperfusion time points(3h,12h,24h,72h). Preoperative treatment and anesthesia methords were as the same as the ischemia-reperfusion group in SO group, dissociated but not blocked hepatic pedicle, took samples at different time points.3. Detection mothods and indexes①The changes of pathology under light microscope:Specimens of rat liver and small intestine (about 5cm up-jejunum organization) were fixed by 10% formalin, dehydrated by gradient ethanol, embed by paraffin, then routine sected and observed by rutine HE staining.②The ultrastructure changes under electron microscopy:The samples of liver and small intestine which detected by electron microscope were put into 3% glutaraldehyde, fixed for 20min, then embedd by SPURR medium. Specimens were observed by scanning electron microscope.③Immunohistochemistry methods to detected the expressions of MIP-la and HIF-la.:Test kit purchased from Wuhan Boster Biological Products Company. The appearation of brown-yellow granules in cytoplasm was considered as the standard of MIP-la positive expression; the criterion of HIF-la positive expression was the appearation of cytoplasmic brown granules or a small amount of nuclear staining.10 visions (200/H) of each slice were taken for the quantitative analysis of the positive products(using HPIAS-1000 high-resolution color pathological image analysis system), the positive units were recorded (positive unit, PU), and the average level of PU represented the amount of positive products expression. 4. Statistical analysisAll data were recorded as x±s, basic statistics were calculated. Factorial design ANOVE was used to analyze the main effects and interaction of all groups. The difference of every groups was tested by LSD method. Bivariate correlation was used to analyze (Bivariate) the correlation between two variables. SPSS13.0 statistical software was used for statistical analysis, P<0.05 represents statistical significance.Results1. The observation of HE staining under light microscope:The structure of the liver was basically integrity in CO and SO groups. Hepatocytes apparent mild edema and sinusoidal gap began to narrow at 45min time limit; swollen hepatocytes,. translucent cytoplasm, and slacked reticular appeared at 3h; diffuse swollen hepatocytes, spotty necrosis with acidophilic body formation could be seen at 12h within the hepatic lobules; the hepatocytes were seriously damaged at 24h, reflceted as the distorted hepatic lobules and the damaged liver panels with portion of necrosis; but the narrow of sinusoidal space reduced and hepatic lobule reconstructed at 72h after reperfusion. The structure of the small intestine was integrity in the CO group. The expanded subepithelial spaces, congested capillaries, separated epitheliums and lamina proprias, mild impairment glands all could be seen at the reperfusion time point of Oh; but glands damaged significantly at 3h, accompanied with some of the top hair off. The epitheliums obviously separated with lamina propriasand most of the glands damaged, capillaries also began to expand at 12h; obviously villus shedding appeared at 24h, part of the lamina propria digested and decomposited. Although there was still some structural damage at 72h of the small intestine, but the structure basically restored to normal.2. The changes of ultrastructures under electron microscopy:The ultrastructures of the liver and small intestine were basically integrity in CO and SO groups. Mitochondria and endoplasmic reticulums slightly swollen at the beginning of the reperfusion; mitochondria turned shorter, intumesce got worse, nuclear chromatin condensation and margination were also detected at 3h after reperfusion; nucleus shrinked, glycogen disappeared at 12h after reperfusion; pyknosis death and neonatal hepotocytes could be seen at 24h after reperfusion; the ultrastructure of liver cells basicly restored to normal at 72h after reperfusion, but the mitochondrions were still slightly swollen. Gland of intestine epithelial cells could be seen mild impairment, and mitochondrial cristae turned thicker at 3h after reperfusion; significiently swollen mitochondria and agglutinated nuclears could be seen at 12h after reperfusion; mitochondrial cristaes were bubbly dissoluted, even flocculent scattered at 24h after reperfusion; but the ultrastructure of small intestine cells basicly returned to normal at 72h after reperfusion.3. There were also no significant expressions of MIP-la in the liver and small intestine tissues between CO and SO group of all time points, no significant difference between the two gurops (P> 0.05). Positive expressions could be seen at sinusoids and central veins in liver at the begining of the reperfusion, then gradually increased with the reperfusion time prolonged, peak at 12h after reperfusion. The positive expression numbers were significantly declined but still high at 72h time point. In the small intestine tissues, the positive expressions of MIP-1a appeared since the early reperfusion, reached the peak at 24h after reperfusion. Despite decline, but the number of positive expressions still high at 72h after reperfusion. The MIP-la expressed mainly in lamina propria, its expression could also be seen at muscular layers around vascular. The expressions of MIP-la in IR group were statistically significant compared with CO and SO group.4. There were no significant expressions of HIF-la in the liver and small intestine tissues within CO and SO groups at all time points, no significant difference between the two groups (P> 0.05). The positive expressions at cytoplasm could be seen at periportal capillaries, micro-duct in IR at Oh group, and wildly expressed at 12h after reperfusion. With the reperfusion time extending, the expressions of positive cells gradually reduced, and basically back to the normal level at 72h after reperfusion. In the tissues of small intestine, HIF-la expressed at the beginning of reperfusion and concentrated in the mucosal cells. They gradually increased with the time, reached the peak at 24h after reperfusion, and reduced to the initial level at 72h after reperfusion. The expressions of HIF-1a in IR group were statistically significant compared with the CO and SO group.5. There was a significant positive correlation between MIP-la and HIF-la in the liver tissues(rp=0.72 P<0.05) and small intestine tissues(rp=0.97 P<0.05).ConclusionsThe structures and cell functions of liver are damaged and aggravated, when the blood supply of liver restored after hepatic ischemia. In this process a large number of inflammatory cells infiltrate and release inflammatory mediators to involve in tissue injury. HIF-la expresses in a variety of organizations under the state of hypoxic stress and plays a role on tissue cells by regulating the expression of other molecules. MIP-1a belongs to CC chemokine family, which can be secreted by liver Kuffer cells, cause an accumulation of inflammatory cells, and promote the inflammatory injury of tissues.HIF-la can combine and regulate the expression of related genes, make an enhanced adaptability to huyoxia state. Its enhanced expression may be connected with a variety of cells apoptosis. In this experiment, the expressions of HIF-la were fine in CO and SO groups in liver and small intestine tissues. After ischemia-reperfusion, the expressions of HIF-1a were increased. The first expression site of HIF-la may be related to the poor blood supply and hypoxia state of liver sinusoidal space, micro-duct, central venous. While in the small intestine, the mucosal cells microvilli have the worst blood supply, and most vulnerable to the injury which caused by hypoxic-ischemic, so they becomes the first expression place of HIF-1a.The positive correlation between HIF-1a and MIP-1a indicated that HIF-1a may be an important expression regulator of MIP-la, it can promote the expression of MIP-la, then enhance the process of inflammatory reaction. Studies have been shown that HIF-1a can increase the spontaneous expression of inflammatory proteins and enhance the dermatitis reaction, and involved in the differentiation process of bone marrow cells. The difference expression peak phase of HIF-la and MIP-la at the small intestine and liver tissues may because of the formation of microthrombus in gastrointestinal tract micro-circulatory system. So after the reperfusion of hepatic portal, there is still some ischemic and hypoxic conditions, which affect the recover of the gastrointestinal tract inflammatory injury.The results indicated that the hepatic ischemia cause not only the liver substantive and non-parenchymal cells injury, but also gastrointestinal system injury. At the beginning of the reperfusion, oxygen free radicals play an important role in the injury of live. The mionectic state of gastrointestinal was aggravated by the congestion of portal vein, with the time of hepatic pedical blocking extending. Oxyradicals produced by small intestine brought about the small intestinal hypoxia and inflammatory injury by promote lipid peroxidation, neutrophil aggregation and inhibit mitochondrial function to accelerate cellular energy consumption. Furthermore, the congestive injury of digestive tract may be also an important reason for the injury of liver after hepatic reperfusion. Because oxyradical and various inflammatory mediators produced by digestive system may increase the injury of liver after hepatic reperfusion. The injury of liver and gastrointestin reinforce each other resulting in HIRI and oxidative stress in the body.The pathological changes of liver and small intestine at different phases of HIRI and the variational expressions of HIF-la/MIP-la suggest that:hepatic ischemia and reperfusion can cause intestinal injury; liver and small intestine injury induced by the liver ischemia and reperfusion relate with inflammatory cell accumulation and hypoxic stress. Hypoxia and inflammatory reactions collaborative contribute to the liver and gastrointestine stress response. HIF-la and MIP-la play an important role in HIRI. It is may be an useful method to mitigate HIRI by improving the state of hypoxia stress and inflammation response.
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