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With the advances in modern clinical surgery, liver ischemia-reperfusion injury (hepatic ischemia andreperfusion injury, HIRI) more and more important role in liver surgery and liver transplantation, its mechanism of injury is greater. The decrease in hepatic blood flow and subsequent reperfusion period microcirculation disorders are leading to hypoxic environment inside the cell, will inevitably lead to an important regulator of protein to maintain the body's oxygen balance hypoxia inducible factor -1 (hypoxia inducible factor-1, HIF- 1) expression change. Schedule of hepatic ischemia-reperfusion of HIF-1 expression characteristics, and its significance is unclear, rarely reported in the literature. Therefore, this study was designed to explore new study hepatic ischemia-reperfusion period, the relationship between the expression of HIF-spatial and temporal expression characteristics and role of significance, and its expression HIRI process cell damage and apoptosis, prevention and treatment of hepatic ischemia reperfusion injury direction. Experiment HIF-1α in rat liver ischemia time-expression changes in purpose and significance of the establishment of rat liver ischemia model, detection of HIF-1α and Caspase-3 protein expression changes in the liver different ischemic time limit and explore significance. Two methods. Healthy SD rats were randomly divided into normal group (5) 35, the sham group (15), ischemia group (15), sham group and ischemia group, according to different ischemic time limit 30min, 45min, 60min three sub-groups, each group of five rats, rats were prepared total hepatic ischemia model. Blood samples were extracted by the liver and inferior vena cava about 2ml, serum ALT values. Rat liver samples drawn by 10% formalin fixed, electron microscopy specimens derived fixed after 20min after placed in a pre-configured 3% glutaraldehyde repair blocks is based. The 4.HE staining and immunohistochemistry according test kit steps, rabbit anti-rat HIF-1α and Caspase-3 in concentration 1:100, were purchased from Wuhan Boster Biological Engineering Co., Ltd.. 5 light microscope and electron microscope observation of histopathology, immunohistochemistry SABC method HIF-1α and Caspase-3 expression in the liver different ischemic time limit HPIAS-1000 high-resolution color pathological image analysis system for quantitative detection . 6 All measurement data application SPSS 11.5 for statistical analysis to take factorial design variance analysis analysis of main effects and interaction, LSD test among groups difference, P <0.05 considered statistically significant. Three results and a normal group and sham group HIF-1α expression, the differences are not statistically significant (P> 0.05). Various time limits ischemia group compared with the sham group, and its results are statistically significant differences (P <0.01). HIF-1α protein expression, since ischemia 30min after a significant increase, with gradual increase and extend the time limit with ischemic. Visible on the regional distribution of ischemic 30min HIF-1α protein is expressed mainly in the portal area portal vein, capillaries, micro peribiliary cells, some cells were strongly positive expression. Ischemia 45min express area gradually spread interlobular central venous ischemia 60min, HIF-1α protein expression can be seen in the central vein endothelial cells and their surrounding liver cells. Normal group and sham group Caspase-3 protein expression amount is less, compared to no significant difference (P> 0.05). Of the time limit points ischemia group compared with the sham group, a statistically significant difference (P <0.01). With the extension of ischemic time limit, each ischemia group Caspase-3 expression was statistically no difference (P> 0.05). Positive cells were seen in the endothelial cells of the liver sinusoidal shape. 3 the ischemic group serum ALT water average higher than the normal group and sham group, ALT gradually increased with increasing ischemic time limit, histopathological damage was also found aggravating trend. Four to discuss the experimental results show that the simple surgical injury did not affect the blood supply to the liver, does not cause the damage of the liver tissue and cells, it is at a normal level of serum ALT values, as hypoxia regulatory factors HIF-1α protein the expression level is extremely low, the amount of apoptosis execution of Caspase-3 protein expression at baseline level. Once the liver suffered ischemic injury, that HIF-1α protein stress significantly upregulated HIF-1α protein accumulation and the extent of changes in liver function and cell morphology injury was certain, so acute ischemic period of HIF-1α expression intensity can be used as a sensitive index to reflect the extent of hypoxia injury. Rats subjected to sudden 30min hepatic ischemia, can make a significant expression of Caspase-3 upregulated activation of the apoptotic program. Therefore, how to prevent the upregulation and activation of Caspase-3 protein in the liver sinusoidal endothelial cells, one can be used as reduce liver ischemia injury prevention direction. Second experiment HIF-1α in rat liver ischemia and 45min reperfusion the differing deadlines investigate the expression and significance of a purpose to establish the rat hepatic ischemia-reperfusion injury model, using immunohistochemistry SABC method HIF-1α and Caspase-3 protein in hepatic ischemia the 45min reperfusion time-expression changes and explore its significance, and to observe the impact of of Salvia pretreatment expression. Two methods 1. Healthy SD rats were randomly divided into 80 normal group (5), sham group (25), ischemia-reperfusion group (25), Salvia treatment group (25), sham group ischemia-reperfusion group and Danshen treated by reperfusion period (0h, 3h, 12h, 24h, 72h) is divided into five sub-groups, each group of five rats. Model preparation, drawing method, HE staining and immunohistochemical staining step, observation methods and statistical processing, the same experiment. After three 1.HIF-1α positive expression in the normal group and sham group very weak, both no significant difference (P> 0.05), HIF-1α expression significantly increased after ischemia to reperfusion 12h reached its peak with the same period in the sham group showed a significant difference (P <0.01), falling back to baseline levels after 24h of reperfusion. Salvia treated with reperfusion 0h, 3h can upregulate the expression of HIF-1α, HIF-1α expression reduced reperfusion 12h, ischemia reperfusion group were statistically significant (P <0.05) during the same period to 24h of reperfusion also fell back to baseline levels. 3.Caspase-3 expression was also no difference between the normal group and sham group between the two (P> 0.05), and Caspase-3 expression after ischemia rapid increase to peak at 24h reperfusion, sham-operated with the same period The group have significant difference (P <0.01), falling back to baseline levels 72 hours after reperfusion. Salvia treated ischemia-reperfusion group compared with the same period were lower, both with a statistically significant difference (P <0.05). Pathological morphology visible liver after ischemic injury, with prolonged reperfusion period, tissue and cell damage gradually intensified to reach the pinnacle of the damage at 24h reperfusion to reperfusion the 72h injury gradually returned to normal, Salvia treatment group compared with the same time limit points reperfusion group light damage. The four conclusions liver ischemic injury and reperfusion injury is a close contact with the extremely complex pathophysiological process, a variety of factors involved in HIF-1α and Caspase-3 is also involved in this process. The experimental rat liver ischemia 45min reperfusion within 0 ~ 72h HIF-1α, caspase-3 and pathomorphological damage observed over the same period, the analysis concluded that the process may experience the following four the: ① hypoxic stress period (0 ~ 3h), HIF-1α and Caspase-3 expression is rapidly increasing, microscope, cell and organelle swelling, liver sinusoidal space narrowing, reflecting this period the liver tissue of hypoxic injury and oxidative damage stress response occurs rapidly. ② overreaction period (12h), the expression of HIF-1α in this period to continue to enhance and reached the peak at 12h, and spread to the liver, Caspase-3 expression also continue to enhance the microscope, part of the regional cell swelling more apparent, the cell nucleus visible shrinkage of the part of the region. ③ continue injury period (12 to 24h) of HIF-1α protein expression gradually weakened, close to the baseline level at 24h, and Caspase-3 continue to enhance the expression of up to 24h peak microscope, the tissue damage of this period The gradually increased tissue damage around 24h heaviest. ④ recovery period (24h ~ 72h), this period of Caspase-3 protein also gradually fell back to baseline levels of the liver tissue is in the recovery phase of the injury, is also visible on the proliferation and Reconstruction of liver tissue morphology. The experimental results show that the hypoxic stress period of HIF-1α expression Salvia hepatic ischemia-reperfusion, can up to inhibit subsequent overreaction of the HIF-1α protein expression levels, suggesting that Salvia by HIF-1α expression balanced regulatory role, to play the role of anti-anoxia, anti injury. Salvia also by inhibiting the activation and expression of apoptotic factors, improve liver damage stress adaptation, increase cell survival opportunities, thereby protecting the liver, the specific mechanisms and the link takes another further study.
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