黄芪甲苷预处理对大鼠肝脏缺血再灌注损伤及自噬的影响

发布时间：2018-07-26 12:10

【摘要】：目的肝癌、肝移植等肝脏外科手术往往面临肝脏缺血再灌注损伤的难题,明显增加了肝脏外科手术的难度和风险。本实验在建立大鼠肝脏缺血再灌注模型的基础上,术前予黄芪甲苷预处理。通过对术后4 h、8 h、16 h血清转氨酶、炎性相关因子的检测及肝组织显微结构的观察,判断黄芪甲苷预处理对大鼠肝脏缺血再灌注损伤是否具有保护作用。用Western Blot及免疫组化染色分析各组大鼠肝组织中自噬相关蛋白Beclin-1的表达;电镜下观察各组大鼠肝脏细胞内自噬小体数量的变化。探讨缺血再灌注后大鼠肝脏自噬水平的变化,以及黄芪甲苷预处理对大鼠肝脏缺血再灌注损伤可能存在的作用机制,为减轻患者肝脏缺血再灌注损伤提供潜在的理论依据。方法实验分组:90只纯系SD大鼠,随机分为假手术组(Sham组),肝脏缺血再灌注组(HIRI组)、黄芪甲苷组(干预组),每组30只。再按术后4h、8h、16h三个时间段,每个时间段各10只。Sham组大鼠行腹腔注射麻醉后,腹部正中纵形切口,分离肝蒂,不建立肝脏缺血再灌注模型。HIRI组和干预组大鼠麻醉后,建立肝脏缺血再灌注模型。干预组大鼠术前予黄芪甲苷溶液腹腔注射,1次/天,连续7天。观察指标:各组大鼠分别在术后4 h、8 h、16 h采取血液和肝脏组织标本。用全自动生化分析仪检测各组大鼠血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)水平;用ELISA法测定大鼠血清炎性相关因子TNF-α,IL-6水平;使用苏木精-尹红(HE)染色后,观察各组肝脏组织病理学变化;Western Blot及免疫组化染色分析肝组织中自噬相关蛋白Beclin-1的表达;电镜下观察肝脏细胞内自噬小体数量的变化。结果1.血清中转氨酶水平的变化:同Sham组大鼠比较,HIRI组大鼠血清中ALT和AST水平在术后4 h、8 h、16 h均明显升高(P0.05),于再灌注后8 h时达到顶峰。与HIRI组大鼠比较,干预组大鼠血清ALT和AST水平在术后4 h、8 h、16 h均明显降低(P0.05)。2.血清中炎性相关因子水平的变化:同Sham组大鼠比较,HIRI组大鼠血清中TNF-α,IL-6水平在术后4h、8h、16h均明显升高(P0.05),于再灌注后8h达到顶峰。与HIRI组大鼠比较,干预组大鼠血清TNF-α,IL-6水平在术后4h、8h、16h均明显降低(P0.05)。3.肝组织HE染色后观察:Sham组大鼠肝细胞索排列整齐、肝小叶结构清晰、中央静脉区无淤血、无炎性细胞浸润。HIRI组大鼠在再灌注后4 h、8 h、16 h,出现了不同程度的肝细胞索排列紊乱、中央静脉区淤血、肝小叶结构模糊、炎性细胞浸润和肝细胞的水肿、坏死,其中以再灌注后8h的损伤最为严重。与HIRI组大鼠相比,干预组大鼠的肝脏组织病理变化在术后4 h、8 h、16 h均有明显改善。4.肝组织自噬相关蛋白Beclin-1表达水平的变化:同Sham组大鼠比较,HIRI组大鼠Beclin-1蛋白的灰度比在术后4h、8h、16h均明显升高(P0.05),于再灌注后8h达到顶峰。与HIRI组大鼠比较,干预组大鼠Beclin-1蛋白的灰度比在术后4h、8h、16h均明显降低(P0.05)。5.免疫组化染色分析肝组织中Beclin-I的表达:与Sham组大鼠相比,HIRI组大鼠肝脏组织Beclin-1的平均标记指数(LI)在术后8 h明显升高(P0.05)。与HIRI组大鼠相比,干预组大鼠肝脏组织Beclin-1平均LI在术后8 h明显降低(P0.05)。6.肝脏细胞自噬小体数量的变化:与Sham组大鼠比较,HIRI组大鼠肝脏细胞的自噬小体数量在术后8 h明显上升(P0.05);与HIRI组大鼠比较,干预组大鼠肝脏细胞的自噬小体在术后8 h明显减少(P0.05)。结论本实验结果提示黄芪甲苷预处理可以降低大鼠肝脏缺血再灌注后转氨酶水平并减轻炎症反应,同时肝组织HE染色观察结果也显示黄芪甲苷预处理能明显减轻大鼠肝脏的病理损伤程度,由此推断黄芪甲苷预处理能够减轻大鼠肝脏缺血再灌注损伤。本研究还发现缺血再灌注后的大鼠肝脏组织Beclin-1呈高表达状态,且肝细胞内自噬小体数量也明显升高,而黄芪甲苷预处理可以明显降低自噬相关蛋白Beclin-1在蛋白水平和组织水平的表达,并且也减少了肝脏细胞内自噬小体的数量,表明黄芪甲苷可能通过降低大鼠肝脏自噬水平来保护大鼠肝缺血再灌注损伤。本研究为黄芪甲苷减轻肝脏缺血再灌注损伤提供了新的理论基础,具有潜在的临床应用前景。
[Abstract]:Objective liver surgery, such as liver cancer and liver transplantation, often faces the problem of liver ischemia reperfusion injury, which obviously increases the difficulty and risk of liver surgery. On the basis of establishing rat liver ischemia-reperfusion model, this experiment was pretreated by Astragalus glucoside preoperatively. By 4 h, 8 h, 16 h serum aminotransferase, inflammatory related cause On the basis of Western Blot and immunohistochemical staining, the expression of autophagy related protein Beclin-1 in liver tissues of rats was analyzed by using Western Blot and immunohistochemistry. The number of autophagic bodies in the liver cells of each group was observed under electron microscope. Changes of autophagy in rat liver after ischemia and reperfusion, and the possible mechanism of astragalin preconditioning on hepatic ischemia reperfusion injury in rats to provide a potential theoretical basis for alleviating liver ischemia reperfusion injury in the patients. Methods the experimental group: 90 pure SD rats were randomly divided into sham operation group (group Sham). Liver ischemia reperfusion group (HIRI group), astragalin group (intervention group), 30 rats in each group, and then three time periods of 4h, 8h, 16h after operation, 10 rats in each time period, each of the.Sham groups were injected into the abdominal cavity, the abdominal median longitudinal incision, the liver pedicle, the liver ischemia reperfusion model.HIRI group and the intervention group were not established, and the liver deficiency was established. Blood reperfusion model. The rats in the intervention group were given Astragalus methoside solution intraperitoneally before operation, 1 times / day for 7 days. The indexes of blood and liver tissue were taken at 4 h, 8 h and 16 h after operation. The serum alanine transaminase (ALT), cereal transaminase (AST) level in the serum of each group was detected by automatic biochemical analyzer, and the level of glutarine transaminase (AST) was measured by the total automatic biochemical analyzer. The ELISA method was used to determine the level of the rats. Rat serum inflammatory related factor TNF- alpha, IL-6 level; after hematoxylin Yin Hong (HE) staining, the pathological changes of liver tissues were observed; Western Blot and immunohistochemical staining were used to analyze the expression of autophagic protein Beclin-1 in liver tissue; the changes in the number of autophagic corpuscles in liver cells were observed under electron microscopy. Results 1. serum transaminase water was observed. Compared with the Sham group, the level of ALT and AST in the serum of HIRI rats was 4 h, 8 h and 16 h significantly increased (P0.05), and reached the peak at 8 h after reperfusion. Compared with the HIRI group, the serum ALT and AST levels in the intervention group were 4, 8 and 16. The levels of inflammatory related factors in the serum were significantly decreased. Compared with the Sham group, the level of TNF- alpha and IL-6 in the serum of HIRI rats increased significantly (P0.05) after the operation (P0.05), and the 8h reached the peak after reperfusion. Compared with the HIRI group, the serum TNF- alpha was observed in the intervention group, and the IL-6 level was obviously decreased after the operation. The structure of hepatic lobule was clear and there was no blood stasis in the central venous area. The rats in the.HIRI group without inflammatory cell infiltration were 4 h, 8 h and 16 h after reperfusion. There were different degrees of disorder of hepatic cell cord arrangement, the congestion of the central venous area, the blurred structure of hepatic lobule, the infiltration of inflammatory cells and the edema and necrosis of the hepatocytes, among which the most serious injury was 8h after reperfusion. Compared with the HIRI group, the pathological changes in the liver tissue of the rats in the intervention group were 4 h, 8 h and 16 h significantly improved the level of the expression of autophagy related protein Beclin-1 expression in.4. liver tissue. Compared with the Sham group, the gray level of Beclin-1 protein in HIRI group rats was significantly higher than that in 4h, 8h, and reached the peak after reperfusion. Compared with the HIRI group, the gray level of Beclin-1 protein in the intervention group was significantly lower than that of 4h, 8h and 16h after the operation (P0.05).5. immunohistochemical staining analysis of the expression of Beclin-I in the liver tissue: compared with the Sham group, the Beclin-1 average marker index (LI) in the liver tissue of the HIRI group was significantly higher than the 8 after the operation. The average LI of Beclin-1 in the liver tissue of the rats in the intervention group decreased significantly at 8 h after operation (P0.05), the number of autophagic corpuscles in.6. liver cells: compared with the Sham group, the number of autophagic corpuscles in the liver cells of the HIRI group increased significantly (P0.05) after the operation (P0.05). Compared with the HIRI group, the autophagic corpuscle of the liver cells in the intervention group was 8 h after the operation. P0.05. Conclusion the results of this experiment suggest that astragaloside pretreatment can reduce the level of aminotransferase and reduce the inflammatory response after liver ischemia and reperfusion in rats. The results of HE staining in liver tissue also show that astragaloside preconditioning can significantly reduce the degree of pathological injury in the liver of rats. The liver ischemia-reperfusion injury in rats was alleviated. The high expression of Beclin-1 in the liver tissues of rats after ischemia-reperfusion was also found, and the number of autophagic corpuscles in the liver cells was also significantly increased, and the expression of autophagy related protein Beclin-1 at protein level and tissue level could be significantly reduced, and the expression of autophagic related protein was decreased. The number of autophagic bodies in the liver cells indicates that astragaloside may protect the rat liver from ischemia reperfusion injury by lowering the level of autophagy in the rat liver. This study provides a new theoretical basis for alleviating liver ischemia reperfusion injury by Astragalus membranaceus, and has potential clinical application prospects.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R657.3
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本文编号：2145969