黄芪甲苷对肾血管性高血压大鼠主动脉内皮细胞线粒体损伤的保护研究
发布时间：2018-08-01 10:37
【摘要】：背景 血管壁是由内层内皮细胞、中层平滑肌细胞、外层结缔组织构成的。血管内皮细胞(EC)是位于血液与血管组织之间的一层扁平细胞,作为血液和组织之间的屏障,不仅来维持血管壁的完整,完成血液和组织液的代谢交换,还可合成和分泌多种生物活性物质,以保证血管正常的收缩和舒张功能,起到调节血管张力、调节血流、血压的作用。除此之外,它还具有调节凝血与抗凝平衡、防止血栓形成、抑制血管壁细胞的游走和增殖等多种重要的生理功能。血管内皮细胞衰老、凋亡与再生的平衡对正常血管的功能维持具有极其重要的作用。线粒体作为细胞内的重要细胞器,除了合成ATP为组织细胞提供能量外,还调控制细胞程序性死亡、以及细胞衰老等多种病理生理的代谢过程。线粒体作为细胞供能的主要细胞器很容易受到机体内部和外界各种不良因素的攻击,血管内皮细胞线粒体的功能障碍或损伤,必然会导致血管内皮正常生理功能的维持。因此,保护血管内皮细胞线粒体的损伤在防治心血管疾病中有重要意义。 黄芪甲苷(Astragaloside IV, AS-IV)是从中药黄芪中分离得到的具有多种药理活性的皂苷类化合物,是黄芪主要活性成分之一,具有抗氧化特性,有调节机体免疫力、保护组织器官、降低血糖、抗细胞凋亡和抗炎抗病毒等药理作用。研究表明,AS-IV能够通过提高线粒体膜电位减轻线粒体损伤,保护视网膜神经节细胞,还可通过降低线粒体氧化应激产物来减少心肌细胞的凋亡[1-2]。因此,我们进一步推测AS-IV对主动脉内皮细胞线粒体损伤具有保护功能,但AS-IV对线粒体保护作用的确切机制尚不明确。 目的 本研究旨在证实AS-IV对肾血管性高血压大鼠主动脉内皮细胞线粒体损伤的保护作用并探索其可能机制,将有益于开发AS-IV药物作用靶点。 方法 1、肾血管性高血压大鼠模型的建立及血压监测 清洁级SD大鼠麻醉固定后暴露左侧肾脏,钝性分离左侧肾动脉,于肾动脉中段放置银夹(内径0.2mmm),缩窄肾动脉。术后分笼,置于室温,苏醒后给予常规饮食。采用大小鼠无创血压仪BP-2000系统,经套尾法分别于术前、术后每周测量各组大鼠血压,每只大鼠血压测量10次,取平均值；术后2周收缩压140mmHg(1mmHg=0.133kPa)作为高血压模型制作成功标准,用于进一步实验。 2、实验分组与治疗 实验分为假手术组(SHAM组)、两肾一夹组(2K1C组)、黄芪甲苷低剂量组(AST-L组,1.0mg/(kg-d))、黄芪甲苷高剂量组(AST-H组,5.0mg/(kg-d))、洛沙坦钾组(LOS组,10mg/(kg-d))、黄芪甲苷高剂量+洛沙坦钾组(AST-H+LOS组,5.0mg/(kg-d)+10mg/(kg-d))。AST-L组、AST-H组、LOS组和AST-H+LOS组于术后2周腹腔注射给药,SHAM组和2K1C组给予等体积生理盐水腹腔注射。 3、通过电子透射显微镜观察大鼠胸主动脉内皮细胞线粒体 通过透射电镜观察大鼠胸主动脉内皮细胞线粒体内外膜的清晰度、线粒体嵴的完整性、线粒体基质电子密度的高低变化及线粒体肿胀情况。 4、应用免疫组化染色法观察大鼠主动脉内皮细胞锰-超氧化物歧化酶(SOD2)的表达 大鼠主动脉内皮细胞SOD2免疫组化检测,采用形态学图像分析系统Image-ProPlusVersion6.0软件进行主动脉内皮细胞阳性染色面积和总光密度值的测量,计算得出平均光密度值(IOD/area),以平均光密度值代表SOD2的表达水平。 结果 1、鼠尾法测定大鼠血压显示,手术前各组大鼠基础血压差异无统计学意义(P0.05)。术后两周末,2K1C组、AS-IV低剂量组、AS-IV高剂量组、LOS组、AS-IV高剂量+LOS组与SHAM组比较收缩压明显升高,差异有统计学意义(P0.01)。治疗两周后,LOS组、AS-IV高剂量+LOS组与模型组比较收缩压均有明显降低,差异有统计学意义(P0.01); LOS组、AS-IV高剂量+LOS组与AS-IV低剂量组比较降压效果差异有统计学意义(P0.05); AS-IV低剂量组与AS-IV高剂量组相比较降压效果差异无统计学意义(P0.05); LOS组、AS-IV高剂量+LOS组与AS-IV高剂量组相比较降压效果差异无统计学意义(P0.05)。 2、电镜下可见,SHAM组主动脉内皮细胞线粒体双层膜清晰完整,线粒体嵴、嵴间隙清楚,基质电子密度正常。2K1C组主动脉内皮细胞线粒体的嵴断裂缺失,仅留部分残端,可见明显肿胀、空泡化。AS-IV低剂量组线粒体的嵴模糊不清,有轻度空泡化。AS-IV高剂量组线粒体嵴清楚,但有残端,空泡化明显减轻。LOS组、AS-IV高剂量+LOS组与SHAM组相比较主动脉内皮细胞线粒体超微结构无明显差别。 3、免疫组化结果显示：与SHAM组比较,2K1C组大鼠主动脉内皮细胞SOD2蛋白表达水平显著降低(P0.01)；与2K1C组比较,SHAM组、AS-IV高剂量组、LOS组与AS-IV高剂量+LOS组大鼠主动脉内皮细胞SOD2蛋白表达水平显著增加(P0.05)；经低剂量黄芪甲苷(AS-IV低剂量组)治疗后,与2K1C组比较、大鼠主动脉内皮细胞SOD2蛋白表达水平变化不明显(P0.05)；AS-IV高剂量组、LOS组与AS-IV高剂量+LOS组有显著疗效,三组之间大鼠主动脉内皮细胞SOD2蛋白表达水平相比较差异无统计学意义(P0.05)。 结论 AS-IV对肾血管性高血压大鼠主动脉内皮细胞线粒体的损伤有保护作用,上调主动脉内皮细胞SOD2表达可能是其途径之一。
[Abstract]:background
Vascular wall is composed of inner layer endothelial cells, middle smooth muscle cells and outer connective tissue. Vascular endothelial cell (EC) is a flat cell between blood and vascular tissue. As a barrier between blood and tissue, it not only maintains the integrity of the blood vessel wall, but also completes the metabolic exchange of blood and tissue fluid, and can also synthesize and secrete more. Biological active substances, to ensure the normal vasoconstriction and diastolic function of blood vessels, play the role of regulating vascular tension, regulating blood flow and blood pressure. In addition, it also has many important physiological functions, such as regulating coagulation and anticoagulant balance, preventing thrombosis, inhibiting the walking and proliferation of vascular wall cells, vascular endothelial cells aging, apoptosis and The regenerative balance plays an important role in maintaining the function of normal blood vessels. As an important cell organelle in cells, mitochondria regulate cell programmed cell death, as well as cell aging, as well as cell senescence. Mitochondria act as the main organelles for cell energy supply in addition to the synthesis of ATP to provide energy for tissue cells. It is easy to be attacked by various adverse factors inside and outside the body. The dysfunction or damage of the mitochondria of vascular endothelial cells will inevitably lead to the maintenance of normal physiological functions of the vascular endothelial cells. Therefore, it is of great significance to protect the damage of mitochondria of vascular endothelial cells in the prevention and treatment of cardiovascular diseases.
Astragaloside IV (AS-IV) is a saponins with various pharmacological activities isolated from Astragalus membranaceus. It is one of the main active components of Astragalus, and has the antioxidant properties. It can regulate the immunity of the body, protect the tissues and organs, reduce the blood sugar, anti apoptosis and anti-inflammatory and antiviral effects. The research shows that AS-IV We can reduce mitochondrial damage by improving mitochondrial membrane potential, protect retinal ganglion cells, and reduce mitochondrial oxidative stress products to reduce the apoptosis [1-2]. of cardiomyocytes, so we further speculate that AS-IV has protective function on the mitochondrial damage of aortic endothelial cells, but AS-IV protects the mitochondria. The cutting mechanism is not yet clear.
objective
The purpose of this study is to confirm the protective effect of AS-IV on the mitochondrial damage of aortic endothelial cells in renovascular hypertensive rats and to explore its possible mechanism, which will be beneficial to the development of the target target of AS-IV drugs.
Method
1, establishment of a renovascular hypertensive rat model and blood pressure monitoring.
SD rats were exposed to the left kidney after anesthesia and fixation, and the left renal artery was separated from the left renal artery in the middle part of the renal artery. The renal artery was placed in the middle part of the renal artery, and the renal artery narrowed. After the operation, the cage was placed at room temperature and was given to the routine diet after awakening. The rat blood pressure instrument BP-2000 system was used to measure the blood of each group. Pressure, the blood pressure of each rat was measured 10 times, and the mean value was taken. The systolic pressure of 140mmHg (1mmHg=0.133kPa) after 2 weeks was used as the successful standard for making the hypertension model, and used for further experiment.
2, experimental grouping and treatment
The experiment was divided into sham operation group (group SHAM), two kidney one clip group (group 2K1C), low dose group of astragaloside (group AST-L, 1.0mg/ (kg-d)), high dose group of astragalin (AST-H group, 5.0mg/ (kg-d)), losartan potassium group (LOS group, 10mg/ (kg-d)), high dose of astragalin + losartan potassium group (AST-H+LOS group) Group H+LOS was injected intraperitoneally 2 weeks after operation. Group SHAM and group 2K1C were given intraperitoneal injection of equal volume of normal saline.
3, the mitochondria of rat thoracic aorta endothelial cells were observed by electron transmission microscope.
The articulation of the inner and outer membrane of the rat thoracic aorta endothelial cells, the integrity of the mitochondrial crista, the change of the mitochondrial matrix electron density and the swelling of the mitochondria were observed by transmission electron microscopy.
4, immunohistochemical staining was used to observe the expression of manganese superoxide dismutase (SOD2) in rat aortic endothelial cells.
SOD2 immunohistochemical detection was used in rat aortic endothelial cells. The morphological image analysis system Image-ProPlusVersion6.0 software was used to measure the positive staining area and total light density of the aortic endothelial cells. The mean light density (IOD/area) was calculated, and the mean light density value represented the expression level of SOD2.
Result
1, the rat tail method was used to determine the blood pressure in rats. There was no significant difference in the basic blood pressure of rats in each group before operation (P0.05). At the end of the two week, group 2K1C, AS-IV low dose group, AS-IV high dose group, LOS group, AS-IV high dose +LOS group were significantly higher than SHAM group, the difference was statistically significant (P0.01). Two weeks after treatment, LOS group, AS-IV high dose +LO The systolic pressure of the S group was significantly lower than that in the model group (P0.01). In group LOS, there was a significant difference between the high dose +LOS group of AS-IV and the low dose AS-IV group (P0.05), and there was no significant difference between the low dose group of AS-IV and the high dose group of AS-IV (P0.05), while the LOS group, AS-IV high dosage, was no significant difference (P0.05). There was no significant difference between the two groups in the hypotensive effect compared with the AS-IV high dose group (P0.05).
2, under electron microscope, the mitochondrial membrane of the aortic endothelial cells in the SHAM group was clear and complete, the crista and the ridge gap were clear. The ridge fracture of the mitochondria of the aorta endothelial cells in the normal.2K1C group was absent, and the crista was obviously swollen. The cristae of the mitochondria in the vacuolated.AS-IV low dose group was blurred and there was a slight vacuolization. The mitochondrial crista in the high dose group of AS-IV was clear, but there was a residual end, and vacuolization obviously alleviated the.LOS group. There was no significant difference in the ultrastructure of the aortic endothelial cells in the high dose AS-IV +LOS group compared with the SHAM group.
3, the immunohistochemical results showed that compared with the SHAM group, the expression level of SOD2 protein in the aortic endothelial cells of the 2K1C group was significantly lower (P0.01). Compared with the 2K1C group, the SHAM group, the high dose AS-IV group, the LOS group and the AS-IV high dose +LOS group increased significantly the expression of SOD2 protein in the aortic endothelial cells (P0.05), and the low dose astragaloside (astragaloside). After the low dose group, the SOD2 protein expression level in the aortic endothelial cells of the rats was not significantly changed (P0.05) compared with the 2K1C group (P0.05), and there was a significant effect in the high dose group of AS-IV and the high dose +LOS group in the AS-IV group. There was no significant difference in the SOD2 protein expression level of the aortic endothelial cells between the three groups (P0.05).
conclusion
AS-IV can protect the mitochondrial damage of aortic endothelial cells in renovascular hypertensive rats. Up regulation of the expression of SOD2 in the aortic endothelial cells may be one of the pathways.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R544.1
本文编号：2157299
[Abstract]:background
Vascular wall is composed of inner layer endothelial cells, middle smooth muscle cells and outer connective tissue. Vascular endothelial cell (EC) is a flat cell between blood and vascular tissue. As a barrier between blood and tissue, it not only maintains the integrity of the blood vessel wall, but also completes the metabolic exchange of blood and tissue fluid, and can also synthesize and secrete more. Biological active substances, to ensure the normal vasoconstriction and diastolic function of blood vessels, play the role of regulating vascular tension, regulating blood flow and blood pressure. In addition, it also has many important physiological functions, such as regulating coagulation and anticoagulant balance, preventing thrombosis, inhibiting the walking and proliferation of vascular wall cells, vascular endothelial cells aging, apoptosis and The regenerative balance plays an important role in maintaining the function of normal blood vessels. As an important cell organelle in cells, mitochondria regulate cell programmed cell death, as well as cell aging, as well as cell senescence. Mitochondria act as the main organelles for cell energy supply in addition to the synthesis of ATP to provide energy for tissue cells. It is easy to be attacked by various adverse factors inside and outside the body. The dysfunction or damage of the mitochondria of vascular endothelial cells will inevitably lead to the maintenance of normal physiological functions of the vascular endothelial cells. Therefore, it is of great significance to protect the damage of mitochondria of vascular endothelial cells in the prevention and treatment of cardiovascular diseases.
Astragaloside IV (AS-IV) is a saponins with various pharmacological activities isolated from Astragalus membranaceus. It is one of the main active components of Astragalus, and has the antioxidant properties. It can regulate the immunity of the body, protect the tissues and organs, reduce the blood sugar, anti apoptosis and anti-inflammatory and antiviral effects. The research shows that AS-IV We can reduce mitochondrial damage by improving mitochondrial membrane potential, protect retinal ganglion cells, and reduce mitochondrial oxidative stress products to reduce the apoptosis [1-2]. of cardiomyocytes, so we further speculate that AS-IV has protective function on the mitochondrial damage of aortic endothelial cells, but AS-IV protects the mitochondria. The cutting mechanism is not yet clear.
objective
The purpose of this study is to confirm the protective effect of AS-IV on the mitochondrial damage of aortic endothelial cells in renovascular hypertensive rats and to explore its possible mechanism, which will be beneficial to the development of the target target of AS-IV drugs.
Method
1, establishment of a renovascular hypertensive rat model and blood pressure monitoring.
SD rats were exposed to the left kidney after anesthesia and fixation, and the left renal artery was separated from the left renal artery in the middle part of the renal artery. The renal artery was placed in the middle part of the renal artery, and the renal artery narrowed. After the operation, the cage was placed at room temperature and was given to the routine diet after awakening. The rat blood pressure instrument BP-2000 system was used to measure the blood of each group. Pressure, the blood pressure of each rat was measured 10 times, and the mean value was taken. The systolic pressure of 140mmHg (1mmHg=0.133kPa) after 2 weeks was used as the successful standard for making the hypertension model, and used for further experiment.
2, experimental grouping and treatment
The experiment was divided into sham operation group (group SHAM), two kidney one clip group (group 2K1C), low dose group of astragaloside (group AST-L, 1.0mg/ (kg-d)), high dose group of astragalin (AST-H group, 5.0mg/ (kg-d)), losartan potassium group (LOS group, 10mg/ (kg-d)), high dose of astragalin + losartan potassium group (AST-H+LOS group) Group H+LOS was injected intraperitoneally 2 weeks after operation. Group SHAM and group 2K1C were given intraperitoneal injection of equal volume of normal saline.
3, the mitochondria of rat thoracic aorta endothelial cells were observed by electron transmission microscope.
The articulation of the inner and outer membrane of the rat thoracic aorta endothelial cells, the integrity of the mitochondrial crista, the change of the mitochondrial matrix electron density and the swelling of the mitochondria were observed by transmission electron microscopy.
4, immunohistochemical staining was used to observe the expression of manganese superoxide dismutase (SOD2) in rat aortic endothelial cells.
SOD2 immunohistochemical detection was used in rat aortic endothelial cells. The morphological image analysis system Image-ProPlusVersion6.0 software was used to measure the positive staining area and total light density of the aortic endothelial cells. The mean light density (IOD/area) was calculated, and the mean light density value represented the expression level of SOD2.
Result
1, the rat tail method was used to determine the blood pressure in rats. There was no significant difference in the basic blood pressure of rats in each group before operation (P0.05). At the end of the two week, group 2K1C, AS-IV low dose group, AS-IV high dose group, LOS group, AS-IV high dose +LOS group were significantly higher than SHAM group, the difference was statistically significant (P0.01). Two weeks after treatment, LOS group, AS-IV high dose +LO The systolic pressure of the S group was significantly lower than that in the model group (P0.01). In group LOS, there was a significant difference between the high dose +LOS group of AS-IV and the low dose AS-IV group (P0.05), and there was no significant difference between the low dose group of AS-IV and the high dose group of AS-IV (P0.05), while the LOS group, AS-IV high dosage, was no significant difference (P0.05). There was no significant difference between the two groups in the hypotensive effect compared with the AS-IV high dose group (P0.05).
2, under electron microscope, the mitochondrial membrane of the aortic endothelial cells in the SHAM group was clear and complete, the crista and the ridge gap were clear. The ridge fracture of the mitochondria of the aorta endothelial cells in the normal.2K1C group was absent, and the crista was obviously swollen. The cristae of the mitochondria in the vacuolated.AS-IV low dose group was blurred and there was a slight vacuolization. The mitochondrial crista in the high dose group of AS-IV was clear, but there was a residual end, and vacuolization obviously alleviated the.LOS group. There was no significant difference in the ultrastructure of the aortic endothelial cells in the high dose AS-IV +LOS group compared with the SHAM group.
3, the immunohistochemical results showed that compared with the SHAM group, the expression level of SOD2 protein in the aortic endothelial cells of the 2K1C group was significantly lower (P0.01). Compared with the 2K1C group, the SHAM group, the high dose AS-IV group, the LOS group and the AS-IV high dose +LOS group increased significantly the expression of SOD2 protein in the aortic endothelial cells (P0.05), and the low dose astragaloside (astragaloside). After the low dose group, the SOD2 protein expression level in the aortic endothelial cells of the rats was not significantly changed (P0.05) compared with the 2K1C group (P0.05), and there was a significant effect in the high dose group of AS-IV and the high dose +LOS group in the AS-IV group. There was no significant difference in the SOD2 protein expression level of the aortic endothelial cells between the three groups (P0.05).
conclusion
AS-IV can protect the mitochondrial damage of aortic endothelial cells in renovascular hypertensive rats. Up regulation of the expression of SOD2 in the aortic endothelial cells may be one of the pathways.
【学位授予单位】：南京医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R544.1
【参考文献】
相关期刊论文 前2条
1 郑彩云;;黄芪降压作用的实验研究[J];光明中医;2010年04期
2 郑敏,杨宏杰,张丹,陈咸川;黄芪提取液清除自由基的实验研究[J];山东中医杂志;2004年12期
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