黃芪甲苷對腎血管性高血壓大鼠主動脈內皮細胞線粒體損傷的保護研究
發(fā)布時間：2018-08-01 10:37
【摘要】：背景 血管壁是由內層內皮細胞、中層平滑肌細胞、外層結締組織構成的。血管內皮細胞(EC)是位于血液與血管組織之間的一層扁平細胞,作為血液和組織之間的屏障,不僅來維持血管壁的完整,完成血液和組織液的代謝交換,還可合成和分泌多種生物活性物質,以保證血管正常的收縮和舒張功能,起到調節(jié)血管張力、調節(jié)血流、血壓的作用。除此之外,它還具有調節(jié)凝血與抗凝平衡、防止血栓形成、抑制血管壁細胞的游走和增殖等多種重要的生理功能。血管內皮細胞衰老、凋亡與再生的平衡對正常血管的功能維持具有極其重要的作用。線粒體作為細胞內的重要細胞器,除了合成ATP為組織細胞提供能量外,還調控制細胞程序性死亡、以及細胞衰老等多種病理生理的代謝過程。線粒體作為細胞供能的主要細胞器很容易受到機體內部和外界各種不良因素的攻擊,血管內皮細胞線粒體的功能障礙或損傷,必然會導致血管內皮正常生理功能的維持。因此,保護血管內皮細胞線粒體的損傷在防治心血管疾病中有重要意義。 黃芪甲苷(Astragaloside IV, AS-IV)是從中藥黃芪中分離得到的具有多種藥理活性的皂苷類化合物,是黃芪主要活性成分之一,具有抗氧化特性,有調節(jié)機體免疫力、保護組織器官、降低血糖、抗細胞凋亡和抗炎抗病毒等藥理作用。研究表明,AS-IV能夠通過提高線粒體膜電位減輕線粒體損傷,保護視網膜神經節(jié)細胞,還可通過降低線粒體氧化應激產物來減少心肌細胞的凋亡[1-2]。因此,我們進一步推測AS-IV對主動脈內皮細胞線粒體損傷具有保護功能,但AS-IV對線粒體保護作用的確切機制尚不明確。 目的 本研究旨在證實AS-IV對腎血管性高血壓大鼠主動脈內皮細胞線粒體損傷的保護作用并探索其可能機制,將有益于開發(fā)AS-IV藥物作用靶點。 方法 1、腎血管性高血壓大鼠模型的建立及血壓監(jiān)測 清潔級SD大鼠麻醉固定后暴露左側腎臟,鈍性分離左側腎動脈,于腎動脈中段放置銀夾(內徑0.2mmm),縮窄腎動脈。術后分籠,置于室溫,蘇醒后給予常規(guī)飲食。采用大小鼠無創(chuàng)血壓儀BP-2000系統(tǒng),經套尾法分別于術前、術后每周測量各組大鼠血壓,每只大鼠血壓測量10次,取平均值；術后2周收縮壓140mmHg(1mmHg=0.133kPa)作為高血壓模型制作成功標準,用于進一步實驗。 2、實驗分組與治療 實驗分為假手術組(SHAM組)、兩腎一夾組(2K1C組)、黃芪甲苷低劑量組(AST-L組,1.0mg/(kg-d))、黃芪甲苷高劑量組(AST-H組,5.0mg/(kg-d))、洛沙坦鉀組(LOS組,10mg/(kg-d))、黃芪甲苷高劑量+洛沙坦鉀組(AST-H+LOS組,5.0mg/(kg-d)+10mg/(kg-d))。AST-L組、AST-H組、LOS組和AST-H+LOS組于術后2周腹腔注射給藥,SHAM組和2K1C組給予等體積生理鹽水腹腔注射。 3、通過電子透射顯微鏡觀察大鼠胸主動脈內皮細胞線粒體 通過透射電鏡觀察大鼠胸主動脈內皮細胞線粒體內外膜的清晰度、線粒體嵴的完整性、線粒體基質電子密度的高低變化及線粒體腫脹情況。 4、應用免疫組化染色法觀察大鼠主動脈內皮細胞錳-超氧化物歧化酶(SOD2)的表達 大鼠主動脈內皮細胞SOD2免疫組化檢測,采用形態(tài)學圖像分析系統(tǒng)Image-ProPlusVersion6.0軟件進行主動脈內皮細胞陽性染色面積和總光密度值的測量,計算得出平均光密度值(IOD/area),以平均光密度值代表SOD2的表達水平。 結果 1、鼠尾法測定大鼠血壓顯示,手術前各組大鼠基礎血壓差異無統(tǒng)計學意義(P0.05)。術后兩周末,2K1C組、AS-IV低劑量組、AS-IV高劑量組、LOS組、AS-IV高劑量+LOS組與SHAM組比較收縮壓明顯升高,差異有統(tǒng)計學意義(P0.01)。治療兩周后,LOS組、AS-IV高劑量+LOS組與模型組比較收縮壓均有明顯降低,差異有統(tǒng)計學意義(P0.01); LOS組、AS-IV高劑量+LOS組與AS-IV低劑量組比較降壓效果差異有統(tǒng)計學意義(P0.05); AS-IV低劑量組與AS-IV高劑量組相比較降壓效果差異無統(tǒng)計學意義(P0.05); LOS組、AS-IV高劑量+LOS組與AS-IV高劑量組相比較降壓效果差異無統(tǒng)計學意義(P0.05)。 2、電鏡下可見,SHAM組主動脈內皮細胞線粒體雙層膜清晰完整,線粒體嵴、嵴間隙清楚,基質電子密度正常。2K1C組主動脈內皮細胞線粒體的嵴斷裂缺失,僅留部分殘端,可見明顯腫脹、空泡化。AS-IV低劑量組線粒體的嵴模糊不清,有輕度空泡化。AS-IV高劑量組線粒體嵴清楚,但有殘端,空泡化明顯減輕。LOS組、AS-IV高劑量+LOS組與SHAM組相比較主動脈內皮細胞線粒體超微結構無明顯差別。 3、免疫組化結果顯示：與SHAM組比較,2K1C組大鼠主動脈內皮細胞SOD2蛋白表達水平顯著降低(P0.01)；與2K1C組比較,SHAM組、AS-IV高劑量組、LOS組與AS-IV高劑量+LOS組大鼠主動脈內皮細胞SOD2蛋白表達水平顯著增加(P0.05)；經低劑量黃芪甲苷(AS-IV低劑量組)治療后,與2K1C組比較、大鼠主動脈內皮細胞SOD2蛋白表達水平變化不明顯(P0.05)；AS-IV高劑量組、LOS組與AS-IV高劑量+LOS組有顯著療效,三組之間大鼠主動脈內皮細胞SOD2蛋白表達水平相比較差異無統(tǒng)計學意義(P0.05)。 結論 AS-IV對腎血管性高血壓大鼠主動脈內皮細胞線粒體的損傷有保護作用,上調主動脈內皮細胞SOD2表達可能是其途徑之一。
[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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R544.1
本文編號：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.
【學位授予單位】：南京醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R544.1
【參考文獻】
相關期刊論文 前2條
1 鄭彩云;;黃芪降壓作用的實驗研究[J];光明中醫(yī);2010年04期
2 鄭敏,楊宏杰,張丹,陳咸川;黃芪提取液清除自由基的實驗研究[J];山東中醫(yī)雜志;2004年12期
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