Effect Of Astragaloside â…£ On Mitochondrial Injury Of Aortic Endothelial Cell From Renovascular Hypertensive Rats

BackgroundThe blood vessel wall is composed of inner endothelial cells, smooth muscle cells in the middle, the outer layer of connective tissue. Vascular endothelial cell (EC) is a single squamous epithelial cell layer and an anticoagulant barrier between the vessel wall and blood. EC are involved in the immune and inflammatory response, coagulation, growth regulation, production of extracellular matrix components, and is a modulator of blood flow and blood vessel tone. The balance of vascular endothelial cells’senescence, apoptosis and regeneration plays an extremely important role on maintaining on the normal function of blood vessels. Mitochondria are important organelles within the cells, which have the synthesis of ATP to provide energy for cells, and also control programmed cell death, and aging, and other physiological and pathological metabolic processes. Thus, protecting mitochondrial injury of vascular endothelial cells is important on the prevention of cardiovascular disease.Astragaloside (Astragaloside IV, AS-IV) is a saponin compound with a variety of pharmacological activity isolated from Astragalus and is one of an active ingredient of Astragalus, which has antioxidant properties, regulating immunity, protecting tissues and organs, reducing blood sugar, anti-apoptotic and anti-inflammatory anti-virus and other pharmacological effects. Studies have shown that AS-IV can reduce mitochondrial damage protection of retinal ganglion cells by increasing mitochondrial membrane potential, but also by reducing mitochondrial oxidative stress products to reduce apoptosis of cardiomyocytes. Therefore, we further speculate that AS-IV has a protective function on mitochondrial damage, but the exact mechanism of AS-IV for mitochondrial protective effect is not clear.ObjectiveThis study aimed to confirm astragaloside IV protects mitochondrial injury of aortic endothelial cell from renovascular hypertensive rats, and to explore its possible mechanism, which benefits for the development of AS-IV drug targets.Methods1、Establishment of renovascular hypertensive rats and the blood pressure monitoringThe SD rats anesthetized were fixed and expose the left kidney, dissecting the left renal artery and placing silver clip (inner diameter:0.2mm) in the middle of the renal artery. The SD rats were givied regular diet after waking within room temperature. The rat blood pressures were measured by BP-2000system weekly after surgery. Blood pressure gets10times’ measurements of each rat and counting the average. Systolic blood pressure>140mmHg (1mmHg=0.133kPa) after2weeks as successful hypertensive model for further experiments.2、The experimental groups and treatmentThe rats were divided into sham operation group (SHAM group), two kidney one clip group (2K1C group), low-dose astragaloside group (AST-L group,1.0mg/(kg·d)), high-dose astragaloside group (AST-Hgroup,5.0mg/(kg·d)), losartan potassium group (LOS group,10mg/(kg·d)), high-dose astragaloside+losartan potassium group (AST-H+LOS group,5.0mg/(kg·d)+10mg/(kg·d)). AST-L group, AST-H group, LOS group and AST-H+LOS group give intraperitoneal injection after2weeks, SHAM group and2K1C group received an equal volume of normal saline.3^Observing endothelial cell mitochondria of rat aortic by transmission electron microscopyObserving the clarity of inner and outer membranes in mitochondrial, the integrity of mitochondria, changes with electron density of mitochondrial matrix or swelling.4、Observing The expression of Mn-Superoxide Dismutase (SOD2) of aortic endothelium with immunohistochemical technique.SOD2was observed in rat aortic endothelial cells with immunohistochemical technique, measuring positive staining area and the optical density value of total aortic endothelial cells using morphological image analysis system Image-Pro Plus Version6.0software, counting the mean optical density values (IOD/area), with an average optical density values as expression levels of SOD2.Results1、Calculating the basis of blood pressure before surgery, the rats showed no significant difference (P>0.05). After two weeks, compared with SHAM group, the systolic blood pressure of2K1C group, low-dose AS-IV group, high-dose AS-IV group, LOS group, or high-dose AS-IV+LOS group and was significantly higher, and the difference was significant statistically (P<0.01). After giving two weeks’ drug intervention, a little effect on systolic blood pressure for AST-L group; the systolic blood pressure of AST-H group, LOS group and AST-H+LOS group decreased obviously, especially AST-H+LOS group(P<0.05).2、Aortic endothelial cells mitochondrial membrane of SHAM group is clear and complete, and mitochondria electron density matrix is normal by electron microscope. Aortic endothelial cell mitochondria of2K1C group appeared cristae fracture、cristae disappeared, and swelling and obvious vacuolation; the degree of mitochondrial damage was significantly alleviated in AST-Hgroup, LOS group and AST-H+LOS group rats. LOS group or AS-IV+LOS high-dose group compared with SHAM group aortic endothelial cells mitochondrial ultrastructure had no significant difference.3、Immunohistochemical result reveal that compared with the Sham group, the average optical density value of SOD2protein decreased significantly in aortic endothelial cells on2K1C group (P<0.01). Compared with2k1c group, the average optical density value of SOD2protein increased significantly in aortic endothelial cells on the AST-H、LOS group and AST-H+LOS (P<0.05). Compared with the2K1C group, the average optical density value of SOD2protein on AST-L group has no significant difference (P>0.05). During the AST-H、LOS group or AST-H+LOS has no significant difference (P>0.05).ConclusionAstragaloside Ⅳ reverses mitochondrial injury of aortic endothelial cell from renovascular hypertensive rats, which may be linked to its ability to increase SOD2expression in the aortic endothelium of the rats.