[The protective effects of Astragaloside â £ on diastolic function of rat thoracic aortic rings impaired by microvesicles].

OBJECTIVES: To investigate the effects of Astragaloside IV (AST) on diastolic function of rat thoracic aorta rings which was injured by microvesicles derived from hypoxia/reoxygenation (H/R)-treated human umbilical vein endothelial cells (HUVECs), and the mechanism of AST. METHODS: H/R-induced endothelial microvesicles (H/R-EMVs) were generated from cultured HUVECs in vitro under the condition of hypoxia for 12 hour/Reoxygenation for 4 hour, H/R-EMVs were stored in D-Hank's solution. Male Wistar rats were underwent thoracotomy, the thoracic aorta with intact endothelium were carefully removed and cut into 3~4 mm rings. The experiment was divided into six groups. H/R-EMVs group:thoracic aortic rings of rats were incubated in culture medium and treated with H/R-EMVs in a final concentration of 10µg/ml; different doses of AST groups:thoracic aortic rings of rats were treated with 10, 20, 40, 60 mg/L AST co-incubated with 10µg/ml H/R-EMVs respectively; control group were treated with the same volume of D-Hank's solution. Duration of incubation was 4 h, each group was tested in five replicate aortic rings. Effects of AST on endothelium-dependent relaxation were detected. The production of nitric oxide (NO) and the level of endothelial NO synthase (eNOS), phosphorylated eNOS (p-eNOS, Ser-1177), serine/threonine kinase (Akt), phosphorylated Akt (p-Akt, Ser-473), extracellular regulated protein kinases (ERK1/2) and phosphorylated ERK1/2 (p-ERK1/2, Thr202/Tyr204) of rat thoracic aortic rings were detected. RESULTS: Tenµg/ml H/R-EMVs could impaire the relaxation of rat thoracic aortic rings significantly (P<0.01). Compared with H/R-EMVs group, relaxation of rat thoracic aortic rings was increased by 20, 40 and 60 mg/L AST in a concentration-dependent manner (P<0.01), the level of NO production was also enhanced (P<0.05, P<0.01). The level of t-eNOS, t-Akt and ERK1/2 was not changed, but the level of p-eNOS, p-Akt and p-ERK1/2 increased by the treatment with AST (P<0.01). CONCLUSIONS: AST could effectively ameliorate endotheliumdependent relaxation of rat thoracic aortic rings impaired by H/R-EMVs in a concentration-dependent manner, the mechanism might involve the increase in production of NO, and the protein level of p-eNOS, p-Akt and p-ERK1/2.

[Effects of circulating microvesicles derived from myocardial ischemic preconditioning on myocardial ischemia/reperfusion injury in rats].

OBJECTIVE: To investigate the effects of circulating microvesicles (MVs) derived from ischemic preconditioning (IPC) on myocardial ischemia/reperfusion (I/R) injury in rats and explore the underlying mechanism. METHODS: To establish the IPC model, the rats were subjected to brief cycles of left anterior descending (LAD) coronary occlusion and reperfusion. The blood was drawn from abdominal aorta once the operation was finished. IPC-MVs were isolated by ultracentrifugation from the peripheral blood and characterized by flow cytometry. The myocardial I/R model of rats was established in vivo. Rats were injected via the femoral vein with IPC-MVs at 7 mg/kg. Morphological changes of myocardium were observed microscopically after HE staining. Apoptosis of myocardial cells was detected with TUNEL assay. Myocardial infarct size was detected by TTC staining. Moreover, activity of plasma lactate dehydrogenase (LDH) was tested by colorimetry. The activity of caspase 3 in myocardium was assayed with spectrophotometry. Expression levels of Bcl-2 and Bax protein were examined with Western blot. RESULTS: The concentration of IPC-MVs, which was detected by flow cytometry, was 4380±745 cells/µl. Compared with I/R group, IPC-MVs alleviated the damage of tissues in I/R injured rats significantly. The myocardial infarct size and the cardiomyocyte apoptotic index were obviously decreased after IPC-MVs treatment (P<0.01, respectively). The activity of plasma LDH was significantly decreased in IPC-MVs treated rats (P<0.01). Moreover, the activity of caspase 3 was markedly decreased after IPC-MVs treatment (P<0.01). In addition, the expression of Bcl-2 was increased (P<0.01), the expression of Bax was decreased (P<0.01), the ratio of Bcl-2/Bax was significantly increased after IPC-MVs treatment (P<0.01). CONCLUSIONS: IPC-MVs protected myocardial against I/R injury by up-regulating the expression of Bcl-2 protein, down-regulating the expression of Bax protein, increasing the ratio of Bcl-2/Bax and decreasing cleavage of caspase 3.

Flow cytometric analysis of circulating microvesicles derived from myocardial Ischemic preconditioning and cardioprotection of Ischemia/reperfusion Injury in rats.

OBJECTIVE: To establish a flow cytometric method to detect the alteration of phenotypes and concentration of circulating microvesicles (MVs) from myocardial ischemic preconditioning (IPC) treated rats (IPC-MVs), and to investigate the effects of IPC-MVs on ischemia/reperfusion (I/R) injury in rats. METHODS: Myocardial IPC was elicited by three.cycles of 5-min ischemia and 5-min reperfusion of the left anterior descending (LAD) coronary artery. Platelet-free plasma (PFP) was isolated through two steps of centrifugation at room temperature from the peripheral blood, and IPC-MVs were isolated by ultracentrifugation from PFR PFP was incubated with anti-CD61, anti-CD144, anti-CD45 and anti-Erythroid Cells, and added 1, 2 µm latex beads to calibrate and absolutely count by flow cytometry. For functional research, I/R injury was induced by 30-min ischemia and 120-min reperfusion of LAD. IPC-MVs 7 mg/kg were infused via the femoral vein in myocardial I/R injured rats. Mean arterial blood pressure (MAP), heart rate (HR) and ST-segment of electro-cardiogram (ECG) were monitored throughout the experiment. Changes of myocardial morphology were observed after hematoxylin-eosin (HE) staining. The activity of plasma lactate dehydrogenase (LDH) was tested by Microplate Reader. Myocardial infarct size was measured by TTC staining. RESULTS: Total IPC-MVs and different phenotypes, including platelet-derived MVs (PMVs), endothelial cell-derived MVs (EMVs), leucocyte-derived MVs (LMVs) and erythrocyte-derived MVs (RMVs) were all isolated which were identified membrane vesicles (<1 Vm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats (P<0.05, respectively). In addition, at the end of 120-min reperfusion in I/R injured rats, IPC-MVs markedly increased HR (P<0.01), decreased ST-segment and LDH activity (P < 0.05, P < 0.01). The damage of myocardium was obviously alleviated and myocardial infarct size was significantly lowered after IPC-MVs treatment (P < 0.01). CONCLUSION: The method of flow cytometry was successfully established to detect the phenotypes and concentration alteration of IPC-MVs, including PMVs, EMVs, LMVs and RMVs. Furthermore, circulating IPC-MVs protected myocardium against I/R injury in rats.