| BackgroundMyocardial ischemia-reperfusion injury is a difficulty during reperfusion treatment in acute myocardial infarction of coronary heart disease. At present, it was generally regarded that myocardial ischemia-reperfusion injury belonged to an inflammation like reaction. The fact that the no-reflow phenomenon after cardiac microvascular reperfusion was caused by the adhesion between vascular endothelial cells and leukocyte cells was one of the important reasons resulting in the failure of reperfusion treatment. Animal experimental studies were found that astragalus polysaccharides(APS) has the ability of against myocardial ischemia-reperfusion injury, but there are still no reports about the effect of APS on cell adhesion molecule expression and leukocyte adhesion to endothelial cells during the myocardial rererfusion injury.ObjectiveTo observe the role of APS on adhesion between cardiac microvascular endothelial cells and polymorphonuclear leukocyte(PMN) from the aspect of expression of protein and gene in cell adhesion molecule and its transcriptional regulation, in order to investigate the mechanism of APS against myocardial ischemia-reperfusion injury.Methods1 The culture of human cardiac microvascular endothelial cells(HCMEC): Establish a stable cell experiment platform after the recovery, training, passages of cardiac microvascular endothelial cells.2 To establish ischemia-reperfusion injury model of HCMEC: Ischemia/reperfusion was simulated by oxygen-glucose deprivation and recovery method. The survival changes of HCMEC was detected by means of MTT after HCMEC was treated with different glucose-free medium and hypoxia for different time respectively. The injury time of HCMEC was determined in this method of hypoxia and reperfusion, and ischemia-reperfusion injury model of HCMEC was established in vitro.3 The cell proliferous experiment of APS on normal cultured HCMEC: Detect the cell activity changes by MTT method. Observe the cell proliferation of normal cultured HCMEC that was treated with different concentrations of and different time, to determine the safe and effective concentration of APS.4 The effect of APS on neutrophil adhesion to HCMEC after ischemia-reperfusion injury: On the basis of ischemia-reperfusion injury mode of HCMEC in vitro, the PMN was measured by rose Bengal staining, and the effect of APS on adhesion between them was observed.5 Effect of APS on protein and gene expression of ICAM-1 and VCAM-1 in HCMEC after ischemia-reperfusion injury: Expression changes of ICAM-1 and VCAM-1 protein were observed by the method of immunocytochemical staining and the levels of ICAM-1 and VCAM-1mRNA were detected by the means of real time fluorescence quantitative PCR.6 Effect of APS on protein and gene expression of NF-κB in HCMEC after ischemia-reperfusion injury: The positive expression change after nuclear translocation of activated NF-κB was observed by immunocytochemical staining and the level of NF-κB mRNA was detected by the method of real time fluorescence quantitative PCR.Results1 The HCMEC bodies were pachynsis and transparent and showed in rhombus and polygon shapes, cell nuclear was small and round after recovered. HCMEC was monolayer growth and showed a cobblestone appearance.2 HCMEC appeared evident damage after hypoxia for 4h, 6h, 8h in four different glucose-free modeling liquid in vitro (P <0.01), and the longer of hypoxia, the smaller of OD value; cell OD value has a further descending trend in the D-Hank's group and Earle group after 16h reperfusion.3 The proliferation of HCMEC was lower (P <0.05 or 0.01) when concentration of APS≥5000μg·mL-1, and higher (P <0.05 or 0.01) when the concentration of APS in 18.78μg·mL-1-1250μg·mL-1. There was no obvious changes in proliferation when treated with 2500μg·mL-1 APS. Compared with control group, there was no evident change in the proliferation of HCMEC when treated separately with 25, 50, 100μg·mL-1 of APS for 4h, 6h, 8h.4 Adhesion number of PMN was slightly increased after hypoxia alone of HCMEC in 1h, 2h. Compared with control group, adhesion was increased significantly (P <0.05) after reperfusion; PMN adhered to endothelial cells were lower (P <0.05) in the groups of simple hypoxia 3h and reperfusion 6h after hypoxia 3h. APS could reduce the adhesion between the PMN and endothelial cells after ischemia-reperfusion injury, and the high, medium dose groups had significant differences compared with the model group (P <0.01).5 Protein expression of VCAM-1 can be inhibited by each dose of APS groups (P <0.01), and high-dose group can decrease ICAM-1 protein expression (P <0.05). Compared with model group, the ICAM-1, VCAM-1 mRNA expression were significantly inhibited by each dose group of APS(P <0.05 or 0.01), except the ICAM-1 gene transcription in low-dose group. 6 APS could significantly reduce the nuclear translocation after NF-κB activation observed by optical microscopy. Compared with the model group, mRNA expression of NF-κB p65 in APS medium dose group was decreased significantly (P <0.05), measured by fluorescent real-time quantitative PCR.Conclusion1 In a certain range of dosage, APS could promote the proliferation of normal cultured HCMEC, but it needs longer time to take action.2 APS has the role of inhibition of cell adhesion after reperfusion injury both on early and late stage, and there are only some differences between dose and efects.3 APS can reduce the protein and mRNA expression of ICAM-1, VCAM-1, especially the VCAM-1.4 APS can significantly reduce the nuclear translocation after NF-κB activation, and effectively decrease the gene expression of NF-κB.Through this study, the effect of against myocardial ischemia rererfusion injury of APS was further validated. Moreover, the protective effect of APS within a certain dosage on original cultured HCMEC was first confirmed in vitro after ischemia reperfusion injury. Its mechanism was related with promoting the proliferation of HCMEC, inhibiting the expression and activation of NF-κB in HCMEC, reducineg the gene and protein expression of ICAM-1, VCAM-1, therefore, decreasing PMN adhesion to HCMEC.
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