Effect And Mechanism Of Salidroside Analogues On Inhibiting Hypoxia Injury To EAhy926 Endothelial Cells

Objective: To study the protective effect of salidroside analogues on hypoxia injury human umbilical vein endothelial cells EAhy926, and to explore its possible mechanism.Methods:1 Concentration determination and experiment grouping1.1 Logarithmic growth phase cells were tested by MTT assay, and LDH activity was also measured to observe the protective effect of salidroside analogues on EAhy926 endothelial cells, and to determine the concentration of the test administration of salidroside analogues.1.2 The cells were randomly divided into normal control group（Control）, hypoxia model group（Model）, salidroside positive drug group（H） and the Salidroside analogues intervention group.1.3 By depriving the sugar and oxygen in the culture medium, and substituted O2 with N₂ in normal culture environment, EAhy926 endothelial hypoxia model was established.2 Effect of salidroside analogues on inhibiting hypoxia injury to EAhy926 endothelial cell2.1 Morphology changes under hypoxic conditions was observed through the light microscope and HE staining, respectively.2.2 Cell viability under hypoxic conditions was observed by trypan blue staining.2.3 The protective effect of salidroside analogues under hypoxic conditions on EAhy926 cellular induced by lipid peroxidation injury was investigated by Malondialdehyde（MDA） content and total cellular superoxide dismutase（T-SOD） vitality. MDA content was determined by the thiobarbituric acid method（TBA method）, and T-SOD vitality was measured by xanthine oxidase（hydroxylamine） method.3 Mechanism of salidroside analogues on inhibiting hypoxia injury to EAhy926 endothelial cells3.1 Determination of endothelial nitric oxide synthase（e NOS） activity by enzyme-linked immunosorbent assays（ELISA）.3.2 HIF-1α m RNA and VEGF m RNA expression differences were observed in all experimental groups by RT-PCR.3.3 HIF-1α, VEGF and p VHL protein expression differences were observed in each experimental group by Western Blot.Results:1 The optimal concentration of salidroside analoguesMTT assay results showed that, compared with control group cells, model group cell activity was significantly lower（P<0.01）. Compared with the model group cell, salidroside analoguess（1×10-4- 1×10-10mol·L-1） cell vitality were significantly increased（P<0.01）. Among them, salidroside analogues 1×10-6mol·L-1 concentration group had the highest cell viability（P<0.01）. Cell viability was correlated with the salidroside analogues dose in the 1×10-6 mol·L-1-1×10-10 mol·L-1 concentration range.LDH activity results showed that LDH activity was significantly increased in model group compared with the control group cells（P<0.01）. Compared with the model group, LDH activity was significantly lower in salidroside analoguess group（1×10-4-1×10-10mol·L-1）（P<0.01）. LDH activity was decreased with the increasing salidroside analogues dose in 1×10-6 mol·L-1-1×10-10 mol·L-1 concentration range.Therefore, high, medium and low salidroside analogues dose selected in this experiment were: 1×10-6 mol·L-1, 1×10-7 mol·L-1, 1×10-8 mol·L-1, respectively.2 Effect of salidroside analogues on inhibiting hypoxia injury to EAhy926 endothelial cell2.1 morphology changesInverted phase contrast microscope observation showed that normal cultured cells adherent growth, cobblestone-like covered bottom,cell body plump, polygonal or flat cell into short spindle. Hypoxia model group cells were showing poor adherent, cell shrinkage, partially detached. After salidroside analogues and positive drug（salidroside） intervention, cell morphology improved significantly.HE staining showed that, compared with control cells, model group cell body were smaller, nuclear condensation and stained, the cell gap increased. After salidroside analogues and positive drug（salidroside） intervention, cell structure and morphology were significantly improved.2.2 Placental blue stainingPlacenta blue staining showed that, compared with control cells, model group cell survival rate was significantly lower（P<0.01）. Compared with model group, salidroside analogues and positive drug（salidroside） group cell survival rate were significantly higher（P<0.01）.2.3 Intracellular T-SOD activity and MDA content testCompared with control group cells, SOD activity was significantly decreased（P<0.01） and MDA content was significantly increased（P<0.01） in model group. After salidroside analogues and positive drug（salidroside） intervention, a significant increase in intracellular SOD activity（P<0.01） and significantly reduce in the production of MDA（high dose P<0.01, middle and low dose P<0.05）.3 Mechanism of Salidroside analogues on inhibiting hypoxia injury to EAhy926 endothelial cells3.1 ELISA assays of endothelial nitric oxide synthase（e NOS） activityCompared to model group cells, control group cells e NOS activity was significantly decreased（P<0.01）. Salidroside analogues and positive drug（salidroside） intervention group cells had a significant increase（P<0.01） compared to model group.3.2 HIF-1α m RNA, VEGF m RNA expressionRT-PCR results showed that a significant increase expression of HIF-1α m RNA, VEGF m RNA in the model group（P<0.01） compared with controlgroup. The expression of HIF-1α m RNA was significantly decreased（P <0.01）, while the expression of VEGF m RNA was significantly increased（P <0.01） in salidroside analogues and positive drug（salidroside） intervention group.3.3 HIF-related factor protein expressionWestern Blot results showed that model group HIF-1α, VEGF protein expression was significantly increased（P<0.01）; p VHL protein expression levels were significantly lower（P<0.01） compared with control group. Compared with model group, after positive drug（salidroside） and salidroside analogues intervention, HIF-1α protein expression decreased（P <0.01）; VEGF protein expression was increased（positive drug, high dose, P<0.01）; p VHL protein expression levels were significantly increased（P<0.01）.Conclusion: Salidroside analogues have a significant protective effect on hypoxia induced vascular endothelial cell injury, its possible mechanism maybe decrease oxidative damage, increase e NOS activity, and regulating the expression of HIF-related factors.