The Effect Of Androgen In The Repairing Of Vascular Endothelial Cell After Injury And Their Internal Molecular Signaling Mechanism

[Objective]To observe the protective role of androgen in the process of reparation of vascular endothelial cell after injury, and study the internal molecule signal pathway. To investigate simply the clinical feasibility of androgen replacement therapy of protecting vascular endothelial cell.[Methods]1. Cell experimentECV-304cells were pretreated with different concentrations of dihydrotestosterone (0.01,0.1,1μM) for2h, followed by exposure to100μM H2O2for18h. Cells' morphology were observed with fluorescence microscope. Cell viability and apoptosis were evaluated by MTT and flow cytometry labeled with Annexin V-FITC/PI. Finally, the expression of cleaved caspase-3, caspase-9and p-phospho-p38was assayed by Western blot.2. Animal experimentMale rabbits were castrated and its right iliac artery were injured with Foley's tube, thus establishing the qualified animal model. Animals in experiment group were supplemented with different concentration of Testosterone Undecamoate (3mg/kg, 6mg/kg,12mg/kg). Light microscope and scanning electron microscope were used to observe the reparation of vascular intima and endothelial cell.[Results]1. Cell morphology(fluorescence microscope)①Normal group and DHT control group:cell grow up adherence, equal in size, all in fusiform shape with complete cell membrane, no obvious apoptosis evidence.■H2O2injury group:most cells show obvious apoptotic morphology change, such as body cell shrinkage and break away from others, cytoplasma condensed with complete cell membrane, nuclear membrane and nucleoli damage.③DHT pretreatment group:cells show anti-apoptosis evidence, in high concentration group, cells'shape were near the cells in normal group.2. Cells'viability (MTT)There was no obvious difference in cytoactive between normal group and DHT control group. Cells'viability decreased significantly in H2O2group, while recoved gradually after DHT pretreatment. And in high concentration DHT pretreatment group, cells'viability were near the cells in normal group.3. Cells'apoptosis detected with flow cytometry (labeled with Annexin V-FITC/PI)The ratio of apoptotic cell in normal group and DHT control group was only2.95%and3.92%. After H2O2injury the ratio reached up to49.96%, while different concentration DHT pretreatment reduced the ration to33.54%,20.04%and6.76%. The ratio of apoptotic cell in high concentration DHT group was near the ratio of normal group.4. Detection of caspase-3, caspase-9and p-p38MAPK(Western blot)Western blot assay showed that H2O2treatment caused high expression of cleaved caspase-3, caspase-9and p-p38MAPK, while DHT pretreatment obviously inhibited the expression in a dose dependent manner.5. Effect of androgen replacement therapy on the VEC repairmen(Animal experiment)Observation with light microscope showed that intima hyperplasia obviously after injury of Foley's tube comparing with normal intima. Different concentration of Testosterone Undecamoate replacement inhibited the hyperplasy of intima. Observation with scanning electron microscope showed that VEC in normal group were all in fusiform shape, equal in size and in order arrangement. Castration and low androgen level caused the complete injury of VEC. While Testosterone Undecamoate replacement promote the recovery of cells in dose dependent manner.[Conclusion]Androgen have the effect of inhibiting apoptosis and promoting recovery after injury in vascular endothelial cell in a dose dependent manner. This effect was associated with the down-regulation of cleaved caspase-9, caspase-3and p-p38MAPK. In animal experiment, androgen replacement therapy show the effect of protecting VEC and promoting intima reparation.