Silymarin Protects Against The Effect Of Rapamycin On Number And Activity Of Endothelial Progenitor Cells

Objective: Through the mobilization of peripheral blood EPCs (endothelial progenitorof the cells) to the local vascular or by transplanting cultured EPCs in vitro to repairinjured endothelium become one of the new treatment strategies of ischemic diseases.Rapamycin-eluting stents can inhibit DNA synthesis and proliferation of vascular smoothmuscle after PTCA. It played a important role of preventing restenosis. Inhibition ofre-endothelialization of vascular and thrombosis formation affect the long-term prognosisof patients with stent implantation. These effects be associated with the inhibition ofvascular endothelial cells migration and inhibitting EPCs migration, proliferation, andintegrated into the the intimal. Recent studies have found that silymarin can inhibit thesenescence of endothelial progenitor cells, and reduce anti-proliferative effect ofrapamycin on EPCs, but whether silymarin can also improve other inhibitory effects ofrapamycin on EPCs is unclear. Based on the above studies, we culture and identify EPCs invitro, and then study the impact of rapamycin on EPCs proliferation and migration. Wealso observe silymarin’s effect of protecting EPCs under rapamycin intervention to furtheroptimize the therapeutic effect of EPCs,and reduce thrombotic events after drug-elutingstent implantation.Methods:1. Isolation, culture and identification of EPCs: The use of density gradientcentrifugation for isolating EPCs. Identification methods including morphology, measuringcellular and molecular markers (CD34, CD133, KDR) by flow cytometry and laserscanning confocal microscope to observe the fuction of the cellular uptake of ac-LDL andbinding of UEA-I.2. Effects of rapamycin on the number and function of EPCs: InterveneEPCs with different concentrations of rapamycin (0,0.1ng/ml,1ng/ml,10ng/ml,100ng/ml) for24hours and different time (0hours,6hours,12hours,24hours,48hours),then detect proliferation and migration of EPCs.3. Effects of silymarin on the number andfunction of EPCs: Intervene EPCs with different concentrations of silymarin (0,25μg/ml,50μg/ml,100μg/ml) for24hours and detect proliferation, migration and apoptosis ofEPCs.4. The protective effects of Silymarin on EPCs under rapamycin intervention:Added rapamycin (1ng/ml) and silymarin (25μg/ml、50μg/ml、100μg/ml) in EPCs for24hours, then detect proliferation, migration, apoptosis and angiogenesis ability.5. Statisticalapproach: The data were expressed as mean±standard deviation among groups werecompared using single factor analysis of variance, homogeneity of variance with SNK method; heterogeneity of variance using Dunnett’s method. The data were analyzed usingSPSS18.0statistical software, P<0.05was considered statistically significant.Results:1. Identification of EPCs: Cultured for24hours, some cells adherent,3dayslater, most of the adherent cells, colony formation, cells were round, after7days, the cellswere spindle;7days later, the cells covered with plates. Cultured for7days, the expressionof CD34, CD133and KDR was positive, expression rates were as follows:(35.5±4.6)%,(18.4±3.8)%,(88.6±5.1)%. By confocal microscopy, cells uptake of DiI-Ac-LDL, can alsobe combined with FITC-UEA-I Double staining positive rate of cells is more than80%.This proves that the cultured cells are EPCs.2. Effects of rapamycin on the number andfunction of EPCs: Compared with the control group, rapamycin (1ng/ml,10ng/ml,100ng/ml) can inhibit the proliferation and migration of EPCs in a concentration and timedependent(n=6, P<0.05).3. Effects of silymarin on the number and function of EPCs:Compared with the control group, silymarin (50μg/ml,100μg/ml) can enhance theproliferation and migration of EPCs, inhibition of apoptosis in a concentration dependentmanner(n=6, P<0.05).4. The protective effects of Silymarin on EPCs under rapamycinintervention: By adding rapamycin (1ng/ml) and silymarin (25μg/ml、50μg/ml、100μg/ml) to intervene in the24hours, silymarin can inhibit pro-apoptotic role of rapamycinon EPCs, and reverse the inhibition of rapamycin on proliferation, migration andangiogenesis of EPCs(n=6, P<0.05).Conclusion:1. Rapamycin can inhibit the proliferation and migration of EPCs in aconcentration and time dependent.2. Silymarin can enhance the proliferation andmigration of EPCs, inhibition of apoptosis in a concentration dependent manner.3.Silymarin can inhibit pro-apoptotic role of rapamycin on EPCs, and reverse the inhibitionof rapamycin on proliferation, migration and angiogenesis of EPCs.