Effects Of Qihong Decoction Drug-containing Serum On Numbers And Function And Nitric Oxide Synthase Of Peripheral Blood-derived Endothelial Progenitor Cells

Objective:To investigate the effects of Qihong decoction drug-containing serum on numbers and function and nitric oxide synthase of of endothelial progenitor cells(EPCs)derived from peripheral blood.Methods:1 To prepare Qihong decoction drug-containing serumTwelve Wistar male rats were randomly divided into four groups:Qihong decoction high dosage group,medium dosage group, low dosage group and control group.The different dosage Qihong decoction and equal quantity of normal saline were administrated in each group.After 7 days,blood was collected from heart and the drug-containing serum and control serum were prepared.2 Culture and identification of human EPCs from peripheral bloodThe mononuclear cells (MNCs)were isolated from human peripheral blood by density gradient centrifugation. Cells were plated on culture dishes coated with human fibronectin and maintained in Medium 199.After 7d of culture,adherent cells were collected.EPCs were characterized by laser confocal microscope and demonstrated by expressing CD34 and CD 133 using flow cytometry.3 Effects of Qihong Decoction Drug-containing Serum on numbers,function of EPCsAfter cultured for 7d,EPCs were randomized into 4 groups:control group,Qihong decoction low dosage group,medium dosage group,high dosage group. The cultured EPCs were incubated with different cultivate liquid (include different concentration drug-containing serum and control serum respectively)for different periods(24 h,48 h and 72 h).The morphology of the cells was observed under reverse microscope, numbers of EPCs was assessed by cell counting, migration were assayed with modified Boyden chamber assay, secretion of NO of EPC was assessed by the nitrate reductase method,the NOS dtecting kit was also performed to determine the NOS expression after curcumin interference.Results:1 Characteristics of EPCsTotle MNCs isolated were small round cells,and cultured for 4days resulted in cellcolony.After 7days culture,the MNCs showed as spindles-shaped,endothelial cells-like morphology. EPCs were characterized as adherent cells with double positive for Dil-LDL-uptake and lectin binding with laser scanning confocal microscope. Further the cells were demonstrated by expressing CD34+(35.65±2.47)%and CD133+(13.13±1.39)%using flow cytometry.2 Effects of Qihong Decoction Drug-containing Serum on numbers,function and the activity of EPCsCompared with control group,the numbers, the function of migration, the secretion of nitric oxide and the activity of nitric oxide synthase of endothelial progenitor cells were remarkably increased by Qihong decoction low and medium and high dose group,showing a time-effect relation (P<0.05). Incubated for 24h,the Qihong decoction high dosage group had the most prominent effect on the secretion of nitric oxide and the activity of nitric oxide synthase of EPCs(P<0.05). Incubated for 48h or 72h, the Qihong decoction medium dosage group had the most obvious effect on the secretion of nitric oxide and the activity of nitric oxide synthase of EPCs. the Qihong decoction medium dosage group had significant effect on the numbers and the function of migration of EPCs(P<0.05). Effect of the Qihong decoction medium dosage group on the number and the secretion of nitric oxide and the activity of nitric oxide synthase of EPCs begined at 24h and reached peak at 48h(P<0.05). Effect of the Qihong decoction medium dosage group on the function of migration begined at 24h and reached peak at 72 h(P<0.05).Conclusion:1. Qihong decoction increases the numbers and the activity of nitric oxide synthase. Qihong decoction promote the migration and the secretion of nitric oxide of EPCs. The Qihong decoction medium dosage group had most steady effection on the number and function and NOS of EPCs.The effection was the most prominent at 48h.2,Qihong decoction increases the activity of nitric oxide synthase,then promote the secretion of nitric oxide of EPCs,which may be the basis of EPCs number increase and migration.3,Qihong decoction drug-containing serum can premote the numbers and function of EPCs derived from peripheral blood.The results may be parts of the mechanisms of Qihong decoction to prevent and treat vascular endothelial injury/dysfunction-related disease.