The Effects Of Salidrosid And Salvianolic Acid B On Proliferation, Migration And Apoptosis Of Human Endothelial Progenitor Cells

BackgroundPeripheral blood, umbilical cord blood and bone marrow contain a subtype of functional progenitor cells which can differentiate into mature endothelial cells, participate in new vessel formation in postnatal subjects. Thus, these cells are termed as endothelial progenitor cells (EPCs). When vessel injury occurs, EPCs are released from bone marrow, migrate to the site of injury, incorporate into the vessel wall, differentiate into mature endothelial cells, repair the injured vessel, participate the neovascularization and restore or improve the blood flow of the ischemic tissue. Transplantation of bone marrow components containing EPCs or EPCs expanded in vitro may be a potent treatment for ischemic diseases. The number and function of EPCs in peripheral blood may also be used to estimate the risk of cardiovascular diseases and predict the prognosis of such diseases. Pharmacological intervention to enhance the number and function of EPCs in peripheral blood could be a new method to prevent and treat cardiovascular diseases. So it's important to search drugs which can increase the number of EPCs and improve their functions in vivo or in vitro therefore to improve the effect of autologous EPCs transplantation or explore a new way to prevent and cure cardiovascular diseases.Chinese herb Rhodiola sacra and its active component Salidrosid along with Salvia miltiorrhiza and it's active component Salvianolic acid B show protective effects on cardiovascular system. They might have the potential to regulate EPCs.AimWe plan to observe the effects of Salidrosid and Salvianolic acid B on proliferation, migration and apoptosis of EPCs cultured in vitro. If they do have effects on EPCs, we would further study the possible mechanism. We hope tounderstand the effects of these two components on EPCs in vitro and make a fundament for future researches of other Chinese herbs' effects on EPCs. Based upon these researches, we hope to develop some valuable drugs to mobilize EPCs to sites of injury and provide a new strategy to prevent and cure ischemic diseases.Methods1, Neovascularization of myocardial infarction rat model by glycoside components of Rhodiola sacra and Salvianolic acid BMyocardial infarction rat model was established by ligating the left coronary descendent anterior artery. Rats were divided randomly to 7 groups with 10 rats in each group: high/medium/low dose glycoside components of Rhodiola sacra(10g·kg^-1, 5g·kg^-1 and 2.5g·kg^-1, respectively) ; Salvianolic acid B (1g·kg^-1); simvastatin (3.3mg·kg^-1) ; Sham (saline) and model (saline). Rats were fed once per day and killed after 14 days' treatment.Measurement of infarct size: 0.3cm thick infarct parts of left ventricles were cut off along the vertical axis. They were fixed, embedded and stained with HE regularly. The infarct size was measured by IMS image analyzing system.Measurement of expression of CD34 in myocardium: regular immunohistochemistry method was used and quantitative analysis of expression of CD34 in infarct and surrounding areas was performed by IMS image analyzing system2, Isolation, culture and identification of human EPCsTotal mononuclear cells (MNCs) were isolated from peripheral blood of healthy human volunteers by density gradient centrifugation. MNCs (1.5 × 10⁶/well) were plated in 0.8 ml endothelial culture medium on fibronectin coated 24-well plates. After 4 days of culture, medium was changed and suspended cells were aspirated away. Up to the 7^th day after isolation, adherent cells were stained with Dil-acLDL and FITC-UEA-1 Lectin to be identified whether they were EPCs. FACS analysis of surface antigen CD34 and VEGFR-2 was performed. The 7th day cells were digested by 0.25% trypsin and 3 × 10⁴ cells/well were planted on fibronectin coated 96-well plates.3, Effects of Salidrosid and Salvianolic acid B on proliferation of human EPCsXTT chromatometry was used to assess the proliferation of EPCs. First we decided the optimal reaction time of XTT by analyze the correlation between XTT reactiontime and corresponding OD value. Then we decided the optimal planting cell density by analyze the correlation between cell density and corresponding XTT OD value. The optimal drug intervention time was decided accordingly. Finally, we observed the effects of 4μg/ml, 8μg/ml, 16μg/ml Salidrosid and 5μg/ml, 10μg/ml, 20μg/ml, 100μg/ml Salvianolic acid B on proliferation of human EPCs.4, Effects of Salidrosid and Salvianolic acid B on migration of human EPCs Transwell system was used to test the cell migration. Fibronectin was coated onboth the upper and down sides of the porous membranes of Transwell inserts. Cells cultured for 7 days were trypsinized and 3 × 10⁴ cells were plated in the upper chamber of each inserts. 600 μ l culture medium was added into the lower chamber. After 24h culturing, 8μg/ml Salidrosid and 10μg/ml Salvianolic acid B were added to the upper chamber and VEGF to the lower chamber reaching the concentration of 500ng/ml. After another 12h culturing, the down sides of the porous membranes of Transwell inserts was fixed, stained and mounted. Independent investigators evaluated the number of cells of each membrane by counting 9 randomly selected high-power fields and the mean value was calculated.5, Effects of Salidrosid and Salvianolic acid B on apoptosis of human EPCsFACS analysis of DNA contents was used to reflect cell apoptosis. EPC apoptosis was induced by depletion of serum and growth factors. 8μg/ml Salidrosid and 10μg/ml Salvianolic acid B were added to the M199 medium. After 24h inducing, cells were digested by 1 mmol/L EDTA/PBS, fixed with fixation solution and stained by PI. Apoptosis was then assessed by FACS.6, Study of the mechanism of pro-proliferation effects by Salidrosid and Salvianolic acid B on EPCsPI3K inhibitor LY294002, Wortmannin and MEK1 inhibitor PD98059 were used. Groups were: control; 8μg/ml Salidrosid; Salidrosid +10μM LY294002; Salidrosid + 10μM Wortmannin; Salidrosid+10μM PD98059; 10μg/ml Salvianolic acid B; Salvianolic acid B +10μM LY294002; Salvianolic acid B+10μM Wortmannin; Salvianolic acid B+ 10μM PD98059. After 24h culturing, cell proliferation was tested by XTT chromatometry.Results1, Neovascularization of myocardial infarction rat model by glycoside components of Rhodiola sacra and Salvianolic acid BDifferent dose intervention of glycoside components of Rhodiola sacra and Salvianolic acid B all could decrease the infarct size, P<0.01. The effects of high dose glycoside components of Rhodiola sacra and Salvianolic acid B were same in magnitude, P>0.05 and were as strong as simvastatin, P>0.05. While the typical dose-effect relationship was not found in glycoside components of Rhodiola sacra, High dose was better.Different dose intervention of glycoside components of Rhod Aiola sacra and Salvianolic acid B all could increase CD34 expression, P<0.05 or 0.01 and the effects were as strong as simvastatin, P>0.05.2, Isolation, culture and identification of human EPCsHuman PMNC were isolated by density gradient centrifugation. After 7 days culture, adherent cells were spindle shaped. They could take Dil-acLDL and bind with FITC-UEA-1 Lectin at the same time which was a character of endothelial cells. Some cells express CD34 and VEGFR. The passaged cells appeared to be bar-like alignment.3, Effects of Salidrosid and Salvianolic acid B on proliferation of human EPCs Different concentration (4μg/ml, 8μg/ml and 16μg/ml) intervention of Salidrosid allcould increase the proliferation of EPCs in vitro, P<0.01. There was no dose-effect relationship, P>0.05. 10μg/ml, 20μg/ml Salvianolic acid B also could increase the proliferation of EPCs, P<0.01, 10μg/ml was better than 20μg/ml, P<0.05. At 5μg/ml, Salvianolic acid B had no impact, P>0.05, while 100μg/ml could suppress the proliferation of EPCs, P<0.01.4, Effects of Salidrosid and Salvianolic acid B on migration of human EPCsEPCs moved through the pores of Transwell inserts by the attraction of VEGF in the lower chamber and attached on the Fibronectin-coated lower sides of the inserts. These migrated cells appeared to be spindle, sliver or polygon shaped and arranged irregularly. Salidrosid and Salvianolic acid B both could improve the migration of EPCs, P<0.05. And the effects were similar in magnitude, P>0.05.5, Effects of Salidrosid and Salvianolic acid B on apoptosis of human EPCsUnder cultural conditions without serum, VEGF, bFGF, IGF-1 and EGF, the control group had high apoptosis rate. While Salidrosid could reduce the apoptosis rate, P<0.01. Salvianolic acid B also could reduce the rate, P<0.05. Salidrosid and Salvianolic acid B both could suppress the apoptosis of EPCs. The impact of Salvianolic acid B was weaker than Salidrosid, P<0.05.6, Study of the mechanism of pro-proliferation effects by Salidrosid and Salvianolic acid B on EPCsPI3K inhibitorLY294002 and Wortmannin could antagonize the effects of Salidrosid and Salvianolic acid B on proliferation of human EPCs, P<0.01; The antagonistic effect of Wortmannin reduced the proliferation of EPCs to the level of the control group, P>0.05; LY294002's was even lower than the control one, P<0.01. MAPK inhibitor PD98059 had the similar antagonistic effcts, P<0.05. It reduced the proliferation of EPCs to the level of the control group, P>0.05. The study showed that Salidrosid and Salvianolic acid B's impacts on the proliferation of EPCs in vitro were partly via PI3K and MAPK signaling pathways.ConclusionSalidrosid and Salvianolic acid B can improve the proliferation and migration of human EPCs in vitro and suppress EPC apoptosis as well. These effects are partly via PI3K and MAPK signaling pathways. The influence of improving neovascularization in myocardial infarct rats by glycoside components of Rhodiola sacra and Salvianolic acid B may be related to these effects.