Studies About Effects Of Stanniocalcin 1 On The Proliferation And Migration Of Endothelial Progenitor Cells And The Mechanisminvolvedin PI3K/Akt/eNOS Signaling Pathway

Background:Vascular endothelium damagedin atherosclerosis(AS)is the major pathological process occurred in cardiovascular diseases,which is contributed to the initiation and development of atherosclerosis.The damage of vascular endothelium may result from the long-time stimulating of hypercholesterolemiaor the process of percutaneous coronary intervention treatment.Endothelium injury could accelerate the process of plaque formation and vascular stenosis,leading to vascular thrombosis in the end.Thus,studying the mechanism of vascular endothelial injury and repairment is important for developing the treatment of AS,as well as improving the interventionstrategy.Stanniocalcin(STC)is a glycoprotein hormone involved in regulating calcium/ phosphorus metabolism,which is firstly identified in the gill of fish,.In recent years,STC is also found in mammals and plays an important role in many diseases,such ascancer,cardiovascular and nervous system diseases.STC family is composed of STC1 and STC2,which are widely expressed in various tissues of human body.STC2 mainly interacts with metal ions to regulate levels of calcium and phosphorus.On the other hand,STC1 is one of the few hormones regulating mitochondrial function.By binding to the receptor on the mitochondria,STC1 could offer many benefits,such as anti-oxidation,anti-inflammation,protecting myocardium and endothelium.Furthermore,recent studies have focus on its potential effect in cardiovascular diseases.However,it is still unclear whether STC1 has any effects on the function of endothelial progenitor cells(EPCs)Thus,our research designed to observe the effects as well as study the mechanism of STC1 on the proliferation and migration inEPCs.Methods:1.The expression of STC1 in spleen-derived EPCs1.1 Culturing and identification of EPCsHealthy SD rats were dislocated executed.Spleens were collected in aseptic condition.Mononuclearcells(MNCs)were obtained through the density gradient centrifugation and then cultured in low glucose DMEM medium with 10% high quality fetal bovine serum.The morphology of cells was observed by phase contrast microscopy after 5-7 days.DiI-ac-LDL uptake and FITC-UEA-I binding assay were performed to confirm cells were EPCs.1.2 STC1 were expressed in EPC1.2.1 RT-PCR detected the m RNA level of STC1 in EPCsTotal RNA was extracted from EPCs(7 days),RT-PCR was performed to measure STC1 m RNA level.1.2.2 Western blot detected the protein expression of STC1 in EPCsTotal protein was extracted from EPCs(7 days).Western blot was performed to measure STC1 protein level.2.The effects of rh STC1 on EPCsbiological functions2.1 The effects of rhSTC1 on EPCs proliferationEPCs were treated with rhSTC1 at different concentrations(20?50?100 ?g/ml)for 24 h,the proliferation was measured by CCK-8.2.2 The effects of rhSTC1 on EPCs migrationEPCs were treated with rhSTC1 at different concentrations(20?50?100 ?g/ml)for 24 h.Transwell chamber was used to detect the migration level of EPCs.3.EPCs biological function after endogenous STC1 was silenced3.1 Transfectionof siRNA STC1 and negative control in EPCsThe siRNA transfection was carried out when EPCs were cultured for 5days and the cell density was 60-80%.The siRNA and Lipofectamine? 2000 were prepared as liquid A and B,then were mixed together before transfection.3.2 Western blot detected the efficiency of siRNA48 h after transfection,total protein of EPCs was extracted.STC1 expression was detected by western blot among control group,si RNA STC1 group and negative control group.3.3 The effects of silencing endogenous STC1 by siRNA on EPCs proliferationEPCs were measured by CCK-8 to detect the change of proliferation on 48 h after transfection with STC1 siRNA.3.4 The effects of silencing endogenous STC1 by si RNA on EPCs migrationTranswell chamber assay was performed to detect EPCs migration on 48 h after EPCs were transfected with STC1 si RNA.4.Explore the role of PI3K/Akt/e NOS signaling pathway in STC1-induced effects on EPCs biological functionsTo clarify whether the PI3K/Akt/e NOS signaling pathway is involved in the STC1-induced effects on the biological function of EPCs,we first detect the effect of STC1 on Akt and e NOS expression and phosphorylation.Then we detect changes in rhSTC1-induced effects on the EPCs proliferation and migration when PI3K/Akt/e NOS signal pathway was inhibited by PI3 K inhibitor LY294002 and e NOS inhibitor L-NAME.4.1 EPCs were treated with rhSTC1 and silencing STC1 by siRNA to detectexpression and phosphorylation ofthe major component of PI3K/Akt/eNOS signal pathwayTotal protein of EPCs was extracted when EPCs were treated with rh STC1 for 24 h or STC1 si RNA for 48 h,Western blot was performed to detect expression and phosphorylation of Akt and e NOS.4.2 The effects on rhSTC1-induced EPCs proliferation and migration when PI3K/ Akt/ e NOS signaling pathway was inhibitedEPCs was pretreated with PI3 K inhibitor LY294002 and e NOS inhibitor L-NAME for 30 min before the treatment of 50?g/ml rh STC1.Then the changes of EPCs proliferation and migration were detected.Results:1.STC1 expressed in spleen-derived EPCs1.1 Identification of EPCs1.1.1 The morphological featuresof EPCsUnder the microscope,the isolated rat spleen-derived EPCs cultured for 2 days were oval,transparent and good refractive.When cultured for 4 days,EPCs became larger and oval or short spindle,triangle.No cell protrusions and no obvious cell colonies were seen.The EPCs became larger in volume in 7days,and were elliptic,long shuttle,with a small number of cell processes.The cell body was transparent,and the refraction weakened while contour was enhanced.1.1.2 Fluorescence double-dye identificationSplenic primary mononuclear cells were isolated and cultured for 5-7 days,then were incubated with Dil-ac LDL/FITC-UEA-1 dye and observed under laser confocal microscope.Cells with red fluorescent had the ability to absorb Dil-ac LDL;Green fluorescent meant cells could combine with FITC-UEA-1.In the image fusion,double-dyed cells were the EPCs.Double-dye positive cells were counted at random fields(n = 5),and the percentage was 88.5%±5.2%.1.2 RT-PCR detected the mRNA level of STC1 in EPCsThe STC1 mRNA was detected by RT-PCR in EPCs.1.3 Western blot detected the expression of STC1Total protein was extracted from EPCs,and STC1 protein was measured by western blot.2.Exogenous rh STC1 promoted the EPCs proliferation and migration in a dose-dependentmanner.When EPCs were treated with exogenous rh STC1 at different concentrations(20?50?100 ?g/ml)for 24 h,the proliferation and migration of EPCs were increased in a dose-dependentmanner.3.Silencing endogenous STC1 by si RNA inhibited proliferation and migration of EPCsThe proliferation and migration abilityof EPCs were reduced when STC1 was knocked down by si RNA for 48 h.4.Exogenous rh STC1 increased the phosphorylation levels of Akt and e NOS of EPCsDifferent concentrations of exogenous rhSTC1(20 ? 50 ? 100 ?g/ml)increased phosphorylation levels of Akt and e NOS of EPCs.5.Silencing endogenous STC1 of EPCs decreased the phosphorylation levels of Akt and eNOSWhen EPCs were treated with si RNA to inhibit endogenous STC1 expression,the phosphorylation levels of Akt and e NOS were decreased6.Blocking PI3K/Akt/e NOS signaling pathways decreased rh STC1-induced EPCs proliferation and migrationWhen EPCs were pretreated with PI3 K inhibitor LY294002 or e NOS inhibitor L-NAME before the treatment of rhSTC1,rh STC1-induced proliferation and migration were decreased significantly.Conclusions:1.STC1 mRNA and protein were expressed in rat spleen-derived EPCs.2.Exogenous rh STC1 promoted EPCs proliferation and migration in a dosedependentmanner.3.Silencing endogenous STC1 by si RNA inhibited the proliferation and migration of EPCs.4.Activation of PI3K/Akt/eNOS signaling pathway was involved in STC1-induced proliferation and migration of EPCs.