| Objective: Aim To investigate the effects of soluble epoxide hydrolase inhibitor(sEHi)12-(3-adamantan-1-y1-ureido)-dodecanoic acid(AUDA)in regulating the proliferation,apoptosis and Akt phosphorylation(p-Akt)of endothelial progenitor cells(EPCs)in peripheral blood of patients with carotid stenosis(CS)levels.Methods: ⑴ Enrolling 40 in-patients of CS and 30 healthy people,all the enrolled people conducted Digital Subtraction Angiography(DSA).⑵ Exsanguinating 20 ml arterial blood from the femoral artery of the enrolled people in the procedures of cerebrovascular DSA.The mononuclear cells were isolated by density gradient centrifugation and were seeded at a density of 5×106/pole in the 24-well culture plates coated by human fibronectin.Then the mononuclear cells were incubated in 37℃,5% CO2 incubator.After 4 days of culture,the non-adherent cells were washed and cultured for 7 days for experimental use⑶ Identification of endothelial progenitor cells:The expression of CD34 and CD133 was detected by flow cytometry,the take in of acetylated low density lipoprotein(Dil-acLDL)and binding to Fluorescein isothiocyanate Ulex europaeus agglutinin(FITC-UEA-I)ability was detected by laser scanning confocal microscope(LSCM).⑷ MTT assay and Annexin/PI double staining were used to detect the proliferation and apoptosis differenceof EPCsin patients with carotid artery stenosis and the control group.⑸ The effects of AUDA(0 μmol /L,0.1μmol /L,1μmol /L,10μmol /L)on proliferation and apoptosis of CS group were observed by MTT assay and Annexin/PI double staining method.⑹ MTT assay,Annexin/PI double staining and Western blot were used to detect the proliferation、apoptosis and the expression of p-Akt in EPCs treated with AUDA or the PI3K/Akt blocking agent LY294002.Results: ⑴ Freshly isolated peripheral blood mononuclear cells were round,refraction,and suspended in the culture medium,cultured to 4 days,the cells became larger,adherent growth,short spindle,Washing non adherent cells with PBS,cultured to 7 days,EPCs showed a large number of spindle shaped,and showed colony like growth.⑵ Flow cytometry showed that both CD34 and CD133 molecules were expressed on the surface of EPCs.Under the laser confocal microscope,EPCs can not only display the red fluorescence after Dil-ac LDL uptake,but also display the green fluorescence after binding to FITC-UEA-I.⑶ Compared with the control group,the EPCs from CS patients’ s proliferation decreased,apoptosis increased(P<0.05);compared with the untreated group(0 μmol/L),AUDA group showed dose-dependently increase the proliferation of EPCs,reduce the degree of apoptosis.⑷ Compared with the AUDA group,AUDA+LY294002 group and LY294002 group’s EPCs proliferation increased,apoptosis decreased(P<0.05),AUDA+LY294002 group compared with LY294002 group,EPCs’ s proliferation increased and apoptosis decreased(P<0.05),untreated group and AUDA+LY294002 group,EPCs proliferation,apoptosis degree has no significant difference(P>0.05).⑸ The level of p-Akt in the control group was higher than that in untreated group(P<0.05),the AUDA group’s p-Akt degree was higher than that in AUDA+ LY294002 group,LY294002 group(P<0.05).The level of p-Akt in the untreated group and AUDA+LY294002 group was no significant difference(P>0.05).Conclusion: ⑴ Compared with the control group,the proliferation of EPCs in peripheral blood of CS patients decreased and the degree of apoptosis increased.⑵ AUDA could promote the proliferation and inhibit the apotosisof EPCs in CS patients in a dose-dependent manner.⑶ AUDA partially activates PI3K/Akt signaling pathway to promote EPCs proliferation and inhibit apoptosis.⑷ Confirmed that AUDA has the role of anti-carotid atherosclerosis,which is expected to become a class of treatment of carotid artery stenosis of new drugs. |
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