ABSTRACT
Objective: To construct the sponge vector which can target microRNA-186 (miR-186), and to create a stable EA. hy926 cell line that can knockdown miR-186. Methods: The miR-186 sponge sequence was chemically synthesized and cloned into lentiviral vector FV040. Then the FV040-miR-186-sponge recombinant plasmid together with the helper plasmids were cotransfected into the HEK293T cells by using lipofectamine 2000 to package lentivirus and the viral titer was determined. The FV040-control lentivirus (designated as the control group), and the FV040-miR-186-sponge (designated as the experiment group) were used to infect EA. hy926 cells for establishing stable cell lines. The fluorescent quantitative PCR (qPCR) method was used to detect the relative expression levels of miR-186 in the EA. hy926 cells in blank group, FV040-control group and FV040-miR-186 sponge group, respectively. Results: The cloned target sequence was identical with the designed miR-186 sponge sequence. The green fluorescence protein (GFP) was observed in the infected EA. hy926 cells. The lentivirus titre of viruses in the FV040-control group was 2×108 TU middot; mL-1, and which was 6 × 108 TU middot; mL-1 in FV040-miR- 186 sponge group. The EA. hy926 cell line stably expressed miR-186-sponge was established successfully and the infection rate was as high as 95%. The qPCR results indicated that the relative expression level of miR-186 in the EA. hy926 cells in FV040-miR-186-sponge group was lower than those in blank control group and FV040-control group (P<0. 01). Conclusion: The miR-186-sponge vector is successfully constructed, and the EA. hy926-miR- 186-sponge cell line with the stably decreased expression of miR-186 is established successfully.
ABSTRACT
Aim To investigate the protective effect of allicin against EA. hy926 endothelial cell injury in-duced by PM2. 5 and the possible mechanism. Meth-ods The samples of fine particulate matter ( PM2. 5 ) were collected and made into suspension. Different concentrations of PM2. 5 ( 20 , 200 , 400 mg · L-1 ) were added to EA. hy926 cell. The viability and apop-tosis of EA. hy926 cell, the protein levels of p-ERK1/2, Bax and Bcl-2 in the EA. hy926 cell, the contents of tumor necrosis factor-α( TNF-α) , interleukin-6 ( IL-6 ) , and malonaldehyde ( MDA ) , the activities of su-peroxide dismutase ( SOD ) and lactic dehydrogenase ( LDH) in the EA. hy926 cell culture supernatant were measured by MTT assay, flow cytometry, Western blot, enzyme-linked immunosorbent assay ( ELISA ) and colorimetry, respectively. Allicin at different con-centrations(5,20,40 mg·L-1 ) or a specific inhibitor of ERK1/2 signaling pathway PD98059 ( 20 μmol · L-1 ) was added into the EA. hy926 cell to observe the effect of allicin. Results Compared with control group, PM2. 5 significantly increased the apoptosis, the contents of TNF-α, IL-6 and MDA, the activity of LDH, the protein levels of p-ERK1/2 and Bax/Bcl-2 ratio, but decreased the viability and SOD activity in the EA. hy926 cell(P<0. 05). Compared with PM2. 5 group, allicin significantly decreased the apoptosis, the contents of TNF-α, IL-6 and MDA, the activity of LDH, the protein levels of p-ERK1/2 and Bax/Bcl-2 ratio, but increased the viability and SOD activity in the EA. hy926 cell ( P <0. 05 ) . Conclusion Allicin displays a significant protective effect against EA. hy926 endothelial cell injury induced by PM2 . 5 and its mechanism may be related to the attenuations of in-flammation and oxidative stress via the inhibition of ERK1/2 pathway.
ABSTRACT
Zinc plays a protective role in anti-atherosclerosis but the clear mechanism has not been proposed yet. In the present study, we evaluated whether zinc modulates atherosclerotic markers, VACM-1 and ICAM-1 and cell viability both in endothelial cells in vitro and mouse aortic cell viability ex vivo. In study 1, as in vitro model, endothelial EA.hy926 cells were treated with TNFalpha for 5 hours for inducing oxidative stress, and then treated with Zn-adequacy (15 micrometer Zn) or Zn-deficiency (0 micrometer Zn) for 6 hours. Pro-atherosclerosis factors, VCAM-1 and ICAM-1 mRNA expression and cell viability was measured. In study 2, as ex vivo model, mouse aorta ring was used. Mourse aorta was removed and cut in ring then, cultured in a 96-well plate. Aortic ring was treated with various TNFalpha (0-30 mg/ml) and intracellular zinc chelator, N, N, N', N', -tetrakis (2-pyridylmethyl) ethylenediamine (TPEN, 0-30 microM) for cellular zinc depletion for 2 days and then cell viability was measured. The results showed that in in vitro study, Zn-adequate group induced more VCAM-1 & ICAM-1 mRNA expression than Zn-deficient group during 6-hour zinc treatment post-5 hour TNF-alpha treatment, unexpectedly. These results might be cautiously interpreted that zinc would biologically induce the early expression of anti-oxidative stress through the increased adhesion molecule expression for reducing atherosclerotic action, particularly under the present 6-hour zinc treatment. In ex vivo, mouse aortic ring cell viability was decreased as TNF-alpha and TPEN levels increased, which suggests that mouse aortic blood vessel cell viability was decreased, when oxidative stress increases and cellular zinc level decreases. Taken together, it can be suggested that zinc may have a protective role in anti-atherosclerosis by cell viability in endothelial cells and aorta tissue. Further study is needed to clarify how pro-atherosclerosis molecule expression is modulated by zinc.