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Objective: To construct the RHBDF2 gene over-expression lentivirus vector and to establish the KA. hy926 cells stably expressing RHBDF2, and to provide the evidence for the construction of RHBDF2 gene over-expression lentivirus vector and the establishment of RHBDF2 cells stably expressing RHBDF2. Methods: According to the sequence of RHBDF2 gene provided by NCBI, and the primers were designed and synthesized; the RHBDF2 gene was amplified by PCR method, and the target gene was cloned into the entry vector by Gateway cloning technology, and then subcloned into the lentivirus vector pLV [Exp]-EGFP to construct the recombinant lentivirus plasmid pLV I Exp]-EGFP-RHBDF2; the lentivirus expression vector plasmid pLV I Exp]-EGFP and the recombinant lentivirus plasmid pLV I Exp]-EGFP-RHBDF2 were co-transfected into the HEK293T cells with the virus-assisted packaging plasmids to package the lentivirus and the titer of the lentivirus was detected. The EA. hy926 cells infected with pLV [Exp]-EGFP-control were used as control group and the EA. hy926 cells infected with pLV [Exp]-EGFP-RHBDF2 were used as experiment group. The EA. hy926 cells stably expressing RHBDF2 were screened by puromycin. The fluorescent quantitative PCR (qPCR) and Western blotting methods were used to detect the expression levels of RHBDF2 mRNA and protein in the EA. hy926 cells in control group and experiment group. Results: The enzyme digestion electrophoresis and sequencing results showed that the gene sequence of the EA. hy926 cells over-expression lentivirus vector in experiment group was completely consistent with the designed and synthesized sequence. The lentivirus titer in control group was 1 X 10 TU • mL , and the lentivirus titer in experiment group was 3X 10 TU • mL . The EA. hy926 cells were successfully infected with the lentivirus under fluorescence microscope and the infection efficiency was above 95%. The qPCR detection results showed that the expression level of RHBDF2 mRNA in the EA. hy926 cells in experiment group was higher than that in control group (P'<0.01). The Western blotting results showed that the expression level of RHBDF2 protein in the EA. hy926 cells in experiment group was higher than that in control group ( P < 0. 05 ). Conclusion: The lentivirus vector over-expressing RHBDF2 is successfully constructed∗ and the EA. hy926 cell line stably up-regulating the expression of RHBDF2 is established by using pLV [Exp]-EGFP-RHBDF2 lentivirus.
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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.
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Objective: To investigate the effect and mechanism of cinnamaldehyde on the angiogenesis of diabetic retinopathy, and the effect of cinnamaldehyde on vascular endothelial growth factor (VEGF) induced proliferation, migration, tube formation and Janus kinase 2/signal transducer and activator of transcription 3 (JAK2/STAT3) pathway of EA.hy 926 cells were observed. Method:EA.hy 926 cells were divided into normal control group, model group (7 μg·L-1 VEGF), and VEGF+cinnamaldehyde group (60, 90, 120, 150 μmol·L-1). The methyl thiazolyl tetrazolium (MTT) assay and scratch test were used to observe the effect of cinnamaldehyde on the proliferation and migration of EA. hy 926 cells induced by VEGF. EA. hy 926 cells were divided into normal control group, model group (7 μg·L-1 VEGF), and VEGF+cinnamaldehyde group (90, 150 μmol·L-1). The tube formation experiment was used to observe the effect of cinnamaldehyde on the tube formation of EA. hy 926 cells induced by VEGF. EA. hy 926 cells were divided into normal control group, model group (7 μg·L-1 VEGF), VEGF+AG490 group (50 μmol·L-1), VEGF+cinnamaldehyde group (90 μmol·L-1), VEGF+cinnamaldehyde group (150 μmol·L-1), and VEGF+cinnamaldehyde group (150 μmol·L-1)+AG490 group (50 μmol·L-1). Western Blot method was used to explore the effect of cinnamaldehyde on the JAK2/STAT3 signaling pathway in EA.hy 926 cells induced by VEGF. Result:Compared with the control group, model group obviously promoted the proliferation and migration of EA.hy 926 cells(P-1) significantly suppressed VEGF-induced proliferation and migration of EA.hy 926 cells (P-1) showed an obvious inhibitory effect on the number of nodes, junctions and meshes of tubules (PPPP-1) significantly reduced the expressions of P-JAK2, P-STAT3, STAT3 proteins (P-1) obviously reduced the expressions of p-STAT3 and STAT3 proteins (PPConclusion:Cinnamaldehyde showed a significantly inhibitory effect on the proliferation, migration and tube formation of VEGF-induced EA.hy 926 cells, which was related to the inhibition of the activation of JAK2/STAT3 pathway.
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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.
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Objective To observe the mechanism of action of different extracts ofTangwang Mingmu Granules on high glucose induced VEGF and IL-1α gene and protein expressions in vascular endothelial cells.Methods Human umbilical vein endothelial cells EA.hy926 were divided into six groups: blank, high glucose,Tangwang Mingmu Granules, extract 1 (glycoside and flavonoid), extract 2 (organic acid and polysaccharides) and extract 3 (alkaloids) groups. High concentration glucose was used to establish the high glucose model in EA.hy926 cells. The expressions of VEGF and IL-1α mRNA were detected by semi-quantitative RT-PCR. The contents of VEGF and IL-1α protein were tested by ELISA.Results The gene expression and protein levels of VEGF and IL-1α were significantly up-regulated under the high glucose condition (P extract 3> extract 2.Conclusion The action intensity of glycosides and flavonoids, alkaloids, organic acids and polysaccharides on VEGF and IL-1α expression inTangwang Mingmu Granules weakens in sequence.
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OBJECTIVE:To study the inhibitory effects of berberine on EA.hy926 human umbilical vein endothelial cells (EA. hy926 cells) injury induced by particulates with no more than 2.5 μm air aerodynamic diameter in atmospheric (PM2.5),and its p38 mitogen-activated protein kinase(MAPK)signal pathway mechanism. METHODS:PM2.5 samples were collected and hatched EA.hy926 cells with concentrations of 0(blank control),20,200 and 400 mg/L for 24 h. The survival rate and apoptosis rate of cells,contents of IL-6,TNF-α and MDA,activities of SOD and LDH,protein levels of p-p38 MAPK,Bcl-2 and Bcl-2 associated X protein (Bax) were detected. The above indexes of EA.hy926 cells in blank control group,PM2.5 group (200 mg/L PM2.5), p38 MAPK pathway-specific blocker SB203580 group (20 μmol/L SB203580+200 mg/L PM2.5),berberine low-,medium- and high-concentrations groups(5,10,20 μmol/L berberine+200 mg/L PM2.5)were also determined. RESULTS:Compared with blank control,survival rate of cells,SOD activity and Bcl protein decreased after 200,400 mg/L PM2.5 hatched;apoptosis rate of cells, contents of IL-6,TNF-α and MDA,LDH activity,protein levels of p-p38 MAPK and Bax increased (P<0.05),in concentra-tion-dependent manner. Compared with PM2.5 group,survival rate of cells,SOD activity and Bcl-2 protein increased in berberine medium-,high-concentrations groups and SB203580 group;apoptosis rate of cells,contents of IL-6,TNF-α and MDA,LDH ac-tivity,protein levels of p-p38 MAPK and Bax decreased (P<0.05). CONCLUSIONS:Berberine attenuates PM2.5-induced EA. hy926 cells injury via the inhibition of p38 MAPK pathway.
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Objective To observe the effect of GPR30 agonist G1 on high glucose-induced endoplasmic reticulum stress ( ERS) in endothelial EA .hy926 cells.Methods EA.hy926 endothelial cells were divided into three groups:nor-mal control group (Con, 17.51 mmol /L glucose), high glucose (HG, 33.3 mmol /L), high glucose +G1 group (HG+G1, HG +1 μmol/L G1).The apoptosis rate of endothelial cells was measured by flow cytometry , the protein expres-sion changes of ERS related molecules Bip , IRE1, PERK and apoptotic molecules Bax , Bcl-2 were measured by Western blot, the mRNA expressions of Bip and CHOP were measured by RT-PCR assay.Results Compared with Con group , the apoptosis in HG group was significantly increased (P <0.01), Bip, IRE1, PERK and apoptotic molecule Bax were upreg-ulateded (P <0.05, P<0.01 or P <0.001), Bcl-2 downregulatted (P <0.01) and Bip mRNA, CHOP mRNA expres-sion were upregulated (P <0.001 and P<0.01).Compared with the HG group, apoptosis rate in HG +G1 group was significantly lower (P <0.05), BIP, IRE1, PERK and apoptotic molecules Ba.0 downregulated ( P <0.05 or P <0.01), Bcl-2 expressions was increased (P <0.05), Bip mRNA and CHOP mRNA expression were decreased (P<0.001 or P<0.01).Conclusion GPR30 agonist G-1 inhibits EA.hy926 ERS in endothelial cells.
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Angiogenesis, the development of new capillary vessels, has a host of clinical manifestations. The identification of agents that increase or decrease angiogenesis is of great pharmaceutical interest. Classically, in vitro angiogenesis utilizes human umbilical vein endothelial cells (HUVEC) grown in matrigel. This valid and simple method has the drawbacks that each cell population is distinct and the constraint of obtaining primary source material. Herein we utilize the established EA.hy926 endothelial cell line as our model for in vitro angiogenesis and present a novel formula to quantify endothelial cell remodeling to identify pro- and anti-angiogenic agents. Furthermore, our technique details the procedures to identify and quantify compounds that have the capacity to generate pro- or anti-angiogenic factors when given to non-endothelial cells, which we define herein as angiogenic potential. In conclusion, we propose a novel formula that we are confident accurately reflects the degree of in vitro angiogenesis allowing the quantification of prospective angiogenic compounds.
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Humanos , Inductores de la Angiogénesis/farmacología , Colágeno/farmacología , Células Endoteliales/efectos de los fármacos , Laminina/farmacología , Neovascularización Fisiológica/efectos de los fármacos , Proteoglicanos/farmacología , Línea Celular , Combinación de Medicamentos , Evaluación Preclínica de Medicamentos , Neovascularización Fisiológica/fisiologíaRESUMEN
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.