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1.
Journal of Zhejiang University. Science. B ; (12): 348-365, 2021.
Artigo em Inglês | WPRIM | ID: wpr-880743

RESUMO

This study probed the protective effect of recombinant

2.
Chinese Journal of Microbiology and Immunology ; (12): 432-439, 2019.
Artigo em Chinês | WPRIM | ID: wpr-756218

RESUMO

Objective To study the influences on the production of major inflammatory cytokines after co-culturing macrophages with human umbilical vein endothelial cells ( HUVECs) that were infected with dengue virus type 2 (DENV-2). Methods Density gradient centrifugation was used to isolate periph-eral blood mononuclear cells ( PBMC) from concentrated human leukocytes. Adherent monocytes in culture flasks were obtained and stimulated with macrophage colony-stimulating factor ( M-CSF) to prepare macro-phages. The purity of CD14+CD11b+ cells was measured by flow cytometry. Changes in the expression of NS1 at mRNA level in HUVECs were detected by real-time PCR following DENV-2 infection. DENV-2-in-fected HUVECs were co-culture with macrophages in Transwell chambers. A control group was set up by pre-treating HUVECs with sphingosine-1-phosphate (S1P) type 1 (S1P1)-specific receptor agonist CYM-5442 for 24 h to remove the drug before infection and then co-culturing the infected cells with macrophages. Real-time PCR was used to detect the expression at mRNA level of IL-6 and IL-8 in HUVECs and IL-6, IL-8, TNF-α and IL-1β in macrophages. A double-antibody sandwich ELISA was used to detect the expression of above cytokines in culture supernatants. Results After HUVECs were infected with DENV-2, expression of NS1 gene at mRNA level gradually increased to the peak at 24 h (2. 66±0. 53, P<0. 05) and then de-creased. The purity of macrophages detected by flow cytometry was (89. 16±2. 07) %. Expression of IL-6 and IL-8 at mRNA level in DENV-2-infected HUVECs was up-regulated. The peak values reached at 24 h of IL-6 and IL-8 expression were 16. 10±0. 17 and 29. 76±0. 58, while the expression levels at 24 h in the un-infected group were 1. 46±0. 67 and 1. 60±0. 54, respectively. Expression of IL-6, IL-8, TNF-αand IL-1βat mRNA level in DENV-2-infected macrophages was increased significantly. The levels of IL-6, IL-8, TNF-αand IL-1β expression at 24 h were 45. 82±3. 72, 52. 34±1. 69 (12 h), 8. 94±1. 75 and 30. 96±1. 44 in the infected macrophages, and 1. 16±0. 22, 1. 15±0. 21, 1. 11±0. 09 and 1. 47±0. 31 in the uninfected group. Expression of these cytokines was decreased at every time points after co-culturing of DENV-2-infec-ted HUVECs with macrophages, but still significantly higher than that in the uninfected group. In the co-cul-ture group with DENV-2 infection, CYM-5442 pretreatment significantly decreased the expression at mRNA level of IL-6 and IL-8 in HUVECs (P<0. 01) and that of IL-6, IL-8, TNF-αand IL-1βin macrophages (P<0. 01). Conclusions DENV-2 could infect primary HUVECs, and then activate macrophages to promote the secretion of large amounts of IL-6, IL-8, TNF-αand IL-1β. Moreover, the activated macrophages could reduce the production of inflammatory cytokines in HUVECs to a certain extent.

3.
China Journal of Chinese Materia Medica ; (24): 4311-4316, 2018.
Artigo em Chinês | WPRIM | ID: wpr-775342

RESUMO

The aim of this paper was to observe the effect of gambogenic acid on angiogenesis of lung cancer and its preliminary mechanism. After culturing lung adenocarcinoma A549 cells, the conditioned medium was treated with gambogenic acid and then used to culture human umbilical vein endothelial cells (HUVECs) to establish the indirect contact cell co-culture system. A two-dimensional culture model of HUVEC was established with matrigel to observe the effect of gambogenic acid on angiogenesis. DAPI staining was used to observe the morphological changes in HUVEC cells after treatment with gambogenic acid under the fluorescence microscope. Annexin V-FITC/PI staining and flow cytometry analysis were used to determine gambogenic acid's effect on HUVEC cell apoptosis rate. The protein expressions of PI3K, p-PI3K, Akt, p-Akt were measured by Western blot. PTEN-siRNA was transfected into cells, and RT-PCR was used to detect the expression levels of PI3K and Akt genes. Gambogenic acid can significantly inhibit angiogenesis, and its inhibitory effect was dose-dependent. DAPI staining showed apoptotic morphological features of HUVEC cells under fluorescence microscope. Annexin V-FITC/PI staining showed that gambogenic acid induced apoptosis in HUVECs. The results of Western blot showed that the expressions of p-PI3K and p-Akt protein were down-regulated with gambogenic acid, while the expressions of PI3K and Akt protein was insignificant. The results of RT-PCR indicated that the expressions of PI3K and Akt protein were up-regulated by PTEN siRNA. Gambogenic acid can inhibit angiogenesis in lung cancer in vitro, and the mechanism of inhibiting angiogenesis may be related to the PI3K/Akt signaling pathway.


Assuntos
Humanos , Células A549 , Apoptose , Técnicas de Cocultura , Células Endoteliais da Veia Umbilical Humana , Neoplasias Pulmonares , Tratamento Farmacológico , Patologia , Neovascularização Patológica , Patologia , PTEN Fosfo-Hidrolase , Genética , Fosfatidilinositol 3-Quinases , Metabolismo , Proteínas Proto-Oncogênicas c-akt , Metabolismo , Transfecção , Xantenos , Farmacologia
4.
China Journal of Chinese Materia Medica ; (24): 3564-3571, 2017.
Artigo em Chinês | WPRIM | ID: wpr-335818

RESUMO

This study focuses on the protective effect of germacrone on human umbilical vein endothelial cells(HUVECs) damaged by H2O2-induced oxidative stress and its possible mechanisms. The oxidative damage model was established by using 500 μmol•L⁻¹ H2O2 to treat HUVECs for 3 hours, and then protected with different concentrations of germacrone for 24 hours. The effect of germacrone on cell viability of HUVECs damaged by H2O2 was detected by MTT. The contents of PGI2, TXB2, ET-1, t-PA, PAI-1, TNF-α and IL-6 were detected by ELISA. The content of NO was detected by using nitrate reductase method. Colorimetry was used to detect NOS and GSH-Px. The contents of MDA, SOD and LDH were detected by TBA, WST-1 and microplate respectively. Apoptosis was observed by Hoechst 33258 fluorescent staining. The mRNA expressions of Bax, Bcl-2 and Caspase-3 in cells were detected by RT-PCR. The results showed that the cell damage rate was 52% after treated with 500 μmol•L⁻¹ H2O2 for 3 hours. The cell activity was increasing with the rise of germacrone concentration within the range of 20-200 mol•L⁻¹. Compared with normal group, the contents of PGI2, NO, T-NOS, t-PA, SOD, GSH-Px and Bcl-2 mRNA expressions were lower after damaged with H2O2. The contents of PAI-1, ET-1, IL-6, TNF-α, TXB2, LDH, MDA, Bax mRNA and Caspase-3 mRNA expressions were increased. Compared with model group, the contents of PGI2, NO, T-NOS, t-PA, SOD, GSH-Px and Bcl-2 mRNA expressions were increased after treated with germacrone. The contents of PAI-1, ET-1, IL-6, TNF-α, TXB2, LDH, MDA, Bax mRNA and Caspase-3 mRNA expressions were lower after treated with germacrone. According to Hoechst 33258 fluorescence staining, compared with normal group, the cell membrane and the nucleus showed strong dense blue fluorescence, and the number of cells significantly decreased in model group. Compared with model group, blue fluorescence intensity decreased in drug group. The above findings demonstrate that germacrone may improve the effect on HUVECs damaged by H2O2-induced oxidative stress by resisting oxidation and inhibiting cell apoptosis.

5.
Chinese Journal of Natural Medicines (English Ed.) ; (6): 8-14, 2014.
Artigo em Inglês | WPRIM | ID: wpr-812316

RESUMO

AIM@#The compound B19 (C21H22O5) is a newly synthesized, mono-carbonyl analog of curcumin that has exhibited potential antitumor effects. This present study was performed to identify the anti-angiogenic activity of this compound.@*METHODS AND RESULTS@#B19 inhibited migration and tube formation of human umbilical vein endothelial cells, and arrested microvessel outgrowth from rat aortic rings. In addition, B19 suppressed the neovascularization of chicken chorioallantoic membrane. Mechanistic studies revealed that B19 suppressed the downstream protein kinase activation of vascular endothelial growth factor (VEGF) by decreasing phosphorylated forms of serine/threonine kinase Akt, extracellular signal-regulated kinase, and p38 mitogen-activated protein kinase, with or without stimulating vascular endothelial growth factor (VEGF).@*CONCLUSIONS@#B19 exerted anti-angiogenic activity in vitro and ex vivo, which suggests that it merits further investigation as a promising anticancer angiogenesis compound.


Assuntos
Animais , Humanos , Ratos , Inibidores da Angiogênese , Química , Farmacologia , Aorta , Metabolismo , Movimento Celular , Curcumina , Farmacologia , MAP Quinases Reguladas por Sinal Extracelular , Genética , Metabolismo , Células Endoteliais da Veia Umbilical Humana , Biologia Celular , Metabolismo , Técnicas In Vitro , Ratos Sprague-Dawley , Fator A de Crescimento do Endotélio Vascular , Genética , Metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular , Genética , Metabolismo
6.
Chinese Pharmacological Bulletin ; (12)2003.
Artigo em Chinês | WPRIM | ID: wpr-557433

RESUMO

Aim To investigate the protecting role and mechanisms of crude flavones of pueraria loblata(FP) on human umbilical vein endothelial cells(HUVEC) in high glucose medium.Methods ①HUVECs were incubated in 20% FCS RPMI-1640 culture medium containing 0,5,10,30,60,90,120 mmol?L~(-1) glucose for 48 hours.The proliferation of HUVEC was analyzed with MTT method(490 nm).② HUVECs were incubated in 20% FCS RPMI-1640 culture medium containing 0 or 30 mmol?L~(-1) glucose,either alone or in the presence of FP(final concentration 10~(-3),10~(-4),10~(-5),10~(-6) mol?L~(-1) respectively) for 48 hours.At the same time,30 mmol?L~(-1) mannitol was added.The proliferation of HUVEC was analyzed using MTT method.The cell cycle of HUVEC was analyzed using flowcytometry.The contents of SOD,NO and ICAM-1 in HUVEC supernatant were measured using test kits.Results HUVEC in 5 mmol?L~(-1) glucose medium proliferated well,but the proliferation of HUVEC in 10,30,60,90 and 120 mmol?L~(-1) glucose media was disturbed,showing a negative correlation.Proliferation of HUVEC was inhibited in 30 mmol?L~(-1) glucose,compared with that in 0 mmol?L~(-1) glucose and 30 mmol?L~(-1) mannitol(P

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