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Objective: To prepare the cationic solid lipid nanoparticles (Que/mR150 SLNs) co-loaded with quercetin (Que) and microRNA-150 (mR150) and investigate the preparation process, then assess its in vitro release, cell uptake capacity and safety of ocular administration. Method: First, thin-film dispersion method was used to prepare quercetin-encapsulated cationic solid lipid nanoparticles (Que-SLNs), and the preparation process was optimized based on the particle size, PDI and encapsulation rate; Using electrostatic adsorption method to co-load mR150 in nanoparticles (Que/mR150 SLNs), and the adsorption efficiency of the miRNA by the nanoparticles was examined by agarose gel electrophoresis experiment; The in vitro release performance of quercetin in Que/mR150 SLNs was investigated; The effect of Que/mR150 SLNs on the proliferation of HUVEC of human umbilical vein endothelial cells was measured by MTT method, and fluorescence labeling was used to observe their uptake in HUVEC; And the irritancy of Que/mR150 SLNs to rabbit eyes was examined by pathological tissue sections of rabbit eyes. Result: After process optimization, the cationic nano Que-SLNs had good drug-loading, particle size distribution and stability. The appearance of the cationic nano-Que-SLNs was spherical, and it could be kept stable for two months. The quercetin encapsulation rate was (85.25 ± 1.29)%, the drug load was (1.67 ± 0.02)%, the average particle size was (110.00 ± 2.10) nm, and the Zeta potential is (53.2 ± 5.12) mV; The in vitro drug release results showed that the release of quercetin in the nanoparticles was slow, and the cumulative release amount within 48 h was about (80.69 ± 1.29)%; When the mass ratio of dioctadecyl dimethyl ammonium bromide to mR150 (DDAB/RNA) of different cationic materials was 6:1, the cationic solid lipid nanoparticles basically encapsulated mR150 completely with little effect on its particle size and potential. MTT experiments showed that blank nanometer mass concentration of 50-150 mg/L had no significant proliferation toxicity on HUVEC cells; Cell uptake experiments showed that Cy5 and coumarin-6 dual fluorescently labeled and co-loaded nanometers could effectively enter HUVEC cells; Pathological tissues of rabbit eyes showed that Que/mR150 SLNs had no obvious damage to the eyes. Conclusion: The preparation process of Que/mR150 SLNs solid lipid nanoparticles is stable and reliable, with good reproducibility, storage stability and good biological safety, which is conducive to the efficient delivery of quercetin and mR150 into HUVEC cells, which provides the ideas for the treatment of diseases related to angiogenesis
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Objective: To determine the expression of TAZ and its role in angiogenesis in gastric carcinoma. Methods: Immunohistochemical staining was performed to investigate the expression of TAZ and to determine whether a direct relationship exists between TAZ and β-catenin. Transfection with TAZ overexpression plasmid in MKN28 cells was conducted to induce exogenous expression of TAZ and a TAZ knockdown plasmid was transfected into MGC803 cells to reduce TAZ levels. The effects on endothelial cell formation, proliferation, and migration were determined by Matrigel three-dimensional culture, MTT proliferation assay and Transwell migration assay. In addition, the expression of TAZ and β-catenin in transfected gastric cancer cells was detected by Western blot. Results: Immunohistochemistry showed that TAZ protein was expressed in 64 of 150 gastric cancer sample tissues (43%), TAZ was localized in the nucleus, and its expression was associated with tumor grade, TNM stage, metastasis, and microvessel density (MVD) (P<0.05). In addition, the expression frequency of β-catenin in the TAZ positive group was 67.2%, which was significantly higher than that in the TAZ negative group, and the expression of TAZ was positively correlated with β-catenin. After transfection, TAZ overexpression increased the expression of β-catenin and enhanced HUVECs tube formation, proliferation, and migration. In the MGC803 cells transfected with the knockdown plasmid, β-catenin levels were decreased and HUVECs motility was inhibited. Conclusions: TAZ may promote angiogenesis in gastric cancer by promoting β-catenin expression.
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Objective To study the protective effect of Panax notoginseng saponins(PNS)and its components Rg1 and Rb1 on oxygen-glucose deprivation/reoxygenation(OGD/Reox)-induced tight junction damage. Methods Anaerobic box were used to induce OGD in HUVEC cells for 6 h followed by reoxygenation for 24 h. Transepithelial/endothelial electrical resistance(TEER)and cell permeability were detected,immunefluorescence was used to observe the ZO-1 and claudin-5 protein expression. Results PNS 20,40 mg/L and ginsenoside Rb1 significantly inhibited the OGD/Reox-induced decreased tight junction resistance,and the increased cell permeability(P< 0.05). PNS 20,40 mg/L and ginsenoside Rb1 partly restored the inter-cellular tight junctions which were regularly arranged on the cell membrane, and the cells displayed cobble stone-like arrangement. Conclusions PNS ameliorates ischemia-induced vascular endothelial cell tight junction damage via MMP-9 and VEGF/VEGFR2 signaling pathway. Rb1 is one of the effective monomer components.
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To investigate the protective effects and mechanism of Polygonum orientale flower extract on H₂O₂-induced oxidative damage of human umbilical vein endothelial cells (HUVEC), H₂O₂ was used to induce the oxidativestress damage on HUVEC cells and efforts were made to screen the low, medium and high drug concentrations of P.orientale flower extract. Cell viability was detected by the MTS assay. The content of lactate dehydrogenase (LDH) and malondialdehyde (MDA), and the activities of superoxidedimutase (SOD) and catalase (CAT) were detected by biochemical kits. The mRNA and protein levels of Bax, Bcl-2 were detected respectively by quantitative real time polymerase chain reaction (qRT-PCR) and Western blot. The protein level of cleaved caspase-3 was detected by Western blot. According to the results, the viability of HUVEC cells was reduced to around 55% after being treated with 120 μmol·L⁻¹ H₂O₂ for 0.5 h. Treatment of H₂O₂ also could increase LDH leakage rate and MDA content and attenuate the activities of SOD and CAT, up-regulate the expression level of Bax and cleaved caspase-3, and down-regulate the expression level of Bcl-2. As compared with H₂O₂ model group, P.orientale flower extract of 50-200 mg·L⁻¹ could increase the viability of HUVEC cells, reduce LDH release and MDA content, enhance the activities of SOD and CAT, down-regulate pro-apoptotic protein cleaved caspase-3 and Bax, and up-regulate apoptosis inhibitory protein Bcl-2. In summary, P.orientale flower extract showed a protective effect on H₂O₂-induced HUVEC cells injury, which may result from enhancing the cell capability of clearing the oxygen free radial, decreasing the production of lipid peroxidation and inhibiting apoptosis.
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OBJECTIVE Angiogenesis therapy has attracted interest as a potential treatment for hepatocellular carcinoma (HCC). In this study, we investigated the anti-proliferative activities and anti-angiogenesis effects of saikosaponins (SS)-b on hepatocellular carcinoma (HCC) and its regulation on VEGF/ERK/HIF-1α signal pathway. METHODS H22 hepatoma-bearing mice model and HepG-2 cells were used to study the anti-tumor and anti-angiogenesis effects of SS-b in vivo and in vitro. Pathological change of tumor tissue was observed by HE staining, the microvascular changes were detected by immunohistochemical method. The effects of SS-b on angiogenesis were examined by using the chick embryo chorioallantoic membrane (CAM) model. The effects of SS- b on proliferation, migration and invasion were investigated by MTT assay, scratch wound healing assay and transwell assay inhuman umbilical vein endothelial cell (HUVEC) and HepG2 cells in vitro. Vascular endothelial growth factor (VEGF), matrix metalloproteinase-2/9(MMP-2/9), hypoxia-inducible factor-1α (HIF-1α) expression and the phosphorylation of extracellular regulated kinase(ERK) were analyzed using RT-PCR and Western-blot. RESULTS SS-b effectively inhibited the tumor growth of H22 mice in vivo. The inhibitory rate of tumor was 49.1%, 50.7%, 66.1% in SS-b 5, 10 and 20 mg·kg-1 group respectively. HE staining results showed that SS-b induced tumor necrosis and nuclear dissolution in H22 mice. Moreover, SS-b also reduced the number of microvessels of tumor tissue in H22 mice significantly and suppressed the angiogenesis of CAM induced by b-FGF. SS-b had an obvious inhibitory effect on cell proliferation, migration and invasion of HUVEC cells and HepG-2 cells. These effects were associated with down-regulation of the expression of MMP2/9 and suppression of VEGF/ERK/HIF-1α signaling in H22 mice and Hep-G2 cells. CONCLUSION Our findings showed that SS-b exerts anti-tumor effects by inhibit?ing tumor angiogenesis via regulating VEGF/ERK/HIF-1α signal pathway in vivo and in vitro.