Effects Of Celery Glycosides And 3 - Methoxy Cateolin On Ang Ⅱ - Induced Oxidative Damage In HUVECs

In this paper, the angiotensin Ⅱ (Ang Ⅱ) was used to induce human umbilical vein endothelial cells (HUVECs) to establish oxidative damage model so as to simulate hypertension model in vitro. The repair capacity of apiin and 3’-methoxy apiin evaluated by investigating cell morphology, cell activity, microstructure, apoptosis and detecting cell function markers the content of malondialdehyde (MDA) and nitric oxide (NO), the activity of nitric oxide synthase (NOS) and superoxide dismutase (SOD). The influence of apiin for the signal path of extracellular protein kinase (ERK1/2) was researched by real-time fluorescent quantitative PCR (real-time PCR) and protein imprinting method (Western Blot). The repair approach of apiin in vascular endothelial cells was clarified. This paper provide the theoretical basis for celery flavonoid on adjusting blood pressure and the suggestions for hypertension patients on daily meals. The main experimental results and conclusions are as follows:(1)HUVECs were used as model cells, Ang Ⅱ as an inducer, through investigating the concentration of the inducer and the induction time effecting on HUVECs oxidative stress injury to choose the concentration of the inducer and the induction time. The experiment showed that Ang Ⅱ could significantly induce oxidative damage of endothelial cells, mainly for cell morphology changed, organelles and cell membrane structure damaged, cell activity decreased, apoptosis rate increased, and lipid peroxidation increased, meanwhile the secretion function of cells decreased, such as NO release rate, the activity of eNOS and SOD. Results showed that 1 μmol/L Ang Ⅱ induced 12 h could be successful set up HUVECs oxidative damage model, at this time, the cell activity was 65.11%, NO content was 43.57 μmol/L, TNOS activity was 6.99 U/mgprot, eNOS activity was 1.89 U/mgprot, MDA content was 7.46 nmol/mgprot, SOD activity was 144.88 U/mgprot, cell apoptosis rate was 41.5%. Microstructure showed that nuclear membrane shrivel, nucleolus disappeared, intracytoplasmic appear a large number of air bubbles, mitochondria changed small or swollen, some rough endoplasmic reticulum expansion, and lost part of ribosome, endoplasmic reticulum expansion, but the cells did not collapse, still maintained cell structure.(2)Apiin and 3’-methoxy apiin were used as the research objects, through the same technology to inspection of the repair of the model. Results showed that both could increase the cell vitality, increase the NO release quantity, scavenging free radicals, reduce the production of peroxide in the cell, reduce the cells apoptosis rate, repair organelles and cell membrane structure, etc, that explain both have some repaire effect on the damage of endothelial cells, and have a certain concentration of dependencies. But experiments showed that apiin repair effection was greater than 3’-methoxy apiin. Specific results as follows:50 μmol/L apiin repair 12 h, cell vitality was 79.07%, NO content was 139.22 μmol/L, TNOS activity was 26.32 U/mgprot, eNOS activity was 16.32 U/mgprot, MDA content was 5.47 nmol/mgprot, SOD vitality was 51.51 U/mgprot, apoptosis rate dropped to 14.7%. Microstructure showed basic complete cell nucleolus, intracytoplasmic vacuoles, rough endoplasmic reticulum and ribosome number no less, no obvious pathological changes. This explain apiin in the body could be through the above ways to lower blood pressure, and have a certain concentration of dose dependent effect.50 μmol/L 3’-methoxy apiin repair 12 h, cell vitality was 64.50%, NO content was 78.88μmol/L, TNOS vitality was 19.94 U/mgprot, eNOS activity was 12.41 U/mgprot, MDA content was 7.93 nmol/mgprot, SOD vitality was 44.32 U/mgprot, apoptosis rate dropped to 41.8%. Microstructure showed that the decrease in the number of organelles, and there are still some mitochondria swollen. This suggested that 3’-methoxy apiinalso has the function of relieving blood pressure symptoms in a certain extent, but the effect was inferior to apiin. So we chose 50 μmol/L apiin and repaired 12 h to study the role of signaling pathways of ERK1/2.(3)ERK1/2 signaling pathway is one of the signaling pathways which associated with hypertension. The experiment showed that apiin mainly inhibiting mRNA expression of ERKl and ERK2 and reduced by 7.84% and 22.28% respectively. Phosphorylation protein expression of ERK1 and ERK2 reduced by 30.91% and 11.33% respectively. Which explained apiin regulating blood pressure by inhibiting the mRNA and protein expression of ERK1 and ERK2.The experiment also found apiin can restrain mRNA and protein expression of the downstream products ACE, thus reduced the damage of cells, at the same time, apiin can activate mRNA and protein expression of eNOS to increase the release of NO, so as to repair the injury of endothelial cells. The effect after join ERK1/2 specific inhibitor U0126 is more obvious.