| Objective:To explore the protective effects of Atrial Natriuretic Peptide (ANP) Injection on the cultured human umbilial vein endothelial cells injury induced by hypoxia-reoxygenation.Methods:Get the cryopreservated human umbilical vein endothelial cells obtained to recover and subcultured.When cultured to the third generation, inoculated to 24 well culture plates at the same concentration, cultured for one day under the normal circumstances with the normal culture medium, the old medium changed with the prepared culture medium next day. Three experimental groups:1.the normal control group (n=6);2.hypoxia-reoxygenation group (n=6):cells were cultured in the hypoxia box for 6 hours, then reoxygenation for 6h; 3. hypoxia-reoxygenation plus different concentrations of ANP Group:0.001μg/ml (n=6);0.005μg/ml (n=6); 0.01μg/ml (n=6);0.05μg/ml (n=6);0.1μg/ml (n=6), and also hypoxia cultured for 6h and reoxygenation 6h; observed under inverted microscope in the cell morphology, cell culture supernatant obtained in each group were measured malondialdehyde (MDA), lactate dehydrogenase (LDH), nitric oxide (NO) and endothelin (ET) content, the final results of statistical analysis conducted by SPSS13.0 software.Results:1.Changes of Cell morphological:In the hypoxia-reoxygenation group, endothelial cells were irregular shape, some cells hadn't clear cell boundaries, cell gap became widened, and the cells ablate and cell debris were shown. While the Cell morphology in ANP intervention group became less deformation, the cell gap was less widened, shedding cells decereased, and with the drug concentration increasing, morphology tended to be more normal.2.The dehydrogenase (LDH) activity in hypoxia-reoxygenation group was significantly higher compared with the control group (69.35±5.66 U/L vs 31.04±3.43 U/L, P<0.01);LDH activity in drug intervention group was significantly decreased compared with hypoxic reoxygenation(P<0.01).MDA content in hypoxia-reoxygenation group was significantly higher than that in the control group (5.94±0.58nmol/ml vsl.69±0.16nmol/ml,P<0.01),but MDA content in drug intervention group was significantly lower than that in hypoxia-reoxygenation group(P<0.01),during the range of ANP concentration from 0.001μg/ml to 0.05μg/ml), MDA content in the culture medium concentration showed negative correlation with ANP concentration in the ANP group (P<0.01).Levels of intracellular SOD in hypoxia-reoxygenation group were significantly lower than that in the control group(15.74±2.17 U/Lvs 47.08±4.23U/L, P<0.01)and was significantly higher in drug treatment group than in hypoxia-reoxygenation group (P<0.01),When ANP concentration range from 0.001μg/ml to 0.05μg/ml,each sub-group correlation analysis showed medium concentration of SOD content was positively correlated with ANP concentration in ANP intervention group (P<0.01); Nitric oxide(NO) levels in cell culture medium in hypoxia group was significantly lower than that in the control group (36.81±3.78μmol/Lvs 89.78±7.01μmol/L, P <0.01),ET-1 content was significantly higher than that in the control group(1368.64±99.07pg/Lvs305.52±35.30pg/L,P<0.01);Correlation analysis showed that: ANP concentration ranged from 0.001μg/ml to 0.05μg/ml, in each sub-group of ANP intervention group, NO levels were positively correlated with ANP(P<0.01), while the concentration of ET-1 concentration was negatively correlated with ANP concentration (P<0.01).Conclusion:①Hypoxia-reoxygenation induced human umbilical vein endothelial cell shrinkage and round cell gap widened, broken cell loss, cell apoptosis.ANP made the morphological structure of the cells become normal.②ANP could reduce the concentration of LDH in culture medium, and has a protective effect on the cell membrane;ANP could reduce the MDA content in the culture medium and increased SOD content from human umbilical vein endothelial cell, thus inhibiting the oxidative damage on endothelial cells induced by hypoxia-reoxygenation.③ANP improved the content of NO and reduced the content of ET-1 after human umbilical vein endothelial cells undergoing hypoxia-reoxygenation injury, thus played important role in protection of the endothelial function.
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