The Down-regulated Expression Of Caveolin-1 Plays A Potential Role In Coronary Artery Spasm In Vitro By Mediating NO Production In LPS-induced HUVECs Injury

Objective To investigate the effects of the down-regulated expression of Caveolin-1(Cav-1) in the production of nitric oxide(NO)and endothelin-1(ET-1)in primary human umbilical vein endothelial cells(HUVECs)under the environment of coronary spasm induced by lipopolysaccharide(LPS)and acetylcholine(Ach) treatment.Methods Morphology and immunofluorescence for anti-factor Ⅷ antibody to identify HUVECs. Three pairs of Cav-1 small interfering RNA(si RNA)were transfected into the HUVECS cells. Western blotting was used to detect the expression of Cav-1 protein.HUVECs were incubated in LPS of different concentrations at 0,10,25,50,75, and 100μg/m L. Cell viability was measured by CCK8 assay and the inhibition of superoxide dismutase(SOD) was measured by SOD kit. The change of [Ca2+]i was detected by using divert fluorescence microscopy loaded with Fura4-AM. Cells were divided into eight groups as follow:(1)HUVECs/si-NC;(2)HUVECs/si-NC+LPS;(3)HUVECs/si-NC+Ach;(4)HUVECs/si-NC+LPS+Ach;(5)HUVECs/si Cav-1;(6)HUVECs/si Cav-1+LPS;(7)HUVECs/si Cav-1+Ach;(8)HUVECs/si Cav-1+LPS+Ach. The NO content of cell supernatant was measured by nitrate reductase method, and the ET-1 ELISA Kits was used to test the content of ET-1.Results The primary HUVECs started to be attached to the plates after about 1h of culture, showing a typical cobblestone-like appearance under the inverted microscopy and positive immunofluorescence for anti-factor VIII antibody.The results from Westernblotting revealed that Cav-1 si RNA notably down regulated the expression of Cav-1 protein, especially si Cav-1(2).LPS-induced cell viability increased in a dose-dependent manner for 24 h.With a LPS concentration at 75μg/ml, we found a significant decrease in cell viability compared to that at other concentrations of LPS in group HUVECs/si-NC and HUVECs/si Cav-1. The SOD inhibition of HUVECs/si-NC and HUVECs/si Cav-1 group which indirectly reflects cell injury significantly decrease by 75μg/m L of LPS, when compared to that without LPS group of HUVECs/si-NC and HUVECs/si Cav-1,separately. By stimulating the HUVECs/si-NC group with Ach, the level of [Ca2+]i increased more than that HUVECs/si-NC group with LPS and Ach. Under the stimulation of Ach, the content of NO increased in injured HUVECs/si Cav-1 cells, which was higher than that in injured HUVECs/si-NC cells(P<0.05). We did not find any obvious change in the content of ET-1 among different cells groups when stimulated with Ach in injured cell.Conclusion Under the environment with LPS and Ach stimulations, the down-regulated expression of Cav-1 plays a key role in NO production, which provides a potential prevention and therapeutic target in an efficient management of coronary spasm.