[Effect of alpha-zearalanol upon the expression of HO-1 gene and the cytosolic free calcium level in tumor necrosis factor alpha-stimulated human endothelial cell].
OBJECTIVE
To investigate the effects of alpha-zearalanol on the expression of heme oxygenase-1 (HO-1) gene and cytosolic free calcium level in the tumor necrosis factor alpha (TNF-alpha)-stimulated human umbilical vein endothelial cell (HUVEC) and the mechanisms involved.


METHODS
The siRNA expression vector for p47(phox) was constructed and used to block the NADPH oxidase in the HUVEC. The intracellular ROS production was detected by using 2, 7-dichlorofluorescin diacetate as probe. The mRNA expression of the HO-1 was determined by semiquantitative RT-PCR and the protein expression was measured by immunocytochemistry analysis. The level of cytosolic free calcium was determined by using Fluo-3/AM as probe with laser confocal microscope.


RESULTS
TNF-alpha stimulation caused ROS output increased by 155% of control (228 +/- 51 vs 89 +/- 24, P < 0.05); alpha-zearalanol was able to reduce the production of ROS in a dose-dependent manner. Knock down of the p47(phox) subunit for NADPH oxidase by siRNA abolished the production of ROS. TNF-alpha stimulation caused HO-1 mRNA increased by 145% of control (0.88 +/- 0.10 vs 0.36 +/- 0.11, P < 0.01), and also obviously increased HO-1 protein expression; alpha-zearalanol inhibited the expression of HO-1 mRNA in a dose-dependent manner; Pretreatment with alpha-ZAL and p47(phox) siRNA both attenuated TNFalpha-induced HO-1 protein expression. The treatment of TNF-alpha for 24 hours up-regulated cytosolic free calcium level by 179% (107.3 +/- 4.9 vs 38.5 +/- 0.6, P < 0.01); Pretreatment with alpha-ZAL and p47(phox) siRNA depressed TNFalpha-induced cytosolic free calcium level by 46% (58.5 +/- 0.3 vs 107.3 +/- 4.9, P < 0.01) and 57% (46.3 +/- 2.1 vs 107.3 +/- 4.9, P < 0.01) respectively.


CONCLUSIONS
ROS only partly mediated HO-1; expression in the TNF-alpha-stimulated HUVEC; alpha-ZAL has a potent inhibitory effect on the HO-1 expression and cytosolic free calcium level in the TNF-alpha-stimulated HUVEC, mainly through the inhibition of ROS generation derived from NADPH oxidase.
Yu
Xiao-hong
XH
Department of Cardiology, First Affiliated Hospital, Dalian Medical University, Dalian 116011, China.
Liu
Jun
J
Zhang
Yan
Y
Wang
Xiao-ming
XM
D002118:Calcium / Q000378:metabolism; D002478:Cells, Cultured; D042783:Endothelial Cells / Q000187:drug effects* / Q000378:metabolism*; D051547:Heme Oxygenase-1 / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D019255:NADPH Oxidases / Q000378:metabolism; D019897:Periplasm / Q000737:chemistry; D034741:RNA, Small Interfering / Q000378:metabolism; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D015029:Zeranol / Q000494:pharmacology*
D034741:RNA, Small Interfering; D014409:Tumor Necrosis Factor-alpha; D015029:Zeranol; D051547:Heme Oxygenase-1; D019255:NADPH Oxidases; D002118:Calcium