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JOURNAL ARTICLE
RESEARCH SUPPORT, NON-U.S. GOV'T
[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].
Zhonghua Yi Xue za Zhi [Chinese medical journal] 2009 December 16
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.
CONCLUSION: 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.
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.
CONCLUSION: 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.
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