Phytoestrogen α-Zearalanol attenuates homocysteine-induced apoptosis in human umbilical vein endothelial cells

Abstract

Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases. The enhanced nitrative stress plays an important role in homocysteine-induced endothelial dysfunction. Previous studies have showed that phytoestrogen α -zearalanol alleviated endothelial injury in ovariectomized hyperhomocysteinemic rats; however, the underlying mechanism remains to be clarified. This study was to investigate the effects of α -zearalanol on homocysteine-induced endothelial apoptosis in vitro and explore the possible role of nitrative stress in these effects. Results showed that homocysteine (500 μ mol/L, 24 h) induced the apoptosis of human umbilical vein endothelial cells (HUVECs) obviously, and this effect was significantly attenuated by pretreatment with α -zearalanol (10(-8)~10(-6) mol/L). Moreover, α -zearalanol downregulated proapoptotic protein Bax, upregulated antiapoptotic proteins Bcl-2 and Bcl-XL, and decreased the expression and activity of caspase-9. These findings demonstrated that α -zearalanol could effectively alleviate homocysteine-induced endothelial apoptosis, and this antiapoptosis effect might be related to the inhibition of the intrinsic pathway. Western blot indicated an enhanced 3-nitrotyrosine expression in HUVECs when challenged with homocysteine, which was attenuated by pretreatment with α -zearalanol. This result implied that inhibition of nitrative stress might play a role in the protective effect of α -zearalanol on endothelial cells. Such discovery may shed a novel light on the antiatherogenic activities of α -zearalanol in hyperhomocysteinemia.

Figure 1

Characterization of primarily cultured human umbilical vein endothelial cells (HUVECs). (a) After 5 days of feeding, cells isolated from human umbilical cord vein showed a typical, cobblestone-like morphology of endothelial cells. (b) The markers of endothelial cells (eNOS and VE-cadherin) were positive with immunofluorescence staining (×100).

Figure 2

Pretreatment with α-ZAL improved the deceased cell viability induced by homocysteine with methyl thiazolyl-tetrazolium (MTT) assay in HUVECs. (a) Treatment with different concentrations of homocysteine on HUVECs for 24 h decreased cell viability in a dose-dependent manner, which became apparent at 500 μmol/L. (b) Treatment with 500 μmol/L homocysteine on HUVECs decreased cell viability in a time-dependent manner, which became apparent at 24 h. (c) Pretreatment with α-ZAL or 17β-E₂ (10⁻⁸~10⁻⁶ mol/L) could significantly improve the decreased cell viability induced by homocysteine (500 μmol/L, 24 h), which was similar to 17β-E₂. Data were presented as mean ± SD. *P < 0.05 versus Vehicle, ^# P < 0.05 versus Hcy alone. α-ZAL: α-zearalanol; 17β-E₂: 17β-estradiol; Hcy: homocysteine.

Figure 3

Pretreatment with α-ZAL attenuated apoptosis of homocysteine-challenged HUVECs—TUNEL fluorescence staining. The number of TUNEL-positive cells was significantly increased after treatment with 500 μmol/L homocysteine for 24 h. Pretreatment with α-ZAL could attenuate the increased number of TUNEL-positive cells (only showing the picture of the highest concentration 10⁻⁶ mol/L). Data were presented as mean ± SD. *P < 0.05 versus Vehicle, ^# P < 0.05 versus Hcy alone. α-ZAL: α-zearalanol; 17β-E₂: 17β-estradiol; Hcy: homocysteine.

Figure 4

Pretreatment with α-ZAL attenuated apoptosis of homocysteine-challenged HUVECs—caspase-3/cleaved caspase-3 expression (Western blot). The expression of caspase-3 and cleaved caspase-3 was significantly increased after treatment with 500 μmol/L homocysteine for 24 h. Pretreatment with α-ZAL could attenuate this effect. (a) Expression of caspase-3. (b) Expression of cleaved caspase-3. Data were presented as mean ± SD. *P < 0.05 versus Vehicle, ^# P < 0.05 versus Hcy alone. α-ZAL: α-zearalanol; 17β-E₂: 17β-estradiol; Hcy: homocysteine.

Figure 5

Pretreatment with α-ZAL reduced the expression and activity of caspase-9 in homocysteine-challenged HUVECs. The expression and activity of caspase-9 were significantly increased after treatment with 500 μmol/L homocysteine for 24 h. Pretreatment with α-ZAL could attenuate this effect. (a) Expression of caspase-9 (Western blot). (b) Activity of caspase-9 (chemiluminescence). Data were presented as mean ± SD. *P < 0.05 versus Vehicle, ^# P < 0.05 versus Hcy alone. α-ZAL: α-zearalanol; 17β-E₂: 17β-estradiol; Hcy: homocysteine.

Figure 6

Pretreatment with α-ZAL reduced the expression of proapoptotic protein Bax in homocysteine-challenged HUVECs. The expression of proapoptotic protein Bax was significantly increased after treatment with 500 μmol/L homocysteine for 24 h. Pretreatment with α-ZAL could reduce the expression of Bax. (a) Immunohistochemistry staining (only showing the picture of the highest concentration 10⁻⁶ mol/L). (b) Western blot. Data were presented as mean ± SD. *P < 0.05 versus Vehicle, ^# P < 0.05 versus Hcy alone. α-ZAL: α-zearalanol; 17β-E₂: 17β-estradiol; Hcy: homocysteine.

Figure 7

Pretreatment with α-ZAL enhanced the expression of prosurvival protein Bcl-2 in homocysteine-challenged HUVECs (immunohistochemistry staining, only showing the picture of the highest concentration 10⁻⁶ mol/L). The expression of prosurvival protein Bcl-2 was significantly decreased after treatment with 500 μmol/L homocysteine for 24 h. Pretreatment with α-ZAL could enhance the expression of Bcl-2. Data were presented as mean ± SD. *P < 0.05 versus Vehicle, ^# P < 0.05 versus Hcy alone. α-ZAL: α-zearalanol; 17β-E₂: 17β-estradiol; Hcy: homocysteine.

Figure 8

Pretreatment with α-ZAL enhanced the expression of prosurvival protein Bcl-XL in homocysteine-challenged HUVECs (immunohistochemistry staining, only showing the picture of the highest concentration 10⁻⁶ mol/L). The expression of prosurvival protein Bcl-XL was significantly decreased after treatment with 500 μmol/L homocysteine for 24 h. Pretreatment with α-ZAL could enhance the expression of Bcl-XL. Data were presented as mean ± SD. *P < 0.05 versus Vehicle, ^# P < 0.05 versus Hcy alone. α-ZAL: α-zearalanol; 17β-E₂: 17β-estradiol; Hcy: homocysteine.

Figure 9

Pretreatment with α-ZAL reduced the expression of 3-nitrotyrosine in homocysteine-challenged HUVECs (Western blot). The expression of 3-nitrotyrosine was significantly increased after treatment with 500 μmol/L homocysteine for 24 h. Pretreatment with α-ZAL could attenuate this effect. Data were presented as mean ± SD. *P < 0.05 versus Vehicle, ^# P < 0.05 versus Hcy alone. α-ZAL: α-zearalanol; 17β-E₂: 17β-estradiol; Hcy: homocysteine; 3-NT: 3-nitrotyrosine.