Effects of oestradiol on progesterone induced NADH/NADPH oxidase activities, ROS generation, lipid peroxidation, proliferation, αSMA expression, and intracellular levels of TGF-β1 mRNA and protein in cultured rat HSCs with hydrogen peroxide exposure
| NADH oxidase (mmol/min/mg protein) | NADPH oxidase (mmol/min/mg protein) | ROS (channel number/mg protein) | MDA (mmol/mg protein) | BrdU incorporation (% of control) | αSMA (relative protein level) | TGF-β1 mRNA (arbitrary unit) | TGF-β1 protein (pg/mg protein) | |
|---|---|---|---|---|---|---|---|---|
| On culture day seven, oxidative stress was induced in HSCs by exposure to 10−5 mol/l hydrogen peroxide (oxidative stress) for 24 hours in the pretreatment in serum-free DMEM with and without progesterone (10−6 mol/l) and/or oestradiol (10−8–10−7 mol/l) in the presence and absence of 10−6 mol/l ICI 182,780 or 10−6 mol/l DPI for 24 hours. NADH/NADPH oxidase activities, the generation of ROS and MDA were then compared with cultures treated with progesterone alone after hydrogen peroxide exposure. DNA synthesis and TGF-β1 mRNA level were evaluated at 1 hour, and αSMA expression and TGF-β1 protein level at 24 hours. DNA synthesis in the presence of PDGF-BB (5 ng/ml), as a positive control, was 148 (SD 9)%. Results of densitometric analysis are presented as mean percentages of β-actin signal intensity of for αSMA expression. The levels of TGF-β1 gene expression were quantitatively analysed by real-time PCR, and the results are expressed in arbitrary units. Values are means (SD) for six dishes. *p<0.05 compared with cultures after hydrogen peroxide exposure (oxidative stress). †p<0.05 compared with cultures treated with progesterone alone after hydrogen peroxide exposure. | ||||||||
| None | 28.9 (5.5)*† | 8.1 (2.1)*† | 7.7 (1.1)*† | 0.52 (0.14)*† | 100 (6)*† | 63 (15)*† | 0.60 (0.10)*† | 15.6 (3.5)*† |
| + Oxidative stress only | 78.3 (14.8) | 27.2 (5.2) | 20.9 (4.0) | 1.44 (0.23) | 155 (10) | 129 (24) | 1.00 | 33.3 (6.7) |
| + Oxidative stress | 31.4 (5.4)*† | 9.0 (2.1)*† | 7.8 (1.1)*† | 0.55 (0.11)*† | 108 (7)*† | 64 (15)*† | 0.62 (0.11)*† | 16.3 (3.6)*† |
| + DPI 10−6 mol/l | ||||||||
| + Oxidative stress | 111.1 (17.3)* | 35.1 (6.2)* | 29.5 (4.5)* | 1.92 (0.32)* | 202 (20)* | 193 (37)* | 1.44 (0.21)* | 51.2 (10.1)* |
| + Progesterone 10−6 mol/l | ||||||||
| + Oxidative stress | 32.3 (5.6)*† | 9.4 (2.5)*† | 8.5 (1.2)*† | 0.58 (0.14)*† | 109 (7)*† | 66 (13)*† | 0.64 (0.13)*† | 16.5 (3.5)*† |
| + Progesterone 10−6 mol/l | ||||||||
| + DPI 10−6 mol/l | ||||||||
| + Oxidative stress | 73.6 (13.2)† | 22.4 (4.3)† | 19.0 (3.5)† | 1.24 (0.22)† | 165 (12)† | 130 (23)† | 1.09 (0.15)† | 40.4 (9.1)† |
| + Progesterone 10−6 mol/l | ||||||||
| + Estradiol 10−8 mol/l | ||||||||
| + Oxidative stress | 50.2 (13.3)*† | 14.5 (4.2)*† | 13.3 (2.8)*† | 0.81 (0.18)*† | 122 (11)*† | 84 (20)*† | 0.70 (0.15)*† | 26.7 (6.2)*† |
| + Progesterone 10−6 mol/l | ||||||||
| + Estradiol 10−7 mol/l | ||||||||
| + Oxidative stress | 110.5 (16.1)* | 35.7 (6.4)* | 28.4 (4.3)* | 1.87 (0.28)* | 222 (25)* | 186 (38)* | 1.59 (0.30)* | 52.6 (10.7)* |
| + Progesterone 10−6 mol/l | ||||||||
| + Oestradiol 10−7 mol/l | ||||||||
| + ICI | ||||||||
| + Oxidative stress | 33.8 (6.1)*† | 9.9 (2.5)*† | 8.3 (1.5)*† | 0.59 (0.15)*† | 110 (8)*† | 68 (15)*† | 0.65 (0.13)*† | 17.1 (3.7)*† |
| + Progesterone 10−6 mol/l | ||||||||
| + Oestradiol 10−7 mol/l | ||||||||
| + DPI 10−6 mol/l | ||||||||