Fig. 5
Testosterone replacement therapy attenuated the high BP induced by hypoxia. A Male rats were castrated on day 1. Gonadectomized rats were injected subcutaneously with testosterone propionate (20 mg/kg b.w.) on day 7 and day 12. On day 15, the rats were treated with 10% O2 for 24 h. B Serum testosterone levels were measured by ELISA (n = 7, mean ± SEM, *p ≤ 0.05 by two-way ANOVA). C BP was measured from the rat tail artery. The baseline BP was the average value from 10 days of repeated testing. The final values were obtained by subtraction of the baseline from the test values (n = 7, mean ± SEM). D Rat aortic tissue was fixed and stained with anti-NRF1 and anti-ACE antibodies. E The intensity of NRF1 and ACE was quantified with ImageJ (n = 7, mean ± SEM, **p ≤ 0.01, ***p ≤ 0.001 by two-way ANOVA). F The expression of Ace, Edn1, and Nrf1 was detected by qRT-PCR (n = 7, mean ± SEM, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.0001 by two-way ANOVA). G–J HUVECs were incubated with the indicated concentration of testosterone for 24 h under 1% O2. Ang-2 in the supernatant was measured by ELISA (G, mean ± SD, **p ≤ 0.01, ***p ≤ 0.001 by two-way ANOVA). The expression of ACE, EDN1, NRF1, and VEGFA was measured by qRT-PCR (H–J, mean ± SD, *p ≤ 0.05, **p ≤ 0.01 by two-way ANOVA). Nor normoxia, Hyp hypoxia. K–M L132 cells were cotreated with 200 μM CoCl2 and testosterone at the indicated concentrations. The protein levels of HIF-1α and NRF1 were measured by western blot analysis (K), and the mRNA levels of NRF1 and ACE were detected by qRT-PCR (L, M, mean ± SD, two-way ANOVA). N L132 cells were transfected with NRF1 siRNA for 48 h and then subjected to hypoxia treatments. The mRNA level of ACE was detected by qRT-PCR (L, mean ± SD, ***p ≤ 0.001 by two-way ANOVA)
