Losartan improves erectile function through suppression of corporal apoptosis and oxidative stress in rats with cavernous nerve injury

Abstract

This study aimed to investigate the functional and morphological changes in the corpus cavernosum after cavernous nerve (CN) injury or neurectomy and then reveal whether treatment with the angiotensin II Type 1 receptor antagonist losartan would improve erectile function as well as its potential mechanisms. A total of 48 10-week-old Sprague-Dawley male rats, weighing 300-350 g, were randomly divided into the following four groups (n = 12 per group): sham operation (Sham) group, bilateral cavernous nerve injury (BCNI) group, losartan-treated BCNI (BCNI + Losartan) group, and bilateral cavernous neurectomy (Neurectomy) group. Losartan was administered once daily by oral gavage at a dose of 30 mg kg^-1 day^-1 for 4 weeks starting on the day of surgery. The BCNI and the Neurectomy groups exhibited decreases in erectile response and increases in apoptosis and oxidative stress, compared with the Sham group. Treatment with losartan could have a modest effect on erectile function and significantly prevent corporal apoptosis and oxidative stress. The phospho-B-cell lymphoma 2 (Bcl-2)-associated death promoter (p-Bad)/Bad and phospho-the protein kinase B (p-AKT)/AKT ratios were substantially lower, while the Bcl-2-associated X protein (Bax)/Bcl-2 ratio, nuclear factor erythroid 2-related factor 2 (Nrf2)/Kelch-like ECH-associated protein 1 (Keap-1), transforming growth factor-β 1 (TGF-β 1) and heme oxygenase-1 (HO-1) levels, and caspase-3 activity were higher in the BCNI and Neurectomy groups than in the Sham group. After 4 weeks of daily administration with losartan, these expression levels were remarkably attenuated compared with the BCNI group. Taken together, our results suggested that early administration of losartan after CN injury could slightly improve erectile function and significantly reduce corporal apoptosis and oxidative stress by inhibiting the Akt/Bad/Bax/caspase-3 and Nrf2/Keap-1 pathways.

Keywords: angiotensin II; apoptosis; erectile dysfunction; fibrosis; losartan; oxidative stress.

Figure 1

Anatomy of the MPG and CN. (a) MPG and CNs were identified dorsolateral to the prostate by blunt dissection. (b) Dissociated CNs were shown by blunt dissection. Scale bars = 5 mm. MPG: major pelvic ganglion; CN: cavernous nerve.

Figure 2

Ultrastructural changes of CN by TEM. (a) The Sham group; (b) the BCNI group; (c) the Neurectomy group. Black arrows represent damaged myelin sheath and red arrow represents intact myelin sheath. Scale bars=5 μm. BCNI: bilateral cavernous nerve injury; CN: cavernous nerve; TEM: transmission electron microscopy.

Figure 3

Evaluation of erectile response at 10.0 V. (a) The Sham group, the BCNI group, the BCNI + Losartan group, and the neurectomy group; n = 12 in each group. (b) Results of cavernous electrostimulation in the four experimental groups are expressed as ICP/MAP. Bar graphs represent mean ± standard deviation. ^**P < 0.01, the indicated group compared with the Sham group; ^#P < 0.05 and ^##P < 0.01, the indicated group compared with the BCNI group. BCNI: bilateral cavernous nerve injury; ICP: intracavernosal pressure; MAP: mean arterial pressure.

Figure 4

Detection of fibrosis by Masson's trichrome staining, cavernous α-SMA immunohistochemical staining and western blot. (a) Masson's trichrome staining; (b) bar graph of smooth muscle/collagen; (c) cavernous α-SMA immunohistochemical staining; (d) bar graph of cavernous α-SMA immunohistochemical staining; (e) the expression of TGF-β1 in corpus cavernosum of Western blot; (f) bar graph of the expression of TGF-β1 in corpus cavernosum. Scale bars = 50 μm; n = 6 in each group. Representative micrographs of Masson's trichrome staining show smooth muscle component as red areas, collagen component as blue areas (×200). Representative micrographs of cavernous α-SMA immunohistochemical staining show penile smooth muscle alpha-actin as brown areas (×200). Data of western blot analysis are shown as the fold changes over the control group. Bar graphs represent mean ± standard deviation. ^*P < 0.05 and ^**P < 0.01, the indicated group compared with the Sham group; ^#P < 0.05 and ^##P < 0.01, the indicated group compared with the BCNI group. α-SMA: alpha smooth muscle actin; BCNI: bilateral cavernous nerve injury; TGF-β1: transforming growth factor beta 1.

Figure 5

Detection of apoptosis by TUNEL, cavernous caspase-3 immunohistochemical staining and western blot. (a) The TUNEL method; (b) bar graph of apoptotic index; (c) cavernous caspase-3 immunohistochemical staining; (d) bar graph of caspase-3 positive area; (e) western blot of the Akt/Bad/Bax/caspase-3 pathways; (f) bar graph of relative p-AKT/AKT expression; (g) bar graph of relative p-Bad/Bad expression; (h) bar graph of relative Bax/Bcl-2 expression; (i) bar graph of relative caspase-3 activity; scale bars = 50 μm; n = 6 in each group. Representative micrographs of TUNEL show apoptotic cells as black-brown dots (×400). Representative micrographs of cavernous caspase-3 immunohistochemical staining show apoptotic cells as brown areas (×200). Bar graph depicts caspase-3 activity in penile tissues measured by a Caspase-3 Activity Assay Kit, which was calculated according to a standard curve and normalized by the protein concentration. Data of western blot analysis and caspase-3 activity assay are shown as the fold changes over the control group. Bar graphs represent mean ± standard deviation. ^*P < 0.05 and ^**P < 0.01, the indicated group compared with the Sham group; ^#P < 0.05 and ^##P < 0.01, the indicated group compared with the BCNI group. BCNI: bilateral cavernous nerve injury; TUNEL: terminal dexynucleotidyl transferase(TdT)-mediated dUTP nick end labeling; Bcl-2: B-cell lymphoma 2; Bax: Bcl-2-associated X protein; Bad: Bcl-2-associated death promoter; p-Bad: phosphor-Bad; AKT: the protein kinase B; p-AKT: phosphor-AKT.

Figure 6

Detection of oxidative stress by cavernous Nrf2, HO-1 immunohistochemical staining and western blot. (a) Cavernous Nrf2 immunohistochemical staining; (b) bar graph of Nrf2 positive area; (c) cavernous HO-1 immunohistochemical staining; (d) bar graph of HO-1 positive area; (e) western blot of the Nrf2/Keap-1 pathways; (f) bar graph of relative Nrf2/Keap-1 expression; (g) bar graph of relative HO-1 expression; scale bars = 50 μm; n = 6 in each group. Representative micrographs of cavernous Nrf2 immunohistochemical staining show areas of oxidative stress as brown (×200). Representative micrographs of cavernous HO-1 immunohistochemical staining show areas of oxidative stress as brown (×200). Data of western blot analysis are shown as the fold changes over the control group. Bar graphs represent mean ± standard deviation. ^*P < 0.05 and ^**P < 0.01, the indicated group compared with the Sham group; ^#P < 0.05 and ^##P < 0.01, the indicated group compared with the BCNI group. BCNI: bilateral cavernous nerve injury; HO-1: heme oxygenase-1; Keap-1: Kelch-like ECH associated protein 1; Nrf2: nuclear factor erythroid 2-related factor 2.