Argonaute 2 restored erectile function and corpus cavernosum mitochondrial function by reducing apoptosis in a mouse model of cavernous nerve injury

Abstract

Purpose: To determine whether the overexpression of the Argonaute RNA-induced silencing complex catalytic component 2 (Ago2) improves erectile function in mice after cavernous nerve injury (CNI).

Materials and methods: Lentiviruses containing Ago2 open reading frame (ORF) mouse clone (Ago2 O/E) were used to overexpress Ago2, and lentiviruses ORF negative control particles (NC) were used as a negative control. Three days before preparing the CNI model, we injected lentiviruses into the penises of 8-week-old male C57BL/6 mice. Animals were then divided into four groups: the sham operation control group and the CNI+phosphate-buffered saline, CNI+NC, and CNI+Ago2 O/E groups. One week later, erectile function was assessed by electrically stimulating cavernous nerves bilaterally and obtaining intracavernous pressure parameters. Penile tissue was also collected for molecular mechanism studies.

Results: Ago2 overexpression improved erectile function in mice after CNI-induced erectile dysfunction (ED). Immunofluorescence staining and Western blot analysis showed that under Ago2 overexpressing conditions, the contents of endothelial cells, pericytes, and neuronal cells increased in the penile tissues of CNI mice, and this was attributed to reduced apoptosis and ROS production. In addition, we also found that Ago2 overexpression could restore penile mitochondrial function, thereby improving erectile function in CNI-induced ED mice.

Conclusions: Our findings demonstrate that Ago2 overexpression can reduce penile cell apoptosis, restore penile mitochondrial function, and improve erectile function in CNI-induced ED mice.

Keywords: Apoptosis; Argonaute proteins; Mitochondria dysfunction; Nerve regeneration; Reactive oxygen species.

Fig. 1. Ago2 overexpression improved erectile function in CNI mice. (A) Schematic of the experimental procedure. (B) Representative ICP responses of sham (7 days) and CNI (7 days) mice stimulated at 10 days after an intracavernous injection with PBS (20 µL), lentiviruses containing ORF negative control particles (NC, 5×10⁴ IFU/mice), or lentiviruses containing an ORF mouse clone of Ago2 (Ago2 O/E, 5×10⁴ IFU/mice). (C, D) Ratios of mean maximum and total ICPs (area under the curve) to MSBP were calculated for each group and results are presented as mean±standard error of mean (n=8). ^***p<0.001. CNI, cavernous nerve injury; ICP, intracavernous pressure; PBS, phosphate-buffered saline; ORF, open reading frame; IFU, infectious units; MSBP, mean systolic blood pressure; n.s., not significant.

Fig. 2. Ago2 overexpression increased cavernous endothelial and pericyte numbers in CNI mice. (A) Immunofluorescence staining for CD31 (an endothelial cell marker, green) and NG2 (a pericyte marker, red) in cavernous tissues from sham, CNI mice after an intracavernous injection with PBS (20 µL), lentiviruses containing ORF negative control particles (NC, 5×10⁴ IFU/mice), or lentiviruses containing an ORF mouse clone of Ago2 (Ago2 O/E, 5×10⁴ IFU/mice). Nuclei were labeled with DAPI (blue). Scale bar=100 µm. (B, C) Quantification of CD31 and NG2 immunopositive areas in cavernous tissue was performed using ImageJ software relative to the sham control. Results are presented as mean±standard error of mean (n=7). ^***p<0.001. The relative ratio in the Sham group was defined as 1. CNI, cavernous nerve injury; PBS, phosphate-buffered saline; ORF, open reading frame; IFU, infectious units; DAPI, 4,6-diamidino-2-phenylindole; n.s., not significant.

Fig. 3. Ago2 overexpression induced nerve regeneration in CNI mice. (A) Immunofluorescence staining for nNOS (green) and NF (red) in DNB from sham, CNI mice after an intracavernous injection with PBS (20 µL), lentiviruses containing ORF negative control particles (NC, 5×10⁴ IFU/mice), or lentiviruses containing an ORF mouse clone of Ago2 (Ago2 O/E, 5×10⁴ IFU/mice). Nuclei were labeled with DAPI (blue). Scale bar=25 µm. (B, C) Quantification of nNOS and NF immunopositive areas in cavernous tissue was performed using ImageJ software, and results are presented as mean±standard error of mean (SEM) (n=7). ^***p<0.001. (D) Representative western blots for Ago2, nNOS, BDNF, and NT-3 in cavernous tissues of mice in the groups mentioned above. β-Actin was used as the internal control. (E-H) Normalized band intensity values were quantified using ImageJ software relative to the sham control. Results are presented as mean±SEM (n=4). ^*p<0.05; ^**p<0.01; ^***p<0.001. The relative ratio in the Sham group was defined as 1. CNI, cavernous nerve injury; nNOS, neuronal nitric oxide synthase; NF, neurofilament; DNB, dorsal nerve bundles; PBS, phosphate-buffered saline; ORF, open reading frame; IFU, infectious units; DAPI, 4,6-diamidino-2-phenylindole; BDNF, brain-derived neurotrophic factor; NT-3, neurotrophin-3; n.s., not significant.

Fig. 4. Ago2 overexpression reduced apoptosis in CNI mice. (A) TUNEL assay of cavernous tissues from sham, CNI mice after an intracavernous injection with PBS (20 µL), lentiviruses containing ORF negative control particles (NC, 5×10⁴ IFU/mice), or lentiviruses containing an ORF mouse clone of Ago2 (Ago2 O/E, 5×10⁴ IFU/mice). Nuclei were labeled with DAPI (blue). Scale bar=100 µm. (B) Representative western blots for Bcl-2, Bax, phosphor-c-Jun/c-Jun (p-c-Jun/c-Jun), and phosphor-JNK/JNK (p-JNK/JNK) in cavernous tissues of mice in the groups mentioned above. β-Actin was used as the internal control. (C) Quantification of the number of apoptotic cells in cavernous tissue was performed using ImageJ software relative to the sham control. Results are presented as mean±standard error of mean (SEM) (n=7). ^***p<0.001. (D-G) Normalized band intensity values were quantified by ImageJ software and results are presented as mean±SEM (n=4). ^*p<0.05; ^**p<0.01. The relative ratio in the Sham group was defined as 1. CNI, cavernous nerve injury; TUNEL, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labeling; PBS, phosphate-buffered saline; ORF, open reading frame; IFU, infectious units; DAPI, 4,6-diamidino-2-phenylindole; n.s., not significant.

Fig. 5. Ago2 overexpression reduced ROS production in CNI mice. (A) Immunofluorescence staining for nitrotyrosine (green, peroxynitrite) and in situ detection of hydroethidine (red, superoxide anion) in cavernous tissue from sham, CNI mice after an intracavernous injection with PBS (20 µL), lentiviruses containing ORF negative control particles (NC, 5×10⁴ IFU/mice), or lentiviruses containing an ORF mouse clone of Ago2 (Ago2 O/E, 5×10⁴ IFU/mice). Nuclei were labeled with DAPI (blue). Scale bar=100 µm. (B, C) Quantification of nitrotyrosine and hydroethidine immunopositive areas in cavernous tissue was performed using ImageJ software relative to the sham control. Results are presented as mean±standard error of mean (SEM) (n=5). ^***p<0.001. (D) Representative western blots for iNOS and p47phox in cavernous tissue with the same groups as mentioned above. β-Actin was used as the internal control. (E, F) Normalized band intensity values were quantified using ImageJ software relative to the sham control. Results are presented as mean±SEM (n=4). ^*p<0.05; ^**p<0.01; ^***p<0.001. The relative ratio in the Sham group was defined as 1. CNI, cavernous nerve injury; PBS, phosphate-buffered saline; ORF, open reading frame; IFU, infectious units; DAPI, 4,6-diamidino-2-phenylindole; n.s., not significant.

Fig. 6. Ago2 overexpression restored mitochondrial dysfunction in CNI mice. (A) Immunofluorescence staining with MitoSOX red (for mitochondria superoxide) in cavernous tissues from sham, CNI mice after an intracavernous injection with PBS (20 µL), lentiviruses containing ORF negative control particles (NC, 5×10⁴ IFU/mice), or lentiviruses containing an ORF mouse clone of Ago2 (Ago2 O/E, 5×10⁴ IFU/mice). Nuclei were labeled with DAPI (blue). Scale bar=100 µm. (B) Quantification of MitoSOX immunopositive areas in cavernous tissue was performed using ImageJ software and results are presented as mean±standard error of mean (SEM) (n=7). ^**p<0.01; ^***p<0.001. (C) Ratios of mitochondria ATP levels in cavernous tissue were determined using an ATP assay kit and the groups mentioned above (n=5). ^**p<0.01; ^***p<0.001. (D) Representative western blots for MT-ND1 and MT-CO1 in the cavernous tissues of mice in the same groups. β-Actin was used as the internal control. (E, F) Normalized band intensity values were quantified using ImageJ software relative to the sham control. Results are presented as mean±SEM (n=4). ^*p<0.05; ^**p<0.01; ^***p<0.001. The relative ratio in the Sham group was defined as 1. CNI, cavernous nerve injury; PBS, phosphate-buffered saline; ORF, open reading frame; IFU, infectious units; DAPI, 4,6-diamidino-2-phenylindole; n.s., not significant.