Nitro-oleic acid (NO2-OA) ameliorates erectile dysfunction in a rat model of diabetes mellitus via modulation of fibrosis, inflammation and autophagy

Abstract

Background: Inflammation, fibrosis and autophagy represent closely related factors associated with the pathogenesis of diabetes mellitus erectile dysfunction (DMED). In this study, the therapeutic effect of nitro-oleic acid (NO₂-OA) in a streptozotocin-induced rat model of DMED was evaluated.

Methods: Sixty rats were randomly divided into four groups: control, DMED, DMED + Vehicle and DMED + NO₂-OA. DMED was induced by intraperitoneal injection of streptozotocin in male rats. Blood glucose and body weight were measured every 2 weeks. After 4 weeks of NO₂-OA treatment, erectile function was measured by electrical stimulation of cavernous nerve (CN). Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), enzyme-linked immunosorbent assay (ELISA), immunofluorescence and Masson's trichrome staining were used to verify the related factors and protein expression levels.

Results: We found that NO₂-OA could significantly increase erectile pressure in the corpus cavernosum of DMED rats. Results of western blot, confocal immunofluorescence and qRT-PCR assays revealed that NO₂-OA significantly reduced inflammatory factors and the expression of nuclear factor kappa B (NF-κB). In addition, Masson staining results indicated that NO₂-OA significantly reduced the display of fibrotic tissue in the corpus cavernosum. These beneficial effects may be related to reductions in the expression of transforming growth factor-β1 (TGF-β1) and connective tissue growth factor (CTGF) and the increase in the expression of α-smooth muscle actin (α-SMA). Finally, NO₂-OA treatment increased the expression of the autophagy marker, LC3, while P62 was decreased, effects suggesting that one of the underlying mechanisms of NO₂-OA may involve an activation of the PI3K/AKT/mTOR pathway to enhance the capacity for autophagy within this tissue.

Conclusions: NO₂-OA enhances erectile function within a rat model of DMED by inhibiting inflammation and fibrosis along with activating autophagy.

Keywords: Diabetes mellitus erectile dysfunction (DMED); autophagy; fibrosis; inflammation; nitro-oleic acid (NO2-OA).

Figure 1

Effects of NO₂-OA on metabolic parameters and erectile dysfunction in DMED rats. (A) Flow chart illustrating timeline of the experiment. (B) Body weights. (C) Blood glucose levels. (D) Representative traces of ICP in control, DMED, DMED + Vehicle and DMED + NO₂-OA rats. (E) Maximal ICP/MAP ratios. The data are presented as means ± SEMs. N=6 per group. ****, P<0.0001 compared with control group; ^###, P<0.001 compared with DMED group. DM, diabetes mellitus; DMED, diabetes mellitus erectile dysfunction; NO₂-OA, nitro-oleic acid; PCR, polymerase chain reaction; ICP, intra-cavernous pressure; MAP, mean systemic arterial pressure; SEM, standard error of mean.

Figure 2

Effects of NO₂-OA on reducing fibrosis in DMED rats. (A) Representative western blots and (B) protein expressions of TGF-β1, CTGF and α-SMA. (C) Masson trichrome staining (red, smooth muscle; and blue, collagen areas). (D) Ratio of smooth muscle to collagen in Masson’s trichrome-stained corpus cavernosum tissue. Representative immunofluorescence and quantification of (E,F) TGF-β1, (G,H) CTGF, and (I,J) α-SMA within the corpus cavernosum of the four groups. Scale bar is 50 µm. N=6 per group. **, P<0.01; ***, P<0.001; ****, P<0.0001 compared with the control group. ^#, P<0.05; ^##, P<0.01; ^####, P<0.0001 compared with the DMED group. TGF-β1, transforming growth factor-β1; CTGF, connective tissue growth factor; α-SMA, α-smooth muscle actin; DMED, diabetes mellitus erectile dysfunction; NO₂-OA, nitro-oleic acid.

Figure 3

Effects of NO₂-OA on autophagy in DMED rats. (A) Representative western blot and protein expressions of (B) PI3K, (C) AKT, (D) mTOR, (E) p62 and (F) LC3. N=6 per group. **, P<0.01; ***, P<0.001; ****, P<0.0001 compared with the control group. ^#, P<0.05; ^##, P<0.01; ^####, P<0.0001 compared with the DMED group. DMED, diabetes mellitus erectile dysfunction; NO₂-OA, nitro-oleic acid; PI3K, phosphoinositide 3-kinase; AKT, protein kinase B; mTOR, mammalian target of rapamycin; LC3, microtubule-associated protein 1 light chain 3.

Figure 4

Effects of NO₂-OA on inflammation in DMED rats. (A) Representative western blot and (B) protein expression of NF-κB. qPCR assay results for mRNA expression levels of (C) TNF-α, (D) IFN-γ, (E) IL-6 and (F) IL-1β. Representative (G) immunofluorescence (×200) and (H) quantification of CD63 within the corpus cavernosum of the four groups. N=6 per group. ***, P<0.001; ****, P<0.0001 compared with the control group. ^##, P<0.01; ^###, P<0.001; ^####, P<0.0001 compared with the DMED group. NF-κB, nuclear factor kappa-B; TNF-α, tumor necrosis factor-α; IFN-γ, interferon-γ; IL, interleukin; DMED, diabetes mellitus erectile dysfunction; NO₂-OA, nitro-oleic acid; qPCR, quantitative polymerase chain reaction.