Hydroxyl fasudil, an inhibitor of Rho signaling, improves erectile function in diabetic rats: a role for neuronal ROCK

Abstract

Introduction: The pathogenesis of diabetic erectile dysfunction (ED) includes neuropathy, but the molecular basis for neurogenic ED is incompletely understood. The RhoA/ROCK pathway has been implicated in diabetic neuropathy and in ED, but its role in diabetic neurogenic ED is not known.

Aims: The aim of this study was to determine whether hydroxyl fasudil, a ROCK inhibitor, affects diabetic neuropathy-related ED.

Methods: Type 1 diabetes mellitus was induced in male rats by streptozotocin (75 mg/kg, intraperitoneally). After 8 weeks, diabetic rats were administered hydroxyl fasudil, a selective ROCK inhibitor (10 mg/kg/day, intraperitoneally) or vehicle, for 4 weeks. Age-matched control, nondiabetic, rats were treated intraperitoneally for 4 weeks with saline. At week 12, after a 2 day washout, neuro-stimulated erectile function was evaluated. Major pelvic ganglia (MPG) were collected for Western blot analysis of RhoA, ROCK-1, ROCK-2, phospho (P)-AKT (Ser(473) ), and P-phosphatase and tensin homolog (P-PTEN) (Ser(380) /Thr(382/383) ).

Main outcome measures: Effect of ROCK inhibitor hydroxyl fasudil on erectile function and ROCK/P-AKT/P-PTEN pathway in the MPG of diabetic rats.

Results: Erectile response was significantly (P < 0.05) reduced in diabetic rats compared with nondiabetic rats and was preserved (P < 0.05) in diabetic rats treated with hydroxyl fasudil. In diabetic rats, RhoA and ROCK-2 protein expressions in MPG were increased (P < 0.05) and remained increased in hydroxyl fasudil-treated rats. P-AKT (Ser(473) ) expression was decreased (P < 0.05), whereas P-PTEN (Ser(380) /Thr(382/383) ) expression was increased (P < 0.05) in MPG of diabetic rats compared with nondiabetic rats, and both were reversed (P < 0.05) in diabetic rats treated with hydroxyl fasudil.

Conclusion: Improved erectile function and restored P-AKT and P-PTEN in the MPG with hydroxyl fasudil treatment suggest the role of Rho signaling via PTEN/AKT pathway in neurogenic diabetic ED.

Keywords: Apoptosis; Diabetic Neuropathy; Major Pelvic Ganglia; P-AKT; P-PTEN.

Figure 1

Effect of diabetes and hydroxyl fasudil on penile erection. Erectile response to electrical stimulation of the cavernous nerve is indicated by maximal ICP/MAP corrected for baseline (A) and total ICP/MAP corrected for baseline (B) in nondiabetic rats, diabetic rats, and diabetic rats treated with hydroxyl fasudil (10 mg/kg/day). Each bar represents the mean ± SEM of 6-8 rats. *P<0.05 vs nondiabetic, ^#P<0.05 vs diabetic. NonDiab = nondiabetic; Diab = diabetic; Diab + HF = diabetic treated with hydroxyl fasudil; AU = arbitrary units

Figure 2

Effect of diabetes and hydroxyl fasudil on RhoA (A), ROCK-2 (B), and ROCK-1 (C) protein expressions in the MPG. Upper panels are representative Western immunoblots of RhoA, ROCK-2, ROCK-1, and β-tubulin in nondiabetic rats, diabetic rats, and diabetic rats treated with hydroxyl fasudil (10 mg/kg/day). Lower panel represents quantitative analysis of the proteins in the same treatment groups. Each bar represents the mean ± SEM of 6-8 rats. *P<0.05 vs nondiabetic. NonDiab = nondiabetic; Diab = diabetic; Diab + HF = diabetic treated with hydroxyl fasudil.

Figure 3

Effect of diabetes and hydroxyl fasudil on P-AKT (Ser⁴⁷³)/AKT protein expression in the MPG. Upper panels are representative Western immunoblots of P-AKT (Ser⁴⁷³) and AKT in nondiabetic rats, diabetic rats, and diabetic rats treated with hydroxyl fasudil (10 mg/kg/day). Lower panel represents quantitative analysis of P-AKT/AKT in the same treatment groups. Each bar represents the mean ± SEM of 6-8 rats. *P<0.05 vs nondiabetic, ^#P<0.05 vs diabetic. NonDiab = nondiabetic; Diab = diabetic; Diab + HF = diabetic treated with hydroxyl fasudil.

Figure 4

Effect of diabetes and hydroxyl fasudil on P-PTEN (Ser³⁸⁰/Thr^382/383)/PTEN protein expression in the MPG. Upper panels are representative Western immunoblots of P-PTEN (Ser³⁸⁰/Thr^382/383) and PTEN in nondiabetic rats, diabetic rats, and diabetic rats treated with hydroxyl fasudil (10 mg/kg/day). Lower panel represents quantitative analysis of P-PTEN/PTEN in the same treatment groups. Each bar represents the mean ± SEM of 6-8 rats. *P<0.05 vs nondiabetic, ^#P<0.05 vs diabetic. NonDiab = nondiabetic; Diab = diabetic; Diab + HF = diabetic treated with hydroxyl fasudil.