Restoration of Cavernous Veno-Occlusive Function through Chronic Administration of a Jun-Amino Terminal Kinase Inhibitor and a LIM-Kinase 2 Inhibitor by Suppressing Cavernous Apoptosis and Fibrosis in a Rat Model of Cavernous Nerve Injury: A Comparison with a Phosphodiesterase Type 5 Inhibitor

Abstract

Purpose To determine if chronic administration of Jun-amino terminal kinase (JNK)-inhibitors and LIM-kinase 2 (LIMK2)-inhibitors from the immediate post-injury period in a rat model of cavernous-nerve-crush-injury could normalize cavernous-veno-occlusive-function, and to compare it with phosphodiesterase type 5 (PDE5)-inhibitors. Materials and Methods A total of 75 12-week-old male Sprague–Dawley-rats were randomized into five groups: sham-surgery (S), cavernous-nerve-crush-injury (I), cavernous-nerve-crush-injury treated with 10.0 mg/kg LIMK2-inhibitor (L) or 10.0 mg/kg JNK-inhibitor and 10.0 mg/kg LIMK2-inhibitor (J+L) or 20.0 mg/kg udenafil (P) for five-weeks. Five-weeks after surgery, dynamic-infusion-cavernosometry, histological-studies, caspase-3-activity-assay, and Western-blot were investigated. Results Group-I had lower papaverine-response, higher maintenance-rate and higher drop-rate, compared to Group-S. Group-L, Group-J+L and Group-P showed improvement in the three dynamic-infusion-cavernosometry parameters. The papaverine-response and drop-rate in Group-J+L and Group-P recovered to sham-control level, but those in Group-L did not. Regarding apoptosis, Group-I had decreased content of α-smooth-muscle-actin, increased caspase-3 activity and increased cJun-phosphorylation. The cJun-phosphorylation improved only in Group-J+L. The α-smooth-muscle-actin content and caspase-3-activity in Group-J+L and Group-P improved, but those in Group-L were not. Regarding fibrosis, Group-I had decreased smooth muscle (SM)/collagen-ratio, increased protein-expression of fibronectin, and increased Cofilin-phosphorylation. Cofilin-phosphorylation was normalized in Group-L and Group-J+L, but not in Group-P. SM/collagen-ratio and protein-expression of fibronectin in Group-L, Group-J+L and Group-P improved. Conclusions Our data indicate that chronic inhibition of JNK and LIMK2 can restore cavernous-veno-occlusive-function by suppressing cavernous-apoptosis and cavernous-fibrosis, comparable to the results by PDE5-inhibitors. Chronic inhibition of JNK and LIMK2 might be a potential mechanism-specific targeted therapy for cavernous-veno-occlusive-dysfunction induced by cavernous nerve-injury.

Combination Therapy with a JNK Inhibitor and Hepatocyte Growth Factor for Restoration of Erectile Function in a Rat Model of Cavernosal Nerve Injury: Comparison with a JNK Inhibitor Alone or Hepatocyte Growth Factor Alone

“…Although molecular pathways of cavernosal apoptosis induced by CN injuries are still poorly understood, several previous studies, including a previous study of ours, have reported that a few apoptotic pathways including Sonic hedgehog, Rho-kinase, and JNK pathways, are involved in cavernosal apoptosis following CN injury [ 24 , 25 , 26 , 27 ]. Under this background, our previous studies reported that JNK pathway inhibition, which was known to play a critical role in apoptosis through both nucleus- targeted and mitochondria-targeted signaling, improved erectile function in a CNCI rat model [ 11 , 14 , 15 , 28 ]. However, we thought that both anti-apoptotic and regenerative treatment would be needed to completely preserve the structural integrity of the penis and to recover from CN injury-induced ED, given that the treatment alone for suppressing the structural alterations (apoptosis, fibrosis, or both) of the corpus cavernosum did not normalize erectile function in a CN injury rat model, according to our previous studies [ 11 , 14 , 15 ].…”

“…Under this background, our previous studies reported that JNK pathway inhibition, which was known to play a critical role in apoptosis through both nucleus- targeted and mitochondria-targeted signaling, improved erectile function in a CNCI rat model [ 11 , 14 , 15 , 28 ]. However, we thought that both anti-apoptotic and regenerative treatment would be needed to completely preserve the structural integrity of the penis and to recover from CN injury-induced ED, given that the treatment alone for suppressing the structural alterations (apoptosis, fibrosis, or both) of the corpus cavernosum did not normalize erectile function in a CN injury rat model, according to our previous studies [ 11 , 14 , 15 ]. In accordance with this hypothesis, the present study showed that treatment with a JNK inhibitor alone for two weeks starting in the immediate postinjury period partially restored PECAM-1 protein expression, eNOS phosphorylation, and the erectile response to electrostimulation in a CNCI-induced ED rat model.…”

“…A total of 70 male Sprague-Dawley rats were randomized into five groups ( n = 14 in each group), as follows: (S group) rats that underwent sham surgery, (I group) those that underwent CNCI, (J group) those that received once daily intraperitoneal administration of 10.0 mg/kg JNK inhibitors (SP600125, Abcam, Cambridge, UK), (H group) those that received twice weekly intracavernosal administration of 4.2 μg (in PBS 20 μL) recombinant human (rh)-HGF (R&D Systems, Minneapolis, MN, USA), and (J+H group) those that received both once daily intraperitoneal administration of 10.0 mg/kg of JNK inhibitors and twice weekly intracavernosal administration of 4.2 μg (in PBS 20 μL) rh-HGF [ 11 , 14 , 15 , 17 , 19 ]. They were 13 weeks old and weighed 353–411 g at the time of surgery.…”

“…They received daily intraperitoneal administration of saline vehicles and twice-weekly intracavernosal administration of saline vehicles from the day of the surgery. For the I, J, H, and J+H group rats, we crushed the bilateral CNs 4–5 mm below the MPG with mechanical compression (two 80-s applications of pressure), using the microsurgical clamp to mimic nerve-sparing RP, as previously described [ 14 , 15 ]. The I group rats received daily intraperitoneal administration of saline vehicles and twice-weekly intracavernosal administration of saline vehicles starting on the day of the CNCI.…”

“…According to our previous studies, either Jun N-terminal kinase (JNK) inhibition alone or LIM-kinase 2 (LIMK2) inhibition alone partially restored erectile function in a rat model of CN injury [ 11 , 12 , 13 ]. In this context, our recent study tested whether the combination of anti-apoptotic effects induced by Jun N-terminal kinase (JNK) inhibition and anti-fibrotic effects of LIM-kinase 2 (LIMK2) inhibition could restore erectile function by rectifying the JNK-driven and LIMK2-driven pathways related to apoptosis and fibrosis in a rat CN crush injury model (CNCI) [ 14 , 15 ]. However, erectile response to electrostimulation or cavernosal venoocclusive function was not restored to normal control levels by the combination therapy in the nerve-injured rats.…”