Nerve growth factor (ngf)

The following reagents were obtained from Sigma (St. Louis, MO, USA); acetic acid, bicinchoninic acid solution, bovine serum albumin, copper(II) sulfate pentahydrate, Cresyl violet acetate, 3,3′-diamino-benzidine (DAB), radioimmunoprecipitation assay (RIPA), scopolamine hydrobromide, skim milk, and 9-amino-1,2,3,4-tetrahydroacridine hydrochloride hydrate (THA). The CREB, phospho-CREB, NGF, BDNF, beta-actin, and secondary horseradish peroxidase (HRP)-conjugated antibodies used for western blotting were obtained from Abcam (Cambridge, MA) and Santa Cruz Biotechnology (Santa Cruz, CA); the doublecortin (DCX), biotinylated secondary antibodies, and avidin-biotin peroxidase complex used for immunohistochemical staining were obtained from Abcam (Cambridge, MA), Santa Cruz Biotechnology (Santa Cruz, CA) and Vector Laboratories (Burlingame, CA).

Quercetin (CAS NO: 117-39-5, purity > 95%, Figure 1A) was purchased from Sigma-Aldrich. Kits for detecting SOD and MDA were purchased from Nanjing Jiancheng Bioengineering Institute. The antibodies for PSD93, PSD95, SIRT1, Caspase3, Bax, Bcl2, PERK, P-PERK, eIF2α, P-eIF2α, IRE-1α, P-IRE-1α, BIP, PDI were provided by Cell Signaling Technology (MA, USA). Anti-β-actin, BDNF, NGF, ATF6 were purchased from Abcam, Inc (Cambridge, England).

Animals were sacrificed by intraperitoneal injection with sodium pentobarbital (50 mg/kg), followed by bilateral thoracotomy. Penile midshaft tissues were freshly harvested and fixed [23 (link)], and immunofluorescence staining was performed as previously described [31 (link)]. The corpus cavernosum paraffin sections were immunostained with VEGF (diluted 1 : 200 Santa Cruz Biotechnologies, Santa Cruz, US), neuronal nitric oxide synthase (nNOS, diluted 1 : 200 Santa Cruz Biotechnologies, Santa Cruz, CA), brain-derived neurotrophic factor (BDNF, diluted 1 : 200; Abcam, Cambridge, UK), nerve growth factor (NGF, diluted 1 : 200; Abcam, Cambridge, UK), stromal cell-derived factor-1 (SDF-1 diluted 1 : 200; Abcam, Cambridge, UK), and neuron-specific β-III tubulin (diluted 1 : 200; Abcam, Cambridge, UK) and mounted with 4,6-diamidino-2-phenylindole (DAPI; Vector Laboratories Inc., Burlingame, CA) to stain the nuclei. The collagen and vascular smooth muscle was evaluated after tissue staining with the Masson trichrome technique using the method previously described [32 (link)]. Digital images were obtained using a Zeiss LSM 510 Meta confocal microscope (Zeiss, Oberkochen, Germany), and the mean intensity was calculated using ZEN 2009 (Zeiss).

Ventricles were harvested quickly, washed with saline, and stored at −80 °C until the next step. Proteins of frozen tissues were lysed with lysis buffer (20 mM Tris-HCl, 1 mM Na₃VO₄, and 5 mM NaF), then separated by 8–12% SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membranes. After 5% skimmed milk blocking, PVDF membranes were incubated with primary antibodies overnight, such as Cav1.2 (1:1000; Abcam), NGF (1:200; Abcam), Kv4.2 (1:1000; Abcam), and GAPDH (1:10,000; Abcam), followed by incubation with appropriate secondary antibody (peroxidase-coupled goat anti-rabbit or goat anti-mouse, 1:1000, Abcam). Membranes were analyzed by an Image-Quant ECL Imager (GE Healthcare, Chicago, IL).

The human PC cell lines AsPc‐1 and Panc‐1 were obtained from the American Type Culture Collection and cultured in DMEM supplemented with 10% foetal bovine serum (FBS) and 1% antibiotics/antimycotics in a humidified 5% CO₂ atmosphere at 37°C. Antibodies against MMP‐9, vimentin, E‐cadherin, NGF, TrkA, p‐AKT, AKT, GSK and p‐GSK were purchased from Abcam. Recombinant NGF was obtained from R&D Systems.

The human PC cell lines AsPc-1 and Panc-1 were obtained from the American Type Culture Collection (Manassas, VA) and cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic in a humidified 5% CO₂ atmosphere at 37°C. Antibodies against SHH, SMO, PTCH, Gli1, Gli2, NGF, BDNF, GDNF, TRPV1, SP and CGRP were purchased from Abcam, USA. Recombinant sonic hedgehog, NGF and BDNF were obtained from R & D Systems (Minneapolis, MN). Capsaicin was purchased from Sigma, USA.