The near-infrared dye IR-61 restores erectile function in a streptozotocin-induced diabetes model via mitochondrial protection / 亚洲男科学杂志(英文版)

This study aimed to evaluate the therapeutic effect of IR-61, a novel mitochondrial heptamethine cyanine dye with antioxidant effects, on diabetes mellitus-induced erectile dysfunction (DMED). Eight-week-old male Sprague-Dawley rats were intraperitoneally injected with streptozotocin (STZ) to induce type 1 diabetes. Eight weeks after STZ injection, all rats were divided into three groups: the control group, DM group, and DM + IR-61 group. In the DM + IR-61 group, the rats were administered IR-61 (1.6 mg kg

Lentiviral vectors carrying siRNA inhibit S1PR3 gene expression in the corpus cavernosum smooth muscle cells of rats with spontaneous hypertension / 中华男科学杂志

Objective@#To screen lentiviral vectors carrying siRNA which can specifically down-regulate the gene expression of the sphingosine-1-phosphate receptor 3 (S1PR3) in the corpus cavernosum smooth muscle (CCSM) cells of rats with spontaneous hypertension (SHT) and investigate the influence of the vectors on the signaling pathways of ROCK1, ROCK2 and eNOS in the CCSM cells of SHT rats.@*METHODS@#Using the S1PR3 mRNA sequence of the rat as an interfering target, we designed and synthesized three pairs of siRNA sequences (siRNA1, 2 and 3) targeting S1PR3 and one pair of negative control, and then constructed and packaged them into lentiviral vectors. We cultured the CCSM cells of SHT and Wistar-Kyoto (WKY) rats in vitro and randomly divided them into groups A (SHT untransfected control), B (SHT transfected and carrying negative control virus), C (SHT transfected and carrying siRNA1 targeting S1PR3), D (SHT transfected and carrying siRNA2 targeting S1PR3), E (SHT transfected and carrying siRNA3 targeting S1PR3), and F (WKY untransfected control). With the multiplicity of infection (MOI) = 60, we transfected the CCSM cells of the SHT rats with the lentiviral vector and then determined the expression of the green fluorescent protein (GFP) as well as the mRNA and protein expressions of S1PR3, ROCK1, ROCK2 and eNOS in the CCSM cells of the SHT and WKY rats by RT-PCR and Western blot.@*RESULTS@#Gene sequencing proved the successful construction of the lentiviral vector. The transfection efficiency of the CCSM cells of the rats was >80% in groups B, C, D and E. Compared with group A, the mRNA and protein expressions of S1PR3, ROCK1 and ROCK2 exhibited no significant difference in group B but were remarkably decreased in groups C, D, E and F (P0.05) but remarkably lower than those in group F (P0.05) but markedly increased in groups A, B, C and D (P< 0.05), while those of eNOS remarkably decreased in groups A, B, C, D and E (P< 0.05).@*CONCLUSIONS@#The three constructed lentiviral vectors carrying siRNA targeting different loci of the S1PR3 gene could significantly inhibit the expression of S1P3 as well as RhoA/Rho kinase signaling pathways in the CCSM cells of SHT rats, and the vector carrying siRNA3 exhibited the highest inhibitory effect.

Quercetin Relaxed the Smooth Muscle of Rabbit Penile Corpus Cavernosum by Activating the NO-cGMP Signaling Pathway

The aim of this study was to investigate the effect and action mechanism of quercetin on penile corpus cavernosum smooth muscle (PCCSM). PCCSM precontracted with phenylephrine (Phe) was treated with four different concentrations of quercetin (10−7, 10−6, 10−5 and 10−4 M). PCCSM were preincubated with N-Nitro-L-arginine methyl ester hydrochloride (L-NAME) and 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) to block nitric oxide synthase and guanylate cyclase, respectively. The changes in PCCSM tension were recorded, and cyclic nucleotides in the perfusate were measured by radioimmunoassay. The interactions of quercetin with phosphodiesterase type 5 inhibitors (PDE5-Is) such as sildenafil, udenafil and mirodenafil, were also evaluated. PCCSM relaxation induced by quercetin occurred in a concentrationdependent manner. The application of quercetin to PCCSM pre-treated with L-NAME and ODQ significantly inhibited the relaxation. Quercetin significantly increased cGMP in the perfusate. Furthermore, quercetin enhanced PDE5-Is-induced relaxation of PCCSM. Quercetin relaxed the PCCSM by activating the NO-cGMP signaling pathway, and it may be a therapeutic candidate or an alternative treatment for patients with erectile dysfunction who do not completely respond to PDE5-Is.

Effect of salidroside on the expression of connexin43 in the corpus cavernosum smooth muscle cells of hypoxic rats / 中华男科学杂志

<p><b>Objective</b>To investigate the effect of salidroside on the expression of the connexin43 (Cx43) protein in the corpus cavernosum smooth muscle cells (CCSMCs) of hypoxic SD rats.</p><p><b>METHODS</b>CCSMCs were cultured in vitro and identified by immunofluorescence staining. The cells were divided into six groups: normal control (21% O2), hypoxia (1% O2), hypoxia+salidroside (HS) 8 μg/ml,HS 16 μg/ml, HS 32 μg/ml, and HS 64 μg/ml, and cultured for 48 hours. Then the relative expression of Cx43 in different groups was detected by Western blot.</p><p><b>RESULTS</b>The in vitro cultured CCSMCs grew well and 90% of the cells showed positivity for α-SMA and desmin on immunohistochemistry. Salidroside ≤64 μg/ml produced no obvious toxicity on the CCSMCs. The expressions of Cx43 and phosphorylated proteins were dramatically increased in the hypoxia group as compared with the normal control (P<0.01 and P<0.05). The HS groups all showed significantly higher expression of Cx43 than the hypoxia group (P<0.01), but the phosphorylation rate of the Cx43 proteins was remarkably decreased (P<0.01).</p><p><b>CONCLUSIONS</b>Hypoxia increases the expression of Cx43 in the CCSMCs of SD rats. Salidroside ≤64 μg/ml cannot reverse the hypoxia-induced change but can reduce the dephosphorylation of Cx43 in CCSMCs. It is deduced that salidroside can protect CCSMCs by decreasing the phosphorylation of Cx43 and suppressing hypoxia-induced formation of the gap junction channel.</p>

Autophagy and phenotypic modulation of corpus cavernosum smooth muscle cells in hypoxia-induced erectile dysfunction / 中华男科学杂志

The correlation between hypoxia and erectile dysfunction (ED) has been universally acknowledged for decades in the academic world. The phenotypic modulation of corpus cavernosum smooth muscle cells (CCSMCs) is regarded as one of the factors of hypoxia-induced ED, but the underlying mechanisms remain unclear. Recent researches show some correlation between autophagy and phenotypic modulation of CCSMCs, which may be associated with the overexpressions of PDGF, TGF-β, and vasoactive factors in the organism following hypoxia.

Effects of Insulin-Like Growth Factor-1 on Proliferation of Human Corpus Cavernosal Smooth Muscle Cells / 대한남성과학회지

PURPOSE: Insulin-like growth factor-1 (IGF-1) promotes the proliferation and migration of penile cavernous smooth muscle cells in the rat. The goals of this study were to evaluate the expression of IGF-1 and IGF-1 receptor (IGF-1R) in human corpus cavernosal smooth muscle cells (HCCSMCs) and to investigate the effect of IGF-1 on the proliferation of these cells in vitro. MATERIALS AND METHODS: The HCCSMCs were cultured from five impotent patients. The smooth muscle cells were identified by immunofluorescent stain. Total RNA was extracted, and IGF-1 gene expression was determined by RT-PCR. The IGF-1R immunoreactivity was examined by fluorescent Immunocytochemistry. Cell growth was assessed with a novel proliferation assay based on bioreduction of the fluorescent dye, Alamar blue. RESULTS: Endogenous IGF-1 mRNA expression was detected by RT-PCR analysis. The HCCSMCs were positive for IGF-IR. The proliferation of HCCSMCs increased with the dose in response IGF-1 in the culture medium in concentrations up to 100 ng/ml. CONCLUSIONS: Both IGF-1 and IGF-1R are expressed endogenously in human corpus cavernosal smooth muscle cells. IGF-1 promotes the proliferation of these cells in vitro.

Role of Rho-Kinase Activity in Angiotensin II-Induced Contraction of Corpus Cavernosum Smooth Muscle in the Rabbit / 대한남성과학회지

PURPOSE: RhoA/Rho-kinase regulates vascular tone via a calcium sensitization mechanism. Stimulation of the AT1 receptor by angiotensin (ANG) II activates the Rho A/Rho-kinase signaling pathway. However, its role in corpus cavernosum smooth muscle (PCSM) has not been known. MATERIALS AND METHODS: Isometric tension measurements were performed in rabbit PCSM using a selective Rho-kinase inhibitor, (+)-(R)-trans-4-(1-aminoethyl)-N-(4-pyridyl) cyclohexanecarboxamide (Y-27632), and a selective myosin light chain kinase (MLCK) inhibitor, 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine hydrochloride (ML7). RESULTS: Y-27632 significantly attenuated contractions induced by ANG II in a dose-dependent fashion. However, ML7 did not affect the contractile response to ANG II except at high concentration. Y-27632 inhibited contraction in response to phenylephrine (PhE), but ML7 did not. A nitric oxide synthase inhibitor, NG-nitro-L-arginine-methyl ester, did not affect Y-27632-induced relaxation of the strip contracted with PhE. CONCLUSIONS: A G-protein-coupled increase in myofilament Ca2+ sensitivity, mediated through the RhoA/Rho-kinase signaling pathway, is involved in the regulation of the PCSM tone induced by ANG II. The RhoA/Rho-kinase pathway acts in AGN II-induced contraction independent of the NO pathway.

Physiologic Role of K+ Channels in Relaxation of Rabbit Corpus Cavernosal Tissue / 대한남성과학회지

PURPOSE: Ion channels play key roles in determining smooth muscle tone by setting the membrane potential and allowing Ca2+ influx. Potassium channels may be important in modulating corporal smooth muscle tone. In this study, we investigated the effects of potassium channels in the rabbit corpus cavernosal smooth muscle by blocking them with various agents. MATERIALS AND METHODS: Strips of rabbit corpus cavernosum were prepared for mounting and isometric tension measurement in an organ bath. On cavernosal strips contracted with phenylephrine (PHE), sodium nitroprusside (SNP) was applied in increasing concentrations from 10(-7)M to 10(-4)M, causing dose-dependent relaxation. The effects of various potassium channel blockers on SNP-induced relaxation were then evaluated by measuring the tension of the cavernosal strips. The potassium channel blockers used were tetraethyl ammonium (TEA), charybdotoxin, gliben clamide, and apamin. RESULTS: The relaxation responses to SNP of the corporal preparations contracted in response to PHE were significantly attenuated by TEA (10(-2)M) and charybdotoxin (10(-7)M), with no significant difference observed between the two drugs. The SNP-induced relaxation responses were not significantly attenuated by glibenclamide (10(-5)M) or apamin (10(-5)M). CONCLUSIONS: These results suggest that maxi-K+ channels play an important role in corpus cavernosal relaxation. The KATP channel and small-conductance KCa channel are thought to be unrelated to corpus cavernosal smooth muscle relaxation.