ABSTRACT
Type 2 diabetes mellitus (T2DM) is a common cause of erectile dysfunction (ED). It has been demonstrated that G protein-coupled receptor kinase 2 (GRK2) overexpression contributes to diabetic endothelial dysfunction and oxidative stress, which also underlies ED in T2DM. We hypothesized that GRK2 overexpressed and attenuated endothelial function of the cavernosal tissue in a rat model of T2DM. T2DM rats were established by feeding with a high-fat diet (HFD) for 2 weeks and then administering two intraperitoneal (IP) injections of a low dose of streptozotocin (STZ), followed by continuous feeding with a HFD for 6 weeks. GRK2 was inhibited by IP injection of paroxetine, a selective GRK2 inhibitor, after STZ injection. Insulin challenge tests, intracavernous pressure (ICP), GRK2 expression, the protein kinase B (Akt)/endothelial nitric oxide synthase (eNOS) pathway, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunit gp91phox, nitric oxide (NO), reactive oxygen species (ROS) production, and apoptosis in cavernosal tissue were examined. Less response to insulin injection was observed in T2DM rats 2 weeks after HFD. Markedly increased GRK2 expression, along with impaired Akt/eNOS pathway, reduced NO production, increased gp91phox expression and ROS generation, increased apoptosis and impaired erectile function were found in T2DM rats. Inhibition of GRK2 with paroxetine ameliorated Akt/eNOS signaling, restored NO production, downregulated NADPH oxidase, subsequently inhibited ROS generation and apoptosis, and ultimately preserved erectile function. These results indicated that GRK2 upregulation may be an important mechanism underlying T2DM ED, and GRK2 inhibition may be a potential therapeutic strategy for T2DM ED.
ABSTRACT
<p><b>OBJECTIVE</b>To investigate the effect of phosphodiesterase type 5 (PDE5) small interfering RNA (siRNA) on cyclic guanosine monophosphatethe (cGMP) in the smooth muscle cells of human corpus cavernosum, and to provide laboratory evidence for the application of the RNA interference (RNAi) technique for the treatment of erectile dysfunction.</p><p><b>METHODS</b>The recombinant adenovirus rAd5-shRNA-PDE5A3 expressing three pairs of specific shRNA was constructed successfully. The smooth muscle cells of human corpus cavernosum were divided into an experimental, a negative control and a blank control group, and transfected respectively with rAd5-shRNA-PDE5A3, adenovirus rAd5-mock and phosphate buffered saline. The concentration of cGMP was measured by radioimmunoassay at 24, 48 and 72 hours after transfection, and the effect of rAd5-shRNA-PDE5A3 was detected on the cGMP in the smooth muscle cells of the corpus cavernosum.</p><p><b>RESULTS</b>The cGMP level in the smooth muscle cells of the corpus cavernosum was significantly higher in the rAd5-shRNA-PDE5A3 group than in the rAd5-mock control and blank control groups (P < 0.05), most significantly at 72 hours after transfection.</p><p><b>CONCLUSION</b>The rAd5-shRNA-PDE5A3 can obviously increase the cGMP level in the smooth muscle cells of human corpus cavernosum, and enhance the inhibition of the PDE5 gene.</p>
Subject(s)
Humans , Male , Adenoviridae , Cells, Cultured , Cyclic GMP , Metabolism , Cyclic Nucleotide Phosphodiesterases, Type 5 , Genetics , Erectile Dysfunction , Genetics , Genetic Vectors , Myocytes, Smooth Muscle , Metabolism , Penis , Cell Biology , RNA, Small InterferingABSTRACT
<p><b>OBJECTIVE</b>To observe the influence of recombinant adenovirus-mediated PDE5-shRNAs on the free calcium level in rat penile smooth muscle cells and to explore the feasibility of gene therapy for erectile dysfunction (ED).</p><p><b>METHODS</b>Smooth muscle cells of the rat corpus cavernosum were transfected with constructed rAd-rPDE5-shRNAs and then dyed with the calcium fluorescent probe Fluo-3/AM at 24, 48 and 72 hours. The dynamic changes of the calcium fluorescence intensity were observed under the laser scanning confocal microscope (LSCM). The relative level of intracellular calcium was determined by fluorescence indexes.</p><p><b>RESULTS</b>The fluorescence indexes of calcium at 24, 48 and 72 hours were 829.3 +/- 7.8, 801.5 +/- 9.5 and 856.3 +/- 8.7 in the rAd-rPDES-shRNAs group, significantly lower than in the rAd-mock (1106.3 +/- 10.8, 1121.3 +/- 10.2 and 1058.5 +/- 12.1) and blank control group (1076.6 +/- 9.7, 1133.4 +/- 11.2 and 1104.3 +/- 10.5) (P < 0.05).</p><p><b>CONCLUSION</b>Adenovirus mediated shRNAs of the target PDE5 gene can significantly decrease the intracellular calcium level in the smooth muscle cells of the rat corpus cavernosum.</p>