Effect of icarisid II on diabetic rats with erectile dysfunction and its potential mechanism via assessment of AGEs, autophagy, mTOR and the NO-cGMP pathway

Abstract

Erectile dysfunction (ED) is a major complication of diabetes mellitus. Icariin has been shown to enhance erectile function through its bioactive form, icarisid II. This study investigates the effects of icarisid II on diabetic rats with ED and its potential mechanism via the assessment of advanced glycosylation end products (AGEs), autophagy, mTOR and the NO-cGMP pathway. Icarisid II was extracted from icariin by an enzymatic method. In the control and diabetic ED groups, rats were administered normal saline; in the icarisid II group, rats were administered icarisid II intragastrically. Erectile function was evaluated by measuring intracavernosal pressure/mean arterial pressure (ICP/MAP). AGE concentrations, nitric oxide synthase (NOS) activity and cGMP concentration were assessed by enzyme immunoassay. Cell proliferation was analysed using methyl thiazolyl tetrazolium assay and flow cytometry. Autophagosomes were observed by transmission electron microscopy, monodansylcadaverine staining and GFP-LC3 localisation. The expression of NOS isoforms and key proteins in autophagy were examined by western blot. Our results have shown that Icarisid II increased ICP/MAP values, the smooth muscle cell (SMC) growth curve, S phase and SMC/collagen fibril (SMC/CF) proportions and decreased Beclin 1 (P<0.05). Icarisid II significantly increased the proliferative index and p-p70S6K(Thr389) levels and decreased the numbers of autophagosomes and the levels of LC3-II (P<0.01). Icarisid II decreased AGE concentrations and increased cGMP concentration, NOS activity (P<0.05) and cNOS levels (P<0.01) in the diabetic ED group. Therefore, Icarisid II constitutes a promising compound for diabetic ED and might be involved in the upregulation of SMC proliferation and the NO-cGMP pathway and the downregulation of AGEs, autophagy and the mTOR pathway.

Figure 1

The ratio of intracavernosal pressure and mean arterial pressure (ICP/MAP) was investigated to evaluate erectile function in each group. (a) A Biopac physiograph displays the ICP (red curve) and MAP (black curve) values of representative rats in each group. (b) ICP/MAP levels were analysed with acqKnowledge software (BioPac Systems, Santa Barbara, CA,USA) and ANOVA. ^★★P<0.01, ^★P<0.05 vs. diabetic ED group. ^▴P<0.05 vs. control group.

Figure 2

Morphological analysis of the ratio of smooth muscle cells/collagen fibrils (SMCs/CFs) in the corpus spongiosum from a representative of each group. (a) Masson's trichrome staining shows SMCs (red areas) and CFs (blue areas) of representative rats in each group. (b) The morphometric analysis of the area fraction (SMCs/CFs) was calculated using Leica QWin Pro V2.6 image analysis and processing software (Leica DMIRB, Leica, Wetzlar, Germany). Data are presented as the area fraction means from the 20 visual fields randomly selected from every specimen in each group. The differences were analysed by ANOVA. ^★★P<0.01, ^★P<0.05 vs. diabetic ED group. ^▴P<0.05 vs. control group.

Figure 3

The cell growth curve and cell cycle analysis in the smooth muscle cells (SMCs) from each group. (a) The cell growth curve was assayed from the optical density (o.d.) values using the MTT method. (b) The cell cycle was analysed by flow cytometry. (c) The differences were then analysed by ANOVA. ^★★P<0.01 and ^★P<0.05 vs. the diabetic ED group. PI, proliferative index.

Figure 4

Morphological analysis of autophagy in the smooth muscle cells (SMCs) from each group. (a) Punctate autophagosomes were observed using transmission electron microscopy monodansylcadaverine staining and GFP-LC3 transfection localisation methods. (b) The area fractions (autophagosomes/cytoplasm) were calculated using Leica QWin Pro V2.6 image analysis and processing software. Data were presented as the means of the area fractions (autophagosomes/cytoplasm) in the 50 cells randomly selected from every specimen in each group. The differences were then analysed by ANOVA. ^★★P<0.01 vs. the diabetic erectile dysfunction (ED) group. ^▴P<0.05 vs. the control group.

Figure 5

Expression of mTOR signalling pathway proteins in the corpus cavernosum tissue from each group. (a) Western blot of LC3-I/II, Beclin 1, p70S6K, p-p70S6K(Thr389) and β-actin. (b) The relative expression levels were calculated by normalising to β-actin. The differences were then analysed by ANOVA. ^★★P<0.01 and ^★P<0.05 vs. the diabetic erectile dysfunction (ED) group. ^▴▴P<0.01 and ^▴P<0.05 vs. the control group.

Figure 6

Expression of nitric oxide synthase (NOS) isoforms in the corpus cavernosum tissue from each group. (a) Western blot of nNOS, eNOS and iNOS expression. (b) The relative expression levels were calculated by normalising to β-actin. The differences were then analysed by ANOVA. ^★★P<0.01 vs. the diabetic erectile dysfunction (ED) group. ^▴▴P<0.01 vs. the control group.