Antioxidant icariside II combined with insulin restores erectile function in streptozotocin-induced type 1 diabetic rats

Abstract

Erectile dysfunction (ED) worsens in patients with diabetes mellitus (DM) despite good control of blood glucose level with insulin. Recent studies imply that diabetic vascular stresses (e.g. oxidative stress) persist in spite of glucose normalization, which is defined as metabolic memory. Studies suggest that the interaction between advanced glycation end products (AGEs) and their receptor (RAGE) mediates the development of metabolic memory. To investigate the effects of the antioxidant icariside II plus insulin on erectile function in streptozotocin (STZ)- induced type 1 diabetic rats. Fifty 8-week-old Sprague-Dawley rats were randomly distributed into five groups: normal control, diabetic, insulin-treated diabetic, icariside II-treated diabetic, and insulin plus icariside II-treated diabetic. Diabetes was induced by a single intraperitoneal injection of STZ. Eight weeks after induction of diabetes, icariside II was administered by gastric lavage once a day (5 mg/kg) for 6 weeks; and 2-6 units of intermediate-acting insulin were given to maintain normal glycemia for 6 weeks. The main outcome measures were the ratio of intracavernous pressure (ICP) to mean arterial pressure (MAP); histology of penile endothelial cells and smooth muscle cells; neural nitric oxide synthase, AGEs and RAGE expression; malondialdehyde concentration; superoxide dismutase activity; and apoptosis index. Diabetic rats demonstrated a significantly lower ICP/MAP ratio, reduced penile endothelial cells, reduced smooth muscle cells, increased AGEs and RAGE, and increased apoptosis. Insulin and icariside II monotherapy partially restored erectile function and histological changes. However, the combination therapy group showed significantly better erectile parameters, cytological components and biochemistry, similar to those in the normal control group. These results suggest that, although insulin can effectively control glycemic levels, it does not completely alter the pathological changes in erectile tissues. Better efficacy could be expected with tight glycemic control plus the antioxidant icariside II. The proposed combination therapy might have the potential to eliminate metabolic memory by down-regulating the AGEs-RAGE-oxidative stress axis.

Keywords: diabetes mellitus; erectile dysfunction; icariside II; metabolic memory.

Figure 1

Evaluation of erectile function. Erectile function was measured in control (C), diabetic (D), D+ insulin (I), D+ICA II, and D+I+ICA II groups (n = 10 in each group). Erectile function was measured via increased intracavernous pressure (ICP) in response to electrostimulation of the major pelvic ganglion. ICP was normalized to mean arterial pressure (MAP). MAP is 160 cmH₂O. * denotes P < 0.05 when comparing the two groups under each end of the capped line.

Figure 2

Evaluation of nNOS expression. Penile tissues were examined for nNOS expression in nerve endings. The results are shown in the representative histological images with the red and blue (DAPI) stains indicating nNOS-positive nerves and cell nuclei, respectively. Quantitative data of nNOS expression in each group are shown in the bar graph. Rats in the combination D+I+ICA II group demonstrated significantly increased nNOS compared to diabetic (D) rats, which neared the level of the control (C) group. The insulin (I) and ICA II monotherapy groups demonstrated partially increased nNOS expression. Western blot result of nNOS showed similar trend to immunofluorescence. * denotes P < 0.05 when comparing the two groups under each end of the capped line.

Figure 3

Evaluation of endothelial content. Penile tissues were examined for von Willebrand Factor (vWF) expression, a marker of endothelial cells. The results are shown in the representative histological images with red (vWF) and blue (DAPI) stains indicating the endothelium and cell nuclei, respectively. Quantitative data of vWF expression in cavernous sinusoids are shown in the bar graph. Rats in the combination D+I+ICA II group demonstrated significantly increased vWF expression compared to diabetic (D) rats, nearing the level of the control (C) group. The insulin (I) and ICA II monotherapy groups demonstrated partially increased vWF expression. Western blot result of eNOS showed similar trend to immunofluorescence. * denotes P < 0.05 when comparing the two groups under each end of the capped line.

Figure 4

Evaluation of smooth muscle content. Penile tissues were examined for phalloidin (Pha) staining, a marker of smooth muscle expression, and DAPI, a marker for cell nuclei. The results are shown in the representative histological images with the green and blue stains indicating smooth muscle and cell nuclei, respectively. Quantitative data for cavernous smooth muscle content are shown in the bar graph. Rats in the combination D+I+ICA II group demonstrated significantly increased Pha compared to diabetic (D) rats, nearing that of the control (C) group. The insulin (I) and ICA II monotherapy groups demonstrated partially increased Pha expression. Western blot result of α-SMA showed similar trend to immunofluorescence.* denotes P < 0.05 when comparing the two groups under each end of the capped line.

Figure 5

Evaluation of AGEs deposition. Representative immunohistochemistry images for AGEs staining in the corpus cavernosum from each group. AGEs deposition is demonstrated in penile corpus cavernosum, and predominantly in the endothelium and smooth muscle. * denotes P < 0.05 when comparing the two groups under each end of the capped line. C, control group; D, diabetic group; I, insulin group; ICA II, icariside II.

Figure 6

(A and B) Evaluation of RAGE expression by Western blot. Representative Western blots showing RAGE and β-actin protein levels in corpus cavernosum tissue of five groups. Quantification of protein levels using RAGE/β-actin was expressed as mean ± SD. RAGE expression is normalized in the ICA II monotherapy and combination therapy groups. * denotes P < 0.05 when comparing the two groups under each end of the capped line. C, control group; D, diabetic group; I, insulin group; ICA II, icariside II. (C) Evaluation of SOD activity. SOD activity in corpus cavernosum tissue from each group. Combination therapy normalized SOD activity, bringing it close to that of the control rats. * denotes P < 0.05 when comparing the two groups under each end of the capped line. (D) Evaluation of malondialdehyde (MDA) content. MDA levels in corpus cavernosum tissue from each group are shown. Combination therapy decreased the MDA level to that of control rats. * denotes P < 0.05 when comparing the two groups under each end of the capped line.

Figure 7

Evaluation of apoptosis with TUNEL staining. Representative images for TUNEL staining of corpus cavernosum tissue are shown. The nuclei of all cells were stained by DAPI (blue fluorescence). Apoptotic index is presented as the ratio of apoptotic nuclei to the total number of nuclei counted. Combination therapy significant reduced the apoptotic index to near control levels. * denotes P < 0.05 when comparing the two groups under each end of the capped line. C, control group; D, diabetic group; I, insulin group; ICA II, icariside II.