| Objectives1. To investigate the regulatory effects of androgen on autophagy and apoptosis in corpus cavernosum smooth muscle cells(CCSMCs), especially the regulatory effect of androgen on BECN 1 : Bcl-2 interaction.2. To investigate the regulatory effects of varied concentrations of androgen and activities of androgen receptor(AR) on erectile function modulated by vasoactive intestinal polypeptide(VIP).3. By investigating the effects of varied concentrations of androgen on the expressions of AR and coregulators, and the interactions between AR/coregulators and VIP/c AMP/PKA signaling pathway, thus to find out the erectile function maintenance mechanism in the status of low androgen.Methods1. Male SD rats were randomly divided into six groups: control group(group A), castration group(group B), castration with testosterone undecanoate supplementation group(group C), flutamide supplementation group(group D), castration with flutamide supplementation group(group E), and castration with testosterone undecanoate and flutamide supplementation group(group F).2. The erectile function was determined both in vivo and in vitro, by electric stimulation of the cavernous nerve, and corpus cavernosum strip bath test, respectively.3. The structural change of penis was examined by HE staining, Masson’s trichrome staining and transmission electron microscopy.4. The circulating level of testosterone was determined using enzyme linked immunosorbent assay(ELISA).5. The autophagy and apoptosis level of CCSMCs were observed by transmission electron microscopy and TUNEL staining, respectively.6. Immunohistochemistry and western blotting analysis were performed to determine the indicators of erectile function(e NOS, n NOS), penis structure(α-SMA, TGF-β1), autophagy(LC3, BECN1), apoptosis(Bcl-2, Bax), key molecules of VIP signaling pathway(VPAC2, PDE3 A, Gα s, Gα i), AR and coregulators(CBP, p300, SRC-1, NCo R1, NCo R2).7. The regulatory effects of varied concentrations of androgen and Activities of AR on erectile function modulated by VIP were examined both in vivo and in vitro, by intracavernous injection of VIP, and corpus cavernosum strip bath test, respectively.8. The primary culture of CCSMCs was performed using tissue culture method. Effects of varied concentrations of androgen on the growth of CCSMCs were examined using MTT assay. The effects of varied concentrations of androgen on the expressions of AR and coregulators were examined both in vivo and in vitro, respectively.Results1. Compared with group A, the rats in group B and E had lower penis and prostate weights, decreased smooth muscle/collagen ratio, lower α-SMA and higher TGF-β1 expressions; lower serum testosterone concentration; lower ICP/MAP ratio after cavernous nerve electrostimulation, lower systolic and diastolic capability of corporal strips, and lower expressions of e NOS and n NOS; the serum testosterone concentration was increased in group D, and the levels of other indicators, and various indicators of other groups were between group A and B.2. Compared with control group, the castration group showed significantly decreased autophagosomes, lower expressions of BECN1 and LC3-II; higher apoptotic index, and decreased Bcl-2/Bax ratio.3. After intracavernous injection of VIP, the ICP/MAP ratios in all groups were increased, but there were no significant differences between groups. However, the ratios of p ICP/p MAP(post intracavernous injection of VIP) to b ICP/b MAP(before intracavernous injection of VIP) were different between groups. Descending order was as follows: group E>group B>group D>group C>group A>group F. There was no significant difference of VIP-induced maximum relaxation response between groups. The castrated rats had higher expressions of VPAC2 and Gα s, and lower expressions of PDE3 A and Gα i-2.4. The effects of testosterone on the growth of CCSMCs were dose-dependent. The dihydrotestosterone concentration of 10-5 mol/L had the most significant effect for the growth of CCSMCs. Too low(<10-9 mol/L) or too high(>10-4 mol/L) concentrations of dihydrotestosterone would slow the growth of CCSMCs.5. In vivo study: there was no significant difference of the expression of AR in corpus cavernosum between groups A, B and C. Compared with control group, the castration group showed higher expressions of AR co-activators of CBP, p300 and SRC-1. In vitro study: compared with physiological dose androgen group, the low dose androgen group had lower expression of AR in the CCSMCs, and high dose androgen group had higher expression of AR in the CCSMCs. The low dose androgen group showed lower expressions of AR co-activators of CBP, p300 and SRC-1.Conclusions1. Androgen deprivation attenuates erectile function and induces corporeal fibrosis by inhibiting autophagy and promoting apoptosis of CCSMCs in rats, maybe mainly by regulating BECN 1 : Bcl-2 interaction.2. The erectile effect of VIP may not depend on the concentrations of androgen or activities of AR, making VIP an androgen independent erectile neurotransmitter. The VIP/c AMP/PKA pathway may play a supporting role in the status of normal androgen level, but significant role in the status of low androgen level.3. Androgen plays a complex regulatory role in AR and coregulators. There is cross-talk between VIP/c AMP/PKA and AR/coregulators signaling pathways. Under the low level of androgen, the expression of AR in CCSMCs is unchanged or reduced, but the AR coregulators(CBP, p300, SRC-1) are up-regulated. Aberrant expressions of AR coregulators crossover activate the VIP/c AMP/PKA signaling. Thus, the erectile function is being maintained under low levels of androgen. |
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