| Objective:Androgen and erectile dysfunction(ED)has close relationship.Androgen deficiency complicated with ED may be associated with endothelial dysfunction of penis tissue,eNOS expression and activity downregulated and RhoA/Rho kinase signaling pathway upregulation at low androgen state,but the mechanism of androgen adjusting the two pathways is not clear at present.Upstream regulation of e NOS and RhoA/Rho kinase is associated with Sphingosine-1-phosphate(S1P).S1 P as an extracellular ligand,and the corresponding cell membrane receptor-combination S1 P receptors triggers a corresponding cell signal transduction,S1 P and S1 P receptor binding different play different biological effects.Our previous studies have found that high blood pressure can inhibit eNOS/NO/cGMP signaling pathway by inhibiting the expression of S1P1 in penis,increase S1P2,S1P3 upregulation of activated RhoA/Rho-kinase signaling pathway cause ED.But in the corpus cavernosum S1P-3 expression and the relationship with androgen is unclear.This study comparing S1P-3 in castrated rats,control group and castrated and testosterone replacement therapy group expression in rat corpus cavernosum tissue,to explore the relationship between S1P-3 and low androgen state of ED and influence on erectile function.To improve the state of low testosterone causes ED(especially unfavorable use androgen replacement therapy,such as prostate cancer patients)has important significance.Methods:18 healthy male SD rats,8 weeks old,which were randomly divided into castrates group,controlgroup and testosterone(T)replacement group.After 4 weeks weighed,Anesthesia before weighing,1% sodium pentobarbital anesthetized rats by intraperitoneal injection of 40mg/kg.Then rats were exposed to the left carotid artery,26 G needle which was filled with heparinized saline punctured the carotid artery and connected the pressure converter,continuous monitoring and recording the mean arterial pressure(MAP).24 G needle which was filled with heparinized saline successfully punctured the corpus cavernosum and connected the pressure converter,measuring the intracavernous pressure(ICP).Open the abdominal cavity,free the prostate leaves and find the cavernous pelvic ganglion as a stimulation spot.Electrical stimulation was applied to different species(the stimulus intensity 3V and 5V,amplitude 5ms,12 Hz stimulation frequency,sustained stimulation 35 s,stimulus interval 3min).Pressure transducer recorded ICPmax/MAP value.After testing penile erectile function,take the corpus cavernosum penis and blood samples.Serum testosterone was measured in three groups.S1 P was also measured in three groups.Penile specimens were divided into two parts.One part was stored in-80℃ ultra low temperature freezer preservation,for the analysis of Western-blot and the other part was used for immunohistochemical analysis immediately.The expressions of S1P1-3,eNOS,P-eNOS,ROCK1 and ROCK2 were detected by immunohisto-chemistry and Western-blot.Results:Serum testosterone in castrates group(11.79±1.18ng/dl)was significantly decreased compared with control group(461.65±29.52ng/dl)and castrates with testosterone group(456.77±38.12ng/dl)(P<0.01),whileserum testosterone in control group had no significant differences with castrates with testosterone group.By measuring each group ICPmax/MAP at 0V voltage stimulation,castrates group(0.088±0.014)was significantly lower compared with control group(0.155±0.011)and castrates with testosterone group(0.153±0.012)(P<0.01),while ICPmax/MAP in control group had no significant differences with castrates with testosterone group.By measuring each group ICPmax/MAP at 3V voltage stimulation,castrates group(0.323±0.014)was significantly lower compared with control group(0.711±0.010)and castrates with testosterone group(0.697±0.017)(P<0.01),while ICPmax/MAP in control group had no significant differences with castrates with testosterone group.By measuring each group ICPmax/MAP at 5V voltage stimulation,castrates group(0.432±0.012)was significantly lower compared with control group(0.819±0.024)and castrates with testosterone group(0.763±0.027)(P<0.01),while ICPmax/MAP in control group had no significant differences with castrates with testosterone group.S1 P in castrates group(6.682±0.289ng/ml)also had no significant differences with control group(6.642±0.216ng/ml)and castrates with testosterone group(6.897±0.165ng/ml).The results of immunohistochemistry showed that S1P1 is mainly expressed in the membrane of endothelial cells.S1P2 is mainly expressed in the corpus cavernosum smooth muscle cells and vascular endothelial cells.S1P3 is mainly expressed in the corpus cavernosum smooth muscle cells and vascular endothelial cells.eNOS,P-eNOS is mainly expressedin the vascular endothelial cells and cavernous sinus cavity.ROCK1,ROCK2 is mainly expressed in the cytoplasm of corpus cavernosum smooth muscle cells.Immunolabeling of S1P1-3,eNOS,P-eNOS,ROCK1 and ROCK2 was stained in brown.Immunohistochemical data were analyzed by the Integrated Optical Density(IOD).The expressions of S1P1,e NOS and P-eNOS were significantly decreased in castrates group than control group and castrates with testosterone group(P<0.01).Whlie the expressions of S1P2,S1P3,ROCK1 and ROCK2 were significantly increased in castrates group than control group and castrates with testosterone group(P<0.01).Western blot analysis were analyzed by the Relative Optical Density(ROD).The protein expressions of S1P1,eNOS and P-eNOS were significantly decreased in castrates group than control group and castrates with testosterone group(P<0.01).Whlie the protein expressions of S1P2,S1P3,ROCK1 and ROCK2 were significantly increased in castrates group than control group and castrates with testosterone group(P<0.01).Serum testosterone levels and S1P1/GAPDH absorbance ratio positively correlated(P<0.05);Serum testosterone levels with S1P2,S1P3/GAPDH absorbance ratio negatively correlated(P<0.05).Conclusion:The decreasing of ICPmax/MAP of rats caused by androgen deficiency is possibly associated with the decline of S1P1 in corpora cavernosum which may prohibit the eNOS/NO/cGMP signaling pathway and the increased expression of S1P2 and S1P3 which may activate the RhoA/Rho kinase signal pathway. |
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