| Objective:To investigate whether the low androgen status can affects the erectile function through the intermediate（IKca）and small conductance Ca2+-activated potassium channel 3（SKca3）.Methods:After adaptive feeding for one week,36 healthy SD male rats were completely randomly assigned into six groups:4w-sham group,4w-cast group,4w-cast+T group,8w-sham group,8w-cast group and 8w-cast+T group.The rats in the cast groups and the cast+T groups were all performed surgery castration.The rats in Cast+T group were given testosterone propionate with 3mg/kg subcutaneously every other day after castration,while rats in the other groups were injected with equal amounts of eating vegetable oil.The level of serum testosterone,mean arterial pressure（MAP）and as well as the rat penile corpus maximum intracavernous pressure(ICPmax)were detected at 4 weeks and 8 weeks after treatment.After completing the above detection,the rats were sacrificed to obtain penis specimens according to the experimental animal ethics.Then the penis sample was divided into three parts:the first part for immunohistochemical examination of IKca and SKca3 expression and location in rat penile tissue,the second part for Western blot to detect IKca,SKca3,endothelial nitric oxide synthase（eNOS）and P-eNOS protein expression in rat penile corpus cavernosum tissue,and the last part for SKca3,IKca mRNA expression by RT-qPCR in rat corpus cavernosum.The integral optical density（IOD）of immunohistochemical experiments was measured by Image Pro Plus software to represent the expression level of IKca and SKca3 in rat penile corpus cavernosum tissue.The Quanity One image software was used to measure the relative optical density（OD）of the result band of Western blot to express the expression level of IKca,SKca3,eNOS and P-eNOS proteins.The results of RT-qPCR experiment were calculated by 2-ΔΔCt method to express the relative expression level of SKca3 and IKca mRNA.The data were analyzed by SPSS 19.0 software,and the results were represented by mean±standard deviation.Results:There were no significant difference of MAP in different groups of rats.As compared to the 4w-cham group（21.92±1.90nmol/L）and the 4w-cast+T group（22.18±2.72nmol/L）,serum testosterone levels in the 4w-cast group（1.51±0.27nmol/L）decreased significantly（p<0.01）.Serum testosterone levels in the 8w-cast group（0.37±0.13nmol/L）decreased significantly（p<0.01）as compared to the8w-sham group（21.26±2.69nmol/L）and the 8w-cast+T group（21.52±2.56nmol/L）.Serum testosterone levels in the 8w-cast group decreased significantly compared to the 4w-cast group（p<0.01）.The ratio of ICPmax/MAP under 3-voltage and 5-voltage electrical stimulation decreased significantly in the cast groups（4w-cast group（0.40±0.02,0.56±0.02）,8w-cast group（0.27±0.02,0.42±0.01））compared with the sham groups（4w-sham group（0.65±0.04,0.80±0.02）,8w-sham group（0.65±0.04,0.78±0.02））and the cast+T groups（4w-cast+T group（0.67±0.06,0.81±0.04）,8w-cast+T group（0.67±0.05,0.81±0.01））（p<0.01）as well as in 8w-cast group compared with 4w-cast group（p<0.05）.By immunohistochemical detection,it was found that SKca3 was highly expressed on the membrane of endothelial cells of cavernous sinus and small vessels in the corpus cavernosum,and also in the membrane and the cytoplasm nearby the membrane of the smooth muscle cells that close to the endothelium layer of the cavernous sinus and in the small vessels.IKca showed slightly weaker expression intensity than SKca on the membrane of the endothelial cells,and little expression of IKca was detected in the cytoplasm of smooth muscle cells near the endothelium layer of the cavernous sinus.Versus the sham groups（4w-sham group（1.00±0,1.00±0）,8w-sham group（1.04±0.15,0.97±0.07））and the cast+T groups（4w-cast+T group（1.00±0.02,1.02±0.11）,8w-cast+T group（0.99±0.07,1.04±0.05））,the mRNA expressions of IKca,SKca3 in the cast groups（4w-cast group（0.61±0.06,0.69±0.05）,8w-cast group（0.35±0.02,0.49±0.04））were markedly lower（p<0.01）,and also in 8w-cast group compared with 4w-cast group（p<0.05）,without statistical difference between the control groups and the replacement groups.eNOS,P-eNOS,SKca3,IKca protein relative expressions（target/GAPDH）demonstrated a statistically significant decrease in the cast groups（4w-cast group（0.60±0.07,0.47±0.05,0.67±0.08,0.62±0.05）,8w-cast group（0.42±0.06,0.22±0.07,0.40±0.05,0.44±0.02））compared with the sham groups（4w-sham group（0.82±0.04,0.82±0.01,0.92±0.03,0.90±0.03）,8w-sham group（0.80±0.03,0.79±0.03,0.86±0.04,0.87±0.04））and cast+T groups（4w-cast+T group（0.83±0.01,0.78±0.01,0.88±0.06,0.90±0.04）,8w-cast+Tgroup（0.83±0.04,0.78±0.05,0.85±0.06,0.93±0.04））（p<0.01）,and also decreased significantly in the 8w-cast group compared with the 4w-cast group（p<0.05）.Androgen was positively correlated with the expression of SKca3 and IKca in corpus cavernosum,and the expression of SKca3 and IKca was positively correlated with P-eNOS/eNOS.Conclusion:Low androgen status may inhibit P-eNOS/eNOS by reducing the expression of SK3 and IK channels,resulting in the decrease of ICPmax/MAP. |
