Effects Of Low Androgen Status On The Expression Of P2Y Receptor In Corpus Cavernosum Of Rats

Objective:Erectile dysfunction refers to that when adult male in sexual intercourse with a sexual partner,the penis is persistent or repetitively unable to erect or hard to erect so that it can't complete the sexual behavior that both men and women are satisfied with.Even though ED is a mild chronic benign disease,still may affect the patient's emotional communication with their sexual partners,thus affecting the patient's emotional life,personal self-confidence and happiness of the patient,and has a relatively large negative impact on the overall quality of life of the individual.Animal models are established by surgery and androgen replacement therapy to investigate whether low testosterone levels can modulate eNOS expression and activation in the corpus cavernosum tissue through the P2Y receptor subtype and thereby affect penile erection,which is of great value to provide new therapeutic targets for ED caused by low testosterone in middle-aged and elderly men,prostate cancer patients.Methods:Eight weeks old healthy male Sprague-Dawley rats?n=36?were fed adaptively for 1 week and randomly divided into 6 groups,including castration+testosterone replacement therapy for 4 weeks(group A:cast+testosterone?T?^4-week),castration 4-week group(group B:cast ^4-week),sham4-week group(group C:sham ^4-week),castration+testosterone replacement therapy for 8-week group(group D:cast+T ^8-week),castration 8-week group(E group:cast ^8-week),sham-operated 8-week group(F group:sham ^8-week).cast^4-weekand cast ^8-week rats were castrated by the way of surgery.Testosterone 3mg/kg/2d was injected subcutaneously on cast+T ^4-week and cast+T ^8-week rats 1day after castration.The same dose of vegetable oil was injected subcutaneously in the cast ^4-week,sham ^4-week,cast ^8-week,and sham ^8-week groups.The ICPmax)/?MAP?in each group was measured at 4 and 8 weeks respectively.The m RNA expression of P2Y1,P2Y2,P2Y4 and P2Y6 in the corpus cavernosum of each group was detected by RT-PCR.Western blot was used to detect the expression of eNOS,P-eNOS,P2Y1,P2Y2,P2Y4 and P2Y6in corpus cavernosum of rats in each group.Results:There was no significant difference in body weight(group A cast+T ^4-week:295.33±1.79g?group B cast^4-week:296.35±2.69g?group C sham ^4-week:296.65±1.51g?group D cast+T ^8-week:295.22±2.84g?group E cast ^8-week:297.57±1.53g?group F sham ^8-week:295.37±3.28g)and arterial blood pressure(group A cast+T ^4-week:119.19±5.31mmHg?group B cast ^4-week:119.15±5.31 mmHg?group C sham^4-week:119.21±5.14 mmHg?group D cast+T ^8-week:119.20±5.24 mmHg?group E cast ^8-week:119.29±5.10mmHg?group F sham ^8-week:119.37±5.01mmHg)in each group of rats.The rat serum T levels displayed no significant difference among the groups of cast+T ^4-week?20.46±3.43nmol/L?,sham ^4-week?20.63±3.30nmol/L?,cast+T ^8-week?17.78±2.67nmol/L?,and sham ^8-week?18.57±2.84nmol/L?.However,they showed significant reduction in the group of cast ^4-week(1.35±0.41nmol/L compared with cast+T ^4-week?20.46±3.43nmol/L?or sham ^4-week?20.63±3.30nmol/L??P<0.01?,and displayed significant reduction in the group of cast ^8-week?0.30±0.17nmol/L?compared with cast+T ^8-week?17.78±2.67nmol/L?or sham ^8-week?18.57±2.84nmol/L??P<0.01?,as well as a significant reduction in the group of cast ^8-week?0.30±0.17nmol/L?versus cast ^4-week?1.35±0.41nmol/L?,?P<0.05?.Following the electric stimulation of rat MPG at the intensity of 3 or 5 V,the ICPmax/MAP ratios showed a significant reduction in the group of cast ^4-week?0.39±0.03,0.54±0.04?versus cast+T ^4-week?0.70±0.04,0.83±0.01?or sham^4-week?0.69±0.01,0.82±0.01??P<0.01?;a significant reduction in the group of cast ^8-week?0.26±0.03,0.36±0.01?versus cast+T ^8-week?0.70±0.04,0.82±0.02?or 8 sham ^8-week?0.70±0.02,0.80±0.02??P<0.01?;and significant reduction in the group of cast ^8-week?0.26±0.03,0.36±0.01?versus cast ^4-week?0.39±0.03,0.54±0.04??P<0.05?.Meanwhile,the ICPmax/MAP ratios showed no significant difference among the cast+T ^4-week,sham ^4-week,cast+T ^8-week,and sham ^8-weekgroups.The mRNA levels of four genes displayed no significant difference among the groups of cast+T ^4-week,sham ^4-week,cast+T ^8-week,and sham^8-week,while showed significant reduction in the group of cast ^4-week?0.75±0.08?0.86±0.02?0.73±0.09?0.86±0.03?versus cast+T ^4-week?1.17±0.04?1.18±0.06?1.23±0.12?1.16±0.03?or sham ^4-week?1.12±0.09?1.19±0.03?1.18±0.10?1.16±0.02??p<0.01?,and displayed significant reduction in the group of cast ^8-week?0.51±0.07?0.50±0.03?0.42±0.16?0.59±0.02?versus cast+T ^8-week?1.18±0.04?1.17±0.01?1.20±0.07?1.15±0.03?or sham ^8-week?1.17±0.02?1.16±0.04?1.24±0.02?1.15±0.04??P<0.01?,and significant reduction in the group of cast ^8-week?0.51±0.07?0.50±0.03?0.42±0.16?0.59±0.02?versus cast ^4-week?0.75±0.08?0.86±0.02?0.73±0.09?0.86±0.03??P<0.05?The protein levels of P2Y1,P2Y2,P2Y4,P2Y6,eNOS,and p-eNOS showed no significant difference among the groups of cast+T ^4-week,sham ^4-week,cast+T ^8-week,and sham ^8-week,while they showed significant reduction in the group of cast ^4-week?0.85±0.06?0.88±0.13?0.83±0.10?0.69±0.08?0.82±0.12?0.63±0.17?versus cast+T ^4-week?1.16±0.05?1.23±0.10?1.19±0.14?1.14±0.03?1.18±0.03?1.23±0.02?or sham ^4-week?1.20±0.03?1.21±0.07?1.19±0.16?1.12±0.06?1.18±0.06?1.28±0.07??P<0.01?,displayed significant reduction in the group of cast ^8-week?0.55±0.04?0.48±0.09?0.56±0.23?0.32±0.05?0.55±0.04?0.33±0.21?versus cast+T ^8-week?1.14±0.08?1.21±0.12?1.10±0.09?1.11±0.03?1.17±0.06?1.27±0.07?or sham ^8-week?1.13±0.09?1.14±0.04?1.18±0.18?1.11±0.03?1.18±0.05?1.28±0.10??P<0.01?,and showed a significant reduction in the group of cast ^8-week versus cast ^4-week?P<0.05?According to the correlation analysis,the serum level of testosterone in each group was positively correlated with the expression of P2Y1 protein,the correlation equation was Y=0.705+0.023X,r=0.861,P<0.01;It was also positively related to P2Y2,the correlation equation was Y=0.676+0.026X,r=0.837,P<0.01;It was also positively related to P2Y4,the correlation equation was Y=0.702+0.023X,r=0.764,P<0.01;It was also positively related to P2Y6,the correlation equation was Y=0.509+0.031X,r=0.901,P<0.01.Conclusion:low T levels probably downregulated the expression of the P2Y1,P2Y2,P2Y4,and P2Y6 receptors,suppressing eNOS phosphorylation,eNOS activity,and erectile function.Given that the divergent subtypes of P2Y receptors share the same or similar antigenic determinants,uncertainty in the data may exist.Hence,further confirmation via gene knockout or transfection is required.