| Objective:To study the effect of S1P3 siRNA on erectile function of spontaneous hypertension rats?SHR?,and investigate the influence of the lentiviru vectors on gene expression of S1P3,ROCK1,ROCK2 and eNOS in the penis,corpus cavernosum injection was carried out.Until now,it is not clear that,the changes of RhoA/Rho kinase pathway in the corpus cavernosum smooth muscle cells of SHR rats and erectile function,after transfected the lentiviral vectors carrying siRNA targeting the S1P3 gene,at living body level.In this experiment,the virus transfection models were bult by corpus cavernosum injection.Methods:experimental animals grouping,A:5 male health 12week old WKY rats,15 male health 12 week old SHR rats were randomly divided into3groups,B1:SHR+siRNA;B2:SHR+GFP;C:SHR.Intraperitoneally anesthetized with sodium pentobarbital,Group B1intracavernously injected with 20ul S1P3 siRNA lentiviral vectors?titer of2x108TU/ml?,10ul each side,Group B2 20ul vacant lentiviral vectors?titer of 2x108TU/ml?,10ul each side,at the root of the penis.One week after injection,rats were weighed.To find out the major pelvic ganglion?MPG?on the posterolateral side of the prostate,the head,limbs,tail and scrotum were fixed,and the abdominal cavity along the midline was opened.Thendissect the left common carotid artery and open the RM6240system.After connected to 24G fine needle and intravenous indwelling needle,the pressure transducer were filled with heparin saline.After calibration and zero adjustment,the venous indwelling needle put into the left common carotid artery,and the 24G fine needle punctured the distal side of the corpus cavernosumon the same horizontal line,avoiding the glan and hematoma,firstly the right side,then the left side.The system would automatedly record rats'introcavernous pressure/mean arterial pressure?ICP/MAP?changes.ICPmax/MAP was reckoned as erecile function.The electrical stimulation parameters were set as,voltage0V,3V,5V,amplitude 5ms,frequency 12Hz.Under each voltage,the stimulation last about 45s,and the rest duration about 5min.The ICP and MAP baseline should be stable.The MPG played as the electrical stimulation site.After erectile function evaluation,the corpus cavernosum tissue and the blood was collected.Instantly put the blood into the low temperature high speed centrifuge,take the supernatant as serum and test the testosterone level of each group.Corpus cavernosum tissue was made into frozen sections and observated green fluorescence after DAPI staining under a fluorescent microscope.Under cannel“1”,the nucleus appered blue,and cannel“4”was green.The ratio of the green fluorescent positive cells to the total was counted as the transfective index?TI?.Immunohistochemistry,western-blot and RT-qPCR was used to detect S1P3,ROCK1,ROCK2 and eNOS gene expression in the rat penis.Results:Weight and Testosterone level in each group was not significantly different.Green fluorescence appeared in groupB1?TI>90%?and groupB2?TI>90%?,but not in groupA and groupC.ICPmax/MAP between group A and group B1 had no significant difference.ICPmax/MAP between group C and group B2 also had no significant difference.But group B1 was significantly improved than group B2 after lentiviral injection?P<0.05?.According to Immunohistochemical detection,S1P3mainly expressed in the cytoplasm of rat corpus cavernosum smooth muscle cells,partly on the membrane of endothelial cells of small vessels.ROCK1,ROCK2mainly expressed in the cytoplasm of rat corpus cavernosum smooth muscle cells.eNOS mainly expressed on the membrane of endothelial cells of cavernous sinus and small vessels in the corpus cavernosum.Compared with group C,S1P3,ROCK1,ROCK2 and eNOSexpression in group B2 was not statistically changed;S1P3,ROCK1,ROCK2 expression in group A and group B1were significantly reduced?P<0.05?,but eNOS increased?P<0.05?.Conclusion:S1P3 siRNA can improve erectile function of SHR,through inhibiting gene expression of S1P3 and downregulating RhoA/Rho kinase passway in the corpus cavernosum. |
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