| Objective:Hypertension is closely related to erectile dysfunction(ED),and the main signal pathways are eNOS/NO signal pathway and RhoA/Rho kinase signa l pathway.In the early stage of this study,it was demonstrated that there was a down-regulation of eNOS/NO signaling pathway and up-regulation of RhoA/Rho kinase signaling pathway under hypertension.Sphingosine-1-phosphate(S1P)is involved in the upstream regulation of eNOS and RhoA/Rho kinase.S1P is a kind of biologically active sphingolipid medium.Its combination with its corresponding S1P receptor has biological regulation on various cell types such as vascular endothelial cells and smooth muscle cells,and participates in regulating vascular tone,which can cause contraction and expansion of blood vessels.There are currently 5S1P receptor subtypes,of which sphingosine-1-phosphate 1 S1P1 plays an important role in eNOS-mediated vasodilation,possibly through P13k/Akt signaling.The pathway phosphorylates eNOS at position 1177 after activation of eNOS in vivo,thereby promoting NO biosynthesis and release,and improving erectile function.It is unclear whether it is possible to improve the expression of S1P1 in the corpus cavernosum of hypertensive rats(SHR).This study was to investigate the relationship between the expression of S1P1 and the erectile function in the corpus cavernosum of SHR rats,and to elucidate the relationship between the high expression of S1P1 and eNOS/NO and RhoA/Rho kinase signaling pathways.METHODS:Ten adult healthy male normal hypertension(WKY)rats were randomly divided into WKY group and WKY transfection group.Ten Adult male male SHR rats were randomly divided into SHR group and SHR transfection group.Each group of rats was13 weeks old and weighed about 260-300 g.The WKY transfection group and SHR transfection group were intraperitoneally injected with 1%pentobarbital sodium 30 mg/kg,and the lentivirus carrying the S1P1 gene was injected into both sides of the rat corpus cavernosum.The lentivirus titer was 1×10~9 TU/ml,and each rat was injected with 10?l.The reaction of the rats was carefully observed.After the rats were awake,they were returned to the cage and kept for one week.One week after lentivirus transfection,the ICPmax/MAP value was measured,blood was collected and the corpus cavernosum tissues were taken:the anesthesia dose was calculated for each group before anesthesia,and anesthesia was administered by intraperitoneal injection of 1%sodium pentobarbital at a dose of 30 mg/kg.Fix the anesthetized rats on the operation table,surgically separate and expose the right common carotid artery of the rat,puncture the right common carotid artery with a 26G needle filled with heparin saline,and connect the other side of the needle with pressure transduction.The changes in mean arterial pressure(MAP)of the common carotid artery in rats were continuously monitored.Then,the needle of 24G filled with heparinized saline was inserted into the cavernous body of the rat,and the other side of the needle was connected with a pressure transducer,with stimulation intensity of0V,3V,5V,amplitude of 5ms,stimulation frequency of 12Hz,and different cavernous ganglia.The electrical stimulation of intensity,the duration of stimulation was 45s each time,the stimulation interval was 5 min,and the ICPmax/MAP values of each group were recorded.After the measurement,the rat corpus cavernosum was surgically removed and carefully washed.Blood was collected from the common carotid artery to test the serum testosterone(T)level of each group of rats.The sponge tissues of each group were divided into four parts,which were used for fluorescence staining,Western-blot analysis,immunohistochemical analysis and biochemical detection of nitric oxide(NO)in the sponge.In this experiment,Western blotting and immunohistochemistry were used to detect the expression of eNOS,P-eNOS,ROCK1,ROCK2 and S1P1 in rat corpus cavernosum.RESULTS:ICPmax/MAP:The SHR group at 0V,3V,5V(0.046±0.009,0.223±0.027,0.298±0.020)was significantly lower than the WKY group(0.155±0.015,0.712±0.029,0.765±0.020).(P<0.01),SHR transfection group(0.093±0.006,0.656±0.046,0.744±0.045)was significantly higher than SHR group(P<0.01),WKY transfection group(0.187±0.041,0.768±0.046,0.814±0.042)showed no significant change compared with the WKY group.Serum testosterone levels were WKY group(417.24±22.21 ng/dl),SHR group(409.95±11.8 ng/dl),WKY transfection group(428.82±19.85 ng/dl),SHR transfection group(415.47±11.5 ng/dl)There were no significant differences between the groups.Fluorescent staining showed that the lentivirus distribution of S1P1 gene in each group was transfected into the cavernous endothelial cells.The IOD value was semi-quantitative according to the picture analysis.The transfection rate of WKY transfection group was(86.15±4.02)%,The transfection rate of the SHR transfection group was(89.39±3.14)%.The eNOS,P-eNOS,ROCK1,ROCK2,and S1P1 protein bands in the cavernosal of rats in each group were analyzed by Western blot.The ratio of the target protein to the internal reference(GAPDH)was analyzed.S1P1,P-eNOS protein expression in SHR group and SHR transfection group were lower than WKY group and WKY transfection group(P<0.05),SHR transfection group was significantly higher than SHR group(P<0.01),WKY transfection group was significantly higher than WKY group(P<0.01);ROCK1,ROCK2 protein expression SHR group and SHR transfection.The group was significantly higher than the WKY group and the WKY transfection group(P<0.05),and the SHR transfection group was significantly lower than the SHR group(P<0.01),and the WKY transfection group was significantly lower than WKY group(P<0.01),and there was no significant change in eNOS group.Immunohistochemical localization of eNOS,P-eNOS,ROCK1,ROCK2,S1P1 in the corpus cavernosum of each group:mainly expressed eNOS in vascular endothelial cells and cavernous sinus cavities,and mainly expressed P-eNOS in vascular endothelial cells and cavernous sinus cavity.The cytoplasm of cavernous vascular smooth muscle cells mainly expresses ROCK1 and ROCK2,and vascular endothelial cells mainly express S1P1.Data analysis using integrated optical density(IOD)representation,S1P1,P-eNOS protein expression in SHR group and SHR transfection group were lower than WKY group and WKY transfection group(P<0.05),SHR transfection group was significantly higher than SHR group(P<0.01),WKY transfection group was significantly higher than WKY group(P<0.01);ROCK1,ROCK2 protein expression SHR group and SHR transfection The group was significantly higher than the WKY group and the WKY transfection group(P<0.05),and the SHR transfection group was significantly lower than the SHR group(P<0.01),and the WKY transfection group was significantly lower than WKY group(P<0.01),and there was no significant change in eNOS group.The content of NO in the spongewasWKYgroup(3.95±0.1umol/gprot),SHRgroup(2.42±0.29umol/gprot),WKY transfection group(5.22±0.18umol/gprot),SHR transfection group(3.26±0.44umol/Gprotl).Compared with the SHR group,the NO content in the SHR transfection group was significantly higher(P<0.01),and the WKY transfection group was significantly higher than the WKY group(P<0.01).Results:Up-regulation of S1P1 expression in the corpus cavernosum of spontaneously hypertensive rats may improve erectile function by promoting eNOS/NO signaling pathway and inhibiting RhoA/Rho kinase signaling pathway. |
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