| Objective: Penile erection is a complex physiological process. A variety ofsignaling pathways are involved in penile erection process, including NOS/NOsignaling pathway, RhoA/Rho kinase signaling pathways,HO/CO pathway andhydrogen sulfide pathway. Currently,we find that RhoA/Rho kinasemediatedcalcium signaling system sensitive mechanisms is involved in penile erectionprocess, mediated by calcium-dependent mechanism of corpus cavernosumsmooth muscle contraction. There are two known Rho kinase isoforms:ROCK1and ROCK2. Many diseases can cause erectile dysfunction, and highblood pressure is a major cause of ED. We studied the change of erectilefunction of spontaneously hypertensive rats after blocking expression ofROCK2gene with ROCK2siRNA. Methods:30healthy male12-week-oldSHR and30healthy male12-week-old WKY rats were randomly divided intosix groups：WKY control group（group A）,WKY+GFP group(group B),WKY+siRNA group （group C）, SHR control group（group D）, SHR+GFP group（group E）,SHR+siRNAgroup（group F）. Briefly, rats were intraperitoneallyanesthetized with sodium pentobarbital (50mg/kg). Revealing the penis of rats,group A and group D as the control group, group B and group E were inducedby a single intracavernous injection of20ul GFP (titer of3x108TU/ml) withmicro-syringe,group C and group F were induced by a single intracavernousinjection of20ul siRNA (titer of3x108TU/ml), slowly injected into cavernous with10ul each side.we retained needle for three minutes after injection andhemostasis after removing the needle. One week after injection, rats wereweighed. Briefly, rats were intraperitoneally anesthetized with sodiumpentobarbital (50mg/kg). the left carotid artery was exposed by30G needlepuncture filling with heparinized saline,baroreceptor connected computercontinuous monitoring and recorded MAP.along the dorsal penile skin, we cutuntil the middle of the pubic symphysis and cut to the top of the scrotum inventral for Appearing the penis. Along the ventral midline abdominal cavitywas opened, we found pelvic ganglion (major pelvic ganglion, MPG) outsideof the prostate as the electrical stimulation site.24G needle that was filled withheparinized saline was punctured sponge pressure transducer connector torecord ICP, the stability of the electrical stimulation applied to the MPG(stimulation voltage is3v or5v, frequency is12Hz, amplitude is5ms,durationof one minute, stimulation interval is3min), using the RM6240series ofmulti-channel physiological signal acquisition and processing system recordsrats cavernous pressure/mean arterial pressure (ICP/MAP) changes.2ml heartblood was detected testosterone values. Corpora cavernosa tissue was frozensections and Observated green fluorescence after DAPI staining by afluorescent microscope. The corpora cavernosa tissue was made intohomogenates for detecting of nitric oxide synthase (NOS) activity.Immunohistochemistry and western blot detected the expressions of ROCK2,eNOS and p-eNOS in the corpus cavernosum. Results: Weight and Testosterone value in each group were not significantly different. ICP/MAPamong groups A, group B and C group had no significant difference, while thegroup F was significantly higher than group D and group E (P <0.05).Cavernous green fluorescence was observed in group B, group C, group E andgroup F, but there was no green fluorescence in group A and group D. NOSactivity of cavernous tissue in each group was detected, which in group C wassignificantly higher than group A and group B.NOS activity in group F wassignificantly higher than group D and group F(P <0.05). Immunohistochemicaldetection of the ROCK2mainly expressed in the cytoplasm of rat corpuscavernosum smooth muscle cells, eNOS mainly expressed in vascularendothelial cells, p-eNOS also mainly expressed in vascular endothelial cells.western blot detected ROCK2protein expression and found values in group Cwas significantly lower than that in group A and group B (P <0.05), which ingroup F was significantly higher than group D and group F (P <0.05).TheeNOS and p-eNOS protein in Group C were higher than that in group A andgroup B,and eNOS and p-eNOS in group F were significantly higher thangroup D and group E. Conclusions: SiRNA blocking ROCK2gene can inhibitthe expression of ROCK protein in the cavernous tissue, causing enhance theexpression of eNOS and p-eNOS and significantly enhanced erectile functionin SHR ED rats. |
