The Expression Of Phospho-Erk1/2 And Phospho-Akt1 In Corpus Cavernosum Of Spontaneous Hypertensive Rats

Object:To understand the expression of phospho-Erkl/2 and phospho-Aktl in corpus cavernosum between Spontaneous Hypertensive Rats(SHR) and normatensive rats. Methods:1.Experim-ental animal:Healthy adult male SHR and control group WKY each 8,14 weeks, weight 250～300g.2.Erectile functional examination:before anesthesia, rats weighing, to anesthetize the rats by pentobarbital sodium(30mg/Kg,intraperitoneal injection). Then, to expose the arteria carotis communis,the 30G pin which be filled with hepatinate puncture the arteria carotis communis, and connect the pressure translator, computer monitoring and recording MAP continuously. The rat abdominal midline incision, carefully under the microscope at 10 times free prostate on both sides of the rear to expose the pelvic ganglion, along the surface of prostate and rectum will denonvillies fascia between the cut, find the cavernous nerves in the dorsal prostate (corpus nerve, CN), cotton pad covered with warm saline, protection, maintenance of electrical stimulation used.After the penis exposed to the 24G needle filled with heparin saline into the right corpus cavernosum, connected to another pressure converters, continuous monitoring of intracavernosal internal pressure ((intracavernosal pressure, ICP).The basis of computer records a pressure ICP, will stainless steel electrodes placed in the cavernous nerve, applying different electrical stimulation (stimulation voltage was 3V,5V, frequency 12Hz, amplitude 5ms, sustained 30s～60s), continuous record of electrical stimulation ICP/MAP changes.3. draw the materials and preparation:After determination of erectile function, namely, near the corpus cavernosum penis root interception Department to PBs solution (0.01 moL/L, pH 7.4) washed clean of blood remaining after the urethra and penis glans removed parts of penis specimens divided into two parts, and immediately placed in EP tube, part of the neutral formalin-fixed for immunohistochemical analysis, part of the refrigerator at-70℃Ultra-low temperature preservation for RT-PCR experiments.4. Immunohistochemical determination of P-Erk1/2 and P-Aktl protein expression in rat corpus cavernosum:SP two-step method, penis specimens were embedded in paraffin after conventional treatment by immunohistochemical paraffin section, PBS wash 10min; 0.3% H2O2-methanol treatment 30min; pressure restoration 5min; 10% normal goat serum at room temperature closed 15min; drops plus an anti-in specimens,4℃overnight; PBS wash three times, each time 10 min; dropping the second antibody and incubated at room temperature 30min; DAB color.5. RT-PCR detect the expression of P-Erkl/2mRNA and P-Aktl mRNA in rat corpus cavernosum:Based on a small amount of tissue/cell total RNA extraction kit and molecular cloning manual Guide (Second Edition) to operate within the table in the corpus cavernosum tissue extract total RNA, stored at -80℃Ultra-low temperature refrigerator by equipment for use with the Nanodrop measurement of total RNA concentration, according to conventional RT-PCR steps to complete the RT-PCR, electrophoresis apparatus with a LAS-300 imaging scans, semi-quantitative analysis P-Akt1mRNA on P-Erk1/2mRNA. 6.Statistical Analysis:all datas were expressed as mean±standard error, statistical analysis was accomplished with SPSS 14.0 software, the two groups using t test, P<0.05 for the difference was significant. Results: Compared with the controls, ICP/MAP was lower significantly in the SHR group (P＜0.05), The expression of mRNA of P-ERK1, P-ERK2 and protein of P-ERK1/2 in the SHR group (0.81±0.05, 0.91±0.06 and 47.22±10.05, respectively) was significantly increased than that of in the WKY group (0.42±0.04,0.68±0.14 and 7.49±1.45, respectively) (P＜0.05). The level of mRNA of P-Akt and protein of P-Akt in the SHR group (0.90±0.05 and 11.17±2.21) was not significantly with that of in the WKY group (0.92±0.06 and 10.91±1.86). (P＞0.05).Conclusion:The occurrence of erectile dysfunction in hypertension is in relation to the overexpressing of P-Erkl/2 in the cavernous tissues, but is not obvious correlation with the expressing of P-Aktl.