Hengjun Xiao

Total triterpenoids from Ganoderma Lucidum suppresses prostate cancer cell growth by inducing growth arrest and apoptosis.

SummaryIn this study, one immortalized human normal prostatic epithelial cell line (BPH) and four human prostate cancer cell lines (LNCaP, 22Rv1, PC-3, and DU-145) were treated with Ganoderma Lucidum triterpenoids (GLT) at different doses and for different time periods. Cell viability, apoptosis, and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and -3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.In this study, one immortalized human normal prostatic epithelial cell line (BPH) and four human prostate cancer cell lines (LNCaP, 22Rv1, PC-3, and DU-145) were treated with Ganoderma Lucidum triterpenoids (GLT) at different doses and for different time periods. Cell viability, apoptosis, and cell cycle were analyzed using flow cytometry and chemical assays. Gene expression and binding to DNA were assessed using real-time PCR and Western blotting. It was found that GLT dose-dependently inhibited prostate cancer cell growth through induction of apoptosis and cell cycle arrest at G1 phase. GLT-induced apoptosis was due to activation of Caspases-9 and -3 and turning on the downstream apoptotic events. GLT-induced cell cycle arrest (mainly G1 arrest) was due to up-regulation of p21 expression at the early time and down-regulation of cyclin-dependent kinase 4 (CDK4) and E2F1 expression at the late time. These findings demonstrate that GLT suppresses prostate cancer cell growth by inducing growth arrest and apoptosis, which might suggest that GLT or Ganoderma Lucidum could be used as a potential therapeutic drug for prostate cancer.

Antioxidative Protective Effect of Icariin on the FeSO4/H2O2-damaged Human Sperm Based on Confocal Raman Micro-spectroscopy *

SummaryOxidative stress is implicated in male infertility and significantly higher reactive oxygen species are detected in 25% of infertile males. Although different agents of various alternative medicines, including traditional Chinese medicine, have been tried with varying success, evidence remains limited on whether and how much herbs or supplements might help increase the anti-oxidant ability of the sperm. This study examined the anti-oxidative effects of icariin, a flavonoid isolated from HerbaEpimedii, on the human sperm. We prepared the FeSO4/H2O2-damaged human sperms, which were co-cultured with icariin in vitro, and then observed the changes of the sperm by employing Raman micro-spectroscopy. The results showed that Raman mapping with a 514 nm excitation laser allowed clear differentiation of the nucleus, neck, and, in particular, the mitochondria-rich middle piece of a human sperm cell. The effect of icariin on different organelles of the sperm was quantified by localized spectral Raman signatures obtained within milli-seconds, and icariin could keep the “Raman fingerprint” of the human sperm the same as the control groups, suggesting that icariin could protect the human sperm from being damaged by FeSO4/H2O2. Icariin may serve as a tonifying and replenishing agent of herbal origin for enhancing reproductive functions.Oxidative stress is implicated in male infertility and significantly higher reactive oxygen species are detected in 25% of infertile males. Although different agents of various alternative medicines, including traditional Chinese medicine, have been tried with varying success, evidence remains limited on whether and how much herbs or supplements might help increase the anti-oxidant ability of the sperm. This study examined the anti-oxidative effects of icariin, a flavonoid isolated from Herba Epimedii, on the human sperm. We prepared the FeSO4/H2O2-damaged human sperms, which were co-cultured with icariin in vitro, and then observed the changes of the sperm by employing Raman micro-spectroscopy. The results showed that Raman mapping with a 514 nm excitation laser allowed clear differentiation of the nucleus, neck, and, in particular, the mitochondria-rich middle piece of a human sperm cell. The effect of icariin on different organelles of the sperm was quantified by localized spectral Raman signatures obtained within milli-seconds, and icariin could keep the “Raman fingerprint” of the human sperm the same as the control groups, suggesting that icariin could protect the human sperm from being damaged by FeSO4/H2O2. Icariin may serve as a tonifying and replenishing agent of herbal origin for enhancing reproductive functions.

ORIGINAL RESEARCH–SURGERY: Treatment of Penile Deep Dorsal Venous Leakage of Erectile Dysfunction by Embedding the Deep Dorsal Vein of the Penis: A Single Center Experience with 17 Patients

INTRODUCTION The common surgery for venous leakage was not very successful; unsatisfactory long-term results have reduced the indications for venous surgery for erectile dysfunction (ED). AIMS To assess the outcomes of embedding the deep dorsal vein of the penis (EDDVP), a new surgical technique used in patients with penile deep dorsal venous leakage of ED. METHODS Between December 2001 and November 2007, 17 patients diagnosed with penile deep dorsal venous leakage of ED underwent embedding the deep dorsal vein of the penis. MAIN OUTCOME MEASURES All cases were available for follow up by using the abridged 5-item version of the International Index of Erectile Function (IIEF-5) scoring system and penile color Doppler ultrasound. Dynamic cavernosography were also assessed in three patients at 3 months postoperatively. RESULTS After surgery, 14 patients were able to achieve satisfactory intercourse and three had sufficient erection after oral sildenafil (50-100 mg). The IIEF-5 scoring changed from a preoperative mean IIEF-5 score of 8.8 +/- 3.9 to 20.8 +/- 4.1 (P < 0.05). Peak systolic velocity (average of right and left cavernosal arteries) changed from 41.9 +/- 7.7 cm/second to 44.2 +/- 9.2 cm/second (P > 0.05), resistance index changed from 0.79 +/- 0.1 to 1.00 +/- 0.0 (P < 0.05), and venous velocity changed from 8.4 +/- 4.0 cm/second to 0.0 +/- 0.0 cm/second (P < 0.05). Dynamic cavernosography demonstrated a smooth flow of the deep dorsal vein during the flaccid phase. During the tumescent phase, the deep dorsal vein of the penis was compressed between the dilated sinusoidal spaces and the tunica albuginea and resulted in venous drainage blockade. And then the hardness of erection was improved and maintained. CONCLUSIONS The new surgical technique of EDDVP is a simple operative procedure, which seems to be efficient in the treatment of penile deep dorsal venous leakage of ED.

GLUT1 regulates cell glycolysis and proliferation in prostate cancer

Glucose transporter 1 (GLUT1) plays a critical role in tumorigenesis and tumor progression in multiple cancer types. However, the specific function and clinical significance of GLUT1 in prostate cancer (PCa) are still unclear. Therefore, in this study, we investigated the role of GLUT1 in PCa.

Loupe-assisted versus microscopic varicocelectomy: is there an intraoperative anatomic difference?

The aim of this study was to compare the intraoperative difference in anatomic details between loupe-assisted and microscopic varicocelectomy within the same spermatic cord. Between April 2011 and August 2011, 26 men with 33 sides containing grade 2-3 varicocele were enrolled in this study. First, one surgeon performed the open inguinal varicocelectomy under × 3.5 loupe magnification. The presumed vascular channels and lymphatics were isolated and marked without ligation. Another surgeon then microsurgically dissected and checked the same spermatic cord using an operating microscope to judge the results in terms of the ligation of the internal spermatic veins and the preservation of the arteries and lymphatics. There were significant differences in the average number of internal spermatic arteries (1.51 vs 0.97), internal spermatic veins (5.70 vs 4.39) and lymphatics (3.52 vs 1.61) between the microscope and loupe-assisted procedures (P < 0.001, P < 0.001, P < 0.001, respectively). Meanwhile, in varicocele repair with loupe magnification, an average of 1.30 ± 1.07 (43/33) internal spermatic veins per side were missed, among the overlooked veins, 1.12 ± 0.93 (37/33) were adhered to the preserved testicular artery, as well as 0.55 ± 0.79 lymphatics and 0.36 ± 0.55 arteries that were to be ligated. In conclusion, microscopic varicocelectomy could preserve more internal spermatic arteries and lymphatics and could ligate more veins than the loupe-assisted procedure. To some degree, loupe magnification is inadequate for the reliable identification and dissection of the tiny vessels of the spermatic cord, as most of the overlooked veins were adhered to the preserved testicular artery.

MP81-18 INTRACAVERNOUS INJECTION OF BONE MARROW MESENCHYMAL STEM CELLS MODIFIED WITH PDE5-RNAI IMPROVES ERECTILE DYSFUNCTION IN AGED RATS

replacement), indicates an enrichment of stem cell markers upon castration; CD44, NKX3.1 and ALDH1 isoforms. Microarray analysis has confirmed upregulation of CD44 and ALDH1A1 at castrated state, versus downregulation upon androgen replacement at early time points (24 hours). We isolated CD44þ/-ALDHhigh/low subpopulations by flow cytometry for transcriptome analysis. Castration in the BM-18 model induces an increase in the subpopulations of CD44þ/ALDHlow (from 0.44% to 2.3%) and CD44þ/ALDHhigh (from 0.06% to 0.38%). However, the same subpopulations are not increased in the LAPC-9 model upon castration, while only the CD44-/ALDHhigh subset is enriched (from 3.5% to 7.1%). Organoids derived from bulk BM-18 and LAPC-9 tumors are reestablishing tumor formation upon intraosseous inoculation. CONCLUSIONS: Different androgen-independent cancer stem cell subpopulations may be distinguished by the ALDH activity status in combination with CD44. The androgen-independent cells in the BM-18 androgen-dependent model are reflected by enrichment of CD44 expression, as well as a rare double positive population. In the androgen-independent LAPC-9 model, CD44 expression is decreased potentially reflecting androgen-dependent CD44þ cells, and ALDH activity increased in CD44cells.

AB231. The relationship between cardiovascular disease and erectile dysfunction among aged men in Southern China

Objective To investigate the relationship between ED and CVD. Methods A total of 103 male patients aged 40–70 years old (mean age 58.3 years) with ED in Southern China were recruited. ED was assessed by International Index of Erectile Function 5 (IIEF-5) score. The presences of risk factors for CVD were evaluated by lifestyle questionnaires in those men. Results CVD increased according to severity of ED, adjusted for age, smoking, total cholesterol level, hypertension, and body mass index (P<0.01, by analysis of covariance). Of those patients with CVD in coronary heart disease, hypertension, and dyslipidemia, IIEF-5 scores were reported by 10.9±4.1, 15.2±3.9, and 18.3±3.6, respectively. Low IIEF score (<12) showed a significant increased risk of CVD compared with mild ED (P<0.001). The prevalences of CVD in mild, moderate and severe ED were reported by 5.3% (3/57), 26.5% (9/34), and 41.7% (5/12), respectively. Risk factors for CVD are significantly associated with ED (P<0.01). Conclusions Our results demonstrate that men with ED, especially those patients presenting with moderate-to-severe ED should be identify men at higher risk for CVD events. These findings suggest that ED usually precedes CVD onset, and it might be considered an early marker of symptomatic CVD.

AB115. Effects of target gene expression on ex vivo differentiated mesenchymal stromal cells transfected by lentiviral vector with PDE5 of short hairpin RNA

Objective To investigate the expression of PDE5 in ex vivo differentiation of gene-modified BM-MSCs using the lentiviral vector containing silencing target gene PDE5 of shRNA. Methods SD rat bone marrow-derived MSCs were separated, cultured and purified in vitro by Percoll density gradient centrifugation combined with adherent culture. The third passage rat BM-MSCs were obtained, and were identified by cell surface markers with flow cytometry (FCM). The lentiviral vector carrying silencing target gene PDE5 of shRNA was constructed and then transfected into the third passage rat BM-MSCs. The gene-modified rat BM-MSCs were induced to differentiate into smooth muscle-like cells exposed to VEGF and b-FGF media in vitro. The proliferative ability of these cells was tested by cell counting kit 8 (CCK-8). Smooth muscle-like cells differentiation was assessed by immunofluorescence and then subjected to immunocytochemistry for specific markers of α-smooth muscle actin (α-SMA). Real-time quantitative PCR, immunohistochemical staining and Western blot analysis for PDE5 gene expression in differentiated smooth muscle-like cells were carried out. Results After the conducted lentiviral vector containing PDE5-shRNA was transfected into rat BM-MSCs, the expression of EGFP was detected at 24 h and it became the strongest at 72 h, which FCM showed the transfection efficiency was 91.3% at this time. The EGFP expression rates of rat BM-MSCs transfected with PDE5-shRNA at 3 d, 7 d, 14 d after transduction were 91.3%, 86.1%, and 82.7%, respectively. There was still visible green fluorescence at 28 d after transfection. The gene-modified rat BM-MSCs with PDE5-shRNA were successfully induced to differentiate into smooth muscle-like cells. Transduction of the lentiviral vector PDE5-shRNA into rat BM-MSCs led to down-regulation of PDE5. The expression of PDE5 was reduced 67.2% by PDE5-shRNA compared with the control lentiviral vector. The proliferative property of rat BM-MSCs did not significantly change among those transfected with PDE5-shRNA (P<0.001). The differentiated rat BM-MSCs were identified to smooth muscle-like cells by immunocytochemistry. Conclusions These results suggest that the lentiviral vector with gene-specific silencing PDE5 of shRNA could effectively transfect #I# rat BM-MSCs. They reveal that the gene-modified rat BM-MSCs with PDE5-shRNA could be successfully induced to differentiate into smooth muscle-like cells in vitro and inhibit the expression of target gene PDE5.

AB232. The aetiology and efficacy of the treatment of persistent and recurrent hemospermia

Objective To describe the aetiology and efficacy of the treatment of persistent and recurrent hemospermia as a novel technique of transurethral seminal vesiculoscopy. Methods The clinical data of 290 patients with persistent and recurrent hemospermia (a course of hemospermia persisting more than 6 months) from multiple medical center of South China were analyzed retrospectively. The age ranged from 16 to 69 years (mean 31 years). The definite etiologies of persistent and recurrent hemospermia were confirmed by physical examination including digital rectal examination (DRE), tailored investigations such as blood PSA and clotting time, transrectal ultrasound (TRUS), CT or MRI. Of those with persistent and recurrent hemospermia, 269 patients were successfully performed by transurethral seminal vesiculoscopy since July 2008. The aetiology of the other 21 cases with persistent hemospermia were attributable to cystadenoma of the seminal vesicle [1], tuberculosis of tractus genitalis [1], bleeding risk secondary to liver cirrhosis [3], benign prostatic hyperplasia [12] and prostate cancer [4] confirmed by urogenital instrumentation or prostate biopsy. Results All the patients with persistent and recurrent hemospermia were confirmed by transurethral seminal vesiculoscopy (162 seminal vesiculitis and 108 seminal stone secondary to them, 91 ejaculatory ducts obstruction incompletely, 42 Mullerian cyst, 16 cysts of seminal vesicle, 3 cysts of ejaculatory duct and 12 benign prostatic hyperplasia). The mean operative time was 21 min (range, 5–90 min). There were no complications including injury of urethra and seminal vesicle and postoperative discomforts in the perineal region. The mean follow-up period was 24 months (range, 3–72 months). In those 269 cases, 11 patients were out of follow-up. Hematospermia in 235 cases disappeared and 23 patients respectively recurred in 5 to 60 months after receiving transurethral seminal vesiculoscopy. Of those 23 cases with postoperatively recurrent hemospermia, 12 patients were cured by re-transurethral seminal vesiculoscopy. Conclusions The aetiologies of persistent and recurrent hemospermia are mostly associated with seminal vesiculitis and seminal stone secondary to vesiculitis or ejaculatory ducts obstruction incompletely. Transurethral seminal vesiculoscopy could be an effective diagnostic and therapeutic procedure for it.

AB114. Myogenic differentiation of rat bone mesenchymal stem cells in vitro

Objective To culture and myogenic differentiation of rat bone mesenchymal stem cells (BMSCs) in vitro, and provide the available seed cells for erectile dysfunction (ED) therapy. Methods Rat BMSCs were isolated and cultured from the femur and tibia of Sprague Dawley (SD) rat. Mesenchymal stem cell positive cellular markers CD49d, CD73, CD90, CD105, CD106 and negative cellular markers CD31, CD34 and CD45 were arrayed by flow cytometry analysis. Furthermore, the fourth passage cells were induced and identified by their capacities in the myogenic differentiation. Results The cultured cells expressed mesenchymal stem cell positive cellular markers CD49d, CD73, CD90, CD105 and CD106, and lacked negative cellular markers CD31, CD34 and CD45. Myogenic differentiation cells can be stained with alpha-smooth muscle actin (α-SMA) and desmin, respectively. Conclusions Rat BMSCs have been successfully isolated, cultured and myogenic differentiation in vitro. They could be used as autogenous BMSCs and gene modified BMSCs for ED therapy.