MiR-33b inhibits osteosarcoma cell proliferation through suppression of glycolysis by targeting Lactate Dehydrogenase A (LDHA).

Osteosarcoma (OS) is one of the most common types of malignant bone tumor in adolescent. MicroRNAs (miRNAs) are widely studied regulatory molecules which play important roles in tumor development, differentiation, growth, invasion, chemosensitivity and cellular metabolism. Recently, miR-33b has been reported to act as a tumor suppressor in osteosarcoma. However, the detailed mechanism of miR-33b in regulating osteosarcoma cell proliferation remains unclear. In this study, we detected miR-33b was significantly downregulated in osteosarcoma tissues compared to their matched adjacent nontumor tissues. The decreased expressions of miR-33b were also found in multiple osteosarcoma cell lines, including MG63, Saos-2, U2OS and SOSP-9607 when compared to normal osteoblast cell line hFOB. Overexpression of miR-33b suppressed U2OS cell proliferation and anaerobic glycolysis. We identified Lactate dehydrogenase-A (LDHA) as a direct target of miR-33b in osteosarcoma tumors and cells by Western blot and luciferase assay. Moreover, inhibition of LDHA significantly suppressed glycolysis and cell proliferation of osteosarcoma cells. Restoration of LDHA in miR-33b-overexpressing osteosarcoma cells reversed the suppressive effect of miR-33b on cell proliferation. In addition, we report a significantly negative correlation between LDHA mRNA and miR-33b expression in osteosarcoma tumors: miR-33b is downregulated in OS tumors with high levels of LDHA (92.9%). Meanwhile, high miR-33b expressions were found majorly in OS tumors with low LDHA mRNA levels (82.4%). This study reveals that miR-33b plays a suppressive role in the regulation of osteosarcoma cell proliferation through direct targeting LDHA. The miR-33b/glycolysis/LDHA axis may contribute to development of therapeutic anti-tumor agents for osteosarcoma.

Role of oxidative stress in surgical cavernous nerve injury in a rat model.

This study investigates the role of oxidative stress in surgical cavernous nerve (CN) injury in a rat model. Eighty-four male Sprague-Dawley rats were randomly divided into three groups: group 1, sham-operated rats; group 2, bilateral CN-crushed rats; and group 3, bilateral CN-transection-and-sutured-immediately rats. Oxidative stress was evaluated by malondialdehyde levels, super oxide dismutase (SOD) activities, and glutathione peroxidase (GPX) activities in serum. Erectile function was assessed by CN electrostimulation at 3 months with mean maximal intracavernous pressure (ICP) and maximal ICP per mean arterial pressure. Nerve injury was assessed by toluidine blue staining of CNs and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining of penile tissue. GPX protein expression and nitrotyrosine-3 (NT-3) levels in penile tissue were measured. Erectile function and the number of myelinated axons of CNs and NADPH-diaphorase-positive nerve fibers were statistically decreased between groups, from sham to crush to transection. For markers, both nerve-injury groups showed increased oxidative stress markers at early time points, with the transection group showing greater oxidative stress than the crushed group and values normalizing to sham levels by week 12. GPX expression and NT-3 levels in penile tissue were in concordance with the results of SOD and GPX. These results show that oxidative stress plays an important role in injured CNs, and different methods of CN injury can lead to different degrees of oxidative stress in a rat model.

Somatic mutational analysis of FAK in breast cancer: a novel gain-of-function mutation due to deletion of exon 33.

Focal adhesion kinase (FAK) regulates cell adhesion, migration, proliferation, and survival. We identified a novel splicing mutant, FAK-Del33 (exon 33 deletion, KF437463), in both breast and thyroid cancers through colony sequencing. Considering the low proportion of mutant transcripts in samples, this mutation was detected by TaqMan-MGB probes based qPCR. In total, three in 21 paired breast tissues were identified with the FAK-Del33 mutation, and no mutations were found in the corresponding normal tissues. When introduced into a breast cell line through lentivirus infection, FAK-Del33 regulated cell motility and migration based on a wound healing assay. We demonstrated that the expression of Tyr397 (main auto-phosphorylation of FAK) was strongly increased in FAK-Del33 overexpressed breast tumor cells compared to wild-type following FAK/Src RTK signaling activation. These results suggest a novel and unique role of the FAK-Del33 mutation in FAK/Src signaling in breast cancer with significant implications for metastatic potential.

[Protective effect of grape seed proanthocyanidin on spermatogenesis following testicular torsion/detorsion in mice].

OBJECTIVE: To investigate the protective effect of grape seed proanthocyanidin (GSP) on spermatogenesis following testicular torsion/detorsion in mice. METHODS: Twenty-four healthy male Kunming mice, aged 8 weeks and weighing 25 - 27 g, were randomly divided into a control, a torsion and a treatment group, each containing 8 animals. The unilateral testicular torsion/detorsion model was established in the treatment and torsion groups. Thirty minutes before detorsion, the animals of the treatment group were injected intraperitoneally with 50 mg/kg GSP, and those of the torsion group with normal saline at the same dose, both for 3 days postoperatively. On the 4th day after surgery, ipsilateral orchiectomy were performed to detect histopathological changes, the levels of superoxide dismutase (SOD) and malondialdehyde (MDA), and the apoptotic index (AI) of germ cells in all the mice. RESULTS: Compared with the torsion group, the treated mice showed significantly increased Johnsen score (5.00 +/- 1.85 vs 7.38 +/- 0.92, P < 0.05), seminiferous tubule diameter ([176.50 +/- 1.60]microm vs [178.75 +/- 1.58] microm, P > 0.05), spermatogenic cell layers (3.75 +/- 1.03 vs 5.75 +/- 0.71, P < 0.05) and SOD activity ([29.04 +/- 4.46] U/mg prot vs [52.67 +/- 3.57] U/mg prot, P < 0.05), but remarkably reduced level of MDA ([4.63 +/- 0.05] nmol/mg prot vs [2.91 +/- 0.04] nmol/mg prot, P < 0.05) and AI of germ cells ([40.50 +/- 1.60]% vs [16.25 +/- 1.67] %, P < 0.05). CONCLUSION: Grape seed proanthocyanidin has a protective effect against spermatogenic injury in mice, the mechanisms of which may be related to its actions of scavenging oxygen free radicals, inhibiting lipid peroxidation and improving the antioxidant ability of the body.

[Estradiol stimulates proliferation of prostatic smooth muscle cells via estrogen receptor alpha and IGF1].

OBJECTIVE: To investigate the effects of estrogen receptor alpha (ERa) and insulin-like growth factor 1 (IGF1) on the proliferation of prostatic smooth muscle cells (PSMCs) in vitro. METHODS: The ERalpha shRNA expression frame was subcloned to the pGSadeno adenovirus vector by homologous recombination technology to construct the pGSaaeno-ERalpha vector. After the mouse PSMCs were transfected in vitro by pGSaaeno-ERalpha, the mRNA and protein expression levels of ERalpha were detected by RT-PCR and Western blot respectively. The expression of IGF1 in the ERa-reduced cells was determined by Western blot 6 hours after treatment with 17beta-estradiol (E2) at 10(-8) mol/L. The post-transfection activity of estrogen or exogenous IGF1 in the proliferation of PSMCs was evaluated by MTT chlormetric analysis. RESULTS: After treatment with E2, the proliferation of PSMCs and the expression of the IGF1 gene were significantly increased in the normal control group (P <0.05), but not obviously changed in the ERalpha-siRNA group (P> 0.05). And exogenous IGF1 failed to induce the proliferation of the ERalpha-reduced PSMCs. CONCLUSION: E2 induces the expression of IGF1 via ERalpha, and IGFl, with the interaction of ERalpha, promotes the proliferation of PSMCs.

[Induction of cell cycle arrest in bladder cancer RT4 cells by capsaicin].

OBJECTIVE: To study the effects of capsaicin on the growth of bladder cancer RT4 cell and its potential mechanism. METHODS: Cell counting kit-8 (CCK-8) assay and flow cytometry were employed to observe the effects of capsaicin (50, 100, 150, 200, 250 micromol/L) on cell growth, cell cycle and apoptosis. Capsaicin (0 micromol/L) was used as a control. The effects of mRNA and protein of transient receptor potential cation channel subfamily V 1 (TRPV1) on RT4 cells were tested by RT-PCR and immunofluorescence respectively. And the expressions of cell cycle protein P53, P21, CDK2 were detected by Western blot after the treatment of capsaicin. RESULTS: 100 micromol/L capsaicin significantly decreased the viability of RT4 cell [82.0% +/- 6.2% vs 100.0% +/- 12.4% (control), P = 0.036] while the cell viability was 7.8% +/- 2.9% at 250 micromol/L (P = 0.000). It was in a dose-dependent manner. On the other hand, capsaicin induced the cell cycle arrest of bladder cancer RT4 cells G(0)/G(1) phase in a dose-dependent way. The cell proportion of G(0)/G(1) phase in the control was 37.4% +/- 5.6%, however, it was 72.4% +/- 5.3% at 250 micromol/L (P = 0.000). It was showed that TRPV1 mRNA and protein were expressed in RT4 cells. After a 48-hour treatment with capsaicin, the expressions of P53 and P21 were up-regulated in contrary to the expression of CDK2. CONCLUSION: Capsaicin induces the cell cycle arrest of bladder cancer RT4 cells G(0)/G(1) phase and growth inhibition via TRPV1 receptor by modulating the expression of P53, P21 and CDK2.

[Role of CXCL16/CXCR6 axis in the metastasis of human prostate cancer].

OBJECTIVE: To explore the roles of chemokine CXCL16 and its receptor CXCR6 in the directional invasion of human prostate cancer (PCa). METHODS: The expression of CXCL16/CXCR6 in PCa samples and osseous tissues was determined by immunohistochemistry. The expression of CXCR6 in PC3 and LNCap cells was determined by reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry. Then the effects of CXCL16 upon the migration and invasion of human PC3 and LNCap cells were examined by Matrigel invasion assay. RESULTS: The expression of CXCR6 protein was detected in all clinical PCa samples. But no CXCL16 protein was detected. Positive CXCL16 expression was observed in human osseous tissues. Both PC3 and LNCap cells expressed CXCR6 mRNA (0.38+/-0.054 vs 0.41+/-0.019 respectively) and protein. In addition, CXCL16 could promote the in vitro migration and invasion of PC3 and LNCap cell lines (invading cells 211.50+/-5.60 vs 89.25+/-3.31 respectively). Such a promoting effect of CXCL16 could not be blocked influenced by antiCXCL12 or antiCXCR4. CONCLUSION: CXCL16/CXCR6 axis may be another independent chemokine factor playing a significant role in the metastasis of prostate cancer.

Overexpression of CIRP may reduce testicular damage induced by cryptorchidism.

PURPOSE: To investigate the protective effect of overexpression of cold-inducible RNA-binding protein (CIRP) on testicular damage induced by cryptorchidism. METHODS: Male BALB/c mice were made surgically cryptorchid and CIRP gene was transferred into the cryptorchid testis by in vivo electroporation. Seven or ten days after electroporation, the expression of CIRP, p53 and Fas mRNA and protein were analyzed by reverse-transcription polymerase chain reaction (RT-PCR) and immunoblotting, respectively. Meanwhile, Histopathological changes were observed by light microscope, and flow cytometry was used to detect testicular cell apoptosis. RESULTS: Testicular weights after transfection with pVAX1-CIRP or pVAX1 were 0.083+/-0.005 g and 0.065+/-0.004 g, respectively, on day 7(P < 0.05) and 0.078+/-0.004 g and 0.052+/-0.007 g, on day 10 (P < 0.05). Testicular cell apoptosis after transfection with pVAX1-CIRP or pVAX1 were 9.8+/-1.1 % and 20.7+/-1.3 %, respectively, on day 7 (P < 0.01) and 10.4+/-0.9 % and 27.5+/-1.2 %, on day 10 (P < 0.01). In addition, the expression of CIRP mRNA and protein in the testes transfected with pVAX1-CIRP were both increased (P < 0.05) at each indicated time point. Meanwhile, the expression of p53 was decreased on day 7 (P < 0.05) and Fas was decreased on day 10(P < 0.05). CONCLUSIONS: Overexpression of CIRP may reduce testicular damage induced by cryptorchidism by down-regulating the levels of p53 and Fas.

The effect of platelet-rich plasma on cavernous nerve regeneration in a rat model.

The aim of this study was to investigate the effect of platelet-rich plasma (PRP) on cavernous nerve (CN) regeneration and functional status in a nerve-crush rat model. Twenty-four Sprague-Dawley male rats were randomly divided into three equal groups: eight had a sham operation, eight underwent bilateral nerve crushing with no further intervention and eight underwent bilateral nerve crushing with an immediate application of PRP on the site of injury. Erectile function was assessed by CN electrostimulation at 3 months and nerve regeneration was assessed by toluidine blue staining of CN and nicotinamide adenine dinucleotide phosphate (NADPH)-diaphorase staining of penile tissue. Three months after surgery, in the group that underwent bilateral nerve crushing with no further intervention, the functional evaluation showed a lower mean maximal intracavernous pressure (ICP) and maximal ICP per mean arterial pressure (MAP) with CN stimulation than those in the sham group. In the group with an immediate application of PRP, the mean maximal ICP and maximal ICP/MAP were significantly higher than those in the injured control group. Histologically, the group with the application of PRP had more myelinated axons of CNs and more NADPH-diaphorase-positive nerve fibres than the injured control group but fewer than the sham group. These results show that the application of PRP to the site of CN-crush injury facilitates nerve regeneration and recovery of erectile function. Our research indicates that clinical application of PRP has potential repairing effect on CN and peripheral nerves.

[Protective effects of L-carnitine upon testicular ischemia-reperfusion damage in rats].

OBJECTIVE: To investigate the protective effects of L-carnitine upon testicular ischemia-reperfusion injury in rats. METHODS: Sprague-Dawley rats were divided into 3 groups (n = 10). In those animals undergoing unilateral testicular torsion, right testes were rotated 720 degrees for 2 h. Sham operated group served as a control group. Torsion group underwent 2 h torsion and saline was injected intraperitoneally at 30 min pre-detorsion. Treatment group underwent similar torsion but L-carnitine (500 mg/kg) was infused intraoperatively. The right testes of 5 animals in each group were excised after 4 h reperfusion for measuring the levels of malondialdehyde (MDA) and heat shock protein 70 (HSP70), evaluation of activities of antioxidant enzyme including superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx). Histopathological changes and germ cell apoptosis indices (AI) were determined at 24 h post-detorsion in right testes of the remaining 5 animals in each group. RESULTS: The mean number of apoptotic nuclei per tubule cross section and the malondialdehyde level were significantly lower in treatment group as compared with torsion group [AI( 6.87 +/- 2.47) vs (17.13 +/- 3.56), MDA (160 +/- 15) vs (199 +/- 15) nmol/g]. Activities of antioxidant enzyme and the level of HSP70 were significantly higher in treatment group than those in torsion group [SOD (1638 +/- 153) vs (1078 158) U/g, CAT (317 +/- 28) vs (188 +/- 33) U/g, GPx (667 +/- 94) vs (311 +/- 65) U/g, HSP70 (0.87 +/- 0.13) vs (0.25 +/- 0.04)]. The pathological damage of testes in the treatment group was lighter than that in the torsion group (all P < 0.05). CONCLUSIONS: The administration of L-carnitine exerts a beneficial effect upon testicular ischemia-reperfusion injury. This effect may be achieved through an induced expression of HSP70.

[The effect of diethylstilbestrol on inducing abdominal cryptorchidism and relevant genetic expression in rats].

OBJECTIVE: To study the effect of diethylstilbestrol (DES) at different doses on transabdominal testicular descent in rats and the expression of INSL3 in the testis and HOXA10 in the gubernaculum. METHOD: Fifty E13.5 (embryonic day 13.5) pregnant female SD rats were randomly divided into five groups that received a subcutaneous injection of DMSO, 2.5, 5.0, 10.0 and 20.0 mg/kg DES (group A, B, C, D and E), respectively. Male offspring were killed at E19.5, and then fetal mortality, the degree of transabdominal testicular ascent (DTA) was determined by a stereomicroscope. The mRNA expressions of INSL3 in the testis and HOXA10 in the gubernaculum were determined by RT-PCR. The expression of INSL3 protein was determined by Western blotting. RESULTS: Male fetal mortality in group A, B, C, D, and E were 3.57%, 6.90%, 12.00%, 19.23% and 36.36%, respectively, which showed a dose-effect relationship between DES and the male fatal mortality (r=0.999, P<0.01). DTA in group B, C, D and E were (23.7+/-1.7) U, (38.8+/-1.9) U, (49.3+/-1.8) U and (58.6+/-2.1) U that were significantly larger than that in group A [(8.5+/-1.3) U] (q=46.12, 88.53, 120.44 and 141.37, respectively, P<0.01). There was also a dose-effect relationship between DES and DTA. In group B, C, D, and E, the expression of INSL3 mRNA were 0.9570+/-0.1490, 0.6760+/-0.1380, 0.0170+/-0.0040 and 0.0013+/-0.0003, respectively; the expressions of INSL3 protein were 0.8360+/-0.1520, 0.5310+/-0.1070, 0.0140+/-0.0020 and 0.0011+/-0.0003, respectively, which were significantly larger than the expression of INSL3 mRNA (1.801+/-0.126) and INSL3 protein (1.612+/-0.134) in group A (qmRNA=40.4840, 52.4402, 83.1585 and 82.0582, respectively, and qprotein=38.6151, 52.2747, 77.2756 and 76.1983, respectively, P<0.01). The expression of HOXA10 mRNA in group A, B, C, D, and E were 0.945+/-0.125, 0.940+/-0.119, 0.656+/-0.115, 0.544+/-0.118 and 0.463+/-0.114, respectively. Compared with the expression of HOXA10 mRNA in group A, the expression of group B was not significantly different (q=0.2213, P>0.05), those in other groups were down-regulated significantly (q=12.4304, 17.2477 and 20.2789, respectively, P<0.01). CONCLUSION: DES inhibited transabdominal testicular descent dose-dependently via down-regulating the expression of INSL3. HOXA10 may play no role in low-dosage DES induced intra-abdominal cryptorchidism, but down-regulated HOXA10 mRNA was involved in high-dosage DES induced ones.

[Establishment of PEG10 transgenic mouse and effects of PEG10 on growth, metastasis of transplanted tumor in mice].

OBJECTIVE: To establish PEG10 transgenic mice model and study the effect of PEG10 transgene on tumor growth and metastasis in mice. METHODS: The linearized expression element of pALB-PEG10, which contained mouse albumin promoter, structural gene of PEG10, and polyaenylation signal sequence, was microinjected into 3741 KM mouse fertilized ova. The manipulated embryos were then transplanted into the oviducts of 94 pseudopregnant recipient mice. All the newborn mice were screened by PCR to detect genomic DNA in tail tissue, then PEG10 mRNA and protein expression were detected by RT-PCR and western blot, respectively in the positive mice. Hepatoma cell H22 was subcutaneously inoculated into the right armpit of wild type mice and No.17, No.33 transgenic mice. Tumor size was measured every week. Mice were sacrificed on day 12 and then the tumors were exercised and weighted. Tumors and livers were fixed in formaldehyde and sectioned. The sections were stained with hematoxylin/eosin and examined under microscope. The expression of PEG10 protein was detected with immunohistochemistry method. RESULTS: Among the 43 off-springs, 3 were positive for tail tissue PEG10 gene examination, PEG10 was successfully expressed in the liver of the randomly selected transgenic mouse. H22 tumor grew faster in all the transgenic mice than in wild type mice. The average size and weight of tumors between the transgenic mice and wild type mice were significantly different (P < 0.05). Most tumors in the transgenic mice invaded the surrounding tissues and showed liver metastasis, PEG10 protein was expressed in liver. In contrast, nearly all the tumors in wild type mice were capsulized and PEG10 was not expressed in liver. CONCLUSION: Our results showed that the PEG10 gene could be expressed in the liver of the transgenic mice. PEG10 promotes growth, invasion, and metastasis of transplanted H22 tumors in mice.

[Molecular pathways of germ cell apoptosis following testicular torsion in rats].

OBJECTIVE: To investigate the molecular mechanism of germ cell apoptosis following testicular torsion in rats. METHODS: Healthy male Sprague-Dawley rats (n = 16) were equally randomized into a control and a torsion group and the models of testicular torsion (720 degrees 2 h) were established. Twenty-four hours later, the apoptosis and count of germ cells were determined by flow cytometry, the expressions of Bax, Fas and Fas ligand (FasL) mRNA semiquantitatively analyzed by RT-PCR and the cytochrome C release detected by Western blot. RESULTS: Compared with the control group, there was an obvious increase in the number of apoptotic germ cells, a marked decrease in that of haploid and tetraploid cells and significantly up-regulated expressions of Bax and Fas/FasL mRNA in the torsion group (P <0.01). The Western blot analysis showed that the cytochrome C release was remarkably increased 24 hours after the detorsion. There were significant differences between the two groups (P <0.01). CONCLUSION: There are two major signaling pathways of cell apoptosis following testicular torsion, intercellular and intracellular. Up-regulated expressions of the apoptosis-related molecules Bax and Fas/FasL and increased cytochrome C release may play an important role in germ cell apoptosis following testicular torsion in rats.

[Expression of estrogen receptor beta in benign prostatic hyperplasia complicated by chronic prostatitis].

OBJECTIVE: To observe the expression of estrogen receptor-beta (ERbeta) in benign prostatic hyperplasia (BPH) complicated by chronic prostatitis, and to evaluate the correlation of chronic prostatitis with ERbeta expression. METHODS: Histological sections of prostate tissues were obtained from 60 BPH patients complicated by chronic prostatitis and divided into Group 1 (Grade 1), 2 (Grade 2) and 3 (Grade 3) according to the scores on the inflammation of the prostate tissues using the four-point scale designed by Irani et al. The expression of ERbeta was determined by the immunohistochemical method. RESULTS: There were 24 cases (40%) in Group 1, 21 (35%) in Group 2 and 15 (25%) in Group 3, with no statistically significant differences in age and prostate volume among the three groups (P > 0.05). The expression of ERbeta was significantly decreased in Groups 2 and 3 as compared with Group 1 (P < 0.01). CONCLUSION: The expression of ERbeta is reduced with increased scores on the inflammation of the prostate tissues in BPH patients, and the decreased ERbeta expression may be associated with the inflammatory stimulation of prostatitis.

MicroRNA-30e inhibits adhesion, migration, invasion and cell cycle progression of prostate cancer cells via inhibition of the activation of the MAPK signaling pathway by downregulating CHRM3.

Prostate cancer (PCa) testing is currently based on measurement of serum prostate­specific antigen levels and digital rectal examination, which are limited by a low predictive value and the adverse effects associated with overdiagnosis and overtreatment. Recent studies have reported that the abnormal expression of microRNAs (miRNAs) is associated with the mechanism underlying the development of PCa. Thus, the aim of the present study was to investigate the effects of miR­30e and its target gene, M3 muscarinic acetylcholine receptor (CHRM3), on the adhesion, migration, invasion and cell cycle distribution of PCa cells via the mitogen­activated protein kinase (MAPK) signaling pathway. The differentially expressed genes were screened in the Gene Expression Omnibus database from a gene expression microarray (GSE55945) of PCa. PCa tissues and adjacent tissues were collected from patients with PCa. The PC­3 and DU145 human PCa cell lines were treated with activator, inhibitor and siRNAs. The effects of miR­30e on cell adhesion, migration, invasion and cell cycle distribution with the involvement of CHRM3 and the MAPK signaling pathway were investigated. The bioinformatics results demonstrated that the CHRM3 gene and the MAKP signaling pathway were involved in the progression of PCa, and has­miR­30e was selected for further study. The levels of miR­30e were significantly downregulated, while the levels of CHRM3 were obviously upregulated in PCa. CHRM3 was verified as a target gene of miR­30e. Upregulation of miR­30e and downregulation of CHRM3 decreased the levels of p­P38, p­extracellular signal­regulated kinase, p­c­Jun N­terminal kinase, p­c­fos and p­c­JUN, cell adhesion, migration and invasion ability, and the number of cells in the S phase, while they increased the number of cells in the G0 and G1 phases. The findings of the present study suggest that miR­30e inhibited the adhesion, migration, invasion and cell cycle entry of PCa cells by suppressing the activation of the MAPK signaling pathway and inhibiting CHRM3 expression. Thus, miR­30e may serve as a candidate target for the treatment of PCa.

Neural-like cells from adipose-derived stem cells for cavernous nerve injury in rats.

Although the remaining nerve tissue can regenerate and partly restore erectile function when the cavernous nerve is compressed/severed and function lost, the limited regenerative ability of these nerve tissues often fails to meet clinical needs. Adipose-derived stem cells are easy to obtain and culture, and can differentiate into neural cells. Their proliferation rate is easy to control and they may be used to help restore injured cavernous nerve function. Sprague-Dawley male rats (n = 45) were equally randomized into three groups: fifteen rats as a sham-operated group, fifteen rats as a bilateral nerve crush (BINC) group (with no further intervention), fifteen rats as a BINC with intracavernous injection of one million neural-like cells from adipose-derived stem cells (NAS) (BINC + NAS) group. After 4 weeks, erectile function was assessed by stimulating the cavernous body. The number of myelinated axons in the dorsal cavernous nerve was determined by toluidine blue staining. The area of neuronal nitric oxide synthase-positive fibers in the dorsal penile nerve was measured by immunohistochemical staining. Masson staining was used to analyze the ratio of smooth muscle to collagen in penile tissue. The results demonstrate that maximal intracavernous pressure, the ratio of maximal intracavernous pressure to mean arterial pressure, the numbers of myelinated axons and neuronal nitric oxide synthase-positive fibers in the dorsal penile nerve, and the ratio of smooth muscle to collagen could be increased after cell transplantation. These findings indicate that neural-like cells from adipose-derived stem cells can effectively alleviate cavernous nerve injury and improve erectile function. All animal experiments were approved by the Animal Ethics Committee of Huazhong University of Science and Technology, China (approval No. 2017-1925) on September 15, 2017.

[Effect of platelet rich plasma on the regeneration of cavernous nerve: experiment with rats].

OBJECTIVE: To investigate the effect of platelet rich plasma (PRP) on the regeneration of injured cavernous nerve (CN). METHODS: Blood was collected from the hearts of 6 SD rats to prepare PRP. 24 male adult rats were randomly divided into 3 equal groups: pure suture group undergoing bilateral CN transaction and pure suture immediately, PRP group undergoing bilateral CN transaction + suture + PRP 200 microl to the site of suture, and sham operation group. 3 months later intracavernous pressure (ICP) was measured by CN electrostimulation and then samples of CN were obtained to undergo pathological examination. RESULTS: 3 months later after surgery, the ICP of the pure suture group was (46 +/- 8) cm H2O, significantly lower than that of the sham group [(109 +/- 13) cm H2O, P < 0.01], and that of the PRP group was (94 +/- 13) cm H2O, significantly higher than that of the pure sutured group (P < 0.01), however, still significantly lower than that of the sham operation group (P < 0.05). The number of axons of CN in the PRP group was (121 +/- 16), significantly higher than that of the pure sutured group (70 +/- 14, P < 0.01); however, still significantly lower than that of the sham operation group (181 +/- 21, P < 0.01). CONCLUSION: PRP can promote the regeneration of injured CN and the recovery of erectile function.

[Intraprostatic urinary reflux associated prostatitis caused by partial urethral obstruction in the rat model].

OBJECTIVE: To investigate the pathogenesis of chronic prostatitis / chronic pelvic pain syndrome (CP / CPPS) by constructing the rat model of intraprostatic urinary reflux associated prostatitis caused by partial urethral obstruction. METHODS: Fifty-four SD male rats were divided into an experiment group (n = 30) and a partial urethral obstruction (PUO) sham operation group (n = 24). Shinsuke Takechi's surgical method was adopted to achieve PUO and induce intraprostatic urinary reflux in the experiment group. While in the sham operation control group, the prostates were harvested at 1, 3 and 7 days after release from 3-day PUO, their morphological changes observed with the light microscope and the expression of cyclooxygenase-2 (COX-2) examined by immunohistochemistry. RESULTS: Inflammation was observed in the prostate of the experiment group at 1, 3 and 7 days after release from PUO and alleviated with the passing of time, while the control group remained normal. The expression of COX-2 in the prostate was significantly higher in the experiment group than in the control (P < 0.05) and the staining of COX-2 became stronger with the lapse of time (P < 0.05). CONCLUSION: An animal model of intraprostatic urinary reflux associated prostatitis was constructed. The up-regulated expression of COX-2 induced by intraprostatic urinary reflux may be closely related with the development of CP / CPPS.

Cavernous nerve reconstruction with autologous vein graft and platelet-derived growth factors.

In this study, we investigated the feasibility of using autologous vein graft and platelet-derived growth factors to bridge transected cavernous nerve in a rat model. A short defect in the bilateral cavernous nerve was created and repaired with vein graft from the right jugular vein or vein graft plus platelet-derived growth factors. The 32 rats were divided into four groups, namely Group 1 - no repair as a negative control, Group 2 - vein graft alone, Group 3 - vein graft plus platelet-derived growth factors, and Group 4 - sham operation as a positive control. We evaluated nerve regeneration and functional recovery using retrograde tracing study with FluoroGold, Toluidine blue staining of cavernous nerve, and the intracavernous pressure at 3 months. Three months after surgery, rich FluoroGold-positive cells were observed in the sham and vein graft plus platelet-derived growth factors group, but very few were found in the no repair group. The number of myelinated axons of regenerated cavernous nerve and intracavernous pressure were increased obviously in the two vein graft groups, especially in the vein graft plus platelet-derived growth factors group. These findings confirm the feasibility of using autologous vein as guides for cavernous nerve regeneration, and the regeneration can be further enhanced when the vein is filled with platelet-derived growth factors.

Effects of epidermal growth factor on sperm content and motility of rats with surgically induced varicoceles.

AIM: To investigate the effect of epidermal growth factor (EGF) on the sperm content and motility of the varicocelized rats. METHODS: Forty-eight male Wistar rats were randomly divided into five groups. Experimental varicocele was induced by partial ligation of the left renal vein in the varicocele, the varicocele repair, the varicocele with EGF and the varicocele repair with EGF groups, whereas the control group only received a sham induction of varicocele. Surgical repair of varicocele was performed 4 months later in the varicocele repair and varicocele repair with EGF groups. EGF administration was performed daily by s.c. injection in the varicocele with EGF and varicocele repair with EGF groups at the dose of 10 microg/(kg.day) from the next day of the second surgery. One month later, all animals were killed and bilateral cauda epididymal sperm counts and motility were evaluated. RESULTS: The mean sperm count and percentage of motile spermatozoa were significantly higher bilaterally in the varicocele with EGF group than in the varicocele group (P < 0.05). They were also significantly higher bilaterally in the varicocele repair with EGF group than in the varicocele repair and the varicocele with EGF groups (P < 0.05). CONCLUSION: EGF can improve bilateral epididymal sperm content and motility of the rat with surgically induced varicocele. The administration of EGF in combination with surgical repair is more effective than surgical repair or EGF administration alone. EGF might be useful for the treatment of infertility induced by varicocele.