Cyclophosphamide activates ferroptosis-induced dysfunction of Leydig cells via SMAD2 pathway†.

Cyclophosphamide (CP) is a widely used chemotherapeutic drug and immunosuppressant in the clinic, and the hypoandrogenism caused by CP is receiving more attention. Some studies found that ferroptosis is a new mechanism of cell death closely related to chemotherapeutic drugs and plays a key role in regulating reproductive injuries. The purpose of this study is to explore ferroptosis' role in testicular Leydig cell dysfunction and molecular mechanisms relating to it. In this study, the level of ferroptosis in the mouse model of testicular Leydig cell dysfunction induced by CP was significantly increased and further affected testosterone synthesis. The ferroptosis inhibitors ferrostatin-1 (Fer-1) and iron chelator deferoxamine (DFO) can improve injury induced by CP. The results of immunohistochemistry showed that Fer-1 and DFO could improve the structural disorder of seminiferous tubules and the decrease of the number of Leydig cells in testicular tissue induced by CP. Immunofluorescence and western blot confirmed that Fer-1 and DFO could improve the expression of key enzymes in testosterone synthesis. The activation of SMAD family member 2 (Smad2)/cyclin-dependent kinase inhibitor 1A (Cdkn1a) pathway can improve the ferroptosis of Leydig cells induced by CP and protect the function of Leydig cells. By inhibiting the Smad2/Cdkn1a signal pathway, CP can regulate ferroptosis, resulting in testicular Leydig cell dysfunction. In this study, CP-induced hypoandrogenism is explained theoretically and a potential therapeutic strategy is provided.

Exosomes derived from BMSCs ameliorate cyclophosphamide-induced testosterone deficiency by enhancing the autophagy of Leydig cells via the AMPK-mTOR signaling pathway.

Cyclophosphamide-induced testosterone deficiency (CPTD) during the treatment of cancers and autoimmune disorders severely influences the quality of life of patients. Currently, several guidelines recommend patients suffering from CPTD receive testosterone replacement therapy (TRT). However, TRT has many disadvantages underscoring the requirement for alternative, nontoxic treatment strategies. We previously reported bone marrow mesenchymal stem cells-derived exosomes (BMSCs-exos) could alleviate cyclophosphamide (CP)-induced spermatogenesis dysfunction, highlighting their role in the treatment of male reproductive disorders. Therefore, we further investigated whether BMSCs-exos affect autophagy and testosterone synthesis in Leydig cells (LCs). Here, we examined the effects and probed the molecular mechanisms of BMSCs-exos on CPTD in vivo and in vitro by detecting the expression levels of genes and proteins related to autophagy and testosterone synthesis. Furthermore, the testosterone concentration in serum and cell-conditioned medium, and the photophosphorylation protein levels of adenosine monophosphate-activated protein kinase (AMPK) and mammalian target of rapamycin (mTOR) were measured. Our results suggest that BMSCs-exos could be absorbed by LCs through the blood-testis barrier in mice, promoting autophagy in LCs and improving the CP-induced low serum testosterone levels. BMSCs-exos inhibited cell death in CP-exposed LCs, regulated the AMPK-mTOR signaling pathway to promote autophagy in LCs, and then improved the low testosterone synthesis ability of CP-induced LCs. Moreover, the autophagy inhibitor, 3-methyladenine (3-MA), significantly reversed the therapeutic effects of BMSCs-exos. These findings suggest that BMSCs-exos promote LC autophagy by regulating the AMPK-mTOR signaling pathway, thereby ameliorating CPTD. This study provides novel evidence for the clinical improvement of CPTD using BMSCs-exos.

Plasma cell subtypes analyzed using artificial intelligence algorithm for predicting biochemical recurrence, immune escape potential, and immunotherapy response of prostate cancer.

Background: Plasma cells as an important component of immune microenvironment plays a crucial role in immune escape and are closely related to immune therapy response. However, its role for prostate cancer is rarely understood. In this study, we intend to investigate the value of a new plasma cell molecular subtype for predicting the biochemical recurrence, immune escape and immunotherapy response in prostate cancer. Methods: Gene expression and clinicopathological data were collected from 481 prostate cancer patients in the Cancer Genome Atlas. Then, the immune characteristics of the patients were analyzed based on plasma cell infiltration fractions. The unsupervised clustering based machine learning algorithm was used to identify the molecular subtypes of the plasma cell. And the characteristic genes of plasma cell subtypes were screened out by three types of machine learning models to establish an artificial neural network for predicting plasma cell subtypes. Finally, the prediction artificial neural network of plasma cell infiltration subtypes was validated in an independent cohort of 449 prostate cancer patients from the Gene Expression Omnibus. Results: The plasma cell fraction in prostate cancer was significantly decreased in tumors with high T stage, high Gleason score and lymph node metastasis. In addition, low plasma cell fraction patients had a higher risk of biochemical recurrence. Based on the differential genes of plasma cells, plasma cell infiltration status of PCa patients were divided into two independent molecular subtypes(subtype 1 and subtype 2). Subtype 1 tends to be immunosuppressive plasma cells infiltrating to the PCa region, with a higher likelihood of biochemical recurrence, more active immune microenvironment, and stronger immune escape potential, leading to a poor response to immunotherapy. Subsequently, 10 characteristic genes of plasma cell subtype were screened out by three machine learning algorithms. Finally, an artificial neural network was constructed by those 10 genes to predict the plasma cell subtype of new patients. This artificial neural network was validated in an independent validation set, and the similar results were gained. Conclusions: Plasma cell infiltration subtypes could provide a potent prognostic predictor for prostate cancer and be an option for potential responders to prostate cancer immunotherapy.

ACO1 and IREB2 downregulation confer poor prognosis and correlate with autophagy-related ferroptosis and immune infiltration in KIRC.

Background: ACO1 and IREB2 are two homologous cytosolic regulatory proteins, which sense iron levels and change iron metabolism-linked molecules. These two genes were noticeably decreased in kidney renal clear cell carcinoma (KIRC), which confer poor survival. Meanwhile, there is a paucity of information about the mechanisms and clinical significance of ACO1 and IREB2 downregulation in renal cancers. Methods: The expression profiles of ACO1 and IREB2 were assessed using multiple public data sets via several bioinformatics platforms. Clinical and pathological information was utilized to stratify cohorts for comparison. Patient survival outcomes were evaluated using the Kaplan-Meier plotter, a meta-analysis tool. The correlations of ACO1 and IREB2 with ferroptosis were further evaluated in The Cancer Genome Atlas (TCGA)-KIRC database. Tumor immune infiltration was analyzed using the CIBERSORT, TIMER, and GEPIA data resources. ACO1 antagonist sodium oxalomalate (OMA) and IREB2 inhibitor sodium nitroprusside (SNP) was used to treat renal cancer ACHN cells together with sorafenib. Results: KIRC patients with low ACO1 or IREB2 contents exhibited a remarkably worse survival rate in contrast with those with high expression in Kaplan-Meier survival analyses. Meanwhile, ACO1 and IREB2 regulate autophagy-linked ferroptosis along with immune cell invasion in the tumor microenvironment in KIRC patients. Blocking the activation of these two genes by their inhibitors OMA and SNP ameliorated sorafenib-triggered cell death, supporting that ACO1 and IREB2 could be participated in its cytotoxic influence on renal cancer cells. Conclusion: ACO1 and IREB2 downregulation in renal cancers were correlated with cancer aggressiveness, cellular iron homeostasis, cytotoxic immune cell infiltration, and patient survival outcomes. Our research is integral to verify the possible significance of ACO1 and IREB2 contents as a powerful signature for targeted treatment or novel immunotherapy in clinical settings.

Changes in hip joint contact stress during a gait cycle based on the individualized modeling method of "gait-musculoskeletal system-finite element".

OBJECTIVE: To construct a comprehensive simulation method of "gait-musculoskeletal system (MS)-finite element (FE)" for analysis of hip joint dynamics characteristics and the changes in the contact stress in the hip throughout a gait cycle. METHODS: Two healthy volunteers (male and female) were recruited. The 3D gait trajectories during normal walking and the CT images including the hip and femur of the volunteers were obtained. CT imaging data in the DICOM format were extracted for subjected 3D hip joint reconstruction. The reconstructed 3D model files were used to realize the subject-specific registration of the pelvis and thigh segment of general musculoskeletal model. The captured marker trajectory data were used to drive subject-specific musculoskeletal model to complete inverse dynamic analysis. Results of inverse dynamic analysis were exported and applied as boundary and load settings of the hip joint finite element in ABAQUS. Finally, the finite element analysis (FEA) was performed to analyze contact stress of hip joint during a gait cycle of left foot. RESULTS: In the inverse dynamic analysis, the dynamic changes of the main hip-femoral muscle force with respect to each phase of a single gait cycle were plotted. The hip joint reaction force reached a maximum value of 2.9%BW (body weight) and appeared at the end of the terminal stance phase. Twin peaks appeared at the initial contact phase and the end of the terminal stance phase, respectively. FEA showed the temporal changes in contact stress in the acetabulum. In the visual stress cloud chart, the acetabular contact stress was mainly distributed in the dome of the acetabulum and in the anterolateral area at the top of the femoral head during a single gait cycle. The acetabular contact area was between 293.8 and 998.4 mm2, and the maximum contact area appear at the mid-stance phase or the loading response phase of gait. The maximum contact stress of the acetabulum reached 6.91 MPa for the model 1 and 6.92 MPa for the model 2 at the terminal stance phase. CONCLUSIONS: The "Gait-MS-FE" technology is integrated to construct a comprehensive simulation framework. Based on human gait trajectories and their CT images, individualized simulation modeling can be achieved. Subject-specific gait in combination with an inverse dynamic analysis of the MS provides pre-processing parameters for FE simulation for more accurate biomechanical analysis of hip joint.

[Bone marrow mesenchymal stem cell-derived exosomes protect TM3 Leydig cells from cyclophosphamide-induced injury].

OBJECTIVE: To investigate the effect of exosomes derived from mouse bone marrow mesenchymal stem cells (BMSC) on the injury of TM3 Leydig cells induced by cyclophosphamide (CP). METHODS: The exosomes from BMSCs were extracted by ultrahigh speed centrifugation, and their particle size and morphology observed under the electron microscope, and their typical marker proteins examined by Western blot. The uptake of exosomes by TM3 Leydig cells was observed by co-culturing the exosomes with the TM3 cells. The viability and apoptosis rate of the TM3 cells in the normal control, CP-induction and CP+exosomes groups were detected using the CCK-8 method and flow cytometry respectively. ELISA was used to measure the testosterone (T) level in the cell supernatant, and Western blot adopted to determine the expression level of the steroidogenic acute regulatory (StAR) protein, a key enzyme related to T synthesis. RESULTS: The viability of the TM3 Leydig cells was markedly decreased and the apoptosis rate of the cells remarkably increased in the CP-induction group compared with that in the normal control, but both significantly restored after co-culture with exosomes (P < 0.01 and P < 0.05). The T level in the supernatant and the expression of the StAR protein in the cells were lower in the CP-induction than in the normal control group, but both dramatically increased in the CP+exosomes group (P < 0.01). CONCLUSION: Exosomes from BMSCs and protect TM3 Leydig cells from cyclophosphamide-induced injury and restore the level of testosterone secreted by the TM3 cells to a certain extent.

Activating transcription factor 5 (ATF5) promotes tumorigenic capability and activates the Wnt/b-catenin pathway in bladder cancer.

BACKGROUND: In bladder cancer, up to 70% of patients will relapse after resection within 5 years, in which the mechanism underlying the recurrence remains largely unclear. METHODS: Quantitative real-time PCR, western blot and immunohistochemistry were conducted. The assays of tumor sphere formation and tumor xenograft were further performed to assess the potential biological roles of ATF5 (activating transcription factor 5). Chromatin immunoprecipitation-qPCR and luciferase activity assays were carried out to explore the potential molecular mechanism. A two-tailed paired Student's t-test, χ2 test, Kaplan Meier and Cox regression analyses, and Spearman's rank correlation coefficients were used for statistical analyses. RESULTS: ATF5 is elevated in bladder urothelial cancer (BLCA) tissues, especially in recurrent BLCA, which confers a poor prognosis. Overexpressing ATF5 significantly enhanced, whereas silencing ATF5 inhibited, the capability of tumor sphere formation in bladder cancer cells. Mechanically, ATF5 could directly bind to and stimulate the promoter of DVL1 gene, resulting in activation of Wnt/ß-catenin pathway. CONCLUSIONS: This study provides a novel insight into a portion of the mechanism underlying high recurrence potential of BLCA, presenting ATF5 as a prognostic factor or potential therapeutic target for preventing recurrence in BLCA.

BMI1 activates P-glycoprotein via transcription repression of miR-3682-3p and enhances chemoresistance of bladder cancer cell.

Chemoresistance is the most significant reason for the failure of cancer treatment following radical cystectomy. The response rate to the first-line chemotherapy of cisplatin and gemcitabine does not exceed 50%. In our previous research, elevated BMI1 (B-cell specific Moloney murine leukemia virus integration region 1) expression in bladder cancer conferred poor survival and was associated with chemoresistance. Herein, via analysis of The Cancer Genome Atlas database and validation of clinical samples, BMI1 was elevated in patients with bladder cancer resistant to cisplatin and gemcitabine, which conferred tumor relapse and progression. Consistently, BMI1 was markedly increased in the established cisplatin- and gemcitabine-resistant T24 cells (T24/DDP&GEM). Functionally, BMI1 overexpression dramatically promoted drug efflux, enhanced viability and decreased apoptosis of bladder cancer cells upon treatment with cisplatin or gemcitabine, whereas BMI1 downregulation reversed this effect. Mechanically, upon interaction with p53, BMI1 was recruited on the promoter of miR-3682-3p gene concomitant with an increase in the mono-ubiquitination of histone H2A lysine 119, leading to transcription repression of miR-3682-3p gene followed by derepression of ABCB1 (ATP binding cassette subfamily B member 1) gene. Moreover, suppression of P-glycoprotein by miR-3682-3p mimics or its inhibitor XR-9576, could significantly reverse chemoresistance of T24/DDP&GEM cells. These results provided a novel insight into a portion of the mechanism underlying BMI1-mediated chemoresistance in bladder cancer.

Comprehensive signature analysis of drug metabolism differences in the White, Black and Asian prostate cancer patients.

The drug response sensitivity and related prognosis of prostate cancer varied from races, while the original mechanism remains rarely understood. In this study, the comprehensive signature including transcriptomics, epigenome and single nucleotide polymorphisms (SNPs) of 485 PCa cases- including 415 Whites, 58 Blacks and 12 Asians from the TCGA database were analyzed to investigate the drug metabolism differences between races. We found that Blacks and Whites had a more prominent drug metabolism, cytotoxic therapy resistance, and endocrine therapy resistance than Asians, while Whites were more prominent in drug metabolism, cytotoxic therapy resistance and endocrine therapy resistance than Blacks. Subsequently, the targeted regulation analysis indicated that the racial differences in cytotoxic therapy resistance, endocrine therapy resistance, might originate from drug metabolisms, and 19 drug metabolism-related core genes were confirmed in the multi-omics network for subsequent analysis. Furthermore, we verified that CYP1A1, CYP3A4, CYP2B6, UGT2B17, UGT2B7, UGT1A8, UGT2B11, GAS5, SNHG6, XIST significantly affected antineoplastic drugs sensitivities in PCa cell lines, and these genes also showed good predictive efficiency of drug response and treatment outcomes for PCa in this cohort of patients. These findings revealed a comprehensive signature of drug metabolism differences for the Whites, Blacks and Asians, and it may provide some evidence for making individualized treatment strategies.

A novel robust nomogram based on peripheral monocyte counts for predicting lymph node metastasis of prostate cancer.

Accurate methods for identifying pelvic lymph node metastasis (LNM) of prostate cancer (PCa) prior to surgery are still lacking. We aimed to investigate the predictive value of peripheral monocyte count (PMC) for LNM of PCa in this study. Two hundred and ninety-eight patients from three centers were divided into a training set (n = 125) and a validation set (n = 173). In the training set, the independent predictors of LNM were analyzed using univariate and multivariate logistic regression analyses, and the optimal cutoff value was calculated by the receiver operating characteristic (ROC) curve. The sensitivity and specificity of the optimal cutoff were authenticated in the validation cohort. Finally, a nomogram based on the PMC was constructed for predicting LNM. Multivariate analyses of the training cohort demonstrated that clinical T stage, preoperative Gleason score, and PMC were independent risk factors for LNM. The subsequent ROC analysis showed that the optimal cutoff value of PMC for diagnosing LNM was 0.405 × 109 l-1 with a sensitivity of 60.0% and a specificity of 67.8%. In the validation set, the optimal cutoff value showed significantly higher sensitivity than that of conventional magnetic resonance imaging (MRI) (0.619 vs 0.238, P < 0.001). The nomogram involving PMC, free prostate-specific antigen (fPSA), clinical T stage, preoperative Gleason score, and monocyte-to-lymphocyte ratio (MLR) was generated, which showed a robust predictive capacity for predicting LNM before the operation. Our results indicated that PMC as a single agent, or combined with other clinical parameters, showed a robust predictive capacity for LNM in PCa. It can be employed as a complementary factor for the decision of whether to conduct pelvic lymph node dissection.

Protective effect of bone marrow mesenchymal stem cell-derived exosomes against the reproductive toxicity of cyclophosphamide is associated with the p38MAPK/ERK and AKT signaling pathways.

Spermatogenic dysfunction caused by cyclophosphamide (CP) chemotherapy has seriously influenced the life quality of patients. Unfortunately, treatments for CP-induced testicular spermatogenic dysfunction are limited, and the molecular mechanisms are not fully understood. For the first time, here, we explored the effects of bone marrow mesenchymal stem cell-derived exosomes (BMSC-exos) on CP-induced testicular spermatogenic dysfunction in vitro and in vivo. BMSC-exos could be taken up by spermatogonia (GC1-spg cells). CP-injured GC1-spg cells and BMSC-exos were cocultured at various doses, and then, cell proliferation was measured using 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyl tetrazolium bromide (MTT) assay. In addition, photophosphorylation of extracellular-regulated kinase (ERK), p38 mitogen-activated protein kinase (p38MAPK), and protein kinase B (AKT) proteins was evaluated by western blotting as well as apoptosis in GC1-spg cells measured using flow cytometry. Treatment with BMSC-exos enhanced cell proliferation and reduced apoptosis of CP-injured GCI-spg cells. Phosphorylated levels of ERK, AKT, and p38MAPK proteins were reduced in CP-injured spermatogonia when co-treated with BMSC-exos, indicating that BMSC-exos acted against the reproductive toxicity of CP via the p38MAPK/ERK and AKT signaling pathways. In experiments in vivo, CP-treated rats received BMSC-exos by injection into the tail vein, and testis morphology was compared between treated and control groups. Histology showed that transfusion of BMSC-exos inhibited the pathological changes in CP-injured testes. Thus, BMSC-exos could counteract the reproductive toxicity of CP via the p38MAPK/ERK and AKT signaling pathways. The findings provide a potential treatment for CP-induced male spermatogenic dysfunction using BMSC-exos.

[Effects of silencing the SEMG1 protein on the cycle and apoptosis of GC-1 spg cells].

OBJECTIVE: To investigate the effects of silencing the semenogelin 1 (SEMG1) protein on the cycle and apoptosis of the spermatogonia germ cell line (GC-1 spg). METHODS: SEMG1-specific siRNA was transfected into GC-1 spg cells by lipofectamine 2000 (the siRNA-SEMG1 group), the relative expression levels of the SEMG1 protein in the GC-1 spg cells of the siRNA-SEMG1, blank control and negative control groups were detected by Western blot, and the apoptosis and cycle of the cells in different groups were determined by flow cytometry. RESULTS: The expression of the SEMG1 protein in the GC-1 spg cells was dramatically decreased in the siRNA-SEMG1 group compared with those in the blank and negative control groups (1.80±0.05 vs 2.51±0.13 and 2.50±0.12， P < 0.01), but the apoptosis rate was remarkably higher in the former than in the latter two groups (ï¼»6.77 ± 0.15ï¼½% vs ï¼»0.70 ± 0.06ï¼½% and ï¼»0.8 ± 0.06ï¼½%, P < 0.01). No statistically significant difference was observed in the cell cycles among the three groups (P > 0.05). In addition, Western blot showed that the expression of the caspase-3 protein was significantly higher and that of the BCL2 protein markedly lower in the siRNA-SEMG1 than in the blank and negative control groups (P < 0.05). CONCLUSIONS: SEMG1-specific siRNA can effectively silence the expression of the SEMG1 protein in GC-1 spg cells and promote their apoptosis.

Down regulating PHGDH affects the lactate production of sertoli cells in varicocele.

BACKGROUND: Although varicocele is considered to be one of the leading causes of male infertility, the precise mechanism underlying how varicocele leads to male infertility is not completely understood. We found the lactate concentration on the varicocele side of the patients was decreased compare with peripheral venous blood. In the testicles, the lactate produced by the sertoli cells through the glycolysis pathway provides most of the energy needed for spermatogenesis, the reduction of lactate will affect spermatogenesis. The objective of this study was to investigate the mechanism of this abnormal energy metabolism phenomenon in varicocele. METHODS: In this study, we collected the testicular tissue from patients with varicocele, the glycolysis related proteins PHGDH was identified by iTRAQ proteomics technology. Experimental rat varicocele model was constructed according to our new clip technique, the mRNA and protein expression levels of PHGDH were examined with qRT-PCR and Western blotting. We constructed a sertoli cell of PHGDH down-regulation model, and then detected the glucose consumption, LDH activities and lactate production in the sertoli cells. Western blot was conducted to investigate the effects of PHGDH on the expression of phosphoserine phosphatase (PSPH) and Pyruvate kinase M2 (PKM2). Flow cytometry was used to detect the cell apoptosis and cell cycle in sertoli cells. RESULTS: The results showed that testicular protein PHGDH was down-regulated in patients with varicocele and in experimental rat varicocele model. Down-regulation of PHGDH in sertoli cells significantly decreased the glucose consumption, LDH activities and lactate production in the sertoli cells, indicating that the low expression of PHGDH ultimately led to a decrease in lactate production by affecting the glycolysis. The Western blot results showed that the down-regulation of PHGDH significantly reduced the expression of pathway protein PSPH and PKM2, leading to the reduction of lactate production. Moreover, PHGDH knockdown can promote apoptosis and inhibit cell cycle to affect cell growth. CONCLUSIONS: Overall, we conformed that varicocele lead to the decreasing of testis lactate production. Down-regulation of PHGDH in sertoli cells may mediate the process of abnormal glucose metabolism. Our study provide new insight into the mechanisms underlying metabolism-associated male infertility and suggests a novel therapeutic target for male infertility.

Predictive value of cadherin-11 for subsequent recurrence and progression in non-muscle invasive bladder cancer.

BACKGROUND: Cadherin-11 (CDH11) is a type II cadherin and reported to function as an oncogene in various cancers. Our present study aims to investigate the role of CDH11 in bladder cancer (BCA). METHODS: Bioinformatics analysis was performed in four independent microarray data including 56 non-muscle-invasive bladder cancer (NMIBC) and 132 muscle-invasive bladder cancer (MIBC) tissues from Gene Expression Omnibus to screen out differentially expressed genes. Next, we detected CDH11 expression in BCA specimens and cell lines by qPCR and western blotting assays. Immunohistochemical analyses were performed in 209 paraffin-embedded BCA samples and 30 adjacent normal bladder tissues. RESULTS: Bioinformatics analysis revealed that CDH11 had a higher expression level in MIBC tissues than in NMIBC, which was consistent with our clinical BCA specimens and cell lines at both mRNA and protein levels. Immunohistochemical analysis demonstrated that over-expression of CDH11 was closely related to the histological grade, pT status, tumour size and poor outcomes of BCA patients. What's more, CDH11 (area under curve (AUC) = 0.673 and 0.735) had a better predictive value than E-cadherin (AUC = 0.629 and 0.629) and a similar discrimination with the European Organization for Research and Treatment of Cancer (EORTC) score system (AUC = 0.719 and 0.667) in evaluating potential recurrence and progression of NMIBC. Moreover, combination of CDH11 and EORTC score system was the best predictive model in predicting recurrence of NMIBC (AUC = 0.779) among the three models. CONCLUSIONS: CDH11 was a reliable therapeutic target in BCA and a useful index to predict the possibilities of recurrence and progression in NMIBC patients.

Optimal Starting Age and Baseline Level for Repeat Tests: Economic Concerns of PSA Screening for Chinese Men - 10-Year Experience of a Single Center.

OBJECTIVE: To investigate the optimal age for the baseline serum prostate-specific antigen (PSA) test and for repeat screening and its economic burden in a single center in China. MATERIALS AND METHODS: 35,533 men with PSA screening were retrospectively enrolled in this study. Follow-ups were conducted in 1,586 men with PSA >4 ng/mL, and receiver-operating characteristic (ROC) curves were employed to investigate the optimal cutoffs. RESULTS: ROC analysis indicated that the optimal age for initial PSA screening was 57.5 years (AUC = 0.84), 62.5 years (AUC = 0.902), 60.5 years (AUC = 0.909), and 61.5 years (AUC = 0.890) for individuals with PSA >4 and >10 ng/mL, a diagnosis of prostate cancer (PCa), and clinically significant PCa defined as the focus events, respectively. For Chinese men aged 50-59, 60-69, and >70 years, the initial PSA levels of 1.305 ng/mL (AUC = 0.699), 1.975 ng/mL (AUC = 0.711), and 2.740 ng/mL (AUC = 0.720) might have a PSA velocity >0.75 ng/mL per year during the follow-up. In addition, the total cost amounts to CNY 13,609,260 in these cases, but only 60 of the 35,533 (0.17%) men gained benefit from PSA screening. CONCLUSION: In our opinion, the optimal starting age for initial PSA testing was 57.5 years. The necessity for repeat screening should be based on the first PSA level depending on age. A cost--benefit analysis should be included in population-based screening.

[rotective effect of bone marrow mesenchymal stem cells-derived exosomes against testicular ischemia-reperfusion injury in rats].

OBJECTIVE: To investigate the protective effect of bone marrow mesenchymal stem cells (BMSCs)-derived exosomesagainst testicular ischemia-reperfusion injury (IRI) in rats. METHODS: Rat BMSCs were isolated, cultured and identified in theprimary culture. The exosomes were extracted from the BMSCs and characterized using nanoparticle tracking analysis, transmission electron microscopy, and Western blotting. Twenty-four healthy male SD rats were randomly divided into shamoperation group, testicular IRI with saline treatment group and IRI with exosome treatment group. The contralateral testes ofthe rats were collected for pathological observation, aseessment of superoxide dismutase (SOD) and malondialdehyde (MDA), and detection of HMGB1, caspases-3 and cleaved caspase-3 expressions using Western blotting. RESULTS: We successfullyobtained exosomes from rat BMSCs. Testicular IRI significantly impaired testicular spermatogenesis, which was markedlyimproved by treatment with the exosomes (P < 0.05). Testicular IRI also caused significant increase in the protein expression ofHMGB1, caspase-3 and cleaved caspase-3 in the testicular tissue, and treatment with the exosomes obviously amelioratedthese changes (P < 0.05). CONCLUSIONS: BMSCs-derived exosomes protects against testicular IRI due to the anti-oxidant, antiinflammatory and anti-apoptosis activities of the exosomes.

With a new clip technique surgically inducing varicocele in Sprague-Dawley rats.

BACKGROUND: We introduced and recreated a more consistent and effective experimental varicocele rat model by a new clip technique. METHODS: A total of 40 rats were numbered and randomly assigned to 5 groups of 8 each, including sham surgery (Group I), conventional (Group II) and clip groups with 0.7, 0.8, 0.9 mm gap widths, respectively (Group III, IV, V). All of the rats in each group were sacrificed at 8 weeks after initial surgery, and the rats forming out with less than 1 mm diameter of left spermatic vein or no presence of the pampiniform plexus dilation were excluded from the experimental groups. The left spermatic vein (LSV) diameter, testicular weight, left kidney weight to body weight coefficients, kidney and testicular histology were determined. RESULTS: The baseline mean diameter of the LSV in Group I, II and III was 0.22 ± 0.02, 0.23 ± 0.02 and 0.22 ± 0.03 mm, respectively (P = 0.7504). At 8 weeks after initial surgery, varicocele was successfully created in 6/8 (75%), 7/8 (87.5%), 3/8 (37.5%), 3/8 (37.5%) in GroupII-V, no varicocele was observed in Group I. In Group I, II and III, no pathological changes were observed and the left kidney weight to body weight coefficients showed no significant differences. The diameter of LSV was remarkably increased both in Group II and III compared to Group I (1.72 ± 0.13, 1.57 ± 0.19 and 0.25 ± 0.02, respectively), and Group II and III had a smaller testicular weight than the rats in Group I (1.67 ± 0.05, 1.62 ± 0.06, and 1.92 ± 0.12, respectively). CONCLUSIONS: With a new clip technique, surgically inducing varicocele rat model becomes convenient and safe. This appears to improve the effectiveness of the model and this innovation may allow us to further understand the pathophysiology of varicocele.

MicroRNA-27a-mediated repression of cysteine-rich secretory protein 2 translation in asthenoteratozoospermic patients.

Cysteine-rich secretory protein 2 (CRISP2) is an important protein in spermatozoa that plays roles in modulating sperm flagellar motility, the acrosome reaction, and gamete fusion. Spermatozoa lacking CRISP2 exhibit low sperm motility and abnormal morphology. However, the molecular mechanisms underlying the reduction of CRISP2 in asthenoteratozoospermia (ATZ) remain unknown. In this study, low expression of CRISP2 protein rather than its mRNA was observed in the ejaculated spermatozoa from ATZ patients as compared with normozoospermic males. Subsequently, bioinformatic prediction, luciferase reporter assays, and microRNA-27a (miR-27a) transfection experiments revealed that miR-27a specifically targets CRISP2 by binding to its 3' untranslated region (3'-UTR), suppressing CRISP2 expression posttranscriptionally. Further evidence was provided by the clinical observation of high miR-27a expression in ejaculated spermatozoa from ATZ patients and a negative correlation between miR-27a expression and CRISP2 protein expression. Finally, a retrospective follow-up study supported that both high miR-27a expression and low CRISP2 protein expression were associated with low progressive sperm motility, abnormal morphology, and infertility. This study demonstrates a novel mechanism responsible for reduced CRISP2 expression in ATZ, which may offer a potential therapeutic target for treating male infertility, or for male contraception.

Inhibition of prostate cancer growth by solanine requires the suppression of cell cycle proteins and the activation of ROS/P38 signaling pathway.

Solanine, a naturally steroidal glycoalkaloid in nightshade (Solanum nigrum Linn.), can inhibit proliferation and induce apoptosis of tumor cells. However, the mechanism of solanine-suppressing prostate cancer cell growth remains to be elucidated. This study investigates the inhibition mechanism of solanine on cancer development in vivo and in cultured human prostate cancer cell DU145 in vitro. Results show that solanine injection significantly suppresses the tumor cell growth in xenograft athymic nude mice. Solanine regulates the protein levels of cell cycle proteins, including Cyclin D1, Cyclin E1, CDK2, CDK4, CDK6, and P21 in vivo and in vitro. Also, in cultured DU145 cell, solanine significantly inhibits cell growth. Moreover, the administration of NAC, an active oxygen scavenger, markedly reduces solanine-induced cell death. Blockade of P38 MAPK kinase cannot suppress reactive oxygen species (ROS), but can suppress solanine-induced cell apoptosis. Also, inhibition of ROS by NAC inactivates P38 pathway. Taken together, the data suggest that inhibition of prostate cancer growth by solanine may be through blocking the expression of cell cycle proteins and inducing apoptosis via ROS and activation of P38 pathway. These findings indicate an attractive therapeutic potential of solanine for suppression of prostate cancer.