Identification and validation of the surface proteins FIBG, PDGF-ß, and TGF-ß on serum extracellular vesicles for non-invasive detection of colorectal cancer: experimental study.

OBJECTIVES: The absence of non-invasive biomarkers for the early diagnosis of colorectal cancer (CRC) has contributed to poor prognosis. Extracellular vesicles (EVs) have emerged as promising candidates for cancer monitoring using liquid biopsy. However, the complexity of EVs isolation procedures and absence of clear targets for detecting serum-derived EVs have hindered the clinical application of EVs in early CRC diagnosis. METHODS: In the discovery phase, we conducted a comprehensive 4D-DIA proteomic analysis of serum-derived EVs samples from 37 individuals, performing an initial screening of EVs surface proteins. In the technical validation phase, we developed an extraction-free CRC-EVArray microarray to assess the expression of these potential EVs surface proteins in a multicenter study comprising 404 individuals. In the application phase, we evaluated the diagnostic efficacy of the CRC-EVArray model based on machine-learning algorithms. RESULTS: Through 4D-DIA proteomic analysis, we identified 7 potential EVs surface proteins showing significantly differential expression in CRC patients compared to healthy controls. Utilizing our developed high-throughput CRC-EVArray microarray, we further confirmed the differential expression of 3 EVs surface proteins, FIBG, PDGF-ß and TGF-ß, in a large sample population. Moreover, we established an optimal CRC-EVArray model using the NNET algorithm, demonstrating superior diagnostic efficacy with an AUC of 0.882 in the train set and 0.937 in the test set. Additionally, we predicted the functions and potential origins of these EVs-derived proteins through a series of multi-omics approaches. CONCLUSIONS: Our systematic exploration of surface protein expression profiles on serum-derived EVs has identified FIBG, PDGF-ß, and TGF-ß as novel diagnostic biomarkers for CRC. And the development of CRC-EVArray diagnostic model based on these findings provided an effective tool for the large-scale CRC screening, thus facilitating its translation into clinical practice.

Exosome circATP8A1 induces macrophage M2 polarization by regulating the miR-1-3p/STAT6 axis to promote gastric cancer progression.

Circular RNAs (circRNAs) play important roles in gastric cancer progression but the regulatory role of circRNAs in controlling macrophage function remains elusive. Exosomes serve as cargo for circRNAs and play a crucial role as mediators in facilitating communication between cancer cells and the tumor microenvironment. In this study, we found that circATP8A1, a previously unreported circular RNA, is highly expressed in both gastric cancer tissues and exosomes derived from plasma. Increased circATP8A1 was associated with advanced TNM stage and worse prognosis in patients with gastric cancer. We showed that  the circATP8A1 knockdown significantly inhibited gastric cancer proliferation and invasion in vitro and in vivo. Functionally, exosome circATP8A1 induced the M2 polarization of macrophages through the STAT6 pathway instead of the STAT3 pathway. Mechanistically, circATP8A1 was shown to activate the STAT6 pathway through competitive binding to miR-1-3p, as confirmed by Fluorescence In Situ Hybridization (FISH), RNA immunoprecipitation, RNA pulldown, and Luciferase reporter assays. The reversal of circATP8A1-induced STAT6 pathway activation and macrophage polarization was observed upon blocking miR-1-3p. Macrophages treated with exosomes from gastric cancer cells overexpressing circATP8A1 were able to promote gastric cancer migration, while knockdown of circATP8A1 reversed these effects in vivo. In summary, exosome-derived circATP8A1 from gastric cancer cells induce macrophages M2 polarization via the circATP8A1/miR-1-3p/STAT6 axis, and tumor progression. Our results highlight circATP8A1 as a potential prognostic biomarker and therapeutic target in gastric cancer.

M1 polarization of macrophages promotes stress-induced hair loss via interleukin-18 and interleukin-1ß.

Stress-induced hair loss is a prevalent health concern, with mechanisms that remain unclear, and effective treatment options are not yet available. In this study, we investigated whether stress-induced hair loss was related to an imbalanced immune microenvironment. Screening the skin-infiltrated immune cells in a stressed mouse model, we discovered a significant increase in macrophages upon stress induction. Clearance of macrophages rescues mice from stress-induced hair shedding and depletion of hair follicle stem cells (HFSCs) in the skin, demonstrating the role of macrophages in triggering hair loss in response to stress. Further flow cytometry analysis revealed a significant increase in M1 phenotype macrophages in mice under stressed conditions. In searching for humoral factors mediating stress-induced macrophage polarization, we found that the hormone Norepinephrine (NE) was elevated in the blood of stressed mice. In addition, in-vivo and in-vitro studies confirm that NE can induce macrophage polarization toward M1 through the ß-adrenergic receptor, Adrb2. Transcriptome, enzyme-linked immunosorbent assay (ELISA), and western blot analyses reveal that the NLRP3/caspase-1 inflammasome signaling and its downstream effector interleukin 18 (IL-18) and interleukin 1 beta (IL-1ß) were significantly upregulated in the NE-treated macrophages. However, inhibition of the NE receptor Adrb2 with ICI118551 reversed the upregulation of NLRP3/caspase-1, IL-18, and IL-1ß. Indeed, IL-18 and IL-1ß treatments lead to apoptosis of HFSCs. More importantly, blocking IL-18 and IL-1ß signals reversed HFSCs depletion in skin organoid models and attenuated stress-induced hair shedding in mice. Taken together, this study demonstrates the role of the neural (stress)-endocrine (NE)-immune (M1 macrophages) axis in stress-induced hair shedding and suggestes that IL-18 or IL-1ß may be promising therapeutic targets.

Clinicopathological characteristics and prognosis of adolescents and young adults with gastric cancer after gastrectomy: a propensity score matching analysis.

Background: Gastric cancer (GC) among adolescents and young adults (AYAs, aged 15-39 years) has limited data on clinicopathological characteristics and prognosis. This study aimed to compare the clinicopathological characteristics, perioperative outcomes, and long-term outcomes of AYAs and older adults (OAs, aged > 39 years) with GC who underwent curative gastrectomy. Methods: From January 1994 to June 2019, patients with GC undergoing curative gastrectomy were enrolled and divided into AYA group and OA group. The clinicopathological characteristics, treatment variables, perioperative outcomes and long-term outcomes were compared between the two groups, both before and after propensity score matching (PSM). Results: AYAs had fewer comorbid conditions and were more likely to be females, have normal carcinoembryonic antigen (CEA) levels, poorly differentiated tumors with perineural invasion, and receive adjuvant chemotherapy. AYA patients had lower incidence of postoperative complications and shorter length of postoperative hospital stay than OA patients. No significant differences in postoperative 30-day or 90-day mortality were observed between AYAs and OAs, both before and after PSM. In the entire cohort, AYAs had similar median overall survival (OS) to OAs. However, in the PSM cohort, AYAs had significantly shorter median OS. Young age (15-39 years) was an independent risk factor for OS in GC patients following gastrectomy. Conclusion: The clinicopathological characteristics were significantly different between AYA and OA patients with GC. AYA patients with GC had worse long-term prognosis than OA patients, and young age was an independent risk factor for OS in GC patients following gastrectomy.

Comprehensive analysis to construct a novel immune-related prognostic panel in aging-related gastric cancer based on the lncRNA‒miRNA-mRNA ceRNA network.

Introduction: Gastric cancer (GC) is the fifth frequent malignancy and is responsible for the third leading cause of cancer-related deaths. Gastric cancer is an aging-related disease, with incidence and mortality rates increasing with aging. The development of GC is affected by lncRNAs, miRNAs, and mRNAs at the transcriptional and posttranscriptional levels. This study aimed to establish a prognostic panel for GC based on competing endogenous RNA (ceRNA) networks. Methods: RNA sequences were obtained from the TCGA database. Different expressions of RNAs were scrutinized with the EdgeR package. The ceRNA network was built using the starBase database and the Cytoscape. The prognostic panel was constituted with the LASSO algorithm. We developed a nomogram comprising clinical characteristic and risk score. The receiver operating characteristic (ROC) was used to evaluate the accuracy of the nomogram prediction. Hub RNAs expressions were detected by qPCR, immunohistochemistry and western blot respectively. Clinical relevance and survival analyses were analyzed. The relationship between RNAs and immune infiltrations, as well as immune checkpoints, was analyzed and evaluated using the CIBERSORT, TIMER and TISIDB databases. Results: Four DElncRNAs, 21 DEmiRNAs and 45 DEmRNAs were included in the ceRNA network. A 3-element panel (comprising lncRNA PVT1, hsa-miR-130a-3p and RECK) with poor overall survival (OS) was established and qPCR was applied to validate the expressions of hub RNAs. Hub RNAs were firmly associated with T, M, and N stage. The CIBERSORT database showed that the high lassoScore group exhibited a significantly high ratio of resting memory CD4+ T cells, M2 macrophages and a significantly low ratio of activated memory CD4+ T cells and M1 macrophages. According to the TIMER database, this panel was linked to immune infiltrations and immune cell gene markers. TISIDB database indicated that RECK was positively correlated with immune checkpoints (including CD160, CD244, PDCD1, and TGFBR1). Discussion: A novel triple prognostic panel of GC constructed based on the ceRNA network was associated with clinical prognostic, clinicopathological features, immune infiltrations, immune checkpoints and immune gene markers. This panel might provide potential therapeutic targets for GC and more experimental verification research is needed.

Construction of a hypoxia-immune-related prognostic panel based on integrated single-cell and bulk RNA sequencing analyses in gastric cancer.

Introduction: Gastric cancer (GC) is the fifth most common tumor, contributing to the third-highest number of cancer-related deaths. Hypoxia is a major feature of the tumor microenvironment. This study aimed to explore the influence of hypoxia in GC and establish a hypoxia-related prognostic panel. Methods: The GC scRNA-seq data and bulk RNA-seq data were downloaded from the GEO and TCGA databases, respectively. AddModuleScore() and AUCell() were used to calculate module scores and fractions of enrichment for hypoxia-related gene expression in single cells. Least absolute shrinkage and selection operator cox (LASSO-COX) regression analysis was utilized to build a prognostic panel, and hub RNAs were validated by qPCR. The CIBERSORT algorithm was adopted to evaluate immune infiltration. The finding of immune infiltration was validated by a dual immunohistochemistry staining. The TIDE score, TIS score and ESTIMATE were used to evaluate the immunotherapy predictive efficacy. Results: Hypoxia-related scores were the highest in fibroblasts, and 166 differentially expressed genes were identified. Five hypoxia-related genes were incorporated into the hypoxia-related prognostic panel. 4 hypoxia-related genes (including POSTN, BMP4, MXRA5 and LBH) were significantly upregulated in clinical GC samples compared with the normal group, while APOD expression decreased in GC samples. Similar results were found between cancer-associated fibroblasts (CAFs) and normal fibroblasts (NFs). A high hypoxia score was associated with advanced grade, TNM stage, N stage, and poorer prognosis. Decreased antitumor immune cells and increased cancer-promoting immune cells were found in patients with high hypoxia scores. Dual immunohistochemistry staining showed high expression of CD8 and ACTA2 in gastric cancer tissue. In addition, the high hypoxia score group possessed higher TIDE scores, indicating poor immunotherapy benefit. A high hypoxia score was also firmly related to sensitivity to chemotherapeutic drugs. Discussion: This hypoxia-related prognostic panel may be effective in predicting the clinical prognosis, immune infiltrations, immunotherapy, and chemotherapy in GC.

Gastric cancer clinical characteristics and their altered trends in South China: An epidemiological study with 2,800 cases spanning 26 years.

Background: Gastric cancer (GC) is a serious threat to human health. The clinical GC characteristics in China may be impacted by changes in people's lifestyles and the promotion of early GC (EGC) screening. The present study aims to evaluate the recent trends of GC characteristics in South China and search for hazardous factors limiting the survival time of GC patients. Methods: Data on GC patients that were hospitalized in the Department of Digestive Center, the First Affiliated Hospital, Sun Yat-sen University, from 1994 to 2019 were collected and divided into two categories according to the time when the EGC screening began in China: the PRE group (previous 13 years, 1994-2006) and the PAS group (past 13 years, 2007-2019). Results: We found that, although the 5-year survival rate increased in the PAS group compared with the PRE group (P < 0.0001), patients with age ≥60 years or Borrmann type IV still had a worse prognosis. In the PAS group, the larger percentages of elderly patients and patients with Borrmann type IV in the lymphatic metastases (N1) group (41.0% vs. 51.1%, P = 0.0014) and stage IV subgroup (20.7% vs. 32.2%, P = 0.016), respectively, when compared with the PRE group, may have contributed to the poor outcome of GC. By comparing the odds ratio (OR) of 5-year overall survival (OS) in the two 13-year periods, female sex and T2 turned into risk factors because of a greater proportion of Borrmann type IV or elderly patients in the PAS group (OR = 0.983, 95% CI = 0.723-1.336 vs. OR = 1.277, 95% CI = 1.028-1.586 and OR = 1.545, 95% CI = 0.499-4.775 vs. OR = 2.227, 95% CI = 1.124-4.271, respectively). Conclusions: Despite the GC epidemiology changes, the overall prognosis of GC patients has improved in South China. However, old age and Borrmann type IV are still the major restrictions affecting the survival of GC patients, a situation which calls for additional attention.

Identification and validation of a PD-L1-related signature from mass spectrometry in gastric cancer.

BACKGROUND: According to the guidelines, PD-L1 expression is a critical indicator for guiding immunotherapy application. According to certain studies, regardless of PD-L1 expression, immunotherapy could be advantageous for individuals with gastric cancer. Therefore, new scoring systems or biomarkers are required to enhance treatment strategies. METHODS: Mass spectrometry and machine learning were used to search for strongly related PD-L1 genes, and the NMF approach was then used to separate gastric cancer patients into two categories. Differentially expressed genes (DEGs) between the two subtypes identified in this investigation were utilized to develop the UBscore predictive model, which was verified by the Gene Expression Omnibus (GEO) database. Coimmunoprecipitation, protein expression, and natural killing (NK) cell coculture experiments were conducted to validate the findings. RESULTS: A total of 123 proteins were identified as PD-L1 interactors that are substantially enriched in the proteasome complex at the mRNA level. Using random forest, 30 UPS genes were discovered in the GSE66229 cohort, and ANAPC7 was experimentally verified as one of 123 PD-L1 interactors. Depending on the expression of PD-L1 and ANAPC7, patients were separated into two subgroups with vastly distinct immune infiltration. Low UBscore was related to increased tumor mutation burden (TMB) and microsatellite instability-high (MSI-H). In addition, chemotherapy medications were more effective in individuals with a low UBscore. Finally, we discovered that ANAPC7 might lead to the incidence of immunological escape when cocultured with NK-92 cells. CONCLUSION: According to our analysis of the PD-L1-related signature in GC, the UBscore played a crucial role in prognosis and had a strong relationship with TMB, MSI, and chemotherapeutic drug sensitivity. This research lays the groundwork for improving GC patient prognosis and treatment response.

Crosstalk of Histone and RNA Modifications Identified a Stromal-Activated Subtype with Poor Survival and Resistance to Immunotherapy in Gastric Cancer.

Emerging evidence has revealed the pivotal role of epigenetic modifications in shaping the tumor microenvironment (TME). However, crosstalk between different modification types and their clinical relevance in cancers remain largely unexplored. In this study, using ChIP/MeRIP-seq data of seven human gastric cell lines, we systematically characterized the crosstalk of four epigenetic modification types including H3K4me1, H3K4me3, H3K27ac, and N6-methyladenosine (m6A) and identified a recurrent subtype with high FTO expression and low HDAC1 expression across three independent gastric cancer (GC) cohorts, which we named the epigenetic-modification-dysregulated (EMD) subtype. Patients of the EMD subtype were featured with poor survival, stromal activation, and immune suppression. Extensive relevance to clinical characteristics was observed in the EMD subtype, including the Lauren classification, MSI status, histological grade, TNM stage, the Asian Cancer Research Group classification, and the immune/fibrotic classification. An EMD score was then constructed using WGCNA and ssGSEA algorithms, to precisely recognize the EMD subtype and indicate prognosis and response to immunotherapy in multiple independent GC cohorts. Correlations of the EMD score with tumor mutation burden, tumor purity, aneuploidy score, tumorigenic pathways, TME characteristics, and FTO/HDAC1 ratio were measured. In vitro experiments were performed to demonstrate the correlation between FTO and the epithelial-mesenchymal transition pathway, which suggested FTO as a targetable vulnerability for GC patients with a high EMD score. Altogether, by comprehensively analyzing the epigenetic modification patterns of 1518 GC patients, we identified a novel stromal-activated subtype with poor survival and resistance to immunotherapy, which might benefit from the combined immune checkpoint inhibition therapy with FTO inhibition.

Effects of varicocele and microsurgical varicocelectomy on the metabolites in semen.

The influence of varicocele and microsurgical varicocelectomy on semen quality remains unclear. Few studies have investigated the relationship between semen metabolism and the abnormalities in reproductive function caused by varicocele, however, there is no study on the changes of semen metabolism after microsurgical varicocelectomy. Here, we used the non-targeted and targeted metabolic analysis to investigate the different metabolites in seminal plasma within normal, varicocele, and varicocelectomy groups. We clearly showed that varicocele significantly affects semen metabolism, and microsurgical varicocelectomy can reverse this metabolic abnormality. Moreover, we characterized the landscape of three dipeptides in the seminal plasma of patients with varicocele that have not been identified previously in human tissues or biofluids. Interestingly, the levels of these three dipeptides decreased after microsurgical varicocelectomy coincident with an improvement in semen quality. Western blotting confirmed the downregulation of DPEP3 (dipeptidase 3) in the varicocele group and the upregulation of DPEP3 in the varicocelectomy group. Furthermore, we found that eight metabolites may be helpful to distinguish varicocele patients from normal subjects. Our results may be applied to earlier diagnosis or to predict the outcome of microsurgery for varicocele.

The Prognostic Effect of Multidisciplinary Team Intervention in Patients with Advanced Gastric Cancer.

BACKGROUND: The effect of multidisciplinary team intervention (MDT) on the prognosis of advanced gastric cancer (GC) is still controversial. This study aims to analyze the effect of MDTs on the overall survival time of advanced gastric cancer patients. METHODS: Patients with advanced GC who underwent surgical treatment between 2007 and 2014 were included in the study. They were divided into two groups; the MDT group received MDT treatment and the non-MDT group received conventional treatment. The Kaplan-Meier method was used to compare the overall survival (OS) of the two groups. The prognostic factors of advanced GC were evaluated by multivariate Cox regression analysis. RESULTS: 394 patients were included in our study. Kaplan-Meier survival analysis showed that the prognosis of advanced GC patients with who underwent MDT intervention was better than those without (3-year OS of 55.6% vs. 46.1%, p = 0.005), Multivariate analysis indicated that MDT intervention could reduce mortality (HR = 0.493, p < 0.001). CONCLUSIONS: MDT intervention is an effective measure that improves the survival of patients with advanced GC.

Syntaxin-6, a Reliable Biomarker for Predicting the Prognosis of Patients with Cancer and the Effectiveness of Immunotherapy.

Syntaxin-6 (STX6), a vesicular transport protein, is a direct target of the tumor suppressor gene P53, supporting cancer growth dependent on P53. However, STX6's function in the tumor microenvironment has yet to be reported. In this research, we comprehensively explored the role of the oncogene STX6 in pan-cancer by combining data from several databases, including the Cancer Genome Atlas, CPTAC, cBioPortal, and TIMER. Then, we verified the carcinogenic effect of STX6 in hepatocellular carcinoma (HCC) and colorectal cancer (CRC) through a series of experiments in vitro and in vivo. Bioinformatics analysis demonstrated that STX6 is an oncogene for several cancers and is mainly involved in the cell cycle, epithelial-mesenchymal transition, oxidative phosphorylation, and tumor immune modulation, especially for tumor-associated fibroblasts (CAFs) and NKT cells. Additionally, a high level of STX6 could indicate patients' resistance to immunotherapy. Our own data indicated that the STX6 level was upregulated in HCC and CRC. Knockdown of the STX6 levels could arrest the cell cycle and restrain cell proliferation, migration, and invasion. RNA-seq indicated that STX6 was significantly involved in pathways for cancer, such as the MAPK signal pathway. In a mouse model, knockdown of STX6 inhibited tumor growth and potentiated anti-PD-1 efficacy. In light of the essential roles STX6 plays in carcinogenesis and cancer immunology, it has the potential to be a predictive biomarker and a target for cancer immunotherapy.

Reproductive outcomes of intracytoplasmic sperm injection using testicular sperm and ejaculated sperm in patients with AZFc microdeletions: a systematic review and meta-analysis.

Studies have explored the assisted reproductive technology (ART) outcomes of Y-chromosome azoospermia factor c (AZFc) microdeletions, but the effect of sperm source on intracytoplasmic sperm injection (ICSI) remains unknown. To determine the ART results of ICSI using testicular sperm and ejaculated sperm from males with AZFc microdeletions, we searched Embase, Web of Science, and PubMed to conduct a systematic review and meta-analysis. The first meta-analysis results for 106 cycles in five studies showed no significant differences in the live birth rate between the testicular sperm group and the ejaculated sperm group (risk ratio: 0.97, 95% confidence interval [CI]: 0.73-1.28, P = 0.82). The second meta-analysis of 106 cycles in five studies showed no difference in the abortion rate between the testicular sperm group and ejaculated sperm group (risk ratio: 1.06, 95% CI: 0.54-2.06, P = 0.87). The third meta-analysis of 386 cycles in seven studies showed no significant difference in clinical pregnancy rates between the testicular sperm group and the ejaculated sperm group (risk ratio: 1.24, 95% CI: 0.66-2.34, P = 0.50). Inevitable heterogeneity weakened our results. However, our results indicated that testicular sperm and ejaculated sperm yield similar ART outcomes, representing a meaningful result for clinical treatment. More properly designed studies are needed to further confirm our conclusions.

[Stem cell therapy for non-obstructive azoospermia].

Non-obstructive azoospermia (NOA) is an important factor that causes male infertility. Stem cells are a group of cells capable of self-renewal and multi-directional differentiation, and embryonic stem cells and induced pluripotent stem cells can generate spermatozoa through differentiation, which, however, is confronted with ethical constraints and the risk of tumorigenesis. Spermatogonial stem cells can produce haploid gametes by differentiation but human spermatogonial stem cells are difficult to be cultured in vitro. Mesenchymal stem cells promote spermatogenesis through paracrine activity, and Leydig stem cells act on sperm production by secreting testosterone. 2D co-culture of multiple stem cells and 3D testicular organ culture can promote spermatogenesis by simulating a better spermatogenic microenvironment of the testis. Some progress has been achieved in the treatment of NOA by stem cell therapy despite existing problems and difficulties. This review summarizes the advances in the studies of stem cell therapy for NOA and introduces its application prospects and existing problems so as to provide some reference for the relevant researches and application.

Combined Transplantation of Adipose Tissue-Derived Stem Cells and Endothelial Progenitor Cells Improve Diabetic Erectile Dysfunction in a Rat Model.

Erectile dysfunction (ED) is a common complication in men suffered with diabetic mellitus. Stem cell transplantation is a promising strategy for the treatment of diabetic ED (DED). In this study, we evaluated whether combined transplantation of adipose tissue-derived stem cells (ADSCs) and endothelial progenitor cells (EPCs) could improve the erectile function of the DED rat model. DED rats were induced via intraperitoneal injection of streptozotocin (50 mg/kg), and ED was screened by apomorphine (100 mg/kg). DED rats were divided into 4 groups (n = 14 each): DED, ADSC, EPC, and ADSC/EPC group. Another 14 age-matched male SD rats with normal erectile function were served as the normal group. The normal group and the DED group were received intracavernous injection with phosphate-buffered saline (PBS). And the other groups were received intracavernous injection with ADSCs (1 × 106), EPCs (1 × 106), and ADSCs/EPCs (0.5 × 106/0.5 × 106), respectively. The total intracavernous pressure (ICP) and mean arterial pressure (MAP) were recorded at day 28 after injection. The endothelium, smooth muscle, and penile dorsal nerves were assessed within cavernoursal tissue. On day 28 after injection, the ADSC/EPC group displayed more significantly enhanced ICP and ICP/MAP than the DED or ADSC or EPC group (p < 0.05). Immunofluorescent analysis and western blot demonstrated that the improvement of erectile function in the ADSC/EPC5 group was associated with increased expression of endothelial marker (CD31) and the correction of eNOS-cGMP-NO signaling. More 5-ethynyl-2'-deoxyuridine- (EdU-) positive EPCs could be found lining in the cavernous endothelial layer in the ADSC/EPC group than the EPC group, which was attributed to the paracrine of vascular endothelial growth factor (VEGF) and stromal-derived factor-1 (SDF-1) by ADSCs. Combined transplantation of ADSCs and EPCs has a synergic effect in repairing the endothelial function of DED rats, and the underlying mechanism might be the paracrine of VEGF and SDF-1 by ADSCs, which improves the recruitment and proliferation of EPCs in the cavernosum.

The Anti-Inflammatory and Antioxidative Effects of Ningmitai Capsule in the Experimental Autoimmune Prostatitis Rat Model.

OBJECTIVE: Ningmitai (NMT) capsule has been widely prescribed for the treatment of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS), but the mechanism remains unclear. This study aims to evaluate the therapeutic effects of the NMT capsule in the experimental autoimmune prostatitis (EAP) rat models and explore its possible mechanisms. METHODS: A total of fifty male Sprague Dawley rats were used in this study. Prostate extract was obtained for the induction of EAP rat models. The EAP rats were randomly divided into the model group, NMT low-dose group (0.45 g/kg/d), NMT medium-dose group (0.90 g/kg/d), and NMT high-dose group (1.80 g/kg/d), with six rats per group. Three NMT treatment groups were administered with the NMT capsule by gavage for 30 days. HE staining was used for histopathological analyses of prostate tissues. Western blotting was used to measure the expression of proinflammatory factors IL-1ß and TNF-α. The MDA level was detected to reflect the level of oxidative stress. The bilateral dorsal root ganglia of T3/L1 to S4 were dissected to measure the substance P expression. RESULTS: EAP rat models were successfully constructed, in which extensive infiltration of inflammatory cells was found. Treatment of NMT capsule for 30 days and the infiltration of inflammatory cells were significantly mitigated (P < 0.05), especially in the NMT medium-dose group and NMT high-dose group. Moreover, the expression of IL-1ß and the level of MDA were significantly decreased (P < 0.05). In addition, NMT treatment could significantly alleviate substance P expression in dorsal root ganglia. CONCLUSION: Our findings demonstrate that the NMT capsule can alleviate inflammatory response and oxidative stress and reduce the production of substance P in EAP rats. This provides a theoretical basis for the clinical application of NMT capsule for CP/CPPS treatment.

Inhibiting Necroptosis of Spermatogonial Stem Cell as a Novel Strategy for Male Fertility Preservation.

Fertility preservation is a common concern for male cancer survivors of reproductive age. However, except for testicular tissue cryopreservation, which is not very effective, there is no feasible and precise therapy capable of protecting spermatogenesis for prepubertal boys before or during gonadotoxic treatment. This study aims to investigate the effects of inhibiting necroptosis of spermatogonial stem cell (SSC) in fertility preservation. Male mice 12 weeks of age were used to establish gonadotoxicity with two intraperitoneal injections of busulfan at a total dose of 40 mg kg-1. The mouse model and the primary cultured mouse SSCs were used to characterize the relationship between necroptosis of SSC and gonadotoxicity. Meanwhile, the effects of an inhibitor of necroptosis pathway, RIPA-56, were observed on day 36 in the mouse model of busulfan-induced gonadotoxicity. We found that the number of SSCs was decreased, but the level of necroptosis was upregulated on day 18 after busulfan treatment in testes from gonadotoxic mice. Further experiments in primary cultured cells showed that the necroptosis caused cell death in busulfan-treated SSCs and could be inhibited by RIPA-56. After suppressing the necroptosis of SSCs, the busulfan-induced mice had a decreased loss of spermatogenic cells as shown by histology and an increased Johnsen's score. Moreover, the quantities of SSCs and epididymal spermatozoa were restored after intervention with RIPA-56, indicating a series of beneficial effects by targeting the necroptosis of SSCs in mice undergoing busulfan treatment. In conclusion, our findings reveal that the necroptosis of SSCs plays a critical role in busulfan-induced gonadotoxicity and may be a potential target for male fertility preservation.

Establishing a nonlethal and efficient mouse model of male gonadotoxicity by intraperitoneal busulfan injection.

An ideal animal model of azoospermia would be a powerful tool for the evaluation of spermatogonial stem cell (SSC) transplantation. Busulfan has been commonly used to develop such a model, but 30%-87% of mice die when administered an intraperitoneal injection of 40 mg kg-1. In the present study, hematoxylin and eosin staining, Western blot, immunofluorescence, and quantitative real-time polymerase chain reaction were used to test the effects of busulfan exposure in a mouse model that received two intraperitoneal injections of busulfan at a 3-h interval at different doses (20, 30, and 40 mg kg-1) on day 36 or a dose of 40 mg kg-1 at different time points (0, 9, 18, 27, 36, and 63 days). The survival rate of the mice was 100%. When the mice were treated with 40 mg kg-1 busulfan, dramatic SSC depletion occurred 18 days later and all of the germ cells were cleared by day 36. In addition, the gene expressions of glial cell line-derived neurotrophic factor (GDNF), fibroblast growth factor 2 (FGF2), chemokine (C-X-C Motif) ligand 12 (CXCL12), and colony-stimulating factor 1 (CSF1) were moderately increased by day 36. A 63-day, long-term observation showed the rare restoration of endogenous germ cells in the testes, suggesting that the potential period for SSC transplantation was between day 36 and day 63. Our results demonstrate that the administration of two intraperitoneal injections of busulfan (40 mg kg-1 in total) at a 3-h interval to mice provided a nonlethal and efficient method for recipient preparation in SSC transplantation and could improve treatments for infertility and the understanding of chemotherapy-induced gonadotoxicity.

Transplantation of Human Urine-Derived Stem Cells Ameliorates Erectile Function and Cavernosal Endothelial Function by Promoting Autophagy of Corpus Cavernosal Endothelial Cells in Diabetic Erectile Dysfunction Rats.

AIMS: Cavernosal endothelial dysfunction is one of the factors in developing diabetic erectile dysfunction (DED), but the mechanism of cavernosal endothelial dysfunction is unclear. The present study is aimed at determining the contribution of autophagy in cavernosal endothelial dysfunction of DED rats and explaining the therapeutic effect of urine-derived stem cells (USCs). METHODS: After rat corpus cavernosal vascular endothelial cells (CCECs) were isolated and cultured in vitro, CCECs were treated with advanced glycation end products (AGEs) to mimic the diabetic situation. Autophagy flux, proliferation, and apoptosis of CCECs were determined by mRFP-GFP-LC3 adenovirus infection combined with fluorescence observation and western blot analysis. USCs were isolated from the urine of six healthy male donors, and coculture systems of USCs and CCECs were developed to assess the protective effect of USCs for CCECs in vitro. The contribution of autophagy to the cellular damage in CCECs was evaluated by the autophagic inhibitor, 3-methyladenine (3-MA). Then, DED rats were induced by streptozotocin (50 mg/kg) and screened by apomorphine test (100 µg/kg). In DED rats, USCs or PBS as vehicle was administrated by intracavernous injection (n = 15 per group), and another 15 normal rats served as normal controls. Four weeks after injection, erectile function was evaluated by measuring the intracavernosal pressure (ICP) and mean arterial pressure (MAP). Cavernosal endothelial function and autophagic activity were examined by western blot, immunofluorescence, and transmission electron microscopy. RESULTS: In vitro, AGE-treated CCECs displayed fewer LC3 puncta formation and expressed less LC3-II, Beclin1, and PCNA but expressed more p62 and cleaved-caspase3 than controls (p < 0.05). Coculture of USCs with CCECs demonstrated that USCs were able to protect CCECs from AGE-induced autophagic dysfunction and cellular damage, which could be abolished by 3-MA (p < 0.05). DED rats showed lower ratio of ICP/MAP, reduced expression of endothelial markers, and fewer autophagic vacuoles in the cavernosal endothelium when compared with normal rats (p < 0.05). Intracavernous injection of USCs improved erectile function and cavernosal endothelial function of DED rats (p < 0.05). Most importantly, our data showed that the repaired erectile function and cavernosal endothelial function were the result of restored autophagic activity of the cavernosal endothelium in DED rats (p < 0.05). CONCLUSIONS: Impaired autophagy is involved in the cavernosal endothelial dysfunction and erectile dysfunction of DED rats. Intracavernous injection of USCs upregulates autophagic activity in the cavernosal endothelium, contributing to ameliorating cavernosal endothelial dysfunction and finally improving the erectile dysfunction induced by diabetes.

Urine-Derived Stem Cells Facilitate Endogenous Spermatogenesis Restoration of Busulfan-Induced Nonobstructive Azoospermic Mice by Paracrine Exosomes.

Nonobstructive azoospermia (NOA) is a severe form of male infertility, with limited effective treatments. Urine-derived stem cells (USCs) possess multipotent differentiation capacity and paracrine effects, and participate in tissue repair and regeneration. The aim of this study is to investigate whether the transplantation of USCs or USC exosomes (USC-exos) could promote endogenous spermatogenesis restoration in a busulfan-induced NOA mice model. USCs were cultured and characterized by flow cytometry. High-density USCs were cultured in a hollow fiber bioreactor for exosomes collection. USC-exos were isolated from USCs conditional media and identified by transmission electron microscopy, western blotting, and Flow NanoAnalyzer analysis. USC-exos exhibited sphere- or cup-shaped morphology with a mean diameter of 66.5 ± 16.0 nm, and expressed CD63 and CD9. USCs and USC-exos were transplanted into the interstitial space in the testes of NOA mice per the following groups: normal group; groups treated with no injection, phosphate-buffered saline (PBS), USCs or USC-exos on days 3 and 36 after busulfan administration, respectively. Thirty days after USCs and USC-exos transplantation, spermatogenesis was restored by both USCs and USC-exos in NOA mice 36 days after busulfan treatment as confirmed by immunofluorescence staining and hematoxylin and eosin staining. Moreover, spermatogenic genes (Pou5f1, Prm1, SYCP3, and DAZL) and the spermatogenic protein UCHL1 were significantly increased in both the USCs 36 and USC-exos36 groups compared with the PBS group, as demonstrated using quantitative real-time polymerase chain reaction and western blot analysis. However, the transplantation of USCs or USC-exos at day 3 after busulfan treatment did not improve spermatogenesis in NOA mice. Our study demonstrated that USCs could facilitate endogenous spermatogenesis restoration of busulfan-induced NOA mice through paracrine exosomes but could not protect the mouse testicles at the early stage of destruction caused by busulfan. This study provides a novel insight into the treatment of NOA.