Astragali Radix: comprehensive review of its botany, phytochemistry, pharmacology and clinical application.

Astragali Radix (A. Radix) is the dried root of Astragalus membranaceus var. mongholicus (Bge) Hsiao or Astragalus membranaceus (Fisch.) Bge., belonging to the family Leguminosae, which is mainly distributed in China. A. Radix has been consumed as a tonic in China for more than 2000 years because of its medicinal effects of invigorating the spleen and replenishing qi. Currently, more than 400 natural compounds have been isolated and identified from A. Radix, mainly including saponins, flavonoids, phenylpropanoids, alkaloids, and others. Modern pharmacological studies have shown that A. Radix has anti-tumor, anti-inflammatory, immunomodulatory, anti-atherosclerotic, cardioprotective, anti-hypertensive, and anti-aging effects. It has been clinically used in the treatment of tumors, cardiovascular diseases, and cerebrovascular complications associated with diabetes with few side effects and high safety. This paper reviewed the progress of research on its chemical constituents, pharmacological effects, clinical applications, developing applications, and toxicology, which provides a basis for the better development and utilization of A. Radix.

Gut microbiome in men with chronic prostatitis/chronic pelvic pain syndrome: profiling and its predictive significance.

PURPOSE: To investigate the difference in gut microbiome composition between patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) and healthy controls, and to assess the potential of gut microbiota as predictive markers for CP/CPPS risk. METHODS: The present study included 41 CP/CPPS patients and 43 healthy controls in China. Fecal specimen data were obtained and analysed using 16S rRNA gene sequencing. Alpha and beta-diversity indices, relative microbiome abundances, cluster analysis, and linear discriminant analysis effect size (LEfSe) were employed. Microbial biomarkers were selected for the development of a diagnostic classification model, and the functional prediction was conducted using PICRUSt2. RESULTS: Alpha-diversity measures revealed no statistically significant difference in bacterial community structure between CP/CPPS patients and controls. However, significant differences were observed in the relative abundances of several bacterial genera. Beta-diversity analysis revealed a distinct separation between the two groups. Significant inter-group differences were noted at various taxonomic levels, with specific bacterial genera being significantly different in abundance. The LEfSe analysis indicated that three bacterial species were highly representative and seven bacterial species were low in CP/CPPS patients as compared to the control group. A diagnostic model for CP/CPPS based on microbial biomarkers exhibited good performance. PICRUSt2 functional profiling indicated significant differences in the development and regeneration pathway. CONCLUSION: Significant differences in the gut microbiome composition were found between groups. The study provided a novel diagnostic model for CP/CPPS based on microbiota, presenting promising potential for future therapeutic targets and non-invasive diagnostic biomarkers for CP/CPPS patients.

Nociceptor Neurons are Involved in the Host Response to Escherichia coli Urinary Tract Infections.

Purpose: Urinary tract infections (UTIs) can evoke a rapid host immune response leading to bladder inflammation and epithelial damage. Neuroimmune interactions are critical for regulating immune function in mucosal tissues. Yet the role of nociceptor neurons in bladder host defense has not been well defined. This study aimed to explore the interaction between nociceptor neurons and bladder immune system during UTIs. Methods: In this study, whether uropathogenic Escherichia coli (UPEC) and lipopolysaccharide (LPS) can directly stimulate nociceptor neurons was detected. Female C57BL/6J mice were treated with high dose of capsaicin, a high-affinity TRPV1 agonist, to ablate nociceptor neurons. Bladder inflammation, barrier epithelial function and bladder immune cell infiltration were assessed after UPEC infection. The level of neuropeptide calcitonin gene-related peptide (CGRP) in infected bladder was detected. Furthermore, the effects of CGRP on neutrophils and macrophages were evaluated both in vitro and in vivo. Results: We found that UPEC and its pathogenic factor LPS could directly excite nociceptor neurons, releasing CGRP into infected bladder, which suppressed the recruitment of neutrophils, the polarization of macrophages and the killing function of UPEC. Both Botulinum neurotoxin A (BoNT/A) and BIBN4096 (CGRP antagonism) blocked neuronal inhibition and prevented against UPEC infection. Conclusion: The present study showed a novel mechanism by which UPEC stimulated the secretion of CGRP from nociceptor neurons to suppress innate immunity.

Identification of a Nuclear Mitochondrial-Related Multi-Genes Signature to Predict the Prognosis of Bladder Cancer.

INTRODUCTION: Bladder cancer (BC) is one of the most prevalent urinary cancers, and its management is still a problem causing recurrence and progression, elevating mortality. MATERIALS AND METHODS: We aimed at the nuclear mitochondria-related genes (MTRGs), collected from the MITOMAP: A Human Mitochondrial Genome Database. Meanwhile, the expression profiles and clinical information of BC were downloaded from the Cancer Genome Atlas (TCGA) as a training group. The univariate, multivariate, and the least absolute shrinkage and selection operator (LASSO) Cox regression analyses were used to construct a nuclear mitochondrial-related multi-genes signature and the prognostic nomogram. RESULTS: A total of 17 nuclear MTRGs were identified to be correlated with the overall survival (OS) of BC patients, and a nuclear MTRGs signature based on 16 genes expression was further determined by the LASSO Cox regression analysis. Based on a nuclear MTRGs scoring system, BC patients from the TCGA cohort were divided into high- and low- nuclear MTRGs score groups. Patients with a high nuclear MTRGs score exhibited a significantly poorer outcome (median OS: 92.90 vs 20.20 months, p<0.0001). The nuclear MTRGs signature was further verified in three independent datasets, namely, GSE13507, GSE31684, and GSE32548, from the Gene Expression Omnibus (GEO). The BC patients with a high nuclear MTRGs score had significantly worse survival (median OS in GSE13507: 31.52 vs 98.00 months, p<0.05; GSE31684: 32.85 months vs unreached, p<0.05; GSE32548: unreached vs unreached, p<0.05). Furthermore, muscle-invasive bladder cancer (MIBC) patients had a significantly higher nuclear MTRGs score (p<0.05) than non-muscle-invasive bladder cancer (NMIBC) patients. The integrated signature outperformed each involved MTRG. In addition, a nuclear MTRGs-based nomogram was constructed as a novel prediction prognosis model, whose AUC values for OS at 1, 3, 5 years were 0.76, 0.75, and 0.75, respectively, showing the prognostic nomogram had good and stable predicting ability. Enrichment analyses of the hallmark gene set and KEGG pathway revealed that the E2F targets, G2M checkpoint pathways, and cell cycle had influences on the survival of BC patients. Furthermore, the analysis of tumor microenvironment indicated more CD8+ T cells and higher immune score in patients with high nuclear MTRGs score, which might confer sensitivity to immune checkpoint inhibitors. CONCLUSIONS: Not only could the signature and prognostic nomogram predict the prognosis of BC, but it also had potential therapeutic guidance.

Prognostic Role of the Ubiquitin Proteasome System in Clear Cell Renal Cell Carcinoma: A Bioinformatic Perspective.

Background: Clear cell renal cell carcinoma (ccRCC) is a common malignant tumor of the urinary system. The ubiquitin proteasome system (UPS) plays an important role in the generation, metabolism and survival of tumor. We are aimed to make a comprehensive exploration of the UPS's role in ccRCC with bioinformatic tools, which may contribute to the understanding of UPS in ccRCC, and give insight for further research. Methods: The UPS-related genes (UPSs) were collected by an integrative approach. The expression and clinical data were downloaded from TCGA database. R soft was used to perform the differentially expressed UPSs analysis, functional enrichment analysis. We also estimated prognostic value of each UPS with the help of GEPIA database. Two predicting models were constructed with the differentially expressed UPSs and prognosis-related genes, respectively. The correlations of risk score with clinical characteristics were also evaluated. Data of GSE29609 cohort were obtained from GEO database to validate the prognostic models. Results: We finally identified 91 differentially expressed UPSs, 48 prognosis related genes among them, and constructed a prognostic model with 18 UPSs successfully, the AUC was 0.760. With the help of GEPIA, we found 391 prognosis-related UPSs, accounting for 57.84% of all UPSs. Another prognostic model was constructed with 28 prognosis-related genes of them, and with a better AUC of 0.825. Additionally, our models can also stratify patients into high and low risk groups accurately in GSE29609 cohort. Similar prognostic values of our models were observed in the validated GSE29609 cohort. Conclusions: UPS is dysregulated in ccRCC. UPS related genes have significant prognostic value in ccRCC. Models constructed with UPSs are effective and applicable. An abnormal ubiquitin proteasome system should play an important role in ccRCC and be worthy of further study.

Metformin ameliorates bladder dysfunction in a rat model of partial bladder outlet obstruction.

Alteration of bladder morphology and function was the most important consequence of bladder outlet obstruction (BOO). Using a rat model of partial BOO (pBOO), we found that rats treated with metformin showed lower baseline pressures with a reduced inflammatory reaction in the early phase (2 wk) after pBOO. The NLR family pyrin domain containing 3 inflammasome pathway was inhibited in pBOO rat bladders with treatment of metformin in the early phase. Metformin reduced the activity of NLR family pyrin domain containing 3 in primary urothelial cells. In the chronic phase (9 wk after pBOO), metformin treatment ameliorated bladder fibrosis and improved the reduced compliance. Treatment with metformin suppressed the activation of Smad3 and compensated the diminished autophagy in 9-wk pBOO rat bladders. Autophagy was inhibited with upregulation of profibrotic proteins in primary fibroblasts from chronic pBOO bladders, which could be restored by administration of metformin. The antifibrotic effects of metformin on fibroblasts were diminished after silencing of AMP-activated protein kinase or light chain 3B. In summary, this study elucidates that oral administration of metformin relieves inflammation in the bladder during the early phase of pBOO. Long-term oral administration of metformin can prevent functional and histological changes in the pBOO rat bladder. The current study suggests that metformin might be used to prevent the development of bladder dysfunction secondary to BOO.NEW & NOTEWORTHY The present study in a rat model showed that oral administration of metformin alleviated inflammation following partial bladder outlet obstruction in the early phase and ameliorated bladder fibrosis as well as bladder dysfunction by long-term treatment. Our study indicated that metformin is a potential drug to inhibit bladder remodeling and alleviate bladder dysfunction. Clinical trials are needed to validate the effect of metformin on the bladder dysfunction and bladder fibrosis in the future.

Single-cell transcriptomes of mouse bladder urothelium uncover novel cell type markers and urothelial differentiation characteristics.

OBJECTIVES: Much of the information to date in terms of subtypes and function of bladder urothelial cells were derived from anatomical location or by the expression of a small number of marker genes. To have a comprehensive map of the cellular anatomy of bladder urothelial cells, we performed single-cell RNA sequencing to thoroughly characterize mouse bladder urothelium. MATERIALS AND METHODS: A total of 18,917 single cells from mouse bladder urothelium were analysed by unbiased single-cell RNA sequencing. The expression of the novel cell marker was confirmed by immunofluorescence using urinary tract infection models. RESULTS: Unsupervised clustering analysis identified 8 transcriptionally distinct cell subpopulations from mouse bladder urothelial cells. We discovered a novel type of bladder urothelial cells marked by Plxna4 that may be involved with host response and wound healing. We also found a group of basal-like cells labelled by ASPM that could be the progenitor cells of adult bladder urothelium. ASPM+ urothelial cells are significantly increased after injury by UPEC. In addition, specific transcription factors were found to be associated with urothelial cell differentiation. At the last, a number of interstitial cystitis/bladder pain syndrome-regulating genes were found differentially expressed among different urothelial cell subpopulations. CONCLUSIONS: Our study provides a comprehensive characterization of bladder urothelial cells, which is fundamental to understanding the biology of bladder urothelium and associated bladder disease.

Urinary ATP may be a biomarker for bladder outlet obstruction and its severity in patients with benign prostatic hyperplasia.

BACKGROUND: Urothelial cells release ATP into the urine in response to bladder stretch. Urinary ATP concentration in benign prostatic hyperplasia (BPH) patients was higher compared with asymptomatic controls. In this study, we aimed to explore the possibility that the urinary ATP level could be a non-invasive biomarker for bladder outlet obstruction (BOO) and its severity in BPH patients. METHODS: We included 117 BPH patients who underwent urodynamic studies and 109 asymptomatic controls. Urine samples at normal desire (from patients and controls), instilled fluids at maximum cystometric capacity (capacity fluid), and voided fluids during a pressure-flow study (only from patients) were collected. The ATP concentration in collected samples was measured using a luciferin-luciferase bioluminescence assay and normalized to urine creatinine (ATP/Cr). The degree of BOO was quantified using the BOO index (BOOI). Correlation between urodynamic parameters and urinary ATP concentration was analyzed in BPH patients. RESULTS: Urinary ATP concentration of BPH patients was significantly higher compared with controls (P<0.001). For BPH patients, a significant positive correlation was found between urinary ATP concentration and BOOI (P<0.0001). Although BPH patients with detrusor overactivity or a history of acute urinary retention had increased urinary ATP, a significant positive correlation between ATP and BOOI was also observed in these patients. When BOOI >40 was set as a cutoff point to differentiate BOO from non-BOO patients, the area under the receiver operating characteristic (ROC) curve was 0.77 (P<0.001). CONCLUSIONS: BPH patients with BOO released higher amounts of ATP into the urine. Urinary ATP can be used as a non-invasive biomarker of BOO, and its level may also have a predictive value for the degree of obstruction.

Profiles of Immune Infiltration in Bladder Cancer and its Clinical Significance: an Integrative Genomic Analysis.

Tumor-infiltrating immune cells are closely related to the prognosis of bladder cancer. Analysis of tumor infiltrating immune cells is usually based on immunohistochemical analysis. Since many immune cell marker proteins are not specific for different immune cells, which may induce misleading or incomplete. CIBERSORT is an algorithm to estimate specific cell types in a mixed cell population using gene expression data. In this study, the CIBERSORT algorithm was used to identify the immune cell infiltration signatures. The gene expression profiles, mutation data, and clinical data were collected from The Cancer Genome Atlas (TCGA) database. Unsupervised consensus clustering was used to acquire the immune cell infiltration subtypes of bladder cancer based on the fractions of 22 immune cell types. Four immune cell clusters with different immune infiltrate and mutation characteristics were identified. In addition, this stratification has a prognostic relevance, with cluster 2 having the best outcome, cluster 1 the worst. These clusters showed distinct mRNA expression patterns. The characteristic genes in subtype cluster 1 were mainly involved in cell division, those in subtype cluster 2 were mainly related in antigen processing and presentation, those in subtype cluster 3 were mainly involved in epidermal cell differentiation, and those in subtype cluster 4 were mainly related in the humoral immune response. These differences may affect the development of the bladder cancer, the sensitivity to treatment as well as the prognosis. Through further validation, this study may contribute to the development of personalized therapy and precision medical treatments.

Identification of gene expression profiles and immune cell infiltration signatures between low and high tumor mutation burden groups in bladder cancer.

Bladder cancer is one of the most commonly diagnosed tumors and is results from the accumulation of somatic mutations in the DNA. Tumor mutation burden (TMB) has been associated with cancer immunotherapeutic response. In this study, we attempted to explore the correlation between TMB and cancer prognosis. Identify the different expressed genes and immune cell infiltration signatures between low and high TMB group. Mutation data, gene expression profiles and clinical data were downloaded from The Cancer Genome Atlas (TCGA) database. Patients were divided into high and low TMB groups, allowing differentially expressed genes (DEGs) to be identified. Functional enrichment and protein-protein interaction (PPI) network analysis were used to identify the functions of the DEGs. And immune cell infiltration signatures were evaluated by CIBERSORT algorithm. These results shown that high TMB was significantly associated with prognosis. We obtained a list of TMB related genes which may influence the infiltrations of immune cells. We also found a higher proportion of CD8 T cells, CD4 T cells and NK cells in the high TMB group. Our data suggest that higher TMB tends to promote the infiltrations of T cells and NK cells and patients with higher TMB may achieve a more favorable prognosis in bladder cancer.

Cigarette smoke induces the pyroptosis of urothelial cells through ROS/NLRP3/caspase-1 signaling pathway.

AIMS: Cell death and inflammation are involved in the development of bladder dysfunction. Pyroptosis is programmed cell death, causing cytotoxic effects and local inflammation. As one of the biggest health threats in the world, smoking is also closely related to urinary system diseases. The aims of this study were to investigate the role of NLRP3 inflammasome-mediated pyroptosis in the bladder after cigarette smoke exposure. METHODS: The expression of NLRP3 inflammasome and the activity of caspase-1 in bladder tissue was investigated after cigarette smoke exposure. In vitro, bladder urothelial cells were stimulated by cigarette smoke extract and then the activity of caspase-1 and the expression of NLRP3 inflammasome were measured. The role of oxidative stress was also assessed. RESULTS: The activity of caspase-1 in bladder tissue increased by 50% after cigarette smoke exposure. Cigarette smoke caused oxidative stress injury and the activation of NLRP3 inflammasome. In addition, reactive oxygen species (ROS) inhibitor N-acetyl-cysteine alleviated the pyroptosis of urothelial cells. CONCLUSIONS: Cigarette smoke-induced pyroptosis of bladder tissue by activating ROS/NLRP3/caspase-1 signaling pathway. Inhibition of bladder urothelial cell pyroptosis may be a new approach to alleviate bladder damage caused by smoking.

Cannabinoid receptor 2 activation decreases severity of cyclophosphamide-induced cystitis via regulating autophagy.

PURPOSE: Cannabinoids have been shown to exert analgesic and anti-inflammatory effects, and the effects of cannabinoids are mediated primarily by cannabinoid receptors 1 and 2 (CB1 and CB2). The objective of this study was to determine efficacy and mechanism of CB2 activation on cyclophosphamide (CYP)-induced cystitis in vivo. METHODS: Cystitis was induced by intraperitoneal (IP) injection of CYP in female C57BL/6J mice. Mice were pretreated with CB2 agonist JWH-133 (1 mg/kg, intraperitoneally), CB2 antagonist AM-630 (1 mg/kg, intraperitoneally) or autophagy inhibitor 3-methyladenine (3-MA) (50 mM, intraperitoneally) before IP injection of CYP. Peripheral nociception and spontaneous voiding were investigated in these mice. Bladders were collected, weighed, and processed for real-time polymerase chain reaction, immunoblotting analysis, histological and immunohistochemical analysis. RESULTS: Twenty-four hours after IP injection of CYP, the bladder of CYP-treated mice showed histological evidence of inflammation. The expression of CB2 in bladder was significantly increased in CYP-treated mice. Mechanical sensitivity was significantly increased in CYP-treated mice and CB2 agonist JWH-133 attenuated this effect (P < .05). The number of urine spots was significantly increased after CYP treatment and it was decreased in JWH-133 treated mice (P < .05). Activating CB2 with JWH-133 significantly alleviated bladder tissue inflammatory responses and oxidative stress induced by CYP. Activation of CB2 by JWH-133 increased the expression of LC3-II/LC3-I ratio, and decreased the expression of SQSTM1/p62 in the bladder of cystitis mice, whereas AM-630 induced inverse effects. Further study indicated that JWH-133 could promote autophagy and blocking autophagy by 3-MA dismissed the effort of CB2 in alleviating bladder tissue inflammatory responses and oxidative stress injury. Furthermore, treatment with 3-MA decreased the expression of p-AMPK and induced the phosphorylation of mTOR in the presence of JWH-133 stimulation in cystitis model. CONCLUSIONS: Activation of CB2 decreased severity of CYP-induced cystitis and ameliorated bladder inflammation. CB2 activation is protective in cystitis through the activation of autophagy and AMPK-mTOR pathway may be involved in the initiation of autophagy.

Pyroptosis engagement and bladder urothelial cell-derived exosomes recruit mast cells and induce barrier dysfunction of bladder urothelium after uropathogenic E. coli infection.

The specific regulatory mechanism of bladder urothelial barrier dysfunction after infection with uropathogenic Escherichia coli (UPEC) is still unclear. The cross talk between bladder urothelial cells and mast cells may play an important role during UPEC infection. In this study, the pyroptosis of urothelial cells was investigated after UPEC infection both in vivo and in vitro. The levels of IL-1ß and IL-18 in exosomes derived from bladder urothelial cells after UPEC infection were detected. The role of these processes in the recruitment and activation of mast cells was measured. The mechanism of mast cell-induced disruption of bladder epithelial barrier function was also assessed. We found that UPEC infection induced pyroptosis of bladder urothelial cells and led to the release of IL-1ß and IL-18 in the form of exosomes, which promoted the migration of mast cells. Tryptase secreted by mast cells aggravated the damage to the barrier function of the bladder urothelium by acting on protease-activated receptor 2 (PAR2). Inhibition of pyroptosis or the tryptase-PAR2 axis reduced the disruption of bladder urothelial barrier function and decreased the bacterial burden. The present study supports a novel mechanism by which pyroptosis-dependent release of exosomes from bladder urothelial cells activates mast cells and regulates bladder urothelial barrier function during UPEC infection.

Role of GM-CSF in a mouse model of experimental autoimmune prostatitis.

The etiology of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is still unknown. Granulocyte macrophage colony-stimulating factor (GM-CSF) has been shown to play an important role in the development of autoimmune and inflammatory diseases. Here, we investigated the expression and function of GM-CSF in patients with CP/CPPS and in a mouse model of experimental autoimmune prostatitis (EAP). GM-CSF mRNA levels were detected in expressed prostatic secretions samples from patients with CP/CPPS and in prostate tissue from a mouse model of EAP. The expression of GM-CSF receptor in mouse prostate and dorsal root ganglia were determined using PCR and immunohistochemistry. Behavioral testing and inflammation scoring were performed to evaluate the role of GM-CSF in disease development and symptom severity of EAP using GM-CSF knockout mice. mRNA levels of putative nociceptive and inflammatory markers were measured in the prostate after the induction of EAP. Elevated GM-CSF mRNA levels were observed in expressed prostatic secretions samples from patients with CP/CPPS compared with healthy volunteers. GM-CSF mRNA was also significantly increased in prostate tissue of the EAP mice model. The expression of GM-CSF receptors was confirmed in mouse prostate and dorsal root ganglia. GM-CSF knockout mice showed fewer Infiltrating leukocytes and pain symptoms after the induction of EAP. Deletion of GM-CSF significantly diminished EAP-induced increases of chemokine (C-C motif) ligand 2, chemokine (C-C motif) ligand 3, and nerve growth factor mRNA expression. The results indicated that GM-CSF plays a functional role in the pathogenesis of EAP. GM-CSF may function as a signaling mediator for both inflammation and pain transduction in CP/CPPS.

Role of APLP2 in the prognosis and clinicopathology of renal cell carcinoma.

Identifying diagnostic and prognostic biomarkers is crucial for improved guidance of the treatment of renal cell carcinoma (RCC). Amyloid ß precursor-like protein 2 (APLP2) has been determined to serve an important role in the progression of a number of cancer types. However, the expression and significance of APLP2 in RCC remains unknown. In the present study, it was determined that the expression of APLP2 protein (n=10) and mRNA (n=8) expression was significantly decreased in clear cell RCC (CCRCC) tissues compared with that in matched normal renal tissues. The expression level of APLP2 was significantly associated with high Fuhrman grade, high pT stage, and presence of distant metastasis and lymph node metastasis (P<0.05). Multivariate analysis demonstrated that the expression of APLP2 was a significant independent predictor of disease-specific survival in renal cell carcinoma (P=0.026). Notably, APLP2 expression was significantly associated with disease-specific survival (P<0.001). APLP2 may be used to potentially predict patient prognosis, and to guide clinical diagnosis and treatment in CCRCC.

Ectopic Cushing's syndrome due to retroperitoneal ACTH-producing paragangliomas.

Extra-adrenal pheochromocytomas, or paragangliomas, are rare tumours that derive from extra-adrenal chromaffin cells. Cushing's syndrome (CS) caused by paragangliomas is extremely rare. We report a 53-year-old man with hypertension, diabetes, and symptoms of hypokalemia. Computer tomography (CT) revealed two retroperitoneal masses and bilateral adrenal hyperplasia. Together with the laboratory examinations, ectopic CS caused by multiple paragangliomas was highly suspected. The patient underwent resections of retroperitoneal tumours, left kidney, and left adrenal; postoperative histopathology confirmed two paragangliomas that were both positively stained for adrenocorticotropic hormone (ACTH). He got clinical and biochemical recoveries without any recurrent evidence at the nine-month followup.