Melatonin attenuates prostatic inflammation and pelvic pain via Sirt1-dependent inhibition of the NLRP3 inflammasome in an EAP mouse model.

BACKGROUND: Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common male genitourinary system disease. As a neuroendocrine hormone, melatonin possesses a variety of biological functions, among which its anti-inflammatory effects have recently drawn substantial attention. The purpose of the current research was to study the effect of melatonin on CP/CPPS and the underlying mechanisms using a mouse model of experimental autoimmune prostatitis (EAP). METHODS: The EAP mouse model was successfully established by subcutaneously injecting a mixture of prostate antigen and complete Freund's adjuvant. On Day 42, hematoxylin-eosin staining was used to evaluate the histological appearance of prostate tissues. Chronic pelvic pain development was assessed by suprapubic allodynia. The levels of inflammation-related cytokines, such as interferon-γ, interleukin (IL)-17, and IL-1ß, were detected by enzyme-linked immunosorbent assay. Then, we explored the anti-inflammatory effects of melatonin on CP/CPPS by Western blotting and immunohistochemical staining, by measuring the expression of silent information regulator 1 (Sirt1) and NLRP3 inflammasome-related proteins in EAP mice. RESULTS: The EAP model mice exhibited severe diffuse leukocyte infiltration and significantly increased pelvic pain compared to the control mice. In the melatonin treatment group, the histological appearance of the prostate tissues, pelvic pain development, and the levels of proinflammatory cytokines were significantly alleviated compared to the EAP + dimethyl sulfoxide group. Furthermore, we found that the protective effects of melatonin were achieved through activation of the Sirt1 pathway and downregulation of the NLRP3 inflammasome. CONCLUSIONS: The results indicated that melatonin could attenuate prostate inflammation and pelvic pain by inhibiting the NLRP3 inflammasomes signaling pathway through the activation of Sirt1 in mice with EAP, and these efforts should provide a promising therapeutic strategy for CP/CPPS.

CaMK4-dependent phosphorylation of Akt/mTOR underlies Th17 excessive activation in experimental autoimmune prostatitis.

Chronic prostatitis and chronic pelvic pain syndrome (CP/CPPS) is a complicated syndrome characterized by genitourinary pain in the absence of bacterial infection. Th17 cell-driven autoimmunity has been proposed as a cause of CP/CPPS. However, the factors that promote Th17-driven autoimmunity in experimental autoimmune prostatitis (EAP) and the molecular mechanisms are still largely unknown. Here, we showed that Th17 cells were excessively activated, and blockade of IL-17A could effectively ameliorate various symptoms in EAP. Furthermore, we revealed that calcium/calmodulin-dependent kinase Ⅳ (CaMK4), especially Thr196 p-CaMK4 was increased in the Th17 cells of the EAP group, which were activated by intracellular cytosolic Ca2+ . Pharmacologic and genetic inhibition of CaMK4 decreased the proportion of Th17 cells, and the protein and mRNA level of IL-17A, IL-22, and RORγt. The phosphorylation of CaMK4 was dependent on the increase in intracellular cytosolic Ca2+ concentration in Th17 cells. A mechanistic study demonstrated that inhibition of CaMK4 reduced IL-17A production by decreasing the phosphorylation of Akt-mTOR, which was well accepted to positively regulate Th17 differentiation. Collectively, our results demonstrated that Ca2+ -CaMK4-Akt/mTOR-IL-17A axis inhibition may serve as a promising therapeutic strategy for CP/CPPS.

Abnormal gut microbiota composition is associated with experimental autoimmune prostatitis-induced depressive-like behaviors in mice.

BACKGROUND: Depressive symptoms are found in approximately 78% of chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) patients, but the pathological mechanisms remain unknown. Increasing evidence suggests that abnormal gut microbiota may play an important role in depression. Thus, we aimed to investigate whether gut microbiota contributes to CP/CPPS-associated depression by using a mouse model of experimental autoimmune prostatitis (EAP). METHODS: Male nonobese diabetic mice were immunized twice by subcutaneous injection of prostate antigen and adjuvant. Behavioral tests consisted of an open field test, sucrose preference test, forced swimming tests, and tail suspension test was used to confirm the depression-like symptoms that were induced by EAP. Then, fecal samples were collected, and 16S ribosomal RNA gene sequencing was performed to detect differences in gut microbiota composition between control and EAP group. Additionally, fecal bacteria from the control and EAP mice were transplanted into antibiotics-induced pseudo-germ-free mice to investigate the effects on host behaviors and the composition of gut bacteria. RESULTS: EAP was successfully established and exhibited depressive-like behaviors in mice. The 16S rRNA analysis of fecal samples indicated the abnormal composition of gut microbiota in the EAP mice compared to the control mice. In the fecal microbiota transplant study, antibiotics-treated pseudo-germ-free mice presented depressive states as compared to naïve mice. Fecal bacteria transplant from EAP mice, but not from control mice, into the pseudo-germ-free mice, significantly exaggerated host depression-like behaviors. Moreover, fecal bacteria transplants from control and EAP mice induced distinct alterations in α-diversity and ß-diversity indices. In all, 24 bacteria at six phylogenetic levels were remarkably changed by the fecal bacteria transplantation. CONCLUSIONS: Abnormal gut microbiota composition after EAP induction may contribute to the development of depression in mice. A therapeutic strategy that targets gut microbiota may provide an alternative treatment for alleviating this condition.

High glucose intake exacerbates experimental autoimmune prostatitis through mitochondrial reactive oxygen species-dependent TGF-ß activation-mediated Th17 differentiation.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a common inflammatory immune disease of the urogenital system. High glucose intake is considered to be a potential promoter of autoimmune diseases. However, the influence of high glucose intake on CP/CPPS is unknown. This research aimed to explore the influences of high glucose intake on experimental autoimmune prostatitis (EAP), a valid animal model of CP/CPPS, and the underlying mechanism. NOD mice received 20% glucose water or normal water treatment during EAP induction. EAP severity and Th17 cell responses were evaluated. Then, we explored the effects of an IL-17A neutralizing antibody, an inhibitor of TGF-ß, the reactive oxygen species (ROS) inhibitor NAC, and the mitochondrial ROS (mtROS) antioxidant MitoQ on glucose-fed EAP mice. The results demonstrated that high glucose intake aggravated EAP severity and promoted Th17 cell generation, which could be ameliorated by the neutralization of IL-17A. In vitro experiments showed that high dextrose concentrations promoted Th17 cell differentiation through mtROS-dependent TGF-ß activation. Treatment with TGF-ß blockade, NAC, or MitoQ suppressed Th17 cell generation both in vivo and in vitro, resulting in the amelioration of EAP manifestations caused by high glucose intake. This study revealed that high glucose intake exacerbates EAP through mtROS-dependent TGF-ß activation-mediated Th17 differentiation. Our results may provide insights into the molecular mechanisms underlying the detrimental effects of an environmental factor, such as high glucose intake, on CP/CPPS.

The CXCL10/CXCR3 axis regulates Th1 cell differentiation and migration in experimental autoimmune prostatitis through the PI3K/AKT pathway.

OBJECTIVE: To investigate the mechanism of the CXCL10/CXCR3 axis regulating Th1 cell differentiation and migration through the PI3K/AKT pathway in chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS). METHODS: Experimental autoimmune prostatitis (EAP) model, a well-described and validated animal model of CP/CPPS, was used in our study. After treatment with CXCL10, the severity of EAP and Th1 cell proportion were respectively measured by HE stains, immunohistochemistry, and flow cytometry. Then, the protein expression of the PI3K/AKT pathway in CXCL10/CXCR3-regulated Th1 cell differentiation and migration was evaluated by western blotting. Additionally, by the CXCR3 antagonist AMG487 and the PI3K inhibitor LY294002 applications, the effects of CXCL10/CXCR3 through PI3K/AKT pathway on the Th1 cell differentiation and migration were further assessed. RESULTS: The EAP model was successfully built. CXCL10 increased the proportion of Th1 cells in EAP mice, accompanied by upregulation of the PI3K/AKT pathway. Additionally, the PI3K/AKT pathway was found to be involved in CXCL10/CXCR3 axis-mediated Th1 cell differentiation and migration. CONCLUSIONS: Our investigations indicate that the CXCL10/CXCR3 axis regulates Th1 cell differentiation and migration in EAP through the PI3K/AKT pathway, which provides a new perspective on the immunological mechanisms of CP/CPPS.

BLCA prognostic model creation and validation based on immune gene-metabolic gene combination.

BACKGROUND: Bladder cancer (BLCA) is a prevalent urinary system malignancy. Understanding the interplay of immunological and metabolic genes in BLCA is crucial for prognosis and treatment. METHODS: Immune/metabolism genes were extracted, their expression profiles analyzed. NMF clustering found prognostic genes. Immunocyte infiltration and tumor microenvironment were examined. Risk prognostic signature using Cox/LASSO methods was developed. Immunological Microenvironment and functional enrichment analysis explored. Immunotherapy response and somatic mutations evaluated. RT-qPCR validated gene expression. RESULTS: We investigated these genes in 614 BLCA samples, identifying relevant prognostic genes. We developed a predictive feature and signature comprising 7 genes (POLE2, AHNAK, SHMT2, NR2F1, TFRC, OAS1, CHKB). This immune and metabolism-related gene (IMRG) signature showed superior predictive performance across multiple datasets and was independent of clinical indicators. Immunotherapy response and immune cell infiltration correlated with the risk score. Functional enrichment analysis revealed distinct biological pathways between low- and high-risk groups. The signature demonstrated higher prediction accuracy than other signatures. qRT-PCR confirmed differential gene expression and immunotherapy response. CONCLUSIONS: The model in our work is a novel assessment tool to measure immunotherapy's effectiveness and anticipate BLCA patients' prognosis, offering new avenues for immunological biomarkers and targeted treatments.

Gut Microflora Modulates Th17/Treg Cell Differentiation in Experimental Autoimmune Prostatitis via the Short-Chain Fatty Acid Propionate.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) is a very common urological disorder and has been gradually regarded as an immune-mediated disease. Multiple studies have indicated that the gut microflora plays a pivotal part in immune homeostasis and autoimmune disorder development. However, whether the gut microflora affects the CP/CPPS, and the underlying mechanism behind them remain unclear. Here, we built an experimental autoimmune prostatitis (EAP) mouse model by subcutaneous immunity and identified that its Th17/Treg frequency was imbalanced. Using fecal 16s rRNA sequencing and untargeted/targeted metabolomics, we discovered that the diversity and relative abundance of gut microflora and their metabolites were obviously different between the control and the EAP group. Propionic acid, a kind of short-chain fatty acid (SCFA), was decreased in EAP mice compared to that in controls, and supplementation with propionic acid reduced susceptibility to EAP and corrected the imbalance of Th17/Treg cell differentiation in vivo and in vitro. Furthermore, SCFA receptor G-protein-coupled receptor 43 and intracellular histone deacetylase 6 regulated by propionic acid in Th17 and Treg cells were also evaluated. Lastly, we observed that fecal transplantation from EAP mice induced the decrease of Treg cell frequency in recipient mice. Our data showed that gut dysbiosis contributed to a Th17/Treg differentiation imbalance in EAP via the decrease of metabolite propionic acid and provided valuable immunological groundwork for further intervention in immunologic derangement of CP/CPPS by targeting propionic acid.

Microglial activation and neurobiological alterations in experimental autoimmune prostatitis-induced depressive-like behavior in mice.

BACKGROUND: Patients with chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) frequently show depressive symptoms clinically and increasing evidence indicates a correlation between CP/CPPS and depression. However, the underlying mechanisms of CP/CPPS-related depression remain poorly understood. Here, we sought to determine the role of hippocampal microglial activation and neurobiological changes in a mouse model of experimental autoimmune prostatitis (EAP)-induced depression and the treatment efficacy of Chinese herb extract baicalein. METHODS: EAP was induced through intradermal injection of prostate antigen and adjuvant twice. Then, mice were assessed for affective behaviors in the open field test, elevated plus maze, forced swim test, and tail suspension test. The morphology and function of microglia and astrocytes were detected by immunofluorescence, Western blotting, and transmission electron microscopy. Proinflammatory mediators along with serotonin transporter (SLC6A4/SERT) and indoleamine 2,3-dioxygenase (IDO) were quantified with reverse transcription-polymerase chain reaction (RT­PCR), and serum serotonin concentrations were measured by enzyme-linked immunosorbent assay (ELISA). Proton magnetic resonance spectroscopy (1H-MRS) was performed to measure hippocampal glutamate levels. In addition, baicalein was used in a subset of EAP mice to test its anti-depressant action. RESULTS: EAP was successfully established and induced depressive- and anxiety-like behavior in mice. Increasing levels of co-expressed ionized calcium-binding adapter molecule 1 (Iba1) and glial fibrillary acidic protein (GFAP) and ultrastructural observations suggested microglial activation and reactive astrocytosis in the hippocampus. These activated microglia resulted in increased expressions of multiple proinflammatory cytokines. Simultaneously, EAP mice showed higher gene expressions of SLC6A4 and IDO and lower concentrations of serotonin. 1H-MRS indicated a decrease in the glutamate + glutamine (Glx)/total creatine (tCr) ratio in EAP mice. Furthermore, baicalein treatment alleviated the depressive-like behavior and neuroinflammation by suppressing the nuclear factor-kappa B (NF-κB) pathway. CONCLUSION: Our data indicate that EAP-induced depressive-like behavior is linked to microglia activation and subsequent neurotransmitter metabolism. Moreover, baicalein attenuates behavioral changes by inhibiting neuroinflammation via downregulation of the NF-κB pathway.

MicroRNA expression profile in chronic nonbacterial prostatitis revealed by next-generation small RNA sequencing.

MicroRNAs (miRNAs) are considered to be involved in the pathogenic initiation and progression of chronic nonbacterial prostatitis (CNP); however, the comprehensive expression profile of dysregulated miRNAs, relevant signaling pathways, and core machineries in CNP have not been fully elucidated. In the current research, CNP rat models were established through the intraprostatic injection of carrageenan into the prostate. Then, next-generation sequencing was performed to explore the miRNA expression profile in CNP. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) bioinformatical analyses were conducted to reveal the enriched biological processes, molecular functions, and cellular components and signaling pathways. As a result, 1224, 1039, and 1029 known miRNAs were annotated in prostate tissues from the blank control (BC), normal saline injection (NS), and carrageenan injection (CAR) groups (n = 3 for each group), respectively. Among them, 84 miRNAs (CAR vs BC) and 70 miRNAs (CAR vs NS) with significantly different expression levels were identified. Compared with previously reported miRNAs with altered expression in various inflammatory diseases, the majority of deregulated miRNAs in CNP, such as miR-146b-5p, miR-155-5p, miR-150-5p, and miR-139-5p, showed similar expression patterns. Moreover, bioinformatics analyses have enriched mitogen-activated protein kinase (MAPK), cyclic adenosine monophosphate (cAMP), endocytosis, mammalian target of rapamycin (mTOR), and forkhead box O (FoxO) signaling pathways. These pathways were all involved in immune response, which indicates the critical regulatory role of the immune system in CNP initiation and progression. Our investigation has presented a global view of the differentially expressed miRNAs and potential regulatory networks containing their target genes, which may be helpful for identifying the novel mechanisms of miRNAs in immune regulation and effective target-specific theragnosis for CNP.