Background: Patients with interstitial cystitis/bladder pain syndrome (IC/BPS) often grieve over a low quality of life brought about by chronic pain. In our previous studies, we determined that neuroinflammation of the spinal dorsal horn (SDH) was associated with mechanisms of interstitial cystitis. Moreover, it has been shown that brain-derived neurotrophic factor (BDNF) participates in the regulation of neuroinflammation and pathological pain through BDNF-TrkB signaling; however, whether it plays a role in cyclophosphamide (CYP)-induced cystitis remains unclear. This study aimed to confirm whether BDNF-TrkB signaling modulates neuroinflammation and mechanical allodynia in CYP-induced cystitis and determine how it occurs. Methods: Systemic intraperitoneal injection of CYP was performed to establish a rat cystitis model. BDNF-TrkB signaling was modulated by intraperitoneal injection of the TrkB receptor antagonist, ANA-12, or intrathecal injection of exogenous BDNF. Mechanical allodynia in the suprapubic region was assessed using the von Frey filaments test. The expression of BDNF, TrkB, p-TrkB, Iba1, GFAP, p-p38, p-JNK, IL-1β, and TNF-α in the L6-S1 SDH was measured by Western blotting and immunofluorescence analysis. Results: BDNF-TrkB signaling was upregulated significantly in the SDH after CYP was injected. Similarly, the expressions of Iba1, GFAP, p-p38, p-JNK, IL-1β, and TNF-α in the SDH were all upregulated. Treatment with ANA-12 could attenuate mechanical allodynia, restrain activation of astrocytes and microglia and alleviate neuroinflammation. Besides, the intrathecal injection of exogenous BDNF further decreased the mechanical withdrawal threshold, promoted activation of astrocytes and microglia, and increased the release of TNF-α and IL-1β in the SDH of our CYP-induced cystitis model. Conclusions: In our CYP-induced cystitis model, BDNF promoted the activation of astrocytes and microglia to release TNF-α and IL-1β, aggravating neuroinflammation and leading to mechanical allodynia through BDNF-TrkB-p38/JNK signaling.

Background: Neuroinflammation in spinal dorsal horn (SDH) plays an important role in the pathogenesis of interstitial cystitis/bladder pain syndrome (IC/BPS). Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) exert potent anti-inflammatory activities in the treatment of various diseases. This study aimed to determine the therapeutic effects of MSC-EVs on IC and furtherly investigate the potential mechanism to attenuate neuroinflammation. Methods: Female IC rat model was established by intraperitoneal injection of cyclophosphamide (50 mg/kg, every 3 days for 3 doses). Inhibition of NLRP3 inflammasome was performed by intraperitoneal injection of MCC950 (10 mg/kg). MSC-EVs were isolated from the culture supernatants of human umbilical cord derived MSCs using ultracentrifugation, and then injected intrathecally into IC rats (20 μg in 10 μl PBS, every other day for 3 doses). Suprapubic mechanical allodynia was assessed using up-down method with von Frey filaments, and micturition frequency was examined by urodynamics. The expression of NLRP3 inflammasome components (NLRP3 and Caspase-1), glial cell markers (IBA-1 and GFAP), proinflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-18) and TLR4/NF-κB signal pathway (TLR4, p65 NK-κB and phospho-p65 NK-κB) in L6–S1 SDH was measured by Western blot analysis. The cellular localization of NLRP3 in SDH was detected using immunofluorescence co-staining. Results: NLRP3 inflammasome was activated in neurons in SDH of IC rats. NLRP3 inflammasome activation contributed to activation of glial cells and process of spinal neuroinflammation in IC rats, and was related to suprapubic mechanical allodynia and frequent micturition. Intrathecal injection of MSC-EVs alleviated suprapubic mechanical allodynia and frequent micturition in IC rats, restrained activation of glial cells and attenuated neuroinflammation in SDH. In addition, MSC-EV treatment significantly inhibited activation of both NLRP3 inflammasomes and TLR4/NF-κB signal pathway. Conclusions: NLRP3 inflammasome activation is involved in the neuroinflammation of IC. Intrathecal injection of MSC-EVs alleviates neuroinflammation and mechanical allodynia in IC by inhibiting the activation of NLRP3 inflammasome, and TLR4/NF-κB signal pathway may be the potential regulatory target.

Background: Bladder-related pain symptoms in patients with bladder pain syndrome/interstitial cystitis (BPS/IC) are often accompanied by depression and memory deficits. Magnesium deficiency contributes to neuroinflammation and is associated with pain, depression, and memory deficits. Neuroinflammation is involved in the mechanical allodynia of cyclophosphamide (CYP)-induced cystitis. Magnesium-L-Threonate (L-TAMS) supplementation can attenuate neuroinflammation. This study aimed to determine whether and how L-TAMS influences mechanical allodynia and accompanying depressive symptoms and memory deficits in CYP-induced cystitis. Methods: Injection of CYP (50 mg/kg, intraperitoneally, every 3 days for 3 doses) was used to establish a rat model of BPS/IC. L-TAMS was administered in drinking water (604 mg·kg⁻¹·day⁻¹). Mechanical allodynia in the lower abdomen was assessed with von Frey filaments using the up-down method. Forced swim test (FST) and sucrose preference test (SPT) were used to measure depressive-like behaviors. Novel object recognition test (NORT) was used to detect short-term memory function. Concentrations of Mg²⁺ in serum and cerebrospinal fluid (CSF) were measured by calmagite chronometry. Western blot and immunofluorescence staining measured the expression of tumor necrosis factor-α/nuclear factor-κB (TNF-α/NF-κB), interleukin-1β (IL-1β), and N-methyl-d-aspartate receptor type 2B subunit (NR2B) of the N-methyl-d-aspartate receptor in the L6–S1 spinal dorsal horn (SDH) and hippocampus. Results: Free Mg²⁺ was reduced in the serum and CSF of the CYP-induced cystitis rats on days 8, 12, and 20 after the first CYP injection. Magnesium deficiency in the serum and CSF correlated with the mechanical withdrawal threshold, depressive-like behaviors, and short-term memory deficits (STMD). Oral application of L-TAMS prevented magnesium deficiency and attenuated mechanical allodynia (n = 14) and normalized depressive-like behaviors (n = 10) and STMD (n = 10). The upregulation of TNF-α/NF-κB signaling and IL-1β in the L6–S1 SDH or hippocampus was reversed by L-TAMS. The change in NR2B expression in the SDH and hippocampus in the cystitis model was normalized by L-TAMS. Conclusions: Normalization of magnesium deficiency by L-TAMS attenuated mechanical allodynia, depressive-like behaviors, and STMD in the CYP-induced cystitis model via inhibition of TNF-α/NF-κВ signaling and normalization of NR2B expression. Our study provides evidence that L-TAMS may have therapeutic value for treating pain and comorbid depression or memory deficits in BPS/IC patients.

Background: Bladder cancer (BC) is a commonly occurring malignant tumor of the urinary system, demonstrating high global morbidity and mortality rates. BC currently lacks widely accepted biomarkers and its predictive, preventive, and personalized medicine (PPPM) is still unsatisfactory. N6-methyladenosine (m⁶A) modification and non-coding RNAs (ncRNAs) have been shown to be effective prognostic and immunotherapeutic responsiveness biomarkers and contribute to PPPM for various tumors. However, their role in BC remains unclear. Methods: m⁶A-related ncRNAs (lncRNAs and miRNAs) were identified through a comprehensive analysis of TCGA, starBase, and m6A2Target databases. Using TCGA dataset (training set), univariate and least absolute shrinkage and selection operator (LASSO) regression analyses were performed to develop an m⁶A-related ncRNA–based prognostic risk model. Kaplan-Meier analysis of overall survival (OS) and receiver operating characteristic (ROC) curves were used to verify the prognostic evaluation power of the risk model in the GSE154261 dataset (testing set) from Gene Expression Omnibus (GEO). A nomogram containing independent prognostic factors was developed. Differences in BC clinical characteristics, m⁶A regulators, m⁶A-related ncRNAs, gene expression patterns, and differentially expressed genes (DEGs)–associated molecular networks between the high- and low-risk groups in TCGA dataset were also analyzed. Additionally, the potential applicability of the risk model in the prediction of immunotherapeutic responsiveness was evaluated based on the “IMvigor210CoreBiologies” data set. Results: We identified 183 m⁶A-related ncRNAs, of which 14 were related to OS. LASSO regression analysis was further used to develop a prognostic risk model that included 10 m⁶A-related ncRNAs (BAALC-AS1, MIR324, MIR191, MIR25, AC023509.1, AL021707.1, AC026362.1, GATA2-AS1, AC012065.2, and HCP5). The risk model showed an excellent prognostic evaluation performance in both TCGA and GSE154261 datasets, with ROC curve areas under the curve (AUC) of 0.62 and 0.83, respectively. A nomogram containing 3 independent prognostic factors (risk score, age, and clinical stage) was developed and was found to demonstrate high prognostic prediction accuracy (AUC = 0.83). Moreover, the risk model could also predict BC progression. A higher risk score indicated a higher pathological grade and clinical stage. We identified 1058 DEGs between the high- and low-risk groups in TCGA dataset; these DEGs were involved in 3 molecular network systems, i.e., cellular immune response, cell adhesion, and cellular biological metabolism. Furthermore, the expression levels of 8 m⁶A regulators and 12 m⁶A-related ncRNAs were significantly different between the two groups. Finally, this risk model could be used to predict immunotherapeutic responses. Conclusion: Our study is the first to explore the potential application value of m⁶A-related ncRNAs in BC. The m⁶A-related ncRNA–based risk model demonstrated excellent performance in predicting prognosis and immunotherapeutic responsiveness. Based on this model, in addition to identifying high-risk patients early to provide them with focused attention and targeted prevention, we can also select beneficiaries of immunotherapy to deliver personalized medical services. Furthermore, the m⁶A-related ncRNAs could elucidate the molecular mechanisms of BC and lead to a new direction for the improvement of PPPM for BC.