The role of netrin-1 in the diagnosis and prognosis of bladder pain syndrome /interstitial cystitis: a comparative study.

There were no significant differences in age, BMI, hypertension, diabetes, or menopausal status among the BPS/IC, normal, and OAB groups ( P  > 0.05). The expression level of Netrin-1 in the BPS/IC group was 777.37 ± 268.02 pg/ml , which was significantly different from the other two groups ( P  < 0.001) ( Fig.  1 A ) . There was no significant difference in Netrin-1 expression between the OAB and healthy control groups, as shown in Table  1 . Table 1 Baseline data of BPS/IC, OAB, and normal groups BPS/IC Normal OAB P value Age (years), mean ± SD 50.20±11.80 51.4±10.12 50.70±11.87 0.929 BMI (Kg·m − 2 ), mean ± SD 22.46±3.01 22.97±2.43 22.45±1.94 0.758 Menstruation, n (%) 12 (30%) 10 (50%) 7 (35%) 0.313 Hypertension, n (%) 9 (22.5%) 2 (10%) 5 (25%) 0.453 Diabetes, n (%) 6 (15%) 2 (10%) 3 (15%) 0.854 Netrin-1 (pg/ml) 777.37±268.02 446.19±103.6 a 429.83±145.5 a <0.001 Note: a indicates a statistically significant difference compared to BPS/IC, with p  < 0.05 Abbreviations: Bladder Pain Syndrome /Interstitial Cystitis (BPS/IC); overactive bladder (OAB) Baseline data of BPS/IC, OAB, and normal groups Note: a indicates a statistically significant difference compared to BPS/IC, with p  < 0.05 Abbreviations: Bladder Pain Syndrome /Interstitial Cystitis (BPS/IC); overactive bladder (OAB) The ROC curve for Netrin-1 expression levels in distinguishing BPS/IC from healthy controls is shown in Fig.  1 B, with an AUC of 0.860 (95% CI: 0.768–0.952, P  < 0.001). At a cutoff value of 594.02 pg/ml, the sensitivity was 75%, specificity was 90%, and the accuracy was 75%. Fig. 1 Netrin-1 Expression Levels and ROC Analysis in BPS/IC, OAB, and normal Groups. ( A ) The expression of Netrin-1 in the BPS/IC group was significantly higher than in the Normal and OAB groups, while there was no difference in Netrin-1 expression between the Normal and OAB groups; ( B ) The ROC curve for Netrin-1 as a diagnostic biomarker showed an AUC of 0.860. Abbreviations: Bladder Pain Syndrome /Interstitial Cystitis (BPS/IC); overactive bladder (OAB) Netrin-1 Expression Levels and ROC Analysis in BPS/IC, OAB, and normal Groups. ( A ) The expression of Netrin-1 in the BPS/IC group was significantly higher than in the Normal and OAB groups, while there was no difference in Netrin-1 expression between the Normal and OAB groups; ( B ) The ROC curve for Netrin-1 as a diagnostic biomarker showed an AUC of 0.860. Abbreviations: Bladder Pain Syndrome /Interstitial Cystitis (BPS/IC); overactive bladder (OAB) The expression level of Netrin-1 also showed a certain correlation with the clinical scores of patients. The Urination frequency , ICSI, ICPI, and VAS scores were positively correlated with Netrin-1 expression, with correlation coefficients of 0.629 (95% CI: 0.386–0.790 , P  < 0.01) 0.612 (95% CI: 0.363–0.779 , P  < 0.01) 0.656 (95% CI: 0.39–0.79 , P  < 0.01) and 0.565(95% CI: 0.300–0.750 , P  < 0.01) , respectively, as shown in Fig.  2 A-D. After two months of bladder perfusion therapy , the frequency of urination , ICSI , ICPI , and VAS scores were positively correlated with Netrin-1 expression , with correlation coefficients of 0.581 (95% CI: 0.321–0.760 , P  < 0.01) , 0.593 (95% CI: 0.337–0.768 , P  < 0.01) , 0.609 (95% CI: 0.359–0.778 , P  < 0.01) , and 0.591(95% CI: 0.334–0.766 , P  < 0.01) , respectively , as shown in Fig.  2 E-H. Fig. 2 Correlation analysis of Netrin-1 expression with urination frequency, ICSI, ICPI, and VAS before and after treatment. A - D ) Before treatment, Netrin-1 expression showed a positive correlation with urination frequency, ICSI, ICPI, and VAS; E - H ) After two months of treatment, Netrin-1 expression remained positively correlated with urination frequency, ICSI, ICPI, and VAS Correlation analysis of Netrin-1 expression with urination frequency, ICSI, ICPI, and VAS before and after treatment. A - D ) Before treatment, Netrin-1 expression showed a positive correlation with urination frequency, ICSI, ICPI, and VAS; E - H ) After two months of treatment, Netrin-1 expression remained positively correlated with urination frequency, ICSI, ICPI, and VAS We also compared the Netrin-1 expression levels before and two months after treatment in BPS/IC patients. We found that after symptom relief following treatment, the Netrin-1 expression level in patients decreased to 485.06 ± 135.99 pg/ml, which was significantly lower than before treatment, as shown in Table  2 . Table 2 Comparison of Netrin-1 expression, urination frequency, ICSI, ICPI, and VAS before and after treatment pre-treatment post-treatment t P value Netrin-1 777.37±268.02 485.06±135.99 9.136 <0.001 Urination frequency 14.15±2.79 9.68±1.73 11.316 <0.001 ICSI 11.73±1.84 8.68±1.67 11.19 <0.001 ICPI 10.73±1.38 9.07±2.18 5.736 <0.001 VAS 3.40±1.60 3.25±1.26 0.798 0.43 Abbreviations: Bladder Pain Syndrome /Interstitial Cystitis (BPS/IC); overactive bladder (OAB); Interstitial Cystitis Symptom Index (ICSI); Interstitial Cystitis Problem Index (ICPI); and Visual Analog Scale (VAS) Comparison of Netrin-1 expression, urination frequency, ICSI, ICPI, and VAS before and after treatment Abbreviations: Bladder Pain Syndrome /Interstitial Cystitis (BPS/IC); overactive bladder (OAB); Interstitial Cystitis Symptom Index (ICSI); Interstitial Cystitis Problem Index (ICPI); and Visual Analog Scale (VAS)

Forty patients diagnosed with BPS/IC at our hospital who voluntarily participated in the study were included. The inclusion criteria were as follows: (1) voluntary participation in the study and signed informed consent, with good compliance; (2) age between 18 and 70 years; (3) more than 8 voids per day while awake, with ≥ 2 nocturia episodes and a disease duration of ≥ 6 months; (4) pelvic, perineal, lumbar, or urethral discomfort associated with bladder filling, significantly relieved after urination; (5) bladder capacity < 350 mL; (6) presence of Hunner’s ulcer or glomeruloid hemorrhagic spots under cystoscopy. Additionally, 20 patients with overactive bladder and 20 healthy controls were recruited. The diagnosis of OAB complies with the AUA guidelines [ 12 ]. The normal group consisted of healthy volunteers with no history of BPS/IC , no major chronic diseases such as tumors , and no other chronic inflammatory conditions. The study was approved by the hospital’s ethics committee (Approval No.2024655), and all participants signed informed consent forms. All participants were given a questionnaire to collect baseline data at enrollment. The data included age, gender, menstrual history, and medical history of hypertension and diabetes. For BPS/IC patients, assessments of the Interstitial Cystitis Symptom Index (ICSI) , Interstitial Cystitis Problem Index (ICPI) , and Visual Analog Scale (VAS) were also conducted [ 13 , 14 ]. The O’Leary-Sant ICSI/ICPI questionnaires assess urinary symptoms and pain severity , while the VAS quantifies overall discomfort. A double-checking process was performed to ensure the accuracy of the clinical baseline data. All BPS/IC patients were treatment naïve prior to enrollment and received weekly bladder instillations of heparin + gentamicin + low-molecular-weight heparin sodium after bladder hydrodistension , continuing until two months postoperatively. Venous blood was collected from all participants during episodes of urgent urination. After standing for 30 min, the blood was centrifuged at 3000 rpm for 10 min to separate the plasma. The supernatant was transferred to centrifuge tubes and stored at -80 °C. Additionally, two months after treatment, venous blood was drawn again from BPS/IC patients to measure Netrin-1 levels.The expression of Netrin-1 was measured using an ELISA kit (CSB-E11899h, Cusabio Biotech, Wuhan, China). And performed triplicate measurements for each sample to ensure accuracy and reproducibility. Data analysis was performed using SPSS 26.0 software. Continuous data are presented as means ± standard deviations (x̅ ± s). For data that follow a normal distribution with equal variances, a two-sample t-test was applied; and one-way analysis of variance (ANOVA) was used for analyzing differences among three groups. Categorical data are presented as n (%), and the Chi-square test was used. A P-value of < 0.05 was considered statistically significant.R 3.6.2 software was used to generate violin plots for Netrin-1 expression levels and analyze the diagnostic value of Netrin-1 by calculating the area under the ROC curve (AUC). Spearman’s correlation was applied to assess the relationship between Netrin-1 expression levels and the ICSI, ICPI, and VAS scores.

Netrin-1, as a novel biomarker for BPS/IC, not only demonstrates significant advantages in diagnosing BPS/IC but also holds potential for widespread clinical application, advancing early diagnosis and precision treatment of BPS/IC. However, the mechanisms by which Netrin-1 affects BPS/IC require further investigation.

This study found that Netrin-1 can serve as a diagnostic and prognostic biomarker for BPS/IC, with high sensitivity and specificity. Additionally, the expression level of Netrin-1 was positively correlated with clinical symptoms such as ICSI, ICPI, and VAS in BPS/IC patients, and after two months of treatment, Netrin-1 expression decreased as the patient’s symptoms improved, indicating that Netrin-1 may play an important role in the pathogenesis of BPS/IC. The Netrin family includes Netrin-1, Netrin-2, and Netrin-3, as well as more distantly related members such as Netrin-4, Netrin-G1, and G2. They are characterized by a conserved structure containing an N-terminal laminin-type domain, followed by three epidermal growth factor-like repeat sequences and a small, less conserved, positively charged C-terminal domain [ 15 , 16 ]. The most common receptors for Netrin-1 are the deleted in colorectal cancer (DCC) gene and the UNC5 homologous protein family (UNC5A-D), with other receptors including neogenin, membrane-bound glycoproteins, and more [ 17 , 18 ]. Netrin-1 is widely expressed in the nervous system, lungs, mammary glands, intestinal epithelium, and other tissues [ 19 , 20 ]. Subsequently, Netrin-1 has been increasingly found to be involved in the inflammatory responses of various diseases , such as hypoxia, experimental autoimmune encephalomyelitis, subarachnoid hemorrhage (SAH), rheumatoid arthritis, and chronic renal failure, clarifying its protective regulatory role in the inflammatory process [ 21 , 22 ]. Netrin-1 is highly expressed in vascular endothelial cells, and its receptor UNC-5B is highly expressed in leukocytes. After binding with UNC-5B, Netrin-1 can inhibit the migration and aggregation of leukocytes to sites of inflammation. Netrin-1 reduces inflammation by binding to Unc5b , promoting macrophage survival and retention [ 23 , 24 ]. During the inflammatory response, Netrin-1 can reduce the release of monocyte chemotactic protein-1 by epithelial cells, inhibit the infiltration of macrophages, and thus suppress the migration of inflammatory cells. It can also induce macrophage polarization to the M2 phenotype, which produces anti-inflammatory factors to inhibit the inflammatory response [ 25 ]. Netrin-1 can also significantly reduce the release of pro-inflammatory factors by CD4 + T cells under hypoxic conditions, enhance the effectiveness of nitric oxide, and reduce tissue damage under hypoxia [ 26 ]. Netrin-1 inhibits neuroinflammation and improves neural injury through the peroxisome proliferator-activated receptor γ (PPARγ)/NF-κB pathway. PPARγ is a downstream molecule of Netrin-1, and its activation can reduce inflammation by inhibiting the NF-κB pathway and decreasing the production of inflammatory cytokines [ 27 , 28 ]. Previous studies have reported biomarkers for BPS/IC, such as GP51, antiproliferative factor (APF), and nerve growth factor (NGF), but due to various reasons, these biomarkers have not been further translated into clinical practice [ 29 – 31 ]. For instance, although APF has high sensitivity, its complex detection method limits its clinical application; although nerve growth factor is easy to detect, it can be elevated in cases of urinary stones, urinary tract infections, or bladder outlet obstruction, resulting in low specificity [ 32 , 33 ]. Kuret et al. compared the levels of MMP9 in the serum and urine of IC/BPS patients and found that serum MMP9 levels were associated with disease severity , further confirming that serum biomarkers are more stable than urine biomarkers [ 34 ]. This study found that the AUC for plasma Netrin-1 was 0.860 , with a sensitivity of 75% and specificity of 90%. Furthermore , its detection method is simple and easy to perform. Additionally, it can differentiate BPS/IC from OAB, providing strong support for clinical decision-making and making it highly promotable. The limitation of this study is that the sample size is still small, and it is a single-center clinical trial, which may introduce diagnostic biases and unavoidable selection biases. Further studies with larger sample sizes are needed. Additionally , we did not further distinguish between HIC/BPS and NHIC/BPS. Moreover , we measured Netrin-1 expression in BPS patients during episodes of urinary urgency , but its expression in non-urgent states was not assessed , which requires further investigation.

Bladder pain syndrome/interstitial cystitis (BPS/IC) is a condition lasting more than six months , characterized by chronic pelvic pain, pressure, and bladder discomfort, accompanied by lower urinary tract symptoms such as urinary frequency and urgency [ 1 , 2 ]. This disease significantly impacts patients’ quality of life. The etiology of BPS/IC remains undetermined and is associated with the loss of urothelial barrier function , infections, autoimmune responses, and neuroinflammation, which consequently limits the available treatment options and their efficacy [ 3 – 5 ]. Therefore, a deeper understanding of the mechanisms of BPS/IC, the identification of novel biomarkers, and the development of targeted therapies have become urgent issues to address. Netrin-1 is a member of the laminin-like protein family and was initially considered an effective chemotactic molecule involved in axon guidance and cell migration regulation during embryonic development [ 6 ]. It is widely expressed in the bladder , colorectal tissue , nervous tissue , and neuronal cells , playing important physiological and pathological roles [ 7 , 8 ]. Recent studies have shown that Netrin-1 also functions as a novel anti-inflammatory factor, inhibiting the migration and infiltration of inflammatory cells, regulating inflammatory signaling pathways, and exerting protective effects on tissues and cells [ 9 ]. Furthermore, Netrin-1 has shown significant roles in alleviating pain associated with endometriosis and osteoarthritis [ 10 , 11 ]. Given that inflammation and pain are the two core pathological features of BPS/IC, the role of Netrin-1 in BPS/IC has not yet been studied, and its potential pathophysiological mechanisms merit further exploration. In this study, the expression of plasma Netrin-1 in BPS/IC patients was found to be 777.37 ± 268.02 pg/ml , which was significantly higher than that of the healthy control group 446.19 ± 103.65 pg/ml , with an AUC of 0.860. We found that it can serve not only as a potential biomarker but also as a prognostic indicator of disease severity. The findings provide new perspectives and possibilities for further developing therapeutic targets and strategies for BPS/IC.