Surgical injury to the pelvic nerve by pelvic tumour (bilateral pelvic nerve injury, BPNI) leads to bladder dysfunction, which is clinically common and difficult to treat, and the pathogenesis is still not completely clear. Recently, the role of mechanosensitive ion channel protein PIEZO1 in micturition reflex has received increasing attention[1], but its role in BPNI bladder dysfunction has not been explored. In this study, we intended to establish a rat model of bladder dysfunction caused by BPNI, to explore the changes in the expression and the mechanism of PIEZO1 in the bladder dysfunction caused by pelvic nerve injury, and to provide references for the discovery of novel targets for the treatment of BPNI bladder dysfunction.

Female SD rats were selected, and the bilateral pelvic nerves were squeezed to establish an animal model of BPNI bladder dysfunction, which was randomly divided into the BPNI-1W group, the BPNI-4W group, and the sham-operated group (Sham group). After 1 week of surgery in the BPNI-1W group, and 4 weeks of surgery in the BPNI-4W and Sham groups, bladder tissues were collected after urodynamic and other examinations were carried out respectively. The bladder tissues of rats in the BPNI-1W and Sham groups were subjected to transcriptome sequencing to screen for differentially expressed genes, and the differentially expressed genes were subjected to Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and Gene Set Enrichment Analysis (GSEA) enrichment analyses to screen for the involved signalling pathways. The expression changes and sites of key molecules of PIEZO1 and NLRP3 signalling pathways in bladder tissues were detected using immunofluorescence and Westernblot, and correlation analysis was performed.

Rats in the BPNI-1W group developed postoperative filling incontinence, and the urinary function was partially recovered in the BPNI-4W group, the maximum bladder capacity was (8.84±1.02) and (5.54±1.42) ml in the BPNI-1W group and the BPNI-4W group, respectively, and the residual urine was significantly higher than that of (8.31±1.05) and (4.16±1.37) ml in the Sham group [(3.35±0.39) ml and (0.11±0.03) ml, P<0.05], maximum intravesical pressure in BPNI-1W and BPNI-4W groups were (15.37±1.76) and (21.36±2.98) cmH2O, respectively, and voiding efficiency was (6.17±1.42)% and (26.14±6.13 ) % were significantly lower than those of Sham group [(39.76±3.13) cmH2O, (96.65±0.88) %, P<0.05]. Masson staining showed a significant increase in collagen fibers in the BPNI-4W group compared with the Sham group and the BPNI-1W group, whereas there was no significant change in the collagen content in the BPNI -1W group compared with the Sham group. HE staining showed a thickening of the bladder wall and an increase in vacuolization of the detrusor muscle in the BPNI group compared with the Sham group, and a marked subplasma membrane edema and infiltration of inflammatory cells in the detrusor muscle in the BPNI-1W group; Bladder weight ratio was significantly increased in both groups compared to Sham group. The expression of αSMA and Collagen I in the bladder tissue of the BPNI-4W group was significantly higher than that of the BPNI-1W group and Sham, and there was no significant difference in the expression of αSMA in the bladder tissue of the BPNI-1W and Sham groups. Transcriptome sequencing analysis of the bladder tissues of rats in the Sham and BPNI groups revealed that the differential genes were mainly enriched in mechanical stimulus response, inflammatory response, NLRP3 signalling pathway, calcium transmembrane input, membrane potential and muscle contraction regulation. Validation of the transcriptome sequencing results by Western Blot and immunofluorescence showed that the key molecules of PIEZO1 and NLRP3 signalling pathway, NLRP3, IL-1β, IL-18, gasderminD and caspase1, were highly expressed in the BPNI-1W group. Then, the expression of both PIEZO1 and key molecules of the NLRP3 pathway decreased in the BPNI-4W group to a level that was not significantly different from the Sham group. Correlation analysis revealed that the expression of PIEZO1 was positively correlated with the expression of key molecules in the NLRP3 inflammatory vesicle signalling pathway.

The significant reduction in filling incontinence and voiding efficiency at 1 week after BPNI suggests that the pathophysiological changes of bladder denervation may occur earlier. The lack of complete recovery of voiding function at 4 weeks after BPNI may be related to the difficulty of complete recovery after nerve injury, which is similar to the phenomenon found in the clinic. The incidence and severity of bladder dysfunction in the early postoperative period after pelvic nerve tumour surgery is high and some patients suffer from bladder dysfunction for a long period of time. The expression of key molecules in the PIEZO1 and NLRP3 pathways in bladder tissues increased with the increase in bladder pressure at 1 week after BPNI. The expression of key molecules in the PIEZO1 and NLRP3 pathways decreased with the decrease in bladder pressure as the bladder function was restored at 4 weeks after BPNI to the point that there was no significant difference from that in the Sham group. Previous studies have reported that both PIEZO1 and NLRP3 have important roles in the immune response and are associated with compensatory mechanisms of bladder fibrosis and denervation[2]. The intracellular signals activated by NLRP3 mainly include reactive oxygen species generation, mitochondrial dysfunction, and Ca2+ ion efflux. Miyamoto et al. found that PIEZO1 is indispensable for Ca2+ inward flow induced by uroepithelial cells following inductive stretch stimulation[3]. Therefore, it is reasonable to speculate that pelvic nerve injury bladder hypertension increases intracellular Ca2+ concentration by high expression of PIEZO1, and the increased intracellular Ca2+ may cause mitochondrial damage by inducing the accumulation of mitochondrial ROS and a decrease in membrane potential, which then activates the inflammatory response mediated by the NLRP3 pathway, leading to an increased release of inflammatory factors, such as IL-1β, IL-18 and other inflammatory factors, so that bladder de neurological changes and fibrosis aggravate bladder dysfunction.

By squeezing the bilateral pelvic nerves, a female rat detrusor underactivity model was successfully constructed, which showed increased bladder fibrosis, thickening of the bladder wall and impaired voiding function. Elevated bladder pressure in the early stage of BPNI induced the up-regulation of PIEZO1, which may be activating the NLRP3 pathway through Ca2+/mitochondrial damage causing inflammatory response to aggravate bladder dysfunction after BPNI. Inflammatory response may be an important target for early intervention of bladder dysfunction after BPNI.

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Solis AG, Bielecki P, Steach HR, Sharma L, Harman CCD, Yun S, et al. Mechanosensation of cyclical force by PIEZO1 is essential for innate immunity. Nature. 2019;573:69-74.
Miyamoto T, Mochizuki T, Nakagomi H, Kira S, Watanabe M, Takayama Y, et al. Functional role for Piezo1 in stretch-evoked Ca(2)(+) influx and ATP release in urothelial cell cultures. J Biol Chem. 2014;289:16565-75.

Continence 12S (2024) 101374
DOI: 10.1016/j.cont.2024.101374