Nampt Silencing Inhibits ECM Degradation and Enhances Autophagy in Stress Urinary Incontinence Fibroblasts

Discussion Stress urinary incontinence (SUI) is regarded as an intractable disorder precipitated by the failure of urethral closure function, and exerts a tremendous burden on populations and healthcare facilities across the world [36]. Alteration of ECM production is a critical candidate for SUI incidence and prevalence [37]. Meanwhile, the hard-done work of our peers has shown the involvement of Nampt in a plethora of diseases due to its ability to regulate inflammatory reactions, immune behaviors, cell metabolism, and gene activity [38]. Interestingly, Nampt over-expression was previously indicated to enhance ECM degradation, thereby promoting pathological injuries [39]. In the current study, we elucidated that Nampt silencing enhanced SUI fibroblast autophagy and inhibited ECM degradation. Our study is the first-of-its-kind to shed a light on the effect of Nampt silencing on the inhibition of ECM degradation in SUI fibroblasts by enhancing autophagy.

Furthermore, we employed a novel approach to explore the regulatory factors and their downstream mechanisms in SUI fibroblasts, aiming to improve diagnostic and therapeutic approaches against SUI. Senescence, the process of aging, is well established as one of the causative factors of SUI [40]. Meanwhile, Nampt is also known to participate in senescence-related diseases due to its effects on the mediation of cellular metabolic behaviors, cell death, and re-programming [41]. Herein our study, we noticed that Nampt was highly-expressed in SUI patients and VD-induced SUI rat models. We further isolated fibroblasts from non-SUI patients and treated them with IL-1β to explore the specific role of Nampt in SUI, and uncovered that Nampt was over-expressed in IL-1β-treated fibroblasts. On a separate note, IL-1β activated Nampt expressions were previously indicated to retard articular chondrocyte differentiation and augment osteoarthritis development [42]. Similarly, another study demonstrated that Nampt induced by IL-1β treatment was overexpressed in fibroblasts of periodontitis, and further conferred a promotive effect on inflammatory reaction and alveolar bone disruption [32]. All in all, these evidences are suggestive of the negative effects of Nampt on SUI fibroblasts.

Recent investigations have illustrated that ECM degradation and production can exacerbate SUI through destroying cellular construction and energy metabolic behaviors [19]. Moreover, Nampt is known to regulate cellular biological behaviors and enhance ECM remodeling, which augment endothelial injury to the cardiovascular system [43]. Accordingly, to further elucidate the role of Nampt in ECM degradation of SUI fibroblasts, we injected LV-sh-Nampt into IL-1β-treated fibroblasts to down-regulate the Nampt expression, and found that Nampt silencing brought about increased expressions of COL1A, ACAN, and TIMP-1 and declined MMP-2 and MMP-9 expressions. Consistently, another prior study came indicated that Nampt depletion was associated with TIMP-1 activation, and could be further used to predict retarded ECM production of fibroblasts in diabetic nephropathy [11]. Further in line with our findings, the investigation performed by Peiro C et al. illustrated that Nampt could augment MMP-2 and MMP-9 expressions, while discouraging TIMP-1 levels to sabotage ECM metabolism and defenses in hypertensive patients during pregnancy [44]. Collectively, these findings and evidences indicate that silencing of Nampt can inhibit the IL-1β-induced SUI fibroblast ECM degradation.

On the other hand, Nampt exhaustion is also associated with enhanced autophagy, which results in decreased sepsis-induced acute lung injury [45]. The self-degradation mechanism of autophagy is involved in various pathologies by virtue of catalyzing aging cell loss and eliminating surplus or injured genes [46]. In addition, autophagy was previously associated with limited ECM production and promoted anti-senescent function in intervertebral disc degeneration [47]. Accordingly, we explored the mechanism underlying Nampt-regulation of autophagy in SUI fibroblasts, and discovered that Nampt silencing resulted in improved autophagy as evidenced by elevated LC3 II/I ratio and Beclin-1 expression levels. Similarly, prior studies have noted that suppression of Nampt protein levels brought about elevated LC3 levels, a well-known indicator of autophagy [48]. Moreover, Nampt knockdown was previously revealed to enhance autophagy by improving Beclin-1 levels, which is much in accordance with our findings [49]. Altogether, the aforementioned evidences indicated that Nampt silencing exerted a diminishing effect on IL-1β-treated SUI fibroblast autophagy.

Additionally, to verify the role of autophagy in the mechanism of Nampt in regulating ECM degradation in SUI fibroblasts, autophagy was inhibited with help of 3-MA in fibroblasts with Nampt silencing treatment, which revealed that inhibition of autophagy was associated with decreased expressions of COL1A, ACAN, and TIMP-1 in cells, and increased expressions of MMP-2 and MMP-9. Moreover, another investigation highlighted that during conditions of promoted autophagy, COL2A and ACAN expressions were accordingly activated, which underscores the positive association between COL2A, ACAN expressions and autophagy [50]. Meanwhile, in subjects suffering from endometriosis, reduced levels of autophagy were accompanied by elevated MMP-2 expressions [51]. It is also noteworthy that Nampt was previously shown to enhance the expression of ECM degradation proteins and decelerate autophagy [20]. In a word, it would be plausible to suggest that inhibition of autophagy attenuates the inhibitory effects of Nampt silencing on IL-1β-induced ECM degradation in SUI fibroblasts. However, being the first study to explore the role of Nampt in SUI, vast majority of our experiments were performed at a cellular level, and lacked histological detection of autophagy. In addition, we did not perform enough gain/loss-of-function assays. Nevertheless, we will thrive to define the role of Nampt in SUI with the help of in vivo experiments in our future endeavors.