Nampt Silencing Induces Autophagy in IL-1β-Treated Fibroblasts and Inhibits ECM Degradation

Nampt silencing elicits IL-1β-treated fibroblast autophagy. Previous studies have also suggested that Nampt can influence autophagy [13,14], while changes in autophagy levels are known to affect ECM metabolism [16,35]. Therefore, we speculated that Nampt affected ECM metabolism by modulating autophagy, and subsequently examined the effects of Nampt on autophagy. It was found that IL-1β treatment led to decreased autophagy levels, as evidenced by decreased number of autophagosomes, declined ratio of LC3 II/I, and the downregulated expression of Beclin-1 (p < 0.01, Figure 5a-b), whereas LC3 and Beclin-1 are established as indicators of promoted autophagy [27,28]. Besides, immunofluorescence assay confirmed that IL-1β treatment brought about a reduction in LC3 fluorescence intensity in cells (p < 0.01, Figure 5c). On the other hand, enhanced autophagy levels were documented following Nampt silencing (p < 0.01, Figure 5a-c). Collectively, these findings indicated Nampt silencing evoked IL-1β-treated SUI fibroblast autophagy.

Nampt silencing inhibits IL-1β-induced fibroblast ECM degradation by inducing autophagy

Lastly, to verify the role of autophagy in the mechanism of Nampt in regulating ECM degradation in SUI fibroblasts, we employed 3-MA to inhibit fibroblast autophagy in the LV-sh-Nampt group. As revealed by the results of RT-qPCR and Western blot analysis, following inhibition of autophagy, the expression levels of COL1A, ACAN, and TIMP-1 were all decreased in cells, while those of MMP-2 and MMP-9 were increased (p < 0.05, Figure 6a-b). Results of immunofluorescence assay further illustrated the fluorescence confirmed that inhibition of autophagy reversed the inhibitory effect of Nampt silencing on IL-1β-induced ECM degradation in SUI fibroblasts.