N-acetylserotonin alleviated the expression of interleukin-1β in retinal ischemia-reperfusion rats via the TLR4/NF-κB/NLRP3 pathway

Abstract

This study aimed to explore the effects of N-acetylserotonin (NAS) on the expression of interleukin-1β (IL-1β) in the retina of retinal ischemia-reperfusion injury (RIRI) rats via the toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB)/nod-like receptor pyrin domain containing 3 (NLRP3) signaling pathway. In this study, adult male Sprague Dawley rats were randomly divided into the sham, RIRI, RIRI + NAS and RIRI + TAK-242 + NAS groups. The rats in the RIRI + NAS and RIRI + TAK-242 + NAS groups were intraperitoneally injected with NAS 30 min before and after modeling. TAK-242, a selective TLR4 inhibitor, was administered by intraperitoneal injection in RIRI + TAK-242 + NAS group. The RIRI rat model was established by elevating the intraocular pressure to 110 mmHg for 60 min. The retinal structure and edema were assessed by H&E staining. The expression levels of TLR4, phosphorylated NF-κB (p-NF-κB), NLRP3, cleaved Caspase-1, and IL-1β in the retina of each group were detected using immunohistochemistry and Western blot. The correlations of the differences of TLR4⁺ and cleaved Caspase-1⁺ with IL-1β⁺ cells (between the NAS and the RIRI groups) were analyzed, using linear regression in the RIRI + NAS group. Results showed that thinner retina, more RGCs, and less TLR4⁺, p-NF-κB⁺, NLRP3⁺, cleaved Caspase-1⁺, and IL-1β⁺ cells in the retina were observed in the RIRI + NAS and RIRI + TAK-242 + NAS groups compared with the RIRI group 12 h after RIRI (all P < 0.01). Western blot analysis results showed that the expression of IL-1β in the RIRI + NAS group began to increase 6 h after RIRI, and it reached a high level 12 h after RIRI, and then decreased. And it was lower at each time point in the RIRI + NAS group than in the RIRI group, and there existed significant difference (all P < 0.01). Besides, the expression levels of TLR4, p-NF-κB, NLRP3, and cleaved Caspase-1 proteins in the RIRI + NAS and RIRI + TAK-242 + NAS groups decreased 12 h after RIRI compared with those in the RIRI group (all P < 0.01). The difference in IL-1β⁺ cells was significantly correlated with those of TLR4⁺ and cleaved Caspase-1⁺ cells in the RIRI + NAS group (r² = 0.9054 or 0.7431, P < 0.01). In conclusion, NAS could attenuate the expression of IL-1β by inhibiting the TLR4/NF-κB/NLRP3 signaling pathway, reduce the retina edema, and promote the survival of RGCs, thereby alleviating the retinal injury and exert its neuroprotective effect.

Keywords: Interleukin-1β; N-acetylserotonin; Rats; Retinal ischemia-reperfusion injury; TLR4/NF-κB/NLRP3 signaling pathway.