UFL1 alleviates ER stress and apoptosis stimulated by LPS via blocking the ferroptosis pathway in human granulosa-like cells

Abstract

Ubiquitin-like modifier 1 ligating enzyme 1 (UFL1) is a unique E3 ligase of the UFMylation system. Recent studies have shown that this enzyme plays a crucial role in the processes of endoplasmic reticulum stress (ER stress) and apoptosis. Lipopolysaccharide (LPS) can cause injury to ovarian granule cells and hinder follicular development by triggering ER stress and apoptosis. Our study aimed to investigate the mechanism by which UFL1 alleviates ER stress and apoptosis caused by LPS in human granulosa-like cells (KGNs). In this study, we found that the protein levels of UFL1 were increased obviously under LPS stimulation in KGNs and that ER stress and apoptosis were further aggravated when UFL1 was knocked down; in contrast, these events were rescued when UFL1 was overexpressed. Next, we showed that the levels of ferroptosis-related proteins were relatively altered, accompanied by the accumulation of reactive oxygen species (ROS) and Fe2⁺, following the inhibition of UFL1 expression. In contrast, the overexpression of UFL1 reversed the ferroptosis process by regulating the P53/SLC7A11 (solute carrier family 7, member 11, SLC7A11) system and autophagy in response to LPS stimulation. Furthermore, apoptosis and ER stress in KGNs are rescued by the administration of the ferroptosis inhibitor ferrostatin-1 (Fer-1). Collectively, our research demonstrated a new mechanism for UFL1 that can alleviate ER stress and apoptosis stimulated by LPS; this occurred via the regulation of the ferroptosis pathway in KGNs and may provide a new strategy for research in the field of reproduction.

Keywords: Apoptosis; ER stress; Ferroptosis; UFL1.

Fig. 1

LPS stimulation led to UFL1 elevation, ER stress, and the occurrence of apoptosis. a, b Changes in UFL1 protein levels after 16 h of treatment with different LPS concentrations. c Changes in cell activity under different LPS doses. d–g The effect of different concentrations of LPS on the marker proteins BIP, XBP1s, and CHOP. h–k Changes in the levels of key apoptosis proteins (BAX and BCL-2) under different LPS concentrations. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 2

The inhibition of UFL1 expression aggravated LPS-induced apoptosis and ER stress. a, b UFL1 siRNA changed the expression levels of UFL1 n KGNs. c The cell growth curve in KGNs in which UFL1 had been knocked down. d Cell viability, as detected by CCK-8. e–h Changes in the protein expression of BIP, XBP1s, and CHOP with LPS stimulation in KGNs in which UFL1 had been knocked down. i–k Changes in the protein expression of BAX and BCL-2 with LPS stimulation in KGNs in which UFL1 had been knocked down. l The ratio of BAX/BCL-2 in LPS-stimulated KGNs after UFL1 knockdown. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 3

The overexpression of UFL1 alleviated LPS-induced apoptosis and ER stress. a–b The overexpression of UFL1 in KGNs. c The cell growth curve in KGNs overexpressing UFL1. d Cell viability, as detected by CCK-8 assays. e–h Changes in the protein expression of BIP, XBP1s, and CHOP after the overexpression of UFL1 alone. i–l Changes in the protein expression of BIP, XBP1s, and CHOP under LPS stimulation after UFL1 overexpression. m–o Changes in the protein levels of BAX and BCL-2 under LPS stimulation after UFL1 overexpression. p The ratio of BAX/BCL-2 under LPS stimulation after UFL1 overexpression. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 4

UFL1 knockdown led to ferroptosis and oxidative stress. a) Changes in the protein expression of UFL1, P53, SLC7A11, GPX4, P62, NRF2, LC3, and FHT1 after the silencing of UFL1. b–i Quantitative analysis of UFL1, P53, SLC7A11, GPX4, P62, NRF2, LC3, and FHT1 expression in KGNs in which UFL1 had been knocked down. j The production of ROS in KGNs in which UFL1 had been knocked down. Bar = 200 μm. k FerroOrange was used to detect changes in iron ions (Fe.²⁺⁾ in KGNs in which UFL1 had been knocked down. Bar = 100 μm. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 5

UFL1 regulated the P53/Xc system and autophagy-dependent ferroptosis. a Changes in the protein expression of P53, SLC7A11, GPX4, P62, NRF2, LC3, and FHT1 after UFL1 overexpression. b–h Quantitative analysis of P53, SLC7A11, GPX4, P62, NRF2, LC3, and FHT1 expression after UFL1 overexpression. i The products of ROS after UFL1 overexpression. Bar = 200 μm. j FerroOrange was used to detect changes in iron ions (Fe.²⁺) in KGNs in which UFL1 had been knocked down. Bar = 100 μm. *p < 0.05, **p < 0.01, and ***p < 0.001

Fig. 6

The administration of a ferroptosis inhibitor reduced cell damage caused by LPS stimulation. a–c Changes in the protein expression of SLC7A11 and GPX4 in LPS-stimulated KGNs treated with Fer-1. d Changes in cell activity in LPS-stimulated KGNs treated with Fer-1. e–h Changes in the protein expression levels of BIP, XBP1s, and CHOP in LPS-stimulated KGNs treated with Fer-1. i–l Changes in the protein expression levels of BAX and BCL-2 in LPS-stimulated KGNs treated with Fer-1. *p < 0.05, **p < 0.01, and ***p < 0.001