Inhibition of ACSL4 Attenuates Behavioral Deficits by Regulating Ferroptosis in a Murine Model of Systemic Lupus Erythematosus

Abstract

Neuropsychiatric systemic lupus erythematosus (NPSLE) is a disorder with a poor prognosis characterized by psychiatric and neurological manifestations directly associated with systemic lupus erythematosus (SLE). Neutrophil ferroptosis has been identified as a significant contributor to neutropenia and disease progression in SLE, but its role in NPSLE remains unclear. Female MRL/lpr and MRL/Mpj mice were used. The selective ferroptosis inhibitor liproxstatin-1 and the acyl-CoA synthetase long-chain family member 4 (ACSL4) inhibitor rosiglitazone were administered separately. Assessments included behavioral testing, transmission electron microscopy (TEM), ELISA, Western blotting, RT-PCR, and Nissl staining. Our data showed that neurons in the brain parenchyma undergo ferroptosis, with decreased glutathione peroxidase 4 (GPX4) expression and increased levels of lipid peroxidation indicators and have the typical morphology of ferroptosis confirmed by transmission electron microscopy. Selective ferroptosis inhibitor liproxstatin-1 attenuated the neuropsychiatric manifestations, including depression-like and impulsive behaviors, of MRL/lpr mice. ACSL4 is the main enzyme in lipid metabolism. Our study further found that the utilization of rosiglitazone by inhibiting ACSL4 could also significantly attenuate neuropsychiatric manifestations of MRL/lpr mice. Moreover, blocking ACSL4 might considerably boost GPX4 levels and decrease lipid peroxidation indicators in NPSLE, with reduced neuronal damage, as well as reduced neuroinflammation. This study concluded that inhibiting ACSL4 could facilitate the recuperation of behavioral deficits by suppression of ferroptosis in NPSLE, implying that ACSL4 might be a potential new therapeutic focus for NPSLE.

Keywords: ACSL4; GPX4; ferroptosis; neuropsychiatric syndrome; systemic lupus erythematosus.

Figure 1

Ferroptosis is prevalent in NPSLE. (A) The protein levels of GPX4, COX2, and ACSL4 in MRL/Mpj and MRL/lpr mice at 20 weeks, respectively. (B–D) Quantification of relative expression of GPX4, COX2, and ACSL4; n = 6 in each group. (E,F) Quantification of GPX activity and LPO levels; n = 5 in each group. * p < 0.05, ** p < 0.01 vs. MRL/Mpj. (G) Representative transmission electron microscopy images of MRL/Mpj and MRL/lpr mice are shown. The red arrows show that the outer mitochondrial membrane of neurons has been damaged and that the number of mitochondrial cristae has diminished or vanished. Upper bar = 2 μm, lower bar = 1 μm, n = 3 each group.

Figure 2

Liproxstatin-1 attenuates the neuropsychiatric manifestations of NPSLE. (A) MRL/lpr mice were intraperitoneally administered 10 mg/kg liproxstatin-1 every other day for 12 weeks. (B,C) Quantification of daily food and water intake; n = 6 in each group. (D) Quantification of the tail suspension test; n = 6 in each group. (E–G) Quantification of total distance, center distance, and time spent in the center area during the open field test; n = 6 in each group. (H) The representatibe image of the open field test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. MRL/Mpj + DMSO group; # p < 0.05 vs. MRL/lpr + DMSO group.

Figure 3

Liproxstatin-1 inhibits ferroptosis to alleviate lipid peroxidation in NPSLE. (A) The representative images of the protein levels of COX2 and GPX4. (B,C) Quantification of COX2 and GPX4 relative expression; n = 6 in each group. (D–I) Quantification of GPX activity, LPO level, MDA level, 4-HNE level, GSH level, and SOD level; n = 5 in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. MRL/Mpj + DMSO group; # p < 0.05, ## p < 0.01 vs. MRL/lpr + DMSO group.

Figure 4

Inhibition of ACSL4 ameliorates neuropsychiatric manifestations of NPSLE (A) MRL/lpr mice were administered 10 mg/kg/day rosiglitazone with a normal diet for 12 weeks. (B,C) Quantification of daily food and water intake; n = 6 in each group. (D) Quantification of the tail suspension test; n = 6 in each group. (E–G) Quantification of total distance, center distance, and time spent in the center area in the open field test; n = 6 in each group. (H) The representative image of the open field test. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. MRL/Mpj + ND group; # p < 0.05, ### p < 0.001 vs. MRL/lpr + ND group.

Figure 5

Inhibition of ACSL4 suppresses ferroptosis and lipid peroxidation in NPSLE. (A) The representative images of the protein levels of GPX4 and COX2. (B,C) Quantification of GPX4 and COX2 relative expression; n = 6 in each group. (D–I) Quantification of GPX activity, LPO level, MDA level, 4-HNE level, GSH level, and SOD level; n = 5 in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. MRL/Mpj + ND group; # p < 0.05, ## p < 0.01 vs. MRL/lpr + ND group. (J) Representative transmission electron microscopy images. The red arrows show that the outer mitochondrial membrane of neurons has been damaged and that the number of mitochondrial cristae has diminished or vanished. Bar = 2 μm.

Figure 6

Inhibition of ACSL4 prevents neuronal death and reverses the inflammatory microenvironment in MRL/lpr mice. (A) The representative images of the Nissl staining in the hippocampus and cortex. Bar = 50 μm. (B–E) Quantification of surviving neurons in the hippocampus and cortex; n = 5 in each group. (F–H) Quantification of ELISA levels of inflammatory markers; n = 5 in each group. (I–K) Quantification of relative mRNA levels of inflammatory marker genes; n = 5 in each group. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. MRL/Mpj + ND group; # p < 0.05, ## p < 0.01, ### p < 0.001 vs. MRL/lpr + ND group.

Figure 7

The role of ferroptosis in NPSLE. ACSL4 induces ferroptosis by inhibiting GPX4, which reduces the rise of lipid ROS. By inhibiting ferroptosis, blocking ACSL4 with rosiglitazone greatly accelerates the recovery of behavioral abnormalities in NPSLE.