The Role And Mechanisms Of ACSL4 Promotes Microglia-Mediated Neuroinflammation

Objective:Neuroinflammation is an important factor of neurodegeneration disease such as Parkinson’s disease and Alzheimer’s disease.Microglia play important roles in neuroinflammation under a variety of physiological and pathological conditions.Acyl-CoA synthetase long-chain family member 4(ACSL4)is an important isozyme for polyunsaturated fatty acid(PUFA)metabolism.The role of ACSL4 in LPS-induced inflammation of microglia,and the effects of ACSL4-mediated inflammation on the progression of Parkinson’s disease(PD)are unknown.Here,we aimed to investigate the effect and mechanism of ACSL4 on PD or other neurological diseases involving neuroinflammation.Methods:The expression of NF-κB p65,ERK and JNK signaling pathways in microglia cell line BV-2 stimulated by lipopolysaccharide(LPS)was detected by Western blot.Real Time PCR and enzyme-linked immunosorbent assay(ELISA)were used to detect the production of inflammatory cytokines after BV-2 transfected with si NC and si ACSL4.RNA-sequencing、untargeted lipidomic and flow cytometry was used to detect the process of ferroptosis the mechanism that how the ACSL4 influences the neuroinflammation process.Microglia-neuron co-culture system was used to assess the effect of ACSL4 knockdown on the viability of cortical neurons under LPS stimulation in vitro.The level of ACSL4 in SN region was inhibited by stereotaxic injection of lentivirus transfection method,and the systemic LPS model and acute MPTP model of mice were made to assess the effect of ACSL4 on microglia-mediated neuroinflammation in vivo.The number and activation of microglia cells and the survival rate of tyrosine hydroxylase positive(TH+)neurons were detected by immunofluorescence(IF),the production levels of inflammatory cytokines were detected by qRT-PCR.The rotarod test to assess the motor ability of mouse.Results: We showed that the expression of ACSL4 increased after LPS stimulation and mainly located at cytoplasm;the production of pro-inflammatory cytokines TNFA、IL-6and IL-1b decreased after ACSL4 knockdown in microglia,which was achieved through decreasing NF-κB signal activation which is not depended on ferroptosis manner.Mechanism researches showed that ACSL4 could inhibit Vestigial Like Family Member4(VGLL4)expression to regulate NF-κB activation.On the other hand,ACSL4 could change the lipid composition of cells to mediated the inflammation process.In vitro,knockdown ACSL4 directly reduced neuronal death after co-culture with the conditioned medium of BV2 cells by decreasing the cytokines production.In vivo,ACSL4 knockdown after stereotaxic injection of lentivirus in the SN region could inhibit inflammation process in systemic LPS model and acute MPTP model,which will increase the survival of neurons.Conclusion: These data revealed ACSL4 promotes microglia-mediated neuroinflammation by regulating lipid metabolism and the expression of VGLL4;identified ACSL4 is involved in regulating the inflammatory response that can influence the development of Parkinson’s disease.This study reveals the mechanism of lipid metabolism regulating neuroinflammation and provides important ideas for the treatment of neurodegenerative diseases such as Parkinson’s disease.