Autophagy Impairment Is Involved In Midazolam-Induced Lipid Droplet Accumulation And Consequent Phagocytosis Decrease In BV2 Cells

Background: Anaesthetics allows the safe performance of complex surgical and diagnostic procedures.However,an increasing number of clinical observations and experimental suggest that the use of anaesthetics triggers morpho-functional changes in the central nervous system(CNS),particularly young and old individuals.At present,accumulating evidence indicates that the change of phagocytosis in microglia is involved in the development of central nervous system diseases(CNSDs).Microglia,the phagocytes of the brain,perform a series of functions that require energy consumption,which is crucial for immune surveillance and to maintain normal brain function.Studies have found that microglia can take up and utilize lipid,and that lipid metabolism plays a significance role in maintaining microglial function,especially phagocytosis.However,the effect of anaesthetics on nerve cell lipid utilization,especially microglia,and its potential effects on cell function still unclear.Elucidating the effects of anaesthetics on lipid droplets,which are specific lipid storage organs,and phagocytosis of microglia is crucial to discover a new therapeutic concept for postoperative CNS complications.Methods: In this study,we used immortalized microglial BV2 cells to explore the effects of the commonly used anaesthetic midazolam on BV2 cells lipid droplets and phagocytosis.Moreover,the mechanisms by which autophagy mediates midazolaminduced lipid droplet accumulation was also explored in BV2 cells.After midazolam treatment,lipid droplets in BV2 cells were stained with BODIPY 493/503 fluorescent probe,and the mean fluorescence intensity of each group was detected by flow cytometry,Moreover,the triglyceride levels were further detected by triglyceride quantification kit.For the phagocytosis of BV2 cells,after treated with midazolam for 24 hours,the cultured cells were treated with fluorescent latex beads for 4 hours,and then the phagocytosis of BV2 cells was evaluated by microscope observation and flow cytometry.A long-chain acyl-Co A synthase inhibitor Triacsin C was used to inhibit the formation of lipid droplets,and the phagocytosis of BV2 cells was detected by microscope and flow cytometry.In addition,the levels of autophagy-related proteins p62,LC3,phosphorylated TFEB and total TFEB were detected by western blot,and the fluorescence of m Cherry-GFP-LC3 B was observed by microscope to further evaluate the autophagy flux.Results: Our study found that 15 μM midazolam treatment of BV2 cells for 24 hours increased the mean fluorescence intensity and cellular triglyceride levels of BV2 cells compared to the control group.Microscopy revealed a decrease in phagocytosis of latex beads in BV2 cells treated with 15 μM midazolam for 24 hours compared to the control group.Flow cytometry assays similarly revealed that the mean fluorescence intensity of latex beads decreased after midazolam treatment.After inhibition of lipid droplets formation with Triacsin C,a long-chain acyl coenzyme A synthase inhibitor,the level of phagocytosed latex beads increased in midazolam-treated BV2 cells.In the autophagy assay,western blot assays revealed a significant increase in both p62 and LC3 II protein levels in midazolam-treated BV2 cells.Rapamycin treatment reduced the increase in p62 protein levels induced by midazolam.Observation under confocal microscopy revealed that cells in the midazolam-treated group formed more autophagosomes and fewer autolysosomes,while rapamycin treatment increased the level of autolysosomes.In addition,midazolam treatment significantly increased phosphorylated TFEB protein levels in BV2 cells,while rapamycin decreased phosphorylated TFEB in the midazolam-treated group.In addition,the rapamycin and midazolam co-treatment group decreased the mean fluorescence intensity and triglyceride levels of BV2 cells.Moreover,rapamycin and midazolam co-treatment group phagocytosed more latex beads compared to the sole midazolam treatment group.Conclusion: These findings reveal that midazolam effected the lipid utilization and decreased phagocytosis in microglia,inhibiting the accumulation of lipid droplets partially restores the phagocytosis.Moreover,midazolam impair autophagy,promoting autophagic reverse the lipid droplet accumulation and phagocytosis decrease.This study suggests autophagy is a target for attenuating lipid droplet accumulation,normal degradation of lipid droplets is important for maintaining microglia phagocytosis and attenuating the side effects of midazolam on the CNS.